Extended Data Fig. 6: Characterization of CD200^Hi capillaries: cytometric and transcriptome profiles.

a, Representative flow cytometry plots illustrating the gating strategy to identify CD200^Hi capillaries, Type H vessels, other arterial fractions, and sinusoids. CD200^Hi capillaries (CD200^HiEMCN⁻Sca1⁺CD31⁺CD45⁻) are exclusively found within Sca1^HiCD31^HiCD140b^Int–HiCD45⁻. Type H vessels (EMCN⁺Sca1⁺CD31⁺CD45⁻) are frequently found within Sca1^IntCD31^HiCD140b^Int–HiCD45⁻. Other arterial fractions (EMCN⁻ Sca1⁺CD31⁺CD45⁻) are frequently found within Sca1^IntCD31^IntCD140b^Int–LowCD45⁻. b, Frequencies of: Sca1^HiCD31^HiCD140b^Int–HiCD45⁻; Sca1^IntCD31^HiCD140b^Int–HiCD45⁻; and Sca1^IntCD31^IntCD140b^Int–LowCD45⁻ fractions within: CD200^Hi capillaries; Type H vessels; and other arterial fractions. The experiment included four mice from four independent experiments. The results were analysed by 1-way ANOVA with post hoc test with Bonferroni correction. c, Representative flow cytometric plots illustrating the expression levels of Arl13b-mCherry and Centrin2-GFP within different BM mesenchymal factions in Arl13b-mCherry Centrin2-GFP mice. d, Flow cytometric analysis of CD106, CD54, and LYVE1 expression in CD200^Hi capillaries, Type H vessels, other arterial fractions, and sinusoids. Frequencies of CD106-, CD54-, and LYVE1-expressing cells within each vascular fraction. The results were analysed by 1-way ANOVA with post hoc test with Bonferroni correction. N = 4. e, Multi-dimensional scaling plot showing tight intra-sample variability per sample type across all profiled samples: CD200^Hi capillaries (CD200); EMCN+ Type H vessels (Type H); other arterial fractions (Artery); and sinusoids (Sinusoid). f, Gene set enrichment analysis (GSEA) demonstrating CD200^Hi capillaries upregulate representative gene signatures of sprouting, immature capillaries and Sonic Hedgehog Pathway. CD200^Hi capillaries upregulate the following gene signatures of arterial vessels; immature blood vessels; capillaries; tip cell genes; vasculogenesis; endothelium development; artery development; and Sonic Hedgehog pathway. Normalized enrichment score and q-value are shown in each panel. CD200: CD200^Hi capillaries. Type H: Type H vessels. Artery: other arterial fractions. Sinusoid: Sca1-CD31 + CD45- cells. g, GO enrichment analysis demonstrating the top 30 GO terms. Left: dot plots in biological process (CD200 versus Sinusoid). Middle: dot plots in biological process (CD200 versus Other Arterial). Right: Ridge plots in the cellular compartment (CD200 versus Type H). Various adaptive and innate immunoregulatory genes (blue arrows) and gene signatures in relationship to vascular development and sprouting (pink arrows) are upregulated in CD200^Hi capillaries (CD200) in comparison with Type H vessels (Type H), Other arterial fractions (Other Arterial), and Sinusoids (Sinusoid). The results presented are significant after Benjamini-Hochberg multiple testing correction was applied. h, Unsupervised hierarchical clustering and heatmap demonstrating differential expression levels of genes representative of HSC regulatory molecules and angiogenic receptors in: CD200^Hi capillaries (Cd200); EMCN+ Type H vessels (Type H); other arterial fractions (Artery); and sinusoids. Three biological replicates are shown per sample type. Each row of the heatmap represents the z-score transformed log(CPM) values across all samples (blue, low expression; red, high expression). i, Flow cytometric analysis of expression levels of angiogenic receptors and angiocrine HSC regulators in CD200^Hi capillaries, Type H vessels, other arterial fractions, and sinusoids. Top two rows: representative histograms. Bottom two rows: frequencies of: Tie1-; Tie2-; VEGFR1-; VEGFR2-; VEGFR3-; SCF-; BMP2-; BMP4-; and DLL4-expressing cells. Frequencies were measured in comparison with isotype control antibody staining. MFIs of SCF expression are also shown. The results were analysed by Student’s t-test. N = 4–9/group. The results were pooled from two to three independent experiments. All data are presented as mean ± SD. Asterisks indicate significant differences (p-value < 0.05, *; <0.01, **; <0.001, ***; <0.0001, ****; N.S., >0.05).