The role of tumor suppressor Rb in horizontal neuron development

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DESCRIPTION (provided by applicant): The decision to exit the cell cycle during retinal development must be precisely regulated to ensure that each cell type is generated in the correct proportion. In addition, when cell cycle regulation becomes perturbed during retinogenesis it can lead to microphthalmia, retinal degeneration or retinoblastoma. The Rb family of proteins (Rb, p107 and p130) lie at the heart of the cell cycle machinery that regulates retinal progenitor cell proliferation. Interestingly, we found that, distinct from its role in regulating retinal progenitor cell proliferation, Rb is required for rod photoreceptor development. In addition, Rb family proteins appears to be required in differentiated horizontal cells to prevent them from re-entering the cell cycle and our preliminary data suggest that Rb may be required for the reorganization of horizontal cell processes that occurs during the later stages of differentiation. Specifically, in Rb-deficient retina, horizontal cells extend their processes into the outer nuclear layer (ONL), while in the normal mature retina these processes are restricted to the outer plexiform layer (OPL). In this research proposal, our first aim is to define the role of Rb in horizontal neuron development, by determining whether Rb is required cell autonomously for horizontal cell maturation, or if defects in synaptogenesis in Rb-deficient retina are triggered secondarily by the absence of rods. In addition, we will visualize the maturation of horizontal cells in the absence of rods to determine whether the defects observed in Rb-deficient retinae are caused by a failure of horizontal cells to form appropriate laterally-arranged processes during development, or if they reflect reorganization of the processes after they extend laterally along the outer plexiform layer (OPL). This research will be done primarily in Brazil, at the Biophysics Institute of the Federal University of Rio de Janeiro (IBCCF/UFRJ) in collaboration with Dr. Rodrigo Martins, as an extension of the NIG Grant R01EY014867-06. The project period will be from 07/01/2009 until 06/30/2012. We believe the findings from the proposed experiments will provide essential data for Aim 3 of the parent grant (R01EY014867-06) and will directly contribute to a better understanding of the roles of Rb tumor suppressor in retinogenesis. PUBLIC HEALTH RELEVANCE: The role of the tumor suppressor Rb in horizontal neuron development This proposal aims to establish a scientific collaboration between the Biophysics Institute of the Federal University of Rio de Janeiro, Brazil (IBCCF/UFRJ) and Developmental Neurobiology of St Jude Children's Research Hospital, USA. The research proposed here is essential for the parent grant (R01EY014867-06), but was beyond the scope of the original grant. The LMIC collaborator (Dr. Martins) has worked as a postdoctoral research fellow under the supervision of the PI (Dr. Dyer) for the last 4 years and was recently appointed an Associate Professor of the IBCCF/UFRJ. In Brazil, Dr. Martins will have access to all of the resources of the Laboratory of Neurogenesis led by Dr. Rafael Linden and to all the facilities of the IBCCF/UFRJ. The approval of this project will be instrumental to the continuation of the scientific collaboration between Dr. Martins and Dr. Dyer. We believe that advanced training in sophisticated technologies and access to innovative perspectives and concepts will allow the development of high merit scientific projects in Brazil and will help decreasing the disparity in the research performed in this country as compared to most US-based research centers. Our goal is to build on the acquired training and experience to develop this innovative research project that will lead to a significant advancement of the aims of the parent grant and directly contribute to a better understanding of the roles of Rb tumor suppressor in retinogenesis. In the parent grant aim 3 is focused on elucidating the role of the entire Rb family (Rb, p107 and p130) in horizontal cell maturation. One essential question that is not addressed in the parent grant is whether the Rb family is required cell autonomously for the later stages of horizontal neuron development (e.g. synaptogenesis). Because traditional Rb-knockout mice die in utero, we conditionally inactivated Rb by mating RbLox/- mice14 with Chx10-Cre mice. The transgenic mouse model to be used in this study (Chx10-Cre;RbLox/-) is the only one in which the rod photoreceptors fail to form and do not adopt another cell fate or die. The pattern of recombination of RbLox alleles by the Chx10-Cre transgene is also unique. This retina is chimeric, having apical-basal stripes of Rb-deficient retina, in which Rb has been inactivated, flanked by wild-type stripes of retina. This mosaic distribution of Rb-deficient and wildtype retinal cells facilitates distinction between Rb's cell autonomous roles and non-cell autonomous roles in retinogenesis. We propose a unique combination of classic approaches to retinal synaptogenesis, including electron microscopy, and sophisticated genetic mosaic analysis. We also plan to perform long-term 2-photon live imaging of Rb-deficient retinae, because this cutting-edge approach will allow us for the first time to monitor the continuum of horizontal cell maturation in the absence of rods. These experiments will advance our understanding of fundamental process that lie at the heart of retinal development and are required for vision. They may also provide valuable insight into the mechanisms of synaptogenesis that may ultimately benefit efforts to replace photoreceptors lost to retinal degeneration using cell-based assays.