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，并且它们的轴突包括视神经。涉及视神经的缺陷是儿童失明的重要原因，而RGCs是青光眼发病机制的常见最终靶点。RGCs是在视杯中诞生的第一批神经元，来自多能祖细胞，其后来产生无长突、视锥和视杆光感受器、水平和双极细胞以及Muller胶质细胞。视网膜组织发生的基本机制知之甚少，但被认为涉及内在和外在因素。最近，我们鉴定了小鼠碱性螺旋-环-螺旋（bHLH）转录因子Math 5，其与果蝇无调同源，并且其在视网膜中的特异性表达模式与RGC的产生相关（Brown等，1998）。我们创造了Math 5基因敲除小鼠。纯合子存活，但缺乏RGCs和视神经（Brown et al 2001 a）。它们伴随着视锥光感受器的增加，与细胞命运转变一致。他们的眼睛也缺乏中央动脉和静脉，并表现出新血管形成表型与一些层状改变，但在其他方面表现正常。这种单一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尚未探索。我们的初步数据和进化比较表明，Math 5是下游的Hes 1和上游的Brn 3b（Pou 4f 2）的转录因子在视网膜发生的层次。预期Math 5-/-眼缺乏RGC确定、分化和维持所独特需要的所有mRNA。我们已经表征了人ATH 5直系同源物（Brown等，2001 b），并从患有视神经发育不全、发育不全和青光眼的患者中收集样品用于突变筛选。除Math 5外，已知三个位点控制小鼠RGC的发育或数量-Brn 3b、Nnc 1 QTL和经典的自发突变Bst（腹部斑点和尾部）。与Math 5-/-小鼠相比，Bst/+小鼠具有相似但不太显著的RGC减少，并且具有相当的视网膜新生血管形成表型。使用亚种间回交，我们已经映射到一个-1厘米的间隔在小鼠染色体16，接近Hes 1和桥接两个YAC重叠群的BST。最后，使用lacZ基因敲入等位基因，我们已经确定了一个晚期Math 5表达域在后脑（梯形体）和小脑。这些发现，以及对N2 F2代少数共济失调Math 5突变小鼠的观察，表明Math 5在听觉处理和协调中也可能具有次要作用。 在本提案中，我们的目标是：（1）详细表征成年Math 5眼表型，包括用一组视网膜神经元和血管标记物的组织学分析、ERG和昼夜节律生理学研究以及Math 5-/+杂合子中RGC的定量分析;（2）通过探讨视柄发育的衰减以及神经元、血管、和星形胶质细胞发育，通过使用BrdU出生日期方法直接测试RGC-to-cone细胞命运开关模型，并通过产生Math 5-Cre转基因小鼠来完全定义Math 5阳性前体细胞的谱系并测试特定信号传导途径的作用（例如Notch-Delta）;（3）利用I基因cDNA微阵列确定Math 5的靶基因以及发育和成年RGCs的转录组（Farjo et al.2001）和消减PCR技术;（4）测试ATH 5和POU 4F 2突变在人类视神经发育不全和青光眼中的作用;（5）精细定位和克隆Bst，并在发育层次中相对于Math 5对Bst进行排序;（6）研究Math 5在大鼠后脑和小脑的表达结构域，并探讨Math 5在听觉加工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.