Genetic analysis of hedgehog signalling and the regulation of patched gene expression in vertebrate embryos

This thesis addresses vertebrate Hedgehog (Hh) signalling pathways, and in particular the role played in these pathways by vertebrate homologues of patched (ptc). Experiments using both zebrafish and chickens embryos are described, hh genes encode signalling molecules that have been implicated in a wide variety of developmental patterning events. Ptc has been implicated as the receptor for the Hh signalling pathway and is also a direct target gene of Hh signalling. I describe the isolation and characterisation of full length cDNA sequence for two zebrafish homologues of ptc. The expression of ptcl and ptc2 is described in wild type embryos, in embryos homozygous for a mutation in shh and in embryos that are missing expression of all characterised hh genes in particular regions of the embryo. Analysis of ptc1 and ptc2 expression in embryos in which synthetic RNA from different hh genes has been over-expressed is also described. I have also analysed a number of different zebrafish mutations that affect the development of the embryonic midline or somites aswell as the phenotypic consequences of over-expressing ptc1. Experiments that address the regulation of ptc gene expression in the chicken and a molecular analysis of the chicken mutation talpid3 are also described, talpid3 is a autosomal, pleiotropic, recessive-lethal mutation in an unknown gene. My analysis has concentrated on the limb, though I have undertaken some analysis of gene expression in the trunk, talpid3 has an interesting limb phenotype which resembles the effects predicted for a uniform ectopic expression of shh, or from a partial loss of function of ptc. However, shh is expressed normally in talpid3. I have analysed the expression of several known Shh pathway genes, and the effect on these gene expression patterns of experimental manipulations such as tissue grafts and exposure to ectopic Shh protein