Fig. 3

Transcriptionally and functionally distinct SpleMdCs infiltrate the retina after injury. A Cartoon of the experiments for the analysis of single-cell RNA sequencing. Cells were FACS-sorted from five independent mice. B Expression of representative upregulated genes in SpleMos compared to BMMos in CD11b⁺Ly6G⁻Ly6C^high cells from the spleen, CD11b⁺Ly6G⁻ cells from sham-operated lasered eyes and splenectomized lasered eyes (splenic MdC signature genes marked in red). C Uniform Manifold Approximation and Projection (UMAP) of scRNA-sequencing of CD11b⁺Ly6G⁻Ly6C^high cells from the spleen and the BM; of CD11b⁺Ly6G⁻ cells from eyes of splenectomized, 24 h-lasered mice (SpleX-lasered eyes); and from eyes of sham-operated, 24 h-lasered mice (sham-lasered eye). UMAP of the distribution of spMdCs defined by the transcriptional signature composed of Apoe, Marcks, Filip1l, Metrnl, and Sirpb1c. D UMAPs showing the relative expression of the five spleMdC signature genes. E Functional annotation of the 195 upregulated genes in spMdCs versus the other MPs in laser-injured eyes performed with DAVID for Gene Ontology term (GOTERM) Biological Process and showed a significant enrichment of genes associated with antigen processing, protein transport and phagocytosis pathways. F Known and predicted protein interaction (STRING) of the genes belonging to the significant GO term processes shown in C (blue dot) and the other upregulated genes (red dots) in spMdC (52/195 upregulated genes are represented). G Representative images of RPE flatmounts and quantification of CFSE-stained BMMos or SpleMos 24 h after adoptive transfer to the subretinal space of recipient mice (n = 20; Mann–Whitney test, BMMos vs SpleMos, *p = 0.0468). Scale bar = 50 µm. SpleX: splenectomy; scRNAseq: single-cell RNA sequencing; spleMo: splenic monocyte; BMMo: bone marrow monocyte MdC: monocyte-derived cell; spMdC: splenic monocyte-derived cell