Extended Data Fig. 5: An Mrc1⁺ A2 population in the yolk sac gives rise to both microglia and CAMs.

(a) Gating strategies for yolk sac macrophage progenitors. c-Kit^loCD45⁺ cells (R1, blue) isolated from the wild-type yolk sac at E9.5 were used for scRNA-seq shown in Fig. 3a–d. CX3CR1–GFP⁻c-Kit⁺ A1 and CX3CR1–GFP⁺c-Kit⁻ A2 cells isolated from the yolk sac of Cx3cr1^GFP/+ mice were used for the analysis shown in Fig. 3e, f. Representative plots out of three mice are shown. (b) UMAP plots representing the core signature genes for monocytes (left) and granulocytes (right) in the yolk sac. (c) UMAP plots of genes that are enriched in clusters C2 and C4 (Slc2a3 and Apoa2), or C1, C5 and C6 (Psat1) are shown (483 yolk sac cells). Each dot represents a single cell. (d) UMAP plot of Ccnd2 gene enriched in cluster C3 is shown (483 yolk sac cells). Each dot represents a single cell. (e) Gating strategies for EMPs are shown. c-Kit⁺AA4.1⁺CD45^neg-loF4/80⁻ cells isolated from the Cx3cr1^GFP/+ yolk sac at E8.5 were used for the analysis shown in Fig. 3e, f. Representative plots out of four mice are shown. (f) Representative immunofluorescence images depicting CD206⁻ c-Kit⁺ EMPs in the yolk sac at E8.5 of Cx3cr1^GFP/+ pregnant females. Immunolabelling was performed for c-Kit (red), CD206 (blue) and CD31 for yolk sac endothelia cells (white), respectively. White filled arrowhead indicates CD206⁻ c-Kit⁺ EMPs, and blank white arrowheads indicate CD206⁺ yolk sac cells. Representative pictures out of four yolk sac investigated are shown. Scale bars: 50 μm, 20 μm (inset). (g) Scheme of the experimental set-up. (h) Representative gating strategy for EMPs, A1 and A2 cells isolated from the yolk sac of Mrc1^{CreERT2/CreERT2}R26^tdT/tdT embryos at E10.5. Plots show six individual yolk sacs that were concatenated for representation. (i) Quantification of tdT-expressing EMPs, A1 and A2 cells isolated from the yolk sac of Mrc1^{CreERT2/CreERT2}R26^tdT/tdT embryos at E10.5 (n = 6). (j–l) Representative gating strategy (j) for the quantification of tdT-expressing CD206^– and CD206⁺ macrophages isolated from the brains of Mrc1^{CreERT2/CreERT2}R26^tdT/tdT embryos at E16.5 (k: n = 6) or E17.5 (l: n = 6). (m–o) Representative gating strategy (m) for the quantification of tdT-expressing lineage-restricted progenitors (LRP), short-term (LT-) hematopoietic stem cells (HSC) and long-term (LT-) HSC isolated from the fetal livers of Mrc1^{CreERT2/CreERT2}R26^tdT/tdT embryos at E16.5 (n: n = 6) or E17.5 (o: n = 6). (p) Scheme of the experimental set-up. (q) Quantification of tdT⁺ microglia, pvMΦ and mMΦ in Mrc1^CreERT2/+R26^tdT/tdT mice. Each symbol represents one mouse (n = 4 mice). Three sections/mouse were quantified (means ± s.e.m.).

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