2016 Group Project 4

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2016 Student Projects 
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

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[2][3].
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[3].
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.
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[4].

Origin of name

Differences in patterning of the dentricle 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”.

Function

Neural development

Organogenesis

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

Mechanism

Processing of precursor


Overview of the Hedgehog signalling pathway

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[5]. 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[6]. 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[7].

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[8]. 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[9]. 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

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[10]. In Drosophila melanogaster, the Hh pathway begins when the Hh proteins bind to the extracellular domain of the transmembrane protein known as Patched (PTC) to inactivate it. This inactivation occurs by Hh trapping PTC in an inactive conformational state[11]. 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[12]. 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 [13]. It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) [14].

This overall increased phosphorylation of SMO due to a repression of PTC activity by Hh leads to an accumulation of SMO on the cell surface, which collectively allows for SMO to exert its activity. Most importantly phosphorylation also disrupts intramolecular electrostatic interactions between SMO molecules which switches the molecule into its active conformational state [15]. Generally, SMO has been shown to have very little signal transducing ability, and thus this accumulation allows it to jointly transduce as substantial signal. This occurs by the C terminus of SMO on the intracellular domain interacting with the kinesin like protein Costal-2 (Cos2), which is thought to bind to microtubules in order to acts as a scaffolding protein [16]. Then the kinase known as Fused (Fu) binds to Cos2, which phosphorylates Suppressor of Fused (SUFU) to inhibit it. Without this inhibition SUFU goes onto prevent the translocation of the transcriptional factor Cubitus interruptus (Ci) into the nucleus by leading to its phosphorylation, and subsequently its partial cleavage via the proteosome. The partial cleavage of Ci leaves a lower molecular weight protein known as Ci repressor (CiR) or Ci75, which translocates into the nucleus and acts to repress the target genes of the Hh signalling pathway via Ci[17]. 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[18].

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[19]. 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 [20] [21]. This overall stabalizes the boundaries between the segments of the developing Drosophila melanogaster signalled by the gene engrailed[22].

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

An overview of the hedgehog signalling pathway in Drosophila[23].

Mammals

Although the Hh signaling pathway has been conserved across species to an extent [24], 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[25]. 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. [26]. 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[27]. Other studies have also suggested that Shh has a role in increasing phosphorylation of SMO, which is required for it to accumulate as well on the cilia[28]. Overall the net action of Shh initially is believed to cause accumulation of SMO at the cilia by inhibiting PTCH1 and phosphorylation of SMO.

How SMO acts next is relatively unknown but, it has been shown to promote the disassociation between SUFU and Gli3 transcription factor, which allows Gli to be transported to the nucleus to activate effector genes of the pathway. Gli is normally bound in the cytoplasm to SUFU, where SUFU promotes the partial degradation of Gli into its repressor form, similar to that in the fruit fly model[29]. 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[30]. On the other hand, Kif7 is also thought to play an inhibitory role for the signaling pathway as when it is localized at the base of the cilia in the absence of Hh signaling and traffics Gli factors away from the cilia[31]. This all leads to an accumulation of active Gli transcription factors which migrate to the nucleus in order either inhibit or activate gene transcription in response to Hh signaling.

Animal models

Drosophila melanogaster

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

Upon further research on Drosophila it was found that Hh may play a role in germ cell proliferation and in particular, may control the proliferation and activity of somatic cells found within the germarium, which is the most anterior structure within the Drosophila ovary. [32] 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. [33] 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. [34] In addition, research on Drosophila has indicated the direct effect of Hh signalling on tracheal branch patterning. [35]

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 [36], 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”[37] (Ralph et al., 2005). Through reference to studies performed on chick and mice embryos, it was found that Shh signalling is particularly vital in development of maxillary and frontonasal components of the cranium.

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

Shh knockout mice

Extensive research on Shh knockout mice allowed discovery of the roles of Shh in embryonic development and patterning of the limb buds and sclerotomes, and maintenance of the notochord. [38] These mice expressed defects in the cephalic neural tube with the fusion of telencephalic and optic vesicles. Through this research it is evident that Shh is partially responsible for the subdivision of the eye field through forebrain optic stalk development, along with formation of the ventral midline. It is these forebrain abnormalities established in the absence of Shh that results in congenital malformations of holoprosencephaly, development of a single nasal chamber and other facial defects in humans.

The absence of the vertebral column including, the intervertebral discs and vertebrae along with the medial regions of the ribs were observed within Shh knockout mice. This lack of sclerotome derivatives indicates the role of Shh in maintenance or expansion of sclerotome cell population (Chiang et al., 1996). In addition, though the role of Shh in patterning the anterior-posterior limb axis is evident through anterior limb bud cell death in the absence of Shh, it is also responsible for the development of proximal-distal limb segments, particularly in the patterning of structures at the level of or distal to the latent elbow and knee joints. [39] 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

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 [40]. 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 [41].

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 [42]. 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 [43]. 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.

Cleft Lip and Palate

Diagnosis

Current research

Quiz

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References

  1. ↑ 1.0 1.1 <pubmed>6776413</pubmed>
  2. ↑ <pubmed>7916661</pubmed>
  3. ↑ 3.0 3.1 <pubmed>8269518</pubmed>
  4. ↑ <pubmed> 2679031</pubmed>
  5. ↑ <pubmed>1394430</pubmed>
  6. ↑ <pubmed>21357747</pubmed>
  7. ↑ <pubmed>8824192</pubmed>
  8. ↑ <pubmed>11486055</pubmed>
  9. ↑ <pubmed>9593755</pubmed>
  10. ↑ <pubmed>8595881</pubmed>
  11. ↑ <pubmed>27647915</pubmed>
  12. ↑ <pubmed>15102702</pubmed>
  13. ↑ <pubmed>10966113</pubmed>
  14. ↑ <pubmed>15616566 </pubmed>
  15. ↑ <pubmed>17960137 </pubmed>
  16. ↑ <pubmed>18691888 </pubmed>
  17. ↑ <pubmed>9215627</pubmed>
  18. ↑ <pubmed>10952898 </pubmed>
  19. ↑ <pubmed>9053330</pubmed>
  20. ↑ <pubmed>9502727</pubmed>
  21. ↑ <pubmed>10457026</pubmed>
  22. ↑ <pubmed>3282172</pubmed>
  23. ↑ YouTube. (2016). The Hedgehog signalling pathway in Drosophila online Available at: https://www.youtube.com/watch?v=w1xXD9kss2w
  24. ↑ <pubmed>8595881</pubmed>
  25. ↑ <pubmed>17641202</pubmed>
  26. ↑ <pubmed>PMC1462959</pubmed>
  27. ↑ <pubmed>16136078</pubmed>
  28. ↑ <pubmed>21695114</pubmed>
  29. ↑ <pubmed>20360384</pubmed>
  30. ↑ <pubmed>19592253</pubmed>
  31. ↑ <pubmed>19666503</pubmed>
  32. ↑ 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
  33. ↑ <pubmed>8620839</pubmed>
  34. ↑ <pubmed>10331974</pubmed>
  35. ↑ <pubmed>11290298</pubmed>
  36. ↑ 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/
  37. ↑ <pubmed>15979605</pubmed>
  38. ↑ <pubmed>8837770</pubmed>
  39. ↑ <pubmed>11476582</pubmed>
  40. ↑ <pubmed>9620294</pubmed>
  41. ↑ <pubmed>8681379</pubmed>
  42. ↑ <pubmed>11941477</pubmed>
  43. ↑ <pubmed>20583177</pubmed>
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