Extended Data Fig. 3: MMϕ subset phenotype and function prior to and after weaning.

(A) Representative gating strategy used to identified MMϕ subsets via flow cytometry. (B) Heatmap depicting expression of genes identified by Chakarov et al., within each cluster, cells pooled. (C) Representative contour plots of identified MMϕ subsets at P10 and P56. (D) Relative frequency of Lyve1⁺ MMϕ quantified via immunohistochemistry, in the different micro-anatomical niches of the muscularis externa (n = 4). (E) Phagocytic index of each MMϕ subset phagocytosing pHRodo-labelled apoptotic thymocytes, and representative histograms from the same sample incubated at 4 °C and at 37 °C with phRodo-labelled apoptotic cells (n = 5). (F) Representative confocal images showing CD68+ lysosomes in NA-MMϕ and Lyve1⁺ MMϕ. Images shown are representative of two independent experiments. Scale bar is 20 µm. (G) Relative frequency of NA-MMϕ quantified via immunohistochemistry, in the different micro-anatomical niches of the muscularis externa (n = 4). (H) Dot plot depicting expression of top 5 transcription factors identified via SCENIC within each cluster. Colour of dots represents expression level, while size of dots represents percentage of cells expressing the transcription factor identified. Only genes significantly (p ≤ 0.05) upregulated (logFC ≥ 0.2) are shown. (I) Quantification of the percentage of Ms4a3⁺ Cx3cr1⁺ MMϕ in the muscularis externa throughout early postnatal life and adulthood (n = 5-6). (J) Quantification of the percentage of Ms4a3⁺ Cx3cr1⁺ MMϕ in the identified MMФ subsets in the muscularis externa throughout early postnatal life and adulthood (n = 5-6). (K) Quantification via flow cytometry of population frequency at 8 weeks (P56) and 20 weeks (n = 5). Data are shown as Mean ± SD, analysed by multiple unpaired t-tests (D-G) or two-way ANOVA followed by Tukey’s (J) or Šídák’s (K) multiple comparison. Each data point indicates a separate biological replicate. n = x biologically independent samples.

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