The hedgehog signalling pathway is of major significance, involved not only in the growth and proliferation of the individual cell, but in cell to cell signalling and patterning of the embryo as a whole. It is therefore essential to the survival and appropriate development of the organism that the region of control of the potent morphogen hedgehog be tightly regulated. For this pathway to be held in check an appropriate balance must be maintained between the activities of the hedgehog protein and that of its counterbalancing regulators.
A key and consistently conserved element of control of the hedgehog signalling pathway is the transmembrane domain protein patched. The human homologue of this protein was identified in our, and in an independent laboratory in 1996, as the gene mutated in the familial disorder Naevoid Basal Cell Carcinoma Syndrome (NBCCS). NBCCS patients display a variable phenotype exhibiting the results of both mispatterning such as skeletal abnormalities, and poorly controlled cell proliferation such as a propensity for the growth of tumours.
Patched was identified in the fruit fly Drosophila in 1980 and isolated in flies in 1989, and the hedgehog pathway has from this time been intensively studied due to its wide ranging effects. Although it is accepted patched plays a key role as a regulator of the pathway, the mechanism by which patched exerts its repressive function is still poorly understood. To gain insight into the means by which patched produces its effects it was proposed to functionally dissect the patched protein by modelling mutations. Those mutations modelled were selected from published missense mutations, those which occurred in areas expected to be of high functional significance were made the major focus of this project. Similarly, based on the predicted topology of the patched molecule deletions mutants were constructed removing what where predicted to be critical sections of the protein.
To investigate the functional capacity of the mutated versions of the patched protein it was decided to focus on in-vivo models, which overcome the inherent difficulties of cell-based systems as they are better able to provide the appropriate context for operation of the hedgehog signalling pathway. The chick and the fly have been intensively used as in-vivo model systems for investigating components of this pathway. Hence to examine the effects created by mutating the patched protein these two model systems which were available to our laboratories were utilised.
A collaboration was formed in which Drosophila versions of the mutants were expressed in the fly in another laboratory. The core of this thesis however involves the expression of human forms of the mutants in the chick. Studying the effects of the mutations in chicks has the benefit of this model system having the downstream components of the vertebrate hedgehog signalling pathway which are now known to diverge significantly at the level of nuclear transcription proteins from that of the fly. In-ovo electroporation is rapidly becoming an established technique for investigating the longer term effects of protein overexpression in the embryonic chick, and has recently been used successfully in our group of laboratories. By combining this procedure with the expression of what were judged to be the most biologically relevant mutants this work aims to help elucidate the structure-function relationship of the patched protein.
These studies show that in the context of overexpression in the neural tube these mutants retain a predominantly repressive function with respect to patterning. However there are clear differences in the abilities of each of the mutants to carry out this function. Additionally these results demonstrate that overexpression of the patched protein contributes to apoptosis, however this function is ablated by a single amino acid change. As the functions of patterning and apoptosis in the neural tube were affected by distinct mutations, this thesis provides direct evidence that distinct domains are essential for separate functions of the patched protein. This protein operates in a pathway involved in both normal development and tumourigenesis. A better understanding of the protein could potentially lead to a better understanding of the pathway as a whole and consequently its potential for manipulation in both familial and sporadic disease.