Mechanisms of Lens Morphogenesis and Regeneration

描述（由申请人提供）：为了在损伤后再生功能性组织或替换被病理性疾病的后果破坏的组织，干细胞或祖细胞的分化必须伴随有效的策略以促进组织形态发生。该提案研究了指导细胞和组织形态发生的机制，最终目标是将结果应用于指导组织修复和再生中正常发育过程的重演。这些研究的模型是胚胎透镜，理想的研究形态发生的分子机制，因为它的细胞结构定义了它的功能。透镜的形态发生必须精确地执行，并且其结构必须保持，以便其执行其将图像聚焦在视网膜上的功能。该提议旨在发现负责透镜发育的两个主要形态发生事件的机制：1）建立高度伸长的透镜纤维细胞表型的伸长/会聚延伸机制和2）相邻的透镜纤维细胞被组织成精确的六边形堆积细胞阵列的机制。这些研究集中在N-钙粘蛋白连接及其与不同细胞骨架元素的动态相互作用，作为发育过程中细胞和组织形态发生的关键调节剂。对于这些研究，我们开发了一些重要的培养模型，包括：1）离体模拟白内障手术培养物，其中可以检查透镜上皮细胞在其天然微环境中对临床相关创伤的反应及其再生晶状体样组织的能力，2）透镜上皮细胞的原代培养物，其在培养物中分化以形成微型透镜结构，和3）上皮外植体，其中纤维细胞伸长并组织成高度有序的组织样结构;此外还有N-钙粘蛋白纤维细胞特异性敲除。这些模型提供了强有力的工具，检查N-钙粘蛋白作为一个焦点，其中肌动蛋白微丝，微管和中间丝协调其功能，直接透镜形态发生与以下问题：什么是驱动纤维细胞伸长的机制？相邻的透镜纤维细胞如何相互作用形成功能结构？是什么调节纤维细胞的组织 变成六边形阵列是什么稳定了透镜这种独特的细胞结构？透镜上皮细胞如何重塑其N-钙粘蛋白/细胞骨架相互作用以促进伤口愈合和组织再生？这些拟议的研究的结果，预计将确定负责建立和维持细胞和组织的细胞结构，知识，这将提供指导有效的组织再生在体内的实质性目标的潜在机制。 公共卫生相关性：本提案中的研究探讨了N-钙粘蛋白连接和细胞骨架之间的动态相互作用在指导细胞和组织形态发生中的重要性，重点关注调节发育中细胞形态发生运动的机制以及这些细胞如何排列成组织功能所需的高度有序的结构。这种方法解决了再生医学中最大的挑战之一，即如何在损伤后或对病理性疾病引起的组织损伤做出反应，可以诱导修复细胞进行形态发生分化并与其近邻相互作用以形成新组织。我们的研究有望为促进组织再生提供功能靶点。

DESCRIPTION (provided by applicant): In order to regenerate a functional tissue following an injury or to replace a tissue destroyed by the consequences of a pathological disease, the differentiation of stem or progenitor cells must be accompanied by effective strategies to promote tissue morphogenesis. This proposal investigates the mechanisms that guide cell and tissue morphogenesis with the ultimate goal of applying the results to instruct the recapitulation of normal developmental processes in tissue repair and regeneration. The model for these studies is the embryonic lens, ideal for investigating the molecular mechanisms of morphogenesis because its cytoarchitecture defines its function. Morphogenesis of the lens must be precisely executed and its structure maintained in order for it to perform its function of focusing images on the retina. This proposal aims to discover the mechanisms responsible for the two principal morphogenetic events of lens development: 1) the elongation/convergent extension mechanism that establishes the highly elongated lens fiber cell phenotype and 2) the mechanism by which neighboring lens fiber cells are organized into an exact hexagonally packed cellular array. The studies are focused on N-cadherin junctions and their dynamic interactions with different cytoskeletal elements as key modulators of cell and tissue morphogenesis during development. For these studies we developed a number of important culture models including: 1) ex vivo mock cataract surgery cultures where it is possible to examine the response of the lens epithelium to a clinically relevant wounding in its native microenvironment and its ability to regenerate lens-like tissue, 2) primary cultures of lens epithelial cells that differentiate in culture to form mini-lens structures, and 3) epithelial explnts where fiber cells both elongate and organize into a highly ordered tissue-like structure; in addition to N-cadherin fiber cell-specific knockouts. These models provide powerful tools with which to examine N-cadherin as a focal point where actin microfilaments, microtubules and intermediate filaments coordinate their function to direct lens morphogenesis with the following questions: What are the mechanisms that drive fiber cell elongation? How do neighboring lens fiber cells interact to form a functional structure? What regulates the organization of fiber cells into a hexagonal array? What stabilizes this unique cytoarchitecture of the lens? How do lens epithelial cells remodel their N-cadherin/cytoskeletal interactions for wound healing and tissue regeneration? The results of these proposed studies are expected to identify the underlying mechanisms responsible for establishing and maintaining cell and tissue cytoarchitecture, knowledge of which would provide substantive targets for guiding effective tissue regeneration in vivo. PUBLIC HEALTH RELEVANCE: The studies in this proposal examine the importance of dynamic interactions between N-cadherin junctions and the cytoskeleton in directing both cell and tissue morphogenesis, focusing on mechanisms that regulate the morphogenetic movement of cells in development and how these cells become arranged into highly ordered structures required for tissue function. This approach addresses one of the greatest challenges in Regenerative Medicine, how following an injury or in response to tissue damage resulting from a pathological disease, reparative cells can be induced to undergo morphogenetic differentiation and interact with their near neighbors to form a new tissue. Our studies are expected to provide functional targets for promoting tissue regeneration.