Investigating the role of a gene suspected to be part of the hedgehog signaling pathway in Drosophila

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Investigating the role of a gene suspected to be part of the hedgehog signaling pathway in Drosophila

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Abstract

Though Hedgehog (hh) has many homologues in vertebrates that signal through the same pathway, involving homologue proteins involved in Drosophila, leading to different effects on the organism (though all imply the morphogen feature of hedgehog), we will focus on its signalling pathway in the latter organism. Hh is known to be one of the segment polarity genes expressed during the late phase of embryo development in Drosophila melanogaster. The segment polarity genes, as the name suggests, are the genes encoding proteins that will set up the anterio-posterior polarity of the segments, developmental units of Drosophila embryos, once cellularization has occurred. Hedgehog is a secreted protein that will activate a signal transduction cascade once bound to its receptor. We will first see what is nowadays known about the hedgehog signaling pathway, including what happens in the Hedgehog protein (Hh) and some target genes, though both aspects are not related to the signaling pathway, but are important to understand such a blur event in the patterning of future adult fly. Then we will think about ways to investigate the role of a gene we suspect to be part of the pathway, considering the level of its possible action in this pathway

Introduction.
The hedgehog signalling pathway.
An active signalling protein.
Signals through a currently obscure scheme to a microtubule bound complex.
The target genes.
The possibility of a physical interaction between Smo and Ptc leading to a conformational change.
1. The transport of Smo into intracellular vesicles.
2. The binding of an ihnibitory molecule to Smo mediated by Ptc.
3. Evidences of an Hh signalling pathway non-dependant on Ptc.
Unelucidated part of the pathway.
Conclusion.
Bibliography.

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[...] A way to investigate the role of a gene we suspect to encode a protein activating a kinase acting at this point of the pathway would be to overexpress it in smo- mutants (showing the same phenotype as normal flies when no Hh is bound to Ptc and Smo is repressed). Being in larger amounts than normal when no Hh ligands are available, the protein would be able to activate the kinase and therefore ensuring the proper transduction of the Hh signal. [...]

[...] Speaking of the target genes, Hedgehog pathway is thought to activate the transcription of many different genes involved in upregulating its own pathway, in transmitting its signal to a longer range of cells through expression of intermediaries genes such as wingless, as said before, and in cell growth and proliferation (disrupted Hh pathway can lead to tumorigenesis), which doesn't come as a surprise when we consider its patterning activity. Two main aspects of the pathway are currently being questioned. The first one is how does Ptc exerts its inhibitory effect on Smo. [...]

[...] The sterolation ensures that Hh is localised in the cells where it is needed by giving it the ability of anchoring to the cell membrane by its added cholesterol group. It also allows the Dispatched (Disp) transmembrane protein to secrete Hh outside the cells and the binding to its receptors. The palmytoylation enhances its biological activity, as it has been proved with mutants lacking the palmytoylation that have a 30-fold lower activity. Once activated and secreted, Hh binds to a receptor complex, involving Patched (Ptc) and Smoothened (Smo). [...]

[...] On another hand, if we suspect the gene we found to encode one of the kinases that phosphorylate Fu or Cos2, the simpliest way would be to knock it out, once again using the RNA interference technique. Because the pathway would be disrupted, knocked out flies should not be able to express the Hh target genes. Hh can be found to be called “versatile”. Proportionally to its complicated set of genes it activates and to the many cellular aspects Hh is involved into, its signalling pathway is quite a puzzle for researchers, considering its many levels of regulation and the number of proteins it involves. [...]

[...] We can trace this with adding a lacZ marker to the regulating sequence of the Hh target genes, wingless for exemple, as we know it is secreted when the pathway is activated. Evidences of an Hh signalling pathway non-dependant on Ptc have been shown. This raises the possibility of the existence of another Hh receptor also acting on Smo, like it is the case for vertebrates. If a gene encoding such a protein was suspected, here again knocking it out at the same time as we knock out patched would be a good approach. [...]

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