Genetic Analysis of Retinal Ganglion Cell Function, Development and Disease

描述（由申请人提供）：视网膜神经节细胞（RGC）是青光眼疾病发病机制的主要靶细胞，也是视杯中由有丝分裂祖细胞发育而来的第一种细胞类型。bHLH转录因子Math 5在细胞周期退出后瞬时表达，并且是RGC发生所需的。使用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Math 5+细胞，但只有1/9的Math 5+前体细胞发育成RGC。这些细胞是完全多能的，表达同等水平的Math 5，而不管它们的命运选择如何。在Math 5突变体中，谱系标记的细胞形成除了RGC之外的所有主要细胞类型。因此，Math 5是RGC命运特化的能力因子-必要但不是充分的。神经节细胞的发育需要额外的积极或消极因素。已知Notch信号传导可以抑制RGC命运，但目前尚不清楚Notch是否在祖细胞或有丝分裂后细胞（Math 5之前或之后）中发挥作用，以及Notch是否影响Math 5谱系中的次要命运选择。当我们在Math 5+细胞中激活Notch时，它们最初发育为视网膜神经元，但转分化为Muller胶质细胞。当我们在承诺的光感受器中表达Math 5时，多能性得到恢复。该提案研究了Math 5+细胞的可塑性和在小鼠中形成RGCs所需的下游因子。我们的目标是：（1）为了测试小鼠视网膜中的初生细胞表达Math 5并发育成RGC作为默认命运，以及Notch是Math 5+细胞中RGC命运的主要抑制剂的假设，使用BAC转基因追踪接收Notch信号的Math 5后代的命运，并以快速或缓慢动力学驱动或阻断Math 5+细胞中的Notch，在突变型和野生型小鼠和视网膜外植体中;和（2）通过在光感受器和神经节细胞前体中交叉表达Math 5和Nrl来评估Math 5和Nrl作为RGC和视杆细胞命运的容许和指导因子。公共卫生相关性：我们将研究胚胎眼中控制干细胞形成视网膜神经节细胞的途径。将在转基因小鼠中测试阳性和阴性因子，如Math 5和Notch。我们的研究结果将提高对青光眼、视神经发育不全和视网膜血管增生的认识和治疗，这些都是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.