Brn-3 Transcription Factors in Retinal Development

描述(申请人提供)：视网膜变性疾病对健康构成极大风险。目前，有效的治疗和预防方法仍然难以找到。识别对视网膜神经元的形成和存活至关重要的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开发，并开发通过移植或神经干细胞再生来取代退化的视网膜神经元的方法。这一建议的长期目标是了解BRN-3POU结构域转录因子在视网膜神经发生中的作用，并识别和表征其下游效应基因的作用。BRN-3基因BRN-3a、BRN-3b和BRN-3c共有一个高度保守的功能结构域，它们在神经发生和成体中的表达在很大程度上重叠。小鼠的定向突变研究表明，每个BRN-3基因的缺失会导致特定神经细胞群的丢失，从而导致独特的神经元表型。有趣的是，每个BRN-3突变体中敲除表型的唯一性与其独特的时空表达模式密切相关。在视网膜中，BRN-3基因的表达主要重叠在视网膜神经节细胞(RGCs)中，并且BRN-3b的表达先于BRN-3a和BRN-3c的表达。BRN-3b基因缺失导致约70%的视网膜节细胞终末分化失败和凋亡。BRN-3c的丢失对一小部分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，并探索BRN-3功能的共同分子机制，在本应用中，我们将：1)使用转基因方法检验BRN-3基因的功能等价性。在敲入实验中，BRN-3a和BRN-3c的编码区将被用来取代BRN-3b。将检测敲入的BRN-3基因挽救与BRN-3b敲除相关的视网膜表型的能力；2)确定BRN-3a在RGCs发育中的作用。将分析BRN-3a缺失小鼠或BRN-3b和BRN-3a缺失小鼠的视网膜缺陷；3)在体内鉴定和表征BRN-3b下游基因的作用。我们发现BRN-3b的缺失导致转录因子GFI-1和LMO2的RGC表达减少。GFI-1是内耳毛细胞生存所必需的锌指蛋白。最近，我们已经确定LMO2是第一个在发育中的视网膜的GCL中以低鼻到高时间梯度表达的转录因子。转基因方法将被用来研究它们在视网膜发育中的作用，特别是在视网膜节细胞的轴突寻路和存活方面。

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.