GENETIC ANALYSIS OF RETINAL GANGLION CELL FUNCTION

描述（由申请人提供）：视网膜神经节细胞（RGCs）是脊椎动物眼睛的唯一投射神经元，它们的轴突组成视神经。视神经缺损是儿童致盲的重要原因，而RGCs是青光眼常见的最终发病目标。RGCs是在视杯中诞生的第一批神经元，来自多能祖细胞，后来产生无突细胞、锥体和杆状光感受器、水平细胞和双极细胞以及穆勒神经胶质。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，我们发现了一种小鼠基本螺旋-环-螺旋（bHLH）转录因子Math5，它与果蝇的atonal同源，其在视网膜中的特异性表达模式与RGCs的诞生相关（Brown et al. 1998）。我们创造了Math5基因敲除小鼠。纯合子可以存活，但缺乏RGCs和视神经（Brown et al, 2001a）。它们的视锥光感受器也随之增加，这与细胞命运的转变相一致。他们的眼睛也缺少中心动脉和静脉，并表现出新生血管化表型和一些层流改变，但在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，在视网膜发生的层次结构中，Math5位于Hes1的下游，Brn3b （Pou4f2）的上游。Math5 -/- eyes预计缺乏RGC测定、分化和维持所需的所有mrna。我们鉴定了人类ATH5同源基因（Brown et al. 2001b），并从视神经发育不全、发育不全和青光眼患者中收集样本进行突变筛选。除Math5外，已知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（腹部斑点和尾巴）。与Math5 -/-小鼠相比，Bst/+小鼠的RGCs减少相似，但不太明显，视网膜新生血管表型相似。通过亚种间回交，我们将Bst定位在小鼠16号染色体上a-1 cM的间隔上，靠近Hes1并连接两个YAC序列。最后，利用lacZ敲入等位基因，我们在后脑（梯形体）和小脑中发现了一个晚期Math5表达域。这些发现以及对少量N2F2代共济失调Math5突变小鼠的观察表明，Math5可能在听觉加工和协调中也起次要作用。

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.