2016 Group Project 4-1

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Signalling: 1 Wnt | 2 Notch | 3 FGF Receptor | 4 Hedgehog | 5 T-box | 6 TGF-Beta
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Hedgehog Signalling Pathway

Introduction

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. 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) . 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. 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 and degradation of proteins 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

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.
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.
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.
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.
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.
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.
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.
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.
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.

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

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. 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

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. 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.  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.

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. 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.  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

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. 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. 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 . Overall hedgehog binding leads to inactivation of PTC which occurs by Hh trapping PTC in an inactive conformational state. 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. 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 . It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) .

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 . 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 . 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. 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. 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.

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. 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 . This overall stabalizes the boundaries between the segments of the developing Drosophila melanogaster signalled by the gene engrailed.

<html5media width="560" height="315">https://www.youtube.com/watch?v=w1xXD9kss2w</html5media>

An overview of the hedgehog signalling pathway in Drosophila.

Mammals

Although the Hh signaling pathway has been conserved across species to an extent , 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. 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. 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. 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. 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. 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. 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. 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. 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. 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 . 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 . As a result Gl1 is considered as not entirely essential ,where mice models with no production of Gl1 have shown viability with few defects. 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. 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.