Pancreatic Beta cell development and functional maintenance

描述（由申请人提供）：尽管在过去二十年中对胰腺发育进行了严格的研究，但关于如何获得功能性β细胞的关键问题仍然存在。很明显，一组胰腺祖细胞激活前内分泌基因Ngn 3及其靶点来调节β细胞发育。目前尚不清楚祖细胞何时决定β细胞的命运，以及β细胞的数量在胚胎发生过程中是如何确定的。这些问题与人类糖尿病特别相关。几项研究表明，体外从人ES/iPS细胞分化的内分泌只能产生缺乏必要葡萄糖反应的胰岛素表达细胞，这强调了我们无法获得具有正确β细胞生产能力的适当内分泌祖细胞。此外，在胚胎发生期间可降低内分泌祖细胞数量的宫内因子使人和模型生物体易患晚发性糖尿病，突出了在发育中获得足够数量的胰腺β细胞祖细胞的重要性。我们一直在研究Myt 1和Ngn 3之间的前馈基因表达环，以研究控制β细胞发育和功能的基本机制。我们发现GAP连接和microRNA都可以正向调节Ngn 3表达，既可以调节Ngn 3+细胞的数量，也可以调节每个细胞内Ngn 3的相对水平。这些发现表明，GAP连接介导的通信是必不可少的协调行为的祖细胞的enabeta-cell生产。通过开发一种创新的基于Cre的双向细胞命运图谱，我们还发现，β细胞的命运是在Ngn 3开启时或之前决定的。这种β细胞的命运选择至少部分是通过Myt 1和Ngn 3之间的相互作用介导的。结果，Myt 1 + Ngn 3+祖细胞优选产生β细胞，而Myt 1-Ngn 3+细胞产生α细胞。此外，我们发现表观遗传修饰剂可以调节Myt 1的表达和Myt 1 + Ngn 3+祖细胞的命运，并且Myt 1与已知的染色质修饰剂包括组蛋白去乙酰化酶Sin 3A相互作用。在此，我们建议研究GAP连接介导的信号如何与microRNA和其他表观遗传修饰剂一起调节β细胞的产生。我们将首先测试一种新的假设，即microRNA可以通过GAP连接来协调细胞分化为内分泌命运。然后，我们将研究表观遗传修饰剂如何调节Myt 1的表达，以及Myt 1如何与其他染色质修饰剂（如Sin 3A）相互作用，以指导β细胞对其他内分泌细胞类型的命运选择。常规细胞纯化和遗传操作将用于实现这些目标。我们设想这些机制的研究，以提供有关早期发育事件，可能会影响β细胞功能，在以后的生理学工具的细节。将这些知识转化为人类组织可以直接帮助从人类ES/iPS细胞中衍生功能性胰岛，用于治疗I型糖尿病，并帮助设计增强II型糖尿病β细胞功能的方法。

DESCRIPTION (provided by applicant): Despite the rigorous studies on pancreatic development over the past two decades, key questions remains regarding how functional beta cells can be derived. It is clear that a group of pancreatic progenitors activate proendocrine gene Ngn3 and its targets to regulate beta-cell development. It is not clear when are the progenitors committed to beta-cell fate and how the number of beta cells is determined during embryogenesis. These questions are particularly relevant to human diabetes. Several studies have demonstrated that endocrine differentiation from human ES/iPS cells in vitro can only produce insulin-expressing cells that lack necessary glucose response, underscoring our inability to derive proper endocrine progenitor cells with the right competence for beta-cell production. Furthermore, intrautarine factors that can lower the number of endocrine progenitors during embryogenesis predispose human and model organisms to late onset diabetes, highlighting the significance of obtaining sufficient numbers of pancreatic beta-cell progenitors i development. We have been studying a feed-forward gene expression loop, between Myt1 and Ngn3, to examine the basic mechanisms governing beta-cell development and function. We found that GAP junctions and microRNAs can both positively regulate Ngn3 expression, both the number of Ngn3+ cells and the relatively level of Ngn3 within each cell. These findings suggest that GAP junction-mediated communications are essential to coordinate the behaviors of progenitors for en masse beta-cell production. By developing an innovative bipartite Cre-based cell fate mapping, we also found that beta-cell fates are determined when or before Ngn3 is turned on. This beta-cell fate choice is mediated, at least partly, through the interaction between Myt1 and Ngn3. As a result, Myt1+Ngn3+ progenitor cells preferably give rise to beta cells, whereas Myt1-Ngn3+ cells give rise to alpha cells. Furthermore, we found that epigenetic modifiers can modulate the expression of Myt1 and the fate of the Myt1+Ngn3+ progenitors, and that Myt1 interacts with known chromatin modifiers including histone deacetylase Sin3A. Here in this grant renewal, we propose to investigate how GAP junction-mediated signals in conjunction with microRNAs and other epigenetic modifiers to regulate beta-cell production. We will first test a novel hypothesis that microRNAs can pass through GAP junctions to coordinate cellular differentiation to endocrine fate. We will then examine how epigenetic modifiers modulate Myt1 expression and how Myt1 interacts with other chromatin modifiers, such as Sin3A, to direct beta-cell fate choice over other endocrine cell types. Routine cell purification and genetic manipulations will be utilized to accomplish these goals. We envision these mechanistic studies to provide instrumental details regarding earlier developmental events that could affect beta-cell function in later physiology. Translating these knowledge to human tissues can directly help with derivation of functional islets from human ES/iPS cells for usage in curing Type I diabetes and help with designing ways to enhance beta-cell function for Type II diabetes.