Time-lapse imaging reveals differences in Gata2 dynamics between ECs, BCs, and IAHCs. (A) Mean number of Venus⁺ EHT subset cells per G2V embryo according to their ventral and dorsal aortic location, as determined by microscopy. Venus⁺ HECs, BCs, or IAHCs were counted in the first frame of time-lapse imaging experiments (n = 15, 42 sections) of G2V embryo slices of 150 µm thickness and calculated per embryo (E10, 32–37 SPs). The data represent the mean ± SEM of 15 independent experiments, and dorsal and ventral location were compared using two-way ANOVA with Bonferroni post test (*, P < 0.05). (B) Left: Example of a BC showing decreasing and increasing levels of Venus expression during a 12-h imaging period, imaged at a time interval of 15 min. Middle: Three-dimensional projections (x-y axes and x-z axes) of the same BC as shown in the top panel with time (hours) indicated. Right: Corresponding Venus (green) MFI profile in time. Bar, 25 µm. Sections were stained with anti–CD31 (red) antibody. (C) Venus MFI (averaged over frames 3–12) in single EHT subset cells (HECs, BCs, and IAHCs; n = 15, 718 cells). The data represent the mean ± range. Statistical significance was calculated on the pooled data of 15 independent experiments using Mann–Whitney U test (***, P < 0.0001). (D) Venus MFI in single EHT subset cells plotted according to their dorsal (d) and ventral (v) location (n = 15, 718 cells). The data represent the mean ± range. Statistical significance was calculated on the pooled data of 15 independent experiments using Mann-Whitney U test (*, P < 0.0288; **, P = 0.0020; ***, P < 0.0001). (E) Top: Temporal variation of Venus MFI for individual Venus⁺ cells (colored lines) plotted according to their affiliation to one of the EHT subsets (EC, BC, or IAHC). Bottom: Gray bands represent the standard deviation of the mean MFI of all tracks (black line) over time.