Figure 6

P2-mediated embryonal expression of Runx1 is required for development of FL HPC. (A) RT-PCR analysis of RNA from E13.5 FL cells of WT and P2^neo/neo embryos. P2-mediated transcription in P2^neo/neo was largely attenuated, whereas P1-mediated transcription was apparently unaffected. (B) Colony forming activity of FL hematopoietic stem/progenitor cells. Colonies (>30 cells in size) were scored on day 7 of incubation. Shown are average numbers of colonies per FL ± S.E. of at least three different WT or P2^neo/neo embryos at E12.5 and E13.5. The difference between WT and P2^neo/neo colony number is significant (*) at P < 0.001 by Student's t test. (C and D) Altered expression of surface antigens on P2^neo/neo FL HPC. (C) Expression profiles of Gr-1, CD18, CD11b, CD11a, CD44 and c-Kit, in FL HPC from E17.5 WT and P2^neo/neo embryos. Representative data of at least three different experiments are shown as FACS dot plots; percentages of cells in each quadrant are indicated. Note the 2-fold decrease in the proportions of Gr-1⁺/CD18⁺ and CD11a⁺/CD11b⁺ (left panels) as well as CD44⁺/c-Kit⁺ (right panel), FL cells in P2^neo/neo compared to WT. (D) Proportion of CD34⁺ FL HPC. Profiles of WT and P2^neo/neo FL HPC, filled (blue) and unfilled (red) histograms, respectively, at E14.5, E16.5 and E18.5. Shown are representative results of at least three different experiments. Numbers at the upper right corners are percentages of CD34⁺ cells in WT (upper) and P2^neo/neo (lower). Note the marked decrease in the proportions of P2^neo/neo CD34⁺ HPC. (E) Analysis of committed FL T-cell precursors (TCPs). FACS analysis showing the distribution of B220⁺/c-Kit⁺/CD19^- FL HPC of E15.5 WT and P2^neo/neo embryos. Gated CD19^- precursors (R1) were analyzed for c-Kit and B220 expression. Note the 4-fold decrease in the proportions of c-Kit⁺/B220^low/D19^- TCPs (R2) in P2^neo/neo FL HPC. Data shown on the right are average ± S.E. of four independent experiments using four different mice. The difference between the percentages of TCPs from WT and P2^neo/neo FL HPC is significant at P < 0.005 by Student's t test. (F) Reduced capacity of P2^neo/neo FL HPC to colonize FTOC. Isolated E15.5 FL HPC from WT or P2^neo/neo embryos were set to differentiate, under limiting dilution conditions, for 16 days in E14.5 WT FTOC. Shown are percentage of unsuccessfully populated lobes per cultured cells and the calculated frequencies of three different independent experiments.