Extended Data Fig. 2: MMϕ mature transcriptionally after weaning.
From: Dedicated macrophages organize and maintain the enteric nervous system
(A) Dot plot in αCSF1R-treated mice and controls to show efficient depletion of MMϕ. Cells shown are pre-gated on fsc/ssc, single cells, live and CD45+. (B) Quantification of immune cells in αCSF1R-treated mice (n = 4) and controls (n = 5). Macrophages are defined as CD45+CD11b+CD64+, B cells as CD45+CD3e-CD64−CD11b−CD19+ and T cells as CD45+CD3e+. All populations were pre-gated on fsc/ssc and live cells. Unpaired two-tailed t-tests. Data are shown as Mean ± SD. Each data point indicates a separate biological replicate. *** p < 0.001; ns = not significant. (C) Gating strategy used to isolate Cx3cr1+ MMϕ from the muscularis externa for scRNAseq. (D) UMAP of data prior to exclusion of contaminating cluster (pink). (E) UMAP with heatmap colour-coding to show expression for canonical macrophage markers and CCR2 within the scRNAseq dataset. (F) Violin plots showing the upregulation of extracellular matrix genes in the contaminating cluster (pink). (G) Representative confocal image of the muscularis externa at P10, showing Cx3cr1+ MMϕ embedded within the extracellular matrix (Dcn, Decorin). Image is representative of 3 biological replicates. Scale bar is 20 µm. (H) Volcano plot depicting globally differentially expressed genes at P10 and P56. Coloured dots represent significantly (p < 0.05) upregulated genes over Log2FC > 0.5. (I) Dot plot to depict expression of top 10 upregulated genes within each cluster. Colour of dots represents expression level, while size of dots represents percentage of cells expressing the gene identified. Only genes significantly (p ≤ 0.05) upregulated (logFC ≥ 0.2) are shown. (J) Violin plots showing the expression of Bmp2 in the identified MMϕ clusters. n = x biologically independent samples.
