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

小胶质细胞在眼睛的慢性退行性变中起着关键作用，但它们是发挥神经保护功能还是神经毒性功能仍不清楚。为了阐明这些作用，我正在研究一个斑马鱼退化突变体，它在整个生命周期中经历光感受器退化，但在大约4周大的时候也表现出相关的再生。我用流式细胞术从5天龄(开始退化)和4周龄(开始相关再生)的突变斑马鱼和同胞斑马鱼的眼睛中收集了表达荧光蛋白的小胶质细胞。然后用10x Chromium平台构建文库进行测序，得到测序数据后，使用Seurat等R软件包进行质量控制和聚类分析，以研究5日龄和4周龄小胶质细胞群体的组成。我通过更改集群解析等参数执行了多次分析迭代，以确保解释是健壮的。通过差异表达测试、文献探索和GO浓缩分析，我列出了候选基因列表，我假设这些基因在退化开始和再生开始时控制着小胶质细胞的功能。为了可视化这些候选基因在突变体和兄弟姐妹视网膜中的表达，我开始通过标准化方案和产生原位杂交探针来准备体内分析。

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.