Figure 5
Figure 5. Loss of Zeb2 alters adhesive properties and localization of hematopoietic progenitors in the fetal liver, ablating their mobilization/homing to the bone marrow. (A) Localization of hematopoietic cells in E11.5 wild-type and Zeb2−/ΔTie2-Cre fetal livers using the pan-hematopoietic marker CD45 (100× magnification) showing increased location of CD45+ cells near major blood vessels (arrows) in Zeb2−/ΔTie2-Cre fetal livers. (B) CD31/CD45 double staining of E11.5 wild-type and Zeb2−/ΔTie2-Cre fetal livers (100× and 400× magnification), confirming increased localization of CD45+ cells near CD31+ vasculature (arrows). (C) CD45/CD31 double-immunofluorescence staining (400× magnification) on E16.5 wild-type and Zeb2−/ΔVav-iCre fetal livers showing clustering within the fetal liver at a distance from the CD31+ vasculature (dotted line). (D) Decreased mobilization of hematopoietic cells to bone marrow could be observed in Zeb2−/ΔVav-iCre neonates by analyzing the redness (from hemoglobinized RBCs) and whole-mount X-gal staining of tibias (left panels). Eosin-counterstained histologic sections of these bones (100× magnification) showing decreased numbers of X-Gal–stained Cre-excised hematopoietic cells in Zeb2−/ΔVav-iCre tibias. (E) FACS quantification of hematopoietic progenitors (Lin−cKit+) in the peripheral blood of E12.0 Zeb2−/ΔTie2-Cre and heterozygote embryos relative to the wild-type control littermates. (F) qRT-PCR on total mRNA analyzing the expression levels of integrin β1 and Cxcr4 involved in HSC mobilization from sorted HSCs/HPCs (Lin−cKit) from mutant Zeb2−/ΔTie2-Cre and control E12.0 fetal livers. Two independent sorting experiments were performed and qRT-PCR was done in triplicate on biological replicates (n > 4 per genotype). (G) Zeb2−/ΔTie2-Cre and (H) Zeb2−/ΔVav-iCre cKit+-enriched fetal liver cells show significant increased migration potential toward low (15 ng/mL) and high (150 ng/mL) recombinant mSDF-1a concentrations in an in vitro Transwell migration assay compared with cKit+-enriched fetal liver cells from control littermates. Bars in panels E through H represent mean ± SD; *P < .05; **P < .01; ***P < .001.

Loss of Zeb2 alters adhesive properties and localization of hematopoietic progenitors in the fetal liver, ablating their mobilization/homing to the bone marrow. (A) Localization of hematopoietic cells in E11.5 wild-type and Zeb2−/ΔTie2-Cre fetal livers using the pan-hematopoietic marker CD45 (100× magnification) showing increased location of CD45+ cells near major blood vessels (arrows) in Zeb2−/ΔTie2-Cre fetal livers. (B) CD31/CD45 double staining of E11.5 wild-type and Zeb2−/ΔTie2-Cre fetal livers (100× and 400× magnification), confirming increased localization of CD45+ cells near CD31+ vasculature (arrows). (C) CD45/CD31 double-immunofluorescence staining (400× magnification) on E16.5 wild-type and Zeb2−/ΔVav-iCre fetal livers showing clustering within the fetal liver at a distance from the CD31+ vasculature (dotted line). (D) Decreased mobilization of hematopoietic cells to bone marrow could be observed in Zeb2−/ΔVav-iCre neonates by analyzing the redness (from hemoglobinized RBCs) and whole-mount X-gal staining of tibias (left panels). Eosin-counterstained histologic sections of these bones (100× magnification) showing decreased numbers of X-Gal–stained Cre-excised hematopoietic cells in Zeb2−/ΔVav-iCre tibias. (E) FACS quantification of hematopoietic progenitors (Lin−cKit+) in the peripheral blood of E12.0 Zeb2−/ΔTie2-Cre and heterozygote embryos relative to the wild-type control littermates. (F) qRT-PCR on total mRNA analyzing the expression levels of integrin β1 and Cxcr4 involved in HSC mobilization from sorted HSCs/HPCs (Lin−cKit) from mutant Zeb2−/ΔTie2-Cre and control E12.0 fetal livers. Two independent sorting experiments were performed and qRT-PCR was done in triplicate on biological replicates (n > 4 per genotype). (G) Zeb2−/ΔTie2-Cre and (H) Zeb2−/ΔVav-iCre cKit+-enriched fetal liver cells show significant increased migration potential toward low (15 ng/mL) and high (150 ng/mL) recombinant mSDF-1a concentrations in an in vitro Transwell migration assay compared with cKit+-enriched fetal liver cells from control littermates. Bars in panels E through H represent mean ± SD; *P < .05; **P < .01; ***P < .001.

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