The identity and location of epithelial precursor cells in exocrine pancreas remain unknown. Further characterization of this precursor population would represent a significant advance in our understanding of pancreatic development, pancreatic regeneration and pancreatic cancer. While studies in mice and other rodents have provided important initial insights, these species have significant limitations related to many aspects of genetic manipulation. The zebrafish (Danio rerio) has recently emerged as a central model organism in the study of vertebrate development. The increasingly widespread use of the zebrafish reflects significant advantages with respect to embryo access, simplified techniques for gain-of-function and loss-of-function analysis, and forward genetic capabilities. Recent studies have suggested that many aspects of pancreas development in the zebrafish are conserved with respect to other vertebrate species. Based on a recent NIDDK program announcement requesting proposals for innovative and exploratory research involving pancreatic stem cells and/or novel animal models (PA-01-129), we have embarked on initial studies of zebrafish exocrine pancreas development. The long-term goal of this research program is to identify and rigorously characterize exocrine pancreatic precursors in zebrafish pancreas, ultimately allowing use of this organism as a novel model system for study of human pancreatic disease. Our preliminary studies have involved cloning and characterization of the zebrafish orthologue for PTF1a-p48, a basic helix-loop-helix transcription factor required for normal pancreas development in the mouse. Initial gene expression and p48 morpholino knock-down studies have suggested fundamental and informative differences between zebrafish and mouse with respect to the relationship between endocrine and exocrine precursor pools. Based on these findings, we propose that identification and characterization of p48-positive precursors in zebrafish pancreas will allow investigation of conserved regulatory mechanisms not discernable in the mouse, and that novel regulators of pancreatic development will be identified among genes differentially expressed in wild-type vs. p48 morphant embryos. To test these hypotheses, the following Specific Aims will be pursued: First, to rigorously determine the identity, location, and gene expression profiles of p48-positive precursor cells in developing zebrafish pancreas. Second, to determine the contribution of p48-positive precursors to endocrine and exocrine lineages; and finally, to identify novel p48 target genes responsible for mediating the effects of p48 on early pancreas development. It is anticipated that these studies will provide important new insights regarding regulation of exocrine pancreatic precursor cells, and further establish the zebrafish as a novel model system for the study of epithelial differentiation in exocrine pancreas. ? ?
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