Identifying the progenitors responsible for Beta Cell regeneration in zebrafish

描述（由申请人提供）： 尽管小鼠研究在确定胰腺发育中的分子途径方面具有明显的力量，但小鼠遗传学通常是费力和耗时的。由于在控制脊椎动物胰腺发育的分子机制中存在高度保守性，我们已经转向斑马鱼作为研究β细胞生物学和再生的替代模型系统。利用胰岛素启动子，已经建立了几个转基因斑马鱼品系，它们表达E。大肠杆菌蛋白质，硝基还原酶（NTR），融合到荧光标签（eGFP或mCherry）。硝基还原酶是一种可以将前药甲硝唑转化为细胞毒素的酶。通过加入这种前药，我们表明转基因相关的荧光在表达胰岛素的β细胞丧失的同时熄灭。因此，我们得出结论，我们能够药物依赖性地消融β细胞，并且有数据证明胰岛中的其他细胞不受影响。在去除药物和恢复36小时后，我们记录了荧光的恢复，与两个细胞的再生一致。在建立了这种消融β细胞的新模型系统之后，我们现在有机会筛选能够改变β细胞再生的药物。为了测试这种筛选的可行性，我们将多种化学物质应用于前药依赖性消融后的斑马鱼幼虫，并观察其对再生的影响。DAPT是一种阻断Notch信号通路的化合物。β细胞消融后用DAPT治疗导致再生的β细胞数量显著增加。我们还首次发现Notch基因（notch 1b）在发育中的胰腺细胞中表达。总之，这些数据表明Notch信号通路参与维持内分泌胰腺中的祖细胞库。我们已经概述了一些我们正在追求的方法，利用这些知识来识别，表征和操纵这些祖细胞。希望通过理解祖细胞如何在我们的模型系统中分化成胰岛素产生细胞，将允许开发更好的技术来利用脊椎动物内分泌胰腺的内源性再生能力。这种技术可以作为替代或补充治疗的基础上胰岛移植。在斑马鱼中鉴定负责β细胞再生的祖细胞。我们已经开发了一种新的系统，在该系统中，我们可以特异性地消融转基因斑马鱼胚胎中产生胰岛素的β细胞，并在不久之后观察胰岛素阳性细胞的再生。使用Notch信号通路，我们的目标是识别和操纵负责β细胞再生的祖细胞。希望通过了解祖细胞如何在我们的模型系统中分化为胰岛素产生细胞，将允许开发更好的技术来分化β细胞用于未来的治疗目的。

DESCRIPTION (provided by applicant): ABSTRACT 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.