The role of tumor suppressor Rb in horizontal neuron development

描述（由申请人提供）：视网膜发育过程中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形成过程中受到干扰时，可导致小眼症、视网膜变性或视网膜母细胞瘤。Rb家族蛋白（Rb， p107和p130）是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是视杆光感受器发育所必需的。此外，Rb家族蛋白似乎在分化的水平细胞中是必需的，以防止它们重新进入细胞周期，我们的初步数据表明Rb可能是在分化后期发生的水平细胞过程重组所必需的。具体来说，在rb缺乏的视网膜中，水平细胞将其过程扩展到外核层（ONL），而在正常成熟的视网膜中，这些过程仅限于外丛状层（OPL）。在本研究计划中，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是否在水平细胞成熟中是细胞自主需要的，或者Rb缺陷视网膜中的突触发生缺陷是否由缺乏视杆细胞继发触发。此外，我们将在没有视杆细胞的情况下观察水平细胞的成熟，以确定在rb缺乏视网膜中观察到的缺陷是由于水平细胞在发育过程中未能形成适当的横向排列的突起，还是它们反映了沿外丛状层（OPL）横向延伸后突起的重组。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拨款R01EY014867-06的延伸。项目期限为2009年7月1日至2012年6月30日。我们相信这些实验的发现将为本项目的第三阶段研究（R01EY014867-06）提供必要的数据，并将直接有助于更好地理解Rb肿瘤抑制因子在视网膜形成中的作用。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St Jude儿童研究医院发育神经生物学之间的科学合作。这里提出的研究对于父母资助（R01EY014867-06）是必不可少的，但超出了原始资助的范围。LMIC合作者（Dr. Martins）在PI （Dr. Dyer）的指导下做了4年的博士后，最近被任命为IBCCF/UFRJ的副教授。在巴西，马丁斯博士将可以使用Rafael Linden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马丁斯博士和戴尔博士之间的科学合作的继续。我们认为，尖端技术方面的高级培训以及获得创新观点和概念的机会将允许在巴西发展高价值的科学项目，并将有助于缩小与大多数美国研究中心相比，在这个国家进行的研究的差距。我们的目标是在获得的培训和经验的基础上开发这个创新的研究项目，这将导致父母资助目标的重大进展，并直接有助于更好地理解Rb肿瘤抑制因子在视网膜形成中的作用。在本研究中，目的3集中于阐明Rb家族（Rb、p107和p130）在水平细胞成熟中的作用。在亲本研究中没有解决的一个重要问题是，Rb家族在水平神经元发育的后期阶段（如突触发生）是否需要细胞自主。由于传统的Rb敲除小鼠在子宫内死亡，我们通过将RbLox/-小鼠14与Chx10-Cre小鼠交配来条件地灭活Rb。本研究使用的转基因小鼠模型（Chx10-Cre;RbLox/-）是唯一一种杆状光感受器未形成、未采用另一种细胞命运或死亡的小鼠模型。Chx10-Cre转基因对RbLox等位基因的重组模式也是独一无二的。嵌合视网膜，具有Rb缺失视网膜的顶基条纹，其中Rb已失活，两侧为野生型视网膜条纹。这种Rb缺陷和野生型视网膜细胞的马赛克分布有助于区分Rb在视网膜形成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结合经典方法视网膜突触发生，包括电子显微镜，和复杂的遗传镶嵌分析。我们还计划对rb缺乏视网膜进行长期的双光子实时成像，因为这种尖端的方法将使我们第一次能够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的核心和视力所必需的基本过程的理解。它们也可能为突触发生的机制提供有价值的见解，这可能最终有助于利用基于细胞的测定来替代视网膜变性所失去的光感受器。

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.