Transcriptional control of the endocrine pancreas
Transcription factors are vitally important to the developmental biology of all life. They also define many of the developmental stages of organs within the body. Their expression allows for the activation of developmentally important cues and signalling cascades, through activation of specific subsets of genes. When transcription factor function is disrupted, it can have serious repercussions on the development and function of organ systems. This dysfunction, in many cases, leads to the onset of diseases such as cancer and diabetes. The general objectives of this thesis were to describe the role and function of several transcription factors vitally important in the development and maintenance of the pancreas through three lines of research. Firstly, neurogenin3 (ngn3) is vitally important to the development of the islets of Langerhans. Knocking out this gene prevents mice from developing endocrine cells of the pancreas (islets of Langerhans). This thesis set out to label the ngn3 positive precursor cell population in developing mice, and characterise their phenotype. Detailed immunohistochemistry and fluorescence activated cell sorting coupled with reverse transcriptase (RT)-PCR were employed to fully characterise the EGFP positive cell population at embryonic day (E)15.5 and E l8.5. EGFP positive cells were able to be isolated at both E l5.5 and E l8.5 and were found to co-express endocrine markers both in immunohistochemical and RT-PCR based assays. Marking cells that are developmentally important to pancreas function, and being able to identify their phenotype, may provide clues to markers that could be exploited as targets for future diabetes therapies. Secondly, pancreatic duodenal homeobox-1 (PDX-1) is another critically important transcription factor in both pancreas development and in maintaining the insulin expression of the beta cells in the islets of Langerhans. Preventing PDX-1 function causes severe agenesis of the pancreas, and mutations in the gene cause one form of diabetes. This thesis describes the production of a super-activated form of the PDX-1 protein using the transactivation domain VP 16. The main objective of this work was to drive PDX-1 function in a non-beta cell. Using chromatin immunoprecipitation (ChIP) assays, the PDX-1 VP 16 protein appeared to increase the acetylation state of the insulin promoter in the mouse non-beta cell line (alpha-TCI.6), particularly through altering the acetylation state of the insulin 2 promoter. The use of the PDX-1VP16 protein could provide a potential starting point for research into driving differentiation of stem cells to a pancreatic phenotype. Finally, there are four transcription factors that are known to drive expression of the insulin gene synergistically. They are the homeodomain protein PDX-1, the leucine zipper protein MafA, and the basic helix loop helix heterodimer beta2/E47. All of these proteins have binding sites within 300 bp of the transcriptional start site of the insulin gene. Through the use of ChIP assays this thesis shows, for the first time, the ordered cyclical binding of these four transcription factors on the insulin 2 gene promoter in the mouse beta cell line MIN-6. Understanding how the transcription factors that drive insulin expression interact on the promoter in vivo could be used for future studies to understand the process of insulin gene transcription both in healthy and diabetic patients. This may then shed light on the processes that cause the perturbations in insulin transcription seen with certain forms of diabetes.