Fig 2: A subset of ECs, HEC/EHT, and IAHCs derived from PDGFRß+ precursors(A–D) Immunohistochemistry of E11 PDGFRß-Cre:mTmG AGM stained with NG2 (A and B) and CD31 (C) and at E10 with Runx1 (D).(A and B) Arrowheads: DP cells; stars: GFP+ mesenchymal cells.(C) Dashed lines: presumptive separation between DP, PDGFRß-S, and DN layers; stars: hematopoietic CD31+GFP+ cells.(E–I) Flow cytometry analyses of E10 (n = 7) and E11 (n = 3) PDGFRß-Cre; tdTomato AGMs, percentage of Tomato+/- cells within (E) PDGFRß+ cells, (F) EC (CD45-cKit-CD31+CD41-), (G) HEC/EHT-enriched population (CD45-cKit-CD31+CD41+), (H) IAHC/HSPC (CD31+cKit+), and (I) MP-enriched population (CD45+CD31-) live cells; error bars: SD; *p < 0.05, **p < 0.005, ***p < 0.005, ****p < 0.0005.(J) CFU-Cs on PDGFRß+/- cells sorted from E10 (n = 8) and E11 (n = 6) AGMs (see Table S6). E11 AGM were ckit+; error bars: SD; ***p < 0.005, ****p < 0.0005.(K) CFU-Cs on Tomato+/- cells sorted from PDGFRß-Cre; tdTomato E10 (n = 6) and E11 (n = 4) AGMs (see Table S7); error bars: SD; ***p < 0.005.(L) PCR on E11 unsorted cells from PDGFRß-Cre; tdTomato and +; tdTomato WT littermates heads and from sorted AGM cells to detect Cre recombinase expression (324 bp). Unpaired t tests or Mann-Whitney tests were used for the statistics.

Fig 3: Efficient ASO-mediated transcript suppression in OPCs and oligodendrocytes in vivo.a-b, Immunostaining of HDAC2+ (red) and (a) NG2+ OPCs (green; arrows) in the spinal cord or (b) CC1+ oligodendrocytes (green; arrows) in the corpus callosum from 8-week-old wild-type mice injected with PBS control or Hdac2-targeting ASO, 2 weeks post-injection. Scale bar, 20 µm. c, Depiction of Plp1 pre-mRNA demonstrating the approximate binding locations of two independent ASOs in intron 5 and the 3’ UTR. d, Schematic of the design for ASO experiments in this figure. e, RT-qPCR data showing wild-type spinal cord Plp1 transcript levels, 3 weeks after injection with the indicated ASO doses (10µg, 30µg, and 60µg) or PBS controls at postnatal day 1 (n=2–6 mice). f, RT-qPCR data showing the levels of Plp1 transcript and g, western blot data showing the levels of PLP protein, 3 weeks post-ASO injection (30µg dose) at birth in wild-type (n=3 mice).Individual data points represent the mean value of 4 technical replicates for each biological replicate (e, f) or independent biological replicates (g). Biological replicates (individual mice) indicated by open circles. Graph bars indicate mean ± standard deviation. p-values calculated using one-way ANOVA with Dunnett’s correction. p-values stated for p<0.1, otherwise not significant (n.s). See Supplementary Data 4 for full western blot source images.

Fig 4: Distinct phenotypic and transcriptomic perivascular cell subsets surround the midgestation DA(A) Immunohistochemistry of E11 WT DA, showing NG2 (i, ii), PDGFRß (i, ii), and CD31 (ii) expression. Nuclei were counterstained with DAPI. CV, cardinal veins; NC, notochord.(B) t-distributed stochastic neighbor embedding (t-SNE) plots showing 12 E11 WT cell populations and their numbers (42sp).(C) Violin plots showing the expression of genes used to identify the cell clusters DP, PDGFRß-S, DN, and NG2-S. MP, macrophages; OBC, other blood cells; Ery/EryP, erythroid/progenitors; IAHC, intra-aortic hematopoietic clusters; HEC/EHT, hemogenic endothelial cells/endothelial-to-hematopoietic transition; EC, endothelial cells; SNS, sympathetic nervous system; SkMP, skeletal muscle progenitors.(D and E) Heatmaps showing gene expression of differentially expressed genes encoding surface (D) and intra-/extracellular (E) proteins enriched in each of the NG2+/-PDGFRß+/- populations.(F) Isolation of perivascular cells.(G) Fragments per kilobase of transcript per million mapped reads (FPKM) values of selected genes by bulk RNA-seq.

Fig 5: AVMs following inhibition of Notch signaling in pericytes at P14.(A) Diagram of tamoxifen administration to postnatal control and Rbpji?PC mice and analysis at P14. (B) Confocal images showing P14 retinal vasculature stained with anti-CD31 (white). Right panels show higher magnification of boxed regions, demonstrating vascular abnormalities in the retina of Rbpji?PC mice. Scale bars: 600 µm (left panels) and 300 µm (right panels). (C) Quantification of percentage of vessel area (n = 4), branching points/mm2 (n = 3), and vessel diameter (n = 4–6) in control and Rbpji?PC mice. (D) Images of vasculature perfused with blue latex compound. Perfused vessels were subsequently stained with IB4 and detected using HRP streptavidin. Boxed region highlights AV shunt. Scale bars: 200 µm. (E) High-magnification confocal images of anti-NG2 (white) and anti-CD31 (red) staining of pericytes and the capillaries, respectively. Scale bars: 100 µm. Quantification of percentage of pericyte coverage (n = 3) in control and Rbpji?PC mice. (F) CD31 (red) highlights the arteries and veins, and a-SMA (white) represents vascular smooth muscle cells (vSMCs). Note CD31+ arteries have reduced a-SMA+ vSMCs, as indicated by arrows. Scale bars: 100 µm. Quantification of percentage of vSMC coverage (n = 5) in control and Rbpji?PC mice. Box-and-whisker plots show median, minimum, and maximum values. Data analyzed using unpaired 2-tailed t test with Welch’s correction. a, artery; c, capillary; and v, vein. *P < 0.05, **P < 0.01, ***P < 0.01.