ISWI remodeling complexes in eye development

描述（由申请人提供）：本研究的长期目标是了解染色质重塑如何控制脊椎动物眼和中枢神经系统（CNS）的发育。与所有发育途径一样，眼睛的发育取决于基因表达的空间和时间模式的精确调节。转录激活和抑制发生在染色质的背景下，染色质是将DNA压缩到真核细胞核中的DNA和组蛋白的复合物。染色质重塑酶正向和负向调节染色质中DNA的可及性，从而是基因表达的重要调节剂。模拟开关（ISWI）染色质重塑酶是非洲爪蟾眼睛和中枢神经系统正常发育所必需的，缺乏ISWI的胚胎会发生先天性被膜下白内障。然而，ISWI是体内多种重塑复合物的催化亚基。我们特别感兴趣的是不同的ISWI依赖的染色质重塑酶如何调节胚胎发育过程中的眼睛发育。为了实现这一点，我们将通过抑制每个复合物特有的亚基来评估单个ISWI复合物的作用。我们将开始使用吗啉注射液抑制WSTF（WICH复合物的一个亚基）和CHRAC-17（CHRAC复合物的一个亚基）。我们将评估这些敲除在非洲爪蟾胚胎发育中的影响，特别是在发育中的眼睛内，并通过显微镜检查，切片和精细结构分析以及基因表达分析来分析由此产生的眼睛畸形。这些研究将提供关键的见解染色质重塑在眼睛发育中的作用。这项建议有三个具体目标。目的1是表征非洲爪蟾胚胎缺乏功能性WICH或CHRAC复合物的发育表型，使用吗啉代抑制，解决形态和转录缺陷。目的2是确定WSTF内的保守结构域（布罗莫结构域，PHD指和DDT结构域）对其发育功能的贡献。这一目标将包括注射WSTF的缺失突变体。目的3是通过产生在诱导型和组织特异性启动子控制下表达显性负性ISWI突变体的转基因非洲爪蟾，确定眼发育所需的ISWI活性的阶段。先天性白内障是儿童失明的主要原因。在发育中的青蛙胚胎中缺乏ISWI重塑活性导致脑和眼发育的许多缺陷，包括囊下白内障的形成。我们的工作将有助于理解正常眼发育的机制，并为研究先天性白内障的形成提供一个新的模型。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand how chromatin remodeling controls the development of the vertebrate eye and central nervous system (CNS). As in all developmental pathways, eye development depends upon precise regulation of spatial and temporal patterns of gene expression. Transcription activation and repression occurs in the context of chromatin, a complex of DNA and histone proteins that compacts DNA into the eukaryotic nucleus. Chromatin remodeling enzymes positively and negatively regulate the accessibility of DNA in chromatin, and are thereby essential regulators of gene expression. The Imitation Switch (ISWI) chromatin remodeling enzyme is required for normal development of the eye and CNS in Xenopus laevis, and embryos lacking ISWI develop congenital subcapsular cataracts. However, ISWI is the catalytic subunit of multiple remodeling complexes in vivo. We are specifically interested in how different ISWI-dependent chromatin remodeling enzymes regulate eye development during embryogenesis. To accomplish this, we will assess the roles of individual ISWI complexes by inhibiting subunits unique to each complex. We will begin by using morpholino injection to inhibit WSTF, a subunit of the WICH complex, and CHRAC-17, a subunit of the CHRAC complex. We will assess the effects of these knockdowns in the developing Xenopus embryo, specifically within the developing eye, and analyze the resulting eye malformations by microscopic examination, sectioning and fine structure analysis, and gene expression analysis. These studies will provide critical insights into the role of chromatin remodeling in eye development. This proposal has three specific aims. Aim 1 is to characterize the developmental phenotypes of Xenopus embryos lacking functional WICH or CHRAC complexes, using morpholino inhibition, addressing both morphological and transcriptional defects. Aim 2 is to identify the contributions of the conserved domains within WSTF (bromodomain, PHD finger, and DDT domains) to its developmental functions. This aim will include injection of deletion mutants of WSTF. Aim 3 is to determine the stage(s) during which ISWI activity is required for eye development, by generating transgenic Xenopus that express a dominant-negative ISWI mutant under control of inducible and tissue-specific promoters. Congenital cataracts are the leading cause of blindness in children. Lack of ISWI remodeling activity in developing frog embryos results in numerous defects in brain and eye development, including the formation of subcapsular cataracts. Our work will contribute to the understanding of the mechanisms of normal eye development and provides a new model for studying congenital cataract formation.