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 2 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 Mller 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 2-containing laminins are pro-angiogenic and 3-containing laminins are anti-angiogenic.
Our second aim i s 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 Mller 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 2- 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.
The extracellular matrix (ECM) surrounds cells providing them with environmental signals during development and stabile substrates for attachment and migration throughout life. Many human diseases are caused by disruptions of the ECM including metastatic cancer, proliferative neo-vascular disease, acquired auto-immune diseases, glaucoma and congenital birth defects. Many of the latter affect eye and brain and lead to impaired vision or blindness with mental retardation. This project investigates the role of ECM in retinal development and the results of these studies will lead to new diagnostics for ocular disease, both genetic and acquired, and will lead to the development of new therapeutics.
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