GENETIC ANALYSIS OF RETINAL GANGLION CELL FUNCTION

描述（由申请人提供）：视网膜神经节细胞（RGC）是脊椎动物眼睛的唯一投射神经元，其轴突构成视神经。涉及视神经的缺陷是儿童失明的重要原因，而 RGC 是青光眼发病机制的共同最终目标。 RGC 是视杯中诞生的第一个神经元，来自多能祖细胞，后来产生无长突、视锥细胞和视杆细胞、水平细胞和双极细胞以及米勒神经胶质细胞。视网膜组织发生的机制尚不清楚，但被认为涉及内在和外在因素。最近，我们鉴定了一种小鼠碱性螺旋-环-螺旋（bHLH）转录因子 Math5，它与果蝇无调性同源，其在视网膜中的特定表达模式与 RGC 的诞生相关（Brown 等人，1998）。我们创造了 Math5 基因敲除小鼠。纯合子可以存活，但缺乏 RGC 和视神经 (Brown et al 2001a)。它们的视锥光感受器随之增加，这与细胞命运的转变一致。他们的眼睛也缺乏中央动脉和静脉，并表现出新生血管表型和一些层状改变，但其他方面看起来正常。单个视网膜神经元类的缺失是一个独特的发现，成人和胚胎表型的重要方面仍未被探索。我们的初步数据和进化比较表明，Math5 是 Hes1 的下游和 Brn3b (Pou4f2) 的上游，Brn3b (Pou4f2) 是视网膜发生层次中的转录因子。 Math5 -/- 眼睛预计缺乏 RGC 测定、分化和维持所独特需要的所有 mRNA。我们对人类 ATH5 直向同源物进行了表征（Brown 等人，2001b），并从视神经发育不全、发育不全和青光眼患者收集样本进行突变筛查。除了 Math5 之外，已知还有三个基因座控制小鼠 RGC 的发育或数量：Brn3b、Nnc1 QTL 和经典的自发突变 Bst（腹斑和尾部）。与 Math5 -/- 小鼠相比，Bst/ 小鼠的 RGC 减少相似但不太明显，并且视网膜新生血管形成表型相当。使用亚种间回交，我们将 Bst 映射到小鼠 16 号染色体上的 a-1 cM 间隔，靠近 Hes1 并桥接两个 YAC 重叠群。最后，使用 lacZ 敲入等位基因，我们鉴定了后脑（梯形体）和小脑中的晚期 Math5 表达域。这些发现以及对 N2F2 代中少数共济失调 Math5 突变小鼠的观察表明，Math5 可能在听觉处理和协调中也发挥次要作用。 在本提案中，我们的目标是：（1）详细描述成人Math5眼睛表型的特征，包括使用一组视网膜神经元和血管标记物进行组织学分析、ERG和昼夜节律生理学研究以及Math5-/杂合子中RGC的定量分析； （2）研究Math5 -/-表型的胚胎学基础，通过探索Math5 -/-视网膜中视柄发育的减弱以及神经元、血管和星形胶质细胞发育之间的相互依赖性，通过使用BrdU出生年代测定方法直接测试RGC到视锥细胞命运转换模型，并通过生成Math5-Cre转基因小鼠来全面定义Math5阳性前体细胞的谱系并测试其作用RGC 发育中的特定信号通路（例如 Notch-delta）； (3) 使用 I-基因 cDNA 微阵列 (Farjo et al. 2001) 和消减 PCR 技术定义 Math5 的靶基因以及发育中和成年 RGC 的转录组； (4)测试ATH5和POU4F2突变在人视神经a/发育不全和青光眼中的作用； (5)精细映射和克隆Bst，并在发育层次中相对于Math5对Bst进行排序； (6)表征后脑和小脑Math5表达域，并测试Math5在听觉处理和运动控制中的作用。

DESCRIPTION (provided by applicant): Retinal ganglion cells (RGCs) are the sole projection neurons of the vertebrate eye, and their axons comprise the optic nerve. Defects involving the optic nerve are a significant cause of blindness in childhood and RGCs are the common final target of glaucoma pathogenesis. RGCs are the first neurons born in the optic cup, from multipotent progenitor cells which later give rise to amacrines, cone and rod photoreceptors, horizontal and bipolar cells, and Muller glia. The mechanisms underlying retinal histogenesis are poorly understood, but are thought to involve intrinsic and extrinsic factors. Recently, we identified a mouse basic helix-loop-helix (bHLH) transcription factor, Math5, that is homologous to the Drosophila atonal and whose specific expression pattern in the retina is correlated with birth of RGCs (Brown et al. 1998). We have created Math5 knockout mice. The homozygotes are viable but lack RGCs and optic nerves (Brown et al 2001a). They have a concomitant increase in cone photoreceptors, consistent with a cell-fate shift. Their eyes also lack the central artery and vein, and exhibit a neovascularization phenotype with some laminar alterations, but otherwise appear normal This deletion of a single retinal neuron class is a unique finding, and significant important aspects of the adult and embryonic phenotype remain unexplored. Our preliminary data and evolutionary comparisons suggest that Math5 is downstream of Hes1 and upstream of Brn3b (Pou4f2) the transcription factor in a hierarchy of retinogenesis. Math5 -/- eyes are expected to lack all mRNAs that are uniquely required for RGC determination, differentiation and maintenance. We have characterized the human ATH5 ortholog (Brown et al. 2001b) and collected samples from patients with optic nerve aplasia, hypoplasia and glaucoma for mutation screening. Apart from Math5, three loci are known to control RGC development or number in mice-Brn3b, the Nnc1 QTL, and the classical spontaneous mutation Bst (belly spot and tail). Bst/+ mice have a similar but less marked reduction in RGCs compared to Math5 -/- mice and a comparable retinal neovascularization phenotype. Using an intersubspecific backcross, we have mapped Bst to a-1 cM interval on mouse chromosome 16, close to Hes1 and bridging two YAC contigs. Finally, using the lacZ knock-in allele we have identified a late Math5 expression domain in the hindbrain (trapezoid body) and cerebellum. These findings, and the observation of a small number of ataxic Math5 mutant mice in the N2F2 generation, suggest that Math5 may also have a secondary role in auditory processing and coordination. In this proposal, we aim: (1) to characterize the adult Math5 eye phenotype in detail, including histological analysis with a panel of retinal neuron and vascular markers, ERG and circadian rhythm physiological studies, and quantitative analysis of RGCs in Math5-/+ heterozygotes; (2) to investigate the embryological basis for the Math5 -/- phenotype, by exploring the attenuated optic stalk development and the interdependence between neuronal, vascular, and astrocyte development in Math5 -/- retinas, by using BrdU birth-dating methods to directly test the RGC-to-cone cell-fate switch model, and by generating Math5-Cre transgenic mice to fully define the lineage of Math5-positive precursor cells and test the roles of specific signaling pathways (e.g. Notch-delta) in RGC development; (3) to define target genes for Math5 and the transcriptome of developing and adult RGCs using I-gene cDNA microarrays (Farjo et al. 2001) and subtractive PCR techniques; (4) to test the role of ATH5 and POU4F2 mutations in human optic nerve a/hypoplasia and glaucoma; (5) to finely map and clone Bst, and order Bst relative to Math5 in a developmental hierarchy; and (6) to characterize the hindbrain and cerebellar Math5 expression domains, and test the role of Math5 in auditory processing and motor control.