Role of Extracellular Matrix in Retinal Development and Disease

 描述(由申请人提供)：我们项目的直接目标是了解细胞外基质在视网膜发育和疾病中的作用。在之前的资助时期，我们确定了层粘连蛋白的独特亚型，包含在眼睛和脑中表达的2或LN 3链。在人类中，这两个层粘连蛋白基因的突变会导致自闭症、眼睛发育不全和肾功能障碍。在小鼠中，去除这些基因会导致皮质和眼睛发育不全；后者包括：1)视网膜神经节细胞发育；2)星形胶质细胞迁移和随后的血管发育；3)Müler细胞的亚细胞组织；4)光感受器-双极突触。我们的基本假设是，层粘连蛋白对建立视网膜的三维结构至关重要。具体地说，我们假设层粘连蛋白提供了对血管生成和神经生成至关重要的环境线索。我们的第一个目标是探索层粘连蛋白信号在血管生成发育模板形成中的作用。工作假说是，RGC驱动星形胶质细胞迁移；然后，星形胶质细胞和小胶质细胞之间的相互作用调节内皮发育。我们将使用反向遗传方法，单独或一起删除Lamb2或Lamc3基因，以扰乱这些细胞之间的信号传递。第一组实验将集中在Lamb2-/-和Lamc3-/-动物的空间图案上。我们的第二组实验将解决层粘连蛋白介导的小胶质细胞招募和激活的作用。第三组实验将研究血管生成过程中内皮细胞中层粘连蛋白信号的效应器。我们目前的数据表明，含？2的层粘连蛋白是促血管生成的，而含？3的层粘连蛋白是抗血管生成的。我们的第二个目标是研究层粘连蛋白在神经发生中的作用。我们将检验这一假设，即层粘连蛋白调节放射状组织的祖细胞的顶端-基底极性。我们发表的数据表明，在Lamb2-/-视网膜中，Müler细胞的区域化被破坏。此外，我们的初步数据表明，在Lamb2-/-和Lamc3-/-小鼠中，细胞周期都是失调的。我们的第一组实验将集中在Lamb2-/-和Lamc3-/-视网膜中对称与不对称分裂的调节。接下来，我们将转向对这些小鼠体内重要细胞周期调节因子遗传模式的研究。最后，我们将直接测量Lamb2-/-和Lamc3-/-视网膜的细胞周期调节。我们的初步数据表明，含有2和3的层粘连蛋白是维持增殖状态所必需的。我们的工作与了解视网膜新生血管疾病、胶质增生和增殖性玻璃体视网膜病变的病理生物学有关，因为星形胶质细胞和小胶质细胞在视网膜血管形成和重塑中发挥关键作用。我们在视网膜前体细胞方面的工作将提高我们对视网膜发育的基本理解和对中枢神经系统前体细胞周期调控的理解，并将影响设计用于体外培养视网膜的3D培养系统的开发。

 DESCRIPTION (provided by applicant): The immediate goal of our project is to understand the role of the extracellular matrix in retinal development and disease. In prior funding periods, we identified unique isoforms of laminins, containing either the ß2 or 3 chains that are expressed in the eye and brain. Mutations in these two laminin genes, in humans, result in autism, ocular dysgenesis, and kidney dysfunction. Ablation of these genes, in mouse, produce cortical and ocular dysgenesis; the latter includes disruptions of: 1) retinal ganglion cell development; 2) astrocyte migration and subsequent vascular development; 3) the sub-cellular organization of the Müller cell; 4) the photoreceptor- bipolar synapse. Our fundamental hypothesis is that laminins are critical for establishing the three dimensional structure of the retina. Specifically, we hypothesize that laminins provide environmental cues that are essential for angiogenesis and neurogenesis. Our first aim explores the contributions of laminin signaling in formation of the template for angiogenic development. The working hypothesis is that RGCs drive astrocyte migration; then, interactions between astrocytes and microglia regulate endothelial development. We will use a reverse genetic approach, deleting Lamb2 or Lamc3 genes alone, or together, to disrupt the signaling among these cells. The first set of experiments will focus on the spatial patterning in Lamb2-/- and Lamc3-/- animals. Our second set of experiments will address the role of laminin-mediated recruitment and activation of microglia. The third set of experiments will examine the effectors of laminin signaling in endothelial cells during angiogenesis. Our current data suggest that ß2-containing laminins are pro-angiogenic and ¿3-containing laminins are anti-angiogenic. Our second aim is focused on the role of laminins in neurogenesis. We will examine the hypothesis that laminin regulates apical-basal polarity of the radially organized progenitor. Our published data demonstrate that Müller cell compartmentalization is disrupted in the Lamb2-/- retina. Moreover, our preliminary data demonstrate that the cell cycle is dysregulated in both Lamb2-/- and Lamc3-/- mice. Our first set of experiments will focus on the regulation of symmetric versus asymmetric division in the Lamb2-/- and Lamc3-/- retina. Next, we will turn to a study of the pattern of inheritance of important cell cycle regulators in these same mice. Last, we will measure directly the cell cycle regulation in Lamb2-/- and Lamc3-/- retina. Our preliminary data suggest that ß2- and 3-containing laminins are necessary to preserve the proliferative state. Our work is relevant to an understanding of the pathobiology of retinal neovascular disease, gliosis and proliferative vitreoretinopathy because astrocytes and microglia play critical roles in retinal vascularization and remodeling. Our work on retinal progenitor cells will improve our fundamental understanding of retinal development and our understanding of the regulation of the cell cycle in CNS progenitors and will influence the development of 3D culture systems designed to grow retina ex vivo.