Welcome to the 2014 Embryology Course!
- Each week the individual assessment questions will be displayed in the practical class pages and also added here.
- Copy the assessment items to your own page and provide your answer.
- Note - Some guest assessments may require completion of a worksheet that will be handed in in class with your student name and ID.
|Individual Lab Assessment|
|Lab 12 - Stem Cell Presentation Assessment||More Info|
Lab 1 Assessment
The article above investigates in detail, the acrosome reaction occurring within spermatozoa and whether other roles are involved with [Ca2+]i. This was facilitated by using FM4-64, Fluo-4 AM (fluorescent dye) and Sytox green (represent whether cell death has occurred) as well as progesterone, Ham's F-10 media, FITC-PSA and ionomycin. The three major steps undertaken in the experiment (method) involve the preparation of the human sperm samples, viewing of spermatozoa samples and the use of the scanning electron microscope. The preparation of the sperm samples involved culturing sperm in a Ham’s F-10 medium in an incubator at room temperature and then placed into medium allowing capacitation to occur. Spermatozoa were then suspended in various reagents including the FM4-64 DYE, incubated then viewed under an inverted microscope. The spermatozoa (given progesterone) were then viewed under a scanning electron microscope.
The study found that applying ionomycin with the FM4-64 dye led to a more accurate image forming on the acrosomal region of spermatozoa due to an increase in fluorescence. With this in mind, distribution changes occurred when AR inducers (ionomycin and progesterone) were applied. The acrosomal reaction is heavily dependent on extracellular Ca2+. The staining revealed the formation of tube-like structures on the spermatozoa head. These structures were unique in that developed while capacitation was underway. Also, it was found that progesterone did not affect spontaneous Ca2+ oscillation and therefore no correlation was found.
The aim of the experiment was to identify and discuss how nuerotensin affects the functioning of sperm. The above article utilised an epididymis removed from mice and placed in a human tubal fluid medium containing various reagents. Cumulus oocyte complexes were then isolated from antral follicles and then applied with IVM. NTR1 and NTR2 were then analysed using immunofluorescence in spermatozoa. The oviduct and uterus dissected from four to five mice was then immersed in Bouin’s fixing solution and then viewed under a fluorescence microscope. RT-PCR was then used to analyse cumulus tissues and female reproductive tissue as well as the monitoring of neurotensin levels in samples.
It was found that tyrosine phosphorylation was not enhanced when neurotensin was present. However, NT did speed up the rate at which the acrosome reaction occurred. Furthermore, when expression levels were monitored in female reproductive tissues and cumulus cells, the ampulla displaced a significantly higher expression level compared to the isthmus or uterus. Lastly, when cumulus oocyte complexes in the presence of FSH or EGF, neurotensin released from cumulus was affected greatly (significant increase).
--Mark Hill These are good summaries of the 2 research articles. (5/5)
Where are your submissions for Lab 2 and 3 assessments?
Lab 2 Assessment
Image A shows that trophoblast epithelium such as syncytiotrphoblast (SCT) and cytotrophoblasts are in close proximity to fetal and maternal circulation. Similarly, Image B containing immunohistostaining of the placenta also represents this. Both blood vessels display a layer of endothelial cells forming the endothelium (innermost lining).
Cvitic S, Longtine MS, Hackl H, Wagner K, Nelson MD, et al. (2013) The Human Placental Sexome Differs between Trophoblast Epithelium and Villous Vessel Endothelium. PLoS ONE 8(10): e79233. doi:10.1371/journal.pone.0079233
© 2013 Cvitic 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.
Lab 3 Assessment
On project page (neural development)
Lab 4 Assessment
The article above looks at whether regulatory T lymphocytes cultured with human cord blood stem cells prevents the occurrence of type 1 diabetes in non-obese mice. Diabetes mellitus type 1 is caused by the immune system attacking and destroying beta cells distributed within the islets of Langerhans. Beta cells are imperative for survival as they responsible for producing the peptide hormone insulin, responsible for lowering blood glucose levels. Therefore, the destruction of these insulin producing beta cells by auto-reactive effector T cells is a major issue. It has been noted that Tregs (Regulatory T cells) display an essential role by having an inhibitory effect on auto-reactive effector T cells. Defects or abnormalities in Tregs accelerates the occurrence of type 1 diabetes.
The findings show that by applying purified autologous CD4(+)CD62L(+)co-cultured with human blood stem cells boots the regeneration of beta cells and an increase in beta-cell mass and hence an increase in insulin production and restoring the framework of pancreatic islets. Furthermore a decrease in islet inflammation, re-establishment of blood cytokine balance (Th1/Th2) and inhibiting leukocyte triggered apoptosis in pancreatic islets. The implications of these findings provide as possible strategy in treating type 1 Diabetes in regenerative medicine.
Ductus Arteriosus - shunts pulmonary artery to aorta above heart
Ductus venosus - shunts left umbilical vein to inferior vena cava near the liver
Foramen ovale - located in atrial septum, allowing blood from right atrium to enter left atrium
Lab 5 Assessment
Alveolar Capillary Dysplasia
Alveolar capillary dysplasia is a developmental condition occurring in newborns involving improper positioning of pulmonary veins and unsuccessful formation alveolar capillaries where there is no contact to the alveolar mucosa as well as increased thickness of the alveolar wall as well as reduced distribution of these capillaries. Associated with this is also pulmonary hypertension leading to respiratory distress and can eventually be fatal for newborns as well as lead to other congenital abnormalities.
It has been strongly noted that a genetic abnormality is major contributing factor to the cause of alveolar capillary dysplasia. This involves mutations such as micro- deletions in a specific gene referred to as the FOX1 gene. The FOX1 gene is important in lung development and its vasculature as well as gastrointestinal tract development. The FOX1 gene produces a transcription factor responsible in regulation activity of other genes by binding to specific regions of DNA. A mutation in the FOX1 gene leads to an inactivated protein leading to lung blood vessel abnormalities. Furthermore deletion in genes along the region of 16q24.1 of chromosome 16 can further lead to the progression of this disease.
--Mark Hill (talk) 11:27, 26 August 2014 (EST) This image should also be on your student page.
Lab 6 Assessment
The article looks at whether the occurrence of beta-cell dysfunction is in relation to the peroxisome proliferator-activated receptor-y coactivator-1a or PGC-1a. PGC-1a, co-regulates the activity of glucocorticoids binding onto the GC receptor. It was found that PGC-1a overexpression affected glucose tolerance and glucose stimulated insulin secretion (GSIS). Furthermore, impairment in B-cell gene expression reduced B-cell mass and insulin composition in the pancreas as well as Langerhans cell islets and islet mitotic growth (hypertrophy). A unique finding demonstrated in the article is that PGC-1a overexpression in the adult stage does not affect glucose tolerance and GSIS but only in the fetal stage. Furthermore, neurod1; which has a regulatory effect on insulin gene expression is reduced by PGC-1a overexpression. This transcription factor when leads to the above exhibited effects on GSIS and pancreatic islet number and growth.
Lab 8 Assessment
There are 3 main stages associated to embryonic development of the ovary which includes the development of the indifferent gonad, differentiation of gonad and differentiation of internal genital organs/ducts. It is also important to note that the differentiation of internal genital organs/ducts stage moves from the late embryo to the fetal stage. A timeline is important here in showing this (shown below).
Week 3/4 --> Whilst gastrulation continues, subsequent migration of primordial germ cells also occurs
Week 4 (24 days) --> Important changes to pronephros primordium and intermediate mesoderm
Week 5 (28 days) --> Mesonephros forms when the pronephros degrades
Week 6 (35 days) --> Ureteric bud (Protrudes from the mesonephric duct), metanephros and genital ridge forms
Week 7 (42 days) --> Cloaca division (urogenital sinus, vesica-urotheral portion), gonadal primordium
Week 8 (49 days) --> Paramesonephric duct (later on develops into fallopian tubes), differentiation of gonads
Week 9 (56 days) --> Mullerian ducts fuse together in females
Week 15 (100 days) --> Primary follicles develop in the ovary
In the development of the indifferent gonad stage, the genital ridge and the kidneys arise from the intermediate mesoderm located between the somites and the lateral plate mesoderm. In week 4 of embryonic development, degradation of the pronephric duct with the mesonephric duct remaining. Within the mesonephric duct, tubules form in the mesenchyme and where gonad development occurs. In the second stage, differentiation of the gonad into the ovary, formation of the support cell structure occurs as well gametogenesis. The granulosa cells and theca cells begins to from as these cells are essential for the maturation of the oocyte in the ovaries. When proliferation occurs later on, they will also be important in steroidgenesis as the steroids generated will have amplificatying effect. Germ cells in females become arrested at the diplotene stage and remain like that (into oocytes).In the last stage of embryonic development, the mesenephros degrade with remnants remaining as epoophoron and paroophoron.
Peer Review Assessment
(Individual assessment section in lab 9 states 5 project reports to be written)
Overall structure of this group structure is decent with use of many articles to support information provided. Furthermore, the structure for this project could be improved on as there are minimal grammatical errors. It is also able to explain concepts regarding the development of the respiratory system in the embryonic stages. If concepts can be conveyed effectively to the reader then this indicates that the project group have been able to demonstrate their understanding on their chosen topic. It is important to identify the advantages and disadvantages and these are;
• Images used are effective in further summarising or explaining content. For example the image under the respiratory zone heading is good in that it shows the histological growth of the tissue as the week’s progress on.
• Heading s provided are short and right to the point.
• The table on lung development stages is very effective as it summarises changes that occur in the weeks following embryonic development.
• Abnormalities section has also been clearly written with in-text citations and a variety of research articles used to explain the conditions.
• First and foremost, most images uploaded onto the group project 1 page do not have any descriptions as well an image name to explain what it is. This makes it difficult for the reader to understand what the image is displaying. Also a file has been deleted (400px) indicating that copyright issues have occurred.
• Consistency across the whole project page is not evident. This can be seen as each section has references being placed under paragraphs and then a full reference list is given in the end. So all references need to be placed in the reference list at the end.
• Historic findings are an important section here that needs work on as there isn’t enough information regarding articles from the 18th century being stated.
The introduction to renal development in the embryonic stages has been written well as it clearly summarises this and conveys to the reader what will be covered below. Furthermore, renal development flows well as the information provided is clear and concise further displaying understanding of the topic. An image could be used that can help summarise the renal development stages of the renal system.
• Introduction written well as it summarises the concepts well.
• Some images have a description such as ‘Glomerula number in Smoke Exposed and Control offspring’.
• Headings are short and to the point.
• Abnormalities section written well with in-text citations and the use of footnotes to good effect.
• Overall structure looks good.
• Some images do not have a description of what the image is showing or an image name. For example, the image in the urethra development section.
• Consistency is not followed to great effect such as some images push the writing to the left side and others to the right side.
• Introduction could be updated indicating that these areas in renal development are covered in detail below as well as for more detailed information to be found in articles that have referenced.
• Some references have been placed under headings in the ureter for example. These references should be put in the end so as to maintain structure and consistency.
• Minimal grammatical errors found.
• Ensure that copyright clearance checked on images as one image has been removed and should be taken note of.
• Tabulated form of the timeline could be used or a good summary image.
• Information should be added to the historic findings heading ( 1700’s and on)
The introduction to the gastrointestinal system development is ok but needs work on as to indicate what will be covered below and any other noteworthy information. However, the introduction does briefly describe the parts of the GIT system as they reader may not be aware of all the structures. Overall, sentence structures need to be improved on as they may lead to confusion. In addition to this certain areas of development have been missed out and need to be included. The strengths and weaknesses are covered below;
• The timeline of GIT development in the embryonic period has been well written and is very concise in conveying what happen in each week.
• Originality is quite important as some images have been drawn up and uploaded. These have been done well and indicate that group project contributors are showing dedication.
• Most Images have an image name and an image description.
• Referencing with in-text citations and no random references in between paragraphs.
• Abnormalities section of the GIT system is quite lacking and so more information on defects as well as image should be provided.
• Certain organ development areas are also lacking ad need to be included with those already mentioned.
• More in-text citing should be used and not only centered in some areas.
• A tabulated form of the timeline would be useful as to allow it to be clearer.
• Some footnotes are placed in the wrong areas and should be within paragraphs and not be separated.
• Headings need to be consistent such as using the same font and size.
Progression of ideas in this group project page is great as concepts are easily understood and conveyed to the reader. Furthermore, genital development for both female and males in the embryonic period are excellent summaries. An introduction would further help introduce the reader to what the page will be discussing and other important information. For improvement, technical problems such as text error and image problem fix-up would make the page more credible.
• The use of the video clearly explains the development of the reproductive system as well written-up images indicate dedication and convey information easily.
• Use of bullet points for current research is easier on the eyes to read and improves structure.
• Headings and short and clear.
• Good amount of references used for the project page.
• Current research finding are good but can still be improved.
• Abnormalities section clearly describes the various defects that can occur in both sexes.
• Table summarises timeline quite well but can be improved.
• Consistency in terms references being placed after paragraphs such as system development. These should be placed in the end.
• More in text citations are needed to show up to date links to current research articles.
• Paragraph and heading errors need to be fixed.
• Some images need to have an image and name and description as this may confuse the reader.
• Historic findings has been stated within the project page should be added as well as current research needs to indicate current research articles and the areas to which they are heading to.
• Grammatical error found and should be fixed.
The project page contains a decent amount of information in the introduction section as it introduces what sections of the integumentary system will be covered below, but does not give a brief description of the system. For example, listing of the organs involved as well as function and their changes in position as development progresses in the embryonic period. The content general has been written well as it is clear and concise and readers that are not in the field of embryology are able to understand ideas presented. The use of tables and well-structured images further complements this and hence shows that there is a good project page structure adopted.
• Good images are used to further show ideas and improve understanding.
• Bullet points used to great effect in summarising information and making it easier to read.
• Tables are used to great effect as the timeline is easy to read and right to the point
• Recent findings are an ‘eye opener’ for information as it summarises the article quite well as well the images displaying the results.
• In-text citations are used quite well as they are distributed.
• Consistency needs to be ensured regarding references as some references are written after paragraphs. Hence, they should be put in the reference list at the end of the project page.
• Most images have an image name but some lack an image description.
• Structural error such as some bullet points needs to be fixed up.
• Historic finding research article should be mentioned as they are highly relevant to the current knowledge possessed today.
• References from the embryology website should be used scarcely.
Lab 10 Assessment
The alarmin HMGB-1 influences healing outcomes in fetal skin wounds
In embryonic development, the is highly impwound healing is highly important for normal functioning and thusis highly regulated by genes specifically the action of high-mobility group box-1 (HMGB-1) on fetal tissue. Furthermore, the degree of healing with respect to whether scar tissue forms, varies to a large extent in different stages of embryonic development. The study investigates this comparing the healing of cutaneous tissue in embryonic development day 15 (E15) and the healing of of cutaneous tissue in embryonic development day 18 (E18).
Cutaneous wound healing is not understood in great deal as there are multiple process that are simultaneously occurring, however it has recently been found that alarmins (HMGB-1); endogenous signals that requires no pathogens to stimulate inflammation at certain sites where cell constituents are released into the surrounding extracellular space. Furthermore it has been found that activated immune cells secrete alarmins which can further lead to a cascade-like reaction resulting in further recruitment and activation of immune cells hence leading to inflammation.
The method utilised by the study involved the use of a mouse model of wound healing. 2mm dorsal skin wounds were created in fetuses (FVB/Ntac mice, in uetero) of both day 15 and day 18 of embryonic development through isoflurane anaesthesia. The wounds formed were distinguished through subcutaneous injection of India ink diluted to 10% in PBS solution. The samples were then placed in TBS tissue-freeing media whilst fixed in 10% buffered formalin overnight. Furthermore, immunohistochemistry analysis of HMGB-1 expression and collagen formation with the assessment being based on cell type response.
Further assessment of HGMB-1 expression was required, so 10-μm sections were air dried and placed in acetone. These sections were treated with hydrogen peroxide, methanol, PBS and goat serum with further incubation in anti-HMGB-1 antibody serum ( control slides also formed) . Various solutions and complexes were used to assess expression/formation such as the identification of the immune cell types (mast cells, neutrophils and myofibroblasts) as well as staining kits. Lastly collagen assessment was quantified via masson's trichrome stain as well as picrosiruius red stain (samples compared to each other).
The findings were particularly interesting following the use of the above method. When HMGB-1 immuno-histological staining was conducted, Staining in sample wound E18 was of a greater intensity to sample E15 wound. This indicated greater expression of HMGB-1. Furthermore when staining patterns were investigated, it was found that the staining pattern of HMGB-1 in E18 after 24 hours display loss of staining in keratinocytes around the wound whilst the staining pattern of the E15 wound resembled that of unwounded skin and minimal staining loss of keratinocytes. At 72 hours, the HGMB-1 staining pattern in E15 wounds was similar to that of normal skin relaying that expression and localisation of this alarmin had returned to normal. This suggest that in scar forming E18 wounds, a larger number of keratinocytes leads to increased synthesis of HGMB-1 followed by a long period of time.
The study then investigated whether scar formation was altered via HGMB-1 expression. Through masson's trichome and picrosirius red, minimal release of HMGB-1 indicated no inflammation and scar formation. When recombinant HMGB-1 was added in increasing amounts, a dose-dependant scar size increase was exhibited as well as affecting the the quality of scar formation. Hence, increased expression of HMGB-1 by keratincytes leads to the formation of larger scars, hair follicle loss and also quality of collagen formation being impaired.
Lastly, fibroblast formation and angiogenesis was analysed in relation to HMGB-1 expression. It was found that an increased number of fibroblasts was exhibied within HMGB-1 treated wounds however myofibroblast detection was not seen. Increased angiogenesis was also seen due to HMGB-1 expression where an increase in blood vessels occured in the wound sample in comparison to the controls. When scar tissue formation occurs, sensory detection (discriminative touch, pressure etc) in skin could be affected and thus it is important that wound recovery leads to minimal scar formation.