Figure 1.

The Most Immature AGM HPCs Are cKit^hiCD31^hi

(A) Flow-cytometric dot plots of representative E11 G2V AGM cells analyzed for side scatter (SSC-A) and CD31 expression (left) and cKit and Gata2 (G2) expression of CD31⁺ cells (right). Cell percentage (±SEM) within gated regions. Hi, high; lo, low; int, intermediate; med, medium (lo+int).

(B) Hematopoietic progenitor numbers per lineage per embryo equivalent (ee) of E10 (32–37 somite pairs, sp; n = 4; embryos/experiment: 4, 4, 4, and 4) and E11 (43–48 sp; n = 3; embryos/experiment: 3, 6, 5, and 7) sorted AGM fractions in the colony-forming unit-culture (CFU-C) assay. GEMM, granulocyte-erythroid-macrophage-megakaryocyte; GM, granulocyte-macrophage; M, macrophage; G, granulocyte; BFU-E, burst-forming unit-erythroid. Error bars represent mean ± SEM. For E10 GEMMs: ^∗cKit^hiG2^int versus cKit^loG2^lo/int. For E11 GEMMs: ^∗cKit^hiG2^lo versus cKit^loG2^lo/int, and ^∗∗cKit^hiG2^int versus cKit^loG2^lo/int. For E11 GMs: ^∗cKit^hiG2^int versus cKit^loG2^lo/int.

(C and D) E11 G2V AGM single cells were sorted for (C) CD31⁺cKit^hiG2^int (11 embryos, 41–47 sp) and (D) CD31⁺cKit⁺G2^hi cells (20 embryos; 40–49 sp) and deposited in methylcellulose. CFU-Cs were counted at days 9–11 (total CD31⁺cKit^hiG2^int = 169; total CD31⁺cKit⁺G2^hi = 105). Each CFU (individual dot) was examined for colony type and plotted against log₁₀-normalized mean fluorescence intensities (MFIs) for expression of (Ci and Di) G2, (Cii and Dii) cKit, and (Ciii and Diii) CD31. Red lines indicate mean ± SEM.

(E) HSC activity in sorted AGM fractions as measured by in vivo transplantation (peripheral blood donor chimerism at 4 months post-injection). Cell numbers injected: for cKit^hiG2^lo, 950–1963, 2.9–5.4 ee; for cKit^hiG2^int, 912–2,048, 2.5–6.2 ee; for cKit^loG2^med, 4,530–12,523, 3.3–5.9 ee (42–50 sp; n = 4; embryos/experiment: 7, 9, 12, and 14).