Notch Signaling in Beta Cell Development and Regeneration

描述(由申请人提供)：在早期胰腺器官发生中，Notch是指导内分泌/外分泌决策所必需的，但我们对这一途径在内分泌发育后期阶段的参与仍有很大的认识空白。使用Ngn3-Cre有条件地去除早老素(Notch激活所必需的)，并结合对缺失细胞的谱系追踪，揭示了早老素缺陷的胰岛前体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早老素下游的分子需求，以确定是否涉及β-分泌酶活性和Notch信号。此外，我们还询问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。具体目标1将定义由早老素介导的分子事件，这些事件是Ngn3表达、承诺的内分泌前体发展到成熟细胞所必需的。切迹的参与将使用一种带有谱系追踪的？-分泌酶抑制剂在体外对胚胎雏形进行测试。此外，我们还会问，缺乏RBPj？的细胞是否通过Ngn3-Cre与RBPJ？杂交获得内分泌命运？RBPj？是一种DNA结合蛋白，介导所有四种Notch受体的信号传递。具有Z/EG报告基因的条件等位基因(R.Kopan)(Ngn3-Cre；RBPj？C/c；Z/EG)。如果早老素的？-分泌酶活性不是命运转换的原因，我们将评估替代的早老素功能的参与。目标2将通过在Z/EG背景上用Pax6或胰岛素驱动的CRE去除早老素来定位内分泌程序中依赖早老素的步骤。一个可能的结果是分离分化和增殖缺陷，导致内分泌肿块扩大，而不改变它们原来的内分泌命运。目的3将在条件早老素条件背景下，使用可诱导缺失模型(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的新生和细胞复制中的作用。综上所述，目前的应用建议理解遗传学观察的分子基础：缺乏早老素、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。胰岛形成过程中的发育转换已被发现。提出了用实验来定义这些机制。这些实验的结果可能有助于加速开发基于细胞的胰岛素输送疗法。

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.