The Mechanisms of Islet Cell Specification and Differentiation

描述（由申请人提供）：内分泌胰岛细胞生产解决了几个不同但相关的问题：内分泌承诺，细胞类型规格，胰岛细胞分化和成熟。在这项授权申请中，我们建议利用Myt 1基因活性操作和组合细胞标记来剖析这些过程。Ngn 3在多能胰腺祖细胞中的高表达对于胰岛细胞分化至关重要。由于Ngn 3结合其自身的启动子以抑制转录，因此需要其他激活剂来打破用于稳健Ngn 3表达的自抑制环，这对于内分泌定型是必需的。很明显，Ngn 3-独立机制指定内分泌胰岛细胞类型。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，以产生成熟的功能性胰岛细胞。但协调成熟过程的分子机制尚不清楚。我们在上一个资助期的研究发现了几种可能弥合上述差距的机制。首先，我们已经证明，Myt 1和Ngn 3形成一个前馈表达环，以促进新生。这一发现表明，Myt 1可能在打破Ngn 3自我抑制环路以促进内分泌承诺方面发挥重要作用。第二，失活Myt 1导致异常的内分泌分化和功能。出乎意料的是，失去Myt 1诱导了内分泌区室中Myt 1 L和Myt 3转录的意外补偿激活，强调了基因表达监测控制的质量。第三，分化的胰岛细胞保持Myt 1和Ngn 3的表达，这有助于内分泌成熟和功能，表明启动胰岛细胞新生的遗传网络也可以参与内分泌成熟和功能。第四，Ngn 3 hi细胞是表达高或低水平Myt 1的异质细胞，表明内分泌祖细胞的固有异质性。最后，我们成功地设计了一种新的基于双组分Cre的细胞谱系追踪方法，从而可以特异性地标记共表达双蛋白标记的细胞。这些发现导致，并提供工具来测试，假设Myt 1与Ngn 3相互作用，以促进内分泌承诺和胰岛细胞成熟和差异Myt 1表达分为Ngn 3 hi祖细胞功能不同的亚型。在这项资助提案中，我们将首先使用功能丧失研究确定Myt 1是否是Ngn 3高表达和内分泌成熟所必需的。然后，我们将检查Ngn 3表达细胞中Myt 1的表达是否标志着它们是特定胰岛细胞类型的祖细胞。这些拟议的研究对所有器官中的细胞分化以及与胰腺缺陷/功能障碍相关的翻译努力具有根本意义。 公共卫生相关性：这项拨款建议研究如何结合基因活动促进内分泌胰岛细胞分化，成熟和规范。这些研究结果将为获得功能性胰岛细胞用于糖尿病细胞治疗奠定基础。这也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.