Neuronal differentiation in the retina

背景 为了更深入地了解视网膜发生，我们正在探索发育中视网膜神经元分化的基本调控机制(即表观遗传修饰)。此外，我们着重于视网膜光感受器的发育过程(细胞极性的建立和光感受器-双极细胞突触的形成)和神经节细胞轴突迁移(视盘形成)。这些都是建立成熟的视网膜组织结构和功能组织的关键事件。我们选择了最近文献中显示的候选分子/途径，参与神经系统其他区域的发育过程和/或其他物种的视网膜发育。 结果 1.视网膜神经发生的表观遗传学调控 染色质重塑作为调节基因表达和调节视网膜细胞分化的一种普遍的细胞内分子机制，近年来引起了研究者的广泛关注。DNA甲基化被认为积极参与了视网膜前体细胞功能的建立(例如，低甲基化可以调节光感受器发生过程中IRBP基因的激活)。然而，人们对甲基化对视网膜细胞命运的影响知之甚少。已知的三种DNA甲基化转移酶家族，Dnmt1、DNMT3a和Dnmt3b，部分合作建立和维持基因组DNA甲基化模式。为了研究DNA甲基化在视网膜分化中的作用，我们建立了视网膜特异的条件性KO小鼠，在这些小鼠中，在视网膜发育的后续阶段，通过Six3-Cre、Rx-Cre或Cx-Cre转基因来去除Dnmt1、DNMT3a或Dnmt3b的loxP位点。Dnmt1 2lox/Rx-Cre小鼠视网膜病理已完成。用Six3-Cre切除Dnmt12lox后视网膜发育的分析正在进行中，Dnmt12lox/CRx-Cre小鼠最近才开始进行鉴定。 2.光感受器细胞极性的建立 刺1和刺2是建立果蝇平面细胞极性(PCP)和光感受器极性所必需的组织极性基因。我们推测，与果蝇类似，这两个基因在哺乳动物光感受器极性的启动和维持中起着至关重要的作用。我们通过原位杂交和免疫组织化学方法证实，刺1和刺2在发育中的小鼠视网膜中有表达。我们已经在出生后的视网膜中电穿孔了刺痛1和针对刺痛1的shRNAi，并正在研究获得和失去功能对光感受器极性的影响。条件性刺痛1和2双KO小鼠正在产生，它们的表型分析将补充我们的体内电穿孔实验。 3.视盘形成 基于蛋白质结构的相似性和视盘区的表达模式，我们推测Frizzled5和8作为一个冗余的备份系统来协调Wnt-Frizzled5和8信号，它们的中断可能参与了缺陷性病变的发生。 我们用敲入法研究了Fz8在小鼠视网膜中的表达 贝塔半乳糖报告老鼠。对这些小鼠的详细视网膜电信号分析目前正在进行中。对Fz5和Fz8双KO小鼠的分析将深入了解这些基因功能的冗余。 4.光感受器与双极细胞之间的突触发生 我们在实验室建立了新的基因转移工具，以研究基因功能并直接成像啮齿动物视网膜中的神经发生作为模型系统，目的是了解人类视网膜电路的生成。利用体内电穿孔传递RNA介导的干扰(RNAi)结构来敲除选定的基因，我们正在研究先前被证明受NRL调控的候选基因的作用，并正在评估它们参与杆状光感受器细胞与突触后靶细胞的突触发生。 我们已经生成了报告荧光标记来识别视网膜神经元分化的特定阶段。目前正在筛选专门在球果中表达TdTomato的转基因小鼠的创建者。它们将用于流动分选实验和活体成像。他们将与以细胞特异性方式表达其他荧光蛋白的不同转基因小鼠品系进行繁殖。此外，还建立了NRL-Cre转基因小鼠系，以驱动Cre重组酶的光感受器特异性表达。这些小鼠将允许对光感受器中的基因表达进行有条件的靶向调控，从它们的诞生开始。 意义 我们对神经元分化的研究将进一步加深我们对视网膜发育的理解。它们还将补充我们将干细胞改造成光感受器的努力，并在功能上将它们整合到移植后变性视网膜的破坏结构中(见EY000473-01项目)。

Background In our efforts to gain a deeper understanding of retinogenesis we are exploring fundamental regulatory mechanisms (i.e. epigenetic modifications) underlying neuronal differentiation in the developing retina. Furthermore, we are focusing on developmental processes characteristic of retinal photoreceptors (establishment of cell polarity and photoreceptor-bipolar cell synaptogenesis) and of ganglion cell axonal migration (optic disc formation). These are key events in establishing the mature tissue architecture and functional organization of the retina. We have selected candidate molecules/pathways that have been shown in recent literature to be involved in developmental processes in other areas of the nervous system and/or in retinal development in other species. Results 1. Epigenetics regulation of neurogenesis in the retina Chromatin remodeling has attracted researchers attention in recent years as a general intracellular molecular mechanism modulating gene expression and regulating retina cell differentiation. DNA methylation has been suggested to be actively involved in establishing retinal progenitor cell competence (e.g., hypometylation could modulate IRBP gene activation during photoreceptor genesis). However, little is known about the influence of methylation on retinal cell fate. A family of three known DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, partially cooperate to establish and maintain genomic DNA methylation patterns. To investigate the role of DNA methylation in retinal differentiation retina-specific conditional KO mice have been generated in which Dnmt1, Dnmt3a, or Dnmt3b flanked by loxP sites are ablated at subsequent stages of retinal development by either Six3-Cre, Rx-Cre, or Crx-Cre transgenes. Retinal pathology of Dnmt1 2lox/Rx-Cre mice has been completed. Analysis of retinal development after ablation of Dnmt1 2lox by Six3-Cre is in progress and Dnmt1 2lox/Crx-Cre mice have just recently become available for characterization. 2. Establishment of photoreceptor cell polarity Prickle 1 and 2 are tissue polarity genes that are necessary for the establishment of planar cell polarity (PCP) as well as photoreceptor polarity in Drosophila. We hypothesize that, similar to Drosophila, the two genes play essential roles in initiation and maintenance of photoreceptor polarity in mammals. We confirmed by in situ hybridization and immunohistochemistry that Prickle 1 and 2 are expressed in the developing mouse retina. We have electroporated Prickle 1 and shRNAi against Prickle 1 in vivo in the postnatal retina and are investigating the effects of gain and loss-of-function on photoreceptor polarity. Conditional Prickle 1 and 2 double KO mice are being generated and analysis of their phenotype will complement our in vivo electroporation experiments. 3. Optic disc formation Based on protein structure similarity and on expression pattern in the optic disc region, we hypothesized that Frizzled (Fz)5 and 8 orchestrate Wnt-Frizzled signaling as a redundant back-up system and that their disruption might be involved in the insurgence of coloboma. We have characterized Fz8 expression in the mouse retina using a knock-in beta-gal reporter mouse. A detailed electroretinographic analysis of these mice is currently ongoing. Analysis of Fz5 and Fz8 double KO mice will give insights into the redundancy of these gene functions. 4. Synaptogenesis between photoreceptors and bipolar cells We have established novel gene transfer tools in the laboratory to study gene function and directly image neurogenesis in the rodent retina as a model system, with a goal to understand human retinal circuitry generation. Using in vivo electroporation to deliver RNA-mediated interference (RNAi) constructs to knockdown selected genes, we are investigating the roles of candidates previously shown to be regulated by NRL and are evaluating their involvement in synaptogenesis of rod photoreceptor cells with postsynaptic target cells. We have generated reporter fluorescent tags to identify specific stages of retinal neuronal differentiation. Founders of transgenic mice expressing TdTomato specifically in cones are currently being screened. They will be used for flow sorting experiments and for in vivo imaging. They will be breed with different mouse transgenic lines expressing other fluorochromes in a cell-specific manner. Furthermore, Nrl-Cre transgenic mouse lines have been established driving photoreceptor-specific expression of Cre recombinase. These mice will allow conditional targeted modulation of gene expression in photoreceptors, initiating with their genesis. Significance Our investigations on neuronal differentiation will further our understanding of retinal development. They will also complement our efforts to engineer stem cells into photoreceptors and functionally integrate them into the disrupted architecture of degenerating retinas after transplantation (see project EY000473-01).