GENETIC ANALYSIS OF RETINAL GANGLION CELL FUNCTION

描述(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，它们的轴突组成视神经。累及视神经的缺陷是导致儿童失明的重要原因，而视网膜节细胞是青光眼发病的共同最终靶点。视网膜节细胞是第一批在视杯中诞生的神经元，来自多能前体细胞，这些细胞后来产生长春瑞林、视锥和视杆感受器、水平和双极细胞以及Muller胶质细胞。视网膜组织发生的机制目前知之甚少，但被认为涉及内在和外在因素。最近，我们发现了一个小鼠碱性螺旋-环-螺旋(BHLH)转录因子Math5，它与果蝇无性系同源，其在视网膜中的特异表达模式与RGCs的出生相关(Brown等人)。1998年)。我们已经创造了Math5基因敲除小鼠。纯合子是可存活的，但缺乏视网膜节细胞和视神经(Brown等人，2001a)。它们伴随着锥体感光细胞数量的增加，这与细胞命运的转变是一致的。他们的眼睛也缺乏中央动脉和静脉，表现为新生血管表型，伴有一些板层改变，但其他方面看起来正常。单个视网膜神经元类别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位于Hes1下游和Brn3b(Pou4f2)的上游，Brn3b是视网膜发生层次中的转录因子。Math5-/-Eyes预计会缺少RGC确定、分化和维持所需的所有mRNAs。我们已经表征了人类ATH5直系同源基因(Brown等人)。2001b)，并采集视神经发育不全、发育不良和青光眼患者的样本进行突变筛查。除了Math5，已知有三个基因座控制小鼠RGC的发育或数量-Brn3b，Nnc1 QTL，以及经典的自发突变Bst(腹点和尾巴)。与Math5-/-小鼠和相似的视网膜新生血管表型相比，BST/小鼠的RGC减少相似，但不那么明显。利用亚种间回交，我们已经将BST定位在小鼠16号染色体上-1 cM的区间，接近Hes1并连接了两个YAC重叠群。最后，利用LacZ敲入等位基因，我们在后脑(梯形小体)和小脑中发现了一个晚期Math5表达区域。这些发现，以及对N2F2代少量共济失调Math5突变小鼠的观察，表明Math5可能在听觉处理和协调中也有次要作用。 在这项建议中，我们的目标是：(1)详细描述成年Math5眼的表型，包括一组视网膜神经元和血管标记物的组织学分析，ERG和昼夜节律生理研究，以及Math5-/杂合子RGCs的定量分析；(2)研究Math5-/-表型的胚胎学基础，通过研究Math5-/-视网膜视柄发育减弱以及神经元、血管和星形胶质细胞发育之间的相互依赖，通过BrdU出生测年法直接测试RGC到锥体细胞命运的转换模型，并通过建立Math5-Cre转基因小鼠来充分确定Math5阳性前体细胞的谱系，并检测特定的信号通路(如Notch-Delta)在RGC发育中的作用；(3)利用I-基因基因芯片确定发育和成体视网膜节细胞Math5的靶基因和转录组(Farjo et al.(4)检测ATH5和POU4F2突变在人视神经发育不良和青光眼中的作用；(5)精细定位和克隆BST，并在发育水平上对BST相对于Math5进行排序；(6)研究Math5在后脑和小脑中的表达区域，并检测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.