Hedgehog Signaling in Optic Fissure Morphogenesis and Coloboma

葡萄膜缺损是导致失明的一个重要原因，据估计，这种情况发生在大约1：10,000名活产儿中 全世界。它的特征是眼睛上有一个洞或裂隙，是由于眼球形成或闭合有缺陷造成的 视裂，一种短暂而重要的结构，视网膜轴突通过它离开，血管系统进入眼睛。 尽管它很重要，但我们对控制视神经的细胞和分子机制了解很少。 裂缝发育，尤指形成的关键初始步骤：如果裂缝没有正确形成，它将 而不是接受关闭。保守的Hedgehog(HH)信号通路至关重要：在Gorlin综合征中，过度活跃 在PTCH突变体(斑马鱼中的ptch2)的背景下，HH信号会导致缺陷症。 在过去的资金周期中，我们使用4种方法确定了视裂形成的细胞运动。 维度成像和细胞跟踪。令人惊讶的是，我们发现之前未描述的折叠事件在 腹眼驱动视裂的形成，然而，潜在的细胞形状变化和重排是 未知。在ptch2突变体中，我们发现了过度活跃的hh信号扰乱这一过程的关键机制。 过程中，发现HH信号的下游靶点负责扰乱细胞运动。 因此，我们进行了单细胞RNA-SEQ以确定控制视裂形成的HH靶基因： 基质蛋白多糖(集聚蛋白、营养不良多糖和Syndecan-4)和线粒体基因已经为我们提供了新的 方向。在这里，我们利用斑马鱼独特的光学透明度和快速发展 胚胎直接检查活体视裂的形成。我们最近开发了成像和计算 可视化和定量分析细胞运动、形状和取向驱动动力学的方法 首次发现了视裂的形成，以及扰乱信号通路的分子遗传学方法。在……里面 在这个提案中，我们将揭示直接导致视裂形成的细胞机制，以及 组织-组织相互作用与嗅觉平台的作用，以及内在代谢活动。 我们的假设是，活跃的细胞形状改变和重新定向驱动了视裂的形成，并且 这一过程也依赖于通过基质蛋白多糖与嗅觉定位蛋白的机械偶联 作为细胞内线粒体的动力学。结合分子遗传学、实时成像、定制计算 方法和定量图像分析，我们将在以下具体目标上检验这一假说：(1)建立 (2)决定视裂形成和破坏的形态发生机制 嗅觉定位影响视裂形成的细胞和分子机制； 确定线粒体动力学如何在缺损性视裂的形成及其破坏中起作用。 我们提议的机械性实验将扩大缺陷基因调控网络，提供 告知患者表型的资源，并建立新的潜在治疗目标，对 除戈林综合征外，还有许多人类缺陷症。

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 optic fissure, a transient, yet vital structure through which retinal axons exit and vasculature enters the eye. Despite its importance, we have a poor understanding of the cellular and molecular mechanisms governing optic fissure development, especially the crucial initial step of formation: if the fissure does not form correctly, it will not undergo closure. The conserved Hedgehog (Hh) signaling pathway is crucial: in Gorlin Syndrome, overactive Hh signaling, in the context of the PTCH mutant (ptch2 in zebrafish) causes coloboma. In the past funding cycle, we determined the cell movements underlying optic fissure formation, using 4- dimensional imaging and cell tracking. Surprisingly, we found that a previously undescribed folding event in the ventral eye drives optic fissure formation, however, the underlying cell shape changes and rearrangements are unknown. In the ptch2 mutant, we uncovered key mechanisms by which overactive Hh signaling perturbs this process and found that downstream targets of Hh signaling are responsible for disrupting cell movements. Therefore, we performed single-cell RNA-seq to identify Hh target genes controlling optic fissure formation: matrix proteoglycans (agrin, dystroglycan, and syndecan-4) and mitochondria genes have informed our new directions. Here we take advantage of the unique optical transparency and rapid development of zebrafish embryos to directly examine optic fissure formation in vivo. We recently developed imaging and computational approaches to visualize and quantitatively analyze dynamics of cell movements, shape, and orientation driving optic fissure formation for the first time, as well as molecular genetic methods to perturb signaling pathways. In this proposal, we will uncover the cellular mechanisms directly responsible for optic fissure formation, as well as the role of tissue-tissue interactions with the olfactory placode, and intrinsic metabolic activity. Our hypothesis is that active cell shape changes and reorientation drive optic fissure formation, and this process relies on mechanical coupling with the olfactory placode via matrix proteoglycans, as well as intracellular mitochondrial dynamics. Combining molecular genetics, live imaging, custom computational methods and quantitative image analyses, we will test this hypothesis in the following specific aims: (1) establish the morphogenetic mechanisms driving optic fissure formation and its disruption in coloboma; (2) determine the cellular and molecular mechanisms by which the olfactory placode influences optic fissure formation; and (3) determine how mitochondrial dynamics contribute to optic fissure formation and its disruption in coloboma. Our proposed mechanistic experiments will expand the coloboma gene regulatory network, provide resources to inform patient phenotypes, and establish new potential therapeutic targets with implications for numerous human coloboma conditions in addition to Gorlin Syndrome.