Hippo suppression of NFkB controls pancreas morphogenesis and beta cell fate

超过3千万的美国人已经或已经患有糖尿病。治疗糖尿病的一种创新方法是产生功能性β细胞，这种细胞起源于早期胰腺芽中的多能祖细胞群。这种方法目前正在临床试验中，但一个公认的问题是次优祖细胞生成。虽然调节胰腺后期发育的机制已被很好地定义，但如何调节胰腺祖细胞规格仍不清楚。本提案将使用转基因小鼠模型，以及包括器官移植和胰腺类器官在内的组织培养技术，研究Hippo信号通路下游的遗传途径。生物信息学方法将用于(1)确定Hippo信号是否调节胰腺祖细胞规范、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，这些串扰机制调节胰腺祖细胞规范和β细胞分化。我们发现，删除Lats激酶会失调上皮细胞的增殖，导致异常的形态发生和细胞分化，支持胰腺形态发生与细胞分化密切相关的观点。通过转录谱分析，我们发现Yap1/Taz促进NFκB激活基因，在这里我们将定义相关的机制。在本研究中，我们提出了一个独特的想法，即Hippo通路成分在这一过程中作为一种变阻器来控制NFκB活性水平，塑造产生β细胞的上皮生态位。这个生态位是胚胎胰腺核心的一个特化和短暂的上皮丛。我们假设在正常胰腺中，Lats1/2活性是灭活Yap1/Taz从而抑制异常的NFκB信号的稳态所必需的。胰腺上皮中NFκB活性升高导致EMT启动异常和β细胞命运的丧失，这是由于上皮丛生态位的破坏。我们假设Yap1活性升高至少部分通过泛肽酶Vanin1 （Vnn1）刺激NFκB信号。我们的目标是使用体内和体外模型来研究这些观察结果。我们的假设将通过以下方式进行验证：1)检查Lats1和Lats2的缺失如何影响上皮完整性和上皮-间质转化（EMTs）的启动；2)检查Yap1的过表达是否在单个或集群上皮细胞中模拟Lats1/2双缺失（1/2DKO），以及评估已知在1/2DKO中受影响的下游靶标；3)评估NFkB通路在胰腺上皮稳态中的作用，并检查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祖细胞和β细胞中的转化影响，以治疗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.