Hedgehog Signaling and Cilia in Choroid Fissure Morphogenesis and Coloboma

 描述(由申请人提供)：葡萄膜缺损是一种估计发生在约1：10,000名活产儿中的疾病，是全球范围内导致失明的重要原因。它的特点是有一个洞 或眼部裂隙，由脉络膜裂隙的缺陷形成或闭合引起，脉络膜裂隙是视网膜轴突退出和血管系统进入眼睛的一种短暂但关键的结构。Hedgehog(HH)信号通路对脉络膜裂隙的发育至关重要：该通路上游、下游和内部的突变可导致缺损。有些自相矛盾的是，高激活HH信号的突变以及失活下游靶标的突变都会导致人类的缺陷性疾病，但这些模型背后的特定形态发生缺陷尚不清楚。此外，脊椎动物的HH信号还需要初级纤毛。Colobomata与人类纤毛疾病有关，但尚不清楚纤毛病变突变是激活还是不激活眼睛中的HH信号，或者它们是如何扰乱特定的形态发生过程而导致Coloboma的。斑马鱼为研究这一过程提供了一个理想的模型系统：光学透明性和快速发展为直接观察活体脉络膜裂隙形态提供了独特的机会。我们之前开发了成像和计算技术来跟踪和可视化视杯形态发生过程中细胞的运动。然而，脉络膜裂隙的发展和在缺损中被破坏的具体机制仍然是一个谜。在这项建议中，我们将确定在正常条件下和在Hedgehog驱动的缺陷性斑马鱼模型中脉络膜裂缝形成的机制。我推测，过度活跃的刺猬信号，通过纤毛作用，以及下游效应器的丢失，都会以相反的方式扰乱脉络膜裂隙形成下的细胞运动，从而导致缺损。结合分子遗传学与创新的4维实时成像和计算方法，我们将在以下特定目标对这一假说进行检验：(1)确定ptch2突变引起的过度活跃的HH信号如何扰乱脉络膜裂隙的形成；(2)确定HH下游效应因子Pax2的缺失如何扰乱脉络膜裂隙的发育而导致缺损；以及(3)确定纤毛信号在脉络膜裂隙发育和纤毛病变相关缺损中的作用。这里提出的机制实验将确定正常脉络膜裂隙形成的细胞动力学，以及三种人类缺陷症模型下的特定缺陷，所有这些都影响Hedgehog通路：Gorlin综合征、肾缺损症和Joubert/Coach综合征。我们的工作代表了一种新的策略来理解这种潜在的破坏性视力障碍的病因。

 DESCRIPTION (provided by applicant): Uveal coloboma, a condition estimated to occur in ~1:10,000 live births, is a significant cause of blindness worldwide. It is characterized by a hole or cleft in the eye, and results from defective formation or closure of the choroid fissure, a transient yet critical structure through which retinal axons exit and vasculature enters the eye. The Hedgehog (Hh) signaling pathway is vital for choroid fissure development: mutations upstream, downstream, and within the pathway can result in coloboma. Somewhat paradoxically, mutations that hyperactivate Hh signaling, as well as mutations that inactivate downstream targets, both lead to coloboma in humans, yet the specific morphogenetic defects underlying each of these models are unknown. In addition, primary cilia are required for vertebrate Hh signaling. Colobomata are associated with human ciliopathies, yet it is unclear whether the ciliopathy mutations activate or inactive Hh signaling in the eye, or how they disrupt specific morphogenetic processes to cause coloboma. Zebrafish presents an ideal model system to study this process: optical transparency and rapid development offer a unique opportunity to directly watch the choroid fissure form in vivo. We previously developed imaging and computational techniques to track and visualize cell movements throughout optic cup morphogenesis. However, choroid fissure development and the specific mechanisms disrupted in coloboma remain a mystery. In this proposal, we will determine the mechanisms underlying choroid fissure formation under normal conditions and in Hedgehog-driven zebrafish models of coloboma. I hypothesize that hyperactive Hedgehog signaling, acting through cilia, and loss of downstream effectors both result in coloboma by disrupting, in an opposing manner, cell movements underlying choroid fissure formation. Combining molecular genetics with innovative 4-dimensional live imaging and computational methods, we will test this hypothesis in the following specific aims: (1) determine how hyperactive Hh signaling, as caused by mutation in ptch2, disrupts choroid fissure formation; (2) determine how loss of the Hh downstream effector pax2 disrupts choroid fissure development to cause coloboma; and (3) determine the role of cilia signaling in choroid fissure development and ciliopathy-associated coloboma. The mechanistic experiments proposed here will define the cellular dynamics underlying normal choroid fissure formation, and the specific defects underlying three models of human coloboma, all of which affect the Hedgehog pathway: Gorlin syndrome, renal coloboma syndrome, and Joubert/COACH syndrome. Our work represents a novel strategy to understand the etiology of this potentially devastating vision disorder.