Fig 2: Interstitial stem cells migrate towards PDGF-BB through PDGFRB. (A) Flow cytometry analysis for PDGFRB of fibro/adipogenic progenitors isolated from the skeletal muscle of Pdgfrb-flox mice that were transiently transduced with ASLV-Cre or mock. (B) Crystal violet staining of transwell assays performed with cells from (A). (C) Percentage of migrated cells shown in (B). n = 3–4 independent wells. Experiments were repeated at least three times, yielding similar results. Scale bar, 50 µm.

Fig 3: Flow cytometry gating strategy. Flow cytometry gating strategy for unlasered (A) and lasered (B) mice. Singlets were identified and gated forward. Dead cells were excluded. Live cells were separated into CD45- and CD45+ populations. Endothelial cells (EC) were delineated by CD31+ staining and pericytes were identified by CD140b+ staining in CD45- cells. Eosinophils, neutrophils, NK cells, T cells, and B cells were excluded by negative staining for SiglecF, Ly6G, NK1.1, CD4, CD8, and B220 (Dump gate). CD11b+Dump- cells were separated into CD45dim and CD45high groups. Microglia were identified as CD64+MHCIIlow in the CD45dim group. Macrophages were detected as CD64+MHCII- (MHCII- Macs) and CD64+MHCII+ (MHCII+ Macs) in the CD45high group. Monocytes were delineated as CD64-MHCII-, and dendritic cells (DC) were identified as CD64-MHCII+.

Fig 4: Four distinct Ng2 perivascular populations are present in adult mouse back skin based on Pdgfra and/or Pdgfrß co-expression. (A) Pool of 3 flow cytometry plots of PdgfraH2BGFP; Ng2DsRed double transgenic adult mouse back dermal cells at P21. Live dermal cells co-expressing Ng2 and Pdgfra were assessed for Pdgfrß expression. (B) Immunofluorescence of a dermal blood vessel from PdgfraH2BGFP; Ng2DsRed double transgenic mouse labelled with anti-Pdgfrß (gray). High magnification images revealed the presence of 4 Ng2 perivascular populations. Ng2+ Pdgfra– Pdgfrß– cells can be seen in all high magnification panels. Arrows show I, II: Ng2+ Pdgfra+ Pdgfrß+, III: Ng2+ Pdgfra- Pdgfrß+, IV: Ng2+ Pdgfra+ Pdgfrß– Representative images shown. (C) Graph depicting the percentage of Ng2+ perivascular cells which are Pdgfr-, Pdgfra– Pdgfrß+, Pdgfra+ Pdgfrß-, and Pdgfra+ Pdgfrß+ at P21. (D) Schematic illustrating the percentage contributions of each Ng2+ perivascular subpopulation to the total Ng2+ perivascular population present at P21. (E) Graph depicting the percentage of Ng2+ perivascular cells which express Pdgfra– Pdgfrß+, Pdgfra– Pdgfrß+ and Pdgfra+ Pdgfrß+ in papillary and reticular layers of the dermis at P21. *P=0.05, **P=0.01, ***P=0.001, and ****P=0.0001. N = 3 Scale bars 100 µm. Error bars represent ±SD. Statistical analysis conducted using One-way ANOVA or Multiple T-Tests using the Holm-Sidak method.

Fig 5: Schematic summary showing origins of Ng2+ skin pericytes in unwounded and wounded skin. In unwounded skin, Lrig1 (green), and Ng2 (gray) lineages, labeled at E18.5, give rise to Ng2+ blood vessel resident pericytes in the papillary layer of the dermis whilst Dlk1 (red) and Ng2 lineages give rise to Ng2+ blood vessel resident pericytes in the reticular layer of the dermis. These NG2+ pericytes can differ in their Pdgfr status. In unwounded skin Lrig1 and Dlk1 fibroblast lineages contribute to Ng2+ pericyte populations in a spatially restricted manner. However, in wounded skin, the Lrig1 and NG2 lineages can contribute to pericytes in both layers of the dermis whilst the contribution of the Dlk1 lineage remains spatially restricted to the reticular layer.