Cell-Cell Signaling During Mammalian Early Eye Formation

描述（由申请人提供）：本提案利用小鼠模型研究人类眼部疾病的潜在原因，重点关注Notch信号通路，这在开发过程中是广泛需要的。Notch信号在特定的细胞环境中调节增殖、细胞形状改变、分化、凋亡和干细胞维持。人类配体Jagged1或Notch2受体的显性突变导致Alagille综合征，其中一些患者表现出眼睛畸形。在上一个资助期内，我们发现了一个Jag1- Notch1/2-Rbpj-Hes1核心信号是产前小鼠晶体形成所必需的。缺乏这些基因的小鼠表现出进行性晶状体无晶状体、瞳孔开口丧失和小眼症。利用种系或条件突变等位基因，以及在复杂突变体中缺失两个或多个基因，我们建议研究早老素（Psen）蛋白酶如何调节Notch受体激活，从而释放c端多肽（NICD），该肽在细胞核中复合物以调节下游基因。我们还将探讨Psen基因的其他与notch无关的作用。此外，Notch配体Dll1、Jag1和Jag2在翻译后受泛素化调控。一个或多个泛素分子的加入使信号发送细胞内的配体降解或内吞循环成为目标。我们建议探索两个E3泛素连接酶家族，Neurl和Mib基因在晶状体发育中的作用，目前尚不清楚。最后，我们将确定Hes1和Hes5下游效应基因在Notch调控生长与分化过程中的独立或联合作用。本应用程序中描述的研究将扩展我们对Notch如何促进晶状体祖细胞增殖，促进纤维细胞分化和防止纤维细胞不适当的有丝分裂或过度凋亡的理解。所有实验将采用复杂条件（cre-lox）小鼠菌株，包括双重和三重突变，组织学，免疫组织化学，共聚焦显微镜，原位杂交，小鼠胚胎学，Western blotting， qPCR和PCR基因分型。我们希望为所有细胞和组织的生长、形态发生和分化过程提供新的信息。

DESCRIPTION (provided by applicant): This proposal investigates the underlying causes of human ocular diseases using mouse models, focusing on the Notch signaling pathway, which is broadly required during development. Notch signaling regulates proliferation, cell shape changes, differentiation, and apoptosis and stem cell maintenance in specific cellular contexts. Dominant human mutations in the ligand Jagged1 or Notch2 receptor cause Alagille syndrome, in which some patient's exhibit eye deformities. In the last funding period we discovered a Jag1- Notch1/2-Rbpj-Hes1 core signal is required for prenatal mouse lens formation. Mice lacking each of these genes exhibited progressive postnatal lens aphakia, loss of the pupillary opening and microphthalmia. Using germline or conditional mutant alleles, and deletion of two or more genes in complex mutants, we propose to investigate how Presenilin (Psen) proteases regulate Notch receptor activation to release a c-terminal polypeptide (NICD) that complexes in the nucleus to regulate downstream genes. Additional Notch-independent roles for Psen genes will also be explored. Moreover, the Notch ligands Dll1, Jag1 and Jag2 are posttranslationally regulated by ubiquitination. The addition of one or more ubiquitin molecules targets the ligands for degradation, or endocytic recycling within signal sending cells. We propose to explore the role of two families of E3 ubiquitin ligases, Neurl and Mib genes in the developing lens, for which nothing is currently known. Finally we will determine the independent, and combined, roles of the Hes1 and Hes5 downstream effector genes, during Notch regulation of growth versus differentiation. The studies described in this application will extend our understanding of how Notch promotes lens progenitor cell proliferation, promotes fiber cell differentiation and prevents inappropriate fiber cell mitoses or excess apoptosis. All experiments will employ complex conditional (cre-lox) mouse strains, including double and triple mutants, histology, immunohistochemistry, confocal microscopy, in situ hybridization, mouse embryology, Western blotting, qPCR and PCR genotyping. We hope to contribute new information to the processes of growth, morphogenesis and differentiation, which are fundamental to all cells and tissues.