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Investigation of the role of the primary cilia and centrosome during signalling events in development using the chicken mutant talpid3

Reference	BB/F024347/1
Principal Investigator / Supervisor	Dr Megan Davey
Co-Investigators / Co-Supervisors
Institution	University of Edinburgh
Department	The Roslin Institute
Funding type	Research
Value (£)	1,102,452
Status	Completed
Type	Fellowships
Start date	01/07/2008
End date	28/02/2014
Duration	68 months

Abstract

Primary non-motile cilia (9+0) and their associated centrosomal components, the centriole are essential components of the Wnt, PDGF and Hedgehog (Hh) signalling pathways. Cilia are also important mechanosensory organelles. Defects in genes associated with cilia formation result in a wide range of human developmental disorders, characterised by phenotypes such as central nervous system disorders, kidney failure, blindness and polydactyly. It is known that components of signalling pathways are preferentially concentrated in the cilia but it is unclear how this protein sorting is achieved. Talpid3 is a chicken mutant with a defect in KIAA0586, a gene associated with the centrosome. Talpid3 embryos lack cilia, have developmental defects which resemble phenotypes seen in the human ciliopathies and have a well characterized Hh signalling defect. As developing chicken embryos are readily available and easily to experimentally manipulate, we will use this ciliary mutant in order to understand the mechanisms involved in cilia based intracellular signalling. Using in vitro and in ovo complementation experiments and co-localisation studies, we will observe the effect on cilia and Hh signalling, by manipulating both known components of the Hh pathway and genes involved in ciliogenesis. We will also use this approach to test candidate genes identified as potentially involved in Hh signalling/ciliogenesis in a previous analysis. The mechanosensory role of cilia in the kidney is clear, but the contribution of mechanical stimulation as a driver of morphogenesis is less well understood, although it is known that blood flow contributes to the patterning of vasculature. Talpid3 embryos have defective vascular patterning. We will investigate how the loss of cilia contributes to this phenotype. This work may elucidate the way in which cilia co-ordinate these diverse signalling events to direct morphogenesis.

Summary

It is thought that every cell in our bodies has a small antenna poking out of it, called a primary cilia. For many years biologist thought that the primary cilia was no more than a vestigial organ, something left behind from when we were single-celled organisms swimming through the oceans using a flagella (the type of tail sperm and other motile cells use to move). However now it seems that the primary cilia is more than just a stumpy tail, it is in fact essential to normal development of the embryo. Clues as to the importance of cilia, first came from investigation into human diseases in which motile cilia were absent or impaired. Not surprisingly, these people often suffer from respiratory and reproductive problems, as they don't have cilia to move mucus, eggs or sperm. In addition it was found that many of these people also have situs inversus, a condition when the internal organs such as the heart are found on the wrong side of the body. This suggested that cilia somehow play an important role in the development of the embryo, somehow controlling which side organs should form on. To understanding how cilia could control such important events, recent research has aimed to determine what cilia consist of. Using many different techniques including studying mouse and human mutations which result tell-tale cilia syndromes such as the development of extra fingers and kidney problems, scientists have begun to understand which genes are needed to form normal cilia. By studying precisely what goes wrong in animals which lack cilia, we now understand that cilia are required to control important intracellular signalling events, particularly in pathways important in development and cancers known as the Hedgehog and Wnt pathways. Using high powered imaging techniques, researchers have been able to look inside cilia and have found that some important proteins become highly concentrated in cilia, using cilia as a special compartment in which vital protein interactions cantake place. But we have little idea how this occurs. We study a chicken flock in which some of the embryos fail to hatch. It was discovered that these embryos die in the egg from serious developmental disorders. By examining the anatomy of these embryos known as talpid3, we discovered that important signalling events in the Hedgehog signalling pathway, go awry, leaving embryos with central nervous system, limb, head, blood vessel and bone defects. Excitingly when the defects in talpid3 embryos are compared to those in humans and mice in which cilia are defective there are many areas of similarity and it now seems talpid3 embryos also have defective cilia. Furthermore the gene which causes talpid3, localises in the centrosome, a structure essential for normal cilia development. Talpid3 embryos offer a unique approach to studying cilia function which is difficult to undertake in mice. Because embryos develop outside the mother (in the egg) it is possible to manipulate embryos while they are still developing. We plan to use talpid3 embryos to examine which genes are important in cilia growth, by over expressing or reducing expression of genes known to be important in cilia formation or Hedgehog signalling in the embryos and then examining cilia formation, or patterning of the nervous system controlled by Hedgehog signalling. We can also use both these embryo manipulations and cell culture techniques to see where proteins localise in the cell. Talpid3 embryos also have one other interesting defect, in that they don't form normal veins. Although this could be because of the abnormal Hedgehog signalling in talpid3 we would also like to investigate if this is due to another function of cilia, which can sense mechanical stresses such as flow. We think it is possible that cilia not only act as a focal point for signalling in the cells but may act to coordinate the many varied signalling events that determine how we develop.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Fellowship - Institute Career Path Fellowship (ICPF) [2006-2010]
Funding Scheme	X – not Funded via a specific Funding Scheme

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