Effect of Tie2Cre-mediated deletion of SCL gene in the development and function of definitive hematopoietic stem cells. (A) Total RNA RT-PCR analysis of E12.5 SCLfl/fl Tie2Cre- and SCLfl/fl Tie2Cre+ fetal liver cells. (B-C) FACS analysis of hematopoietic progenitors and stem cells in fetal livers of E12.5 SCLfl/wt Tie2Cre+ and SCLfl/fl Tie2Cre+ fetuses. (D) Total cellularity and hematopoietic colony-forming potential in SCLfl/wt Tie2Cre+ and SCLfl/fl Tie2Cre+ E12.5 fetal livers (experiments: n = 3; embryos fl/wt: n = 5; embryos fl/fl: n = 6; cellularity: P < .08; CFC: P < .05; t test). Data are presented as means and standard deviations. (E) Single-colony 3-primer PCR analysis of SCL gene excision in SCLfl/wt Tie2Cre+ and SCLfl/fl Tie2Cre+ E12.5 fetal liver–derived colonies. (F) Acetylcholinesterase staining of SCLfl/wt Tie2Cre+ and SCLfl/fl Tie2Cre+ fetal liver cultures 10×. Data are presented as means and standard deviations. (G) Percentage contribution of fetal liver–derived CD45+ cells in the peripheral blood of lethally irradiated recipients at 3 to 4 weeks and more than 5 months after transplantation (experiments: n = 3; donor embryos: n = 6 per genotype; recipients: n = 17 per genotype; P value at 3 to 4 weeks < .01; P value at more than 5 months > .25; t test). Data are presented as means and standard deviations. (H) Three-primer PCR analysis of SCL gene excision in peripheral blood from recipient animals, 5 months after transplantation with E12.5 SCLfl/wt Tie2Cre+ and SCLfl/flTie2Cre+ E12.5 fetal liver cells. Note that the wt band in recipients that received fl/fl fetal liver cells is derived from support BM cells. sBM indicates recipients that received support BM only. (I) SCL is vital for the commitment of mesoderm to the hematopoietic lineage, but thereafter is no longer essential for the establishment of the LTR-HSC pool in the fetal liver. Yet SCL is required for proper differentiation of primitive and definitive erythroid (E) cells and megakaryocytes (Me) in the yolk sac and the fetal liver.
