Fate-tracing embryonic hematopoietic stem cells into adulthood. (A) Outline of the experimental design. Timed matings were set up between HSC-SCL-Cre-ER^T males and R26R-EYFP females. Pregnant females were allocated into 3 experimental groups. Group I females did not receive tamoxifen injections, while group II and group III females received tamoxifen injections at E10.5 (1 mg) and E11.5 (2 mg). Group II pregnancies were terminated at E14.5, embryonic fetal liver cells were harvested, analyzed for the expression of Eyfp, and transplanted into lethally irradiated Ly5.1 recipients. Group III neonates were delivered by Caesarean section on day E19.5 and fostered to lactating females. After weaning group I and III, newborn mice were genotyped for the presence of the HSC-SCL-Cre-ER^T and R26R-EYFP transgenes. (B) Fate-mapping transplanted (group II) and in vivo–remaining (group III) marked embryonic LT-HSCs: a proportion of group II (left panel) and group III (right panel) embryonic LT-HSCs was genetically marked (EYFP⁺) by maternal tamoxifen injections. Group II E14.5 fetal liver cells were transplanted into lethally irradiated congenic adult recipients. The proportion of marked fetal liver LT-HSCs was determined by measuring the hematopoietic contribution of these cells within recipients 5 months after transplantation (group II, left panel). In parallel the contribution of in vivo–remaining marked fetal liver LT-HSCs to adult bone marrow hematopoiesis was investigated (group III, right panel). If the proportion of EYFP⁺ hematopoietic cells within group III adults were found to be significantly lower than within recipient adults (group II), this would mean that the de novo generation of HSCs within the embryo was not completed by the time of transplantation (E14.5). If new LT-HSCs were generated after day 14.5 of gestation, these cells would not be marked (absence of tamoxifen) and would therefore decrease the proportion of group III adult EYFP⁺ hematopoietic cells compared to group II. (C) Five months after birth or transplantation, respectively, flow cytometric analysis of Eyfp expression in hematopoietic organs of group I, II, and III mice was carried out. Representative plots of total bone marrow from each group are shown. Percentages represent means. FSC, forward light scatter. (D) Comparison of mean percentages of Eyfp-expressing cells within different hematopoietic populations (bone marrow: total cells, neutrophils [Mac-1⁺Gr-1^high], B cells [B220⁺], erythroid [Ter119⁺], HSCs [KSL]; thymus: double-positive cells [CD4⁺CD8⁺]) of group II (□) and group III mice (▪). No statistical significant difference between the mean percentages of groups II and III was noted. In group II recipients 99.1 ± SD 0.5% of bone marrow cells were donor derived (CD45.2⁺). Error bars indicate standard deviations. Group I (n = 2); group II (n = 4 donor embryos, n = 2 recipients per donor); group III (n = 4).