Phenotypic and functional analyses of CD41-sorted cell fractions of E11 and E12 embryonic tissues. (A-D,J) E11 AGM, (H,I) E12 AGM, and (E-G) E11 YS. (A,E,H) Flow cytometric analysis of E11 AGM, E11 YS, and E12 AGM, respectively. Representative sorting gates (red represents CD41^high; green, CD41^int; and blue, CD41⁻). The percentage of cells in each fraction is indicated. (B,F) In vitro colony-forming unit in culture (CFU-C) analyses show the number of total hematopoietic progenitors per 1000 cells in each CD41-sorted fraction of E11 AGM and E11 YS cells. Each sample was analyzed in triplicate for each dilution. n = 4 for E11 AGM, and n = 2 for E11 YS. (C,G,I) In vivo hematopoietic repopulation analysis of CD41-sorted fractions of E11 AGM, E11 YS, and E12 AGM 4 months after transplantation. Percentage of repopulated mice showing greater than 10% donor chimerism in peripheral blood is shown. Numbers above columns indicate the number of mice repopulated/number of mice transplanted. Dose of injected cells is indicated as embryo equivalents (ee). n = 3 for E11 AGM, n = 4 for E11 YS, and n = 2 for E12 AGM. nd indicates not done. (D) CD41 immunostaining of E11 Ly6A GFP embryo section showing the ventral wall of the aorta. (Top panel) Red fluorescent CD41 expression in hematopoietic cells. (Middle panel) Green fluorescent Ly6A GFP expression in hematopoietic cells and some endothelial cells. (Bottom panel) Merged fluorescence. Yellow represents overlap of CD41 and Ly6A GFP expression in hematopoietic cells closely associated with the aortic endothelium. Image acquisition was from LSM510NLO/FCS confocal microscope (Carl Zeiss BV) with 40×/1.3 NA water objective and Vectashield medium (Vector Laboratories). LSM image software was used (Carl Ziess BV). (J) Representative semiquantitative PCR analysis of hematopoietic tissue DNA from (upper panel) a primary recipient injected with 3 ee of E11 AGM CD41^int cells and (lower panel) peripheral blood DNA from 6 secondary recipients injected with BM cells from the primary recipient 4 months after transplantation. Donor indicates the human β-globin PCR fragment, and Myo indicates the myogenin DNA normalization control PCR fragment. DNA dilution controls (0%-100%) were used to quantitate percentages of donor chimerism that are indicated below each lane. PB indicates peripheral blood; Th, thymus; LN, lymph node; Sp, spleen; M, myeloid (sorted cells from BM); E, erythroid (sorted from BM); L, lymphoid (sorted from BM); B, B lymphoid (sorted from spleen); and T, T lymphoid (sorted from spleen).