The hedgehog pathway has emerged as a key signalling pathway controlling embryonic development and tumorigenesis. This work was undertaken to further elucidate the complex mechanisms regulating hedgehog signalling at the cellular level.
In this study the subcellular localisation of the human hedgehog receptor, Patched (PTCH), was analysed in mammalian cell cultures in order to gain insight into its cellular trafficking, a process which has emerged as a significant factor in the regulation of hedgehog signalling. This involved the detailed analysis of the subcellular localisation of PTCH at both the light and electron microscopy (EM) levels and this work has resulted in the first published EM images of a vertebrate patched protein (Evans et al., 2003). Patched plays roles in hedgehog signalling by receiving and sequestering the ligand in addition to suppressing signal transduction in the absence of ligand and the correct trafficking of Patched is required to fulfil these roles. This analysis has contributed to our understanding of outcomes of inappropriate patched function, namely developmental defects, tumorigenesis and human disease.
During the course of this project the importance of trafficking in hedgehog signalling was further highlighted by the finding that Rab23, a member of the Rab-GTPase family of vesicle transport proteins, acts to negatively regulate hedgehog signalling (Eggenschwiler et al., 2001). This study provides the first description of the subcellular localisation of wild-type and mutant forms of the mouse Rab23 protein. By analogy to other Rab proteins, which function in vesicular transport, we sought to define the localisation of Rab23 within the cell relative to known cellular markers and hedgehog pathway members at both the light and EM levels. This analysis was undertaken in order to provide better understanding of the Rab23-mediated trafficking events significant to the regulation of hedgehog signal transduction. Both the findings relating to the analysis of PTCH and Rab23 subcellular localisation have been published (Evans et al., 2003) and this article appears as part of this thesis.
Given the strong links between cellular cholesterol levels and hedgehog signalling, this study also sought to explore the role of cholesterol in the regulation of this pathway. As part of this study, the genetic lesions responsible for an inborn error of cholesterol biosynthesis, Smith-Lemli-Opitz syndrome (SLOS), were identified in patient samples. These findings describe the mutations found in the gene encoding the last enzyme in the cholesterol biosynthesis pathway, Δ7-dehydrocholesterol reductase (DHCR7), in these SLOS patient samples. These findings were published (Evans et al., 2001) and this article appears as part of this thesis. The cell lines scored for DHCR7 mutations will be used in future studies to gain greater understanding of how cholesterol depletion influences hedgehog signal transduction.
Furthermore, an in vitro assay system was developed to assess the effect of cholesterol depletion on hedgehog signalling by inhibition of cholesterol biosynthesis. These findings demonstrate that upon cholesterol depletion the transduction of the hedgehog signal is inhibited in receiving cells.
The results described in this thesis contribute significantly to our understanding of the role of intracellular trafficking events and sterol levels in the regulation of the hedgehog signalling pathway. The data and resources generated pave the way for a detailed analysis of the intricacies of hedgehog signalling at the cellular level, which will in turn enhance our understanding of the role of hedgehog signalling in embryonic development and disease.