American Society of Hematology

Figure 3

$Figure 3. Analysis of Mef2C−/− embryos documents that specification into primitive and definitive hematopoietic lineages occurs in the absence of Mef2C. (A) Quantitative real-time PCR analysis reveals a reduction of Mef2C expression in hematopoietic tissues of Scl−/− mice, but not in the heart. *P < .05; N = 5. (B) Gross morphologic comparison of E9.0 Scl−/− and Mef2C−/− yolk sac and embryos documents that the complete hematopoietic specification defect in Scl−/− embryos is not recapitulated in Mef2C−/− embryos. Yet the vascular network in Mef2C−/− yolk sacs is poorly developed. pl indicates placenta; ch, caudal half; h, heart. Dotted lines in the embryo proper indicate dissected tissues. (C) FACS analysis of E9.0 yolk sac cells showed that CD71+Ter119+ primitive erythroid cells are generated in the absence of Mef2C, albeit at reduced levels compared with Mef2C+/− controls. Values represent mean frequency plus or minus SD (N = 4). (D) FACS analysis of ckit+CD41+ nascent hematopoietic progenitors demonstrated a striking reduction of progenitors in the caudal half of the embryo proper but not in the placenta or yolk sac. Values represent mean frequency plus or minus SD (Mef2C+/−; N = 4, Mef2C−/−; N = 5). (E) In agreement with the loss of ckit+CD41+ cells shown by FACS, direct plating of dissociated hematopoietic tissues on methylcellulose showed a severe reduction in numbers of clonogenic progenitors per embryo equivalent (ee) in Mef2C−/− caudal half (CH). As expected, colony formation in placenta (PL) and yolk sac (YS) was unaffected. **P < .01 of total colony numbers. (F) Coculture on OP9 stroma before plating on methylcellulose shows that the ability to generate hematopoietic colonies from the Mef2C−/− caudal half was partially rescued. As expected, placenta and yolk sac hematopoietic potential remained comparable with controls. *P < .05 of total colony numbers. (G) FACS analysis of B-lymphoid potential after coculture on OP9 stroma and methylcellulose documents that all hematopoietic tissues in Mef2C−/− embryos possess B-lymphoid potential. Numbers in FACS plots indicate the fraction of B220+ plus or minus SD (n = 3) cells within the CD45 hematopoietic gate.$

Analysis of Mef2C^−/− embryos documents that specification into primitive and definitive hematopoietic lineages occurs in the absence of Mef2C. (A) Quantitative real-time PCR analysis reveals a reduction of Mef2C expression in hematopoietic tissues of Scl^−/− mice, but not in the heart. *P < .05; N = 5. (B) Gross morphologic comparison of E9.0 Scl^−/− and Mef2C^−/− yolk sac and embryos documents that the complete hematopoietic specification defect in Scl^−/− embryos is not recapitulated in Mef2C^−/− embryos. Yet the vascular network in Mef2C^−/− yolk sacs is poorly developed. pl indicates placenta; ch, caudal half; h, heart. Dotted lines in the embryo proper indicate dissected tissues. (C) FACS analysis of E9.0 yolk sac cells showed that CD71⁺Ter119⁺ primitive erythroid cells are generated in the absence of Mef2C, albeit at reduced levels compared with Mef2C^+/− controls. Values represent mean frequency plus or minus SD (N = 4). (D) FACS analysis of ckit⁺CD41⁺ nascent hematopoietic progenitors demonstrated a striking reduction of progenitors in the caudal half of the embryo proper but not in the placenta or yolk sac. Values represent mean frequency plus or minus SD (Mef2C^+/−; N = 4, Mef2C^−/−; N = 5). (E) In agreement with the loss of ckit⁺CD41⁺ cells shown by FACS, direct plating of dissociated hematopoietic tissues on methylcellulose showed a severe reduction in numbers of clonogenic progenitors per embryo equivalent (ee) in Mef2C^−/− caudal half (CH). As expected, colony formation in placenta (PL) and yolk sac (YS) was unaffected. **P < .01 of total colony numbers. (F) Coculture on OP9 stroma before plating on methylcellulose shows that the ability to generate hematopoietic colonies from the Mef2C^−/− caudal half was partially rescued. As expected, placenta and yolk sac hematopoietic potential remained comparable with controls. *P < .05 of total colony numbers. (G) FACS analysis of B-lymphoid potential after coculture on OP9 stroma and methylcellulose documents that all hematopoietic tissues in Mef2C^−/− embryos possess B-lymphoid potential. Numbers in FACS plots indicate the fraction of B220⁺ plus or minus SD (n = 3) cells within the CD45 hematopoietic gate.

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