Fig. 2

Increased NOTCH activation facilitates EHT. a Schematic diagram of experiments. D4 HE cultured in presence of DAPT for 4 days (D4 + 4) or 1 day (D4 + 1), or DMSO (control). CD144⁺ endothelial and CD43⁺ blood cells were analyzed following 4 days of culture. b Flow cytometric analysis demonstrates that NOTCH activation facilitates EHT as evidenced by the decrease in hematopoietic activity when DAPT is added only from D4 to D4 + 1. Representative contour plots from three independent experiments are shown. c Frequencies of endothelial and blood cells in HE cultures treated with DAPT or DMSO (control). Results are mean ± s.e.m. for at least three independent experiments; two-way ANOVA Bonferroni post-hoc test, *p < 0.05, ***p < 0.001. d Single D4 HE cells were FACS-sorted into 96-well plate with OP9, OP9 + DAPT, and OP9-DLL4. Colonies were scored based on CD43 and CD144 expression on D4 + 10 by immunofluorescence and counted by eye. Scale bar represents 100 μm. e Representative flow cytometric cell proliferation analysis and f bar graph conducted with CellTracer shows an increase in the first generation (Gen1) CD43⁺ cells on D4 + 1 and a proportional decrease in Gen1 CD144⁺ endothelial cells, suggesting that the increase in blood cells is due to an increase in EHT and not just proliferating HPs. g Line graph depicting the percent of each generation within the CD43⁺ population on D4 + 4 in each of the NOTCH treatment conditions. Results are mean ± s.e.m. for three independent experiments; two-way ANOVA Bonferroni post-hoc test, *p < 0.05, ***p < 0.001. No significant change of each generation between conditions suggesting that NOTCH does not affect proliferation of HPs. Generation gates in f, g were determined by concatenating D4 to D4 + 4 results and utilizing FlowJo’s proliferation assay