Genetic Analysis of Retinal Ganglion Cell Function, Development and Disease

描述(申请人提供)：视网膜神经节细胞(RGC)是青光眼疾病发病机制的主要靶点，也是第一种从视杯中有丝分裂的前体细胞发育而来的细胞类型。BHLH转录因子Math5在细胞周期退出后瞬时表达，是RGC发生所必需的。利用Cre-lox谱系分析和BAC转基因小鼠，我们已经确定5%的成年视网膜神经元来自有丝分裂后的Math5+细胞，但只有1/9的Math5+前体细胞发育成RGC。这些细胞是完全多能的，无论它们的命运选择如何，它们都表达等量的Math5。在Math5突变体中，谱系标记的细胞形成除RGC以外的所有主要细胞类型。因此，Math5是RGC命运规范的一个能力因素--必要的，但不充分的。神经节细胞的发育还需要其他积极或消极的因素。Notch信号已知可抑制RGC命运，但尚不清楚Notch是否作用于前体细胞或有丝分裂后细胞(Math5之前或之后)，以及Notch是否影响Math5谱系中的次级命运选择。当我们激活Math5+细胞中的Notch时，它们最初作为视网膜神经元发育，但转分化为Muller胶质细胞。当我们在承诺的光感受器中表达Math5时，多能性就恢复了。这项建议研究了Math5+细胞的可塑性以及在小鼠体内形成RGCs所需的下游因素。我们的目标是：(1)验证假设，即小鼠视网膜中第一出生的细胞表达Math5并作为默认命运发育为RGC，并且Notch是Math5+细胞中RGC命运的主要抑制因子，使用BAC转基因来追踪接受Notch信号的Math5后代的命运，并在突变和野生型小鼠和视网膜外植体中以快速或缓慢的动力学驱动或阻止Math5+细胞中的Notch；(2)通过在光感受器和神经节细胞前体中交叉表达Math5和NRL，评估它们作为RGC和视杆细胞命运的允许和指导因子。公共卫生意义：我们将研究胚胎眼干细胞形成视网膜神经节细胞的控制途径。积极和消极的因素，如Math5和Notch，将在转基因小鼠身上进行测试。我们的结果将提高对青光眼、视神经发育不良和视网膜血管增殖的认识和治疗，这些都是导致人类失明的重要原因。

DESCRIPTION (provided by applicant): Retinal ganglion cells (RGCs) are the primary target of glaucoma disease pathogenesis and the first cell type to develop from mitotic progenitors in the optic cup. The bHLH transcription factor Math5 is transiently expressed following cell cycle exit, and required for RGC genesis. Using Cre-lox lineage analysis and BAC transgenic mice, we have determined that 5% of adult retinal neurons derive from post-mitotic Math5+ cells, but only 1 in 9 Math5+ precursors develops into an RGC. These cells are fully multipotent and express equivalent levels of Math5 regardless of their fate choice. In Math5 mutants, the lineage-marked cells form all major cell types except RGCs. Math5 is thus a competence factor - necessary but not sufficient - for RGC fate specification. Additional positive or negative factors are needed for ganglion cell development. Notch signaling is known to inhibit RGC fate, but it is unclear whether Notch acts in progenitors or post-mitotic cells (before or after Math5), and whether Notch affects secondary fate choice in the Math5 lineage. When we activate Notch in Math5+ cells, they initially develop as retinal neurons but transdifferentiate into Muller glia. When we express Math5 in committed photoreceptors, multipotency is restored. This proposal studies the plasticity of Math5+ cells and downstream factors required to form RGCs in mice. We aim: (1) to test the hypotheses that first-born cells in the mouse retina express Math5 and develop into RGCs as a default fate, and that Notch is the major inhibitor of RGC fate in Math5+ cells, using BAC transgenes - to trace fates of Math5 descendants that receive Notch signals, and to drive or block Notch in Math5+ cells with rapid or slow kinetics, in mutant and wild-type mice - and retinal explants; and (2) to evaluate Math5 and the Nrl as permissive and instructive factors for RGC and rod fates, by cross-expressing them in photoreceptor and ganglion cell precursors. PUBLIC HEALTH RELEVANCE: We will study the pathway controlling formation of retinal ganglion cells from stem cells in the embryonic eye. Positive and negative factors, such as Math5 and Notch, will be tested in transgenic mice. Our results will improve the understanding and treatment of glaucoma, optic nerve hypoplasia and retinal vascular proliferation, important causes of human blindness.