Identifying the progenitors responsible for Beta Cell regeneration in zebrafish

描述(由申请人提供)： 摘要确定斑马鱼中负责β细胞再生的前体细胞尽管小鼠研究在确定胰腺发育的分子途径方面具有明显的力量，但小鼠的遗传学通常是费力和耗时的。由于控制脊椎动物胰腺发育的分子机制高度保守，我们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，已经建立了几个斑马鱼转基因品系，它们表达一种融合到荧光标签(EGFP或mCherry)上的大肠杆菌蛋白--硝基还原酶(NTR)。硝基还原酶是一种能将前药甲硝唑转化为细胞毒素的酶。通过加入这种前药，我们发现转基因相关的荧光在表达胰岛素的β细胞丧失的同时被熄灭。因此，我们得出结论，我们能够药物依赖地消融β细胞，并有数据证明胰岛中的其他细胞不受影响。在药物撤除和36小时的恢复后，我们记录了荧光的恢复，这与两个细胞的再生一致。在建立了这个新的消融β细胞的模型系统后，我们现在有机会筛选出可以改变β细胞再生的药物。为了测试这种筛选的可行性，我们在前药依赖消融后的斑马鱼幼体上应用了各种化学物质，并观察了它们对再生的影响。DAPT是一种阻断Notch信号通路的化合物。在β细胞消融后用DAPT治疗，会导致再生的β细胞数量显著增加。我们还首次展示了Notch基因(Notch 1b)在发育中的胰腺中的离散细胞群中表达。综上所述，这些数据表明，Notch信号通路参与维持内分泌胰腺的祖细胞池。我们已经概述了我们正在寻求的一些方法，以利用这一知识来识别、表征和操纵这些祖细胞。希望通过了解在我们的模型系统中祖细胞是如何分化为胰岛素产生细胞的，将有助于开发更好的技术来开发脊椎动物内分泌胰腺的内源性再生能力。这些技术可以作为基于胰岛移植的替代疗法或补充疗法。记述斑马鱼中负责β细胞再生的祖细胞。我们已经开发出一种新的系统，我们可以在转基因斑马鱼胚胎中特异性地去除产生胰岛素的β细胞，并在不久之后观察胰岛素阳性细胞的再生。利用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。希望通过了解在我们的模型系统中祖细胞如何分化为产生胰岛素的细胞，将有助于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.