Identifying progenitors responsible for beta cell regeneration in zebrafish In spite of the obvious power of mouse studies to identify molecular pathways in pancreas development, mouse genetics is often laborious and time consuming. As there is a high degree of conservation in the molecular mechanisms that control vertebrate pancreas development, we have turned to the zebrafish as an alternative model system to study beta cell biology and regeneration. Using the insulin promoter, several transgenic lines of zebrafish have been created that express an E. coli protein, nitroreductase (NTR), fused to fluorescent tags (eGFP or mCherry). Nitroreductase is an enzyme that can convert the prodrug, metronidazole to a cytotoxin. By adding this prodrug, we show that transgene associated fluorescence is extinguished concurrent with a loss of the insulin expressing beta cells. We conclude therefore that we are able to drug dependently ablate beta cells and have data to demonstrate that other cells in the islet are unaffected. Following removal of drug and 36 hours of recovery, we have documented a return of fluorescence, consistent with regeneration of two cells. Having established this novel model system for ablating beta cells, we now have the opportunity to screen for drugs that will modify beta cell regeneration. To test the feasibility of such a screen, we have applied a variety of chemicals to zebrafish larval following prodrug dependent ablation and observed the effects on regeneration. DAPT is a compound that blocks the Notch signaling pathway. Treatment with DAPT following beta cell ablation, leads to a significant increase in the number of beta cells that regenerate. We also show for the first time that a Notch gene (notch 1b) is expressed in a discrete population of cells in the developing pancreas. Together this data suggests that the Notch signaling pathway is involved in maintaining a progenitor pool in the endocrine pancreas. We have outlined a number of methods we are pursuing to utilize this knowledge to identify, characterize and manipulate these progenitor cells. It is hoped that by understanding how progenitors differentiate into insulin producing cells in our model system will allow the development of better techniques to exploit the endogenous regenerative capacity of vertebrate endocrine pancreas. Such techniques could be utilized as an alternative or to complement therapies based on islet transplantation. NARRATIVE Identifying progenitors responsible for beta cell regeneration in zebrafish. We have developed a novel system where we can specifically ablate the insulin producing beta cells, in transgenic zebrafish embryos and shortly afterwards observe the regeneration of insulin positive cells. Using the Notch signaling pathway we aim to identify and manipulate the progenitor cells responsible for beta cell regeneration. It is hoped that by understanding how progenitors differentiate into insulin producing cells in our model system will allow the development of better techniques to differentiate beta cells for therapeutic purposes in the future.
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