Sox18-sustained expression promotes the proliferation of early hematopoietic precursors, whereas Sox17-ectopic expression results in increased cell death. (A) iSox18 or iSox17 CD41⁺CD34⁻ hematopoietic precursors sorted from day 5 EB were cultured for 6 days with (+) or without (−) doxycycline. Total cell count was determined every other day (n = 3). (B) Apoptosis detection by annexin V staining in CD41⁺CD34⁻ cells sorted from iSox17 or iSox18 EBs day 5 and cultured for 24 hours with or without doxycycline. (C) The cell-cycle status was assessed after 1 hour of BrdU pulse on iSox17 embryonic stem (ES) cells cultured for 2 days with or without doxycycline. (D) Apoptosis detection in iSox17 ES cells cultured for 48 hours with or without doxycycline using 7-amino-actinomycin D (7AAD) and annexin V staining. (E) Absolute quantification of Sox7, Sox17, and Sox18 mRNA by real-time PCR was performed on sorted hemangioblast-enriched precursors (Flk1⁺) from day 3 EBs and in day 1 to 4 hemangioblast-derived blast colonies. Absolute quantification of transcripts was calculated using linear regression analysis on standard calibration of cDNA encoding each of these Sox genes. Data are presented as femtomoles per 5 μg of total RNA. (F) Analysis of Sox18-hCD4 expression relative to Flk1 and CD41 expression in day 3 EBs derived from ES-cell clone transgenic for a BAC hCD4-Sox18 construct. (G) Analysis of Sox18-hCD4 and CD41 expression in Flk1⁺ cells sorted from day 3 EBs and cultured for 72 hours in hemangioblast culture condition. (H) Schematic model depicting the expression pattern for Sox7, Sox17, and Sox18 genes during embryonic hematopoiesis.