Stepwise Coordination of Eye Morphogenesis by Extracellular Matrix

 描述（由申请人提供）：眼睛结构的发育缺陷通常是新生儿视力障碍的原因。在视杯形态发生的过程中，视泡通过一系列复杂的细胞和组织重排转化为视杯，神经视网膜和视网膜色素上皮包围着新形成的透镜，从而初步建立了适当的眼睛结构。 最近的进展，在现场成像已经开始揭示视杯形态发生的细胞过程，但这些关键事件的分子控制仍然在很大程度上是未知的。在控制视杯形态发生中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，可以调节细胞存活、运动、极性和组织张力。ECM成分，包括层粘连蛋白和IV型胶原蛋白，围绕整个视杯形态发生在所有脊椎动物的眼睛结构检查日期。某些ECM组分中的突变可导致人类和模式生物体中的特定眼部病理，例如缺损（脉络膜裂隙发育失败）或透镜缺陷。这表明在视杯形态发生的特定事件中对ECM成分有不同的要求，但这还没有被系统地研究。 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体内眼睛形成提供了独特的机会。此外，我们以前开发了4维成像和计算技术，以跟踪和可视化整个视杯形态发生的细胞运动和行为。这使我们处于一个独特的位置，以定量确定特定的基质成分被破坏时产生的特定形态发生缺陷。在这个建议中，我们将确定层粘连蛋白，IV型胶原，和层粘连蛋白胶原交联分子巢蛋白在视杯形态发生的作用。 我假设，顺序步骤的视杯形态港湾不同的要求，基质成分和高阶组装，这些差异的要求下，在人类和模式生物中看到的特定的眼部病理。结合分子遗传学和创新的四维实时成像和计算方法，我们将在以下具体目标中验证这一假设：（1）确定基质成分和超分子组装在脉络膜裂形成中的作用;（2）确定基质成分如何控制视杯内陷，利用一种新的突变体，其中视网膜-晶状体脱粘，（和可能的胶原信号传导）受损;（3）确定基质组分在建立适当的透镜形状和外胚层分离中的作用。本文提出的实验将确定细胞外基质粘附的细胞和分子动力学，这些细胞和分子动力学是光学系统中关键步骤的基础。 杯状体的形态发生，以及基质在每个步骤中执行的特定细胞功能。

 DESCRIPTION (provided by applicant): Developmental defects in eye structure commonly account for visual impairment in newborns. Proper eye structure is initially established during the process of optic cup morphogenesis, during which the optic vesicle transforms into the optic cup via a series of complex cell and tissue rearrangements, with neural retina and retinal pigmented epithelium surrounding the newly formed lens. Recent advances in live imaging have begun to reveal the cellular processes underlying optic cup morphogenesis, yet molecular control of these critical events still remains largely unknown. A compelling candidate to play a role in controlling optic cup morphogenesis is the extracellular matrix (ECM), a complex, glycoprotein-rich layer that can regulate cell survival, movement, polarity, and tissue tension. ECM components, including laminin and type IV collagens, surround eye structures throughout optic cup morphogenesis in all vertebrates examined to date. Mutations in certain ECM components can lead to specific ocular pathologies in humans and model organisms, such as coloboma (failure of choroid fissure development) or lens defects. This suggests distinct requirements for ECM components during specific events in optic cup morphogenesis, yet this has not been examined in a systematic way. Zebrafish presents an ideal model system to study this process: optical transparency and rapid development offer a unique opportunity to directly watch eye formation in vivo. Further, we previously developed 4-dimensional imaging and computational techniques to track and visualize cell movements and behaviors throughout optic cup morphogenesis. This puts us in a unique position to quantitatively determine specific morphogenetic defects arising when particular matrix components are disrupted. In this proposal, we will determine the roles of laminin, type IV collagens, and the laminin-collagen cross-linking molecule nidogen during optic cup morphogenesis. I hypothesize that sequential steps of optic cup morphogenesis harbor distinct requirements for matrix components and higher-order assembly, and that these differential requirements underlie specific ocular pathologies seen in humans and model organisms. Combining molecular genetics with innovative 4-dimensional live imaging and computational methods, we will test this hypothesis in the following specific aims: (1) determine the role of matrix components and supramolecular assembly in choroid fissure formation; (2) determine how matrix components control optic cup invagination, taking advantage of a novel mutant, in which retina-lens de-adhesion (and possibly collagen signaling) are impaired; (3) determine the role of matrix components in establishing proper lens shape and ectoderm separation. The experiments proposed here will define the cellular and molecular dynamics of extracellular matrix adhesion underlying critical steps in optic cup morphogenesis, and the specific cellular functions executed by matrix during each step.