Extracellular matrix remodeling in ocular anterior segment development

描述（由申请人提供）：眼前节发育不全（ASD）包括眼前节形态发生的各种发育障碍，这是一个涉及几种细胞谱系及其相关细胞外基质（ECM）的复杂过程。ECM重塑对眼前节形态发生的贡献尚未成为先前研究的主题。我们最近发现单个Adamts 9等位基因（编码分泌的金属蛋白酶）的缺失导致Peters异常（角膜白斑和持久性透镜柄）和透镜缺陷，包括白内障。ADAMTS 9可能是葡萄糖基转移酶B3 GALTL的主要底物，其在人类彼得斯综合征（PPS）中突变。在PPS中，眼外出生缺陷，包括腭裂和心脏发育异常，其中ADAMTS 9先前已被牵连，伴随彼得斯异常。我们假设，ADAMTS 9分泌的玻璃体血管内皮细胞和视杯，是所需的ECM周转，并可以非自主地作用于透镜和神经嵴细胞，以调节眼前段形态发生。ADAMTS 9切割蛋白聚糖多功能蛋白聚糖，尽管它在眼病（瓦格纳综合征）中起作用，但在眼睛发育过程中对多功能蛋白聚糖知之甚少。此外，初步研究支持ECM分子，纤连蛋白和β 2作为潜在的ADAMTS 9底物的调查。在本提案的目标1中，我们将全面表征体外B3 GALTL对ADAMTS 9的修饰，以及在培养细胞中防止这种修饰的后果。我们将定义由Adamts 9单倍不足引起的ASD的发育、细胞和分子变化。在目标2中，我们将使用一种新的floxed Adamts 9等位基因在血管内皮（Tie 2-Cre）和视杯（ORE）中进行条件性失活，以描述这些特定谱系表达的ADAMTS 9如何驱动眼前段形态发生。我们将专门研究ADAMTS 9是否在体外和眼中与多功能蛋白聚糖、纤连蛋白和纤维蛋白原2相互作用并进行蛋白水解。总之，这些目标提供了从B3 GALTL到眼睛发育的基本原理再到ADAMTS 9分子作用的连续机制。与公共卫生的相关性：ADAMTS 9是Peters异常、PPS或其变体以及其他形式的人类ASD和/或先天性白内障的新候选基因，其致病基因突变未知。它将为迄今为止被忽视的问题提供答案，即ECM动力学如何影响早期眼睛发育以及修饰ECM的关键蛋白酶是什么？它将推进与ASD、儿童青光眼和白内障高度相关的眼和透镜形态发生的基础知识，并提供与成人青光眼和白内障的形成相关的机制，并可能提供成人青光眼和白内障的预防机制。

DESCRIPTION (provided by applicant): Anterior segment dysgenesis (ASD) encompasses a variety of developmental disorders of ocular anterior segment morphogenesis, a complex process involving several cell lineages and their associated extracellular matrix (ECM). The contribution of ECM remodeling to anterior segment morphogenesis has not been the subject of prior investigation. We recently discovered that loss of a single Adamts9 allele (encoding a secreted metalloprotease) resulted in Peters anomaly (corneal leukoma and persistent lens stalk) and lens defects, including cataract. ADAMTS9 is likely a major substrate for the glucosyltransferase B3GALTL, which is mutated in human Peters plus syndrome (PPS). In PPS, extraocular birth defects, including cleft palate and cardiac development anomalies, in which ADAMTS9 had been previously implicated, accompany Peters anomaly. We hypothesize that ADAMTS9 secreted by endothelial cells of the hyaloid vasculature and by the optic cup, is required for ECM turnover and could act non-autonomously on the lens and neural crest cells to regulate anterior segment morphogenesis. ADAMTS9 cleaves the proteoglycan, versican, about which little is known during eye development, despite its role in eye disease (Wagner syndrome). Also, preliminary studies support investigation of the ECM molecules, fibronectin and fibrillin-2 as potential ADAMTS9 substrates. In aim 1 of this proposal, we will comprehensively characterize ADAMTS9 modification by B3GALTL in vitro, and the consequences of preventing this modification in cultured cells. We will define the developmental, cellular and molecular changes underlying ASD resulting from Adamts9 haploinsufficiency. In aim 2, we will use a new floxed Adamts9 allele for conditional inactivation in vascular endothelium (Tie2-Cre), and optic cup (�re), to delineate how ADAMTS9 expressed by these specific lineages drives anterior segment morphogenesis. We will specifically investigate whether ADAMTS9 interacts with and proteolytically processes versican, fibronectin, and fibrillin-2, both in vitro and in the eye. Together, these aims provide a mechanistic continuum from B3GALTL to fundamentals of eye development to molecular actions of ADAMTS9. Relevance to public health: ADAMTS9 is a novel candidate gene for Peters anomaly, PPS or its variants, and other forms of human ASD and/or congenital cataract for which causative gene mutations are unknown. It will provide answers to a hitherto ignored question, i.e. how does ECM dynamics influence early eye development and what are the key proteases that modify the ECM? It will advance fundamental knowledge of ocular and lens morphogenesis that is highly relevant to ASD, childhood glaucoma and cataract, and provides mechanisms pertinent to the formation and possibly, the prevention of adult glaucoma and cataracts.