Hippo suppression of NFkB controls pancreas morphogenesis and beta cell fate

超过3000万美国人患有糖尿病或糖尿病。治疗糖尿病的一种创新方法是产生功能性β细胞，其来源于早期胰腺芽中的多能祖细胞群。这种方法目前处于临床试验中，但公认的问题是次优的祖细胞生成。虽然调控胰腺发育的机制已经被很好地定义，但胰腺祖细胞的特化是如何被调控的仍然不清楚。该提案将使用转基因小鼠模型研究Hippo信号通路下游的遗传通路，以及包括器官外植体和胰腺类器官在内的组织培养技术。生物信息学方法将用于（1）确定Hippo信号传导是否调节胰腺祖细胞特化、形态发生或细胞分化，以及（2）确定Yap 1/Taz和NFκB之间调节胰腺祖细胞特化和β细胞分化的串扰机制。我们已经发现，删除Lats激酶使上皮细胞增殖失调，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。利用转录谱分析，我们发现Yap 1/Taz促进NFκB B激活基因，在这里我们将定义所涉及的机制。在这个提议中，我们提出了一个独特的想法，即Hippo通路组分在此过程中充当变阻器来控制NFκB活性水平，塑造产生β细胞的上皮小生境。这个小生境是胚胎胰腺核心的一个特化的短暂上皮丛。我们假设Lats 1/2活性是维持Yap 1/Taz稳态所必需的，从而抑制正常胰腺中异常的NFκB信号。胰腺上皮中NFκB活性升高导致EMT启动失调和β细胞命运丧失，这是由于上皮丛生态位的破坏。我们推测升高的Yap 1活性至少部分通过泛酰巯基乙胺酶Vanin 1（Vnn 1）刺激NFκB信号传导。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检验：1）检查Lats 1和Lats 2的缺失如何影响上皮完整性和上皮-间充质转化（EMT）的启动; 2）检查Yap 1的过表达是否模拟单个或簇上皮细胞中的Lats 1/2双缺失（1/2DKO），以及评估已知在1/2DKO中受影响的下游靶点;和3）评估NFkB途径在胰腺上皮稳态中的作用，并检查Vnn 1在该过程中的作用。我们希望这项工作将增强胰腺祖细胞和β细胞中下游靶点的翻译影响，目标是治疗性β细胞替代和再生以治疗糖尿病。

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.