Hippo suppression of NFkB controls pancreas morphogenesis and beta cell fate

超过3000万美国人患有前期或现有的糖尿病。治疗糖尿病的一种创新方法是产生功能性的β细胞，这种细胞来自早期胰腺芽中的多能祖细胞群体。这一方法目前正处于临床试验阶段，但一个公认的问题是次优祖细胞生成。虽然调控胰腺后期发育的机制已经被很好地定义，但胰腺前体规范是如何调控的仍然不清楚。这项提议将使用转基因小鼠模型以及包括器官外植体和胰腺类器官在内的组织培养技术来研究河马信号通路下游的遗传路径。生物信息学方法将被用来(1)确定河马信号是否调控胰腺祖细胞的规格、形态发生或细胞分化，以及(2)确定YAP1/TAZ和NFκB之间的串扰机制，这些机制调节胰腺的祖细胞规格和β细胞分化。我们发现，删除LATS激酶会失调上皮细胞的增殖，导致形态发生和细胞分化的异常，支持胰腺形态发生与细胞分化密切相关的观点。利用转录图谱，我们已经发现YAP1/TAZ促进了NFκB激活基因，在这里我们将定义涉及的机制。在这个建议中，我们提出了一个独特的想法，即河马通路组件作为变阻器来控制这一过程中NFκB的活性水平，塑造产生β细胞的上皮生态位。这个壁龛是一个位于胚胎胰腺核心的专门的暂时性上皮丛。我们假设，Lats1/2的活性是稳态失活YAP1/Taz所必需的，从而抑制正常胰腺中异常的NFκB信号。由于上皮丛的破坏，胰腺上皮细胞中的核因子κB活性升高导致β的启动和细胞命运的丧失。我们推测，YAP1活性的升高至少部分地通过泛氨酸酶Vnn1(Vnn1)刺激了NFκB信号。我们的目标将使用体内和体外模型来研究这些观察。我们的假设将通过：1)研究Lats1和Lats2的缺失如何影响上皮完整性和上皮向间充质转变(EMT)的启动；2)检查YAP1的过表达是否模仿单个或簇上皮细胞中的Lats1/2双缺失(1/2DKO)，以及评估已知在1/2DKO中受影响的下游靶点；以及3)评估NFkB通路在胰腺上皮内稳态中的作用，并研究Vnn1在这一过程中的作用。我们希望这项工作将增强下游靶点在胰腺前体细胞和β细胞中的翻译影响，目标是治疗性β细胞替代和再生来治疗糖尿病。

More than 30 million Americans have pre- or existing diabetes. An innovative approach to treat diabetes is to generate functional beta cells, which originate from a multipotent progenitor population in the early pancreas bud. This approach is currently in clinical trials, but a recognized problem is suboptimal progenitor generation. While mechanisms regulating later pancreas development have been well defined, how pancreas progenitor specification is regulated remains unclear. This proposal will examine genetic pathways downstream of the Hippo signaling pathway using transgenic mouse models, as well as tissue culture techniques that include organ explants and pancreatic organoids. Bioinformatic approaches will be used to (1) determine if Hippo signaling regulates pancreatic progenitor specification, morphogenesis, or cell differentiation, and (2) determine crosstalk mechanisms between Yap1/Taz and NFκB that regulate progenitor specification and beta cell differentiation in the pancreas. We have found that deleting the Lats kinases dysregulates epithelial cell proliferation, leading to aberrant morphogenesis and cell differentiation, supporting the idea that pancreatic morphogenesis is closely tied to cell differentiation. Using transcriptional profiling, we have found that Yap1/Taz promote NFκB activator genes and here we will define the mechanisms involved. In this proposal, we propose the unique idea that Hippo pathway components act as a rheostat to control levels of NFκB activity during this process, sculpting the epithelial niche that generates beta cells. This niche is a specialized and transient epithelial plexus at the core of the embryonic pancreas. We hypothesize that Lats1/2 activity is required homeostatically to inactivate Yap1/Taz and thereby suppress aberrant NFκB signaling in the normal pancreas. Elevated NFκB activity in pancreatic epithelium results in dysregulated EMT initiation and loss of β-cell fate, due to disruption of the epithelial plexus niche. We hypothesize that elevated Yap1 activity stimulates NFκB signaling at least in part via the pantetheinase Vanin1 (Vnn1). Our aims will use in vivo and in vitro models to investigate these observations. Our hypotheses will be tested via: 1) an examination of how loss of Lats1 and Lats2 affects epithelial integrity and initiation of epithelial-to-mesenchymal transitions (EMTs); 2) an examination of whether overexpression of Yap1 mimics the Lats1/2 double deletion (1/2DKO) in single or clusters of epithelial cells, as well as an assessment of downstream targets known to be affected in 1/2DKO; and 3) an evaluation of the role of the NFkB pathway in pancreatic epithelial homeostasis and an examination of the role of Vnn1 in this process. We hope this work will enhance translational impact of downstream targets in pancreas progenitors and beta cells, with the goal of therapeutic beta cell replacement and regeneration to treat diabetes.