Investigation of Mammalian Retinal Neuron Development

描述(由申请人提供)：这项提案使用小鼠模型研究人类眼部疾病的潜在原因。我们关注Notch信号通路，它在多种哺乳动物组织中都是至关重要的。特别是，Notch信号调节细胞的增殖、凋亡、细胞形状的改变、分化和干细胞的维持。这一方案中的实验将1)阐明视网膜神经节细胞(RGC)神经发生过程中Notch信号与Math5的上位关系；2)探索Rbpj的多种视网膜神经元表型；3)确定Notch配体Deltalike1在视网膜神经发生过程中的需求。由于Notch信号在发育过程中被广泛使用，大多数Notch通路基因的小鼠已经产生了突变。利用靶向缺失小鼠(完全突变和条件等位基因)，我们建议了解在视网膜神经节细胞和视锥和视杆感光细胞形成过程中对规范Notch信号的要求。一些研究将使用条件(cre-lox)小鼠品系、组织学、免疫组织化学、原位杂交、小鼠胚胎学和PCR基因分型。其他人将使用人类视网膜母细胞系在体外测试调控关系。这些研究将为后生动物的生长、形态发生和分化过程提供新的信息，这些过程是所有后生动物发育的基础。这项工作将对视锥视杆细胞营养不良、视神经再生障碍、发育不良有更好的了解，并有助于视网膜细胞发育的基本机制与基于基因或细胞的视网膜治疗直接相关。这一发现也将广泛用于癌症生物学领域，因为在各种人类肿瘤中都会出现过度激活的Notch1或Hes1表达。公共卫生相关性：这项研究的目标是通过小鼠模型了解哺乳动物视网膜神经元形成的潜在分子机制。我们建议通过研究视网膜形成的哪些方面需要Notch细胞到细胞的信号通路，以及它如何调节Math5 bHLH转录因子来做到这一点。在身体的其他部位，Notch信号控制细胞形状的变化、生长和死亡。出于这些原因，Notch途径的突变可能会导致癌症。对于Notch在视网膜中如何、何时和在哪里起作用的透彻理解还只是表面上的。这些研究将在单细胞水平上提供对视网膜如何发育的更深层次的理解，并有助于更好地设计针对Leber冷凝性黑色素、视杆细胞营养不良、色盲、视神经发育不全/发育不良和青光眼等疾病的疾病治疗方案。

DESCRIPTION (provided by applicant): This proposal investigates the underlying causes of human ocular diseases using mouse models. We focus on the Notch signaling pathway, which is critically required in multiple mammalian tissues. In particular, Notch signaling regulates proliferation, apoptosis, cell shape changes, differentiation and stem cell maintenance. Experiments in this proposal will 1) elucidate the epistatic relationship between Notch signaling and Math5 during retinal ganglion cell (RGC) neurogenesis 2) explore the multiple retinal neuron phenotypes of Rbpj, and 3) define the requirements of the Notch ligand Deltalike1 during retinal neurogenesis. Because Notch signaling is widely employed during development, mouse mutations in most Notch pathway genes have already been created. Using targeted deletion mice (wholly mutant and conditional alleles), we propose to understand the requirements for canonical Notch signaling during retinal ganglion cell and cone and rod photoreceptor formation. Some studies will employ conditional (cre-lox) mouse strains, histology, immunohistochemistry, in situ hybridization, mouse embryology and PCR genotyping. Others will test regulatory relationships in vitro using a human retinoblast cell line. These studies will contribute new information to the processes of growth, morphogenesis and differentiation, which are fundamental to all metazoan development. This work will yield a better understanding of cone-rod dystrophies, optic nerve aplasia, hypoplasia, as well as contribute to basic mechanisms of retinal cell development with direct relevany to gene- or cell-based retinal therapies. Findings here will also be widely useful to the field of cancer biology, since excess activated Notch1 or Hes1 expression occurs in a variety of human tumors. PUBLIC HEALTH RELEVANCE: The goal of this study is to understand the underlying molecular mechanisms of how mammalian retinal neurons forms, using mouse models. We propose to do this by investigating which aspects of retinal formation require the Notch cell-to-cell signaling pathway, and how it regulates the Math5 bHLH transcription factor. In other parts of the body, Notch signaling controls cell shape changes, growth, and death. For these reasons, mutations in the Notch pathway can cause cancer. A thorough understanding of how, when and where Notch acts in the retina has only been addressed superficially. These studies will provide deeper understanding, at the single cell level, of how the retina develops and contribute to the better design of disease therapies for diseases such as Leber's congeital amaurosis, cone-rod dystrophy, color blindness, optic nerve aplasia/hyplasia and glaucoma.