Identification of Novel Downstream Targets of Sonic, Indian and Desert Hedgehog

The hedgehog signalling pathway is one of the key developmental pathways that plays a critical role in embryonic development, tissue organisation, spatial patterning and tumourigenesis. The importance of this pathway is evidenced by the high degree of conservation of the overall structure of this cell-cell signalling pathway across species. However it is becoming more apparent that specific and important differences in hedgehog signalling do exist in various species. One of the most intriguing of these is the advent of multiple hedgehog ligands in vertebrates, Sonic, Indian and Desert hedgehog, compared to the single hedgehog gene in Drosophila. Hedgehog signalling is essential for normal embryonic development, with disruption of this signalling cascade leading to a variety of developmental disorders and a number of human cancers. Dysregulation of hedgehog signalling is a major factor in the formation of basal cell carcinoma and other solid tumours. The identification of downstream target genes is vital for understanding how perturbation of this pathway so alters the cellular and genetic environment, to allow oncogenic transformation. Historically Sonic hedgehog has been the best characterised of the three hedgehog ligands, and very few downstream targets of Indian and Desert hedgehog have been identified. Whilst Sonic hedgehog is widely expressed in a variety of organs and tissues, Indian and Desert hedgehog have exclusive and more spatially restricted expression patterns. As for many molecules, the increase in the number of hedgehog ligands has been proposed to account for the increase in complexity from Drosophila to vertebrates, allowing more precise control over developmental processes. The three vertebrate ligands display a high degree of sequence homology, however until now no studies had been conducted to compare the suite of downstream target genes controlled by each of the three hedgehog ligands. Using a microarray approach, a direct comparison was made between RNA isolated from a hedgehog responsive embryonic mouse cell line treated with either Sonic, Indian or Desert hedgehog proteins or with a control. A number of known Sonic hedgehog targets were identified and evidence for regulation of some of these genes by Indian and Desert hedgehog obtained. I identified 19 novel validated targets of hedgehog signalling and 4 potential targets. All targets were validated by northern blotting using RNA produced by pathway activation achieved by an alternate mechanism, than that used for the microarray analysis. These novel target genes encode molecules involved in a diverse array of functions including transcriptional regulation, enzymatic catalysis, cell cycle control, intracellular trafficking and organisation of the cytoskeletal architecture. A large number of the novel hedgehog target genes identified are implicated in tumourigenesis and many different types of human cancers. Understanding how aberrant hedgehog signalling regulates these novel target genes may improve our understanding of the process of tumour initiation. I have determined that in the same cell type, under the same conditions, Sonic, Indian and Desert hedgehog do not elicit an identical suite of target genes. Multiple genes were identified as targets of all three hedgehog ligands, however, genes were also identified that were targets of one hedgehog ligand, but not of all three. The work in this thesis has expanded the known targets of hedgehog signalling, in particular elucidating downstream targets of Indian and Desert hedgehog that may expand our understanding of their role in development and tumourigenesis.