The Mechanisms of Islet Cell Specification and Differentiation

描述(由申请人提供)：内分泌胰岛细胞的生产解决了几个不同但相关的问题：内分泌承诺、细胞类型指定、胰岛细胞分化和成熟。在这项拨款申请中，我们建议利用Myt1基因活性操纵和组合细胞标记来剖析这些过程。Ngn3在多能胰岛前体细胞中的高表达是胰岛细胞分化的关键。因为Ngn3结合自己的启动子来抑制转录，所以需要其他激活剂来打破自我抑制环，以实现强大的Ngn3表达，这对内分泌承诺是必不可少的。显然，Ngn3不依赖的机制决定了内分泌胰岛细胞的类型。然而，决定每个胰岛细胞前体最终命运的密码仍然未知。最后，新分化的内分泌细胞微调其基因表达模式和细胞结构，以产生成熟的功能胰岛细胞。但协调成熟过程的分子机制尚不清楚。我们在上一次筹资期间的研究揭示了几个有可能弥补上述差距的机制。首先，我们已经证明了Myt1和Ngn3形成一个前馈表达环来促进新生。这一发现表明，Myt1可能在打破Ngn3自我抑制环以促进内分泌承诺方面发挥重要作用。第二，Myt1失活会导致内分泌分化和功能异常。出乎意料的是，Myt1的缺失会导致内分泌室Myt1和Myt3转录的意外补偿性激活，这突显了基因表达监测控制的质量。第三，分化的胰岛细胞维持Myt1和Ngn3的表达，有助于内分泌成熟和功能，提示启动胰岛细胞新生的遗传网络也可能参与内分泌成熟和功能。第四，Ngn3hi细胞是异质性细胞，表达高水平或低水平的Myt1，显示了内分泌祖细胞固有的异质性。最后，我们成功地设计了一种新的基于二分CRE的细胞谱系追踪方法，从而可以对共表达双重蛋白标记的细胞进行特定的标记。这些发现导致并提供了检验工具，即Myt1与Ngn3相互作用促进内分泌承诺和胰岛细胞成熟，并且Myt1的差异表达将Ngn3hi祖细胞划分为功能不同的亚型。在这项拨款提案中，我们将首先通过功能丧失研究来确定Myt1是否是高Ngn3表达和内分泌成熟所必需的。然后，我们将检查在表达Ngn3的细胞中Myt1的表达是否标志着它们是特定类型的胰岛细胞的祖细胞。这些拟议的研究对所有器官的细胞分化以及与胰腺缺陷/功能障碍相关的翻译努力都具有重要意义。 与公共健康相关：这项拨款提案研究了联合基因活动如何促进内分泌细胞分化、成熟和规范。这些研究的结果可能为以细胞为基础的糖尿病治疗获得功能性胰岛细胞奠定基础。它还将为器官发生领域的细胞谱系和细胞类型特异性基因操作研究建立一种新的技术。

DESCRIPTION (provided by applicant): Endocrine islet cell production solves several distinct yet related problems: endocrine commitment, cell type specification, islet cell differentiation, and maturation. In this grant application, we propose to utilize Myt1 gene activity manipulation and combinatorial cell marking to dissect these processes. High Ngn3 expression in the multipotent pancreatic progenitor cells is critical for islet cell differentiation. Because Ngn3 binds its own promoter to inhibit transcription, other activators are required to break the self-inhibitory loop for robust Ngn3 expression, which is essential for endocrine commitment. It is clear that Ngn3-independent mechanisms specify endocrine islet cell type. Yet the code that determines the eventual fate of each islet cell precursors remains unknown. Finally, newly differentiated endocrine cells fine-tune their gene expression pattern and cellular structure to give rise to mature functional islet cells. But the molecular mechanisms that coordinate the maturation process are unclear. Our studies over the previous funding period have uncovered several mechanisms that could potentially bridge some of the above gaps. First, we have demonstrated that Myt1 and Ngn3 form a feed-forward expression loop to promote neogenesis. This finding suggests that Myt1 could play an essential role in breaking the Ngn3 self-inhibitory loop to facilitate endocrine commitment. Second, inactivating Myt1 results in abnormal endocrine differentiation and function. Unexpectedly, losing Myt1 induces an unexpected compensatory activation of Myt1L and Myt3 transcription in the endocrine compartment, underscoring the quality of gene expression surveillance control. Third, differentiated islet cells maintain Myt1 and Ngn3 expression, which contributes to endocrine maturation and function, indicating that genetic networks that initiate islet cell neogenesis could also participate in endocrine maturation and function. Fourth, Ngn3hi cells were heterogeneous cells that express either high or low levels of Myt1, demonstrating the inherent heterogeneity of the endocrine progenitors. Finally, we have successfully devised a novel bipartite-Cre- based cell lineage tracing method, so that cells that co-express dual protein markers could be specifically labeled. These findings lead to, and provide the tool to test, the hypothesis that the Myt1 interacts with Ngn3 to promote endocrine commitment and islet cell maturation and that differential Myt1 expression divides Ngn3hi progenitors into functionally distinct subtypes. In this grant proposal, we will first determine whether Myt1 is required for high Ngn3 expression and endocrine maturation using loss-of-function studies. Then, we will examine whether Myt1 expression in Ngn3-expressing cells marks them as progenitors of specific islet cell types. These proposed studies are of fundamental significance to cell differentiation in all organs, and also to translational efforts related to pancreas defects/malfunction. PUBLIC HEALTH RELEVANCE: This grant proposal studies how combined gene activities promote endocrine islet cell differentiation, maturation and specification. Results from these studies could lay the foundation to derive functional islet cells for cell-based diabetes therapy. It will also establish a novel technology for cell lineage and cell type specific gene manipulation studies in the field of organogenesis.