Regulation of Eye Morphogenesis

描述(申请人提供)：先天性眼畸形，如无眼球、小眼球和眼球缺陷症，在3-4,000人中普遍存在，是全世界超过25%的儿童失明的原因。仅缺陷症就可能占到儿童失明的10%。因此，了解眼球发育的分子机制至关重要。WNTs是一类分泌的、高度保守的糖蛋白家族，控制着发育、疾病和再生过程中的关键过程。WnT信号是非常复杂的；在哺乳动物中，19个Wnt和10个FrizzledR可以激活规范的Wnt/�-连环蛋白途径，以及两个不太明确的非规范途径，Wnt/Ca~(2+)和平面细胞极性。这种复杂性反映在Wnt信号在眼睛发育过程中的不同作用上。在这个提案中，我们旨在解决两个重要的问题：1)非规范的Wnt信号如何调节眼睛的早期发育？2)Wnt信号控制视裂闭合的细胞机制是什么？为了开始梳理这一点，我们将重点放在豪猪(PORCN)的功能上，它介导了WNTS的翻译后修饰。PORCN是一种膜结合的O-酰基转移酶，存在于内质网中，介导棕榈酰化，对Wnt配体的分泌和信号活性至关重要。人类疾病局灶性真皮发育不全(FDH，Goltz综合征)是一种X连锁的罕见显性疾病，由PORCN突变引起。大约20%的外佣患者出现小眼球、无眼症和缺损(MAC)，以及其他几种严重的发育缺陷。虽然PORCN突变会导致MAC，但我们目前还不清楚PORCN/WNT缺失是如何导致严重的眼畸形，如无眼症和缺陷性眼病。利用PORCN的时间和组织特异性失活、潜在靶点的识别和不同的体外方法，我们建议识别调节视裂闭合的细胞相互作用(目标1)，研究非典型Wnt信号在眼场和视泡形成中的作用(目标2)。在目标3中，我们将研究小GTP酶CDC42在视泡外溢和视杯形态发生中的作用。我们的方法将确定Wnt信号在眼睛发育过程中的新角色，这对治疗和再生努力将是重要的。人类无眼症和缺陷性眼病的病因很复杂，可能是几个因素的干扰造成的。本文提出的研究将促进我们了解在视裂关闭的早期阶段和闭合期间控制眼睛形态发生的细胞和分子机制。

DESCRIPTION (provided by applicant): Congenital ocular malformations such as anophthalmia, microphthalmia and coloboma are prevalent in ~1 in 3-4,000 individuals and are the cause for over 25% of childhood blindness worldwide. Coloboma alone may account up to 10% of childhood blindness. Therefore, it is vitally important to understand the molecular mechanisms underlying ocular development. Wnts belong to a family of secreted, highly conserved glycoproteins that control key processes during development, disease and regeneration. Wnt signaling is very complex; in mammals, 19 Wnts and 10 Frizzled receptors have been identified that can activate the canonical, Wnt/�-catenin pathway and two less well-defined non-canonical pathways, Wnt/Ca2+ and Planar Cell Polarity. This complexity is reflected by the different roles of Wnt signaling during eye development. In this proposal, we aim to address two important questions; 1) How does non-canonical Wnt signaling regulate early eye development? 2) What is the cellular mechanism by which Wnt signaling controls closure of the optic fissure? To begin to tease this apart, we are focusing on the function of Porcupine (Porcn) that mediates posttranslational modification of Wnts. Porcn is a membrane-bound O-acyltransferase that resides in the endoplasmatic reticulum and mediates palmitoylation critical for the secretion and signaling activity of Wnt ligands. The human disease Focal Dermal Hypoplasia (FDH, Goltz Syndrome) is an X-linked, rare dominant disorder caused by mutations in PORCN. About 20% of FDH patients exhibit microphthalmia, anophthalmia, and coloboma (MAC), among several other severe developmental defects. While Porcn mutations cause MAC, we have no current understanding how depletion of Porcn/Wnt results in severe ocular malformations such as anophthalmia and coloboma. Using temporal and tissue-specific inactivation of Porcn, identification of potential targets and diverse in vitro approaches, we propose to identify the cellular interactions regulating closure of the optic fissure (Aim 1), investigate the role of non-canonical Wnt signaling during eye field and optic vesicle formation (Aim 2). In Aim 3, we will investigate the role of the small GTPase Cdc42 in optic vesicle evagination and optic cup morphogenesis. Our approach will identify novel roles for Wnt signaling during eye development that will be important for treatment and regenerative efforts. The etiology of anophthalmia and coloboma in humans is complex and can result from disruption of several factors. The studies proposed here will advance our knowledge toward an understanding of the cellular and molecular mechanisms controlling eye morphogenesis at the earliest stages and during closure of the optic fissure.