Figure 5.
Identification of 2 different RUNX1 subpopulations in the subaortic mesenchyme. (A) Left: experimental design of E10.5 AGM hanging drop reaggregation cultures with LIN (CD45+TER119+) depleted, or LIN and MES (LIN−CDH5+Runx1b:RFP+) depleted cells. CFU assays were performed after 4 days of culture. Right: Bar graph depicting average colony number ± SEM. The number of assayed AGMs are indicated in the bars. ***P < .001 (2-taild Student t test). (B) Heatmap illustrating the top differentially expressed genes between the C9:MES-PDGFRa and C10:MES-SMA. Genes are clustered based on Pearson correlation and cells are grouped based on the in silico clusters. Genes coding for secreted proteins are highlighted in bold. (C-D) Top left: immunofluorescence (IF) staining of formalin-fixed paraffin-embedded (FFPE) E10.5 AGM sections for CD31 (red), RUNX1 (green) and SMA (yellow, C), or PDGFRA (gray, D). DA, dorsal aorta. Bottom left: ×100 magnification of the boxed region in the top panel. Right: quantification of SMA (C) and PDGFRA (D) positive cells within the top field of view. (E) Density plot depicting the distance of RUNX1+SMA+ and RUNX1+PDGFRA+ cells in relation to the endothelial (CD31+) cells lining the DA. (F) Violin plot combined with box plot showing the normalized expression (logcounts) of Pleiotrophin (Ptn) in Runx1 heterozygous (HET, Runx1bRFP/WT) or Runx1 knockout (KO, Runx1bRFP/RFP) C9 and C10 clusters. Medians are shown as a solid black line, gray boxes indicate the upper (75%) and lower (25%) percentile. (G) Representative 35 hpf confocal images of no morpholino control and ptn morpholino treated (100 μM) Tg(cd41:eGFP//kdrl:caax:mCherry) zebrafish embryos. Emerging HSPC (cd41, kdr double-positive cells) in the 35 hpf AGM are indicated with white arrows. (H) Quantification of the emerging HSPC (kdrl+cd41+) in the 35 hpf AGM of embryos treated with no morpholino (no MO), control scrambled morpholino (100 μM), and ptn morpholino (10 and 100 μM). Individual fish (dots) and mean ± SEM are shown. *P < .05, **P < .01 (2-tailed, unpaired Student t test). NS, not significant; SEM, standard error of the mean.

Identification of 2 different RUNX1 subpopulations in the subaortic mesenchyme. (A) Left: experimental design of E10.5 AGM hanging drop reaggregation cultures with LIN (CD45+TER119+) depleted, or LIN and MES (LIN−CDH5+Runx1b:RFP+) depleted cells. CFU assays were performed after 4 days of culture. Right: Bar graph depicting average colony number ± SEM. The number of assayed AGMs are indicated in the bars. ***P < .001 (2-taild Student t test). (B) Heatmap illustrating the top differentially expressed genes between the C9:MES-PDGFRa and C10:MES-SMA. Genes are clustered based on Pearson correlation and cells are grouped based on the in silico clusters. Genes coding for secreted proteins are highlighted in bold. (C-D) Top left: immunofluorescence (IF) staining of formalin-fixed paraffin-embedded (FFPE) E10.5 AGM sections for CD31 (red), RUNX1 (green) and SMA (yellow, C), or PDGFRA (gray, D). DA, dorsal aorta. Bottom left: ×100 magnification of the boxed region in the top panel. Right: quantification of SMA (C) and PDGFRA (D) positive cells within the top field of view. (E) Density plot depicting the distance of RUNX1+SMA+ and RUNX1+PDGFRA+ cells in relation to the endothelial (CD31+) cells lining the DA. (F) Violin plot combined with box plot showing the normalized expression (logcounts) of Pleiotrophin (Ptn) in Runx1 heterozygous (HET, Runx1bRFP/WT) or Runx1 knockout (KO, Runx1bRFP/RFP) C9 and C10 clusters. Medians are shown as a solid black line, gray boxes indicate the upper (75%) and lower (25%) percentile. (G) Representative 35 hpf confocal images of no morpholino control and ptn morpholino treated (100 μM) Tg(cd41:eGFP//kdrl:caax:mCherry) zebrafish embryos. Emerging HSPC (cd41, kdr double-positive cells) in the 35 hpf AGM are indicated with white arrows. (H) Quantification of the emerging HSPC (kdrl+cd41+) in the 35 hpf AGM of embryos treated with no morpholino (no MO), control scrambled morpholino (100 μM), and ptn morpholino (10 and 100 μM). Individual fish (dots) and mean ± SEM are shown. *P < .05, **P < .01 (2-tailed, unpaired Student t test). NS, not significant; SEM, standard error of the mean.

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