Identification and characterization of 2 types of erythroid progenitors that express GATA-1 at distinct levels by Norio Suzuki, Naruyoshi Suwabe, Osamu Ohneda, Naoshi Obara, Shigehiko Imagawa, Xiaoqing Pan, Hozumi Motohashi, and Masayuki Yamamoto Blood Volume 102(10):3575-3583 November 15, 2003 ©2003 by American Society of Hematology

Expression analyses of GFP and GATA-1 in hematopoietic progenitors. Expression analyses of GFP and GATA-1 in hematopoietic progenitors. (A) The Lin- fraction from the bone marrow of the G1-HRD-GFP transgenic mouse was stained with APC-conjugated anti-c-Kit antibody and analyzed by FACS. The percentage of cells in each quadrangle is shown. (B) Immunostaining of intracellular GATA-1 in the sorted cells. Almost all GFP+ cells in the Lin- fraction were GATA-1 positive (top panel). Most GFP- cells in the Lin- fraction were GATA-1-negative (middle). The Lin-/c-Kit+/GFP- fraction contains approximately half of GATA-1-positive cells (bottom panel). Norio Suzuki et al. Blood 2003;102:3575-3583 ©2003 by American Society of Hematology

Colony assay of GFP+ cells in the bone marrow. Colony assay of GFP+ cells in the bone marrow. (A) The Lin-/c-Kit+ fraction is subdivided into 4 fractions with the expression of GFP and CD71. The percentage of cells in each quadrangle is shown. Cells in each fraction were sorted and cultured with only Epo (B), Epo and SCF (C), or Epo, SCF IL-6, and IL-3 (D). Colonies were counted 3 days (B) or 7 days (C, D) after culturing. (E) A colony derived from CFU-E in the CD71+/GFP+ fraction was stained by benzidine (left panel, blue staining), and some of the cells in the colony were detected by GFP expression under the fluorescent microscope (right panel). (F) A mixed colony derived from a CD71-/GFP+ cell was observed by bright (top panel) and fluorescent (middle panel) microscopy. GFP expression was detected in red cells (erythrocytes [E]) and large cells (megakaryocytes [Mk]) but not in small white cells (granulocytes [G]) and large extended cells (macrophages [Mc]). The top and middle panels were merged in the bottom panel (F). Original magnifications, × 200 (E) and × 100 (F). Norio Suzuki et al. Blood 2003;102:3575-3583 ©2003 by American Society of Hematology

Gene expression and morphology of GFP+ progenitor cells. Gene expression and morphology of GFP+progenitor cells. (A) RNA was extracted from the sorted cells in the indicated fractions, and RT-PCR was performed to detect GATA-1, GATA-2, EpoR, c-mpl, and IL-7R expression. The bone marrow Lin-/c-Kit+ hematopoietic progenitor fraction was subdivided into 4 fractions (lanes 1-4). EEP and LEP fractions are represented in lanes 3 and 4, respectively. GFP- (lane 5) and GFP+ (lane 6) cells in the Lin-/c-Kit- fractions also are shown. (B) The morphology of the sorted cells are shown by Wright-Giemsa staining. Proerythroblast-like cells were detected in the LEP fraction (GFP+/CD71+; i,ii). It is likely that erythroid progenitors are i, ii, iii, iv, v, and vi in the LEP and EEP (GFP+/CD71-) fractions. All cells in the GFP-/CD71+ fraction contained azurophil granules (xvi-xx). Norio Suzuki et al. Blood 2003;102:3575-3583 ©2003 by American Society of Hematology

Quantitative analyses of GATA-1 and GATA-2 mRNAs during erythroid differentiation. Quantitative analyses of GATA-1 and GATA-2 mRNAs during erythroid differentiation. (A) Bone marrow cells were analyzed for CD71 and Ter119 expression. Cells in open boxes (i-iii) were sorted and analyzed for GATA-1 expression level in B. The percentage of cells in each box is shown. (B) The relative GATA-1 and GATA-2 mRNA levels at different stages of erythroid cell development were measured by quantitative RT-PCR and normalized to the level of GAPDH mRNA. Erythroid differentiation is indicated by the x-axis from left to right. Norio Suzuki et al. Blood 2003;102:3575-3583 ©2003 by American Society of Hematology

FACS analysis and colony assay of the normal and anemic spleen of G1-HRD-GFP transgenic mouse. FACS analysis and colony assay of the normal and anemic spleen ofG1-HRD-GFPtransgenic mouse. (A) Spleen mononucleated cells (MNCs) were obtained from G1-HRD-GFP transgenic mice before (PHZ-, left) and after (PHZ+, right) injection of phenylhydrazine (PHZ). The MNC, Lin- fraction, and Lin-/c-Kit+ fraction were analyzed by FACS (top, middle, and bottom panels, respectively). The percentage in each quadrangle is shown. The dotted lines show the mean intensities of CD71 expression in CD71+/GFP+ fractions (bottom panels). (B) Results of colony assay in the normal and anemic spleens. Two thousand cells from the indicated fractions were analyzed. Fractions i and ii are shown by open boxes in A. (C) The cell number of the erythroid fractions of the spleen were increased by PHZ injection. Fold increases between the PHZ-induced anemic spleens, and normal spleens are indicated in this graph. The results are shown with standard deviations. Norio Suzuki et al. Blood 2003;102:3575-3583 ©2003 by American Society of Hematology

Analyses of normal and abnormal fetal erythropoiesis by the G1-HRD-GFP transgene. Analyses of normal and abnormal fetal erythropoiesis by theG1-HRD-GFPtransgene. (A) Total (left) and c-Kit+ (right) cells from wild-type (top) and EpoR-null (bottom) E12.5 fetal livers with G1-HRD-GFP transgene were analyzed by FACS. The percentage in each quadrangle is shown. The means of the CD71 expression levels in the CD71+ fractions from each embryo are indicated by dotted lines. (B) Colony assay of sorted cells from G1-HRD-GFP transgenic fetal liver. (C) Result of the quantitative RT-PCR analysis of GATA-1 in EpoR+/+::G1-HRD-GFP+ (closed bars) and EpoR-/-::G1-HRD-GFP+ (hatched bars) fetal liver cell fractions. The GATA-1 mRNA levels were normalized to the level of GAPDH mRNA. The results are shown with standard deviations. Norio Suzuki et al. Blood 2003;102:3575-3583 ©2003 by American Society of Hematology

Measurement of intracellular free calcium concentration upon exposure to Epo and ionomycin in the bone marrow cell fractions. Measurement of intracellular free calcium concentration upon exposure to Epo and ionomycin in the bone marrow cell fractions. Bone marrow cells from G1-HRD-GFP transgenic mice were analyzed for their intracellular free calcium concentration after supplementation of Epo (10 U/mL) or ionomycin (3 μg/mL) by Indo-1 flow cytometry. The percentages of cells containing IFC in each fraction are indicated. Norio Suzuki et al. Blood 2003;102:3575-3583 ©2003 by American Society of Hematology

Summary of the GATA-1 expression profile during erythroid differentiation. Summary of the GATA-1 expression profile during erythroid differentiation. The thickness of the bar indicates the level of gene expression. GATA-1 expression starts at the bipotential (erythrocyte and megakaryocyte) progenitor stage (EMP, erythroid/megakaryocytic precursors) and increases upon differentiation into the proerythroblast. Sca1, c-Kit, and GATA-2 were expressed in hematopoietic stem cells (HSCs) and common myeloid progenitors (CMPs), and their expressions decrease with the progression of differentiation. 30 When the expression level of GATA-1 decreases in Ter119+ erythroblasts, hemoglobin (Hb) accumulates in the cells.31 Pro-EB indicates proerythroblast; Baso-EB, basophilic erythroblast; Poly-EB, polychromatic erythroblast; Ortho-EB, orthochromatic erythroblast; RBC, red blood cell. Norio Suzuki et al. Blood 2003;102:3575-3583 ©2003 by American Society of Hematology