Expression and domain-specific function of GATA-2 during differentiation of the hematopoietic precursor cells in midgestation mouse embryos by Naoko Minegishi, Norio Suzuki, Tomomasa Yokomizo, Xiaoqing Pan, Tetsuhiro Fujimoto, Satoru Takahashi, Takahiko Hara, Atsushi Miyajima, Shin-ichi Nishikawa, and Masayuki Yamamoto Blood Volume 102(3):896-905 August 1, 2003 ©2003 by American Society of Hematology

GFP is expressed in transgenic and knock-in mouse embryos. GFP is expressed in transgenic and knock-in mouse embryos. (A) Structure of 7.0IS GFP and 3.1ISIGII GFP transgenic constructs. Top line represents the structure of the mouse GATA-2 gene. Black arrow and blue arrow indicate the transcription and translation initiation sites, respectively. The map of the murine GATA-2 gene, modified to express EGFP in knock-in mouse, is also shown. (B-C) TG-GFP expression profile of the 3.1ISIGII GFP transgenic mouse E10.5 embryo (line no. 685). Panel C shows a ventral view of the AGM region. Asterisk and m indicate the positions of dorsal aorta and mesonephros, respectively. (D) Immunostaining with anti-GFP antibody confirming the TG-GFP expression (brown) in the lining of the aortic wall and in the cells ventral to the dorsal aorta (red arrow) of an E11.5 embryo (line no. 685). (E) Outline of the planar orientation of E8.0 embryo.17 (F) Confocal microscopic image of the region corresponding to the red square in panel E of line no. 798 transgenic embryo. Broken line in panel F indicates the position of the dorsal aorta. (G) Sagittal image of E9.0 embryo (line no. 798). (H-I) KI-GFP expression in the E10.5 KI-heterozygous embryo. Panel I shows the AGM region of the KI heterozygous embryo. Asterisk and m indicate the position of the dorsal aorta and mesonephros, respectively. (J) Anti-GFP antibody staining of the transverse section of the aorta of E11.5 embryo. (K) KI-GFP fluorescence along the dorsal aorta of E8.0 KI-heterozygous embryo. (L) Immunostaining of E11.5 AGM region with FITC anti–GATA-2 antibody (left), and with control rat antibody (right). (M) Immunohistochemical staining with anti–GATA-2 antibody showing endogenous GATA-2 expression in the lining of the dorsal aorta and in neighboring mesenchymal cells of an E11.5 embryo. See “Materials and methods.” Original magnification for panels D, J, and M is × 100. Naoko Minegishi et al. Blood 2003;102:896-905 ©2003 by American Society of Hematology

TG-GFP+ and KI-GFP+ cells in the AGM region express CD34 and PCLP1 antigens. TG-GFP+ and KI-GFP+ cells in the AGM region express CD34 and PCLP1 antigens. (A) TG-GFP expression in the AGM region of E11.5 embryo (line no. 685). The cells in the region between 2 yellow lines were analyzed. Original magnification, × 50. (B-C) Flow cytometric analysis of 3.1ISIGII GFP transgenic embryos (line no. 685, B) and KI-heterozygous embryos (C). Naoko Minegishi et al. Blood 2003;102:896-905 ©2003 by American Society of Hematology

TG-GFP+ cells from the AGM region have hematogenic activity. TG-GFP+ cells from the AGM region have hematogenic activity. (A) TG-GFP+/CD45+ and TG-GFP+/CD45- cell fractions of the AGM region were sorted by FACS. (B-C) Phase-contrast microscopy analysis of hematopoietic colonies with a cobblestone-like appearance after 4 days of coculture with OP-9 cells. Cells in panel B are from the TG-GFP+/CD45+ fraction, while cells in panel C are from the TG-GFP+/CD45- fraction. The number of colony-forming cells is shown in Table 1. (D) Appearance of TG-GFP+/CD45- cell colonies. TG-GFP+/CD45- cells were cultured in medium containing OSM, SCF, b-FGF, and LIF. Note that colonies contain both adherent cells (dark cells) and floating cells (bright cells). (E) Flow cytometric analysis of the cells in panel D. Majority of the cells show abundant CD45 expression without GFP fluorescence. Original magnification, panels B-D, × 100. Naoko Minegishi et al. Blood 2003;102:896-905 ©2003 by American Society of Hematology

GATA-2 is expressed in CD45- cells in the primary cultured AGM region. GATA-2 is expressed in CD45- cells in the primary cultured AGM region. (A) Flow cytometric analysis of 7-day–cultured AGM cells of KI-heterozygous embryos. (B) RT-PCR analysis of the cells in each sorted fraction from the primary culture of the wild-type AGM cells. Representative results of 2 of 4 mRNA analyses are shown. Naoko Minegishi et al. Blood 2003;102:896-905 ©2003 by American Society of Hematology

Retroviral expression of GATA-2 in hematogenic cells suppresses hematopoietic cell production. Retroviral expression of GATA-2 in hematogenic cells suppresses hematopoietic cell production. (A) Structure of the MSCV–GATA-2–IRES-EGFP and MSCV-IRES-EGFP retrovirus vectors. (B) Western blotting analysis showing modest GATA-2 expression in LO cells8 infected with the MSCV–GATA-2–IRES-EGFP virus (lane 2) and low level endogenous GATA-2 expression in the cells infected with MSCV-IRES-EGFP virus (lane 3). P815 mast cells were used as a positive control for this experiment (lanes 1). (C-D) Fluorescent microscopy analysis of V-GFP+ cells 9 days after culture. Cells infected by MSCV-IRES-EGFP (C) and MSCV–GATA-2–IRES-EGFP (D) are shown. The red arrow in panel C indicates the round-shaped V-GFP+ cells detached from the dishes. (E) Surface-marker profile of V-GFP+ cells infected with MSCV–GATA-2–IRES-EGFP virus or MSCV-IRES-EGFP virus. At day 7, 5.6% or 3.7% of cells were infected by MSCV–GATA-2–IRES-EGFP V-GFP+ virus or MSCV-IRES-EGFP virus, respectively. The bottom 3 panels demonstrate the CD34 and PCLP1 expression on the V-GFP+ cells at day 7. (F) The percentage of CD45+ cells. CD45+ cells in the fraction infected with either MSCV–GATA-2–IRES-EGFP virus (•) or MSCV-IRES-EGFP virus (○) in primary AGM culture. Original magnification, panels C-D, × 100. Naoko Minegishi et al. Blood 2003;102:896-905 ©2003 by American Society of Hematology

GATA-2 gene dosage has an influence on preservation of hematogenic precursor cells. GATA-2 gene dosage has an influence on preservation of hematogenic precursor cells. (A-C) Immunohistochemical analysis of the E9.5 yolk sacs of a wild-type (A), KI-heterozygous (B), and KI-homozygous (C) conceptuses with CD45 antibody. These embryos have 27 to 31 somites. A significantly increased number of CD45+ cells were observed in the homozygous mutants. Original magnification, panels A-C, × 100. (D) The number of CD45+ cells/500 μm2of wild-type, KI-homozygous (4 embryos each), and KI-heterozygous (3 embryos) yolk sacs are presented as the mean ± standard deviation. (E) Hematopoietic cell production of AGM cells from 3 E11.5 KI-heterozygous mutant embryos and their wild-type littermates. The percentages of CD45+ (▦) and CD34+ (□) cells harvested on day 10 of culture are plotted. Naoko Minegishi et al. Blood 2003;102:896-905 ©2003 by American Society of Hematology

The function of GATA-2 is domain-specific in primary cultures of AGM cells. The function of GATA-2 is domain-specific in primary cultures of AGM cells. (A) Schematic representation of the GATA-2 domain mutants integrated into the genome of MSCV-IRES-EGFP virus. (B) Western blot analysis of infected LO cells with anti–GATA-2 antibodies, C-20 and RC.1.1.1, and anti-GFP antibody. Note comparable expression of each mutant protein. (C) Numbers of CD45+ cells in cultures of AGM cells infected with each virus. Results are shown relative to control cultures. Each point and bar represents the mean ± standard deviation of 3 independent experiments. The results of del236 and VP-F are 5.21 ± 3.33 and 1.99 ± 0.72, respectively; and del236 and VP-F resulted in significantly higher numbers of CD45+ cells than wild-type GATA-2 (P < .05, Student t test). The significance of the differences was confirmed by repeated experiments (wild-type del 236, n = 12, P < .0001; wild-type VP, n = 7, P < .0001; Student t test). Naoko Minegishi et al. Blood 2003;102:896-905 ©2003 by American Society of Hematology

GATA-2 functions as a gatekeeper in hematogenic precursor cells. GATA-2 functions as a gatekeeper in hematogenic precursor cells. GATA-2 is expressed in endothelial cells in the early stage of angiogenesis, and TG-GFP specifically recapitulated GATA-2 expression in endothelial and/or mesenchymal cells with the potential to undergo hematopoietic differentiation. The high-level expression of GATA-2 in hematogenic precursor cells blocks subsequent differentiation of the cells into hematopoietic lineages. Cells with a light-blue cytoplasm represent endothelial cells. Cells with rhomboid shape and spheroid shape represent mesenchymal and hematopoietic cells, respectively. The blue nuclei and the green cytoplasm represent the cells expressing GATA-2 and TG-GFP, respectively. Naoko Minegishi et al. Blood 2003;102:896-905 ©2003 by American Society of Hematology