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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”.

== Mechanism ==

=== Processing of precursor ===

<br>[[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'' ====
<br>[[File:Hh signalling pathway.jpg|thumb|600px|Diagrammatic representation of the steps and components involved in the hedgehog signalling pathway in both ''Drosophila'' and mammals.]]

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). Such binding is thought to be greatly enhanced by the presence of proteins known as interference hedgehog (ihog) and brother of ihog (boi), which have shown to be essential to allowing hedgehog binding to inactivate PTC<ref name="p20048000 "><pubmed>20048000 </pubmed></ref>. It has also been demonstrated heparin sulfate proteoglycans (HSPG) such as dally also aid in the modulation of hedgehog signalling, where they act to localise and transport the hedgehog protein to the target receptors<ref name="p14602684"><pubmed>14602684</pubmed></ref> <ref name="p10549295"><pubmed>10549295</pubmed></ref>. Overall hedgehog binding leads to inactivation of PTC which 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. Importantly the 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 a 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>. Such interactions induce the formation of a complex including the kinase known as Fused (Fu) and 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>. Activation of SMO also promotes disassociation of present SUFU-Fu-Cos2-Ci complex which which generally when fully intact leads to cleavage of Ci into its repressor form<ref name="p15063184"><pubmed>15063184</pubmed></ref><ref name="p17881487"><pubmed>17881487</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>.
<br><br>
<html5media width="560" height="315">https://www.youtube.com/watch?v=w1xXD9kss2w</html5media>
<br><br>
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>. Similarities also exist between signalling in the vertebrates and ''Drosophila''. This includes the surface proteins such as Cdo and Brother of cdo (Boc), which act in a similar fashion to ihog and boi in enhancing the binding of the hedgehog protein to PTC<ref><pubmed>16647304</pubmed></ref>. HSPGs such as gylcipans (GPC) 1, 2, 3, 4, 5 and 6 are also thought to play a role in hedgehog signalling in vertebrates with regards to stimulaiton or inhibition of the pathway depending on the isoform<ref><pubmed>26627558</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 via kinases such as CK1, which causes it to accumulate 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 Gli 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 signalling pathway as when it is localized at the base of the cilia in the absence of Hh signaling, where it acts to traffic Gli transcription 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. These Gli transcription factors include Gli1, Gli2, and Gli3, where Gli2 and Gli3 in their activated form are the primary mediators of the pathway, and also act to repress the target genes when hedgehog signalling is absent. Gli3 is considered to be the major repressor of the pathway when the full length version of the Gli3 protein is partially cleaved into its repressor form <ref><pubmed>10433919</pubmed></ref>. Gli1 on the other hand is one of the targets of hedgehog signalling promoted by Gli2 and Gli3 activators, and acts to reinforce the hedgehog pathway through a positive feedback loop <ref><pubmed>10075717</pubmed></ref>. As a result Gl1 is considered as not entirely essential ,where mice models with no production of Gl1 have shown viability with few defects<ref><pubmed>10725236</pubmed></ref>. That being said, other studies have shown the importance of Gl1 as a downstream target of sonic hedgehog, where it is thought to induce ventral neural tube development, as it is on the only Gli factor present in the floor plate during gastrulation<ref><pubmed>9216996</pubmed></ref>. Overall the interactions between such transcription factors are still relatively unknown, where further research is required to elaborate on the specific roles in the hedgehog pathway they each have.

== 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 ===

====Research background====
Research on the role of the notochord, a cartilaginous structure derived from the mesoderm, initially observed its ability to induce the floor plate within the neural tube of chick embryos<ref name="PMID3354847"><pubmed>3354847</pubmed></ref>. This induction of a floor plate occurred in the ventral midline of the neural tube, where the floor plate itself is thought to have signalling effects regarding patterning of the spinal cord.  Alongside this, research also noted that the floor plate and notochord collectively had signalling effects that patterned the differentiation of the neural cells in the neural tube<ref name="PMID1991324"><pubmed>1991324</pubmed></ref>. Such observations lead to research being directed at how this patterning of the neural tube is mediated through the notochord. It had been suggested that the morphogen known as Shh was to play due to its high expression within the notochord and floor plate. It also was showed to activate floor plate expressed genes when ectopically expressed in the mouse CNS<ref name="PMID7916661"><pubmed>7916661</pubmed></ref>. Furthermore, observations regarding the Shh also lead to implicating it in the differentiation of distinct cell types in the ventral neural tube such as motor neurons, alongside the already identified floor plate<ref name="PMID8124714"><pubmed>8124714</pubmed></ref>. Studies looking specifically at the targets of Shh signalling in the neural tube also identified that using antibodies to block the protein would lead to blocking of the induction of the motor neurons, and that applying Shh to explants induced floor plate and motor neurons in a variety of vertebrate models<ref name="PMID7753196 "><pubmed>7753196 </pubmed></ref>. This for the most part formed the basis of research on the specific patterning effects of Shh secreted from the notochord and floor plate on the ventral neural tube.

====Patterning of ventral nerve cell types in the neural tube====
[[File: Regions of varying neural cell types in ventral neural tube.jpg|thumb|400px|Different areas of ventral nerve cell types including inter-neurons and motor neurons on the neural tube induced by the graded signalling of Shh secreted from the floor plate and notochord.]]
As stated, the inductive signals from the notochord in the form of Shh <ref name="PMID7753196"><pubmed>7753196 </pubmed></ref> are primarily responsible for the formation of the floor plate in the ventral midline of the neural tube during embryonic development<ref name="PMID3354847"><pubmed>3354847</pubmed></ref>. The floor plate itself acts in the same fashion as the notochord with regards to expression of Shh for mediatating its signal. Collectively it is thought that the notochord and the floor plate provide a Shh graded response, where there is a gradient of concentrations along the dorsoventral axis of the developing neural tube<ref name="PMID9598380"><pubmed>9598380</pubmed></ref>. As a result, it is believed that the degree of Hh signalling on a cell type is responsible for the differentiation of progenitor cells it to various types. Specifically, with regards to the floor plate, the distinct local signal, given the close proximity of the notochord to the floor plate induction site is responsible for inducing the floor plate<ref name="PMID9598380"><pubmed>9598380</pubmed></ref>. It is thought that through interacting with the receptor PTC, the pathway activates the transcription of hepatocyte nuclear factor 3β (HNF-3β), a gene that has been shown to be commonly expressed in the floor plate. Such a gene is activated by the transcription factors Gli1 and Gli2. Thus HNF-3β is considered to be the downstream target of Shh signalling leading to the induction of the floor plate<ref name="PMID9118802"><pubmed>9118802</pubmed></ref>. It is also believed that HNF-3β is essential for notochord formation as well, as seen in mice models with HNF-3β knocked out, leading to dorsoventral patterning issues<ref name="PMID8069909"><pubmed>8069909</pubmed></ref>. Overall the induced floor plate acts to further aid the notochord in producing a graded Shh response to pattern the ventral neural tube<ref name="PMID1991324"><pubmed>1991324</pubmed></ref>.

With regards to the patterning of the ventral cell types, Shh is able to differentiation the progenitors within the neural tubes into 5 different classes, which include interneurons V0, V1, V2, V3 and motor neurons. As stated previously, it is thought that such differentiation is via the gradient of Shh along the dorsoventral axis, where more ventrally there is a higher concentration of Shh gradually decreasing as you move dorsally<ref name="PMID9598380"><pubmed>9598380</pubmed></ref>. This gradient acts upon the neural progenitors which cause the expression or inhibition of homeodomain transcription factors depending on the cell type they will differentiate to, acting as intermediaries for the Shh signalling to promote patterning<ref name="PMID15936325"><pubmed>15936325</pubmed></ref>. In the context of these transcription factors and hedgehog signalling, studies have shown that at very low concentrations of Shh initially inhibits the Pax7 transcriptional factor, which allow for the formation of the general ventral population of neural progenitors<ref name="PMID8929535"><pubmed>8929535</pubmed></ref>. The ventral progenitor cells will now express varying different factors in response to the graded Shh signalling leading to the defined boundaries of the 5 different nerve cell types. These boundaries are produced by the interaction of these factors, which are split into two classes, class I and class II. The class I factors are Shh repressed and include Pax7, Irx3, Dbx1, Dbx2, and Pax6, while class II consists of Shh induced proteins such as Nkx6.1 and Nkx2.2<ref name="PMID10830170"><pubmed>10830170</pubmed></ref>.  The classes act to repress one another, and when expressed in varying concentrations in response to the interactions between one another and Shh signalling, various distinct domains which represent the various ventral cell types are formed. Specifically, Nkx2.2 identifies the V3 interneurons (most ventral) <ref name="PMID10217145"><pubmed>10217145</pubmed></ref>, while induction of MNR2 and Lim3 via Nkx6.1 in the motor neuron domain produces the motor neurons<ref name="PMID9778248"><pubmed>9778248</pubmed></ref>. Furthermore, Nkx6.1 from the motor neuron domain acts to induce Lim3, while Irx3 in the V2 region inhibits MNR2 to prevent formation of motor neurons to pattern the region containing V2<ref name="PMID10830170"><pubmed>10830170</pubmed></ref>. Finally, Dbx1 is critical for V0 interneuron generation (most dorsal) and Dbx2 is for V1 generation<ref name="PMID11239429"><pubmed>11239429</pubmed></ref>. As described above there are complex interactions between homeobox transcription factors that are activated or inhibited by Shh signalling and are expressed at different concentration given the concentration gradient of Shh. These come together as a whole to produce very distinct regions in the ventral neural tube containing different cell types.