Transcriptional Regulation of Retinal Development

描述(申请人提供)：本申请的长期目标是了解调节不同视网膜细胞类型的确定、分化和维持的分子基础。许多转录因子已被证明在脊椎动物视网膜发生过程中起关键作用。其中包括POU结构域转录因子Brn3b，它是视网膜神经节细胞发育所必需的。Barhl2同源结构域转录因子也可能在视网膜发育中发挥重要作用，因为它在视网膜内核层的神经节细胞和神经元中表达。 本申请中概述的研究旨在提供综合的方法来解决脊椎动物视网膜发育的基本机制，使用Brn3b和Barhl2转录因子作为我们分析的模型。我们将追求三个具体目标：1)研究Bm3b控制视网膜神经节细胞分化和存活的机制。这些研究旨在通过“候选基因”方法和微阵列技术识别Brn3b下游基因，然后测试其生物学意义；ii)识别与Brn3b相互作用的蛋白质并确定其功能特征。我们的目标是鉴定Brn3b结合蛋白，这些蛋白可能在视网膜发育过程中调节其靶基因的选择和激活。将使用酵母双杂交筛选方法分离Bm3b的新蛋白质伙伴。通过研究它们对Brn3b的DNA结合和转录特性的影响，以及它们在Brn3b介导的视网膜神经节细胞分化中的表达模式和作用，来研究它们的功能相关性；iii)在体内研究Barhl2基因在视网膜发育中的作用。两种互补的方法将被用来分析Barhl2在视网膜形成过程中的体内功能。一种是功能丧失的方法，涉及定向基因破坏，以产生Barhl2缺陷的小鼠。另一种是通过逆转录病毒介导的Barhl2在鸡和小鼠视网膜中过表达的“功能获得”方法。总之，这些拟议的研究将为管理哺乳动物视网膜发育的分子调控网络提供重要的见解，并可能为更好地理解和治疗某些视网膜疾病提供基础。

DESCRIPTION (provided by applicant): The long-term objective of this application is to understand the molecular bases that regulate the determination, differentiation and maintenance of different retinal cell types. A number of transcription factors have been shown to play a key role during vertebrate retinogenesis. Among them is the POU domain transcription factor Brn3b which is critically required for the development of retinal ganglion cells. The Barhl2 homeodomain transcription factor may also play an important role in retinal development as it is expressed in ganglion cells as well as in neurons within the inner nuclear layer of the retina. The studies outlined in this application are designed to provide integrated approaches to address the fundamental mechanisms governing vertebrate retinal development, using the Brn3b and Barhl2 transcription factors as models for our analyses. Three specific aims will be pursued: i) to investigate the mechanisms by which Bm3b controls the differentiation and survival of retinal ganglion cells. These studies aim to identify Brn3b downstream genes by a "candidate gene" approach as well as the microarray technology followed by tests of their biological significance; ii) to identify and functionally characterize proteins interacting with Brn3b. We aim to identify Brn3b binding proteins that may regulate the selection and activation of its target genes during retinal development. Novel protein partners for Bm3b will be isolated using a yeast two-hybrid screening approach. Their functional relevance will be investigated by studying their effects on DNA-binding and transcriptional properties of Brn3b, and by examining their expression patterns and effects on Brn3b-mediated retinal ganglion cell differentiation; iii) to study in vivo the role of Barhl2 gene during retinal development. Two complementary approaches will be employed to analyze the in vivo functions of Barhl2 during retinogenesis. One is a loss-of-function approach involving targeted gene disruption to produce mice deficient for Barhl2. The other is a "gain-of-function" approach involving retrovirus-mediated overexpression of Barhl2 in the chick and mouse retina. Together, these proposed studies will provide important insights into the molecular regulatory networks that govern mammalian retinal development and may provide the foundation for better understanding and treatment of certain retinal disorders.