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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This page is an undergraduate science embryology student project and may contain inaccuracies in either descriptions or acknowledgements.
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<pubmed>23719536</pubmed>

Hedgehog signalling pathway

Hh Signalling Pathway.jpg

History

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

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[1]. 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[2]. 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[3].

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[4]. 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[5]. 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[6]. 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[7]. 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[8]. 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 [9]. It is thought that the phosphorylation required to activate SMO are dependent on protein kinase A (PKA) and casein kinase I (CKI) [10].

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 [11]. 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 [12]. 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[13]. 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[14].

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[15]. 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 [16] [17]. This overall stabalizes the boundaries between the segments of the developing Drosophila melanogaster signalled by the gene engrailed[18].

Vertebrates

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. [19] 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. [20] 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. [21] In addition, research on Drosophila has indicated the direct effect of Hh signalling on tracheal branch patterning. [22]

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 [23], 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”[24] (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. [25] 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. [26] 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

Human disease

Holoprosencephaly

Cleft Lip and Palate

Cancer

Diagnosis

Current research

References

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  10. <pubmed>15616566 </pubmed>
  11. <pubmed>17960137 </pubmed>
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  13. <pubmed>9215627</pubmed>
  14. <pubmed>10952898 </pubmed>
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  17. <pubmed>10457026</pubmed>
  18. <pubmed>3282172</pubmed>
  19. 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
  20. <pubmed>8620839</pubmed>
  21. <pubmed>10331974</pubmed>
  22. <pubmed>11290298</pubmed>
  23. 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/
  24. <pubmed>15979605</pubmed>
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  26. <pubmed>11476582</pubmed>