MEPs prospectively isolated from Gata1^low marrow generate many mast cells when cultured under BMMC conditions. (A) Fold increase observed after 7 days in cultures seeded with either MEPs (CD34^lowCD16/CD32^low), CMPs (CD34⁺CD16/CD32^low), MPs (T1/ST2⁻), or MCPs (T1/ST2⁺) isolated from the marrow of wild-type and Gata1^low littermates, as indicated. By definition, wild-type MPs are a mixture of CMPs, MEPs, and GMPs. In the case of the Gata1^low mice, MP^low and MP^high cells (Figure 2B) were independently cultured. All of the cultures were seeded with 10³ cells/mL and stimulated with SCF plus IL-3. Results are presented as mean (± SD) of at least 3 separate experiments per experimental group. (B) Flow cytometry analysis for the presence of cells with the mastocytic phenotype in day 7 cultures of MEPs, CMPs, MPs, and MCPs, prospectively isolated from the marrow of wild-type and Gata1^low mice, as indicated. The c-Kit^highT1/ST2^high antigenic profile corresponds to that of mast cell precursors and the c-Kit^highFcϵRI⁺ one to that of mature mast cells.^6,21  Results from a representative experiment are shown. The mean (± SD) frequency obtained in at least 3 separate cultures per experimental point is reported in Table S2. (C) Quantitative RT-PCR analysis for the expression of Gata1 and of mast cell-specific genes by c-Kit^highFc_RI⁺ cells obtained after 7 days in cultures of the different progenitor cells prospectively isolated from the marrow of either wild-type or Gata1^low mice, as indicated. The mast cell-specific genes analyzed were represented by MMCP-6, a marker for early stages of mast cell maturation,³⁸  and MC-CPA and MMCP-7, markers for late differentiation stages of serosal²⁴  and dermal³⁹  mast cells, respectively. The expression profile of mast cells obtained from wild-type MCPs, for its low MMCP-7 level, is similar to that reported to be expressed by murine mast cells obtained in BMMCs.²⁴  On the other hand, the high level of expression of MMCP-7 in mast cells obtained from wild-type MCPs suggests that this population might be preferentially enriched for precursors of serosal mast cells. The mast cells obtained from all of the Gata1^low progenitors expressed a similar profile, resembling that of mast cells derived from wild-type CMPs. All of the sorted populations analyzed were more than 95% pure, on reanalyses (not shown). Results are expressed and mean (± SD) 2^−ΔCt obtained in at least 3 experiments per group. Single and double asterisks indicate expression levels statistically different (P < .01) from that of the progeny of CMPs purified from the same animal and between the progeny of Gata1^low cells and those of the corresponding wild-type progenitor cells.