Microglia and myeloid cell populations of the developing mouse retina

Microglia make important contributions to central nervous system (CNS) development, but the breadth of their distinct developmental functions remain poorly understood. The mouse retina has been a key model system for understanding fundamental mechanisms controlling assembly of the CNS. To gain insight into where and how microglia might influence retinal development, here we identified molecularly unique myeloid cell populations that are selectively present during development, and characterized their anatomical locations. Development-specific transcriptional states were identified using single-cell (sc) and single-nucleus (sn) RNA-sequencing (RNA-seq) across multiple timepoints: postnatal day (P)5, P10, and P17. Transcriptional states were validated in vivo by histological staining for key RNA and/or protein markers. Overall design: For scRNA-seq, retinal myeloid cells were purified by flow cytometry from Cx3cr1-YFP mice at three timepoints: P5, P10, and P17. Two repilcates were collected at each timepoint, although one P5 replicate failed and is not included here. Each replicate comprised all of the pups from a single litter (10-14 retinas from 5-7 mice). One of the P10 replicates included a cocktail of transcriptional/translational inhibitors to suppress ex vivo gene activation. For snRNA-seq, myeloid cell nuclei were fluorescently labeled using Cx3cr1-CreER; lox-stop-lox-Sun1GFP mice. GFP+ nuclei from P5 and P10 retinas were purified by flow cytometry. One replicate (i.e. litter) was run for each snRNA-seq timepoint.