2016 Group Project 4

2016-10-25T11:36:09Z

Z5019880: /* Drosophila melanogaster */

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= Hedgehog signalling pathway =
==Introduction==
[[File:Hedgehog.jpg|thumb|300px|An image of a hedgehog, the animal of which the Hh proteins are named after.]]
The Hedgehog (Hh) signalling pathway is an important part of the early embryo with regards to the patterning and development of the nervous system, limbs and the cranio-facial region in vertebrates and the polarization of the segments in the ''Drosophila'' embryo. It was first identified in 1980 by Christiane Nüsslein-Volhard and Eric Wieschaus, alongside a group of other genes regulating segment polarity in ''Drosophila'' embryonic development<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. The pathway itself consists of Hh proteins, where there are three identified homologs in vertebrates, being sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (DHH) <ref name="PMID7916661"><pubmed>7916661</pubmed></ref>. All homologs are expressed at varying levels within different tissues in the body, and also act as a form of redundancy to an extent between one another.

The signaling pathway is highly conserved between various species<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. and acts via a G protein coupled receptor like receptor, which uninhibited via the action of the Hh proteins. The pathway overall acts via a balance of dephosphorylation<ref name="PMID10966113"><pubmed>10966113</pubmed></ref> and degradation of proteins<ref name="PMID15102702"><pubmed>15102702</pubmed></ref> in order to regulate transcription factor activity to express various genes to carry out its action. The Hh pathway is also highly implicated when it comes to abnormalities in development, as it is implicated in diseases such as holoprosencephaly and cleft lip and palate. Overall Hh signaling pathways play a large role in embryological development, which will be discussed in detail on this page.

== History ==
{| class="pretty table"
| '''1980'''
|Christiane Nüsslein-Volhard and Eric Wieschaus first identified a group of genes including those related to the Hedgehog signalling pathway and linked them to the segmentation and planning of the embryo in ''Drosophila melanogaster'' by introducing mutagenic substances to the developing embryo<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>.
|-
|'''1993'''
|Multiple researchers including Andrew P. McMahon and Clifford Tabin discovered three equivalent homologs in vertebrates of the ''Drosophila melanogaster'' hedgehog gene, known as sonic hedgehog (Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh), by looking at DNA sequences similar to that of the gene in the fruit fly<ref><pubmed>7916661</pubmed></ref>.
|-
|'''1993'''
|Clifford Tabin and his lab identified the role of Shh in localising the limb bud, where they identified protein to be expressed within a region of the limb bud known as the zone of polarising activity (ZPA), showing that SHH is sufficient to induce the production of a ZPA, and thus limb bud formation in chick embryos<ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
|-
|'''1995'''
|Sonic hedgehog secreted by the notochord was identified to induce ventral cell types in the neural tube during embryonic development, most notably the floor plate cells and the motor neurons as shown in chick embryos. Such differentiation between the two cell types is thought to be a matter of sonic hedgehog concentrations experienced by the neural plate cells<ref name="PMID7736596"><pubmed>7736596</pubmed></ref>.
|-
|'''1995'''
|A shared Nobel prize in physiology or medicine was awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their researching regarding the identification of the developmental genes relating to the formation and patterning of the early embryo via genes.
|-
|'''1996'''
|Studies showed that human homologs of the Patched gene, a component of the hedgehog pathway, is a key gene that is mutated in Gorlin Syndrome, which characterised by a predisposition for basal cell carcinomas, the most common cancers in humans and developmental abnormalities<ref name="PMID8658145"><pubmed>8658145 </pubmed></ref>.
|-
|'''1999'''
|Researchers implicated Indian hedgehog as a key signalling molecule for the maturation and differentiation of prehypertrophic chondrocytes, where Indian hedgehog null mice were shown to have a lack of mature chondrocytes and no development of osteoblast cells in endochondrial bone. This was thought to be due to Indian hedgehog having a feedback signal controlling parathyroid hormone related protein, which regulates endochondrial bone development<ref name="PMID10465785"><pubmed>10465785</pubmed></ref>.
|-
|'''2003'''
|Sonic hedgehog was identified to regulate the proliferation of adult neural stem cells as shown in the hippocampus of rats and in vitro for neural progenitor cells isolated from the hippocampus<ref name="PMID12469128"><pubmed>12469128</pubmed></ref>.
|-
|'''2003'''
|Studies on Shh function found that it can act as a chemoattractant in the developing embryo spinal cord, to help guide commissural axons to the midline of the floor plate<ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
|-
|'''2009'''
|Mouse models showed that reduction in hedgehog signalling allows for transient improved response to chemotherapy with regards to mouse models of human pancreatic ductal adenocarcinoma, a cancer with one of the highest mortality rates in humans<ref name="PMID19460966"><pubmed>19460966</pubmed></ref>.
|}

This time line regarding the discoveries in the hedgehog signalling pathway is by no means exhaustive, but a selection of relatively large discoveries that had a significant impact in the field on the basis of citations.

== Origin of name ==

[[File:Bands of denticles in normal and Hh mutant Drosophila embryo.jpeg|thumb|250px|Differences in patterning of the denticle bands in normal and mutated hedgehog gene variant ''Drosophilia'' embryo]]

The name hedgehog came about when Christiane Nüsslein-Volhard and Eric Wieschaus first identified the group of genes controlling polarization of the segments in the ''Drosophila'' early embryo. They noticed that when they mutated the Hh gene, making it non-functional, that the bands of denticles formed during the early ''Drosophila'' embryo formation became more diffuse as opposed to being distinct normally. The denticles would all clump together forming a single patch on the surface of the embryo, as opposed to normally outlining the distinct segments of the embryo as bands<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. This gave the embryo spiny and prickly look similar to that of the back of a hedgehog.

From there on in the gene was called the hedgehog gene where homologs in vertebrates discovered along the line kept to this naming structure giving various species of hedgehog names to the genes such as Indian hedgehog, and desert hedgehog, with the exception being sonic hedgehog, which is named after a fictional character in the video game “Sonic the Hedgehog”.

== Function ==
===Overview===
{| class="pretty table"
|'''Hedgehog protein homolog'''
|'''Function'''
|-
| Sonic hedgehog [http://www.omim.org/entry/600725 OMIM entry] ||
*Expressed in zone of polarizing activity of the limb bud where it plays a role in helping to pattern the anterior and posterior aspect of the limbs <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
*Based on time and concentration of SHH due to diffusion from the point of secretion in the limb bud, SHH also plays a role in determining digit identity <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>.
*Acts as a chemoattractant to guide commissural axons in the floor plate of the neural tube towards the midline <ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
*Patterns the developing cusps of the teeth and their relative positions in the jaw, and is also essential for the growth of the teeth <ref name="PMID11044393"><pubmed>11044393</pubmed></ref>.
*SHH released from the notochord acts to induce formation of the floor plate, motor neurons, and dopaminergic neurons, as well as signalling the induction of the ventral cell types in the neural tube <ref name="PMID8790332"><pubmed>8790332</pubmed></ref>.
|-
| Desert hedgehog [http://www.omim.org/entry/605423 OMIM entry]||
*Plays a possible role in regulating spermatogenesis as due to the highly localised expression in the testes by pre-Sertoli cells, and the fact that mice with no DHH present are infertile, with no mature spermatozoa <ref name="PMID8805249"><pubmed>8805249</pubmed></ref>.
*DHH derived from Schwann cells signal development of peripheral nerves, by inducing the production of a connective tissue sheath around them <ref name="PMID10482238"><pubmed>10482238</pubmed></ref>.
|-
| Indian hedgehog [http://www.omim.org/entry/600726 OMIM entry]||
*Aid in the signalling of proliferating chondrocytes to undergo hypertrophic differentiation during endochondral ossification<ref name="PMID8662546"><pubmed> 8662546</pubmed></ref>.
*Plays a key role in yolk sac angiogenesis, as seen in studies where an absence of IHH leads to death of the embryo due to poor yolk sac vasculature <ref name="PMID11807029"><pubmed>11807029</pubmed></ref>.
|}

===Limb development===

====Research background====

The hedgehog signalling pathway plays a significant role with regards to the embryonic development of the limbs in vertebrates, specifically SHH. For a while the mechanism of which the limb was patterned on the anterior to posterior axis was quite unknown. Early studies had shown that grafting posterior wing bud cells to the anterior region of the wing bud in a chick embryo, an equal amount of extra digits was produced to that of which was normally produced, and in a mirror image pattern<ref name="PMID4390734"><pubmed>4390734</pubmed></ref>. This showed that the pattern of which the digits were produced were polarized with respect to that of the graft from the limb bud, which has become to be described as the zone of polarizing activity (ZPA). Further studies mapped the ZPA, by showing the area of the wing bud on the chick with the highest polarizing activity to be the posterior margin of the wing bud<ref name="PMID4719010"><pubmed>4719010</pubmed></ref>. Furthermore, studies later on found that the amount or concentration of ZPA cells grafted played a role in the amount of digits induced and how polarized they were<ref name="PMID7453825"><pubmed>7453825</pubmed></ref>. This ultimately gave rise to studies regarding a potential morphogen, which could potentially provide this concentration dependent activity of polarization when it comes to the limb bud. Studies to find this morphogen led to the discover of the ability for retinoic acid to mimic the polarizing action of the ZPA when applied locally to the limb bud<ref name="PMID7070499"><pubmed>7070499</pubmed></ref>. This was found to be due to the induction of a second morphogen known as SHH, where when cells that express SHH are grafted onto the limb bud, polarized digit development is induced<ref name="PMID7720566"><pubmed>7720566</pubmed></ref>.

====Polarizing activity of sonic hedgehog====
[[File: Mechanims of patterning the digits on the posterior to anterior axis of the limb bud by sonic hedgehog.jpg|thumb|450px|Regions of the limb bud and the digits they form based on patterning of the posterior to anterior axis of the limb bud by mechanisms mediated by sonic hedgehog.]]
More recent studies have sought to elucidate how SHH mediates such a polarizing effect in terms of digit identification and its underlying mechanism. It has been suggested that such an effect is heavily concentration dependent similar to that of when transplanting the chick wing bud ZPA cells, acting in a dose dependent matter, due to the ability of SHH to modulate signaling at long ranges via diffusion<ref name="PMID11389830"><pubmed>11389830</pubmed></ref>. This implies that a gradient of varying concentrations of SHH diffused through the mesenchyme of the limb bud leads to the identification of posterior and anterior digits.

Current research has also suggested that not only is the concentration gradient of SHH important for the patterning of the digits, but also the temporal gradient, being the length of time of exposure of SHH to the mesenchyme of the limb bud. Studies have shown in mice that of the 5 digits, polarization from the posterior end, being digit 5, to the anterior end being digit 1 are due to different mechanisms. Digits 5 and 4 have been shown to be comprised of the SHH expressing cells and thus both experience maximal levels of SHH, and thus cannot be distinguished on the basis of a concentration gradient. As a result, temporal gradients are employed, where the expression of SHH in both regions that become the digits vary, where studies have shown that SHH expression in the primordium of digit 5 is maintained longer than that in digit 4 gradient <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>. When it comes to digit 3 and 2, there is differentiation based on the concentration gradient of SHH from the ZPA, where due to the ZPA being further away to digit 2 than 3, the concentration of SHH will be higher at 3<ref name="PMID15315763"><pubmed>15315763</pubmed></ref> <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>. In terms of digit 1, the levels of SHH are so low at this point that it is said to be SHH independent, and actually reliant on the absence of SHH for normal differentiation. With regards to how SHH actually mediates its action, it is thought to be by a balance between the transcription factor Gli3 and SHH expression, which activates Gli1, Gli2 and Gli3 repressor, effectively counteracting Gli3. Gli3 is considered to promote the inhibition of digit formation and identification, while the downstream targets of SHH are thought to promote it<ref name="PMID17400206"><pubmed>17400206</pubmed></ref>.

Other research has also shown the possibility of SHH signaling as a way to prime the posterior mesenchyme and induce the production of bone morphogenetic protein 2 (bmp2), which is thought to be the morphogen creating the chemical gradient for digit identification<ref name="PMID10704381"><pubmed>10704381</pubmed></ref>. Overall the mechanism at which SHH work to pattern the posterior anterior axis of the limb bud is still relatively unknown, and further research is being conducted to elucidate how the underlying mechanisms such as the downstream targets of SHH affect digit formation.

=== Neural development ===

=== Organogenesis ===
The Hedgehog Signalling pathway is a pathway sending information to embryonic cells which plays an especially important role in the regulation of organogenesis. These processes include the organization of the brain (craniofacial) and the growth of appendages with further studies implicating the Hedgehog Signalling pathway in the development of the small intestine, lungs and the pancreas.

== Mechanism ==

=== Processing of precursor ===
 [[File:Processing of Hh precurosr.jpg|thumb|250px|Summary of the steps in the processing of the Hh protein precursor into a functional signalling molecule]]
Hedgehog (Hh) proteins are produced as precursors, which must be processed by the cells producing it before being able to perform its signalling function. The process by which the Hh protein is processed begins with its signal sequence at its N terminus directing the translocation of the precursor into the endoplasmic reticulum, where it is removed via signal peptidase<ref><pubmed>1394430</pubmed></ref>. This process allows the C terminus of the Hh protein to catalyze the cleavage and addition of cholesterol on itself to form a C terminal processing domain and an N terminal processing domain associated with a cholesterol group on its C terminus<ref><pubmed>21357747</pubmed></ref>. The portion associated with the cholesterol will go on to form the signalling molecule, while the C terminal processing domain has no known signalling function<ref><pubmed>8824192</pubmed></ref>.

At this point the Hh protein associated with the cholesterol is able to perform its signalling action, but further modification is still required to ensure efficient signalling. This occurs when the cholesterol group attached to the Hh protein associates with the plasma membrane of the cell, which allows for the addition of a palmitic acid moiety to the N terminal of the protein by an acyl-transferase known as skinny hedgehog<ref><pubmed>11486055</pubmed></ref>. Studies have shown that such an addition allows for an increase in potency in signalling of Hh proteins of 30-fold over its form without palmitic acid added<ref><pubmed>9593755</pubmed></ref>. From this point the Hh protein is now fully active and can either remain associated to the plasma membrane of the cell for autocrine action or be secreted for paracrine action.

=== Mechanism of signalling ===

==== ''Drosophila melanogaster'' ====
 [[File:Hh Signalling Pathway.jpg|thumb|600px|Steps regarding the processing of the hh protein precursor into the fully functional signalling molecule.]]

The Hh signaling pathway has been well studied in ''Drosophila melanogaster'', and has been shown the be conserved to an extent across it and mammals making the species a suitable model for Hh signalling in humans<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. In ''Drosophila melanogaster'', the Hh pathway begins when the Hh proteins bind to the extracellular domain of the transmembrane protein known as Patched (PTC) to inactivate it. This inactivation occurs by Hh trapping PTC in an inactive conformational state<ref><pubmed>27647915</pubmed></ref>. After binding of Hh occurs to PTC and it is inactivated, the receptor and Hh protein is thought to be endocytosed by the cell where they undergo lysosomal degradation in order to limit Hh concentration, thus limiting its spread to other cells and PTC activity<ref name="PMID15102702"><pubmed>15102702</pubmed></ref>. In the absence of Hh, PTC acts to suppress the expression of the Smoothened (SMO), a G-protein coupled receptor like receptor, and thus its signaling. How PTC achieves this inhibition of SMO is currently unclear. Studies have suggested that due to the highly phosphorylated nature of SMO when active, that PTC acts to dephosphorylate SMO in order to repress its signaling. It is thus that binding of Hh to PTC reduces PTCs ability to promote dephosphorylation of SMO, leading to its increased activity and expression on the cell surface <ref name="PMID10966113"><pubmed>10966113</pubmed></ref>. It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) <ref><pubmed>15616566 </pubmed></ref>.

This overall increased phosphorylation of SMO due to a repression of PTC activity by Hh leads to an accumulation of SMO on the cell surface, which collectively allows for SMO to exert its activity. Most importantly phosphorylation also disrupts intramolecular electrostatic interactions between SMO molecules which switches the molecule into its active conformational state <ref><pubmed>17960137 </pubmed></ref>. Generally, SMO has been shown to have very little signal transducing ability, and thus this accumulation allows it to jointly transduce as substantial signal. This occurs by the C terminus of SMO on the intracellular domain interacting with the kinesin like protein Costal-2 (Cos2), which is thought to bind to microtubules in order to acts as a scaffolding protein <ref><pubmed>18691888 </pubmed></ref>. Then the kinase known as Fused (Fu) binds to Cos2, which phosphorylates Suppressor of Fused (SUFU) to inhibit it. Without this inhibition SUFU goes onto prevent the translocation of the transcriptional factor Cubitus interruptus (Ci) into the nucleus by leading to its phosphorylation, and subsequently its partial cleavage via the proteosome. The partial cleavage of Ci leaves a lower molecular weight protein known as Ci repressor (CiR) or Ci75, which translocates into the nucleus and acts to repress the target genes of the Hh signalling pathway via Ci<ref><pubmed>9215627</pubmed></ref>. Thus overall inhibition of SUFU increases the amount of Ci entering into the nucleus of the cell, and activating transcription of target genes of the Hh pathway<ref><pubmed>10952898 </pubmed></ref>.

At this point Ci is able to activate the transcription of various genes, which include importantly the ''ptc'' gene, which encodes the PTC receptor. It is thus as a result of this, that PTC expression will be increased in response to Hh pathway induction, which negatively feeds back to reduce Ci signaling induction, in order to maintain homeostasis and regulate the intensity and duration of the signaling from Hh<ref><pubmed>9053330</pubmed></ref>. Furthermore the ''wg'' gene, encoding the wingless protein is also activated by Ci, which leads to activation of the Wnt pathway via the Frizzled receptor in adjacent cells expressing the gene ''engrailed'' <ref><pubmed>9502727</pubmed></ref> <ref><pubmed>10457026</pubmed></ref>. This overall stabalizes the boundaries between the segments of the developing ''Drosophila melanogaster'' signalled by the gene ''engrailed''<ref><pubmed>3282172</pubmed></ref>.
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=w1xXD9kss2w</html5media>
 
An overview of the hedgehog signalling pathway in ''Drosophila''<ref>YouTube. (2016). The Hedgehog signalling pathway in Drosophila online Available at: https://www.youtube.com/watch?v=w1xXD9kss2w</ref>.

==== Mammals ====

Although the Hh signaling pathway has been conserved across species to an extent <ref name="PMID8595881"><pubmed>8595881</pubmed></ref>, key differences exist when observing the pathway within mammalian tissue in contrast to that which has been studied in ''Drosophila melanogaster''. In mammalian cells and all other vertebrates, Hh signalling is dependent on an organelle known as the primary cilia, which are projections outwards from the cells surface. The importance of such an organelle in the signalling pathway comes as a result of PTC1, one of the two PTC receptor homologs in mammals, that binds to the Hh homolog Sonic Hh (Shh), exists within the cilia<ref><pubmed>17641202</pubmed></ref>. The mechanism of action of SMO inhibition and activation is poorly understood currently, but evidence has suggested that PTC1 acts to inhibit SMO when unbound to Shh by acting as a pump to remove oxysterols from the cilia into the extracellular space. <ref><pubmed>PMC1462959</pubmed></ref>. These oxysterols are normally thought to bind and accumulate around the SMO receptors, which in turn prevents internalization and deactivation of SMO, leading to it accumulating on the apical primary cilia<ref><pubmed>16136078</pubmed></ref>. Other studies have also suggested that Shh has a role in increasing phosphorylation of SMO, which is required for it to accumulate as well on the cilia<ref><pubmed>21695114</pubmed></ref>. Overall the net action of Shh initially is believed to cause accumulation of SMO at the cilia by inhibiting PTCH1 and phosphorylation of SMO.

How SMO acts next is relatively unknown but, it has been shown to promote the disassociation between SUFU and Gli3 transcription factor, which allows Gli to be transported to the nucleus to activate effector genes of the pathway. Gli is normally bound in the cytoplasm to SUFU, where SUFU promotes the partial degradation of Gli into its repressor form, similar to that in the fruit fly model<ref><pubmed>20360384</pubmed></ref>. Alongside this, Kif7, a Cos2 homolog, has been shown to migrate to the apex of the cilium in response to SMO accumulation at the cilium, where it has been postulated to also promote disassociation of SUFU and Gli<ref><pubmed>19592253</pubmed></ref>. On the other hand, Kif7 is also thought to play an inhibitory role for the signaling pathway as when it is localized at the base of the cilia in the absence of Hh signaling and traffics Gli factors away from the cilia<ref><pubmed>19666503</pubmed></ref>. This all leads to an accumulation of active Gli transcription factors which migrate to the nucleus in order either inhibit or activate gene transcription in response to Hh signaling.

== Animal models ==

=== ''Drosophila melanogaster'' ===
 [[File:Hedgehog signalling on plane polarity of denticles in Drosophila.png|thumb|400px|Image of denticles on the ''drosophila'' embryo used as a model to research plane polarization of cells by looking at the orientation of the denticles on the ''drosophila'' embryo in response to many signalling molecules including hedgehog.]]

Hedgehog (Hh) protein signal was initially discovered through experimentation on the fruit fly, "Drosophila melanogaster". It is through this model that we are able to discover not only the functional components of this pathway but also understand its role in embryonic development. Through application of this information on the human biological system we are able to find the cause and thus potential treatments of defects and diseases caused by interruption or mutation of the Hedgehog signalling pathway.

Upon further research on Drosophila it was found that Hh may play a role in germ cell proliferation and in particular, may control the proliferation and activity of somatic cells found within the germarium, which is the most anterior structure within the Drosophila ovary. <ref>Ovaries (Drosophila) definition. (2016). Groups.molbiosci.northwestern.edu. Retrieved 10 September 2016, from http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-D/Drosophila_Ovaries.html</ref> Thus, the Hh signalling pathway is vital in egg chamber formation and its consequent envelopment, budding and polarisation. It has also been observed that somatic cell proliferation is dependent on this pathway and thus, the number of pre-follicle cells is subject to the effect of the Hh signalling pathway. <ref><pubmed>8620839</pubmed></ref> Further studies have also indicated the role played by this pathway in activating the Epidermal growth factor receptor (EGFR) signalling pathway as seen in the induction of EGFR by Hh in Drosophila head development. <ref><pubmed>10331974</pubmed></ref> In addition, research on Drosophila has indicated the direct effect of Hh signalling on tracheal branch patterning. <ref><pubmed>11290298</pubmed></ref>

Despite ongoing research on "Drosophila melanogaster" to uncover the workings of the Hedgehog signalling pathway, further research is needed to confirm and further current findings. In addition, it is evident that it is through the study of these fruit flies that we are able to gain a basis of understanding of the causes of certain human diseases and thus propel research into treatments for sufferers.

===Blockage of Shh Signalling in Forebrain Neuroectoderm of Chick Embryos===

The Hedgehog signalling pathway plays a significant role in embryonic development, particularly of the forebrain. Due to its role in development of craniofacial features by contributing to the epithelia of the frontonasal, maxillary, and pharyngeal ectoderm, a disruption in this pathway can result in a variety of birth deformities including holoprosencephaly (HPE), where the prosencephalon (forebrain) fails to divide into 2 separate hemispheres, telencephalon and diencephalon <ref>Holoprosencephaly - NORD (National Organization for Rare Disorders). (2016). NORD (National Organization for Rare Disorders). Retrieved 10 September 2016, from http://rarediseases.org/rare-diseases/holoprosencephaly/</ref>, as well as cleft lip and palate. The function of Sonic Hedgehog (Shh) and its signalling pathway on the formation of forebrain neuroectoderm was studied in chick embryos.

It was found that the disruption of Shh signalling in the neural tube of chick embryos resulted in the lack of division of the forebrain into the diencephalon and telencephalon. This is in fact, as stated earlier, the fundamental cause of the rare condition, holoprosencephaly. It was discovered that the Shh signalling pathway in the diencephalon was responsible for gene expression in the telencephalon.

In addition, through experimentation on chick embryos it was found that through Shh signalling the development of the forebrain regulates and controls facial morphogenesis, particularly of the upper and middle face. Therefore, interference with Shh signalling in the forebrain prevents this intrinsic communication, thus hindering Shh expression in facial ectoderm. This results in brain malformation accompanied by facial disfiguration as seen in patients suffering from HPE. Other malformations caused by blockage of Shh signalling in craniofacial development include, hypotelorism (decreased space between the orbits), growth restriction as well as cleft lip and palate as mentioned previously.

A commonality discovered among all forms of Shh signalling disruption was two facial defects, “the loss of mediolateral expansion of the face and absence of proximodistal outgrowth of the frontonasal prominence”<ref><pubmed>15979605</pubmed></ref> (Ralph et al., 2005). Through reference to studies performed on chick and mice embryos, it was found that Shh signalling is particularly vital in development of maxillary and frontonasal components of the cranium.

This breakthrough study on the inhibition of Shh signalling in chick embryos has significantly filled the gap in our understanding of the Sonic Hedgehog signalling pathway. It is evident that this pathway is crucial in the development of the forebrain and in turn regulates and controls the development of the facial skeleton. This is further proven through the observation that in the event of Shh signalling inhibition there is a development of craniofacial malformations.

=== Shh knockout mice ===

Extensive research on Shh knockout mice allowed discovery of the roles of Shh in embryonic development and patterning of the limb buds and sclerotomes, and maintenance of the notochord. <ref><pubmed>8837770</pubmed></ref> These mice expressed defects in the cephalic neural tube with the fusion of telencephalic and optic vesicles. Through this research it is evident that Shh is partially responsible for the subdivision of the eye field through forebrain optic stalk development, along with formation of the ventral midline. It is these forebrain abnormalities established in the absence of Shh that results in congenital malformations of holoprosencephaly, development of a single nasal chamber and other facial defects in humans.

The absence of the vertebral column including, the intervertebral discs and vertebrae along with the medial regions of the ribs were observed within Shh knockout mice. This lack of sclerotome derivatives indicates the role of Shh in maintenance or expansion of sclerotome cell population (Chiang et al., 1996). In addition, though the role of Shh in patterning the anterior-posterior limb axis is evident through anterior limb bud cell death in the absence of Shh, it is also responsible for the development of proximal-distal limb segments, particularly in the patterning of structures at the level of or distal to the latent elbow and knee joints. <ref><pubmed>11476582</pubmed></ref> This was perceived after observing the incomplete formation of distal limb structures with abnormal anterior-posterior axis formation (Chiang et al., 2001). Thus, Shh knockout mice provide great insight into the functions of Shh in embryonic development and the abnormalities formed in the absence of this signalling protein.

== Clinical Significance ==

===Gorlin Syndrome===
[[File:1 week year old newborn girl with cleft lip and palate.png|thumb|An image of a 1 week year old newborn girl with cleft lip and palate]]
Gorlin syndrome, also known as nevoid basal cell carcinoma syndrome, is a heritable disease resulting from heterozygousity in Hh receptor, Patched (PTC). The PTC gene encodes a protein which binds sonic hedgehog (Shh) and it is this binding which inhibits the Hh signalling pathway. However mutation of PTC leads to activation of the pathway <ref><pubmed>9620294</pubmed></ref>. The syndrome is characteristic of skin cancer basal cell carcinoma and cerebellum cancer medulloblastoma. 1-2% of medulloblastomas and 0.5% of basal cell carcinomas are attributable to the disease. Patients present with craniofacial and brain abnormalities such as cleft palate, strabismus, macrocephaly, abnormal development of the corpus callosum and frontal bossing, with an overall overgrown appearance. Skeletal defects such as of the shoulder, ribs and vertebrae are often seen, as well immobile thumbs and polydactyly, which is a deformity of the hand or feet in which they have one or more extra fingers or toes. Apart from physical anomalies, patients have an increased risk of tumours throughout the body including cardiac, ovarian fibromas, ovarian dermoid cysts, meningiomas, and fibrosarcomas. Additionally with time, patients eventually undergo intracranial calcification and dyskeratotic pitting of the hands and feet whereby skin cells prematurely convert to keratin <ref><pubmed>8681379</pubmed></ref>.

===Holoprosencephaly===
 [[File:Semilobar holoprosencephaly.jpg|thumb|240px|MRI of fore brain depicting semilobar holoprosencephaly]]
Holoprosencephaly (HPE) is a congenital disease caused by incomplete division of the prosencephalon (embryonic forebrain) into separate lobes of the cerebral hemispheres. The Shh gene has been identified as a HPE-causing gene however recent evidence has also investigated into the PTC gene, which acts to repress Shh signalling. A gain in repressive action of PTC is seen, leading to decreased Shh signalling, and thus HPE <ref><pubmed>11941477</pubmed></ref>. Clinical expression is variable between patients and is dependent upon the 3 forms of increasing severity: lobar, semi-lobar and alobar HPE, where patients can present with right and left ventricles however with a continuous frontal cortex in lobar HPE, to carrying a single cerebral ventricle in alobar HPE. Along with forebrain abnormalities, facial anomalies are also seen including midline cleft lip and/or flat nose, ocular hypotelorism characteristic of a small distance between the eyes. In severe rare cases of cyclopia, a non-functioning nose may be observed in the form of proboscis and the patient may present with a single eye at the root of the nose <ref><pubmed>20583177</pubmed></ref>. Furthermore, patients with HPE often develop a large number of medical issues including epilepsy, mental retardation, thyroid and adrenal hypoplasia due to lack of hypothalamus or pituitary gland development, and diabetes insipidus.

===Medulloblastoma===
Medulloblastoma is a cancerous tumour of the cerebellum where the cells of origin are the granular cell precursors found in the neonatal cerebellum, explaining its high prevalence in children <ref><pubmed>19171780</pubmed></ref>. Commonly, medulloblastomas are found deep in the cerebellum along the midline, however with desmoplastic medulloblastomas, they are found more laterally and superficial. As seen previously with Gorlin syndrome and holoprosencephaly, PTC is also associated with the development of medulloblastoma, and misregulation of Hh-PTC signalling in cells of the external germinal layer, the layer found on the surface of the cerebellum, is thought to give rise to the tumour. In experimental studies, it has been discovered that PTC is highly transcribed in medulloblastomas and in approximately 30% of PTC heterozygous mice the tumour develops <ref><pubmed>9205058</pubmed></ref> <ref><pubmed>9262482</pubmed></ref>. In patients with medulloblastoma, often children, the most common symptoms seen are vomiting, ataxia, psychomotor regression, drowsiness and anorexia. With age, patients are seen to get more frequent headaches, and show psychological symptoms such as behavioural problems, poor performance in school, and anxiety. In patients exhibiting life-threatening symptoms due to increased intracranial hypertension, bradycardia, convulsions and coma are often seen <ref><pubmed>21537925</pubmed></ref>.

Watch below for a breakdown on Hedgehog's role in Tumour Development!
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=gFWO3I-oqsE</html5media>
 
An overview of the Hedgehog Signalling Pathway in Tumour Development<ref>YouTube. (2016). The Hedgehog Signaling in the Tumor Microenvironment - Research Hedgehog online Available at: https://www.youtube.com/watch?v=gFWO3I-oqsE</ref>.

== Current research ==

==Quiz==

<quiz display=simple>

{1. Which of the following are the reasons that are thought to allow for the patterning of digits in vertebrates.
|type="[]"}
+  Concentration gradient of sonic hedgehog.
+  Temporal gradient of sonic hedgehog.
-  The varying homologs of the hedgehog proteins .
-  The length of the hedgehog molecule.
+  No sonic hedgehog signalling.
-  The type of cholesterol attached to the hedgehog molecule.
|| For differentiation of the individual digits in mice to occur it is thought that each digit requires a different signalling mechanism. Digits 5 and 4 which are most posterior, are derived from the region around the zone of polarising area which contain Shh secreting cells, meaning these regions will experience a saturation of Shh signalling and cannot be differentiated by Shh concentration. As a result it is thought that these digits are identfied based on the temporal gradient, meaning that the length of which Shh is expressed in these regions differentiates the digits from one another with digit 5 expressing Shh for longer. Digits 3 and 2 are further away but still receive Shh signalling due to the diffusible nature of Shh from the ZPA, which forms a concentration gradient. This means the the concentration at digit 2 will be less than digit 3 due to being further away from the ZPA and thus can be differentiated from one another based on this concentration. Finally the digit 1 is differentiated on the basis of not receiving any Shh signalling at all. Thus it is a combination of concentration gradient of Shh, temporal gradient of Shh, and independence from Shh.

{2. Which of the following are correct with regards to the hedgehog signalling pathway in ''Drosophila''.
|type="[]"}
- It occurs at the cillum on the cell surface.
- Hedgehog protein binding to the Patched receptor activates it to inhibit Smoothened.
+ Hedgehog protein binding to the Patched recetpor inhibits it to activate Smoothened.
+ Inhibition of SUFU is critical in allowing Ci to translocate into the nucles and activate downstream targets of the hedgehog pathway.
- The pathway contains 3 homologs including sonic hedgehog, Indian hedgehog, and desert hedgehog.
- The hedgehog protein is initially produced in its fully active form.
|| In terms of how the hedgehog protein acts to produce its effect, it must first bind to Patched which activity is inhibiting Smoothened in the absence of the hedgehog protein. The protein acts to trap Patched in its inactive confirmational state cause internalisation and eventually degradation of the receptor. This relives the dephosporylation of the Smoothened, usually induced by Patched, allowing it to accumulate phosphate groups and thus activate. With regards to Ci translocation, SUFU usually inhibits this by leading to phosphorylation of Ci and eventually its cleavage into Ci repressor. During the activation of the hedgehog pathway there is a binding of Fused to Cos2 which phosphorylates SUFU to inactivate it, allowing Ci to translocate into the nucleus and activate downstream targets.

{Question 3 What are some of the roles of the Hedgehog signalling pathway in embryonic development as discovered through animal models? (select one or more options)
|type="[]"}
-   Development of the hindbrain
+   Activates the Epidermal growth factor receptor (EGFR) signalling pathway
+   Development of craniofacial features
-   Development of the spinal nerves and meninges
+   Development and patterning of the limb buds and sclerotomes
|| The Hedgehog signalling pathway plays a significant role in forebrain, not hindbrain, development. Absence of this pathway results in holoprosencephaly (HPE), the lack of division of the forebrain into its 2 separate hemispheres, telencephalon and diencephalon.

Studies on Drosophila melanogaster have indicated the role of the Hedgehog signalling pathway in the activation of the EGFR pathway in Drosophila head development.

This pathway also plays a significant role in the development of craniofacial features, as it contributes to the epithelia of the frontonasal, maxillary, and pharyngeal ectoderm. Blockage of Shh signalling in chick embryos provides inside into the birth defects associated with lack of Shh involvement in craniofacial development. These defects include holoprosencephaly and, cleft lip and palate.

The role of Hh signalling pathway in development of spinal nerves and meninges has not been observed within animal models but as discussed above, this pathway plays a significant role in other areas of embryonic development, such as forebrain and craniofacial development, germ cell proliferation, subdivision of the eye field, production of sclerotomes and patterning of limb buds.

The role of this pathway in development and patterning of limb buds and sclerotomes was observed through experimentation on Shh knockout mice. The absence of this pathway resulted in a lack of vertebral column development. This deficiency of sclerotome derivatives indicates the role of Shh in maintenance or expansion of sclerotome cell population. Anterior limb bud cell death was also observed in these mice, indicating the role of Shh in patterning the anterior-posterior limb axis and even the proximal-distal limb segments, particularly in patterning of structures at the level of or distal to the latent elbow and knee joints.

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==Glossary==

{| class="pretty table"
|-
|'''Term'''
|'''Definition'''
|-
|Acyl-transferase
|A protein responsible for catalyzing the transfer of a palmitic acid moiety to the N-terminus of the hedgehog signalling molecule.
|-
|Casein kinase I (CKI)
|A kinase that is thought to phosphorylate the Smoothened receptor to activate it in the hedgehog signalling pathway.
|-
|Cubitus interruptus (Ci)
|A transcription factor found in the hedgehog signalling pathway of ''Drosophila'', which binds to promoter regions on the DNA in the nucleus to activate target genes of the pathway.
|-
|Costal-2 (Cos2)
|A kinesin like protein which interacts with the C terminus of Smoothened and acts like a scaffolding protein and allows proteins such as Fused to bind to it.
|-
|Denticle
|Bristle like projections off the segments on the ''Drosophila'' embryo.
|-
|Desert hedgehog (Dhh)
|A homolog of the ''Drosophila'' hedgehog protein, which is expressed most prominently in the gonads of vertebrates, where it plays a possible role in regulating spermatogenesis.
|-
|Frizzled
|A receptor that is part of the Wnt signalling pathway, which binds the protein wingless in order to activate signalling cascades.
|-
|Fused (Fu)
|A kinase that binds to Costal-2 and acts to inhbit Suppressor of Fused by phosphorylating it in the hedgehog signalling pathway.
|-
|Gli
|A family of proteins that acts as transcription factors that were originally isolated in human gliboblastoma. The proteins are important mediators in activating target genes for the hedgehog signalling pathway in vertebrates.
|-
|Hedgehog (Hh) protein
|A signalling protein widely expressed in the tissue during embryonic development of vertebrates and some other species such as ''Drosophila'', and acts to pattern and develop tissue. The protein is the primary mediator of the pathway.
|-
|Indian hedgehog (Ihh)
|A signalling protein that is a homolog of the ''Drosophila'' hedgehog protein, that primarily acts in regions where there is endochondrial ossification to aid in the maturation of chondrocytes.
|-
|Kif7
|A homolog of Costal-2 in vertebrates, which acts to regulate the downstream signalling of the hedgehog pathway by causing the disassociation of SUFU and Gli.
|-
|Limb bud
|A structure formed during early embryonic development consisting of ectoderm and mesenchyme representing the early limbs, where signalling processes in the bud will eventually lead to the out growth and patterning of the bud.
|-
|Patched (PTC)
|Patched is a transmembrane receptor expressed on cells that are sensitive to signalling via the hedgehog pathway, where its extracellular domain binds to the hedgehog protein to inactivate it. The receptor it self without hedgehog binding actively inhibits Smoothened.
|-
|Primary cilia
|An organelle that consists of an outward projection from the cell surface, which is thought to be critical for hedgehog signalling in vertebrates as it houses many components of the pathway.
|-
|Protein kinase A (PKA)
|A kinase that is thought to phosphorylate the Smoothened receptor to activate it in hedgehog signalling.
|-
|Smoothened (SMO)
|A G-protein coupled receptor like receptor integral to the hedgehog signalling pathway and acts to set in motion a series of events that lead to the activation of transcriptional factors that activate target genes of the hedgehog pathway.
|-
|Sonic hedgehog (Shh)
|The most widely expressed homolog of the ''Drosophila'' hedgehog protein found in vertebrates, playing a key role in embryonic development of humans.
|-
|Suppressor of Fused (SUFU)
|A protein in the hedgehog signalling pathway which when active, inhibits the translocation of Cubitus interruptus into the nucleus by leading to its phosphorylation, and eventually degradation via the proteosome.
|-
|Wingless protein
|A protein produced as a downstream target of the hedgehog signalling pathway in ''Drosophila'', which activates the Wnt pathway by binding to the Frizzled receptor.
|-
|Zone of polarizing activity (ZPA)
|The posterior region of the limb bud mesenchyme, which aids in the signalling of it to pattern the posterior anterior axis with respect to the digits through mediators such as sonic hedgehog.
|-
|}

===Additional Glossary Links===
[[A|A]] | [[B|B]] | [[C|C]] | [[D|D]] | [[E|E]] | [[F|F]] | [[G|G]] | [[H|H]] | [[I|I]] | [[J|J]] | [[K|K]] | [[L|L]] | [[M|M]] | [[N|N]] | [[O|O]] | [[P|P]] | [[Q|Q]] | [[R|R]] | [[S|S]] | [[T|T]] | [[U|U]] | [[V|V]] | [[W|W]] | [[X|X]] | [[Y|Y]] | [[Z|Z]] | [[Numbers|#]]

== References ==

2016 Group Project 4

2016-10-25T11:33:28Z

Z5019880:

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


= Hedgehog signalling pathway =
==Introduction==
[[File:Hedgehog.jpg|thumb|300px|An image of a hedgehog, the animal of which the Hh proteins are named after.]]
The Hedgehog (Hh) signalling pathway is an important part of the early embryo with regards to the patterning and development of the nervous system, limbs and the cranio-facial region in vertebrates and the polarization of the segments in the ''Drosophila'' embryo. It was first identified in 1980 by Christiane Nüsslein-Volhard and Eric Wieschaus, alongside a group of other genes regulating segment polarity in ''Drosophila'' embryonic development<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. The pathway itself consists of Hh proteins, where there are three identified homologs in vertebrates, being sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (DHH) <ref name="PMID7916661"><pubmed>7916661</pubmed></ref>. All homologs are expressed at varying levels within different tissues in the body, and also act as a form of redundancy to an extent between one another.

The signaling pathway is highly conserved between various species<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. and acts via a G protein coupled receptor like receptor, which uninhibited via the action of the Hh proteins. The pathway overall acts via a balance of dephosphorylation<ref name="PMID10966113"><pubmed>10966113</pubmed></ref> and degradation of proteins<ref name="PMID15102702"><pubmed>15102702</pubmed></ref> in order to regulate transcription factor activity to express various genes to carry out its action. The Hh pathway is also highly implicated when it comes to abnormalities in development, as it is implicated in diseases such as holoprosencephaly and cleft lip and palate. Overall Hh signaling pathways play a large role in embryological development, which will be discussed in detail on this page.

== History ==
{| class="pretty table"
| '''1980'''
|Christiane Nüsslein-Volhard and Eric Wieschaus first identified a group of genes including those related to the Hedgehog signalling pathway and linked them to the segmentation and planning of the embryo in ''Drosophila melanogaster'' by introducing mutagenic substances to the developing embryo<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>.
|-
|'''1993'''
|Multiple researchers including Andrew P. McMahon and Clifford Tabin discovered three equivalent homologs in vertebrates of the ''Drosophila melanogaster'' hedgehog gene, known as sonic hedgehog (Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh), by looking at DNA sequences similar to that of the gene in the fruit fly<ref><pubmed>7916661</pubmed></ref>.
|-
|'''1993'''
|Clifford Tabin and his lab identified the role of Shh in localising the limb bud, where they identified protein to be expressed within a region of the limb bud known as the zone of polarising activity (ZPA), showing that SHH is sufficient to induce the production of a ZPA, and thus limb bud formation in chick embryos<ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
|-
|'''1995'''
|Sonic hedgehog secreted by the notochord was identified to induce ventral cell types in the neural tube during embryonic development, most notably the floor plate cells and the motor neurons as shown in chick embryos. Such differentiation between the two cell types is thought to be a matter of sonic hedgehog concentrations experienced by the neural plate cells<ref name="PMID7736596"><pubmed>7736596</pubmed></ref>.
|-
|'''1995'''
|A shared Nobel prize in physiology or medicine was awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their researching regarding the identification of the developmental genes relating to the formation and patterning of the early embryo via genes.
|-
|'''1996'''
|Studies showed that human homologs of the Patched gene, a component of the hedgehog pathway, is a key gene that is mutated in Gorlin Syndrome, which characterised by a predisposition for basal cell carcinomas, the most common cancers in humans and developmental abnormalities<ref name="PMID8658145"><pubmed>8658145 </pubmed></ref>.
|-
|'''1999'''
|Researchers implicated Indian hedgehog as a key signalling molecule for the maturation and differentiation of prehypertrophic chondrocytes, where Indian hedgehog null mice were shown to have a lack of mature chondrocytes and no development of osteoblast cells in endochondrial bone. This was thought to be due to Indian hedgehog having a feedback signal controlling parathyroid hormone related protein, which regulates endochondrial bone development<ref name="PMID10465785"><pubmed>10465785</pubmed></ref>.
|-
|'''2003'''
|Sonic hedgehog was identified to regulate the proliferation of adult neural stem cells as shown in the hippocampus of rats and in vitro for neural progenitor cells isolated from the hippocampus<ref name="PMID12469128"><pubmed>12469128</pubmed></ref>.
|-
|'''2003'''
|Studies on Shh function found that it can act as a chemoattractant in the developing embryo spinal cord, to help guide commissural axons to the midline of the floor plate<ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
|-
|'''2009'''
|Mouse models showed that reduction in hedgehog signalling allows for transient improved response to chemotherapy with regards to mouse models of human pancreatic ductal adenocarcinoma, a cancer with one of the highest mortality rates in humans<ref name="PMID19460966"><pubmed>19460966</pubmed></ref>.
|}

This time line regarding the discoveries in the hedgehog signalling pathway is by no means exhaustive, but a selection of relatively large discoveries that had a significant impact in the field on the basis of citations.

== Origin of name ==

[[File:Bands of denticles in normal and Hh mutant Drosophila embryo.jpeg|thumb|250px|Differences in patterning of the denticle bands in normal and mutated hedgehog gene variant ''Drosophilia'' embryo]]

The name hedgehog came about when Christiane Nüsslein-Volhard and Eric Wieschaus first identified the group of genes controlling polarization of the segments in the ''Drosophila'' early embryo. They noticed that when they mutated the Hh gene, making it non-functional, that the bands of denticles formed during the early ''Drosophila'' embryo formation became more diffuse as opposed to being distinct normally. The denticles would all clump together forming a single patch on the surface of the embryo, as opposed to normally outlining the distinct segments of the embryo as bands<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. This gave the embryo spiny and prickly look similar to that of the back of a hedgehog.

From there on in the gene was called the hedgehog gene where homologs in vertebrates discovered along the line kept to this naming structure giving various species of hedgehog names to the genes such as Indian hedgehog, and desert hedgehog, with the exception being sonic hedgehog, which is named after a fictional character in the video game “Sonic the Hedgehog”.

== Function ==
===Overview===
{| class="pretty table"
|'''Hedgehog protein homolog'''
|'''Function'''
|-
| Sonic hedgehog [http://www.omim.org/entry/600725 OMIM entry] ||
*Expressed in zone of polarizing activity of the limb bud where it plays a role in helping to pattern the anterior and posterior aspect of the limbs <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
*Based on time and concentration of SHH due to diffusion from the point of secretion in the limb bud, SHH also plays a role in determining digit identity <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>.
*Acts as a chemoattractant to guide commissural axons in the floor plate of the neural tube towards the midline <ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
*Patterns the developing cusps of the teeth and their relative positions in the jaw, and is also essential for the growth of the teeth <ref name="PMID11044393"><pubmed>11044393</pubmed></ref>.
*SHH released from the notochord acts to induce formation of the floor plate, motor neurons, and dopaminergic neurons, as well as signalling the induction of the ventral cell types in the neural tube <ref name="PMID8790332"><pubmed>8790332</pubmed></ref>.
|-
| Desert hedgehog [http://www.omim.org/entry/605423 OMIM entry]||
*Plays a possible role in regulating spermatogenesis as due to the highly localised expression in the testes by pre-Sertoli cells, and the fact that mice with no DHH present are infertile, with no mature spermatozoa <ref name="PMID8805249"><pubmed>8805249</pubmed></ref>.
*DHH derived from Schwann cells signal development of peripheral nerves, by inducing the production of a connective tissue sheath around them <ref name="PMID10482238"><pubmed>10482238</pubmed></ref>.
|-
| Indian hedgehog [http://www.omim.org/entry/600726 OMIM entry]||
*Aid in the signalling of proliferating chondrocytes to undergo hypertrophic differentiation during endochondral ossification<ref name="PMID8662546"><pubmed> 8662546</pubmed></ref>.
*Plays a key role in yolk sac angiogenesis, as seen in studies where an absence of IHH leads to death of the embryo due to poor yolk sac vasculature <ref name="PMID11807029"><pubmed>11807029</pubmed></ref>.
|}

===Limb development===

====Research background====

The hedgehog signalling pathway plays a significant role with regards to the embryonic development of the limbs in vertebrates, specifically SHH. For a while the mechanism of which the limb was patterned on the anterior to posterior axis was quite unknown. Early studies had shown that grafting posterior wing bud cells to the anterior region of the wing bud in a chick embryo, an equal amount of extra digits was produced to that of which was normally produced, and in a mirror image pattern<ref name="PMID4390734"><pubmed>4390734</pubmed></ref>. This showed that the pattern of which the digits were produced were polarized with respect to that of the graft from the limb bud, which has become to be described as the zone of polarizing activity (ZPA). Further studies mapped the ZPA, by showing the area of the wing bud on the chick with the highest polarizing activity to be the posterior margin of the wing bud<ref name="PMID4719010"><pubmed>4719010</pubmed></ref>. Furthermore, studies later on found that the amount or concentration of ZPA cells grafted played a role in the amount of digits induced and how polarized they were<ref name="PMID7453825"><pubmed>7453825</pubmed></ref>. This ultimately gave rise to studies regarding a potential morphogen, which could potentially provide this concentration dependent activity of polarization when it comes to the limb bud. Studies to find this morphogen led to the discover of the ability for retinoic acid to mimic the polarizing action of the ZPA when applied locally to the limb bud<ref name="PMID7070499"><pubmed>7070499</pubmed></ref>. This was found to be due to the induction of a second morphogen known as SHH, where when cells that express SHH are grafted onto the limb bud, polarized digit development is induced<ref name="PMID7720566"><pubmed>7720566</pubmed></ref>.

====Polarizing activity of sonic hedgehog====
[[File: Mechanims of patterning the digits on the posterior to anterior axis of the limb bud by sonic hedgehog.jpg|thumb|450px|Regions of the limb bud and the digits they form based on patterning of the posterior to anterior axis of the limb bud by mechanisms mediated by sonic hedgehog.]]
More recent studies have sought to elucidate how SHH mediates such a polarizing effect in terms of digit identification and its underlying mechanism. It has been suggested that such an effect is heavily concentration dependent similar to that of when transplanting the chick wing bud ZPA cells, acting in a dose dependent matter, due to the ability of SHH to modulate signaling at long ranges via diffusion<ref name="PMID11389830"><pubmed>11389830</pubmed></ref>. This implies that a gradient of varying concentrations of SHH diffused through the mesenchyme of the limb bud leads to the identification of posterior and anterior digits.

Current research has also suggested that not only is the concentration gradient of SHH important for the patterning of the digits, but also the temporal gradient, being the length of time of exposure of SHH to the mesenchyme of the limb bud. Studies have shown in mice that of the 5 digits, polarization from the posterior end, being digit 5, to the anterior end being digit 1 are due to different mechanisms. Digits 5 and 4 have been shown to be comprised of the SHH expressing cells and thus both experience maximal levels of SHH, and thus cannot be distinguished on the basis of a concentration gradient. As a result, temporal gradients are employed, where the expression of SHH in both regions that become the digits vary, where studies have shown that SHH expression in the primordium of digit 5 is maintained longer than that in digit 4 gradient <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>. When it comes to digit 3 and 2, there is differentiation based on the concentration gradient of SHH from the ZPA, where due to the ZPA being further away to digit 2 than 3, the concentration of SHH will be higher at 3<ref name="PMID15315763"><pubmed>15315763</pubmed></ref> <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>. In terms of digit 1, the levels of SHH are so low at this point that it is said to be SHH independent, and actually reliant on the absence of SHH for normal differentiation. With regards to how SHH actually mediates its action, it is thought to be by a balance between the transcription factor Gli3 and SHH expression, which activates Gli1, Gli2 and Gli3 repressor, effectively counteracting Gli3. Gli3 is considered to promote the inhibition of digit formation and identification, while the downstream targets of SHH are thought to promote it<ref name="PMID17400206"><pubmed>17400206</pubmed></ref>.

Other research has also shown the possibility of SHH signaling as a way to prime the posterior mesenchyme and induce the production of bone morphogenetic protein 2 (bmp2), which is thought to be the morphogen creating the chemical gradient for digit identification<ref name="PMID10704381"><pubmed>10704381</pubmed></ref>. Overall the mechanism at which SHH work to pattern the posterior anterior axis of the limb bud is still relatively unknown, and further research is being conducted to elucidate how the underlying mechanisms such as the downstream targets of SHH affect digit formation.

=== Neural development ===

=== Organogenesis ===
The Hedgehog Signalling pathway is a pathway sending information to embryonic cells which plays an especially important role in the regulation of organogenesis. These processes include the organization of the brain (craniofacial) and the growth of appendages with further studies implicating the Hedgehog Signalling pathway in the development of the small intestine, lungs and the pancreas.

== Mechanism ==

=== Processing of precursor ===
 [[File:Processing of Hh precurosr.jpg|thumb|250px|Summary of the steps in the processing of the Hh protein precursor into a functional signalling molecule]]
Hedgehog (Hh) proteins are produced as precursors, which must be processed by the cells producing it before being able to perform its signalling function. The process by which the Hh protein is processed begins with its signal sequence at its N terminus directing the translocation of the precursor into the endoplasmic reticulum, where it is removed via signal peptidase<ref><pubmed>1394430</pubmed></ref>. This process allows the C terminus of the Hh protein to catalyze the cleavage and addition of cholesterol on itself to form a C terminal processing domain and an N terminal processing domain associated with a cholesterol group on its C terminus<ref><pubmed>21357747</pubmed></ref>. The portion associated with the cholesterol will go on to form the signalling molecule, while the C terminal processing domain has no known signalling function<ref><pubmed>8824192</pubmed></ref>.

At this point the Hh protein associated with the cholesterol is able to perform its signalling action, but further modification is still required to ensure efficient signalling. This occurs when the cholesterol group attached to the Hh protein associates with the plasma membrane of the cell, which allows for the addition of a palmitic acid moiety to the N terminal of the protein by an acyl-transferase known as skinny hedgehog<ref><pubmed>11486055</pubmed></ref>. Studies have shown that such an addition allows for an increase in potency in signalling of Hh proteins of 30-fold over its form without palmitic acid added<ref><pubmed>9593755</pubmed></ref>. From this point the Hh protein is now fully active and can either remain associated to the plasma membrane of the cell for autocrine action or be secreted for paracrine action.

=== Mechanism of signalling ===

==== ''Drosophila melanogaster'' ====
 [[File:Hh Signalling Pathway.jpg|thumb|600px|Steps regarding the processing of the hh protein precursor into the fully functional signalling molecule.]]

The Hh signaling pathway has been well studied in ''Drosophila melanogaster'', and has been shown the be conserved to an extent across it and mammals making the species a suitable model for Hh signalling in humans<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. In ''Drosophila melanogaster'', the Hh pathway begins when the Hh proteins bind to the extracellular domain of the transmembrane protein known as Patched (PTC) to inactivate it. This inactivation occurs by Hh trapping PTC in an inactive conformational state<ref><pubmed>27647915</pubmed></ref>. After binding of Hh occurs to PTC and it is inactivated, the receptor and Hh protein is thought to be endocytosed by the cell where they undergo lysosomal degradation in order to limit Hh concentration, thus limiting its spread to other cells and PTC activity<ref name="PMID15102702"><pubmed>15102702</pubmed></ref>. In the absence of Hh, PTC acts to suppress the expression of the Smoothened (SMO), a G-protein coupled receptor like receptor, and thus its signaling. How PTC achieves this inhibition of SMO is currently unclear. Studies have suggested that due to the highly phosphorylated nature of SMO when active, that PTC acts to dephosphorylate SMO in order to repress its signaling. It is thus that binding of Hh to PTC reduces PTCs ability to promote dephosphorylation of SMO, leading to its increased activity and expression on the cell surface <ref name="PMID10966113"><pubmed>10966113</pubmed></ref>. It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) <ref><pubmed>15616566 </pubmed></ref>.

This overall increased phosphorylation of SMO due to a repression of PTC activity by Hh leads to an accumulation of SMO on the cell surface, which collectively allows for SMO to exert its activity. Most importantly phosphorylation also disrupts intramolecular electrostatic interactions between SMO molecules which switches the molecule into its active conformational state <ref><pubmed>17960137 </pubmed></ref>. Generally, SMO has been shown to have very little signal transducing ability, and thus this accumulation allows it to jointly transduce as substantial signal. This occurs by the C terminus of SMO on the intracellular domain interacting with the kinesin like protein Costal-2 (Cos2), which is thought to bind to microtubules in order to acts as a scaffolding protein <ref><pubmed>18691888 </pubmed></ref>. Then the kinase known as Fused (Fu) binds to Cos2, which phosphorylates Suppressor of Fused (SUFU) to inhibit it. Without this inhibition SUFU goes onto prevent the translocation of the transcriptional factor Cubitus interruptus (Ci) into the nucleus by leading to its phosphorylation, and subsequently its partial cleavage via the proteosome. The partial cleavage of Ci leaves a lower molecular weight protein known as Ci repressor (CiR) or Ci75, which translocates into the nucleus and acts to repress the target genes of the Hh signalling pathway via Ci<ref><pubmed>9215627</pubmed></ref>. Thus overall inhibition of SUFU increases the amount of Ci entering into the nucleus of the cell, and activating transcription of target genes of the Hh pathway<ref><pubmed>10952898 </pubmed></ref>.

At this point Ci is able to activate the transcription of various genes, which include importantly the ''ptc'' gene, which encodes the PTC receptor. It is thus as a result of this, that PTC expression will be increased in response to Hh pathway induction, which negatively feeds back to reduce Ci signaling induction, in order to maintain homeostasis and regulate the intensity and duration of the signaling from Hh<ref><pubmed>9053330</pubmed></ref>. Furthermore the ''wg'' gene, encoding the wingless protein is also activated by Ci, which leads to activation of the Wnt pathway via the Frizzled receptor in adjacent cells expressing the gene ''engrailed'' <ref><pubmed>9502727</pubmed></ref> <ref><pubmed>10457026</pubmed></ref>. This overall stabalizes the boundaries between the segments of the developing ''Drosophila melanogaster'' signalled by the gene ''engrailed''<ref><pubmed>3282172</pubmed></ref>.
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=w1xXD9kss2w</html5media>
 
An overview of the hedgehog signalling pathway in ''Drosophila''<ref>YouTube. (2016). The Hedgehog signalling pathway in Drosophila online Available at: https://www.youtube.com/watch?v=w1xXD9kss2w</ref>.

==== Mammals ====

Although the Hh signaling pathway has been conserved across species to an extent <ref name="PMID8595881"><pubmed>8595881</pubmed></ref>, key differences exist when observing the pathway within mammalian tissue in contrast to that which has been studied in ''Drosophila melanogaster''. In mammalian cells and all other vertebrates, Hh signalling is dependent on an organelle known as the primary cilia, which are projections outwards from the cells surface. The importance of such an organelle in the signalling pathway comes as a result of PTC1, one of the two PTC receptor homologs in mammals, that binds to the Hh homolog Sonic Hh (Shh), exists within the cilia<ref><pubmed>17641202</pubmed></ref>. The mechanism of action of SMO inhibition and activation is poorly understood currently, but evidence has suggested that PTC1 acts to inhibit SMO when unbound to Shh by acting as a pump to remove oxysterols from the cilia into the extracellular space. <ref><pubmed>PMC1462959</pubmed></ref>. These oxysterols are normally thought to bind and accumulate around the SMO receptors, which in turn prevents internalization and deactivation of SMO, leading to it accumulating on the apical primary cilia<ref><pubmed>16136078</pubmed></ref>. Other studies have also suggested that Shh has a role in increasing phosphorylation of SMO, which is required for it to accumulate as well on the cilia<ref><pubmed>21695114</pubmed></ref>. Overall the net action of Shh initially is believed to cause accumulation of SMO at the cilia by inhibiting PTCH1 and phosphorylation of SMO.

How SMO acts next is relatively unknown but, it has been shown to promote the disassociation between SUFU and Gli3 transcription factor, which allows Gli to be transported to the nucleus to activate effector genes of the pathway. Gli is normally bound in the cytoplasm to SUFU, where SUFU promotes the partial degradation of Gli into its repressor form, similar to that in the fruit fly model<ref><pubmed>20360384</pubmed></ref>. Alongside this, Kif7, a Cos2 homolog, has been shown to migrate to the apex of the cilium in response to SMO accumulation at the cilium, where it has been postulated to also promote disassociation of SUFU and Gli<ref><pubmed>19592253</pubmed></ref>. On the other hand, Kif7 is also thought to play an inhibitory role for the signaling pathway as when it is localized at the base of the cilia in the absence of Hh signaling and traffics Gli factors away from the cilia<ref><pubmed>19666503</pubmed></ref>. This all leads to an accumulation of active Gli transcription factors which migrate to the nucleus in order either inhibit or activate gene transcription in response to Hh signaling.

== Animal models ==

=== ''Drosophila melanogaster'' ===
 [[File:Hedgehog signalling on plane polarity of denticles in Drosophila.png|thumb|400px|Image of the ''Drosophila'' embryo used as a model to research plane polarization of cells by looking at the denticles of the ''drosophila'' embryo in response to many signalling molecules including hedgehog.]]

Hedgehog (Hh) protein signal was initially discovered through experimentation on the fruit fly, "Drosophila melanogaster". It is through this model that we are able to discover not only the functional components of this pathway but also understand its role in embryonic development. Through application of this information on the human biological system we are able to find the cause and thus potential treatments of defects and diseases caused by interruption or mutation of the Hedgehog signalling pathway.

Upon further research on Drosophila it was found that Hh may play a role in germ cell proliferation and in particular, may control the proliferation and activity of somatic cells found within the germarium, which is the most anterior structure within the Drosophila ovary. <ref>Ovaries (Drosophila) definition. (2016). Groups.molbiosci.northwestern.edu. Retrieved 10 September 2016, from http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-D/Drosophila_Ovaries.html</ref> Thus, the Hh signalling pathway is vital in egg chamber formation and its consequent envelopment, budding and polarisation. It has also been observed that somatic cell proliferation is dependent on this pathway and thus, the number of pre-follicle cells is subject to the effect of the Hh signalling pathway. <ref><pubmed>8620839</pubmed></ref> Further studies have also indicated the role played by this pathway in activating the Epidermal growth factor receptor (EGFR) signalling pathway as seen in the induction of EGFR by Hh in Drosophila head development. <ref><pubmed>10331974</pubmed></ref> In addition, research on Drosophila has indicated the direct effect of Hh signalling on tracheal branch patterning. <ref><pubmed>11290298</pubmed></ref>

Despite ongoing research on "Drosophila melanogaster" to uncover the workings of the Hedgehog signalling pathway, further research is needed to confirm and further current findings. In addition, it is evident that it is through the study of these fruit flies that we are able to gain a basis of understanding of the causes of certain human diseases and thus propel research into treatments for sufferers.

===Blockage of Shh Signalling in Forebrain Neuroectoderm of Chick Embryos===

The Hedgehog signalling pathway plays a significant role in embryonic development, particularly of the forebrain. Due to its role in development of craniofacial features by contributing to the epithelia of the frontonasal, maxillary, and pharyngeal ectoderm, a disruption in this pathway can result in a variety of birth deformities including holoprosencephaly (HPE), where the prosencephalon (forebrain) fails to divide into 2 separate hemispheres, telencephalon and diencephalon <ref>Holoprosencephaly - NORD (National Organization for Rare Disorders). (2016). NORD (National Organization for Rare Disorders). Retrieved 10 September 2016, from http://rarediseases.org/rare-diseases/holoprosencephaly/</ref>, as well as cleft lip and palate. The function of Sonic Hedgehog (Shh) and its signalling pathway on the formation of forebrain neuroectoderm was studied in chick embryos.

It was found that the disruption of Shh signalling in the neural tube of chick embryos resulted in the lack of division of the forebrain into the diencephalon and telencephalon. This is in fact, as stated earlier, the fundamental cause of the rare condition, holoprosencephaly. It was discovered that the Shh signalling pathway in the diencephalon was responsible for gene expression in the telencephalon.

In addition, through experimentation on chick embryos it was found that through Shh signalling the development of the forebrain regulates and controls facial morphogenesis, particularly of the upper and middle face. Therefore, interference with Shh signalling in the forebrain prevents this intrinsic communication, thus hindering Shh expression in facial ectoderm. This results in brain malformation accompanied by facial disfiguration as seen in patients suffering from HPE. Other malformations caused by blockage of Shh signalling in craniofacial development include, hypotelorism (decreased space between the orbits), growth restriction as well as cleft lip and palate as mentioned previously.

A commonality discovered among all forms of Shh signalling disruption was two facial defects, “the loss of mediolateral expansion of the face and absence of proximodistal outgrowth of the frontonasal prominence”<ref><pubmed>15979605</pubmed></ref> (Ralph et al., 2005). Through reference to studies performed on chick and mice embryos, it was found that Shh signalling is particularly vital in development of maxillary and frontonasal components of the cranium.

This breakthrough study on the inhibition of Shh signalling in chick embryos has significantly filled the gap in our understanding of the Sonic Hedgehog signalling pathway. It is evident that this pathway is crucial in the development of the forebrain and in turn regulates and controls the development of the facial skeleton. This is further proven through the observation that in the event of Shh signalling inhibition there is a development of craniofacial malformations.

=== Shh knockout mice ===

Extensive research on Shh knockout mice allowed discovery of the roles of Shh in embryonic development and patterning of the limb buds and sclerotomes, and maintenance of the notochord. <ref><pubmed>8837770</pubmed></ref> These mice expressed defects in the cephalic neural tube with the fusion of telencephalic and optic vesicles. Through this research it is evident that Shh is partially responsible for the subdivision of the eye field through forebrain optic stalk development, along with formation of the ventral midline. It is these forebrain abnormalities established in the absence of Shh that results in congenital malformations of holoprosencephaly, development of a single nasal chamber and other facial defects in humans.

The absence of the vertebral column including, the intervertebral discs and vertebrae along with the medial regions of the ribs were observed within Shh knockout mice. This lack of sclerotome derivatives indicates the role of Shh in maintenance or expansion of sclerotome cell population (Chiang et al., 1996). In addition, though the role of Shh in patterning the anterior-posterior limb axis is evident through anterior limb bud cell death in the absence of Shh, it is also responsible for the development of proximal-distal limb segments, particularly in the patterning of structures at the level of or distal to the latent elbow and knee joints. <ref><pubmed>11476582</pubmed></ref> This was perceived after observing the incomplete formation of distal limb structures with abnormal anterior-posterior axis formation (Chiang et al., 2001). Thus, Shh knockout mice provide great insight into the functions of Shh in embryonic development and the abnormalities formed in the absence of this signalling protein.

== Clinical Significance ==

===Gorlin Syndrome===
[[File:1 week year old newborn girl with cleft lip and palate.png|thumb|An image of a 1 week year old newborn girl with cleft lip and palate]]
Gorlin syndrome, also known as nevoid basal cell carcinoma syndrome, is a heritable disease resulting from heterozygousity in Hh receptor, Patched (PTC). The PTC gene encodes a protein which binds sonic hedgehog (Shh) and it is this binding which inhibits the Hh signalling pathway. However mutation of PTC leads to activation of the pathway <ref><pubmed>9620294</pubmed></ref>. The syndrome is characteristic of skin cancer basal cell carcinoma and cerebellum cancer medulloblastoma. 1-2% of medulloblastomas and 0.5% of basal cell carcinomas are attributable to the disease. Patients present with craniofacial and brain abnormalities such as cleft palate, strabismus, macrocephaly, abnormal development of the corpus callosum and frontal bossing, with an overall overgrown appearance. Skeletal defects such as of the shoulder, ribs and vertebrae are often seen, as well immobile thumbs and polydactyly, which is a deformity of the hand or feet in which they have one or more extra fingers or toes. Apart from physical anomalies, patients have an increased risk of tumours throughout the body including cardiac, ovarian fibromas, ovarian dermoid cysts, meningiomas, and fibrosarcomas. Additionally with time, patients eventually undergo intracranial calcification and dyskeratotic pitting of the hands and feet whereby skin cells prematurely convert to keratin <ref><pubmed>8681379</pubmed></ref>.

===Holoprosencephaly===
 [[File:Semilobar holoprosencephaly.jpg|thumb|240px|MRI of fore brain depicting semilobar holoprosencephaly]]
Holoprosencephaly (HPE) is a congenital disease caused by incomplete division of the prosencephalon (embryonic forebrain) into separate lobes of the cerebral hemispheres. The Shh gene has been identified as a HPE-causing gene however recent evidence has also investigated into the PTC gene, which acts to repress Shh signalling. A gain in repressive action of PTC is seen, leading to decreased Shh signalling, and thus HPE <ref><pubmed>11941477</pubmed></ref>. Clinical expression is variable between patients and is dependent upon the 3 forms of increasing severity: lobar, semi-lobar and alobar HPE, where patients can present with right and left ventricles however with a continuous frontal cortex in lobar HPE, to carrying a single cerebral ventricle in alobar HPE. Along with forebrain abnormalities, facial anomalies are also seen including midline cleft lip and/or flat nose, ocular hypotelorism characteristic of a small distance between the eyes. In severe rare cases of cyclopia, a non-functioning nose may be observed in the form of proboscis and the patient may present with a single eye at the root of the nose <ref><pubmed>20583177</pubmed></ref>. Furthermore, patients with HPE often develop a large number of medical issues including epilepsy, mental retardation, thyroid and adrenal hypoplasia due to lack of hypothalamus or pituitary gland development, and diabetes insipidus.

===Medulloblastoma===
Medulloblastoma is a cancerous tumour of the cerebellum where the cells of origin are the granular cell precursors found in the neonatal cerebellum, explaining its high prevalence in children <ref><pubmed>19171780</pubmed></ref>. Commonly, medulloblastomas are found deep in the cerebellum along the midline, however with desmoplastic medulloblastomas, they are found more laterally and superficial. As seen previously with Gorlin syndrome and holoprosencephaly, PTC is also associated with the development of medulloblastoma, and misregulation of Hh-PTC signalling in cells of the external germinal layer, the layer found on the surface of the cerebellum, is thought to give rise to the tumour. In experimental studies, it has been discovered that PTC is highly transcribed in medulloblastomas and in approximately 30% of PTC heterozygous mice the tumour develops <ref><pubmed>9205058</pubmed></ref> <ref><pubmed>9262482</pubmed></ref>. In patients with medulloblastoma, often children, the most common symptoms seen are vomiting, ataxia, psychomotor regression, drowsiness and anorexia. With age, patients are seen to get more frequent headaches, and show psychological symptoms such as behavioural problems, poor performance in school, and anxiety. In patients exhibiting life-threatening symptoms due to increased intracranial hypertension, bradycardia, convulsions and coma are often seen <ref><pubmed>21537925</pubmed></ref>.

Watch below for a breakdown on Hedgehog's role in Tumour Development!
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=gFWO3I-oqsE</html5media>
 
An overview of the Hedgehog Signalling Pathway in Tumour Development<ref>YouTube. (2016). The Hedgehog Signaling in the Tumor Microenvironment - Research Hedgehog online Available at: https://www.youtube.com/watch?v=gFWO3I-oqsE</ref>.

== Current research ==

==Quiz==

<quiz display=simple>

{1. Which of the following are the reasons that are thought to allow for the patterning of digits in vertebrates.
|type="[]"}
+  Concentration gradient of sonic hedgehog.
+  Temporal gradient of sonic hedgehog.
-  The varying homologs of the hedgehog proteins .
-  The length of the hedgehog molecule.
+  No sonic hedgehog signalling.
-  The type of cholesterol attached to the hedgehog molecule.
|| For differentiation of the individual digits in mice to occur it is thought that each digit requires a different signalling mechanism. Digits 5 and 4 which are most posterior, are derived from the region around the zone of polarising area which contain Shh secreting cells, meaning these regions will experience a saturation of Shh signalling and cannot be differentiated by Shh concentration. As a result it is thought that these digits are identfied based on the temporal gradient, meaning that the length of which Shh is expressed in these regions differentiates the digits from one another with digit 5 expressing Shh for longer. Digits 3 and 2 are further away but still receive Shh signalling due to the diffusible nature of Shh from the ZPA, which forms a concentration gradient. This means the the concentration at digit 2 will be less than digit 3 due to being further away from the ZPA and thus can be differentiated from one another based on this concentration. Finally the digit 1 is differentiated on the basis of not receiving any Shh signalling at all. Thus it is a combination of concentration gradient of Shh, temporal gradient of Shh, and independence from Shh.

{2. Which of the following are correct with regards to the hedgehog signalling pathway in ''Drosophila''.
|type="[]"}
- It occurs at the cillum on the cell surface.
- Hedgehog protein binding to the Patched receptor activates it to inhibit Smoothened.
+ Hedgehog protein binding to the Patched recetpor inhibits it to activate Smoothened.
+ Inhibition of SUFU is critical in allowing Ci to translocate into the nucles and activate downstream targets of the hedgehog pathway.
- The pathway contains 3 homologs including sonic hedgehog, Indian hedgehog, and desert hedgehog.
- The hedgehog protein is initially produced in its fully active form.
|| In terms of how the hedgehog protein acts to produce its effect, it must first bind to Patched which activity is inhibiting Smoothened in the absence of the hedgehog protein. The protein acts to trap Patched in its inactive confirmational state cause internalisation and eventually degradation of the receptor. This relives the dephosporylation of the Smoothened, usually induced by Patched, allowing it to accumulate phosphate groups and thus activate. With regards to Ci translocation, SUFU usually inhibits this by leading to phosphorylation of Ci and eventually its cleavage into Ci repressor. During the activation of the hedgehog pathway there is a binding of Fused to Cos2 which phosphorylates SUFU to inactivate it, allowing Ci to translocate into the nucleus and activate downstream targets.

{Question 3 What are some of the roles of the Hedgehog signalling pathway in embryonic development as discovered through animal models? (select one or more options)
|type="[]"}
-   Development of the hindbrain
+   Activates the Epidermal growth factor receptor (EGFR) signalling pathway
+   Development of craniofacial features
-   Development of the spinal nerves and meninges
+   Development and patterning of the limb buds and sclerotomes
|| The Hedgehog signalling pathway plays a significant role in forebrain, not hindbrain, development. Absence of this pathway results in holoprosencephaly (HPE), the lack of division of the forebrain into its 2 separate hemispheres, telencephalon and diencephalon.

Studies on Drosophila melanogaster have indicated the role of the Hedgehog signalling pathway in the activation of the EGFR pathway in Drosophila head development.

This pathway also plays a significant role in the development of craniofacial features, as it contributes to the epithelia of the frontonasal, maxillary, and pharyngeal ectoderm. Blockage of Shh signalling in chick embryos provides inside into the birth defects associated with lack of Shh involvement in craniofacial development. These defects include holoprosencephaly and, cleft lip and palate.

The role of Hh signalling pathway in development of spinal nerves and meninges has not been observed within animal models but as discussed above, this pathway plays a significant role in other areas of embryonic development, such as forebrain and craniofacial development, germ cell proliferation, subdivision of the eye field, production of sclerotomes and patterning of limb buds.

The role of this pathway in development and patterning of limb buds and sclerotomes was observed through experimentation on Shh knockout mice. The absence of this pathway resulted in a lack of vertebral column development. This deficiency of sclerotome derivatives indicates the role of Shh in maintenance or expansion of sclerotome cell population. Anterior limb bud cell death was also observed in these mice, indicating the role of Shh in patterning the anterior-posterior limb axis and even the proximal-distal limb segments, particularly in patterning of structures at the level of or distal to the latent elbow and knee joints.

{Question 4
|type="()"}
+  
-  
-  
-  
||Replace with Answer

{Question 5
|type="()"}
-  
-  
-  
+  
||Replace with Answer

{Question 6
|type="()"}
-  
-  
-  
+  
||Replace with Answer
</quiz>

==Glossary==

{| class="pretty table"
|-
|'''Term'''
|'''Definition'''
|-
|Acyl-transferase
|A protein responsible for catalyzing the transfer of a palmitic acid moiety to the N-terminus of the hedgehog signalling molecule.
|-
|Casein kinase I (CKI)
|A kinase that is thought to phosphorylate the Smoothened receptor to activate it in the hedgehog signalling pathway.
|-
|Cubitus interruptus (Ci)
|A transcription factor found in the hedgehog signalling pathway of ''Drosophila'', which binds to promoter regions on the DNA in the nucleus to activate target genes of the pathway.
|-
|Costal-2 (Cos2)
|A kinesin like protein which interacts with the C terminus of Smoothened and acts like a scaffolding protein and allows proteins such as Fused to bind to it.
|-
|Denticle
|Bristle like projections off the segments on the ''Drosophila'' embryo.
|-
|Desert hedgehog (Dhh)
|A homolog of the ''Drosophila'' hedgehog protein, which is expressed most prominently in the gonads of vertebrates, where it plays a possible role in regulating spermatogenesis.
|-
|Frizzled
|A receptor that is part of the Wnt signalling pathway, which binds the protein wingless in order to activate signalling cascades.
|-
|Fused (Fu)
|A kinase that binds to Costal-2 and acts to inhbit Suppressor of Fused by phosphorylating it in the hedgehog signalling pathway.
|-
|Gli
|A family of proteins that acts as transcription factors that were originally isolated in human gliboblastoma. The proteins are important mediators in activating target genes for the hedgehog signalling pathway in vertebrates.
|-
|Hedgehog (Hh) protein
|A signalling protein widely expressed in the tissue during embryonic development of vertebrates and some other species such as ''Drosophila'', and acts to pattern and develop tissue. The protein is the primary mediator of the pathway.
|-
|Indian hedgehog (Ihh)
|A signalling protein that is a homolog of the ''Drosophila'' hedgehog protein, that primarily acts in regions where there is endochondrial ossification to aid in the maturation of chondrocytes.
|-
|Kif7
|A homolog of Costal-2 in vertebrates, which acts to regulate the downstream signalling of the hedgehog pathway by causing the disassociation of SUFU and Gli.
|-
|Limb bud
|A structure formed during early embryonic development consisting of ectoderm and mesenchyme representing the early limbs, where signalling processes in the bud will eventually lead to the out growth and patterning of the bud.
|-
|Patched (PTC)
|Patched is a transmembrane receptor expressed on cells that are sensitive to signalling via the hedgehog pathway, where its extracellular domain binds to the hedgehog protein to inactivate it. The receptor it self without hedgehog binding actively inhibits Smoothened.
|-
|Primary cilia
|An organelle that consists of an outward projection from the cell surface, which is thought to be critical for hedgehog signalling in vertebrates as it houses many components of the pathway.
|-
|Protein kinase A (PKA)
|A kinase that is thought to phosphorylate the Smoothened receptor to activate it in hedgehog signalling.
|-
|Smoothened (SMO)
|A G-protein coupled receptor like receptor integral to the hedgehog signalling pathway and acts to set in motion a series of events that lead to the activation of transcriptional factors that activate target genes of the hedgehog pathway.
|-
|Sonic hedgehog (Shh)
|The most widely expressed homolog of the ''Drosophila'' hedgehog protein found in vertebrates, playing a key role in embryonic development of humans.
|-
|Suppressor of Fused (SUFU)
|A protein in the hedgehog signalling pathway which when active, inhibits the translocation of Cubitus interruptus into the nucleus by leading to its phosphorylation, and eventually degradation via the proteosome.
|-
|Wingless protein
|A protein produced as a downstream target of the hedgehog signalling pathway in ''Drosophila'', which activates the Wnt pathway by binding to the Frizzled receptor.
|-
|Zone of polarizing activity (ZPA)
|The posterior region of the limb bud mesenchyme, which aids in the signalling of it to pattern the posterior anterior axis with respect to the digits through mediators such as sonic hedgehog.
|-
|}

===Additional Glossary Links===
[[A|A]] | [[B|B]] | [[C|C]] | [[D|D]] | [[E|E]] | [[F|F]] | [[G|G]] | [[H|H]] | [[I|I]] | [[J|J]] | [[K|K]] | [[L|L]] | [[M|M]] | [[N|N]] | [[O|O]] | [[P|P]] | [[Q|Q]] | [[R|R]] | [[S|S]] | [[T|T]] | [[U|U]] | [[V|V]] | [[W|W]] | [[X|X]] | [[Y|Y]] | [[Z|Z]] | [[Numbers|#]]

== References ==

File:Hedgehog signalling on plane polarity of denticles in Drosophila.png

2016-10-25T11:20:26Z

Z5019880:

File:Hedgehog signalling on plane polarity of denticles in Drosophila.png

2016-10-25T11:13:07Z

Z5019880:

File:Planar polarity of denticle on fruit fly embryo in response to hedgehog.TIF

2016-10-25T11:05:53Z

Z5019880:

File:Mechanims of patterning the digits on the posterior to anterior axis of the limb bud by sonic hedgehog.jpg

2016-10-25T10:46:18Z

Z5019880:

Diagram representing the different regions of the limb bud that will differentiate into the digits. The different colours represent varying mechanisms by which sonic hedgehog acts on the cells in the limb bud to identify them as certain digits. The level of concentration of sonic hedgehog in this case is similar to a gradient going along the posterior to anterior axis of the limb bud.

== Copyright ==
Image created by student z5019880, and is free to be redistributed or used in any way outlined under the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/)

== Reference ==
Image was inspired by figure 5 in the reference below.

<pubmed>15315763</pubmed>

{{Student Image}}

2016 Group Project 4

2016-10-25T07:19:01Z

Z5019880: /* Polarizing activity of sonic hedgehog */

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


= Hedgehog signalling pathway =
==Introduction==
[[File:Hedgehog.jpg|thumb|300px|An image of a hedgehog, the animal of which the Hh proteins are named after.]]
The Hedgehog (Hh) signalling pathway is an important part of the early embryo with regards to the patterning and development of the nervous system, limbs and the cranio-facial region in vertebrates and the polarization of the segments in the ''Drosophila'' embryo. It was first identified in 1980 by Christiane Nüsslein-Volhard and Eric Wieschaus, alongside a group of other genes regulating segment polarity in ''Drosophila'' embryonic development<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. The pathway itself consists of Hh proteins, where there are three identified homologs in vertebrates, being sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (DHH) <ref name="PMID7916661"><pubmed>7916661</pubmed></ref>. All homologs are expressed at varying levels within different tissues in the body, and also act as a form of redundancy to an extent between one another.

The signaling pathway is highly conserved between various species<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. and acts via a G protein coupled receptor like receptor, which uninhibited via the action of the Hh proteins. The pathway overall acts via a balance of dephosphorylation<ref name="PMID10966113"><pubmed>10966113</pubmed></ref> and degradation of proteins<ref name="PMID15102702"><pubmed>15102702</pubmed></ref> in order to regulate transcription factor activity to express various genes to carry out its action. The Hh pathway is also highly implicated when it comes to abnormalities in development, as it is implicated in diseases such as holoprosencephaly and cleft lip and palate. Overall Hh signaling pathways play a large role in embryological development, which will be discussed in detail on this page.

== History ==
{| class="pretty table"
| '''1980'''
|Christiane Nüsslein-Volhard and Eric Wieschaus first identified a group of genes including those related to the Hedgehog signalling pathway and linked them to the segmentation and planning of the embryo in ''Drosophila melanogaster'' by introducing mutagenic substances to the developing embryo<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>.
|-
|'''1993'''
|Multiple researchers including Andrew P. McMahon and Clifford Tabin discovered three equivalent homologs in vertebrates of the ''Drosophila melanogaster'' hedgehog gene, known as sonic hedgehog (Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh), by looking at DNA sequences similar to that of the gene in the fruit fly<ref><pubmed>7916661</pubmed></ref>.
|-
|'''1993'''
|Clifford Tabin and his lab identified the role of Shh in localising the limb bud, where they identified protein to be expressed within a region of the limb bud known as the zone of polarising activity (ZPA), showing that SHH is sufficient to induce the production of a ZPA, and thus limb bud formation in chick embryos<ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
|-
|'''1995'''
|Sonic hedgehog secreted by the notochord was identified to induce ventral cell types in the neural tube during embryonic development, most notably the floor plate cells and the motor neurons as shown in chick embryos. Such differentiation between the two cell types is thought to be a matter of sonic hedgehog concentrations experienced by the neural plate cells<ref name="PMID7736596"><pubmed>7736596</pubmed></ref>.
|-
|'''1995'''
|A shared Nobel prize in physiology or medicine was awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their researching regarding the identification of the developmental genes relating to the formation and patterning of the early embryo via genes.
|-
|'''1996'''
|Studies showed that human homologs of the Patched gene, a component of the hedgehog pathway, is a key gene that is mutated in Gorlin Syndrome, which characterised by a predisposition for basal cell carcinomas, the most common cancers in humans and developmental abnormalities<ref name="PMID8658145"><pubmed>8658145 </pubmed></ref>.
|-
|'''1999'''
|Researchers implicated Indian hedgehog as a key signalling molecule for the maturation and differentiation of prehypertrophic chondrocytes, where Indian hedgehog null mice were shown to have a lack of mature chondrocytes and no development of osteoblast cells in endochondrial bone. This was thought to be due to Indian hedgehog having a feedback signal controlling parathyroid hormone related protein, which regulates endochondrial bone development<ref name="PMID10465785"><pubmed>10465785</pubmed></ref>.
|-
|'''2003'''
|Sonic hedgehog was identified to regulate the proliferation of adult neural stem cells as shown in the hippocampus of rats and in vitro for neural progenitor cells isolated from the hippocampus<ref name="PMID12469128"><pubmed>12469128</pubmed></ref>.
|-
|'''2003'''
|Studies on Shh function found that it can act as a chemoattractant in the developing embryo spinal cord, to help guide commissural axons to the midline of the floor plate<ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
|-
|'''2009'''
|Mouse models showed that reduction in hedgehog signalling allows for transient improved response to chemotherapy with regards to mouse models of human pancreatic ductal adenocarcinoma, a cancer with one of the highest mortality rates in humans<ref name="PMID19460966"><pubmed>19460966</pubmed></ref>.
|}

This time line regarding the discoveries in the hedgehog signalling pathway is by no means exhaustive, but a selection of relatively large discoveries that had a significant impact in the field on the basis of citations.

== Origin of name ==

[[File:Bands of denticles in normal and Hh mutant Drosophila embryo.jpeg|thumb|250px|Differences in patterning of the denticle bands in normal and mutated hedgehog gene variant ''Drosophilia'' embryo]]

The name hedgehog came about when Christiane Nüsslein-Volhard and Eric Wieschaus first identified the group of genes controlling polarization of the segments in the ''Drosophila'' early embryo. They noticed that when they mutated the Hh gene, making it non-functional, that the bands of denticles formed during the early ''Drosophila'' embryo formation became more diffuse as opposed to being distinct normally. The denticles would all clump together forming a single patch on the surface of the embryo, as opposed to normally outlining the distinct segments of the embryo as bands<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. This gave the embryo spiny and prickly look similar to that of the back of a hedgehog.

From there on in the gene was called the hedgehog gene where homologs in vertebrates discovered along the line kept to this naming structure giving various species of hedgehog names to the genes such as Indian hedgehog, and desert hedgehog, with the exception being sonic hedgehog, which is named after a fictional character in the video game “Sonic the Hedgehog”.

== Function ==
===Overview===
{| class="pretty table"
|'''Hedgehog protein homolog'''
|'''Function'''
|-
| Sonic hedgehog [http://www.omim.org/entry/600725 OMIM entry] ||
*Expressed in zone of polarizing activity of the limb bud where it plays a role in helping to pattern the anterior and posterior aspect of the limbs <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
*Based on time and concentration of SHH due to diffusion from the point of secretion in the limb bud, SHH also plays a role in determining digit identity <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>.
*Acts as a chemoattractant to guide commissural axons in the floor plate of the neural tube towards the midline <ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
*Patterns the developing cusps of the teeth and their relative positions in the jaw, and is also essential for the growth of the teeth <ref name="PMID11044393"><pubmed>11044393</pubmed></ref>.
*SHH released from the notochord acts to induce formation of the floor plate, motor neurons, and dopaminergic neurons, as well as signalling the induction of the ventral cell types in the neural tube <ref name="PMID8790332"><pubmed>8790332</pubmed></ref>.
|-
| Desert hedgehog [http://www.omim.org/entry/605423 OMIM entry]||
*Plays a possible role in regulating spermatogenesis as due to the highly localised expression in the testes by pre-Sertoli cells, and the fact that mice with no DHH present are infertile, with no mature spermatozoa <ref name="PMID8805249"><pubmed>8805249</pubmed></ref>.
*DHH derived from Schwann cells signal development of peripheral nerves, by inducing the production of a connective tissue sheath around them <ref name="PMID10482238"><pubmed>10482238</pubmed></ref>.
|-
| Indian hedgehog [http://www.omim.org/entry/600726 OMIM entry]||
*Aid in the signalling of proliferating chondrocytes to undergo hypertrophic differentiation during endochondral ossification<ref name="PMID8662546"><pubmed> 8662546</pubmed></ref>.
*Plays a key role in yolk sac angiogenesis, as seen in studies where an absence of IHH leads to death of the embryo due to poor yolk sac vasculature <ref name="PMID11807029"><pubmed>11807029</pubmed></ref>.
|}

===Limb development===

====Research background====

The hedgehog signalling pathway plays a significant role with regards to the embryonic development of the limbs in vertebrates, specifically SHH. For a while the mechanism of which the limb was patterned on the anterior to posterior axis was quite unknown. Early studies had shown that grafting posterior wing bud cells to the anterior region of the wing bud in a chick embryo, an equal amount of extra digits was produced to that of which was normally produced, and in a mirror image pattern<ref name="PMID4390734"><pubmed>4390734</pubmed></ref>. This showed that the pattern of which the digits were produced were polarized with respect to that of the graft from the limb bud, which has become to be described as the zone of polarizing activity (ZPA). Further studies mapped the ZPA, by showing the area of the wing bud on the chick with the highest polarizing activity to be the posterior margin of the wing bud<ref name="PMID4719010"><pubmed>4719010</pubmed></ref>. Furthermore, studies later on found that the amount or concentration of ZPA cells grafted played a role in the amount of digits induced and how polarized they were<ref name="PMID7453825"><pubmed>7453825</pubmed></ref>. This ultimately gave rise to studies regarding a potential morphogen, which could potentially provide this concentration dependent activity of polarization when it comes to the limb bud. Studies to find this morphogen led to the discover of the ability for retinoic acid to mimic the polarizing action of the ZPA when applied locally to the limb bud<ref name="PMID7070499"><pubmed>7070499</pubmed></ref>. This was found to be due to the induction of a second morphogen known as SHH, where when cells that express SHH are grafted onto the limb bud, polarized digit development is induced<ref name="PMID7720566"><pubmed>7720566</pubmed></ref>.

====Polarizing activity of sonic hedgehog====
[[File: Mechanims of patterning the digits on the posterior to anterior axis of the limb bud by sonic hedgehog.jpg|thumb|450px|Regions of the limb bud and the digits they form based on patterning of the posterior to anterior axis of the limb bud by mechanisms mediated by sonic hedgehog.]]
More recent studies have sought to elucidate how SHH mediates such a polarizing effect in terms of digit identification and its underlying mechanism. It has been suggested that such an effect is heavily concentration dependent similar to that of when transplanting the chick wing bud ZPA cells, acting in a dose dependent matter, due to the ability of SHH to modulate signaling at long ranges via diffusion<ref name="PMID11389830"><pubmed>11389830</pubmed></ref>. This implies that a gradient of varying concentrations of SHH diffused through the mesenchyme of the limb bud leads to the identification of posterior and anterior digits.

Current research has also suggested that not only is the concentration gradient of SHH important for the patterning of the digits, but also the temporal gradient, being the length of time of exposure of SHH to the mesenchyme of the limb bud. Studies have shown in mice that of the 5 digits, polarization from the posterior end, being digit 5, to the anterior end being digit 1 are due to different mechanisms. Digits 5 and 4 have been shown to be comprised of the SHH expressing cells and thus both experience maximal levels of SHH, and thus cannot be distinguished on the basis of a concentration gradient. As a result, temporal gradients are employed, where the expression of SHH in both regions that become the digits vary, where studies have shown that SHH expression in the primordium of digit 5 is maintained longer than that in digit 4 gradient <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>. When it comes to digit 3 and 2, there is differentiation based on the concentration gradient of SHH from the ZPA, where due to the ZPA being further away to digit 2 than 3, the concentration of SHH will be higher at 3<ref name="PMID15315763"><pubmed>15315763</pubmed></ref> <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>. In terms of digit 1, the levels of SHH are so low at this point that it is said to be SHH independent, and actually reliant on the absence of SHH for normal differentiation. With regards to how SHH actually mediates its action, it is thought to be by a balance between the transcription factor Gli3 and SHH expression, which activates Gli1, Gli2 and Gli3 repressor, effectively counteracting Gli3. Gli3 is considered to promote the inhibition of digit formation and identification, while the downstream targets of SHH are thought to promote it<ref name="PMID17400206"><pubmed>17400206</pubmed></ref>.

Other research has also shown the possibility of SHH signaling as a way to prime the posterior mesenchyme and induce the production of bone morphogenetic protein 2 (bmp2), which is thought to be the morphogen creating the chemical gradient for digit identification<ref name="PMID10704381"><pubmed>10704381</pubmed></ref>. Overall the mechanism at which SHH work to pattern the posterior anterior axis of the limb bud is still relatively unknown, and further research is being conducted to elucidate how the underlying mechanisms such as the downstream targets of SHH affect digit formation.

=== Neural development ===

=== Organogenesis ===
The Hedgehog Signalling pathway is a pathway sending information to embryonic cells which plays an especially important role in the regulation of organogenesis. These processes include the organization of the brain (craniofacial) and the growth of appendages with further studies implicating the Hedgehog Signalling pathway in the development of the small intestine, lungs and the pancreas.

== Mechanism ==

=== Processing of precursor ===
 [[File:Processing of Hh precurosr.jpg|thumb|250px|Summary of the steps in the processing of the Hh protein precursor into a functional signalling molecule]]
Hedgehog (Hh) proteins are produced as precursors, which must be processed by the cells producing it before being able to perform its signalling function. The process by which the Hh protein is processed begins with its signal sequence at its N terminus directing the translocation of the precursor into the endoplasmic reticulum, where it is removed via signal peptidase<ref><pubmed>1394430</pubmed></ref>. This process allows the C terminus of the Hh protein to catalyze the cleavage and addition of cholesterol on itself to form a C terminal processing domain and an N terminal processing domain associated with a cholesterol group on its C terminus<ref><pubmed>21357747</pubmed></ref>. The portion associated with the cholesterol will go on to form the signalling molecule, while the C terminal processing domain has no known signalling function<ref><pubmed>8824192</pubmed></ref>.

At this point the Hh protein associated with the cholesterol is able to perform its signalling action, but further modification is still required to ensure efficient signalling. This occurs when the cholesterol group attached to the Hh protein associates with the plasma membrane of the cell, which allows for the addition of a palmitic acid moiety to the N terminal of the protein by an acyl-transferase known as skinny hedgehog<ref><pubmed>11486055</pubmed></ref>. Studies have shown that such an addition allows for an increase in potency in signalling of Hh proteins of 30-fold over its form without palmitic acid added<ref><pubmed>9593755</pubmed></ref>. From this point the Hh protein is now fully active and can either remain associated to the plasma membrane of the cell for autocrine action or be secreted for paracrine action.

=== Mechanism of signalling ===

==== ''Drosophila melanogaster'' ====
 [[File:Hh Signalling Pathway.jpg|thumb|600px|Steps regarding the processing of the hh protein precursor into the fully functional signalling molecule.]]

The Hh signaling pathway has been well studied in ''Drosophila melanogaster'', and has been shown the be conserved to an extent across it and mammals making the species a suitable model for Hh signalling in humans<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. In ''Drosophila melanogaster'', the Hh pathway begins when the Hh proteins bind to the extracellular domain of the transmembrane protein known as Patched (PTC) to inactivate it. This inactivation occurs by Hh trapping PTC in an inactive conformational state<ref><pubmed>27647915</pubmed></ref>. After binding of Hh occurs to PTC and it is inactivated, the receptor and Hh protein is thought to be endocytosed by the cell where they undergo lysosomal degradation in order to limit Hh concentration, thus limiting its spread to other cells and PTC activity<ref name="PMID15102702"><pubmed>15102702</pubmed></ref>. In the absence of Hh, PTC acts to suppress the expression of the Smoothened (SMO), a G-protein coupled receptor like receptor, and thus its signaling. How PTC achieves this inhibition of SMO is currently unclear. Studies have suggested that due to the highly phosphorylated nature of SMO when active, that PTC acts to dephosphorylate SMO in order to repress its signaling. It is thus that binding of Hh to PTC reduces PTCs ability to promote dephosphorylation of SMO, leading to its increased activity and expression on the cell surface <ref name="PMID10966113"><pubmed>10966113</pubmed></ref>. It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) <ref><pubmed>15616566 </pubmed></ref>.

This overall increased phosphorylation of SMO due to a repression of PTC activity by Hh leads to an accumulation of SMO on the cell surface, which collectively allows for SMO to exert its activity. Most importantly phosphorylation also disrupts intramolecular electrostatic interactions between SMO molecules which switches the molecule into its active conformational state <ref><pubmed>17960137 </pubmed></ref>. Generally, SMO has been shown to have very little signal transducing ability, and thus this accumulation allows it to jointly transduce as substantial signal. This occurs by the C terminus of SMO on the intracellular domain interacting with the kinesin like protein Costal-2 (Cos2), which is thought to bind to microtubules in order to acts as a scaffolding protein <ref><pubmed>18691888 </pubmed></ref>. Then the kinase known as Fused (Fu) binds to Cos2, which phosphorylates Suppressor of Fused (SUFU) to inhibit it. Without this inhibition SUFU goes onto prevent the translocation of the transcriptional factor Cubitus interruptus (Ci) into the nucleus by leading to its phosphorylation, and subsequently its partial cleavage via the proteosome. The partial cleavage of Ci leaves a lower molecular weight protein known as Ci repressor (CiR) or Ci75, which translocates into the nucleus and acts to repress the target genes of the Hh signalling pathway via Ci<ref><pubmed>9215627</pubmed></ref>. Thus overall inhibition of SUFU increases the amount of Ci entering into the nucleus of the cell, and activating transcription of target genes of the Hh pathway<ref><pubmed>10952898 </pubmed></ref>.

At this point Ci is able to activate the transcription of various genes, which include importantly the ''ptc'' gene, which encodes the PTC receptor. It is thus as a result of this, that PTC expression will be increased in response to Hh pathway induction, which negatively feeds back to reduce Ci signaling induction, in order to maintain homeostasis and regulate the intensity and duration of the signaling from Hh<ref><pubmed>9053330</pubmed></ref>. Furthermore the ''wg'' gene, encoding the wingless protein is also activated by Ci, which leads to activation of the Wnt pathway via the Frizzled receptor in adjacent cells expressing the gene ''engrailed'' <ref><pubmed>9502727</pubmed></ref> <ref><pubmed>10457026</pubmed></ref>. This overall stabalizes the boundaries between the segments of the developing ''Drosophila melanogaster'' signalled by the gene ''engrailed''<ref><pubmed>3282172</pubmed></ref>.
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=w1xXD9kss2w</html5media>
 
An overview of the hedgehog signalling pathway in ''Drosophila''<ref>YouTube. (2016). The Hedgehog signalling pathway in Drosophila online Available at: https://www.youtube.com/watch?v=w1xXD9kss2w</ref>.

==== Mammals ====

Although the Hh signaling pathway has been conserved across species to an extent <ref name="PMID8595881"><pubmed>8595881</pubmed></ref>, key differences exist when observing the pathway within mammalian tissue in contrast to that which has been studied in ''Drosophila melanogaster''. In mammalian cells and all other vertebrates, Hh signalling is dependent on an organelle known as the primary cilia, which are projections outwards from the cells surface. The importance of such an organelle in the signalling pathway comes as a result of PTC1, one of the two PTC receptor homologs in mammals, that binds to the Hh homolog Sonic Hh (Shh), exists within the cilia<ref><pubmed>17641202</pubmed></ref>. The mechanism of action of SMO inhibition and activation is poorly understood currently, but evidence has suggested that PTC1 acts to inhibit SMO when unbound to Shh by acting as a pump to remove oxysterols from the cilia into the extracellular space. <ref><pubmed>PMC1462959</pubmed></ref>. These oxysterols are normally thought to bind and accumulate around the SMO receptors, which in turn prevents internalization and deactivation of SMO, leading to it accumulating on the apical primary cilia<ref><pubmed>16136078</pubmed></ref>. Other studies have also suggested that Shh has a role in increasing phosphorylation of SMO, which is required for it to accumulate as well on the cilia<ref><pubmed>21695114</pubmed></ref>. Overall the net action of Shh initially is believed to cause accumulation of SMO at the cilia by inhibiting PTCH1 and phosphorylation of SMO.

How SMO acts next is relatively unknown but, it has been shown to promote the disassociation between SUFU and Gli3 transcription factor, which allows Gli to be transported to the nucleus to activate effector genes of the pathway. Gli is normally bound in the cytoplasm to SUFU, where SUFU promotes the partial degradation of Gli into its repressor form, similar to that in the fruit fly model<ref><pubmed>20360384</pubmed></ref>. Alongside this, Kif7, a Cos2 homolog, has been shown to migrate to the apex of the cilium in response to SMO accumulation at the cilium, where it has been postulated to also promote disassociation of SUFU and Gli<ref><pubmed>19592253</pubmed></ref>. On the other hand, Kif7 is also thought to play an inhibitory role for the signaling pathway as when it is localized at the base of the cilia in the absence of Hh signaling and traffics Gli factors away from the cilia<ref><pubmed>19666503</pubmed></ref>. This all leads to an accumulation of active Gli transcription factors which migrate to the nucleus in order either inhibit or activate gene transcription in response to Hh signaling.

== Animal models ==

=== ''Drosophila melanogaster'' ===

Hedgehog (Hh) protein signal was initially discovered through experimentation on the fruit fly, "Drosophila melanogaster". It is through this model that we are able to discover not only the functional components of this pathway but also understand its role in embryonic development. Through application of this information on the human biological system we are able to find the cause and thus potential treatments of defects and diseases caused by interruption or mutation of the Hedgehog signalling pathway.

Upon further research on Drosophila it was found that Hh may play a role in germ cell proliferation and in particular, may control the proliferation and activity of somatic cells found within the germarium, which is the most anterior structure within the Drosophila ovary. <ref>Ovaries (Drosophila) definition. (2016). Groups.molbiosci.northwestern.edu. Retrieved 10 September 2016, from http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-D/Drosophila_Ovaries.html</ref> Thus, the Hh signalling pathway is vital in egg chamber formation and its consequent envelopment, budding and polarisation. It has also been observed that somatic cell proliferation is dependent on this pathway and thus, the number of pre-follicle cells is subject to the effect of the Hh signalling pathway. <ref><pubmed>8620839</pubmed></ref> Further studies have also indicated the role played by this pathway in activating the Epidermal growth factor receptor (EGFR) signalling pathway as seen in the induction of EGFR by Hh in Drosophila head development. <ref><pubmed>10331974</pubmed></ref> In addition, research on Drosophila has indicated the direct effect of Hh signalling on tracheal branch patterning. <ref><pubmed>11290298</pubmed></ref>

Despite ongoing research on "Drosophila melanogaster" to uncover the workings of the Hedgehog signalling pathway, further research is needed to confirm and further current findings. In addition, it is evident that it is through the study of these fruit flies that we are able to gain a basis of understanding of the causes of certain human diseases and thus propel research into treatments for sufferers.

===Blockage of Shh Signalling in Forebrain Neuroectoderm of Chick Embryos===

The Hedgehog signalling pathway plays a significant role in embryonic development, particularly of the forebrain. Due to its role in development of craniofacial features by contributing to the epithelia of the frontonasal, maxillary, and pharyngeal ectoderm, a disruption in this pathway can result in a variety of birth deformities including holoprosencephaly (HPE), where the prosencephalon (forebrain) fails to divide into 2 separate hemispheres, telencephalon and diencephalon <ref>Holoprosencephaly - NORD (National Organization for Rare Disorders). (2016). NORD (National Organization for Rare Disorders). Retrieved 10 September 2016, from http://rarediseases.org/rare-diseases/holoprosencephaly/</ref>, as well as cleft lip and palate. The function of Sonic Hedgehog (Shh) and its signalling pathway on the formation of forebrain neuroectoderm was studied in chick embryos.

It was found that the disruption of Shh signalling in the neural tube of chick embryos resulted in the lack of division of the forebrain into the diencephalon and telencephalon. This is in fact, as stated earlier, the fundamental cause of the rare condition, holoprosencephaly. It was discovered that the Shh signalling pathway in the diencephalon was responsible for gene expression in the telencephalon.

In addition, through experimentation on chick embryos it was found that through Shh signalling the development of the forebrain regulates and controls facial morphogenesis, particularly of the upper and middle face. Therefore, interference with Shh signalling in the forebrain prevents this intrinsic communication, thus hindering Shh expression in facial ectoderm. This results in brain malformation accompanied by facial disfiguration as seen in patients suffering from HPE. Other malformations caused by blockage of Shh signalling in craniofacial development include, hypotelorism (decreased space between the orbits), growth restriction as well as cleft lip and palate as mentioned previously.

A commonality discovered among all forms of Shh signalling disruption was two facial defects, “the loss of mediolateral expansion of the face and absence of proximodistal outgrowth of the frontonasal prominence”<ref><pubmed>15979605</pubmed></ref> (Ralph et al., 2005). Through reference to studies performed on chick and mice embryos, it was found that Shh signalling is particularly vital in development of maxillary and frontonasal components of the cranium.

This breakthrough study on the inhibition of Shh signalling in chick embryos has significantly filled the gap in our understanding of the Sonic Hedgehog signalling pathway. It is evident that this pathway is crucial in the development of the forebrain and in turn regulates and controls the development of the facial skeleton. This is further proven through the observation that in the event of Shh signalling inhibition there is a development of craniofacial malformations.

=== Shh knockout mice ===

Extensive research on Shh knockout mice allowed discovery of the roles of Shh in embryonic development and patterning of the limb buds and sclerotomes, and maintenance of the notochord. <ref><pubmed>8837770</pubmed></ref> These mice expressed defects in the cephalic neural tube with the fusion of telencephalic and optic vesicles. Through this research it is evident that Shh is partially responsible for the subdivision of the eye field through forebrain optic stalk development, along with formation of the ventral midline. It is these forebrain abnormalities established in the absence of Shh that results in congenital malformations of holoprosencephaly, development of a single nasal chamber and other facial defects in humans.

The absence of the vertebral column including, the intervertebral discs and vertebrae along with the medial regions of the ribs were observed within Shh knockout mice. This lack of sclerotome derivatives indicates the role of Shh in maintenance or expansion of sclerotome cell population (Chiang et al., 1996). In addition, though the role of Shh in patterning the anterior-posterior limb axis is evident through anterior limb bud cell death in the absence of Shh, it is also responsible for the development of proximal-distal limb segments, particularly in the patterning of structures at the level of or distal to the latent elbow and knee joints. <ref><pubmed>11476582</pubmed></ref> This was perceived after observing the incomplete formation of distal limb structures with abnormal anterior-posterior axis formation (Chiang et al., 2001). Thus, Shh knockout mice provide great insight into the functions of Shh in embryonic development and the abnormalities formed in the absence of this signalling protein.

== Clinical Significance ==

===Gorlin Syndrome===
[[File:1 week year old newborn girl with cleft lip and palate.png|thumb|An image of a 1 week year old newborn girl with cleft lip and palate]]
Gorlin syndrome, also known as nevoid basal cell carcinoma syndrome, is a heritable disease resulting from heterozygousity in Hh receptor, Patched (PTC). The PTC gene encodes a protein which binds sonic hedgehog (Shh) and it is this binding which inhibits the Hh signalling pathway. However mutation of PTC leads to activation of the pathway <ref><pubmed>9620294</pubmed></ref>. The syndrome is characteristic of skin cancer basal cell carcinoma and cerebellum cancer medulloblastoma. 1-2% of medulloblastomas and 0.5% of basal cell carcinomas are attributable to the disease. Patients present with craniofacial and brain abnormalities such as cleft palate, strabismus, macrocephaly, abnormal development of the corpus callosum and frontal bossing, with an overall overgrown appearance. Skeletal defects such as of the shoulder, ribs and vertebrae are often seen, as well immobile thumbs and polydactyly, which is a deformity of the hand or feet in which they have one or more extra fingers or toes. Apart from physical anomalies, patients have an increased risk of tumours throughout the body including cardiac, ovarian fibromas, ovarian dermoid cysts, meningiomas, and fibrosarcomas. Additionally with time, patients eventually undergo intracranial calcification and dyskeratotic pitting of the hands and feet whereby skin cells prematurely convert to keratin <ref><pubmed>8681379</pubmed></ref>.

===Holoprosencephaly===
 [[File:Semilobar holoprosencephaly.jpg|thumb|240px|MRI of fore brain depicting semilobar holoprosencephaly]]
Holoprosencephaly (HPE) is a congenital disease caused by incomplete division of the prosencephalon (embryonic forebrain) into separate lobes of the cerebral hemispheres. The Shh gene has been identified as a HPE-causing gene however recent evidence has also investigated into the PTC gene, which acts to repress Shh signalling. A gain in repressive action of PTC is seen, leading to decreased Shh signalling, and thus HPE <ref><pubmed>11941477</pubmed></ref>. Clinical expression is variable between patients and is dependent upon the 3 forms of increasing severity: lobar, semi-lobar and alobar HPE, where patients can present with right and left ventricles however with a continuous frontal cortex in lobar HPE, to carrying a single cerebral ventricle in alobar HPE. Along with forebrain abnormalities, facial anomalies are also seen including midline cleft lip and/or flat nose, ocular hypotelorism characteristic of a small distance between the eyes. In severe rare cases of cyclopia, a non-functioning nose may be observed in the form of proboscis and the patient may present with a single eye at the root of the nose <ref><pubmed>20583177</pubmed></ref>. Furthermore, patients with HPE often develop a large number of medical issues including epilepsy, mental retardation, thyroid and adrenal hypoplasia due to lack of hypothalamus or pituitary gland development, and diabetes insipidus.

===Medulloblastoma===
Medulloblastoma is a cancerous tumour of the cerebellum where the cells of origin are the granular cell precursors found in the neonatal cerebellum, explaining its high prevalence in children <ref><pubmed>19171780</pubmed></ref>. Commonly, medulloblastomas are found deep in the cerebellum along the midline, however with desmoplastic medulloblastomas, they are found more laterally and superficial. As seen previously with Gorlin syndrome and holoprosencephaly, PTC is also associated with the development of medulloblastoma, and misregulation of Hh-PTC signalling in cells of the external germinal layer, the layer found on the surface of the cerebellum, is thought to give rise to the tumour. In experimental studies, it has been discovered that PTC is highly transcribed in medulloblastomas and in approximately 30% of PTC heterozygous mice the tumour develops <ref><pubmed>9205058</pubmed></ref> <ref><pubmed>9262482</pubmed></ref>. In patients with medulloblastoma, often children, the most common symptoms seen are vomiting, ataxia, psychomotor regression, drowsiness and anorexia. With age, patients are seen to get more frequent headaches, and show psychological symptoms such as behavioural problems, poor performance in school, and anxiety. In patients exhibiting life-threatening symptoms due to increased intracranial hypertension, bradycardia, convulsions and coma are often seen <ref><pubmed>21537925</pubmed></ref>.

Watch below for a breakdown on Hedgehog's role in Tumour Development!
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=gFWO3I-oqsE</html5media>
 
An overview of the Hedgehog Signalling Pathway in Tumour Development<ref>YouTube. (2016). The Hedgehog Signaling in the Tumor Microenvironment - Research Hedgehog online Available at: https://www.youtube.com/watch?v=gFWO3I-oqsE</ref>.

== Current research ==

==Quiz==

<quiz display=simple>

{1. Which of the following are the reasons that are thought to allow for the patterning of digits in vertebrates.
|type="[]"}
+  Concentration gradient of sonic hedgehog.
+  Temporal gradient of sonic hedgehog.
-  The varying homologs of the hedgehog proteins .
-  The length of the hedgehog molecule.
+  No sonic hedgehog signalling.
-  The type of cholesterol attached to the hedgehog molecule.
|| For differentiation of the individual digits in mice to occur it is thought that each digit requires a different signalling mechanism. Digits 5 and 4 which are most posterior, are derived from the region around the zone of polarising area which contain Shh secreting cells, meaning these regions will experience a saturation of Shh signalling and cannot be differentiated by Shh concentration. As a result it is thought that these digits are identfied based on the temporal gradient, meaning that the length of which Shh is expressed in these regions differentiates the digits from one another with digit 5 expressing Shh for longer. Digits 3 and 2 are further away but still receive Shh signalling due to the diffusible nature of Shh from the ZPA, which forms a concentration gradient. This means the the concentration at digit 2 will be less than digit 3 due to being further away from the ZPA and thus can be differentiated from one another based on this concentration. Finally the digit 1 is differentiated on the basis of not receiving any Shh signalling at all. Thus it is a combination of concentration gradient of Shh, temporal gradient of Shh, and independence from Shh.

{2. Which of the following are correct with regards to the hedgehog signalling pathway in ''Drosophila''.
|type="[]"}
- It occurs at the cillum on the cell surface.
- Hedgehog protein binding to the Patched receptor activates it to inhibit Smoothened.
+ Hedgehog protein binding to the Patched recetpor inhibits it to activate Smoothened.
+ Inhibition of SUFU is critical in allowing Ci to translocate into the nucles and activate downstream targets of the hedgehog pathway.
- The pathway contains 3 homologs including sonic hedgehog, Indian hedgehog, and desert hedgehog.
- The hedgehog protein is initially produced in its fully active form.
|| In terms of how the hedgehog protein acts to produce its effect, it must first bind to Patched which activity is inhibiting Smoothened in the absence of the hedgehog protein. The protein acts to trap Patched in its inactive confirmational state cause internalisation and eventually degradation of the receptor. This relives the dephosporylation of the Smoothened, usually induced by Patched, allowing it to accumulate phosphate groups and thus activate. With regards to Ci translocation, SUFU usually inhibits this by leading to phosphorylation of Ci and eventually its cleavage into Ci repressor. During the activation of the hedgehog pathway there is a binding of Fused to Cos2 which phosphorylates SUFU to inactivate it, allowing Ci to translocate into the nucleus and activate downstream targets.

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==Glossary==

{| class="pretty table"
|-
|'''Term'''
|'''Definition'''
|-
|Acyl-transferase
|A protein responsible for catalyzing the transfer of a palmitic acid moiety to the N-terminus of the hedgehog signalling molecule.
|-
|Casein kinase I (CKI)
|A kinase that is thought to phosphorylate the Smoothened receptor to activate it in the hedgehog signalling pathway.
|-
|Cubitus interruptus (Ci)
|A transcription factor found in the hedgehog signalling pathway of ''Drosophila'', which binds to promoter regions on the DNA in the nucleus to activate target genes of the pathway.
|-
|Costal-2 (Cos2)
|A kinesin like protein which interacts with the C terminus of Smoothened and acts like a scaffolding protein and allows proteins such as Fused to bind to it.
|-
|Denticle
|Bristle like projections off the segments on the ''Drosophila'' embryo.
|-
|Desert hedgehog (Dhh)
|A homolog of the ''Drosophila'' hedgehog protein, which is expressed most prominently in the gonads of vertebrates, where it plays a possible role in regulating spermatogenesis.
|-
|Frizzled
|A receptor that is part of the Wnt signalling pathway, which binds the protein wingless in order to activate signalling cascades.
|-
|Fused (Fu)
|A kinase that binds to Costal-2 and acts to inhbit Suppressor of Fused by phosphorylating it in the hedgehog signalling pathway.
|-
|Gli
|A family of proteins that acts as transcription factors that were originally isolated in human gliboblastoma. The proteins are important mediators in activating target genes for the hedgehog signalling pathway in vertebrates.
|-
|Hedgehog (Hh) protein
|A signalling protein widely expressed in the tissue during embryonic development of vertebrates and some other species such as ''Drosophila'', and acts to pattern and develop tissue. The protein is the primary mediator of the pathway.
|-
|Indian hedgehog (Ihh)
|A signalling protein that is a homolog of the ''Drosophila'' hedgehog protein, that primarily acts in regions where there is endochondrial ossification to aid in the maturation of chondrocytes.
|-
|Kif7
|A homolog of Costal-2 in vertebrates, which acts to regulate the downstream signalling of the hedgehog pathway by causing the disassociation of SUFU and Gli.
|-
|Limb bud
|A structure formed during early embryonic development consisting of ectoderm and mesenchyme representing the early limbs, where signalling processes in the bud will eventually lead to the out growth and patterning of the bud.
|-
|Patched (PTC)
|Patched is a transmembrane receptor expressed on cells that are sensitive to signalling via the hedgehog pathway, where its extracellular domain binds to the hedgehog protein to inactivate it. The receptor it self without hedgehog binding actively inhibits Smoothened.
|-
|Primary cilia
|An organelle that consists of an outward projection from the cell surface, which is thought to be critical for hedgehog signalling in vertebrates as it houses many components of the pathway.
|-
|Protein kinase A (PKA)
|A kinase that is thought to phosphorylate the Smoothened receptor to activate it in hedgehog signalling.
|-
|Smoothened (SMO)
|A G-protein coupled receptor like receptor integral to the hedgehog signalling pathway and acts to set in motion a series of events that lead to the activation of transcriptional factors that activate target genes of the hedgehog pathway.
|-
|Sonic hedgehog (Shh)
|The most widely expressed homolog of the ''Drosophila'' hedgehog protein found in vertebrates, playing a key role in embryonic development of humans.
|-
|Suppressor of Fused (SUFU)
|A protein in the hedgehog signalling pathway which when active, inhibits the translocation of Cubitus interruptus into the nucleus by leading to its phosphorylation, and eventually degradation via the proteosome.
|-
|Wingless protein
|A protein produced as a downstream target of the hedgehog signalling pathway in ''Drosophila'', which activates the Wnt pathway by binding to the Frizzled receptor.
|-
|Zone of polarizing activity (ZPA)
|The posterior region of the limb bud mesenchyme, which aids in the signalling of it to pattern the posterior anterior axis with respect to the digits through mediators such as sonic hedgehog.
|-
|}

===Additional Glossary Links===
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== References ==

2016 Group Project 4

2016-10-25T07:16:28Z

Z5019880: /* Polarizing activity of sonic hedgehog */

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


= Hedgehog signalling pathway =
==Introduction==
[[File:Hedgehog.jpg|thumb|300px|An image of a hedgehog, the animal of which the Hh proteins are named after.]]
The Hedgehog (Hh) signalling pathway is an important part of the early embryo with regards to the patterning and development of the nervous system, limbs and the cranio-facial region in vertebrates and the polarization of the segments in the ''Drosophila'' embryo. It was first identified in 1980 by Christiane Nüsslein-Volhard and Eric Wieschaus, alongside a group of other genes regulating segment polarity in ''Drosophila'' embryonic development<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. The pathway itself consists of Hh proteins, where there are three identified homologs in vertebrates, being sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (DHH) <ref name="PMID7916661"><pubmed>7916661</pubmed></ref>. All homologs are expressed at varying levels within different tissues in the body, and also act as a form of redundancy to an extent between one another.

The signaling pathway is highly conserved between various species<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. and acts via a G protein coupled receptor like receptor, which uninhibited via the action of the Hh proteins. The pathway overall acts via a balance of dephosphorylation<ref name="PMID10966113"><pubmed>10966113</pubmed></ref> and degradation of proteins<ref name="PMID15102702"><pubmed>15102702</pubmed></ref> in order to regulate transcription factor activity to express various genes to carry out its action. The Hh pathway is also highly implicated when it comes to abnormalities in development, as it is implicated in diseases such as holoprosencephaly and cleft lip and palate. Overall Hh signaling pathways play a large role in embryological development, which will be discussed in detail on this page.

== History ==
{| class="pretty table"
| '''1980'''
|Christiane Nüsslein-Volhard and Eric Wieschaus first identified a group of genes including those related to the Hedgehog signalling pathway and linked them to the segmentation and planning of the embryo in ''Drosophila melanogaster'' by introducing mutagenic substances to the developing embryo<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>.
|-
|'''1993'''
|Multiple researchers including Andrew P. McMahon and Clifford Tabin discovered three equivalent homologs in vertebrates of the ''Drosophila melanogaster'' hedgehog gene, known as sonic hedgehog (Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh), by looking at DNA sequences similar to that of the gene in the fruit fly<ref><pubmed>7916661</pubmed></ref>.
|-
|'''1993'''
|Clifford Tabin and his lab identified the role of Shh in localising the limb bud, where they identified protein to be expressed within a region of the limb bud known as the zone of polarising activity (ZPA), showing that SHH is sufficient to induce the production of a ZPA, and thus limb bud formation in chick embryos<ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
|-
|'''1995'''
|Sonic hedgehog secreted by the notochord was identified to induce ventral cell types in the neural tube during embryonic development, most notably the floor plate cells and the motor neurons as shown in chick embryos. Such differentiation between the two cell types is thought to be a matter of sonic hedgehog concentrations experienced by the neural plate cells<ref name="PMID7736596"><pubmed>7736596</pubmed></ref>.
|-
|'''1995'''
|A shared Nobel prize in physiology or medicine was awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their researching regarding the identification of the developmental genes relating to the formation and patterning of the early embryo via genes.
|-
|'''1996'''
|Studies showed that human homologs of the Patched gene, a component of the hedgehog pathway, is a key gene that is mutated in Gorlin Syndrome, which characterised by a predisposition for basal cell carcinomas, the most common cancers in humans and developmental abnormalities<ref name="PMID8658145"><pubmed>8658145 </pubmed></ref>.
|-
|'''1999'''
|Researchers implicated Indian hedgehog as a key signalling molecule for the maturation and differentiation of prehypertrophic chondrocytes, where Indian hedgehog null mice were shown to have a lack of mature chondrocytes and no development of osteoblast cells in endochondrial bone. This was thought to be due to Indian hedgehog having a feedback signal controlling parathyroid hormone related protein, which regulates endochondrial bone development<ref name="PMID10465785"><pubmed>10465785</pubmed></ref>.
|-
|'''2003'''
|Sonic hedgehog was identified to regulate the proliferation of adult neural stem cells as shown in the hippocampus of rats and in vitro for neural progenitor cells isolated from the hippocampus<ref name="PMID12469128"><pubmed>12469128</pubmed></ref>.
|-
|'''2003'''
|Studies on Shh function found that it can act as a chemoattractant in the developing embryo spinal cord, to help guide commissural axons to the midline of the floor plate<ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
|-
|'''2009'''
|Mouse models showed that reduction in hedgehog signalling allows for transient improved response to chemotherapy with regards to mouse models of human pancreatic ductal adenocarcinoma, a cancer with one of the highest mortality rates in humans<ref name="PMID19460966"><pubmed>19460966</pubmed></ref>.
|}

This time line regarding the discoveries in the hedgehog signalling pathway is by no means exhaustive, but a selection of relatively large discoveries that had a significant impact in the field on the basis of citations.

== Origin of name ==

[[File:Bands of denticles in normal and Hh mutant Drosophila embryo.jpeg|thumb|250px|Differences in patterning of the denticle bands in normal and mutated hedgehog gene variant ''Drosophilia'' embryo]]

The name hedgehog came about when Christiane Nüsslein-Volhard and Eric Wieschaus first identified the group of genes controlling polarization of the segments in the ''Drosophila'' early embryo. They noticed that when they mutated the Hh gene, making it non-functional, that the bands of denticles formed during the early ''Drosophila'' embryo formation became more diffuse as opposed to being distinct normally. The denticles would all clump together forming a single patch on the surface of the embryo, as opposed to normally outlining the distinct segments of the embryo as bands<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. This gave the embryo spiny and prickly look similar to that of the back of a hedgehog.

From there on in the gene was called the hedgehog gene where homologs in vertebrates discovered along the line kept to this naming structure giving various species of hedgehog names to the genes such as Indian hedgehog, and desert hedgehog, with the exception being sonic hedgehog, which is named after a fictional character in the video game “Sonic the Hedgehog”.

== Function ==
===Overview===
{| class="pretty table"
|'''Hedgehog protein homolog'''
|'''Function'''
|-
| Sonic hedgehog [http://www.omim.org/entry/600725 OMIM entry] ||
*Expressed in zone of polarizing activity of the limb bud where it plays a role in helping to pattern the anterior and posterior aspect of the limbs <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
*Based on time and concentration of SHH due to diffusion from the point of secretion in the limb bud, SHH also plays a role in determining digit identity <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>.
*Acts as a chemoattractant to guide commissural axons in the floor plate of the neural tube towards the midline <ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
*Patterns the developing cusps of the teeth and their relative positions in the jaw, and is also essential for the growth of the teeth <ref name="PMID11044393"><pubmed>11044393</pubmed></ref>.
*SHH released from the notochord acts to induce formation of the floor plate, motor neurons, and dopaminergic neurons, as well as signalling the induction of the ventral cell types in the neural tube <ref name="PMID8790332"><pubmed>8790332</pubmed></ref>.
|-
| Desert hedgehog [http://www.omim.org/entry/605423 OMIM entry]||
*Plays a possible role in regulating spermatogenesis as due to the highly localised expression in the testes by pre-Sertoli cells, and the fact that mice with no DHH present are infertile, with no mature spermatozoa <ref name="PMID8805249"><pubmed>8805249</pubmed></ref>.
*DHH derived from Schwann cells signal development of peripheral nerves, by inducing the production of a connective tissue sheath around them <ref name="PMID10482238"><pubmed>10482238</pubmed></ref>.
|-
| Indian hedgehog [http://www.omim.org/entry/600726 OMIM entry]||
*Aid in the signalling of proliferating chondrocytes to undergo hypertrophic differentiation during endochondral ossification<ref name="PMID8662546"><pubmed> 8662546</pubmed></ref>.
*Plays a key role in yolk sac angiogenesis, as seen in studies where an absence of IHH leads to death of the embryo due to poor yolk sac vasculature <ref name="PMID11807029"><pubmed>11807029</pubmed></ref>.
|}

===Limb development===

====Research background====

The hedgehog signalling pathway plays a significant role with regards to the embryonic development of the limbs in vertebrates, specifically SHH. For a while the mechanism of which the limb was patterned on the anterior to posterior axis was quite unknown. Early studies had shown that grafting posterior wing bud cells to the anterior region of the wing bud in a chick embryo, an equal amount of extra digits was produced to that of which was normally produced, and in a mirror image pattern<ref name="PMID4390734"><pubmed>4390734</pubmed></ref>. This showed that the pattern of which the digits were produced were polarized with respect to that of the graft from the limb bud, which has become to be described as the zone of polarizing activity (ZPA). Further studies mapped the ZPA, by showing the area of the wing bud on the chick with the highest polarizing activity to be the posterior margin of the wing bud<ref name="PMID4719010"><pubmed>4719010</pubmed></ref>. Furthermore, studies later on found that the amount or concentration of ZPA cells grafted played a role in the amount of digits induced and how polarized they were<ref name="PMID7453825"><pubmed>7453825</pubmed></ref>. This ultimately gave rise to studies regarding a potential morphogen, which could potentially provide this concentration dependent activity of polarization when it comes to the limb bud. Studies to find this morphogen led to the discover of the ability for retinoic acid to mimic the polarizing action of the ZPA when applied locally to the limb bud<ref name="PMID7070499"><pubmed>7070499</pubmed></ref>. This was found to be due to the induction of a second morphogen known as SHH, where when cells that express SHH are grafted onto the limb bud, polarized digit development is induced<ref name="PMID7720566"><pubmed>7720566</pubmed></ref>.

====Polarizing activity of sonic hedgehog====
[[File: Mechanisms of patterning the digits on the posterior to anterior axis of the limb bud by sonic hedgehog.jpg|thumb|450px|Regions of the limb bud and the digits they form based on patterning of the posterior to anterior axis of the limb bud by mechanisms mediated by sonic hedgehog.]]
More recent studies have sought to elucidate how SHH mediates such a polarizing effect in terms of digit identification and its underlying mechanism. It has been suggested that such an effect is heavily concentration dependent similar to that of when transplanting the chick wing bud ZPA cells, acting in a dose dependent matter, due to the ability of SHH to modulate signaling at long ranges via diffusion<ref name="PMID11389830"><pubmed>11389830</pubmed></ref>. This implies that a gradient of varying concentrations of SHH diffused through the mesenchyme of the limb bud leads to the identification of posterior and anterior digits.

Current research has also suggested that not only is the concentration gradient of SHH important for the patterning of the digits, but also the temporal gradient, being the length of time of exposure of SHH to the mesenchyme of the limb bud. Studies have shown in mice that of the 5 digits, polarization from the posterior end, being digit 5, to the anterior end being digit 1 are due to different mechanisms. Digits 5 and 4 have been shown to be comprised of the SHH expressing cells and thus both experience maximal levels of SHH, and thus cannot be distinguished on the basis of a concentration gradient. As a result, temporal gradients are employed, where the expression of SHH in both regions that become the digits vary, where studies have shown that SHH expression in the primordium of digit 5 is maintained longer than that in digit 4 gradient <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>. When it comes to digit 3 and 2, there is differentiation based on the concentration gradient of SHH from the ZPA, where due to the ZPA being further away to digit 2 than 3, the concentration of SHH will be higher at 3<ref name="PMID15315763"><pubmed>15315763</pubmed></ref> <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>. In terms of digit 1, the levels of SHH are so low at this point that it is said to be SHH independent, and actually reliant on the absence of SHH for normal differentiation. With regards to how SHH actually mediates its action, it is thought to be by a balance between the transcription factor Gli3 and SHH expression, which activates Gli1, Gli2 and Gli3 repressor, effectively counteracting Gli3. Gli3 is considered to promote the inhibition of digit formation and identification, while the downstream targets of SHH are thought to promote it<ref name="PMID17400206"><pubmed>17400206</pubmed></ref>.

Other research has also shown the possibility of SHH signaling as a way to prime the posterior mesenchyme and induce the production of bone morphogenetic protein 2 (bmp2), which is thought to be the morphogen creating the chemical gradient for digit identification<ref name="PMID10704381"><pubmed>10704381</pubmed></ref>. Overall the mechanism at which SHH work to pattern the posterior anterior axis of the limb bud is still relatively unknown, and further research is being conducted to elucidate how the underlying mechanisms such as the downstream targets of SHH affect digit formation.

=== Neural development ===

=== Organogenesis ===
The Hedgehog Signalling pathway is a pathway sending information to embryonic cells which plays an especially important role in the regulation of organogenesis. These processes include the organization of the brain (craniofacial) and the growth of appendages with further studies implicating the Hedgehog Signalling pathway in the development of the small intestine, lungs and the pancreas.

== Mechanism ==

=== Processing of precursor ===
 [[File:Processing of Hh precurosr.jpg|thumb|250px|Summary of the steps in the processing of the Hh protein precursor into a functional signalling molecule]]
Hedgehog (Hh) proteins are produced as precursors, which must be processed by the cells producing it before being able to perform its signalling function. The process by which the Hh protein is processed begins with its signal sequence at its N terminus directing the translocation of the precursor into the endoplasmic reticulum, where it is removed via signal peptidase<ref><pubmed>1394430</pubmed></ref>. This process allows the C terminus of the Hh protein to catalyze the cleavage and addition of cholesterol on itself to form a C terminal processing domain and an N terminal processing domain associated with a cholesterol group on its C terminus<ref><pubmed>21357747</pubmed></ref>. The portion associated with the cholesterol will go on to form the signalling molecule, while the C terminal processing domain has no known signalling function<ref><pubmed>8824192</pubmed></ref>.

At this point the Hh protein associated with the cholesterol is able to perform its signalling action, but further modification is still required to ensure efficient signalling. This occurs when the cholesterol group attached to the Hh protein associates with the plasma membrane of the cell, which allows for the addition of a palmitic acid moiety to the N terminal of the protein by an acyl-transferase known as skinny hedgehog<ref><pubmed>11486055</pubmed></ref>. Studies have shown that such an addition allows for an increase in potency in signalling of Hh proteins of 30-fold over its form without palmitic acid added<ref><pubmed>9593755</pubmed></ref>. From this point the Hh protein is now fully active and can either remain associated to the plasma membrane of the cell for autocrine action or be secreted for paracrine action.

=== Mechanism of signalling ===

==== ''Drosophila melanogaster'' ====
 [[File:Hh Signalling Pathway.jpg|thumb|600px|Steps regarding the processing of the hh protein precursor into the fully functional signalling molecule.]]

The Hh signaling pathway has been well studied in ''Drosophila melanogaster'', and has been shown the be conserved to an extent across it and mammals making the species a suitable model for Hh signalling in humans<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. In ''Drosophila melanogaster'', the Hh pathway begins when the Hh proteins bind to the extracellular domain of the transmembrane protein known as Patched (PTC) to inactivate it. This inactivation occurs by Hh trapping PTC in an inactive conformational state<ref><pubmed>27647915</pubmed></ref>. After binding of Hh occurs to PTC and it is inactivated, the receptor and Hh protein is thought to be endocytosed by the cell where they undergo lysosomal degradation in order to limit Hh concentration, thus limiting its spread to other cells and PTC activity<ref name="PMID15102702"><pubmed>15102702</pubmed></ref>. In the absence of Hh, PTC acts to suppress the expression of the Smoothened (SMO), a G-protein coupled receptor like receptor, and thus its signaling. How PTC achieves this inhibition of SMO is currently unclear. Studies have suggested that due to the highly phosphorylated nature of SMO when active, that PTC acts to dephosphorylate SMO in order to repress its signaling. It is thus that binding of Hh to PTC reduces PTCs ability to promote dephosphorylation of SMO, leading to its increased activity and expression on the cell surface <ref name="PMID10966113"><pubmed>10966113</pubmed></ref>. It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) <ref><pubmed>15616566 </pubmed></ref>.

This overall increased phosphorylation of SMO due to a repression of PTC activity by Hh leads to an accumulation of SMO on the cell surface, which collectively allows for SMO to exert its activity. Most importantly phosphorylation also disrupts intramolecular electrostatic interactions between SMO molecules which switches the molecule into its active conformational state <ref><pubmed>17960137 </pubmed></ref>. Generally, SMO has been shown to have very little signal transducing ability, and thus this accumulation allows it to jointly transduce as substantial signal. This occurs by the C terminus of SMO on the intracellular domain interacting with the kinesin like protein Costal-2 (Cos2), which is thought to bind to microtubules in order to acts as a scaffolding protein <ref><pubmed>18691888 </pubmed></ref>. Then the kinase known as Fused (Fu) binds to Cos2, which phosphorylates Suppressor of Fused (SUFU) to inhibit it. Without this inhibition SUFU goes onto prevent the translocation of the transcriptional factor Cubitus interruptus (Ci) into the nucleus by leading to its phosphorylation, and subsequently its partial cleavage via the proteosome. The partial cleavage of Ci leaves a lower molecular weight protein known as Ci repressor (CiR) or Ci75, which translocates into the nucleus and acts to repress the target genes of the Hh signalling pathway via Ci<ref><pubmed>9215627</pubmed></ref>. Thus overall inhibition of SUFU increases the amount of Ci entering into the nucleus of the cell, and activating transcription of target genes of the Hh pathway<ref><pubmed>10952898 </pubmed></ref>.

At this point Ci is able to activate the transcription of various genes, which include importantly the ''ptc'' gene, which encodes the PTC receptor. It is thus as a result of this, that PTC expression will be increased in response to Hh pathway induction, which negatively feeds back to reduce Ci signaling induction, in order to maintain homeostasis and regulate the intensity and duration of the signaling from Hh<ref><pubmed>9053330</pubmed></ref>. Furthermore the ''wg'' gene, encoding the wingless protein is also activated by Ci, which leads to activation of the Wnt pathway via the Frizzled receptor in adjacent cells expressing the gene ''engrailed'' <ref><pubmed>9502727</pubmed></ref> <ref><pubmed>10457026</pubmed></ref>. This overall stabalizes the boundaries between the segments of the developing ''Drosophila melanogaster'' signalled by the gene ''engrailed''<ref><pubmed>3282172</pubmed></ref>.
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=w1xXD9kss2w</html5media>
 
An overview of the hedgehog signalling pathway in ''Drosophila''<ref>YouTube. (2016). The Hedgehog signalling pathway in Drosophila online Available at: https://www.youtube.com/watch?v=w1xXD9kss2w</ref>.

==== Mammals ====

Although the Hh signaling pathway has been conserved across species to an extent <ref name="PMID8595881"><pubmed>8595881</pubmed></ref>, key differences exist when observing the pathway within mammalian tissue in contrast to that which has been studied in ''Drosophila melanogaster''. In mammalian cells and all other vertebrates, Hh signalling is dependent on an organelle known as the primary cilia, which are projections outwards from the cells surface. The importance of such an organelle in the signalling pathway comes as a result of PTC1, one of the two PTC receptor homologs in mammals, that binds to the Hh homolog Sonic Hh (Shh), exists within the cilia<ref><pubmed>17641202</pubmed></ref>. The mechanism of action of SMO inhibition and activation is poorly understood currently, but evidence has suggested that PTC1 acts to inhibit SMO when unbound to Shh by acting as a pump to remove oxysterols from the cilia into the extracellular space. <ref><pubmed>PMC1462959</pubmed></ref>. These oxysterols are normally thought to bind and accumulate around the SMO receptors, which in turn prevents internalization and deactivation of SMO, leading to it accumulating on the apical primary cilia<ref><pubmed>16136078</pubmed></ref>. Other studies have also suggested that Shh has a role in increasing phosphorylation of SMO, which is required for it to accumulate as well on the cilia<ref><pubmed>21695114</pubmed></ref>. Overall the net action of Shh initially is believed to cause accumulation of SMO at the cilia by inhibiting PTCH1 and phosphorylation of SMO.

How SMO acts next is relatively unknown but, it has been shown to promote the disassociation between SUFU and Gli3 transcription factor, which allows Gli to be transported to the nucleus to activate effector genes of the pathway. Gli is normally bound in the cytoplasm to SUFU, where SUFU promotes the partial degradation of Gli into its repressor form, similar to that in the fruit fly model<ref><pubmed>20360384</pubmed></ref>. Alongside this, Kif7, a Cos2 homolog, has been shown to migrate to the apex of the cilium in response to SMO accumulation at the cilium, where it has been postulated to also promote disassociation of SUFU and Gli<ref><pubmed>19592253</pubmed></ref>. On the other hand, Kif7 is also thought to play an inhibitory role for the signaling pathway as when it is localized at the base of the cilia in the absence of Hh signaling and traffics Gli factors away from the cilia<ref><pubmed>19666503</pubmed></ref>. This all leads to an accumulation of active Gli transcription factors which migrate to the nucleus in order either inhibit or activate gene transcription in response to Hh signaling.

== Animal models ==

=== ''Drosophila melanogaster'' ===

Hedgehog (Hh) protein signal was initially discovered through experimentation on the fruit fly, "Drosophila melanogaster". It is through this model that we are able to discover not only the functional components of this pathway but also understand its role in embryonic development. Through application of this information on the human biological system we are able to find the cause and thus potential treatments of defects and diseases caused by interruption or mutation of the Hedgehog signalling pathway.

Upon further research on Drosophila it was found that Hh may play a role in germ cell proliferation and in particular, may control the proliferation and activity of somatic cells found within the germarium, which is the most anterior structure within the Drosophila ovary. <ref>Ovaries (Drosophila) definition. (2016). Groups.molbiosci.northwestern.edu. Retrieved 10 September 2016, from http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-D/Drosophila_Ovaries.html</ref> Thus, the Hh signalling pathway is vital in egg chamber formation and its consequent envelopment, budding and polarisation. It has also been observed that somatic cell proliferation is dependent on this pathway and thus, the number of pre-follicle cells is subject to the effect of the Hh signalling pathway. <ref><pubmed>8620839</pubmed></ref> Further studies have also indicated the role played by this pathway in activating the Epidermal growth factor receptor (EGFR) signalling pathway as seen in the induction of EGFR by Hh in Drosophila head development. <ref><pubmed>10331974</pubmed></ref> In addition, research on Drosophila has indicated the direct effect of Hh signalling on tracheal branch patterning. <ref><pubmed>11290298</pubmed></ref>

Despite ongoing research on "Drosophila melanogaster" to uncover the workings of the Hedgehog signalling pathway, further research is needed to confirm and further current findings. In addition, it is evident that it is through the study of these fruit flies that we are able to gain a basis of understanding of the causes of certain human diseases and thus propel research into treatments for sufferers.

===Blockage of Shh Signalling in Forebrain Neuroectoderm of Chick Embryos===

The Hedgehog signalling pathway plays a significant role in embryonic development, particularly of the forebrain. Due to its role in development of craniofacial features by contributing to the epithelia of the frontonasal, maxillary, and pharyngeal ectoderm, a disruption in this pathway can result in a variety of birth deformities including holoprosencephaly (HPE), where the prosencephalon (forebrain) fails to divide into 2 separate hemispheres, telencephalon and diencephalon <ref>Holoprosencephaly - NORD (National Organization for Rare Disorders). (2016). NORD (National Organization for Rare Disorders). Retrieved 10 September 2016, from http://rarediseases.org/rare-diseases/holoprosencephaly/</ref>, as well as cleft lip and palate. The function of Sonic Hedgehog (Shh) and its signalling pathway on the formation of forebrain neuroectoderm was studied in chick embryos.

It was found that the disruption of Shh signalling in the neural tube of chick embryos resulted in the lack of division of the forebrain into the diencephalon and telencephalon. This is in fact, as stated earlier, the fundamental cause of the rare condition, holoprosencephaly. It was discovered that the Shh signalling pathway in the diencephalon was responsible for gene expression in the telencephalon.

In addition, through experimentation on chick embryos it was found that through Shh signalling the development of the forebrain regulates and controls facial morphogenesis, particularly of the upper and middle face. Therefore, interference with Shh signalling in the forebrain prevents this intrinsic communication, thus hindering Shh expression in facial ectoderm. This results in brain malformation accompanied by facial disfiguration as seen in patients suffering from HPE. Other malformations caused by blockage of Shh signalling in craniofacial development include, hypotelorism (decreased space between the orbits), growth restriction as well as cleft lip and palate as mentioned previously.

A commonality discovered among all forms of Shh signalling disruption was two facial defects, “the loss of mediolateral expansion of the face and absence of proximodistal outgrowth of the frontonasal prominence”<ref><pubmed>15979605</pubmed></ref> (Ralph et al., 2005). Through reference to studies performed on chick and mice embryos, it was found that Shh signalling is particularly vital in development of maxillary and frontonasal components of the cranium.

This breakthrough study on the inhibition of Shh signalling in chick embryos has significantly filled the gap in our understanding of the Sonic Hedgehog signalling pathway. It is evident that this pathway is crucial in the development of the forebrain and in turn regulates and controls the development of the facial skeleton. This is further proven through the observation that in the event of Shh signalling inhibition there is a development of craniofacial malformations.

=== Shh knockout mice ===

Extensive research on Shh knockout mice allowed discovery of the roles of Shh in embryonic development and patterning of the limb buds and sclerotomes, and maintenance of the notochord. <ref><pubmed>8837770</pubmed></ref> These mice expressed defects in the cephalic neural tube with the fusion of telencephalic and optic vesicles. Through this research it is evident that Shh is partially responsible for the subdivision of the eye field through forebrain optic stalk development, along with formation of the ventral midline. It is these forebrain abnormalities established in the absence of Shh that results in congenital malformations of holoprosencephaly, development of a single nasal chamber and other facial defects in humans.

The absence of the vertebral column including, the intervertebral discs and vertebrae along with the medial regions of the ribs were observed within Shh knockout mice. This lack of sclerotome derivatives indicates the role of Shh in maintenance or expansion of sclerotome cell population (Chiang et al., 1996). In addition, though the role of Shh in patterning the anterior-posterior limb axis is evident through anterior limb bud cell death in the absence of Shh, it is also responsible for the development of proximal-distal limb segments, particularly in the patterning of structures at the level of or distal to the latent elbow and knee joints. <ref><pubmed>11476582</pubmed></ref> This was perceived after observing the incomplete formation of distal limb structures with abnormal anterior-posterior axis formation (Chiang et al., 2001). Thus, Shh knockout mice provide great insight into the functions of Shh in embryonic development and the abnormalities formed in the absence of this signalling protein.

== Clinical Significance ==

===Gorlin Syndrome===
[[File:1 week year old newborn girl with cleft lip and palate.png|thumb|An image of a 1 week year old newborn girl with cleft lip and palate]]
Gorlin syndrome, also known as nevoid basal cell carcinoma syndrome, is a heritable disease resulting from heterozygousity in Hh receptor, Patched (PTC). The PTC gene encodes a protein which binds sonic hedgehog (Shh) and it is this binding which inhibits the Hh signalling pathway. However mutation of PTC leads to activation of the pathway <ref><pubmed>9620294</pubmed></ref>. The syndrome is characteristic of skin cancer basal cell carcinoma and cerebellum cancer medulloblastoma. 1-2% of medulloblastomas and 0.5% of basal cell carcinomas are attributable to the disease. Patients present with craniofacial and brain abnormalities such as cleft palate, strabismus, macrocephaly, abnormal development of the corpus callosum and frontal bossing, with an overall overgrown appearance. Skeletal defects such as of the shoulder, ribs and vertebrae are often seen, as well immobile thumbs and polydactyly, which is a deformity of the hand or feet in which they have one or more extra fingers or toes. Apart from physical anomalies, patients have an increased risk of tumours throughout the body including cardiac, ovarian fibromas, ovarian dermoid cysts, meningiomas, and fibrosarcomas. Additionally with time, patients eventually undergo intracranial calcification and dyskeratotic pitting of the hands and feet whereby skin cells prematurely convert to keratin <ref><pubmed>8681379</pubmed></ref>.

===Holoprosencephaly===
 [[File:Semilobar holoprosencephaly.jpg|thumb|240px|MRI of fore brain depicting semilobar holoprosencephaly]]
Holoprosencephaly (HPE) is a congenital disease caused by incomplete division of the prosencephalon (embryonic forebrain) into separate lobes of the cerebral hemispheres. The Shh gene has been identified as a HPE-causing gene however recent evidence has also investigated into the PTC gene, which acts to repress Shh signalling. A gain in repressive action of PTC is seen, leading to decreased Shh signalling, and thus HPE <ref><pubmed>11941477</pubmed></ref>. Clinical expression is variable between patients and is dependent upon the 3 forms of increasing severity: lobar, semi-lobar and alobar HPE, where patients can present with right and left ventricles however with a continuous frontal cortex in lobar HPE, to carrying a single cerebral ventricle in alobar HPE. Along with forebrain abnormalities, facial anomalies are also seen including midline cleft lip and/or flat nose, ocular hypotelorism characteristic of a small distance between the eyes. In severe rare cases of cyclopia, a non-functioning nose may be observed in the form of proboscis and the patient may present with a single eye at the root of the nose <ref><pubmed>20583177</pubmed></ref>. Furthermore, patients with HPE often develop a large number of medical issues including epilepsy, mental retardation, thyroid and adrenal hypoplasia due to lack of hypothalamus or pituitary gland development, and diabetes insipidus.

===Medulloblastoma===
Medulloblastoma is a cancerous tumour of the cerebellum where the cells of origin are the granular cell precursors found in the neonatal cerebellum, explaining its high prevalence in children <ref><pubmed>19171780</pubmed></ref>. Commonly, medulloblastomas are found deep in the cerebellum along the midline, however with desmoplastic medulloblastomas, they are found more laterally and superficial. As seen previously with Gorlin syndrome and holoprosencephaly, PTC is also associated with the development of medulloblastoma, and misregulation of Hh-PTC signalling in cells of the external germinal layer, the layer found on the surface of the cerebellum, is thought to give rise to the tumour. In experimental studies, it has been discovered that PTC is highly transcribed in medulloblastomas and in approximately 30% of PTC heterozygous mice the tumour develops <ref><pubmed>9205058</pubmed></ref> <ref><pubmed>9262482</pubmed></ref>. In patients with medulloblastoma, often children, the most common symptoms seen are vomiting, ataxia, psychomotor regression, drowsiness and anorexia. With age, patients are seen to get more frequent headaches, and show psychological symptoms such as behavioural problems, poor performance in school, and anxiety. In patients exhibiting life-threatening symptoms due to increased intracranial hypertension, bradycardia, convulsions and coma are often seen <ref><pubmed>21537925</pubmed></ref>.

Watch below for a breakdown on Hedgehog's role in Tumour Development!
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=gFWO3I-oqsE</html5media>
 
An overview of the Hedgehog Signalling Pathway in Tumour Development<ref>YouTube. (2016). The Hedgehog Signaling in the Tumor Microenvironment - Research Hedgehog online Available at: https://www.youtube.com/watch?v=gFWO3I-oqsE</ref>.

== Current research ==

==Quiz==

<quiz display=simple>

{1. Which of the following are the reasons that are thought to allow for the patterning of digits in vertebrates.
|type="[]"}
+  Concentration gradient of sonic hedgehog.
+  Temporal gradient of sonic hedgehog.
-  The varying homologs of the hedgehog proteins .
-  The length of the hedgehog molecule.
+  No sonic hedgehog signalling.
-  The type of cholesterol attached to the hedgehog molecule.
|| For differentiation of the individual digits in mice to occur it is thought that each digit requires a different signalling mechanism. Digits 5 and 4 which are most posterior, are derived from the region around the zone of polarising area which contain Shh secreting cells, meaning these regions will experience a saturation of Shh signalling and cannot be differentiated by Shh concentration. As a result it is thought that these digits are identfied based on the temporal gradient, meaning that the length of which Shh is expressed in these regions differentiates the digits from one another with digit 5 expressing Shh for longer. Digits 3 and 2 are further away but still receive Shh signalling due to the diffusible nature of Shh from the ZPA, which forms a concentration gradient. This means the the concentration at digit 2 will be less than digit 3 due to being further away from the ZPA and thus can be differentiated from one another based on this concentration. Finally the digit 1 is differentiated on the basis of not receiving any Shh signalling at all. Thus it is a combination of concentration gradient of Shh, temporal gradient of Shh, and independence from Shh.

{2. Which of the following are correct with regards to the hedgehog signalling pathway in ''Drosophila''.
|type="[]"}
- It occurs at the cillum on the cell surface.
- Hedgehog protein binding to the Patched receptor activates it to inhibit Smoothened.
+ Hedgehog protein binding to the Patched recetpor inhibits it to activate Smoothened.
+ Inhibition of SUFU is critical in allowing Ci to translocate into the nucles and activate downstream targets of the hedgehog pathway.
- The pathway contains 3 homologs including sonic hedgehog, Indian hedgehog, and desert hedgehog.
- The hedgehog protein is initially produced in its fully active form.
|| In terms of how the hedgehog protein acts to produce its effect, it must first bind to Patched which activity is inhibiting Smoothened in the absence of the hedgehog protein. The protein acts to trap Patched in its inactive confirmational state cause internalisation and eventually degradation of the receptor. This relives the dephosporylation of the Smoothened, usually induced by Patched, allowing it to accumulate phosphate groups and thus activate. With regards to Ci translocation, SUFU usually inhibits this by leading to phosphorylation of Ci and eventually its cleavage into Ci repressor. During the activation of the hedgehog pathway there is a binding of Fused to Cos2 which phosphorylates SUFU to inactivate it, allowing Ci to translocate into the nucleus and activate downstream targets.

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{Question 5 (select one or more options)
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</quiz>

==Glossary==

{| class="pretty table"
|-
|'''Term'''
|'''Definition'''
|-
|Acyl-transferase
|A protein responsible for catalyzing the transfer of a palmitic acid moiety to the N-terminus of the hedgehog signalling molecule.
|-
|Casein kinase I (CKI)
|A kinase that is thought to phosphorylate the Smoothened receptor to activate it in the hedgehog signalling pathway.
|-
|Cubitus interruptus (Ci)
|A transcription factor found in the hedgehog signalling pathway of ''Drosophila'', which binds to promoter regions on the DNA in the nucleus to activate target genes of the pathway.
|-
|Costal-2 (Cos2)
|A kinesin like protein which interacts with the C terminus of Smoothened and acts like a scaffolding protein and allows proteins such as Fused to bind to it.
|-
|Denticle
|Bristle like projections off the segments on the ''Drosophila'' embryo.
|-
|Desert hedgehog (Dhh)
|A homolog of the ''Drosophila'' hedgehog protein, which is expressed most prominently in the gonads of vertebrates, where it plays a possible role in regulating spermatogenesis.
|-
|Frizzled
|A receptor that is part of the Wnt signalling pathway, which binds the protein wingless in order to activate signalling cascades.
|-
|Fused (Fu)
|A kinase that binds to Costal-2 and acts to inhbit Suppressor of Fused by phosphorylating it in the hedgehog signalling pathway.
|-
|Gli
|A family of proteins that acts as transcription factors that were originally isolated in human gliboblastoma. The proteins are important mediators in activating target genes for the hedgehog signalling pathway in vertebrates.
|-
|Hedgehog (Hh) protein
|A signalling protein widely expressed in the tissue during embryonic development of vertebrates and some other species such as ''Drosophila'', and acts to pattern and develop tissue. The protein is the primary mediator of the pathway.
|-
|Indian hedgehog (Ihh)
|A signalling protein that is a homolog of the ''Drosophila'' hedgehog protein, that primarily acts in regions where there is endochondrial ossification to aid in the maturation of chondrocytes.
|-
|Kif7
|A homolog of Costal-2 in vertebrates, which acts to regulate the downstream signalling of the hedgehog pathway by causing the disassociation of SUFU and Gli.
|-
|Limb bud
|A structure formed during early embryonic development consisting of ectoderm and mesenchyme representing the early limbs, where signalling processes in the bud will eventually lead to the out growth and patterning of the bud.
|-
|Patched (PTC)
|Patched is a transmembrane receptor expressed on cells that are sensitive to signalling via the hedgehog pathway, where its extracellular domain binds to the hedgehog protein to inactivate it. The receptor it self without hedgehog binding actively inhibits Smoothened.
|-
|Primary cilia
|An organelle that consists of an outward projection from the cell surface, which is thought to be critical for hedgehog signalling in vertebrates as it houses many components of the pathway.
|-
|Protein kinase A (PKA)
|A kinase that is thought to phosphorylate the Smoothened receptor to activate it in hedgehog signalling.
|-
|Smoothened (SMO)
|A G-protein coupled receptor like receptor integral to the hedgehog signalling pathway and acts to set in motion a series of events that lead to the activation of transcriptional factors that activate target genes of the hedgehog pathway.
|-
|Sonic hedgehog (Shh)
|The most widely expressed homolog of the ''Drosophila'' hedgehog protein found in vertebrates, playing a key role in embryonic development of humans.
|-
|Suppressor of Fused (SUFU)
|A protein in the hedgehog signalling pathway which when active, inhibits the translocation of Cubitus interruptus into the nucleus by leading to its phosphorylation, and eventually degradation via the proteosome.
|-
|Wingless protein
|A protein produced as a downstream target of the hedgehog signalling pathway in ''Drosophila'', which activates the Wnt pathway by binding to the Frizzled receptor.
|-
|Zone of polarizing activity (ZPA)
|The posterior region of the limb bud mesenchyme, which aids in the signalling of it to pattern the posterior anterior axis with respect to the digits through mediators such as sonic hedgehog.
|-
|}

===Additional Glossary Links===
[[A|A]] | [[B|B]] | [[C|C]] | [[D|D]] | [[E|E]] | [[F|F]] | [[G|G]] | [[H|H]] | [[I|I]] | [[J|J]] | [[K|K]] | [[L|L]] | [[M|M]] | [[N|N]] | [[O|O]] | [[P|P]] | [[Q|Q]] | [[R|R]] | [[S|S]] | [[T|T]] | [[U|U]] | [[V|V]] | [[W|W]] | [[X|X]] | [[Y|Y]] | [[Z|Z]] | [[Numbers|#]]

== References ==

2016-10-24T07:41:56Z

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= Hedgehog signalling pathway =
==Introduction==
[[File:Hedgehog.jpg|thumb|300px|An image of a hedgehog, the animal of which the Hh proteins are named after.]]
The Hedgehog (Hh) signalling pathway is an important part of the early embryo with regards to the patterning and development of the nervous system, limbs and the cranio-facial region in vertebrates and the polarization of the segments in the ''Drosophila'' embryo. It was first identified in 1980 by Christiane Nüsslein-Volhard and Eric Wieschaus, alongside a group of other genes regulating segment polarity in ''Drosophila'' embryonic development<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. The pathway itself consists of Hh proteins, where there are three identified homologs in vertebrates, being sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (DHH) <ref name="PMID7916661"><pubmed>7916661</pubmed></ref>. All homologs are expressed at varying levels within different tissues in the body, and also act as a form of redundancy to an extent between one another.

The signaling pathway is highly conserved between various species<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. and acts via a G protein coupled receptor like receptor, which uninhibited via the action of the Hh proteins. The pathway overall acts via a balance of dephosphorylation<ref name="PMID10966113"><pubmed>10966113</pubmed></ref> and degradation of proteins<ref name="PMID15102702"><pubmed>15102702</pubmed></ref> in order to regulate transcription factor activity to express various genes to carry out its action. The Hh pathway is also highly implicated when it comes to abnormalities in development, as it is implicated in diseases such as holoprosencephaly and cleft lip and palate. Overall Hh signaling pathways play a large role in embryological development, which will be discussed in detail on this page.

== History ==
{| class="pretty table"
| '''1980'''
|Christiane Nüsslein-Volhard and Eric Wieschaus first identified a group of genes including those related to the Hedgehog signalling pathway and linked them to the segmentation and planning of the embryo in ''Drosophila melanogaster'' by introducing mutagenic substances to the developing embryo<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>.
|-
|'''1993'''
|Multiple researchers including Andrew P. McMahon and Clifford Tabin discovered three equivalent homologs in vertebrates of the ''Drosophila melanogaster'' hedgehog gene, known as sonic hedgehog (SHH), desert hedgehog (DHH) and indian hedgehog (IHH), by looking at DNA sequences similar to that of the gene in the fruit fly<ref><pubmed>7916661</pubmed></ref>.
|-
|'''1993'''
|Clifford Tabin and his lab identified the role of SHH in localising the limb bud, where they identified protein to be expressed within a region of the limb bud known as the zone of polarising activity (ZPA), showing that SHH is sufficient to induce the production of a ZPA, and thus limb bud formation in chick embryos<ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
|-
|'''1995'''
|Sonic hedgehog secreted by the notochord was identified to induce ventral cell types in the neural tube during embryonic development, most notably the floor plate cells and the motor neurons as shown in chick embryos. Such differentiation between the two cell types is thought to be a matter of sonic hedgehog concentrations experienced by the neural plate cells<ref name="PMID7736596"><pubmed>7736596</pubmed></ref>.
|-
|'''1995'''
|A shared Nobel prize in physiology or medicine was awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their researching regarding the identification of the developmental genes relating to the formation and patterning of the early embryo via genes.
|-
|'''1996'''
|Studies showed that human homologs of the Patched gene, a component of the hedgehog pathway, is a key gene that is mutated in Gorlin Syndrome, which characterised by a predisposition for basal cell carcinomas, the most common cancers in humans and developmental abnormalities<ref name="PMID8658145"><pubmed>8658145 </pubmed></ref>.
|-
|'''1999'''
|Researchers implicated Indian hedgehog as a key signalling molecule for the maturation and differentiation of prehypertrophic chondrocytes, where Indian hedgehog null mice were shown to have a lack of mature chondrocytes and no development of osteoblast cells in endochondrial bone. This was thought to be due to Indian hedgehog having a feedback signal controlling parathyroid hormone related protein, which regulates endochondrial bone development<ref name="PMID10465785"><pubmed>10465785</pubmed></ref>.
|-
|'''2003'''
|Sonic hedgehog was identified to regulate the proliferation of adult neural stem cells as shown in the hippocampus of rats and in vitro for neural progenitor cells isolated from the hippocampus<ref name="PMID12469128"><pubmed>12469128</pubmed></ref>.
|-
|'''2003'''
|Studies on SHH function found that it can act as a chemoattractant in the developing embryo spinal cord, to help guide commissural axons to the midline of the floor plate<ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
|-
|'''2006'''
|Mouse models showed that reduction in hedgehog signalling allows for transient improved response to chemotherapy with regards to mouse models of human pancreatic ductal adenocarcinoma, a cancer with one of the highest mortality rates in humans<ref name="PMID19460966"><pubmed>19460966</pubmed></ref>.
|}

This time line regarding the discoveries in the hedgehog signalling pathway is by no means exhaustive, but a selection of relatively large discoveries that had a significant impact in the field on the basis of citations.

== Origin of name ==

[[File:Bands of denticles in normal and Hh mutant Drosophila embryo.jpeg|thumb|250px|Differences in patterning of the denticle bands in normal and mutated hedgehog gene variant ''Drosophilia'' embryo]]

The name hedgehog came about when Christiane Nüsslein-Volhard and Eric Wieschaus first identified the group of genes controlling polarization of the segments in the ''Drosophila'' early embryo. They noticed that when they mutated the Hh gene, making it non-functional, that the bands of denticles formed during the early ''Drosophila'' embryo formation became more diffuse as opposed to being distinct normally. The denticles would all clump together forming a single patch on the surface of the embryo, as opposed to normally outlining the distinct segments of the embryo as bands<ref name="PMID6776413"><pubmed>6776413</pubmed></ref>. This gave the embryo spiny and prickly look similar to that of the back of a hedgehog.

From there on in the gene was called the hedgehog gene where homologs in vertebrates discovered along the line kept to this naming structure giving various species of hedgehog names to the genes such as Indian hedgehog, and desert hedgehog, with the exception being sonic hedgehog, which is named after a fictional character in the video game “Sonic the Hedgehog”.

== Function ==
===Overview===
{| class="pretty table"
|'''Hedgehog protein homolog'''
|'''Function'''
|-
| Sonic hedgehog ||
*Expressed in zone of polarizing activity of the limb bud where it plays a role in helping to pattern the anterior and posterior aspect of the limbs <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>.
*Based on time and concentration of SHH due to diffusion from the point of secretion in the limb bud, SHH also plays a role in determining digit identity <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>.
*Acts as a chemoattractant to guide commissural axons in the floor plate of the neural tube towards the midline <ref name="PMID2679031"><pubmed>2679031</pubmed></ref>.
*Patterns the developing cusps of the teeth and their relative positions in the jaw, and is also essential for the growth of the teeth <ref name="PMID11044393"><pubmed>11044393</pubmed></ref>.
*SHH released from the notochord acts to induce formation of the floor plate, motor neurons, and dopaminergic neurons, as well as signalling the induction of the ventral cell types in the neural tube <ref name="PMID8790332"><pubmed>8790332</pubmed></ref>.
|-
| Desert hedgehog ||
*Plays a possible role in regulating spermatogenesis as due to the highly localised expression in the testes by pre-Sertoli cells, and the fact that mice with no DHH present are infertile, with no mature spermatozoa <ref name="PMID8805249"><pubmed>8805249</pubmed></ref>.
*DHH derived from Schwann cells signal development of peripheral nerves, by inducing the production of a connective tissue sheath around them <ref name="PMID10482238"><pubmed>10482238</pubmed></ref>.
|-
| Indian hedgehog ||
*Aid in the signalling of proliferating chondrocytes to undergo hypertrophic differentiation during endochondral ossification<ref name="PMID8662546"><pubmed> 8662546</pubmed></ref>.
*Plays a key role in yolk sac angiogenesis, as seen in studies where an absence of IHH leads to death of the embryo due to poor yolk sac vasculature <ref name="PMID11807029"><pubmed>11807029</pubmed></ref>.
|}

===Limb development===

====Research background====

The hedgehog signalling pathway plays a significant role with regards to the embryonic development of the limbs in vertebrates, specifically SHH. For a while the mechanism of which the limb was patterned on the anterior to posterior axis was quite unknown. Early studies had shown that grafting posterior wing bud cells to the anterior region of the wing bud in a chick embryo, an equal amount of extra digits was produced to that of which was normally produced, and in a mirror image pattern<ref name="PMID4390734"><pubmed>4390734</pubmed></ref>. This showed that the pattern of which the digits were produced were polarized with respect to that of the graft from the limb bud, which has become to be described as the zone of polarizing activity (ZPA). Further studies mapped the ZPA, by showing the area of the wing bud on the chick with the highest polarizing activity to be the posterior margin of the wing bud<ref name="PMID4719010"><pubmed>4719010</pubmed></ref>. Furthermore, studies later on found that the amount or concentration of ZPA cells grafted played a role in the amount of digits induced and how polarized they were<ref name="PMID7453825"><pubmed>7453825</pubmed></ref>. This ultimately gave rise to studies regarding a potential morphogen, which could potentially provide this concentration dependent activity of polarization when it comes to the limb bud. Studies to find this morphogen led to the discover of the ability for retinoic acid to mimic the polarizing action of the ZPA when applied locally to the limb bud<ref name="PMID7070499"><pubmed>7070499</pubmed></ref>. This was found to be due to the induction of a second morphogen known as SHH, where when cells that express SHH are grafted onto the limb bud, polarized digit development is induced<ref name="PMID7720566"><pubmed>7720566</pubmed></ref>.

====Polarizing activity of sonic hedgehog====
[[File:Digit patterning based on time and concentration of SHH exposure.jpg|thumb|450px|Regions of the limb bud and the digits they form into categorised into how they interact with SHH. The red and green dot represent the 5th and 4th digit region respectively.]]
More recent studies have sought to elucidate how SHH mediates such a polarizing effect in terms of digit identification and its underlying mechanism. It has been suggested that such an effect is heavily concentration dependent similar to that of when transplanting the chick wing bud ZPA cells, acting in a dose dependent matter, due to the ability of SHH to modulate signaling at long ranges via diffusion<ref name="PMID11389830"><pubmed>11389830</pubmed></ref>. This implies that a gradient of varying concentrations of SHH diffused through the mesenchyme of the limb bud leads to the identification of posterior and anterior digits.

Current research has also suggested that not only is the concentration gradient of SHH important for the patterning of the digits, but also the temporal gradient, being the length of time of exposure of SHH to the mesenchyme of the limb bud. Studies have shown in mice that of the 5 digits, polarization from the posterior end, being digit 5, to the anterior end being digit 1 are due to different mechanisms. Digits 5 and 4 have been shown to be comprised of the SHH expressing cells and thus both experience maximal levels of SHH, and thus cannot be distinguished on the basis of a concentration gradient. As a result, temporal gradients are employed, where the expression of SHH in both regions that become the digits vary, where studies have shown that SHH expression in the primordium of digit 5 is maintained longer than that in digit 4 gradient <ref name="PMID15315763"><pubmed>15315763</pubmed></ref>. When it comes to digit 3 and 2, there is differentiation based on the concentration gradient of SHH from the ZPA, where due to the ZPA being further away to digit 2 than 3, the concentration of SHH will be higher at 3<ref name="PMID15315763"><pubmed>15315763</pubmed></ref> <ref name="PMID8269518"><pubmed>8269518</pubmed></ref>. In terms of digit 1, the levels of SHH are so low at this point that it is said to be SHH independent, and actually reliant on the absence of SHH for normal differentiation. With regards to how SHH actually mediates its action, it is thought to be by a balance between the transcription factor Gli3 and SHH expression, which activates Gli1, Gli2 and Gli3 repressor, effectively counteracting Gli3. Gli3 is considered to promote the inhibition of digit formation and identification, while the downstream targets of SHH are thought to promote it<ref name="PMID17400206"><pubmed>17400206</pubmed></ref>.

Other research has also shown the possibility of SHH signaling as a way to prime the posterior mesenchyme and induce the production of bone morphogenetic protein 2 (bmp2), which is thought to be the morphogen creating the chemical gradient for digit identification<ref name="PMID10704381"><pubmed>10704381</pubmed></ref>. Overall the mechanism at which SHH work to pattern the posterior anterior axis of the limb bud is still relatively unknown, and further research is being conducted to elucidate how the underlying mechanisms such as the downstream targets of SHH affect digit formation.

=== Neural development ===

=== Organogenesis ===
The Hedgehog Signalling pathway is a pathway sending information to embryonic cells which plays an especially important role in the regulation of organogenesis. These processes include the organization of the brain (craniofacial) and the growth of appendages with further studies implicating the Hedgehog Signalling pathway in the development of the small intestine, lungs and the pancreas.

== Mechanism ==

=== Processing of precursor ===
 [[File:Processing of Hh precurosr.jpg|thumb|250px|Summary of the steps in the processing of the Hh protein precursor into a functional signalling molecule]]
Hedgehog (Hh) proteins are produced as precursors, which must be processed by the cells producing it before being able to perform its signalling function. The process by which the Hh protein is processed begins with its signal sequence at its N terminus directing the translocation of the precursor into the endoplasmic reticulum, where it is removed via signal peptidase<ref><pubmed>1394430</pubmed></ref>. This process allows the C terminus of the Hh protein to catalyze the cleavage and addition of cholesterol on itself to form a C terminal processing domain and an N terminal processing domain associated with a cholesterol group on its C terminus<ref><pubmed>21357747</pubmed></ref>. The portion associated with the cholesterol will go on to form the signalling molecule, while the C terminal processing domain has no known signalling function<ref><pubmed>8824192</pubmed></ref>.

At this point the Hh protein associated with the cholesterol is able to perform its signalling action, but further modification is still required to ensure efficient signalling. This occurs when the cholesterol group attached to the Hh protein associates with the plasma membrane of the cell, which allows for the addition of a palmitic acid moiety to the N terminal of the protein by an acyl-transferase known as skinny hedgehog<ref><pubmed>11486055</pubmed></ref>. Studies have shown that such an addition allows for an increase in potency in signalling of Hh proteins of 30-fold over its form without palmitic acid added<ref><pubmed>9593755</pubmed></ref>. From this point the Hh protein is now fully active and can either remain associated to the plasma membrane of the cell for autocrine action or be secreted for paracrine action.

=== Mechanism of signalling ===

==== ''Drosophila melanogaster'' ====
 [[File:Hh Signalling Pathway.jpg|thumb|600px|Steps regarding the processing of the hh protein precursor into the fully functional signalling molecule.]]

The Hh signaling pathway has been well studied in ''Drosophila melanogaster'', and has been shown the be conserved to an extent across it and mammals making the species a suitable model for Hh signalling in humans<ref name="PMID8595881"><pubmed>8595881</pubmed></ref>. In ''Drosophila melanogaster'', the Hh pathway begins when the Hh proteins bind to the extracellular domain of the transmembrane protein known as Patched (PTC) to inactivate it. This inactivation occurs by Hh trapping PTC in an inactive conformational state<ref><pubmed>27647915</pubmed></ref>. After binding of Hh occurs to PTC and it is inactivated, the receptor and Hh protein is thought to be endocytosed by the cell where they undergo lysosomal degradation in order to limit Hh concentration, thus limiting its spread to other cells and PTC activity<ref name="PMID15102702"><pubmed>15102702</pubmed></ref>. In the absence of Hh, PTC acts to suppress the expression of the Smoothened (SMO), a G-protein coupled receptor like receptor, and thus its signaling. How PTC achieves this inhibition of SMO is currently unclear. Studies have suggested that due to the highly phosphorylated nature of SMO when active, that PTC acts to dephosphorylate SMO in order to repress its signaling. It is thus that binding of Hh to PTC reduces PTCs ability to promote dephosphorylation of SMO, leading to its increased activity and expression on the cell surface <ref name="PMID10966113"><pubmed>10966113</pubmed></ref>. It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) <ref><pubmed>15616566 </pubmed></ref>.

This overall increased phosphorylation of SMO due to a repression of PTC activity by Hh leads to an accumulation of SMO on the cell surface, which collectively allows for SMO to exert its activity. Most importantly phosphorylation also disrupts intramolecular electrostatic interactions between SMO molecules which switches the molecule into its active conformational state <ref><pubmed>17960137 </pubmed></ref>. Generally, SMO has been shown to have very little signal transducing ability, and thus this accumulation allows it to jointly transduce as substantial signal. This occurs by the C terminus of SMO on the intracellular domain interacting with the kinesin like protein Costal-2 (Cos2), which is thought to bind to microtubules in order to acts as a scaffolding protein <ref><pubmed>18691888 </pubmed></ref>. Then the kinase known as Fused (Fu) binds to Cos2, which phosphorylates Suppressor of Fused (SUFU) to inhibit it. Without this inhibition SUFU goes onto prevent the translocation of the transcriptional factor Cubitus interruptus (Ci) into the nucleus by leading to its phosphorylation, and subsequently its partial cleavage via the proteosome. The partial cleavage of Ci leaves a lower molecular weight protein known as Ci repressor (CiR) or Ci75, which translocates into the nucleus and acts to repress the target genes of the Hh signalling pathway via Ci<ref><pubmed>9215627</pubmed></ref>. Thus overall inhibition of SUFU increases the amount of Ci entering into the nucleus of the cell, and activating transcription of target genes of the Hh pathway<ref><pubmed>10952898 </pubmed></ref>.

At this point Ci is able to activate the transcription of various genes, which include importantly the ''ptc'' gene, which encodes the PTC receptor. It is thus as a result of this, that PTC expression will be increased in response to Hh pathway induction, which negatively feeds back to reduce Ci signaling induction, in order to maintain homeostasis and regulate the intensity and duration of the signaling from Hh<ref><pubmed>9053330</pubmed></ref>. Furthermore the ''wg'' gene, encoding the wingless protein is also activated by Ci, which leads to activation of the Wnt pathway via the Frizzled receptor in adjacent cells expressing the gene ''engrailed'' <ref><pubmed>9502727</pubmed></ref> <ref><pubmed>10457026</pubmed></ref>. This overall stabalizes the boundaries between the segments of the developing ''Drosophila melanogaster'' signalled by the gene ''engrailed''<ref><pubmed>3282172</pubmed></ref>.
 
<html5media width="560" height="315">https://www.youtube.com/watch?v=w1xXD9kss2w</html5media>
 
An overview of the hedgehog signalling pathway in ''Drosophila''<ref>YouTube. (2016). The Hedgehog signalling pathway in Drosophila online Available at: https://www.youtube.com/watch?v=w1xXD9kss2w</ref>.

==== Mammals ====

Although the Hh signaling pathway has been conserved across species to an extent <ref name="PMID8595881"><pubmed>8595881</pubmed></ref>, key differences exist when observing the pathway within mammalian tissue in contrast to that which has been studied in ''Drosophila melanogaster''. In mammalian cells and all other vertebrates, Hh signalling is dependent on an organelle known as the primary cilia, which are projections outwards from the cells surface. The importance of such an organelle in the signalling pathway comes as a result of PTC1, one of the two PTC receptor homologs in mammals, that binds to the Hh homolog Sonic Hh (Shh), exists within the cilia<ref><pubmed>17641202</pubmed></ref>. The mechanism of action of SMO inhibition and activation is poorly understood currently, but evidence has suggested that PTC1 acts to inhibit SMO when unbound to Shh by acting as a pump to remove oxysterols from the cilia into the extracellular space. <ref><pubmed>PMC1462959</pubmed></ref>. These oxysterols are normally thought to bind and accumulate around the SMO receptors, which in turn prevents internalization and deactivation of SMO, leading to it accumulating on the apical primary cilia<ref><pubmed>16136078</pubmed></ref>. Other studies have also suggested that Shh has a role in increasing phosphorylation of SMO, which is required for it to accumulate as well on the cilia<ref><pubmed>21695114</pubmed></ref>. Overall the net action of Shh initially is believed to cause accumulation of SMO at the cilia by inhibiting PTCH1 and phosphorylation of SMO.

How SMO acts next is relatively unknown but, it has been shown to promote the disassociation between SUFU and Gli3 transcription factor, which allows Gli to be transported to the nucleus to activate effector genes of the pathway. Gli is normally bound in the cytoplasm to SUFU, where SUFU promotes the partial degradation of Gli into its repressor form, similar to that in the fruit fly model<ref><pubmed>20360384</pubmed></ref>. Alongside this, Kif7, a Cos2 homolog, has been shown to migrate to the apex of the cilium in response to SMO accumulation at the cilium, where it has been postulated to also promote disassociation of SUFU and Gli<ref><pubmed>19592253</pubmed></ref>. On the other hand, Kif7 is also thought to play an inhibitory role for the signaling pathway as when it is localized at the base of the cilia in the absence of Hh signaling and traffics Gli factors away from the cilia<ref><pubmed>19666503</pubmed></ref>. This all leads to an accumulation of active Gli transcription factors which migrate to the nucleus in order either inhibit or activate gene transcription in response to Hh signaling.

== Animal models ==

=== ''Drosophila melanogaster'' ===

Hedgehog (Hh) protein signal was initially discovered through experimentation on the fruit fly, "Drosophila melanogaster". It is through this model that we are able to discover not only the functional components of this pathway but also understand its role in embryonic development. Through application of this information on the human biological system we are able to find the cause and thus potential treatments of defects and diseases caused by interruption or mutation of the Hedgehog signalling pathway.

Upon further research on Drosophila it was found that Hh may play a role in germ cell proliferation and in particular, may control the proliferation and activity of somatic cells found within the germarium, which is the most anterior structure within the Drosophila ovary. <ref>Ovaries (Drosophila) definition. (2016). Groups.molbiosci.northwestern.edu. Retrieved 10 September 2016, from http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-D/Drosophila_Ovaries.html</ref> Thus, the Hh signalling pathway is vital in egg chamber formation and its consequent envelopment, budding and polarisation. It has also been observed that somatic cell proliferation is dependent on this pathway and thus, the number of pre-follicle cells is subject to the effect of the Hh signalling pathway. <ref><pubmed>8620839</pubmed></ref> Further studies have also indicated the role played by this pathway in activating the Epidermal growth factor receptor (EGFR) signalling pathway as seen in the induction of EGFR by Hh in Drosophila head development. <ref><pubmed>10331974</pubmed></ref> In addition, research on Drosophila has indicated the direct effect of Hh signalling on tracheal branch patterning. <ref><pubmed>11290298</pubmed></ref>

Despite ongoing research on "Drosophila melanogaster" to uncover the workings of the Hedgehog signalling pathway, further research is needed to confirm and further current findings. In addition, it is evident that it is through the study of these fruit flies that we are able to gain a basis of understanding of the causes of certain human diseases and thus propel research into treatments for sufferers.

===Blockage of Shh Signalling in Forebrain Neuroectoderm of Chick Embryos===

The Hedgehog signalling pathway plays a significant role in embryonic development, particularly of the forebrain. Due to its role in development of craniofacial features by contributing to the epithelia of the frontonasal, maxillary, and pharyngeal ectoderm, a disruption in this pathway can result in a variety of birth deformities including holoprosencephaly (HPE), where the prosencephalon (forebrain) fails to divide into 2 separate hemispheres, telencephalon and diencephalon <ref>Holoprosencephaly - NORD (National Organization for Rare Disorders). (2016). NORD (National Organization for Rare Disorders). Retrieved 10 September 2016, from http://rarediseases.org/rare-diseases/holoprosencephaly/</ref>, as well as cleft lip and palate. The function of Sonic Hedgehog (Shh) and its signalling pathway on the formation of forebrain neuroectoderm was studied in chick embryos.

It was found that the disruption of Shh signalling in the neural tube of chick embryos resulted in the lack of division of the forebrain into the diencephalon and telencephalon. This is in fact, as stated earlier, the fundamental cause of the rare condition, holoprosencephaly. It was discovered that the Shh signalling pathway in the diencephalon was responsible for gene expression in the telencephalon.

In addition, through experimentation on chick embryos it was found that through Shh signalling the development of the forebrain regulates and controls facial morphogenesis, particularly of the upper and middle face. Therefore, interference with Shh signalling in the forebrain prevents this intrinsic communication, thus hindering Shh expression in facial ectoderm. This results in brain malformation accompanied by facial disfiguration as seen in patients suffering from HPE. Other malformations caused by blockage of Shh signalling in craniofacial development include, hypotelorism (decreased space between the orbits), growth restriction as well as cleft lip and palate as mentioned previously.

A commonality discovered among all forms of Shh signalling disruption was two facial defects, “the loss of mediolateral expansion of the face and absence of proximodistal outgrowth of the frontonasal prominence”<ref><pubmed>15979605</pubmed></ref> (Ralph et al., 2005). Through reference to studies performed on chick and mice embryos, it was found that Shh signalling is particularly vital in development of maxillary and frontonasal components of the cranium.

This breakthrough study on the inhibition of Shh signalling in chick embryos has significantly filled the gap in our understanding of the Sonic Hedgehog signalling pathway. It is evident that this pathway is crucial in the development of the forebrain and in turn regulates and controls the development of the facial skeleton. This is further proven through the observation that in the event of Shh signalling inhibition there is a development of craniofacial malformations.

=== Shh knockout mice ===

Extensive research on Shh knockout mice allowed discovery of the roles of Shh in embryonic development and patterning of the limb buds and sclerotomes, and maintenance of the notochord. <ref><pubmed>8837770</pubmed></ref> These mice expressed defects in the cephalic neural tube with the fusion of telencephalic and optic vesicles. Through this research it is evident that Shh is partially responsible for the subdivision of the eye field through forebrain optic stalk development, along with formation of the ventral midline. It is these forebrain abnormalities established in the absence of Shh that results in congenital malformations of holoprosencephaly, development of a single nasal chamber and other facial defects in humans.

The absence of the vertebral column including, the intervertebral discs and vertebrae along with the medial regions of the ribs were observed within Shh knockout mice. This lack of sclerotome derivatives indicates the role of Shh in maintenance or expansion of sclerotome cell population (Chiang et al., 1996). In addition, though the role of Shh in patterning the anterior-posterior limb axis is evident through anterior limb bud cell death in the absence of Shh, it is also responsible for the development of proximal-distal limb segments, particularly in the patterning of structures at the level of or distal to the latent elbow and knee joints. <ref><pubmed>11476582</pubmed></ref> This was perceived after observing the incomplete formation of distal limb structures with abnormal anterior-posterior axis formation (Chiang et al., 2001). Thus, Shh knockout mice provide great insight into the functions of Shh in embryonic development and the abnormalities formed in the absence of this signalling protein.

== Clinical Significance ==

===Gorlin Syndrome===
[[File:1 week year old newborn girl with cleft lip and palate.png|thumb|An image of a 1 week year old newborn girl with cleft lip and palate]]
Gorlin syndrome, also known as nevoid basal cell carcinoma syndrome, is a heritable disease resulting from heterozygousity in Hh receptor, Patched (PTC). The PTC gene encodes a protein which binds sonic hedgehog (Shh) and it is this binding which inhibits the Hh signalling pathway. However mutation of PTC leads to activation of the pathway <ref><pubmed>9620294</pubmed></ref>. The syndrome is characteristic of skin cancer basal cell carcinoma and cerebellum cancer medulloblastoma. 1-2% of medulloblastomas and 0.5% of basal cell carcinomas are attributable to the disease. Patients present with craniofacial and brain abnormalities such as cleft palate, strabismus, macrocephaly, abnormal development of the corpus callosum and frontal bossing, with an overall overgrown appearance. Skeletal defects such as of the shoulder, ribs and vertebrae are often seen, as well immobile thumbs and polydactyly, which is a deformity of the hand or feet in which they have one or more extra fingers or toes. Apart from physical anomalies, patients have an increased risk of tumours throughout the body including cardiac, ovarian fibromas, ovarian dermoid cysts, meningiomas, and fibrosarcomas. Additionally with time, patients eventually undergo intracranial calcification and dyskeratotic pitting of the hands and feet whereby skin cells prematurely convert to keratin <ref><pubmed>8681379</pubmed></ref>.

===Holoprosencephaly===
 [[File:Semilobar holoprosencephaly.jpg|thumb|240px|MRI of fore brain depicting semilobar holoprosencephaly]]
Holoprosencephaly (HPE) is a congenital disease caused by incomplete division of the prosencephalon (embryonic forebrain) into separate lobes of the cerebral hemispheres. The Shh gene has been identified as a HPE-causing gene however recent evidence has also investigated into the PTC gene, which acts to repress Shh signalling. A gain in repressive action of PTC is seen, leading to decreased Shh signalling, and thus HPE <ref><pubmed>11941477</pubmed></ref>. Clinical expression is variable between patients and is dependent upon the 3 forms of increasing severity: lobar, semi-lobar and alobar HPE, where patients can present with right and left ventricles however with a continuous frontal cortex in lobar HPE, to carrying a single cerebral ventricle in alobar HPE. Along with forebrain abnormalities, facial anomalies are also seen including midline cleft lip and/or flat nose, ocular hypotelorism characteristic of a small distance between the eyes. In severe rare cases of cyclopia, a non-functioning nose may be observed in the form of proboscis and the patient may present with a single eye at the root of the nose <ref><pubmed>20583177</pubmed></ref>. Furthermore, patients with HPE often develop a large number of medical issues including epilepsy, mental retardation, thyroid and adrenal hypoplasia due to lack of hypothalamus or pituitary gland development, and diabetes insipidus.

===Medulloblastoma===
Medulloblastoma is a cancerous tumour of the cerebellum where the cells of origin are the granular cell precursors found in the neonatal cerebellum, explaining its high prevalence in children <ref><pubmed>19171780</pubmed></ref>. Commonly, medulloblastomas are found deep in the cerebellum along the midline, however with desmoplastic medulloblastomas, they are found more laterally and superficial. As seen previously with Gorlin syndrome and holoprosencephaly, PTC is also associated with the development of medulloblastoma, and misregulation of Hh-PTC signalling in cells of the external germinal layer, the layer found on the surface of the cerebellum, is thought to give rise to the tumour. In experimental studies, it has been discovered that PTC is highly transcribed in medulloblastomas and in approximately 30% of PTC heterozygous mice the tumour develops <ref><pubmed>9205058</pubmed></ref> <ref><pubmed>9262482</pubmed></ref>. In patients with medulloblastoma, often children, the most common symptoms seen are vomiting, ataxia, psychomotor regression, drowsiness and anorexia. With age, patients are seen to get more frequent headaches, and show psychological symptoms such as behavioural problems, poor performance in school, and anxiety. In patients exhibiting life-threatening symptoms due to increased intracranial hypertension, bradycardia, convulsions and coma are often seen <ref><pubmed>21537925</pubmed></ref>.

== Current research ==

==Quiz==

<quiz display=simple>

{1. Which of the following are the reasons that are thought to allow for the patterning of digits in vertebrates.
|type="[]"}
+  Concentration gradient of sonic hedgehog.
+  Temporal gradient of sonic hedgehog.
-  The varying homologs of the hedgehog proteins .
-  The length of the hedgehog molecule.
+  No sonic hedgehog signalling.
-  The type of cholesterol attached to the hedgehog molecule.
|| For differentiation of the individual digits in mice to occur it is thought that each digit requires a different signalling mechanism. Digits 5 and 4 which are most posterior, are derived from the region around the zone of polarising area which contain Shh secreting cells, meaning these regions will experience a saturation of Shh signalling and cannot be differentiated by Shh concentration. As a result it is thought that these digits are identfied based on the temporal gradient, meaning that the length of which Shh is expressed in these regions differentiates the digits from one another with digit 5 expressing Shh for longer. Digits 3 and 2 are further away but still receive Shh signalling due to the diffusible nature of Shh from the ZPA, which forms a concentration gradient. This means the the concentration at digit 2 will be less than digit 3 due to being further away from the ZPA and thus can be differentiated from one another based on this concentration. Finally the digit 1 is differentiated on the basis of not receiving any Shh signalling at all. Thus it is a combination of concentration gradient of Shh, temporal gradient of Shh, and independence from Shh.

{2. Which of the following are correct with regards to the hedgehog signalling pathway in ''Drosophila''.
|type="[]"}
- It occurs at the cillum on the cell surface.
- Hedgehog protein binding to the Patched receptor activates it to inhibit Smoothened.
+ Hedgehog protein binding to the Patched recetpor inhibits it to activate Smoothened.
+ Inhibition of SUFU is critical in allowing Ci to translocate into the nucles and activate downstream targets of the hedgehog pathway.
- The pathway contains 3 homologs including sonic hedgehog, Indian hedgehog, and desert hedgehog.
- The hedgehog protein is initially produced in its fully active form.
|| In terms of how the hedgehog protein acts to produce its effect, it must first bind to Patched which activity is inhibiting Smoothened in the absence of the hedgehog protein. The protein acts to trap Patched in its inactive confirmational state cause internalisation and eventually degradation of the receptor. This relives the dephosporylation of the Smoothened, usually induced by Patched, allowing it to accumulate phosphate groups and thus activate. With regards to Ci translocation, SUFU usually inhibits this by leading to phosphorylation of Ci and eventually its cleavage into Ci repressor. During the activation of the hedgehog pathway there is a binding of Fused to Cos2 which phosphorylates SUFU to inactivate it, allowing Ci to translocate into the nucleus and activate downstream targets.

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==Glossary==

{| class="pretty table"
|-
|Term
|Definition
|-
|Desert hedgehog (Dhh)
|A homolog of the ''Drosophila'' hedgehog protein, which is expressed most prominently in the gonads of vertebrates, where it plays a possible role in regulating spermatogenesis.
|-
|Hedgehog (Hh) protein
|A signalling protein widely expressed in the tissue during embryonic development of vertebrates and some other species such as ''Drosophila'', and acts to pattern and develop tissue. The protein is the primary mediator of the pathway.
|-

|Indian hedgehog (Ihh)
|A signalling protein that is a homolog of the ''Drosophila'' hedgehog protein, that primarily acts in regions where there is endochondrial ossification to aid in the maturation of chondrocytes.
|-
|Limb bud
|A structure formed during early embryonic development consisting of ectoderm and mesenchyme representing the early limbs, where signalling processes in the bud will eventually lead to the out growth and patterning of the bud.
|-
|Sonic hedgehog (Shh)
|The most widely expressed homolog of the ''Drosophila'' hedgehog protein found in vertebrates, playing a key role in embryonic development of humans.
|-
|Zone of polarizing activity (ZPA)
|The posterior region of the limb bud mesenchyme, which aids in the signalling of it to pattern the posterior anterior axis with respect to the digits through mediators such as sonic hedgehog.
|-
|}

===Additional Glossary Links===
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== References ==