Stepwise Coordination of Eye Morphogenesis by Extracellular Matrix

项目摘要 眼睛结构的发育缺陷通常是新生儿视力受损的原因。正眼 结构最初是通过视杯形态发生的过程建立的，在此过程中，一系列复杂的 细胞和组织重排将视泡转化为视杯，并带有神经视网膜和视网膜 色素上皮(RPE)包裹新形成的晶状体。 随着成像和计算分析的进步，我们实验室和其他实验室的工作已经开始揭示 然而，视杯形态发生的细胞事件，这些过程的分子控制仍然存在。 人们对此知之甚少。在控制视杯形态发生方面发挥作用的一个引人注目的候选者是 细胞外基质(ECM)，一种复杂的富含糖蛋白的层，调节细胞的生存、运动、信号转导、 和极性。某些细胞外基质成分的突变可导致眼部病理，如缺损， 提示视杯形态发生过程中的特殊要求。了解外部控制 形态发生对有机体的方法和策略也有影响。我们之前的数据表明， 核心分子，层粘连蛋白，在不同的眼睛区域引起不同的细胞反应。我们还发现，单独的 眼睛区域暴露在不同的ECM微环境中，其中一些是通过组织组织组装的 相互作用：具体地说，在RPE周围建立基底膜需要神经脊。这些数据 提示独特的ECM微环境可能是区域性眼部形态发生事件的重要驱动因素。 斑马鱼为研究这一过程提供了一个理想的模型系统：光学透明和快速发展 提供了一个独特的机会，直接观察和分子解剖活体眼睛的形成。我们之前 开发了跟踪和可视化细胞运动的4维成像和计算技术 在整个视杯形态发生过程中，以及最近，视网膜细胞的自动定量分析方法 大小、形状和方向。这使我们在分析特定的形态发生缺陷方面处于独特的地位 当特定的矩阵分量被破坏时。在这项提案中，我们将剖析区域特定角色 眼形态发生过程中的ECM因子，包括硝酸甘油、Tenascin-C、MMP2和Verscan。 我们假设，动态的、特定于区域的ECM微环境可以诱导独特的发育和 来自不同眼祖细胞结构域的形态发生反应驱动视杯形态发生。组合 分子遗传学与创新的4维实时成像和计算方法，我们将对此进行测试 以下具体目标的假设：(1)确定ECM微环境如何控制视网膜 形态发生和组织；(2)确定ECM调节因子如何控制RPE的形态发生；以及 (3)确定组织对特定ECM微环境的贡献的功能要求。这个 拟议的实验将定义细胞外基质活动的时空动力学和不同的细胞功能 在眼睛形成的关键步骤中，由特定区域的ECM因子执行。

Project Summary Developmental defects in eye structure commonly account for visual impairment in newborns. Proper eye structure is initially established via the process of optic cup morphogenesis, during which a series of complex cell and tissue rearrangements transforms the optic vesicle into the optic cup, with neural retina and retinal pigmented epithelium (RPE) enwrapping the newly formed lens. With advances in imaging and computational analysis, work from our lab and others has begun to reveal the cellular events underlying optic cup morphogenesis, however, molecular control of these processes still remains poorly understood. A compelling candidate to play a role in controlling optic cup morphogenesis is the extracellular matrix (ECM), a complex, glycoprotein-rich layer that regulates cell survival, movement, signaling, and polarity. Mutations in certain ECM components can lead to ocular pathologies, such as coloboma, suggesting specific requirements during optic cup morphogenesis. Understanding extrinsic control of morphogenesis also has implications for organoid approaches and strategies. Our previous data indicate that a core molecule, laminin, elicits diverse cellular responses in different eye regions. We also found that separate eye domains are exposed to distinct ECM microenvironments, some of which are assembled via tissue-tissue interactions: specifically, neural crest is required to build basement membrane around the RPE. These data suggest that unique ECM microenvironments may be a crucial driver of regional eye morphogenetic events. Zebrafish provide an ideal model system to study this process: optical transparency and rapid development offer a unique opportunity to directly observe and molecularly dissect eye formation in vivo. We previously developed 4-dimensional imaging and computational techniques to track and visualize cell movements throughout optic cup morphogenesis, and recently, methods for automated quantitative analysis of retinal cell size, shape and orientation. This puts us in a unique position to analyze specific morphogenetic defects arising when particular matrix components are disrupted. In this proposal, we will dissect the region-specific roles of ECM factors, including nidogens, tenascin-C, mmp2, and versican, during eye morphogenesis. We hypothesize that dynamic, region-specific ECM microenvironments elicit unique developmental and morphogenetic responses from distinct eye progenitor domains to drive optic cup morphogenesis. 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 ECM microenvironment controls retina morphogenesis and organization; (2) determine how ECM modulatory factors control RPE morphogenesis; and (3) determine functional requirements for tissue contributions to specific ECM microenvironments. The experiments proposed will define the spatiotemporal dynamics of ECM activity and distinct cellular functions executed by region-specific ECM factors during crucial steps of eye formation.