Figure 4
Figure 4. Zeb2 is not essential for LSK marker–enriched HSC migration to the fetal liver, but blocks definitive hematopoietic differentiation at an early stage. (A) FACS quantification of HSCs (LSK) and early progenitors (LK, Lin−cKit+Sca1−) in wild-type and mutant fetal liver. Relative 2.2-fold increase of HSCs (LSK) and relative 40% decrease of early committed progenitors (LK) in Zeb2−/Tie2-Cre fetal livers compared with wild-type littermates. (B) Quantification of total hematopoietic colonies formed after in vitro differentiation in methylcellulose of cells isolated from various hematopoietic organs: E10.5 yolk sacs, E11.5 AGM, E11.5-E12.0 fetal livers, and E12.0 peripheral blood. Equal amounts of cells per organ were plated in triplicate, and the average number of colonies obtained was normalized to the wild-type controls. For each organ, at least 2 experiments were performed. (C) Quantification of mixed (CFU-GEMM), erythroid (BFU-E), megakaryocyte (CFU-Mk), and granulocyte/monocyte (CFU-G/M) colonies formed after in vitro differentiation of E11.5 fetal livers cells of Zeb2−/ΔTie2-Cre and heterozygote embryos relative to control littermates. (D) Quantification of total (CFU) and mixed (CFU-GEMM) hematopoietic colonies formed after in vitro differentiation of cells isolated from E14.5 fetal livers of Zeb2−/ΔVav-iCre and heterozygote embryos relative to control littermates. (E) In vitro differentiation of wild-type and Zeb2-null ES cells and quantification of hemoglobin-positive (arrows) embryoid bodies at day 9. Shown is the quantification of hematopoietic colonies formed after in vitro differentiation of Zeb2-null embryoid body–derived cells relative to wild-type embryoid body–derived cells. (F) qRT-PCR on total RNA from sorted Lin−cKit+ HSCs/HPCs from mutant and control E12.0 fetal livers, analyzing the mRNA levels of various important hematopoietic transcription factors involved in HSC differentiation. Two independent sorting experiments were performed, and qRT-PCR was done in triplicate on biological replicates (n > 4 per genotype). Bars in A-F represent mean ± SD; *P < .05; **P < .01; ***P < .001.

Zeb2 is not essential for LSK marker–enriched HSC migration to the fetal liver, but blocks definitive hematopoietic differentiation at an early stage. (A) FACS quantification of HSCs (LSK) and early progenitors (LK, Lin−cKit+Sca1−) in wild-type and mutant fetal liver. Relative 2.2-fold increase of HSCs (LSK) and relative 40% decrease of early committed progenitors (LK) in Zeb2−/Tie2-Cre fetal livers compared with wild-type littermates. (B) Quantification of total hematopoietic colonies formed after in vitro differentiation in methylcellulose of cells isolated from various hematopoietic organs: E10.5 yolk sacs, E11.5 AGM, E11.5-E12.0 fetal livers, and E12.0 peripheral blood. Equal amounts of cells per organ were plated in triplicate, and the average number of colonies obtained was normalized to the wild-type controls. For each organ, at least 2 experiments were performed. (C) Quantification of mixed (CFU-GEMM), erythroid (BFU-E), megakaryocyte (CFU-Mk), and granulocyte/monocyte (CFU-G/M) colonies formed after in vitro differentiation of E11.5 fetal livers cells of Zeb2−/ΔTie2-Cre and heterozygote embryos relative to control littermates. (D) Quantification of total (CFU) and mixed (CFU-GEMM) hematopoietic colonies formed after in vitro differentiation of cells isolated from E14.5 fetal livers of Zeb2−/ΔVav-iCre and heterozygote embryos relative to control littermates. (E) In vitro differentiation of wild-type and Zeb2-null ES cells and quantification of hemoglobin-positive (arrows) embryoid bodies at day 9. Shown is the quantification of hematopoietic colonies formed after in vitro differentiation of Zeb2-null embryoid body–derived cells relative to wild-type embryoid body–derived cells. (F) qRT-PCR on total RNA from sorted Lin−cKit+ HSCs/HPCs from mutant and control E12.0 fetal livers, analyzing the mRNA levels of various important hematopoietic transcription factors involved in HSC differentiation. Two independent sorting experiments were performed, and qRT-PCR was done in triplicate on biological replicates (n > 4 per genotype). Bars in A-F represent mean ± SD; *P < .05; **P < .01; ***P < .001.

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