Friend or foe? Investigating the role of microglia in chronic photoreceptor degeneration using zebrafish

是敌是友？

• 批准号: 22KJ3081
• 负责人: Ravishankar Darshini
• 金额: $ 1.09万
• 依托单位: Okinawa Institute of Science and Technology Graduate University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2023
• 资助国家: 日本
• 起止时间: 2023-03-08 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-22KJ3081/
• 关键词: ミクログリア

Microglia play key roles in chronic degenerations in the eye, but whether they perform neuroprotective or neurotoxic functions is still unclear. To clarify these roles, I am studying a zebrafish degeneration mutant that undergoes photoreceptor degeneration throughout its lifespan, but that also exhibits associated regeneration around 4 weeks old. I collected fluorescent protein-expressing microglia from mutant and sibling zebrafish eyes at 5 days old (onset of degeneration) and 4 weeks old (start of associated regeneration) using flow cytometry. The 10x Chromium platform was then used to generate libraries for sequencing, and the samples were sequenced.Once the sequencing data was obtained, I performed quality control and clustering analysis using R packages such as Seurat to investigate the composition of the microglial population at 5 days old and 4 weeks old. I performed several iterations of the analysis by changing parameters such as clustering resolution, to ensure that interpretations were robust. Through differential expression testing, exploration of the literature, and GO enrichment analysis, I made candidate lists of genes that I hypothesize are controlling microglial function at the onset of degeneration and start of regeneration. To visualize the expression of these candidate genes in the mutant and sibling retina, I began to prepare for in vivo analysis by standardizing protocols and generating probes for in situ hybridization.

小胶质细胞在眼睛的慢性变性中起着关键作用，但它们是否具有神经保护或神经毒性功能仍不清楚。为了阐明这些作用，我正在研究一种斑马鱼变性突变体，它在整个生命周期中都经历感光细胞变性，但在4周龄左右也表现出相关的再生。我收集荧光蛋白表达的小胶质细胞突变和同胞斑马鱼眼睛在5天大（开始变性）和4周大（开始相关再生），使用流式细胞术。然后使用10x Chromium平台生成文库进行测序，并对样品进行测序，获得测序数据后，我使用Seurat等R软件包进行质量控制和聚类分析，以调查5日龄和4周龄时小胶质细胞群体的组成。我通过改变参数（如聚类分辨率）进行了几次分析迭代，以确保解释是可靠的。通过差异表达测试、文献探索和GO富集分析，我列出了我假设在变性开始和再生开始时控制小胶质细胞功能的基因的候选列表。为了可视化这些候选基因在突变体和同胞视网膜中的表达，我开始通过标准化方案和生成原位杂交探针来准备体内分析。

项目成果

Roles for microglia in chronic photoreceptor degeneration and associated regeneration caused by pde6c dysfunction in zebrafish

小胶质细胞在斑马鱼 pde6c 功能障碍引起的慢性光感受器变性和相关再生中的作用

• 发表时间: 2022
• 作者: Huang Tsung-Yen;Hirota Masato;Sasaki Daiki;Kalra Rajkumar Singh;Chien Hsiao-Chiao;Tamai Miho;Sarkar Shukla;Mi Yang;Miyagi Mio;Seto Yu;Ishikawa Hiroki;Yang Mi;丹上麻里江;丹上麻里江;戸塚史織;戸塚史織;戸塚史織;玉城福子;玉城福子;玉城 福子;Michael Izumiyama;Darshini Ravishankar
• 通讯作者: Darshini Ravishankar