![Figure 4. +9.5 element-regulated genetic network controls HSC emergence from hemogenic endothelium. (A) Representative flow cytometric plots depicting endothelial (CD31+c-Kit−) and hematopoietic (CD31+c-Kit+) cells in cultured AGM explants. (B) Quantitative analysis of flow cytometry data expressed as percentage of live cells from cultured AGM explants (2 independent experiments and 4 litters: +9.5+/+ [n = 14]; +9.5+/− [n = 17]; +9.5−/− [n = 7]). (C and D) Quantitation of relative expression of HSC-related genes and representative novel genes in endothelial cells and hematopoietic cells from +9.5+/+ AGM explants. (E and F) Heatmap showing quantitation of relative expression of HSC-related genes and novel genes in endothelial cells and hematopoietic cells from +9.5+/+, +9.5+/−, and +9.5−/− AGM explants. (G and H) Cell cycle analysis of endothelial (CD31+c-Kit−) and hematopoietic cells (CD31+c-Kit+) from +9.5+/+, +9.5+/−, and +9.5−/− AGM explants (thr3e independent experiments and 5 litters: +9.5+/+ [n = 10]; +9.5+/− [n = 22]; +9.5−/− [n = 11]). (I and J) Flow cytometric analysis of the percentage of apoptotic cells (PI+Annexin V+) within the endothelial cells (CD31+c-Kit−) and hematopoietic cells (CD31+c-Kit+) from +9.5+/+, +9.5+/−, and +9.5−/− AGM explants (2 independent experiments and 2 litters: +9.5+/+ [n = 3]; +9.5+/− [n = 10]; +9.5−/− [n = 4]). (K) Model describing +9.5 cis-element requirement for HSC emergence from AGM and +9.5 cis-element–dependent genetic networks in AGM endothelial and hematopoietic cells. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed unpaired Student’s t test).](https://cdn.rupress.org/rup/content_public/journal/jem/210/13/10.1084_jem.20130733/4/jem_20130733r_fig4.jpeg?Expires=1767747991&Signature=M1vhWwpwEloV3gCHUTkprGJYsJcigOXoX1oLmICBvTRwOYS~H95l~3VYa~13Ppu4qKTrks2Y3CG6fNKtCqCLJlJdJ0UD-5zFYdC0G0G1xOwgnSsbS03Gzk3u33GJ-SphGprCx58tT492e9xu3u1Zg6JkeWll01-VUSKXhWUH1M7LmWr9mdJXKgej-c8AFhtgB7oKGvJC2A-4nP9EE-6VREI0Z6CQ3KViX~sBchS0LltA9NRTjo6-c6iX-N1hNR9gEGSsHn2R-VpbP49e4~dSa1hxpLC~wD8cQWlc8N369hQKUguWmxL3l1eOoSjj9htP3D7Gi4N~Q5NT93vfgb1j~g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
+9.5 element-regulated genetic network controls HSC emergence from hemogenic endothelium. (A) Representative flow cytometric plots depicting endothelial (CD31+c-Kit−) and hematopoietic (CD31+c-Kit+) cells in cultured AGM explants. (B) Quantitative analysis of flow cytometry data expressed as percentage of live cells from cultured AGM explants (2 independent experiments and 4 litters: +9.5+/+ [n = 14]; +9.5+/− [n = 17]; +9.5−/− [n = 7]). (C and D) Quantitation of relative expression of HSC-related genes and representative novel genes in endothelial cells and hematopoietic cells from +9.5+/+ AGM explants. (E and F) Heatmap showing quantitation of relative expression of HSC-related genes and novel genes in endothelial cells and hematopoietic cells from +9.5+/+, +9.5+/−, and +9.5−/− AGM explants. (G and H) Cell cycle analysis of endothelial (CD31+c-Kit−) and hematopoietic cells (CD31+c-Kit+) from +9.5+/+, +9.5+/−, and +9.5−/− AGM explants (thr3e independent experiments and 5 litters: +9.5+/+ [n = 10]; +9.5+/− [n = 22]; +9.5−/− [n = 11]). (I and J) Flow cytometric analysis of the percentage of apoptotic cells (PI+Annexin V+) within the endothelial cells (CD31+c-Kit−) and hematopoietic cells (CD31+c-Kit+) from +9.5+/+, +9.5+/−, and +9.5−/− AGM explants (2 independent experiments and 2 litters: +9.5+/+ [n = 3]; +9.5+/− [n = 10]; +9.5−/− [n = 4]). (K) Model describing +9.5 cis-element requirement for HSC emergence from AGM and +9.5 cis-element–dependent genetic networks in AGM endothelial and hematopoietic cells. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed unpaired Student’s t test).
