Embryology - User contributions [en-gb]

User:Z5020373

2016-10-28T02:08:59Z

Z5020373: /* Lab 11 */

{{ANAT2341Student2016}}
{{ANAT2341Rebecca2016}}

{|
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] 4 August 2016 - Thank you for adding this content before the lab. I would suggest that rather than using a template that you simply paste on this current page with separate subheadings for each lab/assessment item. Also please no names, just your student number.
|}

==Lab 1==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:36, 5 August 2016 (AEST)

===Lab Demonstrations===

====External Link====

[http://www.smh.com.au/ SMH]

====Internal Link====

https://embryology.med.unsw.edu.au/embryology/index.php/2011_Lab_1

[[2011_Lab_1|ANAT2341 Lab 1]]

[[Student Page]]

====Referencing====
[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486280

===Lab 1 Assessment===
<pubmed>27123200</pubmed>

This research article explores whether choosing to a conduct embryo transfer (ET) on a weekend or weekday will affect the success of clinical pregnancy in IVF procedures. In the study, ET transfers were performed on either weekdays or weekends in patients with similar clinical characteristics, such as age, body-mass index and duration of infertility. Clinical pregnancy was determined using blood pregnancy tests and ultrasound examination and was defined as the “presence of a gestational sac with a foetal heart beat.” After the ETs, the authors found that there was an overall 42.8% success rate of clinical pregnancy in patients from both groups, with a 14.6% increase in the pregnancy rate when weekend ETs where compared to weekday ETs. The study however, did not examine any possible reasons to explain this increase in implantation rate although a few potential factors were discussed from previous findings in this area of research. These included endometrial receptivity which occurs 5 days after the post-ovulatory progesterone surge. The article mentioned that uterine receptivity and implantation could be affected by the junctional zone. The extent of junctional zone contractility differs throughout the ovarian cycle and an increased contractility just before ET has been previously shown to significantly decrease the likelihood of successful implantation. Since weekends are more relaxing than weekdays they suggest a possible correlation between that and reduced junctional zone contractions leading to easier ETs. Therefore from this study, it was concluded that ETs performed during the weekends are more successful than those performed during the weekdays identifying a potential factor that can improve ETs in IVF situations.

{| width=95%
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 14:24, 15 August 2016 (AEST) - This is a good summary of a paper that looks at potential environmental/endocrine effects on reproductive fertility. You needed to put the reference at the top rather than just the PMID number, fix this and you can get this full mark for the exercise.

[mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You did not fix, so I have adjusted the final mark.
| width=100px| Assessment 4/5
|}

==Lab 2==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:41, 12 August 2016 (AEST)

===Lab 2 Assessment===
[[File: Amnion_fold_development_in_chicken_embryos.jpg]]

Chicken embryo amion fold development<ref><pubmed>24647352</pubmed></ref>

{|
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 14:24, 15 August 2016 (AEST) - Very good, the image relates to early development and contains the reference, copyright and student template. (5/5)
|}

==Lab 3==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:53, 19 August 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Mesoderm_Development#Paraxial_Mesoderm|Question 2 - paraxial]]

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]
| Assessment 2.5/5
|}

==Lab 4==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:02, 26 August 2016 (AEST)

===Lab 4 Assessment===

Take the Quiz

Make your selection for all questions before clicking submit.

<quiz display=shuffle>

{How many rotations does the stomach undergo during GIT development in week 4 to 5?
|type="()"}
- 1
+ 2
- 3
- 4

|| The stomach undergoes [[two]] embryonic 90 degree rotations: the first to establish the J-shape that forms the adult stomach body (classic curvature), and the second rotation establishes it in its correct anatomical position.

{Following the degeneration of the buccopharyngeal membrane, the foregut is open to which cavity?
|type="()"}
- The peritoneal cavity
- The chorionic cavity
- The yolk sac
+ The amniotic cavity

|| During week 4 of development, the breakdown of the buccopharangeal membrane exposes the foregut to the amniotic cavity where amniotic fluid is then able to fill the foregut.

{Which one of these is not an abnormality that can occur during the proliferation and re-canalisation of the gut tube?
|type="()"}
- Occlusion
+ Meckel's diverticulum
- Duplication
- Stenosis

||During re-canalisation, if the gut tube does not re-canalise the tube can remain completely occluded. Another senario would be if there is renalisation but it occurs in discrete channels to give rise to duplicated gut tubes. The third abnormality occurs when there is incomplete vasculisation which leads to stenosis or narrowing of the tube. The only abnormality that is not involved in Meckel's diverticulum and is associated with failure of Vitelline duct breakdown leaving a yolk stalk remnant. It is a common abnormality with a prevalence of 1-2% and can lead to infection and possibly affect the rotation of the midgut.

{During week 4 in GIT development:
|type="()"}
- The cloacal membrane is broken down while the buccopharyngeal membrane remains intact
+ The buccopharyngeal membrane is broken down while the cloacal membrane remains intact
- Both the buccopharyngeal and cloacal membranes break down simultaneously
- Both the buccopharyngeal and cloacal membranes remain intact.

||Loss of the buccopharangeal membrane during week 4 allows amniotic fluid into the foregut. The cloacal membrane remains intact and does not break down until the cloaca is divided into urogenital sinuses and the rectum (occurs later in embryonic development.

</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem well designed GIT quiz questions that test topic understanding.
| Assessment 5/5
|}

==Lab 5==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:11, 2 September 2016 (AEST)

===Lab 5 Assessment===
Questionaire completed and submitted
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:17, 9 September 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 11 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab 6==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 9 September 2016 (AEST)

===Lab 6 Assessment===

<pubmed>25382630</pubmed>
Cleft palate arises when the bilateral palatal shelves fail to fuse. Many genetic and environmental factors have been identified to contribute to this deformity. One genetic mutation associated with cleft palate is the loss transforming growth factor-beta receptor (TGF-βR). A recent article by Hill ''et al.'' demonstrated that loss of TGF-βR3 reduced the expression of several ligands and receptors in the TGF-β/BMP family, including three TGF-β ligands and BMP2. During embryonic development, these molecules are involved in cell growth and differentiation.

A loss of TGF-β/BMP signaling, by receptor loss was found to be associated with cleft palate formation due to aberrant cell cycle progression and altered gene expression. Furthermore changes to TGF-β/BMP signaling also interrupted vascular development and remodeling as well as osteogenic differentiation during palate formation.
TGF-βR3 is therefore essential for maintaining the expression of TGF-β and BMP molecules and without this receptor processes of palatal shelf elongation, elevation and fusion are disrupted leading to cleft palate.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Good reference and explanation.
| Assessment 5/5
|}

==Lab 7==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 16 September 2016 (AEST)

===Lab 7 Assessment===

1. What is/are the dystrophin mutation(s)?

**The dystrophin gene is located on the short arm of the X chromosome at position 21.2. <ref>Converse, P.J. (2016) MUSCULAR DYSTROPHY, DUCHENNE TYPE; DMD OMIM http://www.omim.org/entry/310200</ref>
**It is the largest gene found in nature spanning 1.5% of the X-chromosome which is about 2.5 Mb of genomic sequence. <ref><pubmed>11917091</pubmed></ref>
**Its size could explain why it is so susceptible to spontaneous mutations.
**Encodes for Dystrophin protein.
**Mutations such as large deletions (60-70% of DMD cases), large duplications (10% of DMD cases) and point mutations (15-30% of DMD cases) can occur resulting in gene inactivation (therefore loss of function).<ref>https://www.duchenneconnect.org/understanding-genetic-testing/types-of-mutations-in-the-dystrophin-gene.html</ref>

2. What is the function of dystrophin?

**Dystrophin is part of a protein complex that work together to strengthen muscle fibers and protect them from injury as muscles contract and relax.
**Dystrophin complex acts as an anchor, connecting each muscle cell's structural framework (cytoskeleton) with the lattice of proteins and other molecules outside the cell (extracellular matrix).
**May also play a role in cell signaling by interacting with proteins that send and receive chemical signals e.g. in alpha-syntrophin.<ref><pubmed>12082140</pubmed></ref>

3. What other tissues/organs are affected by this disorder?

**Muscle (skeletal, cardiac and smooth): fatigue, difficulty with motor skills
**Respiratory system: pneumonia
**Cardiac system: cardiac myopathy
**Central Nervous system (CNS): neurobehavioural disorders e.g. ADHD and dyslexia <ref><pubmed>13947981</pubmed></ref>

4. What therapies exist for DMD?

**There is no cure available for DMD, and the current interventions are based on preventing further muscle wasting and management of symptoms and complications
**Treatments include:
***Physical therapy
***Orthopedic appliances
***Medication:
****Two corticosteroids mainly used in DMD treatment are Prednisone/Prednisolone and Deflazacort, an oxazoline derivative of prednisolone, administered by two common regimens: daily and intermittent <ref><pubmed>26457695</pubmed></ref>
****Prednisone and prednisolone show an anti-inflammatory effect
***Deflazacort acts on muscle regeneration and differentiation
** Future could include:
**Cell-based therapies using stem cells to replace dystrophin gene (a potential cure).
***Gene therapies to deliver a therapeutic gene to skeletal and cardiac muscle, in order to restore the dystrophin protein PMID <ref><pubmed>7683332</pubmed></ref>
***A study by Nelson et al. in 2016, showed that in vivo CRISPR-Cas9–mediated dystrophin restoration in mdx mouse model of DMD removed the mutated exon 23 from the dystrophin gene and improved muscle structure and function.<ref><pubmed>25123483</pubmed></ref>

5. What animal models are available for muscular dystrophy?
**The most widely used animal model for DMD is the '''mdx mouse''', which has a spontaneous point mutation in exon 23 that causes the absence of the dystrophin protein in the muscle.
**However, other animal models inlcude:<ref><pubmed>11917091</pubmed></ref>
***GRMD dog - Dystrophin-deficient dog
***HFMD cat - Dystrophin-deficient cat (clinically a poor model)
**This animal model allows testing ans screening of potential treatments and without these animal models, and without these animal models we would not have any known therapies today
**For example, mdx ''in vivo'' studies have led to U.S. FDA approval of Exondys 51 (eteplirsen) injection, the first drug approved to treat patients with Duchenne muscular dystrophy (DMD). Exondys 51 is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD. <ref>http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm521263.htm
</ref>
{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Muscular Dystrophy questions have been comprehensively answered and you have cited your sources.
| Assessment 5/5
|}

==Lab 8 Assessment==
Completed the quiz during the lab.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 27 October 2016 - Well done. Q3 gonads determine the other genital development. Q5 theca and interstitial.
| Assessment 6/8
|}

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:07, 23 September 2016 (AEST)

===Lab 8 Assessment===
Group 1: Wnt signalling pathway

Firstly, a proper introduction to the Wnt pathway and its various roles in the developing embryo should be added to the page. Even though the page is focusing on Wnt signalling in fetal skin development I still think the other contributions of Wnt signalling need to be at least mentioned in the intro. Mark also suggested adding a table to show the origins of the pathway and how our knowledge about Wnt signaling has evolved.

The page contains appropriate headings and subheadings to address the different mechanisms of Wnt signalling (canonical, non-canonical and non-canonical Wnt/Ca2+ pathway). The main points for each of these pathways are up on the page which is good. Clearly, these signalling mechanisms are quite complex and adding images could help the reader to visualise how the key molecules in this pathway interact with other molecules to induce downstream effects. But otherwise, the detail in the canonical pathway is adequate for a student to understand.

The references included in each section of the page is evidence of research done to support the content on the page. The page also includes up to date information to reflect current research in this area. This information should be integrated into some sort of discussion to show how this contributes to knowledge about the signalling pathway affects developmental events. Also, all the references need to be cited correctly which I am sure you guys are aware of and will do.

Don’t forget the topic is ‘Signalling in Development’, therefore the focus should be on Wnt signalling in the developing embryo.

Overall, Group 1 has made good progress. Keep it up!

Group 2: Notch signalling pathway

The page has a proper introduction stating the critical functions of Notch signalling, examples of diseases associated with Notch mutations as well as a brief description of the mechanism of Notch signalling. There is also a timeline on the page which none of the other groups have managed to do so good job!

Some headings are missing text but those with content (e.g. canonical pathway section) are covered in extensive detail. The subheadings and headings chosen for the page indicate the group’s in depth understanding of the key components of the Notch signalling pathway. Furthermore, the page contains correct referencing and citation of text and images.

The page also discusses Notch signalling in animal models as well. A recent study was included for Drosophila. What about the other two?

Overall, Group 2 has made excellent progress. Well done!

Group 3: FGFR signalling pathway

The headings and subheadings on the page is used very effectively to aid the progression of information. Through the sequence of the headings, it allows the reader to build their understanding about FGFR signalling. The FGFR page definitely address the topic of this assessment - signalling in development, and links FGF signalling to a number of developmental events. This reflects the large contributions of FGFR in development which the page successfully portrays.

In the overview section, it states: “As shown in the image, an acidic box…”. Make it clear which image you are referring to because I can’t find it.

The table for the subtypes of FGFR has been acknowledged that it is incomplete but it gives a good snapshot to function and associated abnormalities of the different FGFR subtypes.

The page includes a student drawn image which summarises the FGFR signalling pathway. None of the other groups have included a student drawn image so good job! The images uses colours to distinguish particular molecules and shows the downstream signalling events to affect gene transcription in the cell. To me the image is a bit blurry on the page, so maybe change the pixels of the image to make it larger and easier to see?

The page includes a quiz which is clever and will definitely make the page stand out from the other groups.

Overall, Group 3 has made good progress. Good job!

Group 4: Hedgehog signalling pathway

Firstly, the page is missing an introduction to the signalling pathway. There is also text missing under the first few subheadings. Since the hedgehog pathway research began as early as the 1970s, a table including the key events in the Hedgehog research would be interesting to add.

The page includes a nice overview of the Hedgehog pathway captured in the image however, it needs a reference to acknowledge the original source of the image. Consider relocating the image to the mechanism of signalling section. This may help the reader understand the processes better if they have that image there.

Mammals have 3 Hedgehog homologues (DHH, IHH and SHH). I think that is an important point to mention.

Good discussion of animal models since it is one of the key regulators of animal development.
Despite having headings without text. Group 3 has made good progress so far. Keep it up!

Group 6: TGF-β signalling pathway

The page contains a good introduction to the TGF-β superfamily, including examples of other signaling proteins. The images help the reader visualise how TGF-β ligand bring the receptors together in a heterotetrameric complex in which the type II receptors phosphorylate and activate the type I receptors. To be pedantic, the second image still needs to include details of the original source.

Remember that the project is meant to focus on ‘Signalling in Development’ and whilst the page addresses the signalling component it does not discuss TGF-β signalling in the development of the embryo. The second image addresses its role in proliferation, migration, growth arrest and apoptosis. This pubmed article: PMID 19289080 discusses TGF-β signalling in early development, axis formation, and patterning of the embryo.

The page does not use the correct referencing or in-text citations.

Overall, Group 6 has made a good start but more research needs to be done. Keep pushing :)

==Lab 9==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:06, 7 October 2016 (AEDT)

==Lab 11==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:02, 21 October 2016 (AEDT)

===Lab 11 Assessment===
<pubmed>21350179</pubmed>

Transient Regenerative Potential of the Neonatal Mouse Heart primary literature findings:
Based on the fact that urodele amphibians and teleost fish are able to retain the capacity for cardiac regeneration throughout their life, the authors questioned whether the potential for cardiac regeneration mammals is absent, or whether it exists but merely switched off early after birth. They investigated this through observing how the hearts of one day old neonatal mice heart develops. To perform further tests they surgically resected the left ventricular apex to see how the neonatal hearts respond to injury. They were able to see that the the ventricular apex in the mice indeed stimulated a regenerative response that appears to restore the damaged heart to its normal anatomy and function. This regenerative response was marked by cardiomyocyte proliferation with little evidence of hypertrophy or fibrosis, and therefore not a result repair processes. The origin of these cardiomyocytes in the regenerated tissue were determined to be from preexisting cardiomyocytes. The regenerated ventricular apex also had normal systolic function indicating a restoration of normal contractile function. A similar approach was done to seven day old mice and unlike the one day old mice, they displayed extensive fibrosis. Therefore the window for cardiac regeneration is brief in mammals, but there is a capacity to regenerate.

The purpose of the review article is to: "review our current understanding of how cardiomyocyte proliferation is regulated during heart development and regeneration". The primary article clearly relates to the topic of cardiomyocyte proliferation in the context of regeneration, which is why it has been used in the review article. The original research was used to provide evidence to support the statement: "cardiomyocytes in various contexts do harbor endogenous proliferative capabilities, albeit to different degrees". Correlations between the original research and the claims made by the review article include: "various contexts" - which in this case refers to the type of organism in which mice applies; and "endogenous proliferative capabilities" which from the first paragraph, the original research does support.

==Lab 12==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:08, 28 October 2016 (AEDT)

==References==

{{Stem Cell Presentations 2016}}

2016 Group Project 5

2016-10-27T08:32:48Z

Z5020373: /* Abnormalities */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page will give a broad overview of how the T-box signalling pathway works, as well as its importance, it's discovery, abnormalities associated with this transcription factor, and animal models that have been used to study these genes. It is important to note that T-box genes have also been found to regulate patterning and cell fate, cell survival, and/or proliferation. This however will not be covered in this web page. This web page will focus on the importance of T-box genes in embryological limb development.
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information, click here]

====T-Box gene and embryology====
The T-box gene family plays an essential role in controlling embryogenesis in a wide variety of organisms, including many invertebrates, amphibians and mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which, during embryological development, are important in establishing the early body plan and later are important in the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

===Origins of the T-box name===
The founding member of the T-box family is '''brachyura''' which comes from the greek meaning short tail. '''Brakhus''' means short in greek and '''oura''' meaning tail. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered through experimental studies with a short tailed mouse that harboured a mutation which affected it's tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted (see below for more information on the discovery).
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948, taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) was soon studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now, in human and mouse genomes, the gene brachyura is represented by the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
In 1927, a Russian female scientist, Nadine Dobrovolskaïa-Zavadskaïa, successfully isolated a strain from a mouse sample with short tail, which caused by a semidominant heterozygous mutation in a locus. After that she named this mutated locus as T and this experiment is trusted that is the first successful mammalian genetic screening. This blaze a trail of further investigation about the human embryonic genetic coordination and its importance <ref name="PMID11268043 "/>. In follow up experiment, some samples were treated with homozygous T locus and ended in mid-gestational stage with unorganized development of mesoderm and notochord. This shows the T locus is a fundamental gene during gastrulation and gives the very first concepts of notochord on neural tube and somite development. 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek to pay tribute to the earliest finding of this gene<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. However the scientist could not figure out its biochemical role since lack of the researches and evidences about the T-gene products until in 1993, T-gene was revealed as a sequence-specific DNA-binding protein <ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, a modern name according to more scientific findings which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

====Timeline of the discovery of the T-Box gene====
In 1927, the Brachyury (T) locus was introduced to the scientific world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, further embryological defects caused by the T mutation were studied, as well as the importance of the T-box genes in normal signalling pathways and embryonic development.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-bgcolor="#CEDFF2"
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-bgcolor="#CEDFF2"
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-bgcolor="#CEDFF2"
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

=== Quiz 1 ===
<quiz display=simple>

{The founding member of the T-box family is brachyura which comes from the greek meaning short tail.
|type="()"}
+ true
- false
|| True. Brakhus means short in greek and oura means tail.

{How was the brachyury mutation first described in 1927 by Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa?
|type="()"}
- A mutation that affected bone formation in birds
+ A mutation that affected tail length and sacral vertebrae in mice
- A mutation of phalangeal formation in mammals
- A mutation that affected several areas of embryological development. These areas had not yet been identified.
|| Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice

{ The T gene was cloned in 1990.
|type="()"}
-False
+True
|| True

{Where is the Tbx1 gene expressed in embryological development?
|type="()"}
+ Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome
- Hindlimb, mandibular and lung mesenchyme, atrium and body wall
- Splanchnic mesoderm, septum traversum, epicardium
|| The Tbx1 gene is expressed in: Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome

{The mutation of which Tbx gene causes Ulnar-mammary syndrome?
|type="()"}
+ Tbx3
- Tbx4
- Tbx18
|| The mutation of Tbx 3 results in this syndrome.

{The function of Tbx20 is the development the lower limbs and the pelvis in humans
|type="()"}
- true
+ false
|| False.Tbx20 is responsible for Cardiac development and yolk sac vascular remodeling.

</quiz>

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref name="PMID26212321><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>. From a recent research, April 2016, found that tbx3 also take parts in the limb bud formation and followed by the signalling by the expression of tbx4 and tbx5<ref name="PMID27046536"><pubmed>27046536</pubmed></ref> . The video below shows tbx3 absences in a mice forelimb and that forelimb has no joints.

<html5media height="300" width="400">https://static-movie-usa.glencoesoftware.com/mp4/10.7554/646/eb046d787f6ac59d0a76265c25a50b17b0186c42/elife-07897-media1.mp4</html5media>

Adult Tbx3;PrxCre mutant mouse is healthy and mobile despite forelimb deformities.
DOI: http://dx.doi.org/10.7554/eLife.07897.007 <ref name="PMID27046536"/>

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref name="PMID26212321"/> ]]

====Respiratory Development====
[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]
Members of the T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) have been found to be expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have implicated in several developmental events: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In chick embryos, Tbx4 and Fgf10 have been found to co-express in the foregut mesoderm (in a lung field), in a domain that coincides with that of Nkx2.1 in the endoderm (except in its most anterior portion)<ref name="PMID8853987"/>. Studies show that abnormal expression of Tbx4 induces ectopic Fgf10 expression and ectopic buds that express Nkx 2.1 molecules. This suggests that Tbx-Fgf10 interaction plays a role in lung morphogenesis as the Nkx2.1 gene encodes a transcription factor that is expressed during early development of thyroid, lung, and forebrain regions, particularly the basal ganglia and hypothalamus <ref><pubmed>24714694</pubmed></ref>.

Moreover, Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a one-sided loss of lung bud specification and absence of tracheal specification in organ culture. Furthermore, mesenchymal markers Wnt2 and Fgf10 expression, and Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung<ref><pubmed>9916808</pubmed></ref>. This is consistent with findings from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires the ability for the initial budding morphogenesis of primary lung buds<ref name="PMID12588840"><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 deficient mice died soon after birth due to respiratory distress. These offspring have small lungs and show severe abnormalities in tracheal and bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development<ref name="PMID22876201"/>.

====Other developmental events====
TBX signalling is also involved in many other developmental processes. 2 examples are palate development and skeletal muscle fibre-type determination.

In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) demonstrated that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 deficient mice had abnormal epithelial adhesion between the palate and mandible which led to several forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft similar to human conditions<ref><pubmed>22371266</pubmed></ref><ref><pubmed>14585638</pubmed></ref>.

Skeletal muscle comprises of a mosaic pattern of slow oxidative myofibres and fast glycolytic myofibres that influences muscle function and whole body metabolism. The mesodermal transcription factor Tbx15 is specifically expressed in glycolytic myofibres. Inactivation of Tbx15 leads to muscle size reduction due to a decrease in the number of glycolytic fibres, associated with a small increase in the number of oxidative fibres. This shift in fibre composition results in a subsequent shift of substrates from muscle to fat and liver where they are stored as lipids, leading to increased adiposity and glucose intolerance. The mechanism by which this occurs involves the activation of AMP-activated protein kinase (AMPK) signalling and a decrease in insulin growth factor 2 (Igf2) expression. Tbx15 is one of the few known transcription factors that are critical regulators of fibre-type distribution and skeletal muscle metabolism in the embryonic and post-natal period
<ref><pubmed>26299309</pubmed></ref><ref><pubmed>18403917</pubmed></ref>.

=== Quiz 2===
<quiz display=simple>

{In the developing heart, Tbx5 expression can detected as early as stage 12
|type="()"}
+ true
- false
|| True. Tbx5 is detected along the entire rostrocaudal length of the fused heart tube

{Which pathways are the Tbx4 and Tbx5 genes linked to in limb outgrowth regulation?
|type="()"}
- The Notch signalling pathway
+ Fibroblast growth factor and Wnt signalling pathways
- Sonic Hedgehog
- Wnt and sonic hedgehog signalling pathways
|| Fibroblast growth factor and Wnt signalling pathways are closely associated to the signalling associated with Tbx 4 and 5 in limb outgrowth regulation.

{ Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis
|type="()"}
-False
+True
|| True

{Which parts of lung development do the Tbx genes regulate?
|type="()"}
+ lung bud and trachea specification, lunch branching and tracheal/bronchial cartilage formation
- Formation of the tracheal bifurcation only
- Development of intercostal muscles and the respiratory diaphragm
|| The Tbx genes are respobsible for the regulation of lung bud and trachea specification, lung branching morphogenesis, and tracheal/bronchial cartilage formation

{Is the Fgf10 signalling pathway activated upstream or downstream of Tbx4 and 5 in the developing lung?
|type="()"}
- Upstream
+ Downstream
|| Downstream. The Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung

</quiz>

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====
{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>
[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]
|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}

====TBX5/Holt– Oram Syndrome====
[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]
{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]
|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information see here]

[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>
[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

===Quiz 3===

<quiz display=simple>

{TBX3 mutations result in Holt– Oram Syndrome
|type="()"}
+ false
-true
|| False. Mutations of the TBX3 gene leads to ulnar-mammary syndrome.

{Mutations in which gene causes DiGeorge Syndrome
|type="()"}
- Tbx 3
+ Tbx 1
- Tbx 5
- Tbx18
|| Tbx 1

{ Thumb anomaly is an expression of holt-oran syndrome.
|type="()"}
-False
+True
|| True. Other expressions include malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly.

{Which parts of lung development do the Tbx genes regulate?
|type="()"}
+ lung bud and trachea specification, lunch branching and tracheal/bronchial cartilage formation
- Formation of the tracheal bifurcation only
- Development of intercostal muscles and the respiratory diaphragm
|| The Tbx genes are respobsible for the regulation of lung bud and trachea specification, lung branching morphogenesis, and tracheal/bronchial cartilage formation

{Tbx22 is responsible for palate closure, and hence a mutation of this gene leads to cleft palate formation.
|type="()"}
- True
+ False
|| False. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate.

</quiz>

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref> since their genome had been mapped out and in order to perform ethically experiments.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in Placental Mammals: Mouse====
'''''Mus musculus'''''

Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
[[Mouse Development | See more information on Mouse development]]

====T-box in Fish: Zebra fish====
'''''Danio rerio'''''

T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.
[[Zebrafish Development| See more information on Zebrafish Development]]

====T-box in Insects: Fruit fly====
'''''Drosophila melanogaster'''''

The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.
[[Fly Development | See more information on Fly Development]]

====T-Box in Amphibia: Clawed frog====
'''''Xenopus leaves'''''

Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.
[[Frog Development | See more information on Frog Development]]

====T-Box in Aves: Chick====
'''''Gallus gallus domesticus'''''

In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref name="PMID9550719"/>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.
[[Chicken Development | See more information on Chicken Development]]

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development: Wallaby====
'''''Macropus eugenii'''''

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area. The neonate can attach to the teat where it completes its development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.
See also [[Kangaroo Development | See more information on Kangaroo development]]

====T-Box in Amphioxus ====
'''''Branchiostoma lanceolatum'''''

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''AMP-activated protein kinase (AMPK)''': plays a key role as a master regulator of cellular energy homeostasis. Regarded as a cellular energy sensor responding to changing ATP levels. When ATP is low, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid oxidation and autophagy.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Dementia''' : severe impairment or loss of intellectual capacity and personality integration, due to the loss of or damage to neurons in the brain.

'''Heterozygotes''': a hybrid containing genes for two unlike forms of a characteristic, and therefore not breeding true to type.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Homozygote''': an organism with identical pairs of genes with respect to any given pair of hereditary characters, and therefore breeding true for that character.

'''Hypoglycaemia''': an abnormally dropped amount of sugar in the blood

'''Insulin-like growth factor 2 (IGF-2)''': Shares structural similarity to insulin.

'''Limb fields''': areas where the limb buds will develop.

'''Morphogenetic''': the development of structural features of an organism or part.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

===References===

2016 Group Project 5

2016-10-27T08:26:28Z

Z5020373: /* Abnormalities */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page will give a broad overview of how the T-box signalling pathway works, as well as its importance, it's discovery, abnormalities associated with this transcription factor, and animal models that have been used to study these genes. It is important to note that T-box genes have also been found to regulate patterning and cell fate, cell survival, and/or proliferation. This however will not be covered in this web page. This web page will focus on the importance of T-box genes in embryological limb development.
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information, click here]

====T-Box gene and embryology====
The T-box gene family plays an essential role in controlling embryogenesis in a wide variety of organisms, including many invertebrates, amphibians and mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which, during embryological development, are important in establishing the early body plan and later are important in the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

===Origins of the T-box name===
The founding member of the T-box family is '''brachyura''' which comes from the greek meaning short tail. '''Brakhus''' means short in greek and '''oura''' meaning tail. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered through experimental studies with a short tailed mouse that harboured a mutation which affected it's tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted (see below for more information on the discovery).
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948, taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) was soon studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now, in human and mouse genomes, the gene brachyura is represented by the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
In 1927, a Russian female scientist, Nadine Dobrovolskaïa-Zavadskaïa, successfully isolated a strain from a mouse sample with short tail, which caused by a semidominant heterozygous mutation in a locus. After that she named this mutated locus as T and this experiment is trusted that is the first successful mammalian genetic screening. This blaze a trail of further investigation about the human embryonic genetic coordination and its importance <ref name="PMID11268043 "/>. In follow up experiment, some samples were treated with homozygous T locus and ended in mid-gestational stage with unorganized development of mesoderm and notochord. This shows the T locus is a fundamental gene during gastrulation and gives the very first concepts of notochord on neural tube and somite development. 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek to pay tribute to the earliest finding of this gene<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. However the scientist could not figure out its biochemical role since lack of the researches and evidences about the T-gene products until in 1993, T-gene was revealed as a sequence-specific DNA-binding protein <ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, a modern name according to more scientific findings which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

====Timeline of the discovery of the T-Box gene====
In 1927, the Brachyury (T) locus was introduced to the scientific world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, further embryological defects caused by the T mutation were studied, as well as the importance of the T-box genes in normal signalling pathways and embryonic development.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-bgcolor="#CEDFF2"
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-bgcolor="#CEDFF2"
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-bgcolor="#CEDFF2"
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

=== Quiz 1 ===
<quiz display=simple>

{The founding member of the T-box family is brachyura which comes from the greek meaning short tail.
|type="()"}
+ true
- false
|| True. Brakhus means short in greek and oura means tail.

{How was the brachyury mutation first described in 1927 by Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa?
|type="()"}
- A mutation that affected bone formation in birds
+ A mutation that affected tail length and sacral vertebrae in mice
- A mutation of phalangeal formation in mammals
- A mutation that affected several areas of embryological development. These areas had not yet been identified.
|| Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice

{ The T gene was cloned in 1990.
|type="()"}
-False
+True
|| True

{Where is the Tbx1 gene expressed in embryological development?
|type="()"}
+ Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome
- Hindlimb, mandibular and lung mesenchyme, atrium and body wall
- Splanchnic mesoderm, septum traversum, epicardium
|| The Tbx1 gene is expressed in: Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome

{The mutation of which Tbx gene causes Ulnar-mammary syndrome?
|type="()"}
+ Tbx3
- Tbx4
- Tbx18
|| The mutation of Tbx 3 results in this syndrome.

{The function of Tbx20 is the development the lower limbs and the pelvis in humans
|type="()"}
- true
+ false
|| False.Tbx20 is responsible for Cardiac development and yolk sac vascular remodeling.

</quiz>

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref name="PMID26212321><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>. From a recent research, April 2016, found that tbx3 also take parts in the limb bud formation and followed by the signalling by the expression of tbx4 and tbx5<ref name="PMID27046536"><pubmed>27046536</pubmed></ref> . The video below shows tbx3 absences in a mice forelimb and that forelimb has no joints.

<html5media height="300" width="400">https://static-movie-usa.glencoesoftware.com/mp4/10.7554/646/eb046d787f6ac59d0a76265c25a50b17b0186c42/elife-07897-media1.mp4</html5media>

Adult Tbx3;PrxCre mutant mouse is healthy and mobile despite forelimb deformities.
DOI: http://dx.doi.org/10.7554/eLife.07897.007 <ref name="PMID27046536"/>

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref name="PMID26212321"/> ]]

====Respiratory Development====
[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]
Members of the T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) have been found to be expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have implicated in several developmental events: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In chick embryos, Tbx4 and Fgf10 have been found to co-express in the foregut mesoderm (in a lung field), in a domain that coincides with that of Nkx2.1 in the endoderm (except in its most anterior portion)<ref name="PMID8853987"/>. Studies show that abnormal expression of Tbx4 induces ectopic Fgf10 expression and ectopic buds that express Nkx 2.1 molecules. This suggests that Tbx-Fgf10 interaction plays a role in lung morphogenesis as the Nkx2.1 gene encodes a transcription factor that is expressed during early development of thyroid, lung, and forebrain regions, particularly the basal ganglia and hypothalamus <ref><pubmed>24714694</pubmed></ref>.

Moreover, Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a one-sided loss of lung bud specification and absence of tracheal specification in organ culture. Furthermore, mesenchymal markers Wnt2 and Fgf10 expression, and Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung<ref><pubmed>9916808</pubmed></ref>. This is consistent with findings from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires the ability for the initial budding morphogenesis of primary lung buds<ref name="PMID12588840"><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 deficient mice died soon after birth due to respiratory distress. These offspring have small lungs and show severe abnormalities in tracheal and bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development<ref name="PMID22876201"/>.

====Other developmental events====
TBX signalling is also involved in many other developmental processes. 2 examples are palate development and skeletal muscle fibre-type determination.

In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) demonstrated that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 deficient mice had abnormal epithelial adhesion between the palate and mandible which led to several forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft similar to human conditions<ref><pubmed>22371266</pubmed></ref><ref><pubmed>14585638</pubmed></ref>.

Skeletal muscle comprises of a mosaic pattern of slow oxidative myofibres and fast glycolytic myofibres that influences muscle function and whole body metabolism. The mesodermal transcription factor Tbx15 is specifically expressed in glycolytic myofibres. Inactivation of Tbx15 leads to muscle size reduction due to a decrease in the number of glycolytic fibres, associated with a small increase in the number of oxidative fibres. This shift in fibre composition results in a subsequent shift of substrates from muscle to fat and liver where they are stored as lipids, leading to increased adiposity and glucose intolerance. The mechanism by which this occurs involves the activation of AMP-activated protein kinase (AMPK) signalling and a decrease in insulin growth factor 2 (Igf2) expression. Tbx15 is one of the few known transcription factors that are critical regulators of fibre-type distribution and skeletal muscle metabolism in the embryonic and post-natal period
<ref><pubmed>26299309</pubmed></ref><ref><pubmed>18403917</pubmed></ref>.

=== Quiz 2===
<quiz display=simple>

{In the developing heart, Tbx5 expression can detected as early as stage 12
|type="()"}
+ true
- false
|| True. Tbx5 is detected along the entire rostrocaudal length of the fused heart tube

{Which pathways are the Tbx4 and Tbx5 genes linked to in limb outgrowth regulation?
|type="()"}
- The Notch signalling pathway
+ Fibroblast growth factor and Wnt signalling pathways
- Sonic Hedgehog
- Wnt and sonic hedgehog signalling pathways
|| Fibroblast growth factor and Wnt signalling pathways are closely associated to the signalling associated with Tbx 4 and 5 in limb outgrowth regulation.

{ Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis
|type="()"}
-False
+True
|| True

{Which parts of lung development do the Tbx genes regulate?
|type="()"}
+ lung bud and trachea specification, lunch branching and tracheal/bronchial cartilage formation
- Formation of the tracheal bifurcation only
- Development of intercostal muscles and the respiratory diaphragm
|| The Tbx genes are respobsible for the regulation of lung bud and trachea specification, lung branching morphogenesis, and tracheal/bronchial cartilage formation

{Is the Fgf10 signalling pathway activated upstream or downstream of Tbx4 and 5 in the developing lung?
|type="()"}
- Upstream
+ Downstream
|| Downstream. The Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung

</quiz>

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====
{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>
[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====
{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information see here]

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]

===Quiz 3===

<quiz display=simple>

{TBX3 mutations result in Holt– Oram Syndrome
|type="()"}
+ false
-true
|| False. Mutations of the TBX3 gene leads to ulnar-mammary syndrome.

{Mutations in which gene causes DiGeorge Syndrome
|type="()"}
- Tbx 3
+ Tbx 1
- Tbx 5
- Tbx18
|| Tbx 1

{ Thumb anomaly is an expression of holt-oran syndrome.
|type="()"}
-False
+True
|| True. Other expressions include malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly.

{Which parts of lung development do the Tbx genes regulate?
|type="()"}
+ lung bud and trachea specification, lunch branching and tracheal/bronchial cartilage formation
- Formation of the tracheal bifurcation only
- Development of intercostal muscles and the respiratory diaphragm
|| The Tbx genes are respobsible for the regulation of lung bud and trachea specification, lung branching morphogenesis, and tracheal/bronchial cartilage formation

{Tbx22 is responsible for palate closure, and hence a mutation of this gene leads to cleft palate formation.
|type="()"}
- True
+ False
|| False. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate.

</quiz>

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref> since their genome had been mapped out and in order to perform ethically experiments.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in Placental Mammals: Mouse====
'''''Mus musculus'''''

Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
[[Mouse Development | See more information on Mouse development]]

====T-box in Fish: Zebra fish====
'''''Danio rerio'''''

T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.
[[Zebrafish Development| See more information on Zebrafish Development]]

====T-box in Insects: Fruit fly====
'''''Drosophila melanogaster'''''

The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.
[[Fly Development | See more information on Fly Development]]

====T-Box in Amphibia: Clawed frog====
'''''Xenopus leaves'''''

Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.
[[Frog Development | See more information on Frog Development]]

====T-Box in Aves: Chick====
'''''Gallus gallus domesticus'''''

In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref name="PMID9550719"/>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.
[[Chicken Development | See more information on Chicken Development]]

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development: Wallaby====
'''''Macropus eugenii'''''

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area. The neonate can attach to the teat where it completes its development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.
See also [[Kangaroo Development | See more information on Kangaroo development]]

====T-Box in Amphioxus ====
'''''Branchiostoma lanceolatum'''''

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''AMP-activated protein kinase (AMPK)''': plays a key role as a master regulator of cellular energy homeostasis. Regarded as a cellular energy sensor responding to changing ATP levels. When ATP is low, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid oxidation and autophagy.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Dementia''' : severe impairment or loss of intellectual capacity and personality integration, due to the loss of or damage to neurons in the brain.

'''Heterozygotes''': a hybrid containing genes for two unlike forms of a characteristic, and therefore not breeding true to type.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Homozygote''': an organism with identical pairs of genes with respect to any given pair of hereditary characters, and therefore breeding true for that character.

'''Hypoglycaemia''': an abnormally dropped amount of sugar in the blood

'''Insulin-like growth factor 2 (IGF-2)''': Shares structural similarity to insulin.

'''Limb fields''': areas where the limb buds will develop.

'''Morphogenetic''': the development of structural features of an organism or part.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

===References===

2016 Group Project 5

2016-10-27T08:22:02Z

Z5020373: /* Functions of T-box in development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page will give a broad overview of how the T-box signalling pathway works, as well as its importance, it's discovery, abnormalities associated with this transcription factor, and animal models that have been used to study these genes. It is important to note that T-box genes have also been found to regulate patterning and cell fate, cell survival, and/or proliferation. This however will not be covered in this web page. This web page will focus on the importance of T-box genes in embryological limb development.
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information, click here]

====T-Box gene and embryology====
The T-box gene family plays an essential role in controlling embryogenesis in a wide variety of organisms, including many invertebrates, amphibians and mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which, during embryological development, are important in establishing the early body plan and later are important in the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

===Origins of the T-box name===
The founding member of the T-box family is '''brachyura''' which comes from the greek meaning short tail. '''Brakhus''' means short in greek and '''oura''' meaning tail. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered through experimental studies with a short tailed mouse that harboured a mutation which affected it's tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted (see below for more information on the discovery).
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948, taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) was soon studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now, in human and mouse genomes, the gene brachyura is represented by the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
In 1927, a Russian female scientist, Nadine Dobrovolskaïa-Zavadskaïa, successfully isolated a strain from a mouse sample with short tail, which caused by a semidominant heterozygous mutation in a locus. After that she named this mutated locus as T and this experiment is trusted that is the first successful mammalian genetic screening. This blaze a trail of further investigation about the human embryonic genetic coordination and its importance <ref name="PMID11268043 "/>. In follow up experiment, some samples were treated with homozygous T locus and ended in mid-gestational stage with unorganized development of mesoderm and notochord. This shows the T locus is a fundamental gene during gastrulation and gives the very first concepts of notochord on neural tube and somite development. 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek to pay tribute to the earliest finding of this gene<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. However the scientist could not figure out its biochemical role since lack of the researches and evidences about the T-gene products until in 1993, T-gene was revealed as a sequence-specific DNA-binding protein <ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, a modern name according to more scientific findings which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

====Timeline of the discovery of the T-Box gene====
In 1927, the Brachyury (T) locus was introduced to the scientific world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, further embryological defects caused by the T mutation were studied, as well as the importance of the T-box genes in normal signalling pathways and embryonic development.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-bgcolor="#CEDFF2"
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-bgcolor="#CEDFF2"
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-bgcolor="#CEDFF2"
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

=== Quiz 1 ===
<quiz display=simple>

{The founding member of the T-box family is brachyura which comes from the greek meaning short tail.
|type="()"}
+ true
- false
|| True. Brakhus means short in greek and oura means tail.

{How was the brachyury mutation first described in 1927 by Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa?
|type="()"}
- A mutation that affected bone formation in birds
+ A mutation that affected tail length and sacral vertebrae in mice
- A mutation of phalangeal formation in mammals
- A mutation that affected several areas of embryological development. These areas had not yet been identified.
|| Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice

{ The T gene was cloned in 1990.
|type="()"}
-False
+True
|| True

{Where is the Tbx1 gene expressed in embryological development?
|type="()"}
+ Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome
- Hindlimb, mandibular and lung mesenchyme, atrium and body wall
- Splanchnic mesoderm, septum traversum, epicardium
|| The Tbx1 gene is expressed in: Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome

{The mutation of which Tbx gene causes Ulnar-mammary syndrome?
|type="()"}
+ Tbx3
- Tbx4
- Tbx18
|| The mutation of Tbx 3 results in this syndrome.

{The function of Tbx20 is the development the lower limbs and the pelvis in humans
|type="()"}
- true
+ false
|| False.Tbx20 is responsible for Cardiac development and yolk sac vascular remodeling.

</quiz>

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref name="PMID26212321><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>. From a recent research, April 2016, found that tbx3 also take parts in the limb bud formation and followed by the signalling by the expression of tbx4 and tbx5<ref name="PMID27046536"><pubmed>27046536</pubmed></ref> . The video below shows tbx3 absences in a mice forelimb and that forelimb has no joints.

<html5media height="300" width="400">https://static-movie-usa.glencoesoftware.com/mp4/10.7554/646/eb046d787f6ac59d0a76265c25a50b17b0186c42/elife-07897-media1.mp4</html5media>

Adult Tbx3;PrxCre mutant mouse is healthy and mobile despite forelimb deformities.
DOI: http://dx.doi.org/10.7554/eLife.07897.007 <ref name="PMID27046536"/>

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref name="PMID26212321"/> ]]

====Respiratory Development====
[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]
Members of the T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) have been found to be expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have implicated in several developmental events: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In chick embryos, Tbx4 and Fgf10 have been found to co-express in the foregut mesoderm (in a lung field), in a domain that coincides with that of Nkx2.1 in the endoderm (except in its most anterior portion)<ref name="PMID8853987"/>. Studies show that abnormal expression of Tbx4 induces ectopic Fgf10 expression and ectopic buds that express Nkx 2.1 molecules. This suggests that Tbx-Fgf10 interaction plays a role in lung morphogenesis as the Nkx2.1 gene encodes a transcription factor that is expressed during early development of thyroid, lung, and forebrain regions, particularly the basal ganglia and hypothalamus <ref><pubmed>24714694</pubmed></ref>.

Moreover, Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a one-sided loss of lung bud specification and absence of tracheal specification in organ culture. Furthermore, mesenchymal markers Wnt2 and Fgf10 expression, and Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung<ref><pubmed>9916808</pubmed></ref>. This is consistent with findings from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires the ability for the initial budding morphogenesis of primary lung buds<ref name="PMID12588840"><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 deficient mice died soon after birth due to respiratory distress. These offspring have small lungs and show severe abnormalities in tracheal and bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development<ref name="PMID22876201"/>.

====Other developmental events====
TBX signalling is also involved in many other developmental processes. 2 examples are palate development and skeletal muscle fibre-type determination.

In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) demonstrated that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 deficient mice had abnormal epithelial adhesion between the palate and mandible which led to several forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft similar to human conditions<ref><pubmed>22371266</pubmed></ref><ref><pubmed>14585638</pubmed></ref>.

Skeletal muscle comprises of a mosaic pattern of slow oxidative myofibres and fast glycolytic myofibres that influences muscle function and whole body metabolism. The mesodermal transcription factor Tbx15 is specifically expressed in glycolytic myofibres. Inactivation of Tbx15 leads to muscle size reduction due to a decrease in the number of glycolytic fibres, associated with a small increase in the number of oxidative fibres. This shift in fibre composition results in a subsequent shift of substrates from muscle to fat and liver where they are stored as lipids, leading to increased adiposity and glucose intolerance. The mechanism by which this occurs involves the activation of AMP-activated protein kinase (AMPK) signalling and a decrease in insulin growth factor 2 (Igf2) expression. Tbx15 is one of the few known transcription factors that are critical regulators of fibre-type distribution and skeletal muscle metabolism in the embryonic and post-natal period
<ref><pubmed>26299309</pubmed></ref><ref><pubmed>18403917</pubmed></ref>.

=== Quiz 2===
<quiz display=simple>

{In the developing heart, Tbx5 expression can detected as early as stage 12
|type="()"}
+ true
- false
|| True. Tbx5 is detected along the entire rostrocaudal length of the fused heart tube

{Which pathways are the Tbx4 and Tbx5 genes linked to in limb outgrowth regulation?
|type="()"}
- The Notch signalling pathway
+ Fibroblast growth factor and Wnt signalling pathways
- Sonic Hedgehog
- Wnt and sonic hedgehog signalling pathways
|| Fibroblast growth factor and Wnt signalling pathways are closely associated to the signalling associated with Tbx 4 and 5 in limb outgrowth regulation.

{ Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis
|type="()"}
-False
+True
|| True

{Which parts of lung development do the Tbx genes regulate?
|type="()"}
+ lung bud and trachea specification, lunch branching and tracheal/bronchial cartilage formation
- Formation of the tracheal bifurcation only
- Development of intercostal muscles and the respiratory diaphragm
|| The Tbx genes are respobsible for the regulation of lung bud and trachea specification, lung branching morphogenesis, and tracheal/bronchial cartilage formation

{Is the Fgf10 signalling pathway activated upstream or downstream of Tbx4 and 5 in the developing lung?
|type="()"}
- Upstream
+ Downstream
|| Downstream. The Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung

</quiz>

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>
[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information see here]

====TBX22/Cleft Palate====

Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]

===Quiz 3===

<quiz display=simple>

{TBX3 mutations result in Holt– Oram Syndrome
|type="()"}
+ false
-true
|| False. Mutations of the TBX3 gene leads to ulnar-mammary syndrome.

{Mutations in which gene causes DiGeorge Syndrome
|type="()"}
- Tbx 3
+ Tbx 1
- Tbx 5
- Tbx18
|| Tbx 1

{ Thumb anomaly is an expression of holt-oran syndrome.
|type="()"}
-False
+True
|| True. Other expressions include malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly.

{Which parts of lung development do the Tbx genes regulate?
|type="()"}
+ lung bud and trachea specification, lunch branching and tracheal/bronchial cartilage formation
- Formation of the tracheal bifurcation only
- Development of intercostal muscles and the respiratory diaphragm
|| The Tbx genes are respobsible for the regulation of lung bud and trachea specification, lung branching morphogenesis, and tracheal/bronchial cartilage formation

{Tbx22 is responsible for palate closure, and hence a mutation of this gene leads to cleft palate formation.
|type="()"}
- True
+ False
|| False. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate.

</quiz>

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref> since their genome had been mapped out and in order to perform ethically experiments.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in Placental Mammals: Mouse====
'''''Mus musculus'''''

Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
[[Mouse Development | See more information on Mouse development]]

====T-box in Fish: Zebra fish====
'''''Danio rerio'''''

T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.
[[Zebrafish Development| See more information on Zebrafish Development]]

====T-box in Insects: Fruit fly====
'''''Drosophila melanogaster'''''

The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.
[[Fly Development | See more information on Fly Development]]

====T-Box in Amphibia: Clawed frog====
'''''Xenopus leaves'''''

Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.
[[Frog Development | See more information on Frog Development]]

====T-Box in Aves: Chick====
'''''Gallus gallus domesticus'''''

In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref name="PMID9550719"/>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.
[[Chicken Development | See more information on Chicken Development]]

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development: Wallaby====
'''''Macropus eugenii'''''

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area. The neonate can attach to the teat where it completes its development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.
See also [[Kangaroo Development | See more information on Kangaroo development]]

====T-Box in Amphioxus ====
'''''Branchiostoma lanceolatum'''''

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''AMP-activated protein kinase (AMPK)''': plays a key role as a master regulator of cellular energy homeostasis. Regarded as a cellular energy sensor responding to changing ATP levels. When ATP is low, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid oxidation and autophagy.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Dementia''' : severe impairment or loss of intellectual capacity and personality integration, due to the loss of or damage to neurons in the brain.

'''Heterozygotes''': a hybrid containing genes for two unlike forms of a characteristic, and therefore not breeding true to type.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Homozygote''': an organism with identical pairs of genes with respect to any given pair of hereditary characters, and therefore breeding true for that character.

'''Hypoglycaemia''': an abnormally dropped amount of sugar in the blood

'''Insulin-like growth factor 2 (IGF-2)''': Shares structural similarity to insulin.

'''Limb fields''': areas where the limb buds will develop.

'''Morphogenetic''': the development of structural features of an organism or part.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

===References===

2016 Group Project 5

2016-10-27T08:19:44Z

Z5020373:

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page will give a broad overview of how the T-box signalling pathway works, as well as its importance, it's discovery, abnormalities associated with this transcription factor, and animal models that have been used to study these genes. It is important to note that T-box genes have also been found to regulate patterning and cell fate, cell survival, and/or proliferation. This however will not be covered in this web page. This web page will focus on the importance of T-box genes in embryological limb development.
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information, click here]

====T-Box gene and embryology====
The T-box gene family plays an essential role in controlling embryogenesis in a wide variety of organisms, including many invertebrates, amphibians and mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which, during embryological development, are important in establishing the early body plan and later are important in the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

===Origins of the T-box name===
The founding member of the T-box family is '''brachyura''' which comes from the greek meaning short tail. '''Brakhus''' means short in greek and '''oura''' meaning tail. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered through experimental studies with a short tailed mouse that harboured a mutation which affected it's tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted (see below for more information on the discovery).
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948, taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) was soon studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now, in human and mouse genomes, the gene brachyura is represented by the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
In 1927, a Russian female scientist, Nadine Dobrovolskaïa-Zavadskaïa, successfully isolated a strain from a mouse sample with short tail, which caused by a semidominant heterozygous mutation in a locus. After that she named this mutated locus as T and this experiment is trusted that is the first successful mammalian genetic screening. This blaze a trail of further investigation about the human embryonic genetic coordination and its importance <ref name="PMID11268043 "/>. In follow up experiment, some samples were treated with homozygous T locus and ended in mid-gestational stage with unorganized development of mesoderm and notochord. This shows the T locus is a fundamental gene during gastrulation and gives the very first concepts of notochord on neural tube and somite development. 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek to pay tribute to the earliest finding of this gene<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. However the scientist could not figure out its biochemical role since lack of the researches and evidences about the T-gene products until in 1993, T-gene was revealed as a sequence-specific DNA-binding protein <ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, a modern name according to more scientific findings which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

====Timeline of the discovery of the T-Box gene====
In 1927, the Brachyury (T) locus was introduced to the scientific world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, further embryological defects caused by the T mutation were studied, as well as the importance of the T-box genes in normal signalling pathways and embryonic development.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-bgcolor="#CEDFF2"
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-bgcolor="#CEDFF2"
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-bgcolor="#CEDFF2"
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-bgcolor="#CEDFF2"
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

=== Quiz 1 ===
<quiz display=simple>

{The founding member of the T-box family is brachyura which comes from the greek meaning short tail.
|type="()"}
+ true
- false
|| True. Brakhus means short in greek and oura means tail.

{How was the brachyury mutation first described in 1927 by Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa?
|type="()"}
- A mutation that affected bone formation in birds
+ A mutation that affected tail length and sacral vertebrae in mice
- A mutation of phalangeal formation in mammals
- A mutation that affected several areas of embryological development. These areas had not yet been identified.
|| Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice

{ The T gene was cloned in 1990.
|type="()"}
-False
+True
|| True

{Where is the Tbx1 gene expressed in embryological development?
|type="()"}
+ Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome
- Hindlimb, mandibular and lung mesenchyme, atrium and body wall
- Splanchnic mesoderm, septum traversum, epicardium
|| The Tbx1 gene is expressed in: Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome

{The mutation of which Tbx gene causes Ulnar-mammary syndrome?
|type="()"}
+ Tbx3
- Tbx4
- Tbx18
|| The mutation of Tbx 3 results in this syndrome.

{The function of Tbx20 is the development the lower limbs and the pelvis in humans
|type="()"}
- true
+ false
|| False.Tbx20 is responsible for Cardiac development and yolk sac vascular remodeling.

</quiz>

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref name="PMID26212321><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>. From a recent research, April 2016, found that tbx3 also take parts in the limb bud formation and followed by the signalling by the expression of tbx4 and tbx5<ref name="PMID27046536"><pubmed>27046536</pubmed></ref> . The video below shows tbx3 absences in a mice forelimb and that forelimb has no joints.

<html5media height="300" width="400">https://static-movie-usa.glencoesoftware.com/mp4/10.7554/646/eb046d787f6ac59d0a76265c25a50b17b0186c42/elife-07897-media1.mp4</html5media>

Adult Tbx3;PrxCre mutant mouse is healthy and mobile despite forelimb deformities.
DOI: http://dx.doi.org/10.7554/eLife.07897.007 <ref name="PMID27046536"/>

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref name="PMID26212321"/> ]]

====Respiratory Development====
[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]
Members of the T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) have been found to be expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have implicated in several developmental events: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In chick embryos, Tbx4 and Fgf10 have been found to co-express in the foregut mesoderm (in a lung field), in a domain that coincides with that of Nkx2.1 in the endoderm (except in its most anterior portion)<ref name="PMID8853987"/>. Studies show that abnormal expression of Tbx4 induces ectopic Fgf10 expression and ectopic buds that express Nkx 2.1 molecules. This suggests that Tbx-Fgf10 interaction plays a role in lung morphogenesis as the Nkx2.1 gene encodes a transcription factor that is expressed during early development of thyroid, lung, and forebrain regions, particularly the basal ganglia and hypothalamus <ref><pubmed>24714694</pubmed></ref>.

Moreover, Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a one-sided loss of lung bud specification and absence of tracheal specification in organ culture. Furthermore, mesenchymal markers Wnt2 and Fgf10 expression, and Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung<ref><pubmed>9916808</pubmed></ref>. This is consistent with findings from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires the ability for the initial budding morphogenesis of primary lung buds<ref name="PMID12588840"><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 deficient mice died soon after birth due to respiratory distress. These offspring have small lungs and show severe abnormalities in tracheal and bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development<ref name="PMID22876201"/>.

====Other developmental events====
TBX signalling is also involved in many other developmental processes. 2 examples are palate development and skeletal muscle fibre-type determination.

In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) demonstrated that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 deficient mice had abnormal epithelial adhesion between the palate and mandible which led to several forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft similar to human conditions<ref><pubmed>22371266</pubmed></ref><ref><pubmed>14585638</pubmed></ref>.

Skeletal muscle comprises of a mosaic pattern of slow oxidative myofibres and fast glycolytic myofibres that influences muscle function and whole body metabolism. The mesodermal transcription factor Tbx15 is specifically expressed in glycolytic myofibres. Inactivation of Tbx15 leads to muscle size reduction due to a decrease in the number of glycolytic fibres, associated with a small increase in the number of oxidative fibres. This shift in fibre composition results in a subsequent shift of substrates from muscle to fat and liver where they are stored as lipids, leading to increased adiposity and glucose intolerance. The mechanism by which this occurs involves the activation of AMP-activated protein kinase (AMPK) signalling and a decrease in insulin growth factor 2 (Igf2) expression. Tbx15 is one of the few known transcription factors that are critical regulators of fibre-type distribution and skeletal muscle metabolism in the embryonic and post-natal period
<ref><pubmed>26299309</pubmed></ref><ref><pubmed>18403917</pubmed></ref>.

=== Quiz 2===
<quiz display=simple>

{In the developing heart, Tbx5 expression can detected as early as stage 12
|type="()"}
+ true
- false
|| True. Tbx5 is detected along the entire rostrocaudal length of the fused heart tube

{Which pathways are the Tbx4 and Tbx5 genes linked to in limb outgrowth regulation?
|type="()"}
- The Notch signalling pathway
+ Fibroblast growth factor and Wnt signalling pathways
- Sonic Hedgehog
- Wnt and sonic hedgehog signalling pathways
|| Fibroblast growth factor and Wnt signalling pathways are closely associated to the signalling associated with Tbx 4 and 5 in limb outgrowth regulation.

{ Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis
|type="()"}
-False
+True
|| True

{Which parts of lung development do the Tbx genes regulate?
|type="()"}
+ lung bud and trachea specification, lunch branching and tracheal/bronchial cartilage formation
- Formation of the tracheal bifurcation only
- Development of intercostal muscles and the respiratory diaphragm
|| The Tbx genes are respobsible for the regulation of lung bud and trachea specification, lung branching morphogenesis, and tracheal/bronchial cartilage formation

{Is the Fgf10 signalling pathway activated upstream or downstream of Tbx4 and 5 in the developing lung?
|type="()"}
- Upstream
+ Downstream
|| Downstream. The Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung

</quiz>

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>
[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information see here]

====TBX22/Cleft Palate====

Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]

===Quiz 3===

<quiz display=simple>

{TBX3 mutations result in Holt– Oram Syndrome
|type="()"}
+ false
-true
|| False. Mutations of the TBX3 gene leads to ulnar-mammary syndrome.

{Mutations in which gene causes DiGeorge Syndrome
|type="()"}
- Tbx 3
+ Tbx 1
- Tbx 5
- Tbx18
|| Tbx 1

{ Thumb anomaly is an expression of holt-oran syndrome.
|type="()"}
-False
+True
|| True. Other expressions include malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly.

{Which parts of lung development do the Tbx genes regulate?
|type="()"}
+ lung bud and trachea specification, lunch branching and tracheal/bronchial cartilage formation
- Formation of the tracheal bifurcation only
- Development of intercostal muscles and the respiratory diaphragm
|| The Tbx genes are respobsible for the regulation of lung bud and trachea specification, lung branching morphogenesis, and tracheal/bronchial cartilage formation

{Tbx22 is responsible for palate closure, and hence a mutation of this gene leads to cleft palate formation.
|type="()"}
- True
+ False
|| False. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate.

</quiz>

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref> since their genome had been mapped out and in order to perform ethically experiments.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in Placental Mammals: Mouse====
'''''Mus musculus'''''

Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
[[Mouse Development | See more information on Mouse development]]

====T-box in Fish: Zebra fish====
'''''Danio rerio'''''

T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.
[[Zebrafish Development| See more information on Zebrafish Development]]

====T-box in Insects: Fruit fly====
'''''Drosophila melanogaster'''''

The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.
[[Fly Development | See more information on Fly Development]]

====T-Box in Amphibia: Clawed frog====
'''''Xenopus leaves'''''

Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.
[[Frog Development | See more information on Frog Development]]

====T-Box in Aves: Chick====
'''''Gallus gallus domesticus'''''

In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref name="PMID9550719"/>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.
[[Chicken Development | See more information on Chicken Development]]

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development: Wallaby====
'''''Macropus eugenii'''''

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area. The neonate can attach to the teat where it completes its development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.
See also [[Kangaroo Development | See more information on Kangaroo development]]

====T-Box in Amphioxus ====
'''''Branchiostoma lanceolatum'''''

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''AMP-activated protein kinase (AMPK)''': plays a key role as a master regulator of cellular energy homeostasis. Regarded as a cellular energy sensor responding to changing ATP levels. When ATP is low, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid oxidation and autophagy.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Dementia''' : severe impairment or loss of intellectual capacity and personality integration, due to the loss of or damage to neurons in the brain.

'''Heterozygotes''': a hybrid containing genes for two unlike forms of a characteristic, and therefore not breeding true to type.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Homozygote''': an organism with identical pairs of genes with respect to any given pair of hereditary characters, and therefore breeding true for that character.

'''Hypoglycaemia''': an abnormally dropped amount of sugar in the blood

'''Insulin-like growth factor 2 (IGF-2)''': Shares structural similarity to insulin.

'''Limb fields''': areas where the limb buds will develop.

'''Morphogenetic''': the development of structural features of an organism or part.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

===References===

User:Z5020373

2016-10-26T10:20:54Z

Z5020373: /* Lab 11 Assessment */

{{ANAT2341Student2016}}
{{ANAT2341Rebecca2016}}

{|
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] 4 August 2016 - Thank you for adding this content before the lab. I would suggest that rather than using a template that you simply paste on this current page with separate subheadings for each lab/assessment item. Also please no names, just your student number.
|}

==Lab 1==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:36, 5 August 2016 (AEST)

===Lab Demonstrations===

====External Link====

[http://www.smh.com.au/ SMH]

====Internal Link====

https://embryology.med.unsw.edu.au/embryology/index.php/2011_Lab_1

[[2011_Lab_1|ANAT2341 Lab 1]]

[[Student Page]]

====Referencing====
[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486280

===Lab 1 Assessment===
<pubmed>27123200</pubmed>

This research article explores whether choosing to a conduct embryo transfer (ET) on a weekend or weekday will affect the success of clinical pregnancy in IVF procedures. In the study, ET transfers were performed on either weekdays or weekends in patients with similar clinical characteristics, such as age, body-mass index and duration of infertility. Clinical pregnancy was determined using blood pregnancy tests and ultrasound examination and was defined as the “presence of a gestational sac with a foetal heart beat.” After the ETs, the authors found that there was an overall 42.8% success rate of clinical pregnancy in patients from both groups, with a 14.6% increase in the pregnancy rate when weekend ETs where compared to weekday ETs. The study however, did not examine any possible reasons to explain this increase in implantation rate although a few potential factors were discussed from previous findings in this area of research. These included endometrial receptivity which occurs 5 days after the post-ovulatory progesterone surge. The article mentioned that uterine receptivity and implantation could be affected by the junctional zone. The extent of junctional zone contractility differs throughout the ovarian cycle and an increased contractility just before ET has been previously shown to significantly decrease the likelihood of successful implantation. Since weekends are more relaxing than weekdays they suggest a possible correlation between that and reduced junctional zone contractions leading to easier ETs. Therefore from this study, it was concluded that ETs performed during the weekends are more successful than those performed during the weekdays identifying a potential factor that can improve ETs in IVF situations.

{| width=95%
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 14:24, 15 August 2016 (AEST) - This is a good summary of a paper that looks at potential environmental/endocrine effects on reproductive fertility. You needed to put the reference at the top rather than just the PMID number, fix this and you can get this full mark for the exercise.

[mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You did not fix, so I have adjusted the final mark.
| width=100px| Assessment 4/5
|}

==Lab 2==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:41, 12 August 2016 (AEST)

===Lab 2 Assessment===
[[File: Amnion_fold_development_in_chicken_embryos.jpg]]

Chicken embryo amion fold development<ref><pubmed>24647352</pubmed></ref>

{|
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 14:24, 15 August 2016 (AEST) - Very good, the image relates to early development and contains the reference, copyright and student template. (5/5)
|}

==Lab 3==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:53, 19 August 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Mesoderm_Development#Paraxial_Mesoderm|Question 2 - paraxial]]

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]
| Assessment 2.5/5
|}

==Lab 4==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:02, 26 August 2016 (AEST)

===Lab 4 Assessment===

Take the Quiz

Make your selection for all questions before clicking submit.

<quiz display=shuffle>

{How many rotations does the stomach undergo during GIT development in week 4 to 5?
|type="()"}
- 1
+ 2
- 3
- 4

|| The stomach undergoes [[two]] embryonic 90 degree rotations: the first to establish the J-shape that forms the adult stomach body (classic curvature), and the second rotation establishes it in its correct anatomical position.

{Following the degeneration of the buccopharyngeal membrane, the foregut is open to which cavity?
|type="()"}
- The peritoneal cavity
- The chorionic cavity
- The yolk sac
+ The amniotic cavity

|| During week 4 of development, the breakdown of the buccopharangeal membrane exposes the foregut to the amniotic cavity where amniotic fluid is then able to fill the foregut.

{Which one of these is not an abnormality that can occur during the proliferation and re-canalisation of the gut tube?
|type="()"}
- Occlusion
+ Meckel's diverticulum
- Duplication
- Stenosis

||During re-canalisation, if the gut tube does not re-canalise the tube can remain completely occluded. Another senario would be if there is renalisation but it occurs in discrete channels to give rise to duplicated gut tubes. The third abnormality occurs when there is incomplete vasculisation which leads to stenosis or narrowing of the tube. The only abnormality that is not involved in Meckel's diverticulum and is associated with failure of Vitelline duct breakdown leaving a yolk stalk remnant. It is a common abnormality with a prevalence of 1-2% and can lead to infection and possibly affect the rotation of the midgut.

{During week 4 in GIT development:
|type="()"}
- The cloacal membrane is broken down while the buccopharyngeal membrane remains intact
+ The buccopharyngeal membrane is broken down while the cloacal membrane remains intact
- Both the buccopharyngeal and cloacal membranes break down simultaneously
- Both the buccopharyngeal and cloacal membranes remain intact.

||Loss of the buccopharangeal membrane during week 4 allows amniotic fluid into the foregut. The cloacal membrane remains intact and does not break down until the cloaca is divided into urogenital sinuses and the rectum (occurs later in embryonic development.

</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem well designed GIT quiz questions that test topic understanding.
| Assessment 5/5
|}

==Lab 5==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:11, 2 September 2016 (AEST)

===Lab 5 Assessment===
Questionaire completed and submitted
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:17, 9 September 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 11 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab 6==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 9 September 2016 (AEST)

===Lab 6 Assessment===

<pubmed>25382630</pubmed>
Cleft palate arises when the bilateral palatal shelves fail to fuse. Many genetic and environmental factors have been identified to contribute to this deformity. One genetic mutation associated with cleft palate is the loss transforming growth factor-beta receptor (TGF-βR). A recent article by Hill ''et al.'' demonstrated that loss of TGF-βR3 reduced the expression of several ligands and receptors in the TGF-β/BMP family, including three TGF-β ligands and BMP2. During embryonic development, these molecules are involved in cell growth and differentiation.

A loss of TGF-β/BMP signaling, by receptor loss was found to be associated with cleft palate formation due to aberrant cell cycle progression and altered gene expression. Furthermore changes to TGF-β/BMP signaling also interrupted vascular development and remodeling as well as osteogenic differentiation during palate formation.
TGF-βR3 is therefore essential for maintaining the expression of TGF-β and BMP molecules and without this receptor processes of palatal shelf elongation, elevation and fusion are disrupted leading to cleft palate.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Good reference and explanation.
| Assessment 5/5
|}

==Lab 7==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 16 September 2016 (AEST)

===Lab 7 Assessment===

1. What is/are the dystrophin mutation(s)?

**The dystrophin gene is located on the short arm of the X chromosome at position 21.2. <ref>Converse, P.J. (2016) MUSCULAR DYSTROPHY, DUCHENNE TYPE; DMD OMIM http://www.omim.org/entry/310200</ref>
**It is the largest gene found in nature spanning 1.5% of the X-chromosome which is about 2.5 Mb of genomic sequence. <ref><pubmed>11917091</pubmed></ref>
**Its size could explain why it is so susceptible to spontaneous mutations.
**Encodes for Dystrophin protein.
**Mutations such as large deletions (60-70% of DMD cases), large duplications (10% of DMD cases) and point mutations (15-30% of DMD cases) can occur resulting in gene inactivation (therefore loss of function).<ref>https://www.duchenneconnect.org/understanding-genetic-testing/types-of-mutations-in-the-dystrophin-gene.html</ref>

2. What is the function of dystrophin?

**Dystrophin is part of a protein complex that work together to strengthen muscle fibers and protect them from injury as muscles contract and relax.
**Dystrophin complex acts as an anchor, connecting each muscle cell's structural framework (cytoskeleton) with the lattice of proteins and other molecules outside the cell (extracellular matrix).
**May also play a role in cell signaling by interacting with proteins that send and receive chemical signals e.g. in alpha-syntrophin.<ref><pubmed>12082140</pubmed></ref>

3. What other tissues/organs are affected by this disorder?

**Muscle (skeletal, cardiac and smooth): fatigue, difficulty with motor skills
**Respiratory system: pneumonia
**Cardiac system: cardiac myopathy
**Central Nervous system (CNS): neurobehavioural disorders e.g. ADHD and dyslexia <ref><pubmed>13947981</pubmed></ref>

4. What therapies exist for DMD?

**There is no cure available for DMD, and the current interventions are based on preventing further muscle wasting and management of symptoms and complications
**Treatments include:
***Physical therapy
***Orthopedic appliances
***Medication:
****Two corticosteroids mainly used in DMD treatment are Prednisone/Prednisolone and Deflazacort, an oxazoline derivative of prednisolone, administered by two common regimens: daily and intermittent <ref><pubmed>26457695</pubmed></ref>
****Prednisone and prednisolone show an anti-inflammatory effect
***Deflazacort acts on muscle regeneration and differentiation
** Future could include:
**Cell-based therapies using stem cells to replace dystrophin gene (a potential cure).
***Gene therapies to deliver a therapeutic gene to skeletal and cardiac muscle, in order to restore the dystrophin protein PMID <ref><pubmed>7683332</pubmed></ref>
***A study by Nelson et al. in 2016, showed that in vivo CRISPR-Cas9–mediated dystrophin restoration in mdx mouse model of DMD removed the mutated exon 23 from the dystrophin gene and improved muscle structure and function.<ref><pubmed>25123483</pubmed></ref>

5. What animal models are available for muscular dystrophy?
**The most widely used animal model for DMD is the '''mdx mouse''', which has a spontaneous point mutation in exon 23 that causes the absence of the dystrophin protein in the muscle.
**However, other animal models inlcude:<ref><pubmed>11917091</pubmed></ref>
***GRMD dog - Dystrophin-deficient dog
***HFMD cat - Dystrophin-deficient cat (clinically a poor model)
**This animal model allows testing ans screening of potential treatments and without these animal models, and without these animal models we would not have any known therapies today
**For example, mdx ''in vivo'' studies have led to U.S. FDA approval of Exondys 51 (eteplirsen) injection, the first drug approved to treat patients with Duchenne muscular dystrophy (DMD). Exondys 51 is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD. <ref>http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm521263.htm
</ref>
{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Muscular Dystrophy questions have been comprehensively answered and you have cited your sources.
| Assessment 5/5
|}

==Lab 8==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:07, 23 September 2016 (AEST)

===Lab 8 Assessment===
Group 1: Wnt signalling pathway

Firstly, a proper introduction to the Wnt pathway and its various roles in the developing embryo should be added to the page. Even though the page is focusing on Wnt signalling in fetal skin development I still think the other contributions of Wnt signalling need to be at least mentioned in the intro. Mark also suggested adding a table to show the origins of the pathway and how our knowledge about Wnt signaling has evolved.

The page contains appropriate headings and subheadings to address the different mechanisms of Wnt signalling (canonical, non-canonical and non-canonical Wnt/Ca2+ pathway). The main points for each of these pathways are up on the page which is good. Clearly, these signalling mechanisms are quite complex and adding images could help the reader to visualise how the key molecules in this pathway interact with other molecules to induce downstream effects. But otherwise, the detail in the canonical pathway is adequate for a student to understand.

The references included in each section of the page is evidence of research done to support the content on the page. The page also includes up to date information to reflect current research in this area. This information should be integrated into some sort of discussion to show how this contributes to knowledge about the signalling pathway affects developmental events. Also, all the references need to be cited correctly which I am sure you guys are aware of and will do.

Don’t forget the topic is ‘Signalling in Development’, therefore the focus should be on Wnt signalling in the developing embryo.

Overall, Group 1 has made good progress. Keep it up!

Group 2: Notch signalling pathway

The page has a proper introduction stating the critical functions of Notch signalling, examples of diseases associated with Notch mutations as well as a brief description of the mechanism of Notch signalling. There is also a timeline on the page which none of the other groups have managed to do so good job!

Some headings are missing text but those with content (e.g. canonical pathway section) are covered in extensive detail. The subheadings and headings chosen for the page indicate the group’s in depth understanding of the key components of the Notch signalling pathway. Furthermore, the page contains correct referencing and citation of text and images.

The page also discusses Notch signalling in animal models as well. A recent study was included for Drosophila. What about the other two?

Overall, Group 2 has made excellent progress. Well done!

Group 3: FGFR signalling pathway

The headings and subheadings on the page is used very effectively to aid the progression of information. Through the sequence of the headings, it allows the reader to build their understanding about FGFR signalling. The FGFR page definitely address the topic of this assessment - signalling in development, and links FGF signalling to a number of developmental events. This reflects the large contributions of FGFR in development which the page successfully portrays.

In the overview section, it states: “As shown in the image, an acidic box…”. Make it clear which image you are referring to because I can’t find it.

The table for the subtypes of FGFR has been acknowledged that it is incomplete but it gives a good snapshot to function and associated abnormalities of the different FGFR subtypes.

The page includes a student drawn image which summarises the FGFR signalling pathway. None of the other groups have included a student drawn image so good job! The images uses colours to distinguish particular molecules and shows the downstream signalling events to affect gene transcription in the cell. To me the image is a bit blurry on the page, so maybe change the pixels of the image to make it larger and easier to see?

The page includes a quiz which is clever and will definitely make the page stand out from the other groups.

Overall, Group 3 has made good progress. Good job!

Group 4: Hedgehog signalling pathway

Firstly, the page is missing an introduction to the signalling pathway. There is also text missing under the first few subheadings. Since the hedgehog pathway research began as early as the 1970s, a table including the key events in the Hedgehog research would be interesting to add.

The page includes a nice overview of the Hedgehog pathway captured in the image however, it needs a reference to acknowledge the original source of the image. Consider relocating the image to the mechanism of signalling section. This may help the reader understand the processes better if they have that image there.

Mammals have 3 Hedgehog homologues (DHH, IHH and SHH). I think that is an important point to mention.

Good discussion of animal models since it is one of the key regulators of animal development.
Despite having headings without text. Group 3 has made good progress so far. Keep it up!

Group 6: TGF-β signalling pathway

The page contains a good introduction to the TGF-β superfamily, including examples of other signaling proteins. The images help the reader visualise how TGF-β ligand bring the receptors together in a heterotetrameric complex in which the type II receptors phosphorylate and activate the type I receptors. To be pedantic, the second image still needs to include details of the original source.

Remember that the project is meant to focus on ‘Signalling in Development’ and whilst the page addresses the signalling component it does not discuss TGF-β signalling in the development of the embryo. The second image addresses its role in proliferation, migration, growth arrest and apoptosis. This pubmed article: PMID 19289080 discusses TGF-β signalling in early development, axis formation, and patterning of the embryo.

The page does not use the correct referencing or in-text citations.

Overall, Group 6 has made a good start but more research needs to be done. Keep pushing :)

==Lab 9==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:06, 7 October 2016 (AEDT)

==Lab 11==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:02, 21 October 2016 (AEDT)

===Lab 11 Assessment===
<pubmed>21350179</pubmed>

Transient Regenerative Potential of the Neonatal Mouse Heart primary literature findings:
Based on the fact that urodele amphibians and teleost fish are able to retain the capacity for cardiac regeneration throughout their life, the authors questioned whether the potential for cardiac regeneration mammals is absent, or whether it exists but merely switched off early after birth. They investigated this through observing how the hearts of one day old neonatal mice heart develops. To perform further tests they surgically resected the left ventricular apex to see how the neonatal hearts respond to injury. They were able to see that the the ventricular apex in the mice indeed stimulated a regenerative response that appears to restore the damaged heart to its normal anatomy and function. This regenerative response was marked by cardiomyocyte proliferation with little evidence of hypertrophy or fibrosis, and therefore not a result repair processes. The origin of these cardiomyocytes in the regenerated tissue were determined to be from preexisting cardiomyocytes. The regenerated ventricular apex also had normal systolic function indicating a restoration of normal contractile function. A similar approach was done to seven day old mice and unlike the one day old mice, they displayed extensive fibrosis. Therefore the window for cardiac regeneration is brief in mammals, but there is a capacity to regenerate.

The purpose of the review article is to: "review our current understanding of how cardiomyocyte proliferation is regulated during heart development and regeneration". The primary article clearly relates to the topic of cardiomyocyte proliferation in the context of regeneration, which is why it has been used in the review article. The original research was used to provide evidence to support the statement: "cardiomyocytes in various contexts do harbor endogenous proliferative capabilities, albeit to different degrees". Correlations between the original research and the claims made by the review article include: "various contexts" - which in this case refers to the type of organism in which mice applies; and "endogenous proliferative capabilities" which from the first paragraph, the original research does support.

==References==

{{Stem Cell Presentations 2016}}

User:Z5020373

2016-10-26T10:19:53Z

Z5020373: /* Lab 11 Assessment */

{{ANAT2341Student2016}}
{{ANAT2341Rebecca2016}}

{|
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] 4 August 2016 - Thank you for adding this content before the lab. I would suggest that rather than using a template that you simply paste on this current page with separate subheadings for each lab/assessment item. Also please no names, just your student number.
|}

==Lab 1==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:36, 5 August 2016 (AEST)

===Lab Demonstrations===

====External Link====

[http://www.smh.com.au/ SMH]

====Internal Link====

https://embryology.med.unsw.edu.au/embryology/index.php/2011_Lab_1

[[2011_Lab_1|ANAT2341 Lab 1]]

[[Student Page]]

====Referencing====
[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486280

===Lab 1 Assessment===
<pubmed>27123200</pubmed>

This research article explores whether choosing to a conduct embryo transfer (ET) on a weekend or weekday will affect the success of clinical pregnancy in IVF procedures. In the study, ET transfers were performed on either weekdays or weekends in patients with similar clinical characteristics, such as age, body-mass index and duration of infertility. Clinical pregnancy was determined using blood pregnancy tests and ultrasound examination and was defined as the “presence of a gestational sac with a foetal heart beat.” After the ETs, the authors found that there was an overall 42.8% success rate of clinical pregnancy in patients from both groups, with a 14.6% increase in the pregnancy rate when weekend ETs where compared to weekday ETs. The study however, did not examine any possible reasons to explain this increase in implantation rate although a few potential factors were discussed from previous findings in this area of research. These included endometrial receptivity which occurs 5 days after the post-ovulatory progesterone surge. The article mentioned that uterine receptivity and implantation could be affected by the junctional zone. The extent of junctional zone contractility differs throughout the ovarian cycle and an increased contractility just before ET has been previously shown to significantly decrease the likelihood of successful implantation. Since weekends are more relaxing than weekdays they suggest a possible correlation between that and reduced junctional zone contractions leading to easier ETs. Therefore from this study, it was concluded that ETs performed during the weekends are more successful than those performed during the weekdays identifying a potential factor that can improve ETs in IVF situations.

{| width=95%
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 14:24, 15 August 2016 (AEST) - This is a good summary of a paper that looks at potential environmental/endocrine effects on reproductive fertility. You needed to put the reference at the top rather than just the PMID number, fix this and you can get this full mark for the exercise.

[mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You did not fix, so I have adjusted the final mark.
| width=100px| Assessment 4/5
|}

==Lab 2==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:41, 12 August 2016 (AEST)

===Lab 2 Assessment===
[[File: Amnion_fold_development_in_chicken_embryos.jpg]]

Chicken embryo amion fold development<ref><pubmed>24647352</pubmed></ref>

{|
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 14:24, 15 August 2016 (AEST) - Very good, the image relates to early development and contains the reference, copyright and student template. (5/5)
|}

==Lab 3==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:53, 19 August 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Mesoderm_Development#Paraxial_Mesoderm|Question 2 - paraxial]]

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]
| Assessment 2.5/5
|}

==Lab 4==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:02, 26 August 2016 (AEST)

===Lab 4 Assessment===

Take the Quiz

Make your selection for all questions before clicking submit.

<quiz display=shuffle>

{How many rotations does the stomach undergo during GIT development in week 4 to 5?
|type="()"}
- 1
+ 2
- 3
- 4

|| The stomach undergoes [[two]] embryonic 90 degree rotations: the first to establish the J-shape that forms the adult stomach body (classic curvature), and the second rotation establishes it in its correct anatomical position.

{Following the degeneration of the buccopharyngeal membrane, the foregut is open to which cavity?
|type="()"}
- The peritoneal cavity
- The chorionic cavity
- The yolk sac
+ The amniotic cavity

|| During week 4 of development, the breakdown of the buccopharangeal membrane exposes the foregut to the amniotic cavity where amniotic fluid is then able to fill the foregut.

{Which one of these is not an abnormality that can occur during the proliferation and re-canalisation of the gut tube?
|type="()"}
- Occlusion
+ Meckel's diverticulum
- Duplication
- Stenosis

||During re-canalisation, if the gut tube does not re-canalise the tube can remain completely occluded. Another senario would be if there is renalisation but it occurs in discrete channels to give rise to duplicated gut tubes. The third abnormality occurs when there is incomplete vasculisation which leads to stenosis or narrowing of the tube. The only abnormality that is not involved in Meckel's diverticulum and is associated with failure of Vitelline duct breakdown leaving a yolk stalk remnant. It is a common abnormality with a prevalence of 1-2% and can lead to infection and possibly affect the rotation of the midgut.

{During week 4 in GIT development:
|type="()"}
- The cloacal membrane is broken down while the buccopharyngeal membrane remains intact
+ The buccopharyngeal membrane is broken down while the cloacal membrane remains intact
- Both the buccopharyngeal and cloacal membranes break down simultaneously
- Both the buccopharyngeal and cloacal membranes remain intact.

||Loss of the buccopharangeal membrane during week 4 allows amniotic fluid into the foregut. The cloacal membrane remains intact and does not break down until the cloaca is divided into urogenital sinuses and the rectum (occurs later in embryonic development.

</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem well designed GIT quiz questions that test topic understanding.
| Assessment 5/5
|}

==Lab 5==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:11, 2 September 2016 (AEST)

===Lab 5 Assessment===
Questionaire completed and submitted
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:17, 9 September 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 11 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab 6==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 9 September 2016 (AEST)

===Lab 6 Assessment===

<pubmed>25382630</pubmed>
Cleft palate arises when the bilateral palatal shelves fail to fuse. Many genetic and environmental factors have been identified to contribute to this deformity. One genetic mutation associated with cleft palate is the loss transforming growth factor-beta receptor (TGF-βR). A recent article by Hill ''et al.'' demonstrated that loss of TGF-βR3 reduced the expression of several ligands and receptors in the TGF-β/BMP family, including three TGF-β ligands and BMP2. During embryonic development, these molecules are involved in cell growth and differentiation.

A loss of TGF-β/BMP signaling, by receptor loss was found to be associated with cleft palate formation due to aberrant cell cycle progression and altered gene expression. Furthermore changes to TGF-β/BMP signaling also interrupted vascular development and remodeling as well as osteogenic differentiation during palate formation.
TGF-βR3 is therefore essential for maintaining the expression of TGF-β and BMP molecules and without this receptor processes of palatal shelf elongation, elevation and fusion are disrupted leading to cleft palate.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Good reference and explanation.
| Assessment 5/5
|}

==Lab 7==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 16 September 2016 (AEST)

===Lab 7 Assessment===

1. What is/are the dystrophin mutation(s)?

**The dystrophin gene is located on the short arm of the X chromosome at position 21.2. <ref>Converse, P.J. (2016) MUSCULAR DYSTROPHY, DUCHENNE TYPE; DMD OMIM http://www.omim.org/entry/310200</ref>
**It is the largest gene found in nature spanning 1.5% of the X-chromosome which is about 2.5 Mb of genomic sequence. <ref><pubmed>11917091</pubmed></ref>
**Its size could explain why it is so susceptible to spontaneous mutations.
**Encodes for Dystrophin protein.
**Mutations such as large deletions (60-70% of DMD cases), large duplications (10% of DMD cases) and point mutations (15-30% of DMD cases) can occur resulting in gene inactivation (therefore loss of function).<ref>https://www.duchenneconnect.org/understanding-genetic-testing/types-of-mutations-in-the-dystrophin-gene.html</ref>

2. What is the function of dystrophin?

**Dystrophin is part of a protein complex that work together to strengthen muscle fibers and protect them from injury as muscles contract and relax.
**Dystrophin complex acts as an anchor, connecting each muscle cell's structural framework (cytoskeleton) with the lattice of proteins and other molecules outside the cell (extracellular matrix).
**May also play a role in cell signaling by interacting with proteins that send and receive chemical signals e.g. in alpha-syntrophin.<ref><pubmed>12082140</pubmed></ref>

3. What other tissues/organs are affected by this disorder?

**Muscle (skeletal, cardiac and smooth): fatigue, difficulty with motor skills
**Respiratory system: pneumonia
**Cardiac system: cardiac myopathy
**Central Nervous system (CNS): neurobehavioural disorders e.g. ADHD and dyslexia <ref><pubmed>13947981</pubmed></ref>

4. What therapies exist for DMD?

**There is no cure available for DMD, and the current interventions are based on preventing further muscle wasting and management of symptoms and complications
**Treatments include:
***Physical therapy
***Orthopedic appliances
***Medication:
****Two corticosteroids mainly used in DMD treatment are Prednisone/Prednisolone and Deflazacort, an oxazoline derivative of prednisolone, administered by two common regimens: daily and intermittent <ref><pubmed>26457695</pubmed></ref>
****Prednisone and prednisolone show an anti-inflammatory effect
***Deflazacort acts on muscle regeneration and differentiation
** Future could include:
**Cell-based therapies using stem cells to replace dystrophin gene (a potential cure).
***Gene therapies to deliver a therapeutic gene to skeletal and cardiac muscle, in order to restore the dystrophin protein PMID <ref><pubmed>7683332</pubmed></ref>
***A study by Nelson et al. in 2016, showed that in vivo CRISPR-Cas9–mediated dystrophin restoration in mdx mouse model of DMD removed the mutated exon 23 from the dystrophin gene and improved muscle structure and function.<ref><pubmed>25123483</pubmed></ref>

5. What animal models are available for muscular dystrophy?
**The most widely used animal model for DMD is the '''mdx mouse''', which has a spontaneous point mutation in exon 23 that causes the absence of the dystrophin protein in the muscle.
**However, other animal models inlcude:<ref><pubmed>11917091</pubmed></ref>
***GRMD dog - Dystrophin-deficient dog
***HFMD cat - Dystrophin-deficient cat (clinically a poor model)
**This animal model allows testing ans screening of potential treatments and without these animal models, and without these animal models we would not have any known therapies today
**For example, mdx ''in vivo'' studies have led to U.S. FDA approval of Exondys 51 (eteplirsen) injection, the first drug approved to treat patients with Duchenne muscular dystrophy (DMD). Exondys 51 is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD. <ref>http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm521263.htm
</ref>
{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Muscular Dystrophy questions have been comprehensively answered and you have cited your sources.
| Assessment 5/5
|}

==Lab 8==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:07, 23 September 2016 (AEST)

===Lab 8 Assessment===
Group 1: Wnt signalling pathway

Firstly, a proper introduction to the Wnt pathway and its various roles in the developing embryo should be added to the page. Even though the page is focusing on Wnt signalling in fetal skin development I still think the other contributions of Wnt signalling need to be at least mentioned in the intro. Mark also suggested adding a table to show the origins of the pathway and how our knowledge about Wnt signaling has evolved.

The page contains appropriate headings and subheadings to address the different mechanisms of Wnt signalling (canonical, non-canonical and non-canonical Wnt/Ca2+ pathway). The main points for each of these pathways are up on the page which is good. Clearly, these signalling mechanisms are quite complex and adding images could help the reader to visualise how the key molecules in this pathway interact with other molecules to induce downstream effects. But otherwise, the detail in the canonical pathway is adequate for a student to understand.

The references included in each section of the page is evidence of research done to support the content on the page. The page also includes up to date information to reflect current research in this area. This information should be integrated into some sort of discussion to show how this contributes to knowledge about the signalling pathway affects developmental events. Also, all the references need to be cited correctly which I am sure you guys are aware of and will do.

Don’t forget the topic is ‘Signalling in Development’, therefore the focus should be on Wnt signalling in the developing embryo.

Overall, Group 1 has made good progress. Keep it up!

Group 2: Notch signalling pathway

The page has a proper introduction stating the critical functions of Notch signalling, examples of diseases associated with Notch mutations as well as a brief description of the mechanism of Notch signalling. There is also a timeline on the page which none of the other groups have managed to do so good job!

Some headings are missing text but those with content (e.g. canonical pathway section) are covered in extensive detail. The subheadings and headings chosen for the page indicate the group’s in depth understanding of the key components of the Notch signalling pathway. Furthermore, the page contains correct referencing and citation of text and images.

The page also discusses Notch signalling in animal models as well. A recent study was included for Drosophila. What about the other two?

Overall, Group 2 has made excellent progress. Well done!

Group 3: FGFR signalling pathway

The headings and subheadings on the page is used very effectively to aid the progression of information. Through the sequence of the headings, it allows the reader to build their understanding about FGFR signalling. The FGFR page definitely address the topic of this assessment - signalling in development, and links FGF signalling to a number of developmental events. This reflects the large contributions of FGFR in development which the page successfully portrays.

In the overview section, it states: “As shown in the image, an acidic box…”. Make it clear which image you are referring to because I can’t find it.

The table for the subtypes of FGFR has been acknowledged that it is incomplete but it gives a good snapshot to function and associated abnormalities of the different FGFR subtypes.

The page includes a student drawn image which summarises the FGFR signalling pathway. None of the other groups have included a student drawn image so good job! The images uses colours to distinguish particular molecules and shows the downstream signalling events to affect gene transcription in the cell. To me the image is a bit blurry on the page, so maybe change the pixels of the image to make it larger and easier to see?

The page includes a quiz which is clever and will definitely make the page stand out from the other groups.

Overall, Group 3 has made good progress. Good job!

Group 4: Hedgehog signalling pathway

Firstly, the page is missing an introduction to the signalling pathway. There is also text missing under the first few subheadings. Since the hedgehog pathway research began as early as the 1970s, a table including the key events in the Hedgehog research would be interesting to add.

The page includes a nice overview of the Hedgehog pathway captured in the image however, it needs a reference to acknowledge the original source of the image. Consider relocating the image to the mechanism of signalling section. This may help the reader understand the processes better if they have that image there.

Mammals have 3 Hedgehog homologues (DHH, IHH and SHH). I think that is an important point to mention.

Good discussion of animal models since it is one of the key regulators of animal development.
Despite having headings without text. Group 3 has made good progress so far. Keep it up!

Group 6: TGF-β signalling pathway

The page contains a good introduction to the TGF-β superfamily, including examples of other signaling proteins. The images help the reader visualise how TGF-β ligand bring the receptors together in a heterotetrameric complex in which the type II receptors phosphorylate and activate the type I receptors. To be pedantic, the second image still needs to include details of the original source.

Remember that the project is meant to focus on ‘Signalling in Development’ and whilst the page addresses the signalling component it does not discuss TGF-β signalling in the development of the embryo. The second image addresses its role in proliferation, migration, growth arrest and apoptosis. This pubmed article: PMID 19289080 discusses TGF-β signalling in early development, axis formation, and patterning of the embryo.

The page does not use the correct referencing or in-text citations.

Overall, Group 6 has made a good start but more research needs to be done. Keep pushing :)

==Lab 9==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:06, 7 October 2016 (AEDT)

==Lab 11==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:02, 21 October 2016 (AEDT)

===Lab 11 Assessment===
Write a brief summary of the paper's main findings. Then describe how the original research result was used in the review article.
<pubmed>PMID21350179</pubmed>

Transient Regenerative Potential of the Neonatal Mouse Heart primary literature findings:
Based on the fact that urodele amphibians and teleost fish are able to retain the capacity for cardiac regeneration throughout their life, the authors questioned whether the potential for cardiac regeneration mammals is absent, or whether it exists but merely switched off early after birth. They investigated this through observing how the hearts of one day old neonatal mice heart develops. To perform further tests they surgically resected the left ventricular apex to see how the neonatal hearts respond to injury. They were able to see that the the ventricular apex in the mice indeed stimulated a regenerative response that appears to restore the damaged heart to its normal anatomy and function. This regenerative response was marked by cardiomyocyte proliferation with little evidence of hypertrophy or fibrosis, and therefore not a result repair processes. The origin of these cardiomyocytes in the regenerated tissue were determined to be from preexisting cardiomyocytes. The regenerated ventricular apex also had normal systolic function indicating a restoration of normal contractile function. A similar approach was done to seven day old mice and unlike the one day old mice, they displayed extensive fibrosis. Therefore the window for cardiac regeneration is brief in mammals, but there is a capacity to regenerate.

The purpose of the review article is to: "review our current understanding of how cardiomyocyte proliferation is regulated during heart development and regeneration". The primary article clearly relates to the topic of cardiomyocyte proliferation in the context of regeneration, which is why it has been used in the review article. The original research was used to provide evidence to support the statement: "cardiomyocytes in various contexts do harbor endogenous proliferative capabilities, albeit to different degrees". Correlations between the original research and the claims made by the review article include: "various contexts" - which in this case refers to the type of organism in which mice applies; and "endogenous proliferative capabilities" which from the first paragraph, the original research does support.

==References==

{{Stem Cell Presentations 2016}}

User:Z5020373

2016-10-26T09:30:45Z

Z5020373: /* Lab 11 Assessment */

{{ANAT2341Student2016}}
{{ANAT2341Rebecca2016}}

{|
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] 4 August 2016 - Thank you for adding this content before the lab. I would suggest that rather than using a template that you simply paste on this current page with separate subheadings for each lab/assessment item. Also please no names, just your student number.
|}

==Lab 1==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:36, 5 August 2016 (AEST)

===Lab Demonstrations===

====External Link====

[http://www.smh.com.au/ SMH]

====Internal Link====

https://embryology.med.unsw.edu.au/embryology/index.php/2011_Lab_1

[[2011_Lab_1|ANAT2341 Lab 1]]

[[Student Page]]

====Referencing====
[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486280

===Lab 1 Assessment===
<pubmed>27123200</pubmed>

This research article explores whether choosing to a conduct embryo transfer (ET) on a weekend or weekday will affect the success of clinical pregnancy in IVF procedures. In the study, ET transfers were performed on either weekdays or weekends in patients with similar clinical characteristics, such as age, body-mass index and duration of infertility. Clinical pregnancy was determined using blood pregnancy tests and ultrasound examination and was defined as the “presence of a gestational sac with a foetal heart beat.” After the ETs, the authors found that there was an overall 42.8% success rate of clinical pregnancy in patients from both groups, with a 14.6% increase in the pregnancy rate when weekend ETs where compared to weekday ETs. The study however, did not examine any possible reasons to explain this increase in implantation rate although a few potential factors were discussed from previous findings in this area of research. These included endometrial receptivity which occurs 5 days after the post-ovulatory progesterone surge. The article mentioned that uterine receptivity and implantation could be affected by the junctional zone. The extent of junctional zone contractility differs throughout the ovarian cycle and an increased contractility just before ET has been previously shown to significantly decrease the likelihood of successful implantation. Since weekends are more relaxing than weekdays they suggest a possible correlation between that and reduced junctional zone contractions leading to easier ETs. Therefore from this study, it was concluded that ETs performed during the weekends are more successful than those performed during the weekdays identifying a potential factor that can improve ETs in IVF situations.

{| width=95%
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 14:24, 15 August 2016 (AEST) - This is a good summary of a paper that looks at potential environmental/endocrine effects on reproductive fertility. You needed to put the reference at the top rather than just the PMID number, fix this and you can get this full mark for the exercise.

[mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You did not fix, so I have adjusted the final mark.
| width=100px| Assessment 4/5
|}

==Lab 2==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:41, 12 August 2016 (AEST)

===Lab 2 Assessment===
[[File: Amnion_fold_development_in_chicken_embryos.jpg]]

Chicken embryo amion fold development<ref><pubmed>24647352</pubmed></ref>

{|
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 14:24, 15 August 2016 (AEST) - Very good, the image relates to early development and contains the reference, copyright and student template. (5/5)
|}

==Lab 3==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:53, 19 August 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Mesoderm_Development#Paraxial_Mesoderm|Question 2 - paraxial]]

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]
| Assessment 2.5/5
|}

==Lab 4==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:02, 26 August 2016 (AEST)

===Lab 4 Assessment===

Take the Quiz

Make your selection for all questions before clicking submit.

<quiz display=shuffle>

{How many rotations does the stomach undergo during GIT development in week 4 to 5?
|type="()"}
- 1
+ 2
- 3
- 4

|| The stomach undergoes [[two]] embryonic 90 degree rotations: the first to establish the J-shape that forms the adult stomach body (classic curvature), and the second rotation establishes it in its correct anatomical position.

{Following the degeneration of the buccopharyngeal membrane, the foregut is open to which cavity?
|type="()"}
- The peritoneal cavity
- The chorionic cavity
- The yolk sac
+ The amniotic cavity

|| During week 4 of development, the breakdown of the buccopharangeal membrane exposes the foregut to the amniotic cavity where amniotic fluid is then able to fill the foregut.

{Which one of these is not an abnormality that can occur during the proliferation and re-canalisation of the gut tube?
|type="()"}
- Occlusion
+ Meckel's diverticulum
- Duplication
- Stenosis

||During re-canalisation, if the gut tube does not re-canalise the tube can remain completely occluded. Another senario would be if there is renalisation but it occurs in discrete channels to give rise to duplicated gut tubes. The third abnormality occurs when there is incomplete vasculisation which leads to stenosis or narrowing of the tube. The only abnormality that is not involved in Meckel's diverticulum and is associated with failure of Vitelline duct breakdown leaving a yolk stalk remnant. It is a common abnormality with a prevalence of 1-2% and can lead to infection and possibly affect the rotation of the midgut.

{During week 4 in GIT development:
|type="()"}
- The cloacal membrane is broken down while the buccopharyngeal membrane remains intact
+ The buccopharyngeal membrane is broken down while the cloacal membrane remains intact
- Both the buccopharyngeal and cloacal membranes break down simultaneously
- Both the buccopharyngeal and cloacal membranes remain intact.

||Loss of the buccopharangeal membrane during week 4 allows amniotic fluid into the foregut. The cloacal membrane remains intact and does not break down until the cloaca is divided into urogenital sinuses and the rectum (occurs later in embryonic development.

</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem well designed GIT quiz questions that test topic understanding.
| Assessment 5/5
|}

==Lab 5==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:11, 2 September 2016 (AEST)

===Lab 5 Assessment===
Questionaire completed and submitted
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:17, 9 September 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 11 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab 6==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 9 September 2016 (AEST)

===Lab 6 Assessment===

<pubmed>25382630</pubmed>
Cleft palate arises when the bilateral palatal shelves fail to fuse. Many genetic and environmental factors have been identified to contribute to this deformity. One genetic mutation associated with cleft palate is the loss transforming growth factor-beta receptor (TGF-βR). A recent article by Hill ''et al.'' demonstrated that loss of TGF-βR3 reduced the expression of several ligands and receptors in the TGF-β/BMP family, including three TGF-β ligands and BMP2. During embryonic development, these molecules are involved in cell growth and differentiation.

A loss of TGF-β/BMP signaling, by receptor loss was found to be associated with cleft palate formation due to aberrant cell cycle progression and altered gene expression. Furthermore changes to TGF-β/BMP signaling also interrupted vascular development and remodeling as well as osteogenic differentiation during palate formation.
TGF-βR3 is therefore essential for maintaining the expression of TGF-β and BMP molecules and without this receptor processes of palatal shelf elongation, elevation and fusion are disrupted leading to cleft palate.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Good reference and explanation.
| Assessment 5/5
|}

==Lab 7==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 16 September 2016 (AEST)

===Lab 7 Assessment===

1. What is/are the dystrophin mutation(s)?

**The dystrophin gene is located on the short arm of the X chromosome at position 21.2. <ref>Converse, P.J. (2016) MUSCULAR DYSTROPHY, DUCHENNE TYPE; DMD OMIM http://www.omim.org/entry/310200</ref>
**It is the largest gene found in nature spanning 1.5% of the X-chromosome which is about 2.5 Mb of genomic sequence. <ref><pubmed>11917091</pubmed></ref>
**Its size could explain why it is so susceptible to spontaneous mutations.
**Encodes for Dystrophin protein.
**Mutations such as large deletions (60-70% of DMD cases), large duplications (10% of DMD cases) and point mutations (15-30% of DMD cases) can occur resulting in gene inactivation (therefore loss of function).<ref>https://www.duchenneconnect.org/understanding-genetic-testing/types-of-mutations-in-the-dystrophin-gene.html</ref>

2. What is the function of dystrophin?

**Dystrophin is part of a protein complex that work together to strengthen muscle fibers and protect them from injury as muscles contract and relax.
**Dystrophin complex acts as an anchor, connecting each muscle cell's structural framework (cytoskeleton) with the lattice of proteins and other molecules outside the cell (extracellular matrix).
**May also play a role in cell signaling by interacting with proteins that send and receive chemical signals e.g. in alpha-syntrophin.<ref><pubmed>12082140</pubmed></ref>

3. What other tissues/organs are affected by this disorder?

**Muscle (skeletal, cardiac and smooth): fatigue, difficulty with motor skills
**Respiratory system: pneumonia
**Cardiac system: cardiac myopathy
**Central Nervous system (CNS): neurobehavioural disorders e.g. ADHD and dyslexia <ref><pubmed>13947981</pubmed></ref>

4. What therapies exist for DMD?

**There is no cure available for DMD, and the current interventions are based on preventing further muscle wasting and management of symptoms and complications
**Treatments include:
***Physical therapy
***Orthopedic appliances
***Medication:
****Two corticosteroids mainly used in DMD treatment are Prednisone/Prednisolone and Deflazacort, an oxazoline derivative of prednisolone, administered by two common regimens: daily and intermittent <ref><pubmed>26457695</pubmed></ref>
****Prednisone and prednisolone show an anti-inflammatory effect
***Deflazacort acts on muscle regeneration and differentiation
** Future could include:
**Cell-based therapies using stem cells to replace dystrophin gene (a potential cure).
***Gene therapies to deliver a therapeutic gene to skeletal and cardiac muscle, in order to restore the dystrophin protein PMID <ref><pubmed>7683332</pubmed></ref>
***A study by Nelson et al. in 2016, showed that in vivo CRISPR-Cas9–mediated dystrophin restoration in mdx mouse model of DMD removed the mutated exon 23 from the dystrophin gene and improved muscle structure and function.<ref><pubmed>25123483</pubmed></ref>

5. What animal models are available for muscular dystrophy?
**The most widely used animal model for DMD is the '''mdx mouse''', which has a spontaneous point mutation in exon 23 that causes the absence of the dystrophin protein in the muscle.
**However, other animal models inlcude:<ref><pubmed>11917091</pubmed></ref>
***GRMD dog - Dystrophin-deficient dog
***HFMD cat - Dystrophin-deficient cat (clinically a poor model)
**This animal model allows testing ans screening of potential treatments and without these animal models, and without these animal models we would not have any known therapies today
**For example, mdx ''in vivo'' studies have led to U.S. FDA approval of Exondys 51 (eteplirsen) injection, the first drug approved to treat patients with Duchenne muscular dystrophy (DMD). Exondys 51 is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD. <ref>http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm521263.htm
</ref>
{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Muscular Dystrophy questions have been comprehensively answered and you have cited your sources.
| Assessment 5/5
|}

==Lab 8==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:07, 23 September 2016 (AEST)

===Lab 8 Assessment===
Group 1: Wnt signalling pathway

Firstly, a proper introduction to the Wnt pathway and its various roles in the developing embryo should be added to the page. Even though the page is focusing on Wnt signalling in fetal skin development I still think the other contributions of Wnt signalling need to be at least mentioned in the intro. Mark also suggested adding a table to show the origins of the pathway and how our knowledge about Wnt signaling has evolved.

The page contains appropriate headings and subheadings to address the different mechanisms of Wnt signalling (canonical, non-canonical and non-canonical Wnt/Ca2+ pathway). The main points for each of these pathways are up on the page which is good. Clearly, these signalling mechanisms are quite complex and adding images could help the reader to visualise how the key molecules in this pathway interact with other molecules to induce downstream effects. But otherwise, the detail in the canonical pathway is adequate for a student to understand.

The references included in each section of the page is evidence of research done to support the content on the page. The page also includes up to date information to reflect current research in this area. This information should be integrated into some sort of discussion to show how this contributes to knowledge about the signalling pathway affects developmental events. Also, all the references need to be cited correctly which I am sure you guys are aware of and will do.

Don’t forget the topic is ‘Signalling in Development’, therefore the focus should be on Wnt signalling in the developing embryo.

Overall, Group 1 has made good progress. Keep it up!

Group 2: Notch signalling pathway

The page has a proper introduction stating the critical functions of Notch signalling, examples of diseases associated with Notch mutations as well as a brief description of the mechanism of Notch signalling. There is also a timeline on the page which none of the other groups have managed to do so good job!

Some headings are missing text but those with content (e.g. canonical pathway section) are covered in extensive detail. The subheadings and headings chosen for the page indicate the group’s in depth understanding of the key components of the Notch signalling pathway. Furthermore, the page contains correct referencing and citation of text and images.

The page also discusses Notch signalling in animal models as well. A recent study was included for Drosophila. What about the other two?

Overall, Group 2 has made excellent progress. Well done!

Group 3: FGFR signalling pathway

The headings and subheadings on the page is used very effectively to aid the progression of information. Through the sequence of the headings, it allows the reader to build their understanding about FGFR signalling. The FGFR page definitely address the topic of this assessment - signalling in development, and links FGF signalling to a number of developmental events. This reflects the large contributions of FGFR in development which the page successfully portrays.

In the overview section, it states: “As shown in the image, an acidic box…”. Make it clear which image you are referring to because I can’t find it.

The table for the subtypes of FGFR has been acknowledged that it is incomplete but it gives a good snapshot to function and associated abnormalities of the different FGFR subtypes.

The page includes a student drawn image which summarises the FGFR signalling pathway. None of the other groups have included a student drawn image so good job! The images uses colours to distinguish particular molecules and shows the downstream signalling events to affect gene transcription in the cell. To me the image is a bit blurry on the page, so maybe change the pixels of the image to make it larger and easier to see?

The page includes a quiz which is clever and will definitely make the page stand out from the other groups.

Overall, Group 3 has made good progress. Good job!

Group 4: Hedgehog signalling pathway

Firstly, the page is missing an introduction to the signalling pathway. There is also text missing under the first few subheadings. Since the hedgehog pathway research began as early as the 1970s, a table including the key events in the Hedgehog research would be interesting to add.

The page includes a nice overview of the Hedgehog pathway captured in the image however, it needs a reference to acknowledge the original source of the image. Consider relocating the image to the mechanism of signalling section. This may help the reader understand the processes better if they have that image there.

Mammals have 3 Hedgehog homologues (DHH, IHH and SHH). I think that is an important point to mention.

Good discussion of animal models since it is one of the key regulators of animal development.
Despite having headings without text. Group 3 has made good progress so far. Keep it up!

Group 6: TGF-β signalling pathway

The page contains a good introduction to the TGF-β superfamily, including examples of other signaling proteins. The images help the reader visualise how TGF-β ligand bring the receptors together in a heterotetrameric complex in which the type II receptors phosphorylate and activate the type I receptors. To be pedantic, the second image still needs to include details of the original source.

Remember that the project is meant to focus on ‘Signalling in Development’ and whilst the page addresses the signalling component it does not discuss TGF-β signalling in the development of the embryo. The second image addresses its role in proliferation, migration, growth arrest and apoptosis. This pubmed article: PMID 19289080 discusses TGF-β signalling in early development, axis formation, and patterning of the embryo.

The page does not use the correct referencing or in-text citations.

Overall, Group 6 has made a good start but more research needs to be done. Keep pushing :)

==Lab 9==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:06, 7 October 2016 (AEDT)

==Lab 11==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:02, 21 October 2016 (AEDT)

===Lab 11 Assessment===
Write a brief summary of the paper's main findings. Then describe how the original research result was used in the review article.
<ref>PMID21350179</ref>

==References==

{{Stem Cell Presentations 2016}}

2016 Group Project 5

2016-10-26T09:29:06Z

Z5020373: /* Functions of T-box in development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page will give a board overview of how the T-box signalling pathway works, as well as its importance, it's discovery, abnormalities associated with this transcription factor, and animal models that have been used to study these genes. It is important to note that T-box genes have also been found to regulate patterning and cell fate, cell survival, and/or proliferation. This however will not be covered in this web page. This web page will focus on the importance of T-box genes in embryological limb development.
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information, click here]

====T-Box gene and embryology====
The T-box gene family plays an essential role in controlling embryogenesis in a wide variety of organisms, including many invertebrates, amphibians and mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which, during embryological development, are important in establishing the early body plan and later are important in the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek meaning short tail. '''Brakhus''' means short in greek and '''oura''' meaning tail. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered through experimental studies with a short tailed mouse that harboured a mutation which affected it's tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted (see below for more information on the discovery).
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948, taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) was soon studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now, in human and mouse genomes, the gene brachyura is represented by the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The discovery of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref name="PMID11268043 "/>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

====Timeline of the discovery of the T-Box gene====

In 1927, the Brachyury (T) locus was introduced to the scientific world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, further embryological defects caused by the T mutation were studied, as well as the importance of the T-box genes in normal signalling pathways and embryonic development.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]
Members of the T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) have been found to be expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have implicated in several developmental events: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In chick embryos, Tbx4 and Fgf10 have been found to co-express in the foregut mesoderm (in a lung field), in a domain that coincides with that of Nkx2.1 in the endoderm (except in its most anterior portion)<ref name="PMID8853987"/>. Studies show that abnormal expression of Tbx4 induces ectopic Fgf10 expression and ectopic buds that express Nkx 2.1 molecules. This suggests that Tbx-Fgf10 interaction plays a role in lung morphogenesis as the Nkx2.1 gene encodes a transcription factor that is expressed during early development of thyroid, lung, and forebrain regions, particularly the basal ganglia and hypothalamus <ref><pubmed>24714694</pubmed></ref>.

Moreover, Fgf10 genetically interacts with Tbx4 and Tbx5 in lung branching morphogenesis. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a one-sided loss of lung bud specification and absence of tracheal specification in organ culture. Furthermore, mesenchymal markers Wnt2 and Fgf10 expression, and Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung<ref><pubmed>9916808</pubmed></ref>. This is consistent with findings from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires the ability for the initial budding morphogenesis of primary lung buds<ref name="PMID12588840"><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 deficient mice died soon after birth due to respiratory distress. These offspring have small lungs and show severe abnormalities in tracheal and bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development<ref name="PMID22876201"/>.

====Other developmental events====
TBX signalling is also involved in many other developmental processes. 2 examples are palate development and skeletal muscle fibre-type determination.

In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) demonstrated that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 deficient mice had abnormal epithelial adhesion between the palate and mandible which led to several forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft similar to human conditions<ref><pubmed>22371266</pubmed></ref><ref><pubmed>14585638</pubmed></ref>.

Skeletal muscle comprises of a mosaic pattern of slow oxidative myofibres and fast glycolytic myofibres that influences muscle function and whole body metabolism. The mesodermal transcription factor Tbx15 is specifically expressed in glycolytic myofibres. Inactivation of Tbx15 leads to muscle size reduction due to a decrease in the number of glycolytic fibres, associated with a small increase in the number of oxidative fibres. This shift in fibre composition results in a subsequent shift of substrates from muscle to fat and liver where they are stored as lipids, leading to increased adiposity and glucose intolerance. The mechanism by which this occurs involves the activation of AMP-activated protein kinase (AMPK) signalling and a decrease in insulin growth factor 2 (Igf2) expression. Tbx15 is one of the few known transcription factors that are critical regulators of fibre-type distribution and skeletal muscle metabolism in the embryonic and post-natal period
<ref><pubmed>26299309</pubmed></ref><ref><pubmed>18403917</pubmed></ref>.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>
[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}

====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information see here]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref> since their genome had been mapped out and in order to perform ethically experiments.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in Placental Mammals: Mouse====
'''''Mus musculus'''''

Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
[[Mouse Development | See more information on Mouse development]]

====T-box in Fish: Zebra fish====
'''''Danio rerio'''''

T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.
[[Zebrafish Development| See more information on Zebrafish Development]]

====T-box in Insects: Fruit fly====
'''''Drosophila melanogaster'''''

The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.
[[Fly Development | See more information on Fly Development]]

====T-Box in Amphibia: Clawed frog====
'''''Xenopus leaves'''''

Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.
[[Frog Development | See more information on Frog Development]]

====T-Box in Aves: Chick====
'''''Gallus gallus domesticus'''''

In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.
[[Chicken Development | See more information on Chicken Development]]

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development: Wallaby====
'''''Macropus eugenii'''''

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area. The neonate can attach to the teat where it completes its development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.
See also [[Kangaroo Development | See more information on Kangaroo development]]

====T-Box in Amphioxus ====
'''''Branchiostoma lanceolatum'''''

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''AMP-activated protein kinase (AMPK)''': plays a key role as a master regulator of cellular energy homeostasis. Regarded as a cellular energy sensor responding to changing ATP levels. When ATP is low, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid oxidation and autophagy.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Insulin-like growth factor 2 (IGF-2)''': Shares structural similarity to insulin.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

===QUIZ===

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

NO CONTENT YET
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===References===

2016 Group Project 5

2016-10-26T00:25:15Z

Z5020373: /* Glossary */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref name="PMID11268043 "/>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]
Members of the T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) have been found to be expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have implicated in several developmental events: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In chick embryos, Tbx4 and Fgf10 are co-expressed in the foregut mesoderm in the lung field in a domain that coincides with that of Nkx2.1 in the endoderm (except in its most anterior portion). Studies show that misexpression of Tbx4 induces ectopic Fgf10 expression and ectopic buds that express Nkx 2.1 mRNA. Tbx4 therefore influences respiratory endoderm differentiation as the Nkx2.1 gene encodes a transcription factor that is expressed during early development of thyroid, lung, and forebrain regions, particularly the basal ganglia and hypothalamus <ref><pubmed>24714694</pubmed></ref>.

Tbx4 and Tbx5 genetically interact with one another in lung branching morphogenesis. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a one-sided loss of lung bud specification and absence of tracheal specification in organ culture. Moreover, mesenchymal markers Wnt2 and Fgf10 expression, and Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5<ref><pubmed>9916808</pubmed></ref>. This is consistent with findings from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref name="PMID12588840"><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development<ref name="PMID22876201"/>.

====Other developmental events====
TBX signalling is also involved in many other developmental processes. 2 examples are palate development and skeletal muscle fibre-type determination.

In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) demonstrated that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 deficient mice had abnormal epithelial adhesion between the palate and mandible which led to several forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft similar to human conditions<ref><pubmed>PMID22371266</pubmed></ref><ref><pubmed>PMID14585638</pubmed></ref>.

Skeletal muscle comprises of a mosaic pattern of slow oxidative myofibres and fast glycolytic myofibres that influences muscle metabolism, function and whole-body physiology. The mesodermal transcription factor Tbx15 is specifically expressed in glycolytic myofibres. In vivo inactivation of Tbx15 leads to a decrease in muscle size due to a decrease in the number of glycolytic fibres, associated with a small increase in the number of oxidative fibres. This shift in fibre composition results in muscles with slower myofibre contraction and relaxation, and also decreases whole-body oxygen consumption, reduces spontaneous activity, increases adiposity and glucose intolerance. Mechanistically, ablation of Tbx15 leads to activation of AMP-activated protein kinase (AMPK) signalling and a decrease in insulin growth factor 2 (Igf2) expression. Thus, Tbx15 is one of a limited number of transcription factors to be identified with a critical role in regulating fibre-type distribution and skeletal muscle metabolism in the embryonic and post-natal period
<ref><pubmed>PMID26299309</pubmed></ref><ref><pubmed>PMID18403917</pubmed></ref>.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''AMP-activated protein kinase (AMPK)''': plays a key role as a master regulator of cellular energy homeostasis. Regarded as a cellular energy sensor responding to changing ATP levels. When ATP is low, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid oxidation and autophagy.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Insulin-like growth factor 2 (IGF-2)''': Shares structural similarity to insulin.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-26T00:19:34Z

Z5020373: /* Other developmental events */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref name="PMID11268043 "/>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]
Members of the T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) have been found to be expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have implicated in several developmental events: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In chick embryos, Tbx4 and Fgf10 are co-expressed in the foregut mesoderm in the lung field in a domain that coincides with that of Nkx2.1 in the endoderm (except in its most anterior portion). Studies show that misexpression of Tbx4 induces ectopic Fgf10 expression and ectopic buds that express Nkx 2.1 mRNA. Tbx4 therefore influences respiratory endoderm differentiation as the Nkx2.1 gene encodes a transcription factor that is expressed during early development of thyroid, lung, and forebrain regions, particularly the basal ganglia and hypothalamus <ref><pubmed>24714694</pubmed></ref>.

Tbx4 and Tbx5 genetically interact with one another in lung branching morphogenesis. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a one-sided loss of lung bud specification and absence of tracheal specification in organ culture. Moreover, mesenchymal markers Wnt2 and Fgf10 expression, and Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5<ref><pubmed>9916808</pubmed></ref>. This is consistent with findings from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref name="PMID12588840"><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development<ref name="PMID22876201"/>.

====Other developmental events====
TBX signalling is also involved in many other developmental processes. 2 examples are palate development and skeletal muscle fibre-type determination.

In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) demonstrated that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 deficient mice had abnormal epithelial adhesion between the palate and mandible which led to several forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft similar to human conditions<ref><pubmed>PMID22371266</pubmed></ref><ref><pubmed>PMID14585638</pubmed></ref>.

Skeletal muscle comprises of a mosaic pattern of slow oxidative myofibres and fast glycolytic myofibres that influences muscle metabolism, function and whole-body physiology. The mesodermal transcription factor Tbx15 is specifically expressed in glycolytic myofibres. In vivo inactivation of Tbx15 leads to a decrease in muscle size due to a decrease in the number of glycolytic fibres, associated with a small increase in the number of oxidative fibres. This shift in fibre composition results in muscles with slower myofibre contraction and relaxation, and also decreases whole-body oxygen consumption, reduces spontaneous activity, increases adiposity and glucose intolerance. Mechanistically, ablation of Tbx15 leads to activation of AMP-activated protein kinase (AMPK) signalling and a decrease in insulin growth factor 2 (Igf2) expression. Thus, Tbx15 is one of a limited number of transcription factors to be identified with a critical role in regulating fibre-type distribution and skeletal muscle metabolism in the embryonic and post-natal period
<ref><pubmed>PMID26299309</pubmed></ref><ref><pubmed>PMID18403917</pubmed></ref>.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-25T23:50:44Z

Z5020373: /* Respiratory Development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref name="PMID11268043 "/>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]
Members of the T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) have been found to be expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have implicated in several developmental events: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In chick embryos, Tbx4 and Fgf10 are co-expressed in the foregut mesoderm in the lung field in a domain that coincides with that of Nkx2.1 in the endoderm (except in its most anterior portion). Studies show that misexpression of Tbx4 induces ectopic Fgf10 expression and ectopic buds that express Nkx 2.1 mRNA. Tbx4 therefore influences respiratory endoderm differentiation as the Nkx2.1 gene encodes a transcription factor that is expressed during early development of thyroid, lung, and forebrain regions, particularly the basal ganglia and hypothalamus <ref><pubmed>24714694</pubmed></ref>.

Tbx4 and Tbx5 genetically interact with one another in lung branching morphogenesis. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a one-sided loss of lung bud specification and absence of tracheal specification in organ culture. Moreover, mesenchymal markers Wnt2 and Fgf10 expression, and Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5<ref><pubmed>9916808</pubmed></ref>. This is consistent with findings from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref name="PMID12588840"><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development<ref name="PMID22876201"/>.

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-25T11:18:28Z

Z5020373: /* The discovery of T-box genes */

{{ANAT2341Project2016header}}
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{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref name="PMID11268043 "/>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
Members of two T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) are expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have been shown to play important roles in: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

<ref><pubmed>9916808</pubmed></ref>

[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-25T11:14:33Z

Z5020373: /* Origins of the T-box name */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref name="PMID11268043 "><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
Members of two T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) are expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have been shown to play important roles in: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

<ref><pubmed>9916808</pubmed></ref>

[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-25T11:13:14Z

Z5020373: /* Abnormalities */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
Members of two T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) are expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have been shown to play important roles in: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

<ref><pubmed>9916808</pubmed></ref>

[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref name="PMID10235263"/> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-25T11:12:19Z

Z5020373: /* Limb Development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
Members of two T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) are expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have been shown to play important roles in: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

<ref><pubmed>9916808</pubmed></ref>

[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-25T11:10:58Z

Z5020373: /* Limb Development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref name="PMID10235263"><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref name="PMID10235263"/>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
Members of two T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) are expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have been shown to play important roles in: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

<ref><pubmed>9916808</pubmed></ref>

[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-25T11:07:44Z

Z5020373: /* Limb Development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

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{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
{|
|-bgcolor="DDCEF2"
|
* '''The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID16285859><pubmed>16285859</pubmed></ref>'''
|}
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes,
[https://www.ncbi.nlm.nih.gov/pubmed/25294936 For more information see here]

====T-Box gene and embryology====
The T-box gene family, plays an essential role in controlling embryogenesis in a wide variety of organisms (invertebrates, amphibians, mammals) <ref name=PMID14699590><pubmed>14699590</pubmed></ref> . The box-gene family encodes related DNA-binding transcriptional regulators and the genes exhibit diverse patterns of both spatial and temporal expression in the developing embryo.
Studies both in genetic and molecular embryology have shown the importance of these genes in regulating cell fate decisions which later during embryological development are important in establishing the early body plan and later are important during the organogenesis process. <ref name=PMID14699590><pubmed>14699590</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc SEE ABOVE have done this
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>. Unfortunately, the original article had been lost and only the paraphrased version is subsisted.
{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Nadine Dobrovolskaia-Zavadskaia 1948.jpg|600px|thumb|centre| This image is a photograph of Nadezhda Alexandrovna Dobrovolskaïa-Zavadskaïa in 1948 taken from <ref><pubmed>11268043 </pubmed></ref>]]
|}
The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline of the T-box genes since discovery====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes. <ref name=DZ1927/>

Over the following decades, the embryological defects caused by the T mutation were studied.

{| class="wikitable mw-collapsible mw-collapsed"
! Timeline
|-
|'''1990''' - The T gene itself was cloned. <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
|-
|'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
|-
|'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omg". <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
|-
|'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome.<ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
|-
|'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
|-
|'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
|-
|'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d.<ref><pubmed>14550786</pubmed></ref>.
|-
|'''2004''' - With collaboration of other signalling factor, tbx-3 is involved of mammary gland development in mouse model.<ref><pubmed>15255957 </pubmed></ref>
|-
|'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal. <ref><pubmed>15843414</pubmed></ref>.
|-
|'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse.<ref><pubmed>16505380</pubmed></ref>.
|-
|'''2007''' - A clinical human case shown tbx-5 is related to human pericardium agenesis and verified as one of the symptoms of Holt-Oram Syndrome. <ref name="PMID16376438"><pubmed>16376438</pubmed></ref>
|-
|'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology. <ref><pubmed>19494035</pubmed></ref>.
|-
|'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
|-
|'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.
|
|-
|MORE RECENT RESEARCH
|
|PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS
|}

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
|-bgcolor="#cedff2"
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
|-bgcolor="#f5faff"
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina, mammary gland||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
|-bgcolor="#f5faff"
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
|-bgcolor="#f5faff"
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px||thumb|centre|Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate. ]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud. <ref><pubmed>24626928</pubmed></ref><ref><pubmed>12736217</pubmed></ref><ref><pubmed>10235264</pubmed></ref>

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|thumb|centre|Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>]]

====Respiratory Development====
Members of two T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) are expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have been shown to play important roles in: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

<ref><pubmed>9916808</pubmed></ref>

[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 and it is trusted that more disease would be found. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>
[https://omim.org/entry/188400?search=TBX1&highlight=tbx1 For more information see here]

====TBX3/Ulnar-Mammary Syndrome====

{|
|-
| Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

[https://omim.org/entry/181450?search=TBX3&highlight=tbx3 For more information see here]

|
[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]
|}
====TBX5/Holt– Oram Syndrome====

{|
|-
| Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.
[https://omim.org/entry/142900?search=TBX5&highlight=tbx5 For more information see here]

|[[File:Hos.png|200px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

|}

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>
[https://omim.org/entry/201400?search=TBX19&highlight=tbx19 For more information]

====TBX22/Cleft Palate====
{|
|-
| Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

[https://omim.org/entry/303400?search=tbx22&highlight=tbx22 For more information see here]

|[[File:Cleftp.jpg|200px|thumb|right|(A) Normal lip and palate. (B) Unilateral cleft palate. (C) Bilateral cleft palate. (D) Cleft uvula. (E) Submucous cleft palate. <ref name="PMID26973535 "><pubmed>26973535 </pubmed></ref> ]]
|}

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and is also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, and appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref>, and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process <ref name=PMID16285859><pubmed>16285859</pubmed></ref>.

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by singling molecules and also induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems <ref name=PMID16285859><pubmed>16285859</pubmed></ref>. T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation <ref><pubmed>11148447</pubmed></ref>. Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Evolution of T box gene Family.jpg|600px|thumb|left| This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.]]
|}

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in Aves : The Chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: T box in chick.jpg |600px|thumb|left| This image above demonstrates Tbx4 and Tbx5 genes Expression (marked by arrows) of Tbx4 (a, b) and Tbx5 (c, d) in the developing chick embryo at early (a, c) and late (b, d) limb-bud stages. Note that Tbx4 is expressed in the hindlimb and Tbx5 in the forelimb. Image taken from<ref><pubmed>10203826</pubmed></ref>]]
|}

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}
At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====T-Box in Amphioxus (a living chordate)====
The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

In amphioxus two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.
Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Limb fields''': areas where the limb buds will develop.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Phylogenetic''': is the study of the evolutionary history and relationships among individuals or groups of organisms.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

User:Z5020373

2016-10-24T06:54:27Z

Z5020373: /* Lab 11 Assessment */

{{ANAT2341Student2016}}
{{ANAT2341Rebecca2016}}

{|
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] 4 August 2016 - Thank you for adding this content before the lab. I would suggest that rather than using a template that you simply paste on this current page with separate subheadings for each lab/assessment item. Also please no names, just your student number.
|}

==Lab 1==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:36, 5 August 2016 (AEST)

===Lab Demonstrations===

====External Link====

[http://www.smh.com.au/ SMH]

====Internal Link====

https://embryology.med.unsw.edu.au/embryology/index.php/2011_Lab_1

[[2011_Lab_1|ANAT2341 Lab 1]]

[[Student Page]]

====Referencing====
[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486280

===Lab 1 Assessment===
<pubmed>27123200</pubmed>

This research article explores whether choosing to a conduct embryo transfer (ET) on a weekend or weekday will affect the success of clinical pregnancy in IVF procedures. In the study, ET transfers were performed on either weekdays or weekends in patients with similar clinical characteristics, such as age, body-mass index and duration of infertility. Clinical pregnancy was determined using blood pregnancy tests and ultrasound examination and was defined as the “presence of a gestational sac with a foetal heart beat.” After the ETs, the authors found that there was an overall 42.8% success rate of clinical pregnancy in patients from both groups, with a 14.6% increase in the pregnancy rate when weekend ETs where compared to weekday ETs. The study however, did not examine any possible reasons to explain this increase in implantation rate although a few potential factors were discussed from previous findings in this area of research. These included endometrial receptivity which occurs 5 days after the post-ovulatory progesterone surge. The article mentioned that uterine receptivity and implantation could be affected by the junctional zone. The extent of junctional zone contractility differs throughout the ovarian cycle and an increased contractility just before ET has been previously shown to significantly decrease the likelihood of successful implantation. Since weekends are more relaxing than weekdays they suggest a possible correlation between that and reduced junctional zone contractions leading to easier ETs. Therefore from this study, it was concluded that ETs performed during the weekends are more successful than those performed during the weekdays identifying a potential factor that can improve ETs in IVF situations.

{| width=95%
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 14:24, 15 August 2016 (AEST) - This is a good summary of a paper that looks at potential environmental/endocrine effects on reproductive fertility. You needed to put the reference at the top rather than just the PMID number, fix this and you can get this full mark for the exercise.

[mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You did not fix, so I have adjusted the final mark.
| width=100px| Assessment 4/5
|}

==Lab 2==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:41, 12 August 2016 (AEST)

===Lab 2 Assessment===
[[File: Amnion_fold_development_in_chicken_embryos.jpg]]

Chicken embryo amion fold development<ref><pubmed>24647352</pubmed></ref>

{|
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 14:24, 15 August 2016 (AEST) - Very good, the image relates to early development and contains the reference, copyright and student template. (5/5)
|}

==Lab 3==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:53, 19 August 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Mesoderm_Development#Paraxial_Mesoderm|Question 2 - paraxial]]

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]
| Assessment 2.5/5
|}

==Lab 4==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:02, 26 August 2016 (AEST)

===Lab 4 Assessment===

Take the Quiz

Make your selection for all questions before clicking submit.

<quiz display=shuffle>

{How many rotations does the stomach undergo during GIT development in week 4 to 5?
|type="()"}
- 1
+ 2
- 3
- 4

|| The stomach undergoes [[two]] embryonic 90 degree rotations: the first to establish the J-shape that forms the adult stomach body (classic curvature), and the second rotation establishes it in its correct anatomical position.

{Following the degeneration of the buccopharyngeal membrane, the foregut is open to which cavity?
|type="()"}
- The peritoneal cavity
- The chorionic cavity
- The yolk sac
+ The amniotic cavity

|| During week 4 of development, the breakdown of the buccopharangeal membrane exposes the foregut to the amniotic cavity where amniotic fluid is then able to fill the foregut.

{Which one of these is not an abnormality that can occur during the proliferation and re-canalisation of the gut tube?
|type="()"}
- Occlusion
+ Meckel's diverticulum
- Duplication
- Stenosis

||During re-canalisation, if the gut tube does not re-canalise the tube can remain completely occluded. Another senario would be if there is renalisation but it occurs in discrete channels to give rise to duplicated gut tubes. The third abnormality occurs when there is incomplete vasculisation which leads to stenosis or narrowing of the tube. The only abnormality that is not involved in Meckel's diverticulum and is associated with failure of Vitelline duct breakdown leaving a yolk stalk remnant. It is a common abnormality with a prevalence of 1-2% and can lead to infection and possibly affect the rotation of the midgut.

{During week 4 in GIT development:
|type="()"}
- The cloacal membrane is broken down while the buccopharyngeal membrane remains intact
+ The buccopharyngeal membrane is broken down while the cloacal membrane remains intact
- Both the buccopharyngeal and cloacal membranes break down simultaneously
- Both the buccopharyngeal and cloacal membranes remain intact.

||Loss of the buccopharangeal membrane during week 4 allows amniotic fluid into the foregut. The cloacal membrane remains intact and does not break down until the cloaca is divided into urogenital sinuses and the rectum (occurs later in embryonic development.

</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem well designed GIT quiz questions that test topic understanding.
| Assessment 5/5
|}

==Lab 5==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:11, 2 September 2016 (AEST)

===Lab 5 Assessment===
Questionaire completed and submitted
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:17, 9 September 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 11 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab 6==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 9 September 2016 (AEST)

===Lab 6 Assessment===

<pubmed>25382630</pubmed>
Cleft palate arises when the bilateral palatal shelves fail to fuse. Many genetic and environmental factors have been identified to contribute to this deformity. One genetic mutation associated with cleft palate is the loss transforming growth factor-beta receptor (TGF-βR). A recent article by Hill ''et al.'' demonstrated that loss of TGF-βR3 reduced the expression of several ligands and receptors in the TGF-β/BMP family, including three TGF-β ligands and BMP2. During embryonic development, these molecules are involved in cell growth and differentiation.

A loss of TGF-β/BMP signaling, by receptor loss was found to be associated with cleft palate formation due to aberrant cell cycle progression and altered gene expression. Furthermore changes to TGF-β/BMP signaling also interrupted vascular development and remodeling as well as osteogenic differentiation during palate formation.
TGF-βR3 is therefore essential for maintaining the expression of TGF-β and BMP molecules and without this receptor processes of palatal shelf elongation, elevation and fusion are disrupted leading to cleft palate.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Good reference and explanation.
| Assessment 5/5
|}

==Lab 7==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 16 September 2016 (AEST)

===Lab 7 Assessment===

1. What is/are the dystrophin mutation(s)?

**The dystrophin gene is located on the short arm of the X chromosome at position 21.2. <ref>Converse, P.J. (2016) MUSCULAR DYSTROPHY, DUCHENNE TYPE; DMD OMIM http://www.omim.org/entry/310200</ref>
**It is the largest gene found in nature spanning 1.5% of the X-chromosome which is about 2.5 Mb of genomic sequence. <ref><pubmed>11917091</pubmed></ref>
**Its size could explain why it is so susceptible to spontaneous mutations.
**Encodes for Dystrophin protein.
**Mutations such as large deletions (60-70% of DMD cases), large duplications (10% of DMD cases) and point mutations (15-30% of DMD cases) can occur resulting in gene inactivation (therefore loss of function).<ref>https://www.duchenneconnect.org/understanding-genetic-testing/types-of-mutations-in-the-dystrophin-gene.html</ref>

2. What is the function of dystrophin?

**Dystrophin is part of a protein complex that work together to strengthen muscle fibers and protect them from injury as muscles contract and relax.
**Dystrophin complex acts as an anchor, connecting each muscle cell's structural framework (cytoskeleton) with the lattice of proteins and other molecules outside the cell (extracellular matrix).
**May also play a role in cell signaling by interacting with proteins that send and receive chemical signals e.g. in alpha-syntrophin.<ref><pubmed>12082140</pubmed></ref>

3. What other tissues/organs are affected by this disorder?

**Muscle (skeletal, cardiac and smooth): fatigue, difficulty with motor skills
**Respiratory system: pneumonia
**Cardiac system: cardiac myopathy
**Central Nervous system (CNS): neurobehavioural disorders e.g. ADHD and dyslexia <ref><pubmed>13947981</pubmed></ref>

4. What therapies exist for DMD?

**There is no cure available for DMD, and the current interventions are based on preventing further muscle wasting and management of symptoms and complications
**Treatments include:
***Physical therapy
***Orthopedic appliances
***Medication:
****Two corticosteroids mainly used in DMD treatment are Prednisone/Prednisolone and Deflazacort, an oxazoline derivative of prednisolone, administered by two common regimens: daily and intermittent <ref><pubmed>26457695</pubmed></ref>
****Prednisone and prednisolone show an anti-inflammatory effect
***Deflazacort acts on muscle regeneration and differentiation
** Future could include:
**Cell-based therapies using stem cells to replace dystrophin gene (a potential cure).
***Gene therapies to deliver a therapeutic gene to skeletal and cardiac muscle, in order to restore the dystrophin protein PMID <ref><pubmed>7683332</pubmed></ref>
***A study by Nelson et al. in 2016, showed that in vivo CRISPR-Cas9–mediated dystrophin restoration in mdx mouse model of DMD removed the mutated exon 23 from the dystrophin gene and improved muscle structure and function.<ref><pubmed>25123483</pubmed></ref>

5. What animal models are available for muscular dystrophy?
**The most widely used animal model for DMD is the '''mdx mouse''', which has a spontaneous point mutation in exon 23 that causes the absence of the dystrophin protein in the muscle.
**However, other animal models inlcude:<ref><pubmed>11917091</pubmed></ref>
***GRMD dog - Dystrophin-deficient dog
***HFMD cat - Dystrophin-deficient cat (clinically a poor model)
**This animal model allows testing ans screening of potential treatments and without these animal models, and without these animal models we would not have any known therapies today
**For example, mdx ''in vivo'' studies have led to U.S. FDA approval of Exondys 51 (eteplirsen) injection, the first drug approved to treat patients with Duchenne muscular dystrophy (DMD). Exondys 51 is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD. <ref>http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm521263.htm
</ref>
{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Muscular Dystrophy questions have been comprehensively answered and you have cited your sources.
| Assessment 5/5
|}

==Lab 8==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:07, 23 September 2016 (AEST)

===Lab 8 Assessment===
Group 1: Wnt signalling pathway

Firstly, a proper introduction to the Wnt pathway and its various roles in the developing embryo should be added to the page. Even though the page is focusing on Wnt signalling in fetal skin development I still think the other contributions of Wnt signalling need to be at least mentioned in the intro. Mark also suggested adding a table to show the origins of the pathway and how our knowledge about Wnt signaling has evolved.

The page contains appropriate headings and subheadings to address the different mechanisms of Wnt signalling (canonical, non-canonical and non-canonical Wnt/Ca2+ pathway). The main points for each of these pathways are up on the page which is good. Clearly, these signalling mechanisms are quite complex and adding images could help the reader to visualise how the key molecules in this pathway interact with other molecules to induce downstream effects. But otherwise, the detail in the canonical pathway is adequate for a student to understand.

The references included in each section of the page is evidence of research done to support the content on the page. The page also includes up to date information to reflect current research in this area. This information should be integrated into some sort of discussion to show how this contributes to knowledge about the signalling pathway affects developmental events. Also, all the references need to be cited correctly which I am sure you guys are aware of and will do.

Don’t forget the topic is ‘Signalling in Development’, therefore the focus should be on Wnt signalling in the developing embryo.

Overall, Group 1 has made good progress. Keep it up!

Group 2: Notch signalling pathway

The page has a proper introduction stating the critical functions of Notch signalling, examples of diseases associated with Notch mutations as well as a brief description of the mechanism of Notch signalling. There is also a timeline on the page which none of the other groups have managed to do so good job!

Some headings are missing text but those with content (e.g. canonical pathway section) are covered in extensive detail. The subheadings and headings chosen for the page indicate the group’s in depth understanding of the key components of the Notch signalling pathway. Furthermore, the page contains correct referencing and citation of text and images.

The page also discusses Notch signalling in animal models as well. A recent study was included for Drosophila. What about the other two?

Overall, Group 2 has made excellent progress. Well done!

Group 3: FGFR signalling pathway

The headings and subheadings on the page is used very effectively to aid the progression of information. Through the sequence of the headings, it allows the reader to build their understanding about FGFR signalling. The FGFR page definitely address the topic of this assessment - signalling in development, and links FGF signalling to a number of developmental events. This reflects the large contributions of FGFR in development which the page successfully portrays.

In the overview section, it states: “As shown in the image, an acidic box…”. Make it clear which image you are referring to because I can’t find it.

The table for the subtypes of FGFR has been acknowledged that it is incomplete but it gives a good snapshot to function and associated abnormalities of the different FGFR subtypes.

The page includes a student drawn image which summarises the FGFR signalling pathway. None of the other groups have included a student drawn image so good job! The images uses colours to distinguish particular molecules and shows the downstream signalling events to affect gene transcription in the cell. To me the image is a bit blurry on the page, so maybe change the pixels of the image to make it larger and easier to see?

The page includes a quiz which is clever and will definitely make the page stand out from the other groups.

Overall, Group 3 has made good progress. Good job!

Group 4: Hedgehog signalling pathway

Firstly, the page is missing an introduction to the signalling pathway. There is also text missing under the first few subheadings. Since the hedgehog pathway research began as early as the 1970s, a table including the key events in the Hedgehog research would be interesting to add.

The page includes a nice overview of the Hedgehog pathway captured in the image however, it needs a reference to acknowledge the original source of the image. Consider relocating the image to the mechanism of signalling section. This may help the reader understand the processes better if they have that image there.

Mammals have 3 Hedgehog homologues (DHH, IHH and SHH). I think that is an important point to mention.

Good discussion of animal models since it is one of the key regulators of animal development.
Despite having headings without text. Group 3 has made good progress so far. Keep it up!

Group 6: TGF-β signalling pathway

The page contains a good introduction to the TGF-β superfamily, including examples of other signaling proteins. The images help the reader visualise how TGF-β ligand bring the receptors together in a heterotetrameric complex in which the type II receptors phosphorylate and activate the type I receptors. To be pedantic, the second image still needs to include details of the original source.

Remember that the project is meant to focus on ‘Signalling in Development’ and whilst the page addresses the signalling component it does not discuss TGF-β signalling in the development of the embryo. The second image addresses its role in proliferation, migration, growth arrest and apoptosis. This pubmed article: PMID 19289080 discusses TGF-β signalling in early development, axis formation, and patterning of the embryo.

The page does not use the correct referencing or in-text citations.

Overall, Group 6 has made a good start but more research needs to be done. Keep pushing :)

==Lab 9==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:06, 7 October 2016 (AEDT)

==Lab 11==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:02, 21 October 2016 (AEDT)

===Lab 11 Assessment===
Write a brief summary of the paper's main findings. Then describe how the original research result was used in the review article.

==References==

{{Stem Cell Presentations 2016}}

2016 Group Project 5

2016-10-24T06:46:59Z

Z5020373: /* Timeline */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.
----
Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990''' - The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>
----
'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>
----
'''1995''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>
The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>
----
'''1997''' - The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.
----
'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>
----
'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.
----
'''2003''' - 3 mouse Tbx20 splice variants, were cloned and called Tbx20a, Tbx20b, and Tbx20c, and by database analysis they identified a fourth variant, Tbx20d<ref><pubmed>14550786</pubmed></ref>.
----
'''2005''' - A deletion of Tbx20 in mouse was found to be embryonic lethal <ref><pubmed>15843414</pubmed></ref>.
----
'''2006''' - Tbx6 was found to be essential for Mesp2 expression during somitogenesis in mouse<ref><pubmed>16505380</pubmed></ref>.
----
'''2009''' - A Drosophila heart model involving mutation of pannier (pnr) was used to examine the function of GATA4 in adult heart physiology <ref><pubmed>19494035</pubmed></ref>.
----
'''2010''' - Tbx3 showed to significantly improve the quality of induced pluripotent stem (iPS) cells <ref><pubmed>20139965</pubmed></ref>.
----
'''2011''' - TBX6-dependent regulation of SOX2 was demonstrated to determine the fate of axial stem cells <ref><pubmed>21331042</pubmed></ref>.

MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px|centre]]

Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
Members of two T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) are expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have been shown to play important roles in: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

<ref><pubmed>9916808</pubmed></ref>

[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Limb fields''': areas where the limb buds will develop.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T06:04:42Z

Z5020373: /* Respiratory Development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px|centre]]

Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
Members of two T-box gene family (Tbx2/Tbx3 and Tbx4/Tbx5) are expressed in embryonic lung mesenchyme <ref name="PMID8853987"><pubmed>8853987</pubmed></ref> and have been shown to play important roles in: 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal/bronchial cartilage formation <ref name="PMID22876201"><pubmed>22876201</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

<ref><pubmed>9916808</pubmed></ref>

[[File:Tbx in lung and trachea development.png|450px|thumb|right|Tbx in lung and trachea development<ref name="PMID22876201"/>]]

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Limb fields''': areas where the limb buds will develop.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T04:54:29Z

Z5020373: /* Limb Development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px|centre]]

Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>. The role of β-catenin in forelimb initiation, however, has not been studied in detail<ref><pubmed>26212321</pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Limb fields''': areas where the limb buds will develop.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T04:47:58Z

Z5020373: /* Functions of T-box in development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px|centre]]

Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Limb fields''': areas where the limb buds will develop.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T04:46:58Z

Z5020373: /* Cardiac development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

[[File:Cardiac gene induction rates.png|800px|centre]]

Table 2. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Limb fields''': areas where the limb buds will develop.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T04:46:07Z

Z5020373: /* Features of the T-box family */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table 1. adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Limb fields''': areas where the limb buds will develop.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T04:45:07Z

Z5020373: /* Glossary */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

'''Limb fields''': areas where the limb buds will develop.

'''Apical ectodermal ridge (AER)''': a thickened area of pseudo-stratified columnar epithelium at the tip of the developing limb bud. It is a major signaling centre for the developing limb.

'''Zone of Polarising Activity (ZPA)''': a collection of cells at the posterior border of the limb close to the AER, adjacent to the body wall.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

User:Z5020373

2016-10-24T04:42:19Z

Z5020373: /* Lab 11 Assessment */

{{ANAT2341Student2016}}
{{ANAT2341Rebecca2016}}

{|
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] 4 August 2016 - Thank you for adding this content before the lab. I would suggest that rather than using a template that you simply paste on this current page with separate subheadings for each lab/assessment item. Also please no names, just your student number.
|}

==Lab 1==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:36, 5 August 2016 (AEST)

===Lab Demonstrations===

====External Link====

[http://www.smh.com.au/ SMH]

====Internal Link====

https://embryology.med.unsw.edu.au/embryology/index.php/2011_Lab_1

[[2011_Lab_1|ANAT2341 Lab 1]]

[[Student Page]]

====Referencing====
[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486280

===Lab 1 Assessment===
<pubmed>27123200</pubmed>

This research article explores whether choosing to a conduct embryo transfer (ET) on a weekend or weekday will affect the success of clinical pregnancy in IVF procedures. In the study, ET transfers were performed on either weekdays or weekends in patients with similar clinical characteristics, such as age, body-mass index and duration of infertility. Clinical pregnancy was determined using blood pregnancy tests and ultrasound examination and was defined as the “presence of a gestational sac with a foetal heart beat.” After the ETs, the authors found that there was an overall 42.8% success rate of clinical pregnancy in patients from both groups, with a 14.6% increase in the pregnancy rate when weekend ETs where compared to weekday ETs. The study however, did not examine any possible reasons to explain this increase in implantation rate although a few potential factors were discussed from previous findings in this area of research. These included endometrial receptivity which occurs 5 days after the post-ovulatory progesterone surge. The article mentioned that uterine receptivity and implantation could be affected by the junctional zone. The extent of junctional zone contractility differs throughout the ovarian cycle and an increased contractility just before ET has been previously shown to significantly decrease the likelihood of successful implantation. Since weekends are more relaxing than weekdays they suggest a possible correlation between that and reduced junctional zone contractions leading to easier ETs. Therefore from this study, it was concluded that ETs performed during the weekends are more successful than those performed during the weekdays identifying a potential factor that can improve ETs in IVF situations.

{| width=95%
|-bgcolor="FAF5FF"
| [[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 14:24, 15 August 2016 (AEST) - This is a good summary of a paper that looks at potential environmental/endocrine effects on reproductive fertility. You needed to put the reference at the top rather than just the PMID number, fix this and you can get this full mark for the exercise.

[mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You did not fix, so I have adjusted the final mark.
| width=100px| Assessment 4/5
|}

==Lab 2==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:41, 12 August 2016 (AEST)

===Lab 2 Assessment===
[[File: Amnion_fold_development_in_chicken_embryos.jpg]]

Chicken embryo amion fold development<ref><pubmed>24647352</pubmed></ref>

{|
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 14:24, 15 August 2016 (AEST) - Very good, the image relates to early development and contains the reference, copyright and student template. (5/5)
|}

==Lab 3==
===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:53, 19 August 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Mesoderm_Development#Paraxial_Mesoderm|Question 2 - paraxial]]

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]
| Assessment 2.5/5
|}

==Lab 4==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:02, 26 August 2016 (AEST)

===Lab 4 Assessment===

Take the Quiz

Make your selection for all questions before clicking submit.

<quiz display=shuffle>

{How many rotations does the stomach undergo during GIT development in week 4 to 5?
|type="()"}
- 1
+ 2
- 3
- 4

|| The stomach undergoes [[two]] embryonic 90 degree rotations: the first to establish the J-shape that forms the adult stomach body (classic curvature), and the second rotation establishes it in its correct anatomical position.

{Following the degeneration of the buccopharyngeal membrane, the foregut is open to which cavity?
|type="()"}
- The peritoneal cavity
- The chorionic cavity
- The yolk sac
+ The amniotic cavity

|| During week 4 of development, the breakdown of the buccopharangeal membrane exposes the foregut to the amniotic cavity where amniotic fluid is then able to fill the foregut.

{Which one of these is not an abnormality that can occur during the proliferation and re-canalisation of the gut tube?
|type="()"}
- Occlusion
+ Meckel's diverticulum
- Duplication
- Stenosis

||During re-canalisation, if the gut tube does not re-canalise the tube can remain completely occluded. Another senario would be if there is renalisation but it occurs in discrete channels to give rise to duplicated gut tubes. The third abnormality occurs when there is incomplete vasculisation which leads to stenosis or narrowing of the tube. The only abnormality that is not involved in Meckel's diverticulum and is associated with failure of Vitelline duct breakdown leaving a yolk stalk remnant. It is a common abnormality with a prevalence of 1-2% and can lead to infection and possibly affect the rotation of the midgut.

{During week 4 in GIT development:
|type="()"}
- The cloacal membrane is broken down while the buccopharyngeal membrane remains intact
+ The buccopharyngeal membrane is broken down while the cloacal membrane remains intact
- Both the buccopharyngeal and cloacal membranes break down simultaneously
- Both the buccopharyngeal and cloacal membranes remain intact.

||Loss of the buccopharangeal membrane during week 4 allows amniotic fluid into the foregut. The cloacal membrane remains intact and does not break down until the cloaca is divided into urogenital sinuses and the rectum (occurs later in embryonic development.

</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem well designed GIT quiz questions that test topic understanding.
| Assessment 5/5
|}

==Lab 5==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:11, 2 September 2016 (AEST)

===Lab 5 Assessment===
Questionaire completed and submitted
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:17, 9 September 2016 (AEST)

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 11 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab 6==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 9 September 2016 (AEST)

===Lab 6 Assessment===

<pubmed>25382630</pubmed>
Cleft palate arises when the bilateral palatal shelves fail to fuse. Many genetic and environmental factors have been identified to contribute to this deformity. One genetic mutation associated with cleft palate is the loss transforming growth factor-beta receptor (TGF-βR). A recent article by Hill ''et al.'' demonstrated that loss of TGF-βR3 reduced the expression of several ligands and receptors in the TGF-β/BMP family, including three TGF-β ligands and BMP2. During embryonic development, these molecules are involved in cell growth and differentiation.

A loss of TGF-β/BMP signaling, by receptor loss was found to be associated with cleft palate formation due to aberrant cell cycle progression and altered gene expression. Furthermore changes to TGF-β/BMP signaling also interrupted vascular development and remodeling as well as osteogenic differentiation during palate formation.
TGF-βR3 is therefore essential for maintaining the expression of TGF-β and BMP molecules and without this receptor processes of palatal shelf elongation, elevation and fusion are disrupted leading to cleft palate.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Good reference and explanation.
| Assessment 5/5
|}

==Lab 7==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:05, 16 September 2016 (AEST)

===Lab 7 Assessment===

1. What is/are the dystrophin mutation(s)?

**The dystrophin gene is located on the short arm of the X chromosome at position 21.2. <ref>Converse, P.J. (2016) MUSCULAR DYSTROPHY, DUCHENNE TYPE; DMD OMIM http://www.omim.org/entry/310200</ref>
**It is the largest gene found in nature spanning 1.5% of the X-chromosome which is about 2.5 Mb of genomic sequence. <ref><pubmed>11917091</pubmed></ref>
**Its size could explain why it is so susceptible to spontaneous mutations.
**Encodes for Dystrophin protein.
**Mutations such as large deletions (60-70% of DMD cases), large duplications (10% of DMD cases) and point mutations (15-30% of DMD cases) can occur resulting in gene inactivation (therefore loss of function).<ref>https://www.duchenneconnect.org/understanding-genetic-testing/types-of-mutations-in-the-dystrophin-gene.html</ref>

2. What is the function of dystrophin?

**Dystrophin is part of a protein complex that work together to strengthen muscle fibers and protect them from injury as muscles contract and relax.
**Dystrophin complex acts as an anchor, connecting each muscle cell's structural framework (cytoskeleton) with the lattice of proteins and other molecules outside the cell (extracellular matrix).
**May also play a role in cell signaling by interacting with proteins that send and receive chemical signals e.g. in alpha-syntrophin.<ref><pubmed>12082140</pubmed></ref>

3. What other tissues/organs are affected by this disorder?

**Muscle (skeletal, cardiac and smooth): fatigue, difficulty with motor skills
**Respiratory system: pneumonia
**Cardiac system: cardiac myopathy
**Central Nervous system (CNS): neurobehavioural disorders e.g. ADHD and dyslexia <ref><pubmed>13947981</pubmed></ref>

4. What therapies exist for DMD?

**There is no cure available for DMD, and the current interventions are based on preventing further muscle wasting and management of symptoms and complications
**Treatments include:
***Physical therapy
***Orthopedic appliances
***Medication:
****Two corticosteroids mainly used in DMD treatment are Prednisone/Prednisolone and Deflazacort, an oxazoline derivative of prednisolone, administered by two common regimens: daily and intermittent <ref><pubmed>26457695</pubmed></ref>
****Prednisone and prednisolone show an anti-inflammatory effect
***Deflazacort acts on muscle regeneration and differentiation
** Future could include:
**Cell-based therapies using stem cells to replace dystrophin gene (a potential cure).
***Gene therapies to deliver a therapeutic gene to skeletal and cardiac muscle, in order to restore the dystrophin protein PMID <ref><pubmed>7683332</pubmed></ref>
***A study by Nelson et al. in 2016, showed that in vivo CRISPR-Cas9–mediated dystrophin restoration in mdx mouse model of DMD removed the mutated exon 23 from the dystrophin gene and improved muscle structure and function.<ref><pubmed>25123483</pubmed></ref>

5. What animal models are available for muscular dystrophy?
**The most widely used animal model for DMD is the '''mdx mouse''', which has a spontaneous point mutation in exon 23 that causes the absence of the dystrophin protein in the muscle.
**However, other animal models inlcude:<ref><pubmed>11917091</pubmed></ref>
***GRMD dog - Dystrophin-deficient dog
***HFMD cat - Dystrophin-deficient cat (clinically a poor model)
**This animal model allows testing ans screening of potential treatments and without these animal models, and without these animal models we would not have any known therapies today
**For example, mdx ''in vivo'' studies have led to U.S. FDA approval of Exondys 51 (eteplirsen) injection, the first drug approved to treat patients with Duchenne muscular dystrophy (DMD). Exondys 51 is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping, which affects about 13 percent of the population with DMD. <ref>http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm521263.htm
</ref>
{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Muscular Dystrophy questions have been comprehensively answered and you have cited your sources.
| Assessment 5/5
|}

==Lab 8==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:07, 23 September 2016 (AEST)

===Lab 8 Assessment===
Group 1: Wnt signalling pathway

Firstly, a proper introduction to the Wnt pathway and its various roles in the developing embryo should be added to the page. Even though the page is focusing on Wnt signalling in fetal skin development I still think the other contributions of Wnt signalling need to be at least mentioned in the intro. Mark also suggested adding a table to show the origins of the pathway and how our knowledge about Wnt signaling has evolved.

The page contains appropriate headings and subheadings to address the different mechanisms of Wnt signalling (canonical, non-canonical and non-canonical Wnt/Ca2+ pathway). The main points for each of these pathways are up on the page which is good. Clearly, these signalling mechanisms are quite complex and adding images could help the reader to visualise how the key molecules in this pathway interact with other molecules to induce downstream effects. But otherwise, the detail in the canonical pathway is adequate for a student to understand.

The references included in each section of the page is evidence of research done to support the content on the page. The page also includes up to date information to reflect current research in this area. This information should be integrated into some sort of discussion to show how this contributes to knowledge about the signalling pathway affects developmental events. Also, all the references need to be cited correctly which I am sure you guys are aware of and will do.

Don’t forget the topic is ‘Signalling in Development’, therefore the focus should be on Wnt signalling in the developing embryo.

Overall, Group 1 has made good progress. Keep it up!

Group 2: Notch signalling pathway

The page has a proper introduction stating the critical functions of Notch signalling, examples of diseases associated with Notch mutations as well as a brief description of the mechanism of Notch signalling. There is also a timeline on the page which none of the other groups have managed to do so good job!

Some headings are missing text but those with content (e.g. canonical pathway section) are covered in extensive detail. The subheadings and headings chosen for the page indicate the group’s in depth understanding of the key components of the Notch signalling pathway. Furthermore, the page contains correct referencing and citation of text and images.

The page also discusses Notch signalling in animal models as well. A recent study was included for Drosophila. What about the other two?

Overall, Group 2 has made excellent progress. Well done!

Group 3: FGFR signalling pathway

The headings and subheadings on the page is used very effectively to aid the progression of information. Through the sequence of the headings, it allows the reader to build their understanding about FGFR signalling. The FGFR page definitely address the topic of this assessment - signalling in development, and links FGF signalling to a number of developmental events. This reflects the large contributions of FGFR in development which the page successfully portrays.

In the overview section, it states: “As shown in the image, an acidic box…”. Make it clear which image you are referring to because I can’t find it.

The table for the subtypes of FGFR has been acknowledged that it is incomplete but it gives a good snapshot to function and associated abnormalities of the different FGFR subtypes.

The page includes a student drawn image which summarises the FGFR signalling pathway. None of the other groups have included a student drawn image so good job! The images uses colours to distinguish particular molecules and shows the downstream signalling events to affect gene transcription in the cell. To me the image is a bit blurry on the page, so maybe change the pixels of the image to make it larger and easier to see?

The page includes a quiz which is clever and will definitely make the page stand out from the other groups.

Overall, Group 3 has made good progress. Good job!

Group 4: Hedgehog signalling pathway

Firstly, the page is missing an introduction to the signalling pathway. There is also text missing under the first few subheadings. Since the hedgehog pathway research began as early as the 1970s, a table including the key events in the Hedgehog research would be interesting to add.

The page includes a nice overview of the Hedgehog pathway captured in the image however, it needs a reference to acknowledge the original source of the image. Consider relocating the image to the mechanism of signalling section. This may help the reader understand the processes better if they have that image there.

Mammals have 3 Hedgehog homologues (DHH, IHH and SHH). I think that is an important point to mention.

Good discussion of animal models since it is one of the key regulators of animal development.
Despite having headings without text. Group 3 has made good progress so far. Keep it up!

Group 6: TGF-β signalling pathway

The page contains a good introduction to the TGF-β superfamily, including examples of other signaling proteins. The images help the reader visualise how TGF-β ligand bring the receptors together in a heterotetrameric complex in which the type II receptors phosphorylate and activate the type I receptors. To be pedantic, the second image still needs to include details of the original source.

Remember that the project is meant to focus on ‘Signalling in Development’ and whilst the page addresses the signalling component it does not discuss TGF-β signalling in the development of the embryo. The second image addresses its role in proliferation, migration, growth arrest and apoptosis. This pubmed article: PMID 19289080 discusses TGF-β signalling in early development, axis formation, and patterning of the embryo.

The page does not use the correct referencing or in-text citations.

Overall, Group 6 has made a good start but more research needs to be done. Keep pushing :)

==Lab 9==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 13:06, 7 October 2016 (AEDT)

==Lab 11==

===Lab Attendance===
[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:02, 21 October 2016 (AEDT)

===Lab 11 Assessment===
[[Lab 11 Assessment]]

==References==

{{Stem Cell Presentations 2016}}

2016 Group Project 5

2016-10-24T04:08:10Z

Z5020373: /* Features of the T-box family */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/>

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
The following table outlines the functions of some important T-box genes, including their location and associated human diseases.
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T04:06:44Z

Z5020373: /* Limb Development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
The initiation of limb outgrowth involves T-box genes as well as Homeobox (Hox) genes. Specific Hox genes are upregulated by retinoic acid in limb fields which then initiates downstream signaling cascades that ensures correct limb growth along its three axes: anterio-posterior, dorso-ventral and proximo-distal <ref><pubmed>9655805</pubmed></ref>
<ref><pubmed>8625833</pubmed></ref>. An interesting study conducted by Mohanty et al. (1992) amputated the tails of tadpoles to demonstrate that when their tail stumps were treated with retinoic acid they regenerated legs instead of a tails at the site of amputation <ref><pubmed>1731249 </pubmed></ref>.

The initiation of limb outgrowth other transcription factors are expressed to control specific areas of patterning, i.e. forelimb versus hindlimb. Various vertebrate limb models have identified three genes that determine the identity of the developing limb i.e. forelimb or hindlimb. Tbx5 and Tbx4 are T-box family transcription factors specifically expressed in the forelimb and hindlimb, respectively<ref><pubmed>9609833</pubmed></ref><ref><pubmed>10235263</pubmed></ref>. Pitx1, another transcription factor, is expressed in the developing hindlimb, but not the forelimb<ref><pubmed>22071103</pubmed></ref>.

Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the Fibroblast Growth Factor (FGF) and Wnt signaling pathways (more information on these two signalling pathways are described in [[2016 Group Project 3]] and [[2016 Group Project 1]] respectively). Initial activation of fibroblast growth factor-10 (Fgf10) in the lateral plate mesoderm of the forelimb and hindlimb is regulated by Tbx5 and Tbx4 respectively <ref><pubmed>9187149</pubmed></ref>. It has also been found that Tbx5 binding sites have been identified in the Fgf10 promoter sequence in mice and humans <ref><pubmed>12490567</pubmed></ref>. The Fgf10 signals the overlying distal ectoderm to induce Fgf8, which is crucial for the formation of the apical ectodermal ridge (AER) at the tip of the developing limb bud. A positive feedback loop, regulated by the Wnt signalling pathway, is then established between Fgf8 and Fgf10, such that Fgf10 promotes Fgf8 expression and Fgf8 promotes Fgf10 expression. If Fgf10 is flanked in mice the resultant embryos develop without limbs, indicating the importance of this fibroblast growth factor. A deletion of either Tbx5 or Tbx4 will also cause outgrowth defects of limb buds and the the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

The important role of Tbx trancription factors is highlighted in experiments where Tbx5 is knocked out or inactivated. In these studies the embryos that develop do so with complete failure of formation of any elements of the forelimb. These studies further support that Tbx5 interacts with Fgf and Wnt to initiate outgrowth of the limb bud.

A schematic representation of T-box involvement in limb development is outlined in the image below:

[[File:Limb induction-initiation signal 01.jpg|450px|centre]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====T-box in the Mouse====
Brachyury was the first T-box gene to be discovered, and is the most studied gene so far in the T-box gene family. As mentioned above it was discovered in the mouse through a semi-dominant mutation in which heterozygotes have short tails and the homozygotes are distinguished by embryonic lethality. That short tailed heterozygotes have the hall mark short tail and this is why the whole family of T-Box gene family bears the T for tail.
The brachyury gene is responsible for the development of the posterior mesoderm during the gastrulation phase. In homozygote mice, the anterior part of the embryo develops normally, however the axial and posterior mesoderm structures develop abnormally. There is no connection with the allantois and chorion and without a vascular connection between the embryo and the placental and the embryo does not survive <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box in the zebra fish====
T-box orthologs have been recognised in different species. Brachyura expression using genetic analysis was found to be confined to tissues that showed developmental defects in mutants.
In the Zebrafish ortholog the '''zf-T''' is expressed as a nuclear protein in a pattern that resembles mouse T.
A mutant in this gene in the zebrafish is called "no tail" and has a similar phenotype that seen in mouse mutations.

====T-box in ''Drosophilia''====
The Drosophila ortholog of Brachyury is '''dm-Trg''' and mutations in the Drosophila ortholog shows effects in the posterior structures of the fly, which are possibly analogous to the posterior structures found in mammals.

====T-Box in Amphibia: ''Xenopus''====
Using molecular developmental techniques the role of Brachyury was further examined in the frog ''Xenopus laevis''
The ''Xenopus'' homologue of Brachyury, Xbra, was cloned by Smith and coworkers in 1991 <ref name=PMID1717160><pubmed>1717160</pubmed></ref> . This study was based on sequence homology between the frog and mouse sequences, and its expression has shown to represent a true orthologue of the mouse gene with a high degree of similarity in both sequence and expression pattern. In the unfertilised egg, low levels of maternal Xbra are found, but the gene is expressed predominantly at the mid-blastula to neurula stages<ref name=PMID1717160><pubmed>1717160</pubmed></ref>. Studies in ''Xenopus'' have contributed significantly in understanding the mechanism of action of Xbra and the Brachyury gene and have shown that the activation of Xbra in response to mesoderm inducing factors.

====T-Box in the chick====
In the avian model (the chick) the following T-box genes have been isolated '''Tbx-2, Tbx-3, Tbx-4''', and '''Tbx-5''' and, like the mouse homologues, are expressed in the limb regions <ref><pubmed>9550719</pubmed></ref>.
Other Tbox genes '''cTbx2, cTbx3, and cTbx5''' have been found to also be involved in the chick embryo heart development.

====T-Box in amphioxus====
In amphioxuxu two brachyury like genes have been found and are referred to as paralogous genes which are orthologous to vertebrate Brachyury <ref name=PMID9504043><pubmed>9504043</pubmed></ref>.

====T-box gene in Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

At birth marsupials are born with a more developed forelimb than hindlimb. The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. However some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby, and also because gestation is less for didelphid marsupials than for macropods.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T02:25:41Z

Z5020373: /* Functions of T-box in development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110"/>.

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T02:24:16Z

Z5020373: /* Cardiac development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref name="PMID11242110">.

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T02:22:38Z

Z5020373: /* Timeline */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

'''2001''' - It was proposed that TBX1 in humans is a key gene in the etiology of DiGeorge syndrome <ref name="PMID11242110 "><pubmed>11242110 </pubmed></ref>.

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref><pubmed>11242110</pubmed></ref> .

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T02:18:01Z

Z5020373: /* Cardiac development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development <ref name=DZ1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>. '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=DZ1927/>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol '''T''' and '''gene name T'''. However the gene is described as '''brachyury'''.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which contain only one or two T-box genes ancestors , including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See 'Abnormalities' section below for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been implicated in regulating formation and growth of the pharyngeal arch arteries, growth and septation of the outflow tract of the heart, interventricular septation, and conal alignment. Vitelli et al. (2002) showed that homozygous loss of Tbx1 gene can cause severe vascular and heart defects<ref><pubmed>11971873</pubmed></ref> . This is evidenced through the study done by Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref name="PMID11242049"><pubmed>11242049</pubmed></ref>. Another study by Jerome and Papaioannou (2001) revealed that mice with a heterozygous Tbx1 mutation had a high incidence of cardiac outflow tract anomalies. They noticed that this modelled one of the major abnormalities of the human DiGeorge Syndrome/Velocardiofacial syndrome and proposed that TBX1 in humans is a key gene in the etiology of this human disorder (See 'Abnormalities' section below for further info) <ref><pubmed>11242110</pubmed></ref> .

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

A study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 describes for the first time the T Box gene expression in marsupials in the Tammar wallaby (''Macropus eugenii''). This study describes how these genes are also responsible for limb and digit formation in marsupials.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image above demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]
|}

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

{| style="border-spacing: 1px; border: 1px solid white;"
| [[Image: Amphioxus development.gif |600px|thumb|left| This image above demonstrates an overview of amphioxus development and the stages examined in the study by <ref><pubmed>26052418</pubmed></ref>
Amphioxus are classified in the Subphylum Cephalochordate and are approximately 22 mm long and are chordates, and considered to be one of the closest living relatives to all vertebrates.]]
|}

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-24T00:40:55Z

Z5020373: /* Cardiac development */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for a series of T-box proteins, a family of transcription factors that with more than 20 members identified in humans so far, and homologues in many other organisms including different vertebrates. These T-box proteins are termed as transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified that the T-box genes play an important roles of the development of the heart, respiratory system and limbs. Since these genes are involved in the development of certain important regions of human body, mutations of them leads into human gene disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>
This page would give a board overview of how is the T-box signalling pathway worked and its importance, the discovering, abnormalities and animal models that used for researching. It is important to note that T-box genes have also found to regulate the patterning and cell fate, cell survival, and/or proliferation but not be included in this page. For a more comprehensive overview about the role of T-box genes, please click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more.

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development (Dobrovolskaïa-Zavadskaïa, 1927). '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=Dobrovolskaïa-Zavadskaïa1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol T and gene name T. However the gene is described as brachyury.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which one or two T-box genes, including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See section below on Abnormalities for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

In embryonic development, arteries form within pharangeal arches, and subsequently undergo extensive remodeling that ensures proper outflow connection between the heart and systemic and pulmonary circulation. Tbx1 has been heavily implicated in regulating the formation and early remodeling of the pharyngeal arch arteries and a homozygous mutation of Tbx1 causes severe developmental defects of the pharyngeal arches and pouches. Vitelli et al. (2002) suggests that Tbx1 plays at least two roles in pharyngeal development: an early, cell-autonomous role in pharyngeal endoderm growth and patterning, and a later, cell non-autonomous role in supporting the development of pharyngeal arch and pouch derivatives. The latter role may be mediated through signaling to neural crest-derived cells. Lindsey et al. (2001) demonstrated that homozygous Tbx1 mutant embryos do not form pharyngeal arch arteries 3, 4, and 6 <ref><pubmed>11242049</pubmed></ref>.

the growth and septation of the cardiac outflow tract (COT)is an important role of Tbx1.
Furthermore, in the secondary heart field another Tbx gene, Tbx1 has been found to function in both growth and differentiation. Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of Tbx1 that are expressed in secondary heart field mesoderm and associated endoderm, as well as weakly in the outflow tract. Tbx1 -> Fgf8/Fgf10 pathway in driving proliferation in secondary heart field mesoderm, contributing to OFT growth<ref><pubmed>15469978</pubmed></ref>. BMP proteins are also induced in secondary heart field cells proximal to the inflow and outflow poles of the heart<ref><pubmed>15848389</pubmed></ref>. BMPs can induce cardiomyogenic differentiation in collaboration with Fgf8 and can moderate the proliferation of secondary heart field mesoderm. Therefore, a delicate balance between the levels of Fgf and BMP factors appears essential for secondary heart field development<ref><pubmed>15843407</pubmed></ref>.

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

This study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 is the first which describes the T Box gene expression in a marsupial the Tammar wallaby (''Macropus eugenii'') and how these genes are also responsible for limb and digit formation.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

[[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|Demonstration the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale. <ref name=PMID22235805/>]]

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

[[Image:Branchiostoma lanceolatum copy.jpg]]

This photo above is a photo of a Lancelet (or Amphioxus) specimen —Subphylum: Cephalochordate. It was collected in coarse sand sediments (600 µm) on the Belgian continental shelf. Total Length: approximately 22 mm. It is a chordate and considered one of the closest living relatives to all vertebrates. [https://en.wikipedia.org/wiki/Lancelet#/media/File:Branchiostoma_lanceolatum.jpg Image from here]]]

===Glossary===

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Motif''' : a pattern or design

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Transcription factor''': a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-23T23:57:00Z

Z5020373: /* Cardiac development */

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=T-box genes and their signalling pathway=

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for T-box proteins, a family of transcription factors with more than 20 members identified in humans so far, and homologues in many other organisms. These T-box proteins are termed transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified important roles of the T-box genes in the development of the heart, respiratory system and limbs which this page addresses. It is important to note that T-box genes have also found to regulate patterning and cell fate, cell survival, and/or proliferation that have not been included on this page. For a more comprehensive overview about the role of T-box genes click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more. And so, mutations in these genes lead to human disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development (Dobrovolskaïa-Zavadskaïa, 1927). '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=Dobrovolskaïa-Zavadskaïa1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol T and gene name T. However the gene is described as brachyury.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which one or two T-box genes, including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

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Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

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'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

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'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

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'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome <ref name="PMID8988165"><pubmed>8988165</pubmed></ref>.

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'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

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Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects where functional separation of the four cardiac chambers is perturbed<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See section below on Abnormalities for further info).

Moreover, cardiac genes Myh6+ and Tnnt2+ cell induction rate was investigated by Zhou et al. (2012) using combining the expression of various genes. The results of the article are summarised below:

Table 1. Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.
[[File:Cardiac gene induction rates.png|800px|centre]]

The results from this table demonstrates that cardiac marker protein expression of Myh6 and Tnnt2 was only induced by Tbx5+Myocd (T+M) and Tbx5+Gata4+Myocd (T+G+M) combinations in 10T1/2 non-myoblastic cells . This further supports previous findings that Tbx5 and Gata4 interactions are vital activators of cardiac genes and activated the fewest genes associated with non-cardiac processes<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

Furthermore, in the secondary heart field another Tbx gene, Tbx1 has been found to function in both growth and differentiation. Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of Tbx1 that are expressed in secondary heart field mesoderm and associated endoderm, as well as weakly in the outflow tract. Tbx1 -> Fgf8/Fgf10 pathway in driving proliferation in secondary heart field mesoderm, contributing to OFT growth<ref><pubmed>15469978</pubmed></ref>. BMP proteins are also induced in secondary heart field cells proximal to the inflow and outflow poles of the heart<ref><pubmed>15848389</pubmed></ref>. BMPs can induce cardiomyogenic differentiation in collaboration with Fgf8 and can moderate the proliferation of secondary heart field mesoderm. Therefore, a delicate balance between the levels of Fgf and BMP factors appears essential for secondary heart field development<ref><pubmed>15843407</pubmed></ref>.

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

This study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 is the first which describes the T Box gene expression in a marsupial the Tammar wallaby (''Macropus eugenii'') and how these genes are also responsible for limb and digit formation.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

[[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image is from Chew et al. 2012 and demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale.]]

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

[[Image:Branchiostoma lanceolatum copy.jpg]]

This photo above is a photo of a Lancelet (or Amphioxus) specimen —Subphylum: Cephalochordate. It was collected in coarse sand sediments (600 µm) on the Belgian continental shelf. Total Length: approximately 22 mm. It is a chordate and considered one of the closest living relatives to all vertebrates. [https://en.wikipedia.org/wiki/Lancelet#/media/File:Branchiostoma_lanceolatum.jpg Image from here]]]

===Glossary===
'''Homologous''': existence of shared ancestry between a pair of structures, or genes, in different taxa.

'''Homologue''': something homologous.

'''Congenital''': a condition attributable to events prior to birth, could be related to a baby born with disease.

'''Nkx2-5''': a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

'''Wild-type''': refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-23T23:44:23Z

Z5020373: /* Cardiac development */

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{{Group Assessment Criteria table}}

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{{Group Assessment Criteria table}}

==T-box genes and their signalling pathway==

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for T-box proteins, a family of transcription factors with more than 20 members identified in humans so far, and homologues in many other organisms. These T-box proteins are termed transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified important roles of the T-box genes in the development of the heart, respiratory system and limbs which this page addresses. It is important to note that T-box genes have also found to regulate patterning and cell fate, cell survival, and/or proliferation that have not been included on this page. For a more comprehensive overview about the role of T-box genes click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more. And so, mutations in these genes lead to human disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development (Dobrovolskaïa-Zavadskaïa, 1927). '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=Dobrovolskaïa-Zavadskaïa1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol T and gene name T. However the gene is described as brachyury.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which one or two T-box genes, including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome<pubmed>8988165</pubmed></ref>.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects as this transcription factor is essential for functional separation of the four cardiac chambers<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See section below on Abnormalities for further info).

[[File:Cardiac gene induction rates.png|800px]]

Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>. Shows that Myh6 and Tnnt2 cardiac marker protein expression was only induced by Tbx5+Myocd and Tbx5+Gata4+Myocd combinations in 10T1/2 non-myoblastic cells <ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

Furthermore, in the secondary heart field another Tbx gene, Tbx1 has been found to function in both growth and differentiation. Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of Tbx1 that are expressed in secondary heart field mesoderm and associated endoderm, as well as weakly in the outflow tract. Tbx1 -> Fgf8/Fgf10 pathway in driving proliferation in secondary heart field mesoderm, contributing to OFT growth<ref><pubmed>15469978</pubmed></ref>. BMP proteins are also induced in secondary heart field cells proximal to the inflow and outflow poles of the heart<ref><pubmed>15848389</pubmed></ref>. BMPs can induce cardiomyogenic differentiation in collaboration with Fgf8 and can moderate the proliferation of secondary heart field mesoderm. Therefore, a delicate balance between the levels of Fgf and BMP factors appears essential for secondary heart field development<ref><pubmed>15843407</pubmed></ref>.

PMID 11702954
PMID 11572777
PMID 15580613
PMID 16258075
PMID 17460765

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

This study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 is the first which describes the T Box gene expression in a marsupial the Tammar wallaby (''Macropus eugenii'') and how these genes are also responsible for limb and digit formation.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

[[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image is from Chew et al. 2012 and demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale.]]

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

[[Image:Branchiostoma lanceolatum copy.jpg]]

This photo above is a photo of a Lancelet (or Amphioxus) specimen —Subphylum: Cephalochordate. It was collected in coarse sand sediments (600 µm) on the Belgian continental shelf. Total Length: approximately 22 mm. It is a chordate and considered one of the closest living relatives to all vertebrates. [https://en.wikipedia.org/wiki/Lancelet#/media/File:Branchiostoma_lanceolatum.jpg Image from here]]]

===Glossary===
Homologous: existence of shared ancestry between a pair of structures, or genes, in different taxa.

Homologue: something homologous.

Congenital: a condition attributable to events prior to birth, could be related to a baby born with disease.

Nkx2-5: a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

Wild-type: refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

2016 Group Project 5

2016-10-23T23:39:43Z

Z5020373: /* T-box genes and their signalling pathway */

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{{Group Assessment Criteria table}}

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==T-box genes and their signalling pathway==

[[File:TBX_5_3D_strcture.png|250px|thumb|right|Tbx5 3D structure <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>]]
===Introduction===
The T-Box genes encode for T-box proteins, a family of transcription factors with more than 20 members identified in humans so far, and homologues in many other organisms. These T-box proteins are termed transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified important roles of the T-box genes in the development of the heart, respiratory system and limbs which this page addresses. It is important to note that T-box genes have also found to regulate patterning and cell fate, cell survival, and/or proliferation that have not been included on this page. For a more comprehensive overview about the role of T-box genes click: PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more. And so, mutations in these genes lead to human disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development (Dobrovolskaïa-Zavadskaïa, 1927). '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=Dobrovolskaïa-Zavadskaïa1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol T and gene name T. However the gene is described as brachyury.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which one or two T-box genes, including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

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'''1997''' The mapping of the Holt-Oram Syndrome locus was refined to 12q24.1 by fluorescence in situ hybridization using a cosmid containing D12S129, which was tightly linked to Holt-Oram Syndrome<pubmed>8988165</pubmed></ref>.

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'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. The growth of the posterior segment (atria and left ventricle) of the heart is what requires Tbx5, and does not occur in embryos that do not have Tbx5. In contrast, right ventricle and outflow tract development have been shown to be Tbx5-independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.
[[File:Cardiac gene induction rates.png|700px|thumb|right|Multigene transfection efficiency and cardiac genes Myh6+ and Tnnt2+ cell induction rate.<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.]]
The signalling mechanism of TBX5 involves interaction with the cardiac homeobox protein NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors induce activation. Hiroi et al. (2001) proved this by showing that cell lines over-expressing wild-type Tbx5 gene started to expressed more cardiac-specific genes and started to contract earlier. However, cell lines that expressed a mutation in Tbx5 gene did not differentiate into beating cardiomyocytes which indicates that Tbx5 is crucial in cardiomyocyte differentiation.<ref><pubmed>11431700</pubmed></ref>.

GATA4 is a transcription factor essential for heart formation has been known to interact with TBX5 to induce normal cardiac septation. An article by Misra et al. (2014), showed that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle and that a disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation. <ref><pubmed>24858909</pubmed></ref> A mutation of GATA4 can result in human congenital heart defects as this transcription factor is essential for functional separation of the four cardiac chambers<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See section below on Abnormalities for further info).

<ref name="PMID23144723"><pubmed>23144723</pubmed></ref>.

Furthermore, in the secondary heart field another Tbx gene, Tbx1 has been found to function in both growth and differentiation. Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of Tbx1 that are expressed in secondary heart field mesoderm and associated endoderm, as well as weakly in the outflow tract. Tbx1 -> Fgf8/Fgf10 pathway in driving proliferation in secondary heart field mesoderm, contributing to OFT growth<ref><pubmed>15469978</pubmed></ref>. BMP proteins are also induced in secondary heart field cells proximal to the inflow and outflow poles of the heart<ref><pubmed>15848389</pubmed></ref>. BMPs can induce cardiomyogenic differentiation in collaboration with Fgf8 and can moderate the proliferation of secondary heart field mesoderm. Therefore, a delicate balance between the levels of Fgf and BMP factors appears essential for secondary heart field development<ref><pubmed>15843407</pubmed></ref>.

PMID 11702954
PMID 11572777
PMID 15580613
PMID 16258075
PMID 17460765

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

This study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 is the first which describes the T Box gene expression in a marsupial the Tammar wallaby (''Macropus eugenii'') and how these genes are also responsible for limb and digit formation.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

[[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image is from Chew et al. 2012 and demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale.]]

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

[[Image:Branchiostoma lanceolatum copy.jpg]]

This photo above is a photo of a Lancelet (or Amphioxus) specimen —Subphylum: Cephalochordate. It was collected in coarse sand sediments (600 µm) on the Belgian continental shelf. Total Length: approximately 22 mm. It is a chordate and considered one of the closest living relatives to all vertebrates. [https://en.wikipedia.org/wiki/Lancelet#/media/File:Branchiostoma_lanceolatum.jpg Image from here]]]

===Glossary===
Homologous: existence of shared ancestry between a pair of structures, or genes, in different taxa.

Homologue: something homologous.

Congenital: a condition attributable to events prior to birth, could be related to a baby born with disease.

Nkx2-5: a cardiac homeobox protein essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases.

Wild-type: refers to having the gene that encodes the phenotype most common in a particular natural population.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

File:Cardiac gene induction rates.png

2016-10-23T23:34:37Z

Z5020373:

==Cardiac Gene Activation Analysis in Mammalian Non-Myoblasic Cells by Nkx2-5, Tbx5, Gata4 and Myocd==

Table 1. Multigene transfection efficiency and Myh6+ and Tnnt2+ cell induction rate.

===Copyright===
© 2012 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

===Reference===
Citation: Zhou L, Liu Y, Lu L, Lu X, Dixon RAF (2012) Cardiac Gene Activation Analysis in Mammalian Non-Myoblasic Cells by Nkx2-5, Tbx5, Gata4 and Myocd. PLoS ONE 7(10): e48028. doi:10.1371/journal.pone.0048028

PMID 23144723

{{Student Image}}

File:Cardiac gene induction rates.png

2016-10-23T23:33:36Z

Z5020373: Cardiac Gene Activation Analysis in Mammalian Non-Myoblasic Cells by Nkx2-5, Tbx5, Gata4 and Myocd Table 1. Multigene transfection efficiency and Myh6+ and Tnnt2+ cell induction rate. Copyright: © 2012 Zhou et al. This is an open-access article dist...

Cardiac Gene Activation Analysis in Mammalian Non-Myoblasic Cells by Nkx2-5, Tbx5, Gata4 and Myocd

Table 1. Multigene transfection efficiency and Myh6+ and Tnnt2+ cell induction rate.

Copyright: © 2012 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Zhou L, Liu Y, Lu L, Lu X, Dixon RAF (2012) Cardiac Gene Activation Analysis in Mammalian Non-Myoblasic Cells by Nkx2-5, Tbx5, Gata4 and Myocd. PLoS ONE 7(10): e48028. doi:10.1371/journal.pone.0048028

PMID 23144723

2016 Group Project 5

2016-10-23T22:38:48Z

Z5020373: /* Introduction */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

==T-box genes and their signalling pathway==

===Introduction===
The T-Box genes encode for T-box proteins, a family of transcription factors with more than 20 members identified in humans so far, and homologues in many other organisms. These T-box proteins are termed transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified important roles of the T-box genes in the development of the heart and limbs which this page focuses on. It is important to note that T-box genes have also found to regulate patterning and cell fate, cell survival, and/or proliferation. For more comprehensive overview about the role of T-box genes click PMID 25294936 <ref name=PMID25294936><pubmed>25294936</pubmed></ref> to read more. And so, mutations in these genes lead to human disorders including Di-George Syndrome and Holt-Oram syndrome. <ref><pubmed>16285859</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development (Dobrovolskaïa-Zavadskaïa, 1927). '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=Dobrovolskaïa-Zavadskaïa1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol T and gene name T. However the gene is described as brachyury.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which one or two T-box genes, including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====

'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes <ref>Dobrovolskaia-Zavadskaia, N. (1927). Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l'existence d'un caractere hereditaire “non viable”. CR Soc. Biol, 97, 114-116.</ref>.

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990'''

The T gene itself was cloned <ref name="PMID2154694 "><pubmed>2154694 </pubmed></ref>

----

'''1994'''

Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family. <ref name="PMID7774921 "><pubmed>7774921</pubmed></ref>

----

'''1995'''

The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb" <ref name="PMID8530034 "><pubmed>8530034 </pubmed></ref>

The location of tbx-2 was found, 17q21-22 which means in the long arm (q) of chromosome 17, from region 2, band 1 to region 2, band 2. <ref name="PMID8597636"><pubmed>8597636</pubmed></ref>

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively. <ref name="PMID9550719"><pubmed>9550719</pubmed></ref>

----

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

[[File:TBX_5_3D_strcture.png|250px]]
Example of 3D structure. <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA-4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. It is at the stage where growth of the posterior segment (atria and left ventricle) of the heart which requires Tbx5, and does not occur in embryos that lack a Tbx5. In contrast, RV and outflow tract development appears to be Tbx5 independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors lead to synergistic activation<ref><pubmed>11431700</pubmed></ref>. Moreover, GATA4 a transcription factor essential for heart formation has been shown to interact with TBX5. A mutation of GATA4 can result in human congenital heart defects as this transcription factor is essential for functional separation of the four cardiac chambers<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 transcription factor is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See section below on Abnormalities for further info).

Furthermore, in the secondary heart field another Tbx gene, Tbx1 has been found to function in both growth and differentiation. Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of Tbx1 that are expressed in secondary heart field mesoderm and associated endoderm, as well as weakly in the outflow tract. Tbx1 -> Fgf8/Fgf10 pathway in driving proliferation in secondary heart field mesoderm, contributing to OFT growth<ref><pubmed>15469978</pubmed></ref>. BMP proteins are also induced in secondary heart field cells proximal to the inflow and outflow poles of the heart<ref><pubmed>15848389</pubmed></ref>. BMPs can induce cardiomyogenic differentiation in collaboration with Fgf8 and can moderate the proliferation of secondary heart field mesoderm. Therefore, a delicate balance between the levels of Fgf and BMP factors appears essential for secondary heart field development<ref><pubmed>15843407</pubmed></ref>.

PMID 11702954
PMID 11572777
PMID 15580613
PMID 16258075
PMID 17460765

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name="Genetics Home Reference (2016)" >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name="Genetics Home Reference (2016)"/>This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

This study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 is the first which describes the T Box gene expression in a marsupial the Tammar wallaby (''Macropus eugenii'') and how these genes are also responsible for limb and digit formation.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

[[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image is from Chew et al. 2012 and demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale.]]

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

[[Image:Branchiostoma lanceolatum copy.jpg]]

This photo above is a photo of a Lancelet (or Amphioxus) specimen —Subphylum: Cephalochordate. It was collected in coarse sand sediments (600 µm) on the Belgian continental shelf. Total Length: approximately 22 mm. It is a chordate and considered one of the closest living relatives to all vertebrates. [https://en.wikipedia.org/wiki/Lancelet#/media/File:Branchiostoma_lanceolatum.jpg Image from here]]]

===Glossary===
Homologous: existence of shared ancestry between a pair of structures, or genes, in different taxa.

Homologue: something homologous.

Congenital: a condition attributable to events prior to birth, could be related to a baby born with disease.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===

File:TBX 5 3D strcture.png

2016-10-21T04:16:00Z

Z5020373:

==Structural basis of tbx5-dna recognition: the t- box domain in its DNA-bound and -unbound form.==

From pubmed structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644
Image citation: <pubmed>20450920</pubmed>

{{Student Image}}

2016 Group Project 5

2016-10-21T04:15:06Z

Z5020373: /* Features of the T-box family */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

==T-box genes and their signalling pathway==

===Introduction===
The T-Box genes encode for T-box proteins, a family of transcription factors with more than 20 members identified in humans so far, and homologues in many other organisms. These T-box proteins are termed transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified important roles of the T-box genes in the development of the heart and limbs. T-box genes have also found to regulate patterning and cell fate, cell survival, and/or proliferation. And so, mutations in these genes lead to human disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development (Dobrovolskaïa-Zavadskaïa, 1927). '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=Dobrovolskaïa-Zavadskaïa1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol T and gene name T. However the gene is described as brachyury.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which one or two T-box genes, including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====
'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes

----

Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990''' - The T gene itself was cloned

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'''1992''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb"

----

'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family.

----

'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively.

----

Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

[[File:TBX_5_3D_strcture.png|250px]]
Example of 3D structure. <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA-4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. It is at the stage where growth of the posterior segment (atria and left ventricle) of the heart which requires Tbx5, and does not occur in embryos that lack a Tbx5. In contrast, RV and outflow tract development appears to be Tbx5 independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors lead to synergistic activation<ref><pubmed>11431700</pubmed></ref>. Moreover, GATA4 a transcription factor essential for heart formation has been shown to interact with TBX5. A mutation of GATA4 can result in human congenital heart defects as this transcription factor is essential for functional separation of the four cardiac chambers<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 transcription factor is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See section below on Abnormalities for further info).

Furthermore, in the secondary heart field another Tbx gene, Tbx1 has been found to function in both growth and differentiation. Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of Tbx1 that are expressed in secondary heart field mesoderm and associated endoderm, as well as weakly in the outflow tract. Tbx1 -> Fgf8/Fgf10 pathway in driving proliferation in secondary heart field mesoderm, contributing to OFT growth<ref><pubmed>15469978</pubmed></ref>. BMP proteins are also induced in secondary heart field cells proximal to the inflow and outflow poles of the heart<ref><pubmed>15848389</pubmed></ref>. BMPs can induce cardiomyogenic differentiation in collaboration with Fgf8 and can moderate the proliferation of secondary heart field mesoderm. Therefore, a delicate balance between the levels of Fgf and BMP factors appears essential for secondary heart field development<ref><pubmed>15843407</pubmed></ref>.

PMID 11702954
PMID 11572777
PMID 15580613
PMID 16258075
PMID 17460765

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name=Genetics Home Reference (2016) >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name=Genetics Home Reference (2016) >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

This study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 is the first which describes the T Box gene expression in a marsupial the Tammar wallaby (''Macropus eugenii'') and how these genes are also responsible for limb and digit formation.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

[[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image is from Chew et al. 2012 and demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale.]]

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

[[Image:Branchiostoma lanceolatum copy.jpg]]

This photo above is a photo of a Lancelet (or Amphioxus) specimen —Subphylum: Cephalochordate. It was collected in coarse sand sediments (600 µm) on the Belgian continental shelf. Total Length: approximately 22 mm. It is a chordate and considered one of the closest living relatives to all vertebrates. [https://en.wikipedia.org/wiki/Lancelet#/media/File:Branchiostoma_lanceolatum.jpg Image from here]]]

===Glossary===
Homologous: existence of shared ancestry between a pair of structures, or genes, in different taxa.

Homologue: something homologous.

Congenital: a condition attributable to events prior to birth, could be related to a baby born with disease.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===
Check this for doubling up! shows more teamwork and if we've worked together as a team

2016 Group Project 5

2016-10-21T04:14:01Z

Z5020373: /* Features of the T-box family */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

==T-box genes and their signalling pathway==

===Introduction===
The T-Box genes encode for T-box proteins, a family of transcription factors with more than 20 members identified in humans so far, and homologues in many other organisms. These T-box proteins are termed transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified important roles of the T-box genes in the development of the heart and limbs. T-box genes have also found to regulate patterning and cell fate, cell survival, and/or proliferation. And so, mutations in these genes lead to human disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development (Dobrovolskaïa-Zavadskaïa, 1927). '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=Dobrovolskaïa-Zavadskaïa1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol T and gene name T. However the gene is described as brachyury.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which one or two T-box genes, including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====
'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes

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Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990''' - The T gene itself was cloned

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'''1992''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb"

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'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family.

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'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively.

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Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

[[File:TBX_5_3D_strcture.png|450px]]
Example of 3D structure. <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA-4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. It is at the stage where growth of the posterior segment (atria and left ventricle) of the heart which requires Tbx5, and does not occur in embryos that lack a Tbx5. In contrast, RV and outflow tract development appears to be Tbx5 independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors lead to synergistic activation<ref><pubmed>11431700</pubmed></ref>. Moreover, GATA4 a transcription factor essential for heart formation has been shown to interact with TBX5. A mutation of GATA4 can result in human congenital heart defects as this transcription factor is essential for functional separation of the four cardiac chambers<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 transcription factor is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See section below on Abnormalities for further info).

Furthermore, in the secondary heart field another Tbx gene, Tbx1 has been found to function in both growth and differentiation. Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of Tbx1 that are expressed in secondary heart field mesoderm and associated endoderm, as well as weakly in the outflow tract. Tbx1 -> Fgf8/Fgf10 pathway in driving proliferation in secondary heart field mesoderm, contributing to OFT growth<ref><pubmed>15469978</pubmed></ref>. BMP proteins are also induced in secondary heart field cells proximal to the inflow and outflow poles of the heart<ref><pubmed>15848389</pubmed></ref>. BMPs can induce cardiomyogenic differentiation in collaboration with Fgf8 and can moderate the proliferation of secondary heart field mesoderm. Therefore, a delicate balance between the levels of Fgf and BMP factors appears essential for secondary heart field development<ref><pubmed>15843407</pubmed></ref>.

PMID 11702954
PMID 11572777
PMID 15580613
PMID 16258075
PMID 17460765

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name=Genetics Home Reference (2016) >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name=Genetics Home Reference (2016) >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

This study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 is the first which describes the T Box gene expression in a marsupial the Tammar wallaby (''Macropus eugenii'') and how these genes are also responsible for limb and digit formation.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

[[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image is from Chew et al. 2012 and demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale.]]

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

[[Image:Branchiostoma lanceolatum copy.jpg]]

This photo above is a photo of a Lancelet (or Amphioxus) specimen —Subphylum: Cephalochordate. It was collected in coarse sand sediments (600 µm) on the Belgian continental shelf. Total Length: approximately 22 mm. It is a chordate and considered one of the closest living relatives to all vertebrates. [https://en.wikipedia.org/wiki/Lancelet#/media/File:Branchiostoma_lanceolatum.jpg Image from here]]]

===Glossary===
Homologous: existence of shared ancestry between a pair of structures, or genes, in different taxa.

Homologue: something homologous.

Congenital: a condition attributable to events prior to birth, could be related to a baby born with disease.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===
Check this for doubling up! shows more teamwork and if we've worked together as a team

2016 Group Project 5

2016-10-21T04:13:33Z

Z5020373: /* Features of the T-box family */

{{ANAT2341Project2016header}}
{{Group Assessment Criteria table}}

==T-box genes and their signalling pathway==

===Introduction===
The T-Box genes encode for T-box proteins, a family of transcription factors with more than 20 members identified in humans so far, and homologues in many other organisms. These T-box proteins are termed transcription factors because of their ability to regulate the expression and subsequent activity of other genes by binding to DNA. T-box proteins are characterised by a DNA-binding motif known as the T-box that binds DNA. Many researchers have identified important roles of the T-box genes in the development of the heart and limbs. T-box genes have also found to regulate patterning and cell fate, cell survival, and/or proliferation. And so, mutations in these genes lead to human disorders including Di-George Syndrome and Holt-Oram syndrome. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>

Mark's comments
- what the signalling pathway does
- clearly identify exactly what this page will talk about - eg. this pathway in reference to this system/embryonic development etc
- limitations of this page - what we won't discuss
- put an image in straight away to break the copious amounts of text at the start of the page

PMID 9504043

===Origins of the T-box name===

The founding member of the T-box family is '''brachyura''' which comes from the greek and means short tail <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. This gene was discovered following experimental studies with a short tailed mouse that harboured a mutation which affected tail length and embryonic development (Dobrovolskaïa-Zavadskaïa, 1927). '''Brakhus''' means short in greek and '''oura''' meaning tail.

Nadezhda Alexandrovna Dobrovolskaya-Zavadskaya first described the brachyury mutation in 1927 as a mutation that affected tail length and sacral vertebrae in heterozygous mice <ref name=Dobrovolskaïa-Zavadskaïa1927>Dobrovolskaïa-Zavadskaïa, N. (1927). Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire “non viable”. C. R. Seanc. Soc. Biol. 97, 114-116.</ref>.

The brachyury gene (which is also known as T) soon was studied in great detail due to its important role in the development of the notochord and posterior mesoderm. Mutations in T are shown to cause embryonic lethality in homozygote mice and short tails in heterozygote mice <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
Now according to human and mouse genomes, the gene brachyura has the symbol T and gene name T. However the gene is described as brachyury.

===Ancient origins and evolution of the T-box gene family===

T-box gene family is ancient in origin and it is thought to be found in all metazoans <ref name="PMID7920656"><pubmed>7920656</pubmed></ref>.
Due to the increasing availability of sequenced genomes from a diverse group of animal taxa we now know that the origin of the T-box family has been pushed back to unicellular organisms and fungi, in which one or two T-box genes, including T, have been identified. <ref name=PMID24043797><pubmed>24043797</pubmed></ref>

With the analysis of the genomes of bilaterian organisms and representatives of the different phyla, four basal metazoan phyla indicate that T is the most ancient member of the T-box family and that the family has expanded throughout metazoan evolution. <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
In Metazoan evolution it seems that genes were added progressively one by one by gene or genome duplication and some of these genes were lost and gained in specific lineages <ref name=PMID25294936><pubmed>25294936</pubmed></ref>.
In present day or extant vertebrates we now know that the T-box family has radiated throughout the vast vertebrate linages and can be grouped into five subfamilies T, Tbx1, Tbx2, Tbx6 and Tbr1 <ref name=PMID25294936><pubmed>25294936</pubmed></ref>. In the common ancestor of vertebrates and sponges, four of these five subfamilies were already present
<ref name=PMID24043797><pubmed>24043797</pubmed></ref>.

[[File:Brachyury expression in 7.5dpc CD1 mouse embryos.jpg]]

Brachyury expression in 7.5dpc CD1 mouse embryos [https://en.wikipedia.org/wiki/Brachyury#/media/File:Paul_Burridge_Brachyury_in_E7.5.jpg Image from here]

===The discovery of T-box genes===
The story of the T-box genes began in Paris at the Pasteur laboratory in the 1920s with the Russian scientist Nadine Dobrovolskaïa-Zavadskaïa, who embarked on a pioneering screen for X-ray-induced developmental mouse mutants. Her isolation of a mouse strain with a short tail, caused by a semidominant heterozygous mutation in a locus she called T, represented one of the first successful mammalian genetic screens, and provided one of the earliest links between gene activity and cell behaviour during embryogenesis<ref><pubmed>11268043</pubmed></ref>. The mid-gestational death of homozygous T embryos, with perturbed development of the posterior mesoderm and notochord, demonstrated an essential requirement for T during gastrulation, and led to the earliest insights into the inductive influences of notochord on neural tube and somite development. Over 60 years later T, now also known as brachyury, meaning ‘short tail’ in Greek, was cloned in one of the earliest positional cloning efforts in the mouse<ref name="PMID2154694"><pubmed>2154694</pubmed></ref>. At the time, lack of homology in the T-gene product to any previously characterized protein gave no clues as to its biochemical role until, in 1993, it was revealed to be a novel sequence-specific DNA-binding protein<ref><pubmed>8344258</pubmed></ref>. Crystallographic determination of the structure of the DNA-binding domain, now called the Tbox, revealed a new way through which proteins recognize DNA<ref><pubmed>9349824</pubmed></ref>.

TO DO: Produce a timeline for the history of the TBox gene - type TBOx into PubMed and sort the findings by age - use this to see when the first journal articles where released related to TBox and limb development, how this research has improved and developed, where it is heading in the future

====Timeline====
'''1927''' - Brachyury (T) locus was introduced to the world in a report describing the effects of a mutation at this locus on both embryonic viability in homozygotes and the development of the tail in heterozygotes

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Over the following decades, the embryological defects caused by the T mutation were studied.

'''1990''' - The T gene itself was cloned

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'''1992''' - The discovery of sequence homology between the mouse T gene and a newly cloned Drosophila gene called "omb"

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'''1994''' - Bollag and his colleagues showed the existence of a family of T-related genes in the mouse genome, which was christened the T-box gene family.

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'''1998''' - Alison Isaac and the team confirmed that in chicken embryo, Tbx-2 & Tbx-3 are related to both forelimb and hindlimb development, and Tbx-4 & Tbx-5 have limited expression domains in leg and wing respectively.

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Comments - timeline put references - eg. the papers from 1990 etc so that they are useful for other students - this is part of the criteria for the project
MORE RECENT RESEARCH

PROTEIN DATABASE OF PUBMED THERE ARE 3D THINGS OF THE TBOX MEMBERS

===Features of the T-box family===
The defining feature of the T-box gene family is a conserved domain that was first uncovered in the sequence of the mouse T locus, or Brachyury gene<ref name="PMID2154694"/>. This homology domain encodes a polypeptide region that has been named the T-box. <ref name="PMID7920656"/> The following table outlines the functions of some important T-box genes, including their location and associated human diseases.

[[File:Typical tbx protein structure.png]]

[[File:TBX_5_3D_strcture.png]]
Example of 3D structure. <ref> Pubmed structure (2010) Crystal Structure of Human Tbx5 in the DNA-free Form, https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644, accessed 21/10/16</ref>

====Summary of the main T-box genes====
{|border="1"
|-
| T-box gene || Main expression sites during embryogenesis || Function || Abnormalities
|-
| Tbx1 || Pharyngeal endoderm, mesoderm core of the first pharyngeal arch, head mesoderm ventral to hindbrain, sclerotome || Pharyngeal arch arteries development, governs the transition between stem cell quiescence and proliferation in hair follicles, associated with developmental abnormalities with the ear, facial and cardiac outflow || DiGeorge syndrome, Velocardiofacial syndrome, Conotruncal anomaly face syndrome, Tetralogy of Fallot
|-
| Tbx2 || Allantois, non-chanmber myocardium, optic and otic vesicles, naso-facial mesenchyme, limbs, lungs, genitalia ||Potent immortalizing gene that acts by downregulating CDKN2A, regulates Anf expression in chamber myocardium development|| None identified
|-
| Tbx3 || Non-chamber myocardium (sinoatrial region, AV canal and interventricular ring), expressed with TBX2, TBX3, and TBX5 in the embryonic neural retina||Provides positional information important for topographic mapping in differentiation of distinct cell types across the laminar axis of the retina, development of functional ectopic pacemakers, stimulates Nanog, Tbx3 specifies digit III and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade. The fetal lung, kidney, heart, liver, and spleen in humans expresses this gene ||Ulnar-mammary syndrome
|-
| Tbx4 || Hindlimb, mandibular and lung mesenchyme, atrium and body wall||Developmental pathways of the lower limbs and the pelvis in humans || Ischiocoxopodopatellar syndrome, Small patella syndrome
|-
| Tbx5 || Cardiac cresent, heart tube, sinus venous, common atrium, left ventricle (LV) and right ventricle (RV) forelimb, eye || Promotes cardiomyocyte differentiation, interaction with GATA4 cause of human cardiac septal defects||Holt-Oram syndrome
|-
| Tbx18 || Splanchnic mesoderm, septum traversum, epicardium ||Maintain the separation of anterior and posterior somite compartments, specification of ureteral mesenchyme and SMC differentiation in the ureter ||Congenital anomalies of kidney and urinary tract 2
|-
| Tbx20 || Allantois, lateral plate mesoderm, cardiac crescent, heart tube, hindbrain, eye||Cardiac development and yolk sac vascular remodeling ||Atrial septal defect 4
|}
Table adapted from: Table 1. Embryonic expression and mutant phenotypes of mouse cardiac T-box genes <ref><pubmed>16258075</pubmed></ref>

SEE IF WE CAN INSERT IMAGES FROM THE PROTEIN PART OF PUBMED - Mark said something about 3D models/images of these - Link to 3D structure: https://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=81644

===Functions of T-box in development===
T-box genes are a growing family of transcription factors that are expressed in diverse patterns throughout vertebrate development. They have emerged as key players in embryonic patterning, tissue differentiation and morphogenesis, particularly in vertebrates and some specific examples have been described in the following sections.

====Cardiac development====
The heart is one of the first organs to develop and function in vertebrate embryos. Its formation is a complex process and requires contributions from multiple transcription factors, including GATA-4, eHAND, dHAND, Irx4, and TBX genes <ref name="PMID11572777"><pubmed>11572777</pubmed></ref>.

In the developing heart, Tbx5 expression can be first detected at stage 12 along the entire rostrocaudal length of the fused heart tube<ref name="PMID9651516"><pubmed>9651516</pubmed></ref>. Although it is expressed very early in cardiac embryogenesis, Tbx5 is not essential for cardiac crescent formation or for development of the early heart tube. It is at the stage where growth of the posterior segment (atria and left ventricle) of the heart which requires Tbx5, and does not occur in embryos that lack a Tbx5. In contrast, RV and outflow tract development appears to be Tbx5 independent<ref name="PMID11572777"/>. It is interesting to note that the role of TBX5 in the growth and maturation of posterior heart is evolutionarily conserved from amphibia (Xenopus) to mammals<ref name="PMID10079235"><pubmed>10079235</pubmed></ref>.

The signalling mechanism of TBX5 involves interaction with NKX2-5 which synergistically promotes cardiomyocyte differentiation. Both these molecules bind directly to the promoter of the gene encoding cardiac-specific natriuretic peptide precursor type A (NPPA) alongside each other, and the 2 transcription factors lead to synergistic activation<ref><pubmed>11431700</pubmed></ref>. Moreover, GATA4 a transcription factor essential for heart formation has been shown to interact with TBX5. A mutation of GATA4 can result in human congenital heart defects as this transcription factor is essential for functional separation of the four cardiac chambers<ref><pubmed>12845333</pubmed></ref>. Therefore Tbx5 transcription factor is involved in directing gene expression in morphogenetic processes associated with specific chamber formation and any mutations can cause heart septal defects as seen in Holt–Oram syndrome<ref name="PMID10079235"/> (See section below on Abnormalities for further info).

Furthermore, in the secondary heart field another Tbx gene, Tbx1 has been found to function in both growth and differentiation. Fibroblast growth factors (Fgfs) 8 and 10 are key downstream effectors of Tbx1 that are expressed in secondary heart field mesoderm and associated endoderm, as well as weakly in the outflow tract. Tbx1 -> Fgf8/Fgf10 pathway in driving proliferation in secondary heart field mesoderm, contributing to OFT growth<ref><pubmed>15469978</pubmed></ref>. BMP proteins are also induced in secondary heart field cells proximal to the inflow and outflow poles of the heart<ref><pubmed>15848389</pubmed></ref>. BMPs can induce cardiomyogenic differentiation in collaboration with Fgf8 and can moderate the proliferation of secondary heart field mesoderm. Therefore, a delicate balance between the levels of Fgf and BMP factors appears essential for secondary heart field development<ref><pubmed>15843407</pubmed></ref>.

PMID 11702954
PMID 11572777
PMID 15580613
PMID 16258075
PMID 17460765

====Limb Development====
Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations involve four members of the T-box family of transcription factors (Tbx2-Tbx5) as well as other molecules which are expressed in developing limb buds. Limb bud outgrowth is initiated and maintained by establishing a positive feedback loop of FGF signaling comprised of Fgf10 expressed in the lateral plate mesoderm (LPM), inducing the expression of Fgf8 in the overlying, distal ectoderm. Initial expression of Fgf10 in the forelimb- and hindlimb-forming LPM is controlled by Tbx transcription factors, Tbx5 in the forelimb and Tbx4 in the hindlimb, and deletion of either Tbx5 or Tbx4 causes outgrowth defects of limb buds.

The expression of Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. While the expression of Tbx2 and Tbx3 is present in both limbs<ref><pubmed>9609833</pubmed></ref>.

Using the chick model, Tbx genes have been proven to specify posterior digit identity through Shh and BMP signaling. <ref><pubmed>12376101</pubmed></ref>. In particular, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities. Furthermore, RA and Shh both induced Tbx2<ref><pubmed>8269518</pubmed></ref>.

Tbx4 and Tbx5 genes are also implicated in the development of the limbs. They are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Homeobox (Hox) genes<ref><pubmed>8625833</pubmed></ref>. Hox genes may therefore be responsible for regulating expression of these Tbox genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8 <ref><pubmed>7889567</pubmed></ref><ref><pubmed>9435295</pubmed></ref>.

PMID 14723846
PMID 9609833
PMID 9655805
PMID 9550719
PMID 8798150
PMID 11782414
PMID 12490567
PMID 22872086
PMID 12736212
PMID 26212321
PMID 21932311

[[File:Limb induction-initiation signal 01.jpg|450px]]

Close up of Tbx5 role in the Initiation of Limb Bud Formation<ref><pubmed>26212321</pubmed></ref>

http://www.ncbi.nlm.nih.gov/books/NBK10003/
http://dev.biologists.org/content/130/3/623/F1
From the limb development lecture, two major signalling pathways that involve Tbx:
1) FGF signaling (FGF10 and FGF8)
2) Wnt signaling pathway

====Respiratory Development====
In a chicken model, all Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing lung buds and trachea between stages 15–21 <ref name="PMID9651516"/>. In the mouse however, Tbx1 is expressed in lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in lung mesenchyme at E11.5, and Tbx4 and Tbx5 are expressed in both lung and trachea mesenchyme at E12.5 and later<ref><pubmed>8853987</pubmed></ref>.

In lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. In an article by Ripla et al. (2012), they demonstrated that both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea<ref name="PMID22876201"><pubmed>22876201</pubmed></ref>. They showed that a loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. This suggests that Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. This is consistent with finding from Sakiyama et al. (2003) that found out that the Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Tbx4 was found to trigger Fgf10 expression in the lung primordium mesoderm which then acquires an inductive capability for the initial budding morphogenesis of primary lung buds<ref><pubmed>12588840</pubmed></ref>. Of significance, lung-specific Tbx4 heterozygous;Tbx5 conditional null mice died soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings, highlighting the important role of Tbx4 and Tbx5 in respiratory development.

[[File:Tbx in lung and trachea development.png|450px]]

Tbx in lung and trachea development<ref name="PMID22876201"/>

====Other developmental events====
TBX signalling also involved in palate development. In humans, TBX1 mutation is responsible for the major phenotypes of 22q11.2 deletion syndrome (Velo-cardio-facial/DiGeorge syndrome, discussed in Abnormalities section) as well as non-syndromic submucous cleft palate, suggesting that Tbx1 is a regulator of palatogenesis. A study by Funato et al. (2012) revealed that Tbx1 regulates oral epithelial adhesion and palatal development and showed that Tbx1 -/- mice exhibit various forms of cleft palate phenotypes, including submucosal cleft palate and soft palate cleft.

Dorsoventral Patterning of the Mouse Coat by Tbx15 PMID 14737183
Palate Development PMID 22371266

Tbx6 interacting with Ripply for the formation of somite boundaries (in zebrafish) PMID 25725067
Tbx6 is required for the expression of mesp-b and ripply1 in the paraxial mesoderm during somite formation, and for the specification of the central Pax3+/Pax7+ dermomyotome. Mesp-b is necessary and sufficient for central dermomyotome formation, it inhibits myogenic differentiation and promotes dermomyotome development. Ripply1 function is required for maturation and fast muscle fiber differentiation.

Results show that Tbx6 protein has to be removed for the expression of pax3/7 and myoD in the lateral paraxial mesoderm, indicating that Tbx6 and/or Tbx6-dependent genes inhibit maturation of myogenic cells. Downstream of Tbx6, Mesp-ba promotes dermomyotome development.

===Abnormalities===
A number of human disorders have been linked to mutations in T-box genes, confirming their medical importance. They include Holt– Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22. <ref><pubmed>10235264</pubmed></ref> <ref><pubmed>18505863</pubmed></ref> <ref><pubmed>15066124</pubmed></ref>

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 19:37, 1 September 2016 (AEST)

====TBX1/DiGeorge Syndrome====
The TBX1 gene can be mapped on chromosome 21 within the DiGeorge syndrome region. Studies using mice, have shown that the TBX1 gene is haploinsufficient.<ref name=PMID1197183><pubmed>1197183</pubmed></ref> This means that while although mice are diploid organisms, only one functional copy of the gene exists, while the other copy has undergone a mutation, making it inactivated. This single gene is unable to produce sufficient amounts of the protein needed for TBX1 to carry out its function- the development of pouches and pharyngeal arches in these mice. In this study of mice, a deficiency of the TBx1 gene resulted in heart defects, hence suggesting the close association between the TBx1 gene and cardiovascular development.

In this study, a mouse model was used where the relevant region on chromosome 16 of the mice species, corresponding to chromosome 22q11.2, or del22q11, in humans, was deleted. <ref name=McKusick1997>McKusick, V. (1997). T-BOX 1; TBX1, OMIM, accessed 3rd October 2016</ref> These 'mutated' mice exhibited a range of cardiovascular abnormalities and defects, as well as behavioural changes. They also revealed incomplete or mutated pharangeal arch and pouch development. Tbx 1 (7-9) was identified as the gene responsible for these cardiovascular malformations. The normal development of the cochlear and vestibular organs was also observed in these mice. Thus, Tbx1 was also identified as crucial for the development of otic epithelial cells, which then later contribute to the development of these inner ear organs. These defects are also exhibited in the birth defects in humans, hence making this mouse model highly effective and suitable. <ref name=PMID1197183><pubmed>1197183</pubmed></ref>

For more info: https://omim.org/entry/188400?search=TBX1&highlight=tbx1

====TBX3/Ulnar-Mammary Syndrome====

[[File:Ums.jpg|300px|thumb|right|a-c: ums patient and d-f: mother of patient, normal <ref name="PMID19938096"><pubmed>19938096</pubmed></ref> ]]

Mutations of the TBX3 gene leads to ulnar-mammary syndrome, caused by a reduce in the levels of the functional proteins needed for normal development of limbs, mammary glands and other structures. Like DiGeorge Syndrome, this syndrome is a result of the haploinsufficiency of TBX3. This disorder is expressed in abnormalities of the limbs, teeth, genitals and mammary glands. <ref name="PMID3374754"><pubmed>3374754</pubmed></ref> Again, animal models of mice have shown abnormalities in mammary glands, limbs and genitalia, often dying before birth. These abnormalities are often characterised by short, stunted growth of the hindlimbs of mice, as well as missing elements to the forelimb. Images of these can be seen on the right.

On the other hand, when this gene is abundant and over-expressed, cancers in the breast, liver and skin have been seen to develop, as high levels of this gene assist in the development of tumours. Lung cancers, breast cancers, ovarian cancers, bladder cancers and liver tumours have been shown to have high levels of the TBX3 gene. <ref name="PMID3374754"/>

For more info: https://omim.org/entry/181450?search=TBX3&highlight=tbx3

====TBX5/Holt– Oram Syndrome====

[[File:Hos.png|300px|thumb|right|shortened thumb (Fig. 1A). radial flexion (Fig. 1B). enlarged heart (Fig. 1C). <ref name="PMID27652283"><pubmed>27652283</pubmed></ref> ]]

Mutations of the TBX5 gene has been shown to cause defects in cardiac septation and the production of isomers in humans affected with holt-oran syndrome. <ref><pubmed>11161571</pubmed></ref> The TBX5 gene is mutated in affected individuals, on chromosome 12q24. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> Holt-Oram syndrome is an autosomal-dominant disorder, and in humans, is expressed by deformities of the upper limb, the shoulder girdle as well as defects in the septa of the heart. Animal models such as chicks have been used, where the overexpression of this gene in chicken embryos has resulted in incomplete growth of the myocardium and the trabeculae and septa of the heart. In humans, the overexpression and mutation of this gene has also been shown to inhibit normal cardiac development. This is because TBX5 is responsible for the normal proliferation of cells during cardiac development. <ref><pubmed>17534187 </pubmed></ref>Thumb anomaly is another expression of holt-oran syndrome, where the thumb may be completely absent, or may develop as another finger-like digit, not different from the other digits of the finger. Other indicators of this disease are heart and skeletal lesions, patent ductus arteriosus (PDA), malformation of the ventricles in the heart, mitral valve prolapse and superior vena cava anomaly. <ref name=McKusick1986>McKusick, V. (1986). HOLT-ORAM SYNDROME, OMIM, accessed 3rd October 2016</ref> These thumb abnormalities can be seen in the images on the left.

For more info: https://omim.org/entry/142900?search=TBX5&highlight=tbx5

====TBX19/Isolated Adrenocorticotropic Hormone (ACTH) Deficiency====
The TBx19 gene initiates the transcription process of the Proopiomelanocortin (POMC) gene. <ref name=PMID1197183><pubmed>26754976</pubmed></ref> This gene contains the instructions for the synthesis of the proopiomelanocortin protein. This protein is further transformed into smaller peptides which bind to proteins in the body, initiating various signalling pathways throughout the body. When this gene is mutated, congenital isolated adrenocorticotropic hormone deficiency develops, caused by the reduction of the secretion of Isolated adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. This is because ACTH-producing cells of the pituitary gland are damaged and cannot secrete sufficient amounts of adrenocorticotropic hormone. <ref name=Genetics Home Reference (2016) >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This results in severe hypoglycaemia and seizures in neonates. Cholestatic Liver disease also arises from this mutation, blocking or reducing the normal flow of bile through the liver. <ref name=Genetics Home Reference (2016) >Genetics Home Reference (2016). POMC Gene. https://ghr.nlm.nih.gov/gene/POMC. accessed 3rd October 2016</ref> This disease has also been seen to cause a range of other clinical symptoms, including urinary incontinence, gait disturbance and dementia in older patients. Ventricular enlargement in the brain, loss of appetite and vomiting are other symptoms associated with isolated ACTH deficiency. The main treatment for this disease is hormone replacement therapy. <ref name=PMID1197183><pubmed>26754976</pubmed></ref>

For more info: https://omim.org/entry/201400?search=TBX19&highlight=tbx19

====TBX22/Cleft Palate====
Studies on mice with cleft palate have shown that the mutations of the gene encoding TBX22, causing the gene to no longer function. Tbx22 is involved in the development of the intramembranous bone formation of the posterior hard palate, rather than palate closure, and hence a mutation of this gene results in cleft palate. These mutations of the TBX22 gene included frame shifts of the gene, splices, nonsense and missense changes to the gene sequence. TBX22 is regulated by the Mn1 transcription factor, working together to achieve normal palate development. Mutations of Tbx1 and Tbx10 are also responsible for cleft palate. The mutation of this gene also causes ankyloglossia, the development of a short and thick lingual frenulum under the tongue, limiting tongue movement. This can be corrected by surgery. Choanal atresia, a blockage of the nasal airway, was also seen in affected mice. Other developmental mutations can be seen in the incomplete formation of the vomer bone in the skull. <ref><pubmed>19648291</pubmed></ref> <ref><pubmed>18948418</pubmed></ref> <ref><pubmed>17846996</pubmed></ref>

For more info: https://omim.org/entry/303400?search=tbx22&highlight=tbx22
Still get image - Marianne

[[User:Z5039628|Z5039628]] ([[User talk:Z5039628|talk]]) 23:16, 8 September 2016 (AEST)

===Animal models===

The gene brachyury is important in all bilateral organisms (vertebrates- chordates and invertebrates such as mollusca). The brachyury gene is believed to have a conserved role in defining the midline of a bilateral organism <ref><pubmed>15034714</pubmed></ref> and are also fundamental in the establishment of the anterior-posterior axis <ref><pubmed>11880350</pubmed></ref>

It is thought to play a role in the development of organisms in the Phylum Cnidaria, appears to be in defining the blastopore during early development<ref><pubmed>12536320</pubmed></ref>. It is also important during gastrulation where it defines the mesoderm <ref><pubmed>12921737</pubmed></ref> and experiments using tissue culture have demonstrated that the gene brachyury is also important in controlling the velocity of cells as they leave the primitive streak. <ref><pubmed>3327671</pubmed></ref>

Another important role that Brachyury has also been shown to have is to help establish the cervical vertebral blueprint during (human) fetal development. In mammals the number of cervical vertebrae is highly conserved; however a spontaneous vertebral and spinal dysplasia (VSD) mutation in this gene has been associated with the development of six or fewer cervical vertebrae instead of the usual seven. <ref><pubmed>25614605</pubmed></ref>

The T-box gene family have been identified in organisms ranging from hydra to humans and due to extensive research by many investigators , we now know that T-box is important in metazoan development including transcriptional activity, genetic targets, developmental regulatory functions, and associated disease mechanisms (reviewed in Papaioannou, 2001).

====Organisms used in animal models for T-Box====

Animal model studies have been performed in a number of organisms including: ''Drosophila'' (the fruit fly), ''Xenopus'' (a genus of aquatic clawed frog native to sub-Saharan Africa) zebrafish, avians, and mice.
These different animals are used to examine T-box gene regulation in the developmental and disease process (reviewed in Naiche et al., 2005).

T-box genes are involved in the development and patterning of many organ systems and embryonic structures including the heart, limb, eye, central axis, and face. In addition, T-box genes are subject to regulation by or induce the expression of developmentally important signaling molecules, such as retinoic acid (RA), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnts, in different organ systems (reviewed in Naiche et al., 2005). T-box proteins can act as transcriptional activators or repressors with a variety of cofactors to regulate expression of genes involved in cell lineage determination, differentiation, and maturation (reviewed in Tada and Smith, 2001). Overall, T-box genes are integrated into regulatory networks that control patterning, growth, and maturation of many cell types and tissues in the developing embryo.

[[File:Evolution of T box gene Family.jpg]]

This figure is modified from Papaioannou (2014) <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
and shows the subfamilies or classes of genes that have been identified in the different animal groups that are indicated in green in the boxes. What can be demonstrated is that there has been a remarkable conservation of transcription factors between lineages that have been evolving independently since the last common ancestor to metazoans, and many of the T-box gene families have their origin at the base of the tree. However, diversification at the onset of metazoan evolution is evident. T, which is the most ancient T-box gene, is represented in unicellular organisms, as is Tbx7/8, a class not present in Bilateria. Sponges have a diverse set of T-box genes including several that have not been retained in Bilateria, whereas Tbx6 subfamily genes apparently arose in Bilateria. Note that this diagram represents one possible order of divergence of phyla.

====Marsupial forelimb development====

This study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> published in 2012 is the first which describes the T Box gene expression in a marsupial the Tammar wallaby (''Macropus eugenii'') and how these genes are also responsible for limb and digit formation.
Marsupials mammals differ from placental mammals because at birth, neonates are born highly altricial and not very developed. They lack fur, their eyes and ears are not developed and most of the skeleton is still cartilaginous. Another interesting feature that is observed in all marsupial neonates is that the forelimbs are more "developed" than the hindlimb. This is believed to be an adaptation to aid the tiny neonate after birth to climb from the urogenital opening to the pouch or the mammary area, and thus the neonate can attach to the teat where it completes its development- using its forelimbs to help move towards the pouch.

[[Image:Chew et al 2012 Figure 2 Tammar wallaby limb formation.jpg|thumb|alignment|This image is from Chew et al. 2012 and demonstrates the development of tammar fetal limbs at selected stages before birth. (A) day 19, (B) day 20, (C) day 22, (D) day 24 and (E) day 25 (one day before birth). High magnification of the fore- and hindlimb are from samples stored in methanol whilst wholemounts were stored in 70% ethanol. A diagrammatic representation of the fore and hindlimb at day 24 and day 25 is provided showing dorsal and ventral views. All limbs are viewed from the dorsal aspect unless indicated. NB: images not to scale.]]

The more developed forelimb can be observed in the tammar wallaby (''Macropus eugenii'') which clearly demonstrates that the hindlimb development clearly lags behind the forelimb development. Some other marsupials such as the South American didelphid ''Monodelphis domestica'' also known as grey short-tailed opossum, has significantly less difference between forelimb and hindlimb development at birth, however there is still more development in the forelimbs than the hindlimb. This may be because ''Mondelphis domestica'' does not have to climb such a distance to the pouch as the tammar wallaby.
This recent study <ref name=PMID22235805><pubmed>22235805</pubmed></ref> has demonstrated that the key patterning T box genes TBX4, TBX5, PITX1, FGF8, and SHH are also involved in the developing limb buds in the tammar wallaby.
The results show that all the T box genes examined were highly conserved in the tammar wallaby with orthologues from opossum and mouse. TBX4 expression appeared earlier in development in the tammar wallaby than in the mouse, but appeared later in the tammar wallaby than in the opossum. Other results demonstrate that SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The conclusions of this study demonstrate that in kangaroos and wallabies there is a very marked difference in limb size when the forelimb is compared to the hindlimb. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs.

====Evolution of the T-box family: Insights from the living chordate: Amphioxus ====

The '''lancelets''' ( also known as '''amphioxus''' -singular or amphioxi- plural) consist of 32 species of fish-like marine chordates and all are placed in the order Amphioxiformes. They have a distribution in shallow temperate and tropical seas. The amphioxus is a bilaterian cephalochordate and is considered a close relative of vertebrates. They are important in the study of zoology as they provide indications of the evolutionary origin of vertebrates and how vertebrate organisms have evolved. Lancelets have split from vertebrate more than 520 million years ago, however their genomes give us insite on how vertebrates evolved and how vertebrates have employed old genes for new functions.They are regarded as similar to the archetypal vertebrate form.

Phylogenetic analyses indicate that two genome duplications have occurred in the vertebrate linage after cephalochordates diverged so that each amphioxus gene corresponds to two or three vertebrate genes <ref name=PMID25294936><pubmed>25294936</pubmed></ref>
For example the amphioxus gene AmphiTbx1/10 corresponds to two vertebrate T box genes Tbx1 and Tbx10 so these have arose presumably during genome duplications.
AmphiTbx1/10 is expressed in amphioxus during gastrulation in the ventral somites and branchial arches <ref><pubmed>15372236</pubmed></ref> and this corresponds to the mammalian mouse T box gene Tbx1 and the expression of this gene in the ventromedial somites and pharyngeal arches. While the mouse Tbx10 is only expressed in the developing hindbrain <ref><pubmed>12915323</pubmed></ref>
Therefore the function of Tbx1/10 in chordates might originally have been involved in branchial arch patterning and ventral somite specification. These functions are retained by the Tbx1 gene while the Tbx10 has lost its role in pharyngeal arch patterning and instead have gained a novel new role in hindbrain development.

[[Image:Branchiostoma lanceolatum copy.jpg]]

This photo above is a photo of a Lancelet (or Amphioxus) specimen —Subphylum: Cephalochordate. It was collected in coarse sand sediments (600 µm) on the Belgian continental shelf. Total Length: approximately 22 mm. It is a chordate and considered one of the closest living relatives to all vertebrates. [https://en.wikipedia.org/wiki/Lancelet#/media/File:Branchiostoma_lanceolatum.jpg Image from here]]]

===Glossary===
Homologous: existence of shared ancestry between a pair of structures, or genes, in different taxa.

Homologue: something homologous.

Congenital: a condition attributable to events prior to birth, could be related to a baby born with disease.

Glossary doesn't have to be referenced

=== Good places to look ===
PubMed
Biomed Central
OMIM
PNAS
PLOS

DO A QUIZ AT THE BOTTOM OF THE PAGE - maybe collapse it in a tab to make the page look cleaner etc.
to make sure its a comprehensive quiz useful for the student - explain why the correct option is correct and why the other options are wrong

===References===
Check this for doubling up! shows more teamwork and if we've worked together as a team