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Signalling: 1 Wnt | 2 Notch | 3 FGF Receptor | 4 Hedgehog | 5 T-box | 6 TGF-Beta
2016 Group Project Topic - Signaling in Development

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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[1]. 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) [2]. 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[3]. 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[4] and degradation of proteins[5] 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[1].
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[6].
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[7].
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[8].
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[9].
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[10].
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[11].
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[12].
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[13].

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[1]. 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[14]. 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[15].  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[16].

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[17]. 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[18].  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[3]. 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[19]. 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[20] [21]. Overall hedgehog binding leads to inactivation of PTC which occurs by Hh trapping PTC in an inactive conformational state[22]. 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[5]. 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 [4]. It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) [23].

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 [24]. 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 [25]. 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[26]. 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[27]. 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[28][29].

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[30]. 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 [31] [32]. This overall stabalizes the boundaries between the segments of the developing Drosophila melanogaster signalled by the gene engrailed[33].

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

An overview of the hedgehog signalling pathway in Drosophila[34].

Mammals

Although the Hh signaling pathway has been conserved across species to an extent [3], 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[35]. 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[36]. 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[37]. 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[38]. 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[39]. 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[40]. 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[41]. 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[42]. 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[43]. 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 [44]. 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 [45]. As a result Gl1 is considered as not entirely essential ,where mice models with no production of Gl1 have shown viability with few defects[46]. 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[47]. 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

Hedgehog protein homolog Function
Sonic hedgehog 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 [7].
  • 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 [48].
  • Acts as a chemoattractant to guide commissural axons in the floor plate of the neural tube towards the midline [12].
  • Patterns the developing cusps of the teeth and their relative positions in the jaw, and is also essential for the growth of the teeth [49].
  • 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 [50].
Desert hedgehog 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 [51].
  • DHH derived from Schwann cells signal development of peripheral nerves, by inducing the production of a connective tissue sheath around them [52].
Indian hedgehog OMIM entry
  • Aid in the signalling of proliferating chondrocytes to undergo hypertrophic differentiation during endochondral ossification[53].
  • 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 [54].

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[55]. 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[56]. 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[57]. 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[58]. 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[59].

Polarizing activity of sonic hedgehog

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[60]. 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 [48]. 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[48] [7]. 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[61].

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[62]. 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[63]. 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[64]. 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[2]. 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[65]. 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[66]. 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

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 [66] are primarily responsible for the formation of the floor plate in the ventral midline of the neural tube during embryonic development[63]. 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[67]. 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[67]. 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[68]. 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[69]. Overall the induced floor plate acts to further aid the notochord in producing a graded Shh response to pattern the ventral neural tube[64].

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[67]. 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[70]. 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[71]. 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[72]. 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) [73], while induction of MNR2 and Lim3 via Nkx6.1 in the motor neuron domain produces the motor neurons[74]. 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[72]. Finally, Dbx1 is critical for V0 interneuron generation (most dorsal) and Dbx2 is for V1 generation[75]. 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.

Organogenesis

Organogenesis is a term used to describe the process of development of organs within plants or animals. 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, the pancreas as well as the skeleton. Research performed via vertebrate model organisms in recent years has lead to an increased understanding of the role of the endoderm in organogenesis, in particular the respiratory and gastrointestinal tracts. As stated previously, there are three Hedgehog protein homologues; Desert Hedgehog Homologue (DHH), the Indian Hedgehog Homologue (IHH) and the Sonic Hedgehog Homologue (SHH) of which SHH is the most extensively studied.

Previous research has shown that SHH is expressed within the definitive endoderm during the infantile stages of gut organogenesis. Within underlying mesoderm, genes encoding for bone morphogenetic protein 4 (bmp4) and the Abd-B subclass of Hox genes were discovered [76]. Bmp4 is related to Transforming Growth Factor Beta (TGF-β) and functions in normal growth of the visceral mesoderm; studies of knockout mice have shown organ abnormalities upon the removal of said gene [77]. On the other hand, Hox genes are implicated in the regulation of embryonic development. Further research into the nuances of SHH have displayed that SHH by itself allows for induction of bmp4 and Hox genes in the mesoderm itself due to the restriction of SHH expresson to embryonic gut regions alongside the simultaneous detection of proteins [76]. As a result, it is theorized that SHH helps induce epithelial mesenchymal interactions within the process of hindgut organogenesis [78].

SHH is found to play an important role in lung organogenesis, evidence relating lowered levels of SHH signalling with pulmonary hypoplasia [79]. It has been found that there is increased expression of SHH at the site of branching of the lung epithelia, suggesting a regulatory role in the progression of the respiratory tract. This is further supported by ectopic expression of SHH showing increased cell proliferation alongside an inhibition of epithelial branching [80]. Experiments conducted with SHH knockout mice display the characteristics of an improperly separated trachea and esophagus alongside a significant structural downgrade of the lung due to the detrimental effect of respiratory tract organogenesis.

It is well known that DHH is related to testis organogensis, however, it is only recently that researches have elucidated its functions in relation to both Leydig and Sertoli cells. It has been found that DHH signalling causes Leydig Cell differentiaion through the increased expression of Steroidogenic Factor 1 (SF1) and Cholesterol Side Chain Cleavage Enzyme within Protein Patched Homolog 1 (PTCH1) cells which are receptors for SHH outside the testis cordis [81]. DHH help signal molecules which are expressed exclusively in the testis and are thus thought to play part in spermatogenesis. This is evidenced by research showing female DHH knockout mice displaying no phenotype, contrasted to male DHH knockout mice displaying infertility. The fact that DHH is found to be expressed in pre-Sertoli cells also asserts its role in male sexual differentiation. Futher analysis of embryonic and developing mouse testis highlights the role of DHH in regulation of both infantile and later stages of spermatogenesis [51].

Only recently has IHH been implicated in the morphogenesis of the endochondral skeleton, with plenty of factors relating to this subject still yet to be elucidated. IHH signalling has been associated with regulation of chondrocyte maturation through a feedback loop of Parathyroid Hormone related Peptide (PTHrP), occuring at articular surfaces of the skeleton [82]. PTHrP itself has many functions, one of which is the local reabsorption of bone. Studies performed with IHH knockout mice implicate a larger role of IHH in the development of the adult skeleton, mutants showing significant reduction in chondrocyte proliferation. Other important indicators of reduced skeleton developmental capacity include a failure in osteoblast development as well as mistimed chondrocyte maturation [82].

Animal models

Drosophila melanogaster


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. [83] 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. [84] 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. [85] In addition, research on Drosophila has indicated the direct effect of Hh signalling on tracheal branch patterning. [86]

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 [87], 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”[88]. 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. [89] 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 . 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. [90] 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

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 [91]. 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 [92].

Holoprosencephaly


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 [93]. 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 [94]. 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 [95]. 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 [96] [97]. 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 [98].

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[99].


Current research

As a result of the Hedgehog Signalling Pathway's roots in many fundamental processes of embryonic development, there are many pathways that current and future research can take. One such heavily researched pathway today is the implication and subsequent targeting of the Hedgehog Pathway in cancer. What is known is the role of improper activation of the Hedgehog Pathway leading to tumorigenesis and subsequently, basal cell carcinoma or medulloblastoma [100]. It however, must be noted that the exact mechanism of Hedgehog Pathway Signalling mediated carcinogenesis remains a mystery. Organs reliant on the Hedgehog Pathway for development also display imporper activation of this pathway when stricken with cancer and recent research has also linked an increased propensity for the spread of tumour cells with over activation. It can thus be theorized that inhibition of Hedgehog Pathway signalling could in turn lead to the reduction of tumour spread and indeed, prevention of the cancer as a whole. One such orally applicable Hedehog Signalling Pathway antagonist GDC-0449 or Vismodegib has been shown to novelly cause apoptosis within pancreatic cancer stem cells by caspase-3 activation and Poly ADP ribose polymerase (PARP) cleavage [101] . Vismoedgib has also shown promise through a reduction in adverse events in patients with inoperable basal cell carcinoma [102]. With tissue repair and regeneration being on the forefront of discovery in gene therapy, other parallel studies relating to the Hedgehog Signalling Pathway showing promise include the acceleration of liver regeneration after the process of hepatectomy [103], with results showing positive implications for the future of medicine.

Quiz

1 Which of the following are the reasons that are thought to allow for the patterning of digits in vertebrates.

 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.

2 Which of the following are correct with regards to the hedgehog signalling pathway in Drosophila.

 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.

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)

  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

4 A patient presents with crossed eyes, a pronounced forehead and an abnormally large head. Through an ultrasound it was also revealed the patient has an ovarian fibroma. Which of the following is the patient diagnosed with?

  Holoprosencephaly
  Medulloblastoma
  Gorlin Syndrome
  Cowden Syndrome


Glossary

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.
Ataxia Lack of voluntary control and coordination of muscle movement often due to damage of the cerebellum, the region of the brain controlling muscle coordination
Bone morphogenetic protein (bmp) Growth factors allowing for the signalling and subsequent structuring of organic tissue within a body.
Bradycardia Disruption of normal electric impulses controlling the pumping of the heart resulting in a slower heart rate
Brother of ihog (Boi) A surface protein that enhances hedgehog signal transduction through PTC in Drosophila.
Carcinoma A malignant tumour of epithelial tissue of skin or tissues lining body organs
Casein kinase I (CKI) A kinase that is thought to phosphorylate the Smoothened receptor to activate it in the hedgehog signalling pathway.
Cleft Palate An opening or split in the roof of the mouth as a result of the palatal shelves not fusing, most often occurring in embryonic development.
Convulsion Sudden, rapid, involuntary contraction of body muscles repeatedly
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.
Dally An HSPG, which mediates loacalizaiton and transportation of the hedgehog protein to its targets in Drosophila
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.
Desmoplastic A reaction involving growth of dense fibrous tissue around the tumour
Endochrondral Ossification A process by which bone is created within vertebrae.
Epilepsy A neurological disorder whereby nerve cell activity in the brain becomes abnormal resulting in convulsions, or loss of consciousness
Frizzled A receptor that is part of the Wnt signalling pathway, which binds the protein wingless in order to activate signalling cascades.
Frontal bossing A pronounced forehead
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.
Glycipan (GPC) A family of HSPGs which act in the hedgehog signalling pathway of vertebrates to either enhance or inhibit signal transduction depending on the isoform.
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.
Heparin sulfate proteoglycan (HSPG) A proteoglycan ,which is a heavily glycosolated protein, which in the context of hedgehog signalling is thought to aid in the localization and transport of hedgehog proteins to their targets.
Heterozygousity Having different alleles of one gene
Hypoplasia Arrested, incomplete development of an organ or tissue
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.
Interference hedgehog (Ihog) A surface protein that enhances hedgehog signal transduction through PTC in Drosophila.
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.
Macrocephaly Abnormal enlargement of the head due to an enlarged brain or excessive accumulation of cerebrospinal fluid
Organogenesis The process of the development of the ectoderm, endoderm and mesoderm into the organs of an organism.
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.
Strabismus Abnormal alignment of the eyes with eachother resulting in a cross-eyed appearance
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

  1. 1.0 1.1 1.2 <pubmed>6776413</pubmed>
  2. 2.0 2.1 <pubmed>7916661</pubmed>
  3. 3.0 3.1 3.2 <pubmed>8595881</pubmed>
  4. 4.0 4.1 <pubmed>10966113</pubmed>
  5. 5.0 5.1 <pubmed>15102702</pubmed>
  6. <pubmed>7916661</pubmed>
  7. 7.0 7.1 7.2 <pubmed>8269518</pubmed>
  8. <pubmed>7736596</pubmed>
  9. <pubmed>8658145 </pubmed>
  10. <pubmed>10465785</pubmed>
  11. <pubmed>12469128</pubmed>
  12. 12.0 12.1 <pubmed>2679031</pubmed>
  13. <pubmed>19460966</pubmed>
  14. <pubmed>1394430</pubmed>
  15. <pubmed>21357747</pubmed>
  16. <pubmed>8824192</pubmed>
  17. <pubmed>11486055</pubmed>
  18. <pubmed>9593755</pubmed>
  19. <pubmed>20048000 </pubmed>
  20. <pubmed>14602684</pubmed>
  21. <pubmed>10549295</pubmed>
  22. <pubmed>27647915</pubmed>
  23. <pubmed>15616566 </pubmed>
  24. <pubmed>17960137 </pubmed>
  25. <pubmed>18691888 </pubmed>
  26. <pubmed>9215627</pubmed>
  27. <pubmed>10952898 </pubmed>
  28. <pubmed>15063184</pubmed>
  29. <pubmed>17881487</pubmed>
  30. <pubmed>9053330</pubmed>
  31. <pubmed>9502727</pubmed>
  32. <pubmed>10457026</pubmed>
  33. <pubmed>3282172</pubmed>
  34. YouTube. (2016). The Hedgehog signalling pathway in Drosophila online Available at: https://www.youtube.com/watch?v=w1xXD9kss2w
  35. <pubmed>17641202</pubmed>
  36. <pubmed>16647304</pubmed>
  37. <pubmed>26627558</pubmed>
  38. <pubmed>PMC1462959</pubmed>
  39. <pubmed>16136078</pubmed>
  40. <pubmed>21695114</pubmed>
  41. <pubmed>20360384</pubmed>
  42. <pubmed>19592253</pubmed>
  43. <pubmed>19666503</pubmed>
  44. <pubmed>10433919</pubmed>
  45. <pubmed>10075717</pubmed>
  46. <pubmed>10725236</pubmed>
  47. <pubmed>9216996</pubmed>
  48. 48.0 48.1 48.2 <pubmed>15315763</pubmed>
  49. <pubmed>11044393</pubmed>
  50. <pubmed>8790332</pubmed>
  51. 51.0 51.1 <pubmed>8805249</pubmed>
  52. <pubmed>10482238</pubmed>
  53. <pubmed> 8662546</pubmed>
  54. <pubmed>11807029</pubmed>
  55. <pubmed>4390734</pubmed>
  56. <pubmed>4719010</pubmed>
  57. <pubmed>7453825</pubmed>
  58. <pubmed>7070499</pubmed>
  59. <pubmed>7720566</pubmed>
  60. <pubmed>11389830</pubmed>
  61. <pubmed>17400206</pubmed>
  62. <pubmed>10704381</pubmed>
  63. 63.0 63.1 <pubmed>3354847</pubmed>
  64. 64.0 64.1 <pubmed>1991324</pubmed>
  65. <pubmed>8124714</pubmed>
  66. 66.0 66.1 <pubmed>7753196 </pubmed>
  67. 67.0 67.1 67.2 <pubmed>9598380</pubmed>
  68. <pubmed>9118802</pubmed>
  69. <pubmed>8069909</pubmed>
  70. <pubmed>15936325</pubmed>
  71. <pubmed>8929535</pubmed>
  72. 72.0 72.1 <pubmed>10830170</pubmed>
  73. <pubmed>10217145</pubmed>
  74. <pubmed>9778248</pubmed>
  75. <pubmed>11239429</pubmed>
  76. 76.0 76.1 <pubmed>7588051</pubmed>
  77. <pubmed>25401122</pubmed>
  78. <pubmed>17016847</pubmed>
  79. <pubmed>12547712</pubmed>
  80. <pubmed>9768363</pubmed>
  81. <pubmed>186321</pubmed>
  82. 82.0 82.1 <pubmed>316949</pubmed>
  83. 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
  84. <pubmed>8620839</pubmed>
  85. <pubmed>10331974</pubmed>
  86. <pubmed>11290298</pubmed>
  87. 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/
  88. <pubmed>15979605</pubmed>
  89. <pubmed>8837770</pubmed>
  90. <pubmed>11476582</pubmed>
  91. <pubmed>9620294</pubmed>
  92. <pubmed>8681379</pubmed>
  93. <pubmed>11941477</pubmed>
  94. <pubmed>20583177</pubmed>
  95. <pubmed>19171780</pubmed>
  96. <pubmed>9205058</pubmed>
  97. <pubmed>9262482</pubmed>
  98. <pubmed>21537925</pubmed>
  99. YouTube. (2016). The Hedgehog Signaling in the Tumor Microenvironment - Research Hedgehog online Available at: https://www.youtube.com/watch?v=gFWO3I-oqsE
  100. <pubmed>3126020</pubmed>
  101. <pubmed>22087285 </pubmed>
  102. <pubmed>25981813 </pubmed>
  103. <pubmed>27771454</pubmed>