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

TGFαβ信号对脊椎动物视网膜细胞周期和分化的调节

• 批准号: 9171417
• 负责人: Jenny Rae Lenkowski
• 金额: $ 38.19万
• 依托单位: GOUCHER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9171417
• 关键词: Acute; Address; Adult; Antigens; Area; Baltimore; Basic Science; Biological Models; Biology; Biomedical Research; Blindness; Cell Communication; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Cycle Stage; Cell Differentiation process; Cell Proliferation; Cells; Cellular biology; Chemicals; Cicatrix; Clinic; Communities; Cyclin-Dependent Kinase Inhibitor; DNA Sequence Alteration; Development; Developmental Biology; Down-Regulation; Embryo; Equilibrium; Fertilization; Fishes; Fluorescence; G1 Phase; Gene Targeting; Generations; Genes; Genetic; Glial Fibrillary Acidic Protein; Gliosis; Goals; Healed; Human; Immunofluorescence Immunologic; Injury; Institution; Interneurons; Ions; Knowledge; Label; Lesion; Light; Mammals; Measures; Mediating; Metabolic; Mitosis; Muller' s cell; Mutation; Natural regeneration; Nerve Regeneration; Neuraxis; Neurobiology; Neuroepithelial; Neuroglia; Neurons; Pathway interactions; Photoreceptors; Process; Proliferating Cell Nuclear Antigen; Regenerative Medicine; Research; Research Personnel; Research Project Grants; Retina; Retinal; Running; Schools; Science; Science, Technology, Engineering and Mathematics Education; Signal Pathway; Signal Transduction; Stem cells; Structure; Students; System; Technology; Therapeutic; Thymidine; Tissues; Training; Transcription Repressor/Corepressor; Transforming Growth Factor beta; Transgenic Organisms; Ubiquitination; Underrepresented Populations; Vision; Visual impairment; Western Blotting; Woman; Work; Zebrafish; analog; cell type; chemical genetics; college; experience; eye regeneration; healing; in vivo; inhibitor/antagonist; injured; insight; member; mutant; neuroepithelium; novel; outreach; prevent; progenitor; programs; promoter; regenerative; relating to nervous system; research study; response; retinal neuron; retinal regeneration; small molecule inhibitor; summer research; therapeutic target; tool; transcription factor; zebrafish development

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.

项目总结 斑马鱼的视网膜具有与哺乳动物相同的神经元成分和Müler胶质细胞(MG) 视网膜，然而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 旺盛地再生丢失的神经元。哺乳动物体内的MG缺乏这种完全再生的能力。在……里面 斑马鱼视网膜携带转化生长因子β信号转录抑制因子(TGF1)基因突变 和Six3b)，急性轻度损伤后的增殖和再生显著减少 破坏光感受器细胞，这表明这些突变的鱼可以提供对有限的 哺乳动物的神经再生。R15提案中描述的目标将建立在鱼的基础上 探讨转化生长因子β信号增强(在TGF1-/-；Si3b-/-FISH中)和降低转化生长因子β的作用 细胞增殖、细胞周期进程和细胞分化的信号传递(使用小分子抑制剂)。 本研究将斑马鱼作为模型系统进行研究，主要有以下几个方面的好处：1. 可以用于发育研究的大量胚胎，以及2.斑马鱼有 具有非凡的再生神经组织的能力，这是哺乳动物所缺乏的。发展研究将 受精后1至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测 增殖细胞核抗原和细胞周期蛋白依赖性激酶抑制物在细胞退出细胞周期时的特异性表达 采用免疫印迹和免疫荧光技术。而之前的观察表明，转化生长因子β增加 信号导致再生减少，目前尚不清楚这些研究中的Müler胶质细胞是否重新进入 细胞周期的速度较慢和/或产生不同类型神经元的能力降低。 因此，在急性光损伤后，将描述转化生长因子β信号通路成员的表达。 使用免疫荧光，FISH将暴露在一种胸苷类似物中永久标记细胞 新生成的细胞。Müler神经胶质细胞、光感受器和中间神经元产生的数量 在再生过程中将通过共定位细胞类型特异性抗原和类似物标记来定量 使用免疫荧光法。最后，将开发一种新的转基因鱼，使研究人员能够 用荧光标记法区分神经胶质细胞周期各期转化生长因子β 在视网膜再生过程中，信号会阻止细胞周期重新进入或停止细胞周期。这条新奇的鱼线 将是斑马鱼研究界研究神经胶质细胞生物学问题的宝贵工具 贯穿整个中枢神经系统。因为已知转化生长因子β信号转导可以限制细胞增殖和 促进哺乳动物中枢神经系统的疤痕形成，这些在鱼身上的实验将直接告知 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，以减少疤痕，并促进愈合或再生后的急性损伤。