Extracellular matrix remodeling in ocular anterior segment development

描述(申请人提供)：眼前段发育不全(ASD)包括多种眼眼前段形态发生的发育障碍，这是一个涉及多个细胞系及其相关的细胞外基质(ECM)的复杂过程。细胞外基质重塑在前段形态发生中的作用还不是先前研究的主题。我们最近发现，单个Adamts9等位基因(编码一种分泌型金属蛋白酶)的缺失会导致Peter畸形(角膜白斑和持续性晶状体柄)和晶状体缺陷，包括白内障。ADAMTS9可能是葡萄糖转移酶B3GALTL的主要底物，该酶在人类彼得斯综合征(PPS)中发生突变。在PPS中，眼外出生缺陷，包括腭裂和心脏发育异常，以前曾涉及ADAMTS9，伴随着彼得斯畸形。我们推测，ADAMTS9由玻璃体血管内皮细胞和视杯分泌，是细胞外基质转换所必需的，并可能非自主地作用于晶状体和神经脊细胞，调节眼前段的形态发生。ADAMTS9切割蛋白多聚糖，在眼睛发育过程中人们对其知之甚少，尽管它在眼部疾病(瓦格纳综合征)中起到了作用。此外，初步研究支持将细胞外基质分子、纤维连接蛋白和纤维蛋白-2作为潜在的ADAMTS9底物进行研究。在本提案的目标1中，我们将在体外全面表征B3GALTL对ADAMTS9的修饰，以及在培养细胞中阻止这种修饰的后果。我们将定义由Adamts9单倍体不足引起的ASD潜在的发育、细胞和分子变化。在目标2中，我们将使用血管内皮细胞条件性失活的新等位基因(Tie2-Cre)和视杯(�Re)来描述这些特定谱系表达的ADAMTS9是如何驱动眼前节形态发生的。我们将专门研究ADAMTS9在体外和眼睛中是否与Versican、FN和FIG-2相互作用并以蛋白水解性方式处理。总而言之，这些目标提供了从B3GALTL到眼睛发育的基础到ADAMTS9的分子作用的机械连续体。与公众健康相关：ADAMTS9是彼得斯畸形、PPS或其变体以及其他形式的人类自闭症和/或先天性白内障的新候选基因，其原因基因突变尚不清楚。它将为一个迄今被忽视的问题提供答案，即ECM动力学如何影响眼睛的早期发育，以及哪些关键的蛋白水解酶可以改变ECM？它将促进与ASD、儿童青光眼和白内障高度相关的眼和晶状体形态发生的基础知识，并为成人青光眼和白内障的形成和可能的预防提供相关的机制。

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.