The role of tumor suppressor Rb in horizontal neuron development

描述（由申请人提供）：在视网膜发育期间退出细胞周期的决定必须精确调节，以确保每种细胞类型以正确的比例生成。此外，当细胞周期调节在视网膜发生期间受到干扰时，它可导致小眼球、视网膜变性或视网膜母细胞瘤。Rb蛋白家族（Rb，p107和p130）位于调节视网膜祖细胞增殖的细胞周期机制的核心。有趣的是，我们发现，与调节视网膜祖细胞增殖的作用不同，Rb是视杆细胞发育所需的。此外，Rb家族蛋白似乎需要在分化的水平细胞，以防止他们重新进入细胞周期，我们的初步数据表明，Rb可能需要的水平细胞过程中发生的分化后期的重组。具体地，在Rb缺乏的视网膜中，水平细胞将它们的突起延伸到外核层（ONL）中，而在正常成熟的视网膜中，这些突起仅限于外丛状层（OPL）。在这项研究建议中，我们的第一个目标是确定Rb在水平神经元发育中的作用，通过确定Rb是否是水平细胞成熟所需的细胞自主，或者如果Rb缺陷视网膜中的突触发生缺陷是由杆的缺乏继发性触发的。此外，我们将可视化水平细胞的成熟，在没有杆，以确定是否在Rb缺乏视网膜中观察到的缺陷是由水平细胞的失败，形成适当的横向排列的过程中的发展，或者如果他们反映重组后，他们横向沿着外丛状层（OPL）的过程。这项研究将主要在巴西里约热内卢联邦大学生物物理研究所（IBCCF/UFRJ）与Rodrigo Martins博士合作进行，作为NIG赠款R 01 EY 014867 -06的延伸。项目期限为2009年1月7日至2012年6月30日。我们相信，从拟议的实验结果将提供必要的数据，为目标3的母基金（R 01 EY 014867 -06），并将直接有助于更好地了解Rb肿瘤抑制剂在视网膜发生中的作用。公共卫生相关性：肿瘤抑制因子Rb在水平神经元发育中的作用该提案旨在建立巴西里约热内卢联邦大学生物物理研究所（IBCCF/UFRJ）和美国圣犹大儿童研究医院发育神经生物学之间的科学合作。这里提出的研究是必不可少的家长补助金（R 01 EY 014867 -06），但超出了原来的补助金的范围。LMIC合作者（Martins博士）在PI（Dyer博士）的监督下担任博士后研究员已有4年，最近被任命为IBCCF/UFRJ的副教授。在巴西，Martins博士将可以使用Rafael林登博士领导的神经发生实验室的所有资源以及IBCCF/UFRJ的所有设施。该项目的批准将有助于继续马丁斯博士和戴尔博士之间的科学合作。我们相信，先进的技术培训和获得创新的观点和概念将使高价值的科学项目在巴西的发展，并将有助于减少在这个国家进行的研究相比，大多数美国的研究中心的差距。我们的目标是建立在获得的培训和经验，以开发这个创新的研究项目，这将导致一个显着的进步的父母补助金的目标，并直接有助于更好地了解Rb肿瘤抑制基因在视网膜发生中的作用。在母基金中，目标3集中于阐明整个Rb家族（Rb，p107和p130）在水平细胞成熟中的作用。一个基本的问题，是没有解决的父母补助金是Rb家族是否需要细胞自主水平神经元发育的后期阶段（如突触）。由于传统的Rb基因敲除小鼠在子宫内死亡，我们通过将RbLox/-小鼠14与Chx 10-Cre小鼠交配来有条件地灭活Rb。本研究中使用的转基因小鼠模型（Chx 10-Cre;RbLox/-）是唯一一种杆细胞感光器无法形成且不采用另一种细胞命运或死亡的模型。通过Chx 10-Cre转基因重组RbLox等位基因的模式也是独特的。这种视网膜是嵌合体，具有Rb缺陷视网膜的顶部-基底条纹，其中Rb已经失活，两侧是野生型视网膜条纹。Rb缺陷型和野生型视网膜细胞的这种镶嵌分布有助于区分Rb在视网膜发生中的细胞自主作用和非细胞自主作用。我们提出了一个独特的组合的经典方法视网膜突触，包括电子显微镜，和复杂的遗传镶嵌分析。我们还计划对Rb缺乏的视网膜进行长期的双光子活体成像，因为这种尖端的方法将使我们能够首次在没有视杆细胞的情况下监测水平细胞成熟的连续性。这些实验将促进我们对视网膜发育核心和视觉所需的基本过程的理解。它们还可以提供对突触发生机制的有价值的见解，这可能最终有利于使用基于细胞的测定来替代因视网膜变性而丢失的光感受器的努力。

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.