Regulation of the cell cycle and differentiation in the vertebrate retina by TGFÃÂò signaling

项目摘要 斑马鱼视网膜具有与哺乳动物相同的神经元成分和Müller胶质细胞（MG） 视网膜，但在急性视网膜损伤后，斑马鱼MG被激活，并作为组织特异性干细胞， 强大地再生失去的神经元。体内哺乳动物MG缺乏这种完全再生的能力。在 斑马鱼视网膜携带TGFβ信号转录抑制因子（tgif 1）基因突变， 和Six 3b），在急性光损伤后， 破坏感光细胞，这表明这些突变鱼可以提供对有限的 哺乳动物的神经再生本R15提案中描述的目标将建立在鱼的基础上， 研究探索增加的TGFβ信号传导（在tgif 1-/-; six 3b-/-鱼中）和减少的TGFβ 信号传导（使用小分子抑制剂）对增殖、细胞周期进程和细胞分化的影响。 这项研究将使用斑马鱼作为模型系统进行，由于几个好处，包括：1。 可用于发育研究的大量胚胎，以及2.斑马鱼有一个 哺乳动物所缺乏的神经组织再生能力。发展研究将 受精后1 - 5天，视网膜从神经上皮层发育为 功能性视网膜;增殖和细胞周期动力学将通过检查 PCNA和细胞周期蛋白依赖性激酶抑制剂在细胞退出细胞周期时特异性表达 免疫印迹法和免疫荧光法。虽然先前的观察表明，增加TGFβ 信号传导导致再生减少，尚不清楚这些研究中的Müller胶质细胞是否重新进入了 细胞周期以较低的速率和/或是否存在产生不同类型神经元的能力降低。 因此，将描述急性光损伤后TGFβ信号通路成员的表达 使用免疫荧光，鱼将暴露于胸苷类似物，以永久标记细胞 新生成的细胞。Müller胶质细胞、光感受器和中间神经元的数量 将通过共定位细胞类型特异性抗原和类似物标记来定量 使用免疫荧光。最后，一种新的转基因鱼将被开发出来， 使用荧光区分胶质细胞的细胞周期阶段，以解决TGFβ 在视网膜再生期间，信号传导阻止细胞周期再进入或停止细胞周期。这种新颖的鱼线 将是斑马鱼研究团体研究神经胶质细胞生物学问题的宝贵工具 整个中枢神经系统。因为已知TGFβ信号传导限制增殖， 促进哺乳动物中枢神经系统的瘢痕形成，这些鱼类实验将直接告知 哺乳动物系统的生物医学研究，并为人类提供潜在的治疗靶点 视网膜，以尽量减少疤痕和促进愈合或再生后，急性损伤。

PROJECT SUMMARY The zebrafish retina has the same neuronal components and Müller glial cells (MG) as the mammalian retina, yet after an acute retinal injury, zebrafish MG are activated and act as tissue-specific stem cells to robustly regenerate lost neurons. Mammalian MG in vivo lack this capacity for complete regeneration. In the retinas of zebrafish carrying genetic mutations in transcriptional repressors of TGFβ signaling (tgif1 and six3b), there is significantly reduced proliferation and regeneration following an acute light lesion that destroys photoreceptor cells, suggesting that these mutant fish could provide insight into the limited neural regeneration in mammals. The Objectives described in this R15 proposal will build on the fish studies to explore the effects of increased TGFβ signaling (in tgif1-/-;six3b-/- fish) and decreased TGFβ signaling (using small molecule inhibitors) on proliferation, cell cycle progression, and cell differentiation. This research will be performed using zebrafish as a model system due to several benefits including: 1. the large number of embryos that can be used in developmental studies, and 2. that zebrafish have a remarkable capacity for regenerating neural tissue that mammals are lacking. Developmental studies will be from 1 through 5 days post fertilization when the retina develops from a neuroepithelial layer to a functional retina; proliferation and cell cycle dynamics will be measured by examining the expression of PCNA and cyclin-dependent kinase inhibitors specifically expressed when cells are exiting the cell cycle using western blot and immunofluorescence. While previous observations showed that increased TGFβ signaling led to reduced regeneration, it is unknown whether Müller glia in those studies reentered the cell cycle at a lower rate and/or if there is a reduced capacity to generate different types of neurons. Therefore, after an acute light lesion, expression of TGFβ signaling pathway members will be described using immunofluorescence, and fish will be exposed to a thymidine analog to permanently label cells newly regenerated generated cells. The number Müller glia, photoreceptors, and interneurons generated during regeneration will be quantified by colocalizing cell-type specific antigens and the analog label using immunofluorescence. Finally, a novel transgenic fish will be developed that will allow researchers to distinguish between stages of the cell cycle in glial cells using fluorescence to address whether TGFβ signaling prevents cell cycle re-entry or halts the cell cycle during retinal regeneration. This novel fish line will be a valuable tool for the zebrafish research community to examine questions of glial cell biology throughout the central nervous system. Because TGFβ signaling is known to limit proliferation and promote scarring in the mammalian central nervous system, these experiments in fish will directly inform biomedical research in mammalian systems and provide potential therapeutic targets for the human retina to minimize scarring and promote healing or regeneration after an acute damage.