Pancreatic Beta cell development and functional maintenance

描述(由申请人提供)：尽管在过去的二十年里对胰腺发育进行了严格的研究，但关于如何获得功能β细胞的关键问题仍然存在。很明显，一组胰腺前体细胞激活前内分泌基因Ngn3及其靶点来调节β细胞的发育。目前尚不清楚先祖细胞何时决定贝塔细胞的命运，以及在胚胎发育过程中贝塔细胞的数量是如何决定的。这些问题与人类糖尿病特别相关。多项研究表明，人ES/iPS细胞在体外进行内分泌分化只能产生缺乏必要葡萄糖反应的胰岛素表达细胞，这突显了我们无法获得具有适当能力产生β细胞的适当内分泌祖细胞。此外，在胚胎发生过程中可以减少内分泌前体细胞数量的utarine因子使人类和模型生物更容易患上迟发性糖尿病，这突显了获得足够数量的胰岛β细胞前体细胞I发育的重要性。我们一直在研究Myt1和Ngn3之间的前馈基因表达环，以检查控制β细胞发育和功能的基本机制。我们发现，缝隙连接和microRNAs都可以正向调节Ngn3的表达，无论是Ngn3+细胞的数量还是每个细胞内Ngn3的相对水平。这些发现表明，缝隙连接介导的通讯对于协调前体细胞产生大量β细胞的行为是必不可少的。通过开发一种创新的基于两部分CRE的细胞命运图谱，我们还发现，β细胞的命运是在Ngn3被打开时或之前决定的。这种β细胞命运的选择至少部分是通过Myt1和Ngn3之间的相互作用来调节的。因此，Myt1+Ngn3+祖细胞较好地产生β细胞，而Myt1-Ngn3+细胞较好地产生α细胞。此外，我们发现表观遗传修饰物可以调节Myt1的表达和Myt1+Ngn3+祖细胞的命运，并且Myt1与包括组蛋白脱乙酰酶Sin3A在内的已知染色质修饰物相互作用。在这项拨款续期中，我们建议研究缝隙连接如何与microRNAs和其他表观遗传修饰物一起介导信号来调节β细胞的产生。我们将首先测试一个新的假设，即microRNAs可以通过缝隙连接来协调细胞分化和内分泌命运。然后，我们将研究表观遗传修饰物如何调节Myt1的表达，以及Myt1如何与其他染色质修饰物(如Sin3A)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。常规的细胞纯化和遗传操作将被用来实现这些目标。我们设想这些机制研究将提供有关早期发育事件的工具性细节，这些事件可能会影响后期生理学中的β细胞功能。将这些知识转化到人体组织中，可以直接帮助从人类ES/iPS细胞中获得用于治疗I型糖尿病的功能性胰岛，并有助于设计增强II型糖尿病的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.