In response to the recent program announcement (PAR-05-166) """"""""Genetic and genomic analyses of Xenopus"""""""", we are submitting this grant to elucidate the transcriptional regulatory networks operating during Xenopus pancreas development. Our hypothesis is that through the use of microarrays pancreatic lineage-specific genes can be identified by examining the molecular differences between 1) normal and mutant (i.e. those lacking certain pancreatic transcription factors) pancreatic tissues and 2) transdifferentiated and normal livers. Understanding the molecular signals that direct early endodermal progenitors into acinar, ductal or endocrine lineages is essential in order to determine how to generate beta cells from other cell types. We propose to use three different approaches to identify these genetic networks.
In Aim 1, we shall compare and contrast the changes in gene expression in early pancreatic tissue lacking three specific transcription factors, Ptfia, IA-1 or Ngn3, which our lab has identified in Xenopus. This data will be used to generate a transcriptional map of how early endodermal progenitors are specified first to a pancreatic fate (Ptfia), second to an endocrine fate (Ngn3) and third to a beta cell fate (IA-1).
In Aim 2, we will identify the immediate early targets activated during the transdifferentiation of liver to pancreas in transgenic tadpoles by comparing the gene expression profiles of livers expressing either Pdx1-VP16 or Ptf1a-VP16. This will demonstrate what genes must initially be activated in liver cells to generate all pancreatic cells (Pdx1-VP16) or only acinar cells (Ptf1a-VP16), and by comparison only endocrine cells. Once all of the results from Aims 1 and 2 are tabulated, they will provide, via a transcriptional regulatory map, a greater understanding of how individual transcription factors direct pancreas development.
In Aim 3 we propose a slightly different method to identify new inducers of pancreatic fate through an RNA overexpression screen. This gain-of-function approach will be done using our F2 transgenic Elas-GFP X. laevis embryos, thus permitting a quick and easy functional readout simply by looking for ectopic or expanded GFP, indicating a pancreatic fate. Elucidating the transcriptional regulatory networks that operate during pancreatic cell fate specification will greatly increase our understanding of what signals are needed to direct the differentiation/conversion of other cell types into beta cells for the treatment of diabetes.
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