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率约为1：10，000活产的疾病，是全球失明的重要原因。它的特点是一个洞 或裂，并且由脉络膜裂的缺陷形成或闭合引起，脉络膜裂是视网膜轴突通过其离开并且脉管系统通过其进入眼睛的短暂但关键的结构。Hedgehog（Hh）信号通路对于脉络膜裂的发育至关重要：上游、下游和通路内的突变可导致缺损。有些矛盾的是，过度激活Hh信号传导的突变，以及破坏下游靶点的突变，都导致人类的缺损，但这些模型中每一个模型的具体形态发生缺陷尚不清楚。此外，初级纤毛是脊椎动物Hh信号传导所必需的。缺损与人类睫状体病有关，但目前尚不清楚睫状体病突变是否激活或灭活眼睛中的Hh信号传导，或者它们如何破坏特定的形态发生过程以导致缺损。 斑马鱼为研究这一过程提供了理想的模型系统：光学透明性和快速发育为直接观察活体脉络膜裂的形成提供了独特的机会。我们以前开发的成像和计算技术来跟踪和可视化整个视杯形态发生的细胞运动。然而，脉络膜裂的发展和具体的机制中断缺损仍然是一个谜。在这项建议中，我们将确定在正常条件下和刺猬驱动的斑马鱼模型的缺损的脉络膜裂形成的机制。 我推测，过度活跃的刺猬信号，通过纤毛，和下游效应器的损失都导致缺损破坏，以相反的方式，细胞运动脉络膜裂形成的基础。将分子遗传学与创新的四维实时成像和计算方法相结合，我们将在以下具体目标中检验这一假设：（1）确定由ptch 2突变引起的过度活跃的Hh信号传导如何破坏脉络膜裂的形成;（2）确定Hh下游效应子pax 2的缺失如何破坏脉络膜裂的发育以导致缺损;以及（3）确定纤毛信号传导在脉络膜裂发育和纤毛病相关缺损中的作用。 这里提出的机制实验将定义正常脉络膜裂形成的细胞动力学基础，以及三种人类缺损模型的特定缺陷，所有这些都影响Hedgehog通路：Gorlin综合征，肾缺损综合征和Joubert/COACH综合征。我们的工作代表了一种新的策略，以了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.