Notch Signaling in Beta Cell Development and Regeneration

描述（由申请人提供）：Notch在早期胰腺器官发生中指导内分泌/外分泌决定是必需的，但我们对该途径在内分泌发育后期的参与的了解还存在很大差距。使用Ngn3-Cre有条件地去除早老素（Notch激活所必需的），并结合缺失细胞的谱系追踪显示，早老素缺乏的胰岛祖细胞从内分泌转向腺泡样命运。我们建议进行机械实验，确定早老素下游的分子需求，以确定是否？-分泌酶活性和Notch信号参与其中。此外，我们想知道早老素是否只在发育过程中需要，还是在成人的细胞更新过程中也需要。特异性Aim 1将定义由早老素介导的分子事件，这些事件是Ngn3表达所必需的，使内分泌前体向成熟细胞发展。Notch参与将使用？-分泌酶抑制剂在体外对胚胎雏形进行谱系追踪。此外，我们会问细胞是否缺乏RBPj？一种介导所有四种Notch受体信号传导的DNA结合蛋白，通过将Ngn3-Cre与RBPJ？条件等位基因（R. Kopan）带有Z/EG报告基因（Ngn3-Cre; RBPj?c/c; Z/EG）。如果吗?-分泌酶活性的早老素是不负责的命运转换，我们将评估参与替代早老素的功能。目的2将在Z/EG背景下，通过Pax6或胰岛素驱动的Cre去除早老素，在内分泌程序中定位早老素依赖步骤。一种可能的结果是分化和增殖缺陷分离，导致内分泌肿块扩大，而不改变其原有的内分泌命运。目的3将在成人有条件的早老素条件背景下，使用诱导性缺失模型（Ngn3-Cre-ERTM和RIP-Cre-ERTM）确定早老素活性是否参与成熟细胞的维持和/或再生。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，目前的应用建议理解遗传观察的分子基础：早老素缺乏，表达Ngn3的祖细胞被转移到腺泡样命运。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。胰岛形成过程中的一个发育开关已经被发现。提出了实验来确定机理。这些实验的结果可能有助于加速以细胞为基础的胰岛素输送疗法的发展。

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.