Notch Signaling in Beta Cell Development and Regeneration

描述（由申请人提供）：Notch是指导早期胰腺器官发生中内分泌/外分泌决定所必需的，但我们对该途径参与内分泌发育后期阶段的认识存在很大差距。使用Ngn 3-Cre结合缺失细胞的谱系追踪有条件地去除早老素（Notch激活所需的）揭示了早老素缺陷型胰岛祖细胞从内分泌转向腺泡样命运。我们提出的机制实验，将定义的分子要求下游的早老素，以确定是否？-涉及分泌酶活性和Notch信号传导。此外，我们还想知道早老素是否仅在发育过程中需要，还是在成人的细胞更新过程中也需要。具体目标1将定义由早老素介导的分子事件，所述分子事件是Ngn 3表达、定向内分泌前体进展为成熟的细胞所需的。陷波参与将使用？-分泌酶抑制剂在体外胚胎发育中的谱系追踪。此外，我们将询问是否存在RBP 1？缺陷的细胞，一种DNA结合蛋白，介导所有四种Notch受体的信号传导，通过将Ngn 3-Cre与RBPJ交叉获得内分泌命运？条件等位基因（R. Kopan）与Z/EG报告基因（Ngn 3-Cre; RBPj？c/c; Z/EG）。如果？-如果早老素的分泌酶活性与命运转换无关，我们将评估替代早老素功能的参与。目标2将通过在Z/EG背景上用Pax 6或胰岛素驱动的Cre去除早老素，将早老素依赖性步骤定位在内分泌程序中。一个可能的结果是分化和增殖缺陷的分离，导致扩大的内分泌质量而不改变其原始内分泌命运。目的3将确定是否早老素活性参与维持和/或再生成熟的细胞使用诱导缺失模型（Ngn 3-Cre-ERTM和RIP-Cre-ERTM）在成人的条件早老素条件的背景。这些实验的结果将有助于确定早老素活性在细胞前体新生和细胞复制中的作用。总之，本申请提出理解遗传观察的分子基础：早老蛋白缺陷的Ngn 3表达祖细胞转向腺泡样命运。我们将利用新的遗传模型来阐明Notch信号在这一现象中的作用，更重要的是，早老素在胰腺器官发生，维持和再生中的作用。胰岛形成的发育开关已经被发现。实验提出了定义的机制。这些实验的结果可能有助于加速开发用于胰岛素递送的基于细胞的疗法。

DESCRIPTION (provided by applicant): Notch is required for directing the endocrine/exocrine decision in early pancreatic organogenesis, yet there are large gaps in our knowledge of the involvement of this pathway in later stages of endocrine development. Conditional removal of Presenilin (required for Notch activation) using Ngn3-Cre coupled with lineage tracing of deleted cells revealed that Presenilin-deficient islet progenitors are diverted from the endocrine towards an acinar-like fate. We propose mechanistic experiments that will define the molecular requirements downstream of Presenilin to determine if ?-secretase activity and Notch signaling are involved. Additionally, we ask whether Presenilin is only required in development or also during ¿-cell renewal in the adult. Specific Aim 1 will define the molecular events mediated by Presenilins that are required for Ngn3 expressing, committed endocrine precursors to progress to mature ¿-cells. Notch involvement will be tested using a ?- secretase inhibitor with lineage tracing on embryonic rudiments in vitro. Additionally we will ask if cells deficient in RBPj?, a DNA binding protein that mediates signaling by all four Notch receptors, acquire endocrine fates by crossing the Ngn3-Cre with the RBPJ? conditional allele (R. Kopan) with a Z/EG reporter (Ngn3-Cre; RBPj?c/c; Z/EG). If ?-secretase activity of the Presenilins is not responsible for the fate switch, we will assess the involvement of alternate Presenilin functions. Aim 2 will position the Presenilin-dependent step within the endocrine program by removing Presenilin with Pax6- or Insulin-driven Cre on a Z/EG background. One possible outcome would be a separation of the differentiation and proliferation defects resulting in expanded endocrine mass without altering their original endocrine fate. Aim 3 will determine whether Presenilin activity is involved in maintenance and/or regeneration of mature ¿-cells using Inducible deletion models (Ngn3-Cre-ERTM and RIP-Cre-ERTM) on a conditional Presenilin-conditional background in the adult. The results of these experiments will help define the role of Presenilin activity in neogenesis of ¿-cell precursors and in ¿-cell replication. In summary, the current application proposes to understand the molecular basis of a genetic observation: Presenilin-deficient, Ngn3 expressing progenitors are diverted to an acinar-like fate. We will utilize novel genetic models to elucidate the role of Notch signaling in this phenomenon and more importantly, the role of Presenilin in pancreatic organogenesis, maintenance and regeneration. A developmental switch in the formation of pancreatic islets has been uncovered. Experiments are proposed to define the mechanisms. The results of these experiments may serve to accelerate efforts towards the development of cell-based therapies for insulin delivery.