Hedgehog Signaling in Optic Fissure Morphogenesis and Coloboma

视裂形态发生和缺损中的 Hedgehog 信号传导

• 批准号: 10736980
• 负责人: Kristen M Kwan
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736980
• 关键词: Agrin; Automobile Driving; Basal Cell Nevus Syndrome; Blindness; Cell Shape; Cells; Choroid; Coloboma; Computing Methodologies; Coupling; Custom; Defect; Development; Dystroglycan; Embryo; Erinaceidae; Event; Eye; Four-dimensional; Fresh Water; Funding; Genes; Human; Image; Image Analysis; Live Birth; Mechanics; Metabolic; Metabolism; Methods; Microscopy; Mitochondria; Molecular; Molecular Genetics; Morphogenesis; Optics; PTCH gene; Patients; Phenotype; Process; Proteoglycan; Regulation; Resources; Role; Shapes; Signal Pathway; Signal Transduction; Structure; Testing; Time; Tissues; Visualization; Zebrafish; cell motility; experimental study; gene regulatory network; in vivo; mutant; quantitative imaging; retinal axon; single-cell RNA sequencing; smoothened signaling pathway; syndecan-4; teleost fish; therapeutic target; zebrafish development

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.

葡萄膜缺损是导致失明的一个重要原因，估计约1：10，000的活产婴儿会出现这种情况 国际吧它的特征是眼睛上有一个洞或裂缝，并且是由于眼睛的缺陷形成或闭合造成的。 视裂是一种短暂但重要的结构，视网膜轴突通过它退出，血管系统通过它进入眼睛。 尽管它的重要性，我们有一个贫穷的理解细胞和分子机制，控制视神经 裂缝发展，尤其是形成的关键初始步骤：如果裂缝没有正确形成，它将 不要关闭。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ptch 2）的背景下，Hh信号传导导致缺损。 在过去的资助周期中，我们确定了视裂形成的细胞运动，使用4- 三维成像和细胞跟踪。令人惊讶的是，我们发现一个以前未描述的折叠事件， 腹侧眼驱动视裂的形成，然而，潜在的细胞形状变化和重排， 未知在ptch 2突变体中，我们发现了过度活跃的Hh信号干扰这一过程的关键机制。 研究人员发现，Hh信号传导的下游靶点负责破坏细胞运动。 因此，我们进行了单细胞RNA-seq以鉴定控制视裂形成的Hh靶基因： 基质蛋白聚糖（聚集蛋白、肌营养不良蛋白聚糖和多配体蛋白聚糖-4）和线粒体基因为我们的新研究提供了信息。 方向在这里我们利用了斑马鱼独特的光学透明度和快速发育的优势 胚胎直接检查视裂的形成在体内。我们最近开发了成像和计算 可视化和定量分析细胞运动、形状和方向驱动动力学的方法 第一次发现了视裂的形成，以及干扰信号通路的分子遗传学方法。在 这个建议，我们将揭示直接负责视裂形成的细胞机制，以及 组织-组织与嗅觉基板的相互作用以及内在代谢活动的作用。 我们的假设是，活跃的细胞形状变化和重新定向驱动视裂的形成， 该过程也依赖于通过基质蛋白聚糖与嗅觉基板的机械偶联， 细胞内线粒体动力学。结合分子遗传学，实时成像，定制计算 方法和定量图像分析，我们将测试这一假设在以下具体目标：（1）建立 视裂形成和破裂的形态发生机制;（2）确定视裂的形成和破裂的机制。 嗅板影响视裂形成的细胞和分子机制;（3） 确定线粒体动力学如何促进视裂的形成及其在缺损中的破坏。 我们提出的机制实验将扩大缺损基因调控网络，提供 为患者表型提供信息，并建立新的潜在治疗靶点， 除了Gorlin综合征之外，还有许多人类缺损状况。