Figure 4—figure supplement 3.

Expression of Pard3ba is upregulated by dt-Runx1.

(A) Experimental strategy to isolate endothelial cells from Tg(Kdrl:Gal4;UAS:RFP) and Tg(Kdrl:Gal4;UAS:RFP;4xNR:dt-runx1-eGFP) embryos for subsequent gene expression analyses by qRT-PCR. Forty-eight hpf control (left) and dt-runx1 expressing mutant embryos (right) were dissected to isolate trunk regions (delimited by two magenta arrows) that were subsequently treated to dissociate cells and populations of interest were sorted by FACS. n=3 experiments were performed. RNA was isolated from 1900 to 3200 cells for the dt-runx1 mutant (green population) and from 6500 to 9142 cells for control RFP expressing cells (magenta population). (B) Representative images (Imaris 3D-rendering) of RNAscope in situ hybridizations for Pard3ba in 48–50hpf Tg(Kdrl:eGFP) control embryos (left) and dt-runx1 mutant embryos Tg(Kdrl:Gal4;UAS:RFP;4xNR:dt-runx1-eGFP) (right). The aorta is outlined with the white dashed lines. The RNAscope signals were segmented into spots and classified based on their localization: in aortic endothelial and hemogenic cells (magenta spots) or in extra-aortic tissues (grey spots). Scale bars = 10 µm. (C) Hemogenic/EHT cell count per aorta segment, separated based on their pard3ba expression (with, from left to right, 0–4 Par3ba spots). Statistical tests: two-sided unpaired two samples Wilcoxon test, all p-values are displayed. Analysis was carried out on n=5 control embryos and n=6 mutant embryos, 2 aortic segments per embryo.