Figure 2.
Twist1−/−IACs exhibit impaired hematopoietic differentiation ability. (A) Yolk sac and placenta of Twist1−/− mouse embryo at 10.5 dpc. (B) Immunohistochemistry of AGM tissue in Twist1−/− embryos. Cryosections of 10.5-dpc Twist1−/−AGM were costained with c-Kit (an HSPC marker) and CD31 (an endothelial marker) and observed by confocal microscopy. c-Kit (green), CD31 (red), and TOTO-3 iodide (blue) staining are shown. Higher magnification of OA IACs is shown in dashed box. Confocal images were acquired using a 4× (top) and 40× (bottom) objective lens. (C) Flow cytometric analysis of IACs from 10.5-dpc AGM. Bar graph shows percentage of c-Kit+CD31+CD34+ cells per total cells prepared from the AGM region: WT C57BL/6 mice (n = 2), Twist1+/− mice (n = 2), and Twist1−/− mice (n = 2). (D) HSPC CFUs. Cells were prepared from AGM from indicated genotype embryos and cultured 9 days in semisolid medium containing SCF, IL-3, IL-6, and erythropoietin (Epo). The total number of hematopoietic colonies per AGM is shown. Data shown for each genotype are from 3 embryos (n = 3). *P < .05. (E) The number of each type of hematopoietic colony in indicated genotypes. CFU-GEMM (CFU of granulocytes, erythrocytes, monocytes, and macrophages), CFU-GM (CFU of granulocytes and macrophages), CFU-M (CFU of macrophages), CFU-G (CFU of granulocytes), and BFU-E (burst-forming unit of erythroid). Data shown for each genotype are from 3 embryos (n = 3). *P < .05. (F) In vitro B lymphopoiesis assay. Single cells from AGM were cocultured 14 days with OP9 stromal cells in the presence of IL-7. CD19+B220+ cells were analyzed by flow cytometry. Percentages are defined as the number of culture wells containing CD19+B220+ cells relative to total number of culture wells. (G) Erythroid (Gata1 and Klf1) and myeloid (Spi1 and Csf1r) gene expression analysis of IACs prepared from the WT, Twist1+/−, and Twist1−/− AGM region at 10.5 dpc. Comparative expression levels were calculated using the 2−ΔΔCT method. WT IAC sample served as a reference. Data shown are means ± SD of technical triplicate samples.

Twist1−/−IACs exhibit impaired hematopoietic differentiation ability. (A) Yolk sac and placenta of Twist1−/− mouse embryo at 10.5 dpc. (B) Immunohistochemistry of AGM tissue in Twist1−/− embryos. Cryosections of 10.5-dpc Twist1−/−AGM were costained with c-Kit (an HSPC marker) and CD31 (an endothelial marker) and observed by confocal microscopy. c-Kit (green), CD31 (red), and TOTO-3 iodide (blue) staining are shown. Higher magnification of OA IACs is shown in dashed box. Confocal images were acquired using a 4× (top) and 40× (bottom) objective lens. (C) Flow cytometric analysis of IACs from 10.5-dpc AGM. Bar graph shows percentage of c-Kit+CD31+CD34+ cells per total cells prepared from the AGM region: WT C57BL/6 mice (n = 2), Twist1+/− mice (n = 2), and Twist1−/− mice (n = 2). (D) HSPC CFUs. Cells were prepared from AGM from indicated genotype embryos and cultured 9 days in semisolid medium containing SCF, IL-3, IL-6, and erythropoietin (Epo). The total number of hematopoietic colonies per AGM is shown. Data shown for each genotype are from 3 embryos (n = 3). *P < .05. (E) The number of each type of hematopoietic colony in indicated genotypes. CFU-GEMM (CFU of granulocytes, erythrocytes, monocytes, and macrophages), CFU-GM (CFU of granulocytes and macrophages), CFU-M (CFU of macrophages), CFU-G (CFU of granulocytes), and BFU-E (burst-forming unit of erythroid). Data shown for each genotype are from 3 embryos (n = 3). *P < .05. (F) In vitro B lymphopoiesis assay. Single cells from AGM were cocultured 14 days with OP9 stromal cells in the presence of IL-7. CD19+B220+ cells were analyzed by flow cytometry. Percentages are defined as the number of culture wells containing CD19+B220+ cells relative to total number of culture wells. (G) Erythroid (Gata1 and Klf1) and myeloid (Spi1 and Csf1r) gene expression analysis of IACs prepared from the WT, Twist1+/−, and Twist1−/− AGM region at 10.5 dpc. Comparative expression levels were calculated using the 2−ΔΔCT method. WT IAC sample served as a reference. Data shown are means ± SD of technical triplicate samples.

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