Brn-3 Transcription Factors in Retinal Development

描述（申请人提供）：视网膜变性疾病对健康危害很大。目前，有效的治疗和预防方法仍然难以捉摸。识别视网膜神经元形成和存活的重要基因和遗传过程将导致针对这些基因和过程的预防药物的发展，并开发通过移植或再生神经元干细胞来替代退化的视网膜神经元的方法。本研究的长期目标是了解brn - 3po结构域转录因子在视网膜神经发生中的作用，并鉴定和表征其下游效应基因的作用。brn-3基因brn-3a、brn-3b和brn-3c共享一个高度保守的功能域，它们在神经发生和成人中的表达在很大程度上是重叠的。在小鼠中进行的靶向诱变研究表明，每个brn-3基因的缺失会导致特定神经元组的损失，从而导致独特的神经元表型。有趣的是，每个brn-3突变体的敲除表型的独特性与其独特的时空表达模式密切相关。在视网膜中，brn-3基因的表达在视网膜神经节细胞（RGCs）中大多重叠，brn-3b的表达先于brn-3a和brn-3c的表达。brn-3b的缺失导致约70%的RGCs的终末分化失败和凋亡。brn-3c的缺失对一小部分rgc也有类似的影响。为了进一步了解brn-3基因在视网膜神经发生中的作用，探索brn-3功能的共同分子机制，本应用将：1)采用转基因方法检测brn-3基因的功能等效性。在敲入实验中将使用brn-3a和brn-3c的编码区代替brn-3b。敲入brn-3基因挽救与brn-3b敲除相关的视网膜表型的能力将被检验；2)确定brn-3a在rgc发生发展中的作用。分析brn-3a缺失小鼠或brn-3b、brn-3a缺失小鼠视网膜缺陷；3)鉴定和表征brn-3b下游基因在体内的作用。我们已经证明，Brn-3b的缺失导致RGC中转录因子Gfi-1和LMO2的表达减少。Gfi-1是内耳毛细胞存活所必需的锌指蛋白。最近，我们发现LMO2是发育中的视网膜GCL中第一个以低鼻部到高颞部梯度表达的转录因子。转基因方法将用于研究它们在视网膜发育中的作用，特别是RGCs的轴突寻路和存活。

DESCRIPTION (provided by applicant): Retinal degeneration diseases pose great health risks. Currently, the effective cures and prevention methods remain elusive. Identification of genes and genetic processes important for the formation and survival of retinal neurons will lead to the development of prevention drugs targeting these genes and processes and to develop approaches to replace the degenerated retinal neurons by transplantation or regeneration from neuronal stem cells. The long-term objective of this proposal is to understand the role of Brn-3 POU-domain transcription factors in retinal neurogenesis and to identify and characterize the roles of their downstream effect genes. The three brn-3 genes, brn-3a, brn-3b and brn-3c, share a highly conserved functional POU-domain and their expression during neurogenesis and in adult is largely overlapping. Targeted mutagenesis studies in mice have shown that deletion of each brn-3 genes lead to unique neuronal phenotypes with the loss of a selected group of neurons. Intriguingly, the uniqueness of knockout phenotypes in each brn-3 mutant closely correlated to its distinctive spatiotemporal expression pattern. In retina, expression of brn-3 genes is mostly overlapping in retinal ganglion cells (RGCs) and the onset of brn-3b expression precedes those of brn-3a and brn-3c. Deletion of brn-3b results in the terminal differentiation failure and apoptosis of approximate 70% of RGCs. Loss of brn-3c has similar effects on a small percentage of RGCs. To further understand the roles of brn-3 genes in retinal neurogenesis and to explore the common molecular mechanisms of brn-3 function, in this application, we will: 1) use the transgenic approach to test the functional equivalence of brn 3 genes. The coding regions of brn-3a and brn-3c will be used to replace brn-3b in the knock-in experiments. The ability of knock-in brn-3 genes to rescue the retinal phenotypes associated with brn-3b knockout will be examined; 2) determine the role of brn-3a in the development of RGCs. Defects in retina of brn-3a-null mice or mice null for brn-3b and brn-3a will be analyzed; 3) identify and characterize in vivo the role of brn-3b downstream genes. We have shown that loss of Brn-3b leads to the diminished RGC expression of transcription factors Gfi-1 and LMO2. Gfi-1 is a zinc-finger protein required for the survival of inner ear hair cells. Recently, we have identified LMO2 as the first transcription factor expressed in the GCL of developing retina in a low-nasal-to-high-temporal gradient. Transgenic approaches will be used to investigate their roles in retinal development, particularly the axon pathfinding and survival of RGCs.