Cloning and Characterization of Zebrafish Endocrine Pancreas Mutants

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DESCRIPTION (Provided by Applicant): The long-term goal of this application is to develop strategies for ¿ cell based replacement therapy to treat diabetes by understanding the molecular mechanisms regulating ¿ cell specification using zebrafish, a model organism well suited for embryogenic and genetic studies. The investigator conducted a chemical-induced mutagenesis screen to identify the genes that control various aspects of pancreatic ¿ cell formation in zebrafish. In the developing zebrafish pancreas, ¿ cells aggregate into a cluster (islet) at 24 hpf through cell migration. Two mutants, la676 (cheetah) and la572 (minime), have abnormally split morphology of insulin positive cells. Either one exhibits any visible morphological abnormalities. Mnm mutants also show reduced insulin expression. Liver and intestine development is not affected in either mutant. These data have led to the hypothesis that the che and mnm mutations cause cell migration defects and mnm also regulates ¿ cell specification or proliferation. The objective of this proposed study is to identify the che and mnm genes by positional cloning technique. The investigator will focus on, in Specific Aim 1, determine the chromosomal locations of the che and mnm genes based on simple sequence length polymorphism; in Specific Aim 2, define the critical regions harboring the che and mnm genes by high-resolution meiotic mapping; in Specific Aim 3, identify clones from BAC library and genomic contigs that span the che and mnm loci. The candidate genes will be tested by knockdown using morpholino antisense oligos to phenocopy the mutations and analyze the gene mutations by PCR and DNA sequencing. The mutant phenotypes will be rescued by injecting the cDNA or mRNA of candidate genes; in Specific Aim 4, study cell migration defects in detail by time-laps confocal imaging using insulin:GFP transgenic fish; and in Specific Aim 5, examine cell proliferation and cell death in these mutants. These studies will provide further insights into the genetic regulation of endocrine pancreas organogenesis. PROJECT NARRATIVE: This application proposes to clone two genes that regulate cell migration during endocrine ¿ cell development to form islet. Correct formation of islet is critical for ¿ cell function to ensure low amount of insulin secretion during starvation and sufficient amount of insulin secretion after food uptake. These studies will provide invaluable insights on the molecular mechanisms regulating endocrine islet formation.