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THE ROLE OF SOX9 DURING HUMAN PANCREAS DEVELOPMENT
[Thesis]. Manchester, UK: The University of Manchester; 2011.
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Abstract
The work presented in this thesis is a study of human pancreas development. Theprinciple goal of this work is to provide information that can be used in the developmentof treatments for Type 1 Diabetes and in pancreas regeneration methodologies.The transcription factor (TF) sex determining region Y homeobox gene 9 (SOX9) hasbeen identified as a key factor in human pancreas development but its role has not beenwell characterized. The expression of SOX9 during early pancreas development wasanalyzed by immunostaining of fixed embryonic and fetal sections in the context of otherdevelopmentally important TFs. Modulators of SOX9 function, downstream targets andupstream regulatory pathways were investigated in human cell lines using coimmunoprecipitation, small interfering RNA (siRNA) knockdown, quantitativepolymerase chain reaction (qPCR), luciferase assays and small molecule signalingpathway inhibitors.SOX9 was expressed in epithelial progenitors from initial human pancreas specification,but became excluded from the periphery of the epithelium and developing islet cells asdifferentiation proceeded. It was co-expressed with important endocrine and exocrinedifferentiation factors during the early stages of development. Some factors, such asNirenberg and Kim 2, homeobox family member, drosophila, homolog of, 2 (NKX2.2)showed differing expression profile compared to murine development, while thewidespread expression of endocrine factors before expression of the pro-endocrine geneneurogenin 3 (NGN3) suggested that these factors play an important role in initiatingendocrine specification.Two transcription factors, GATA-binding protein 4 (GATA4) and neurogenicdifferentiation 1 (NEUROD1), were found to interact with SOX9 in potentiallydevelopmentally relevant complexes. This prompted the search for downstream targets ofthese transcriptional complexes by in silico analysis, which identified an array of novelpotential downstream targets. Luciferase assay analysis of a subset of these genes showedSOX9 to activate a regulatory region of NGN3, and inhibit the regulatory regions ofcarboxy peptidase A6 (CPA6), v-ets avian erythroblastosis virus E26 oncogene homolog1 (ETS1) and SPONDIN1. An additional target of SOX9, osteopontin (OPN), wasidentified from a microarray of Sox9 knockout mouse pancreata. Investigation of SOX9 and OPN regulation by the Hedgehog signalling pathway (HH) identified both factors tobe regulated by the pathway, suggesting SOX9 may act as a mediator of HH signalling.This is the first study to identify a range of SOX9 targets relevant to human pancreasdevelopment. While further characterization is required this work has provided essentialclues to the function of SOX9, and provides a detailed framework of SOX9 expressionand targets for future pancreatic studies to build upon.