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First published online 13 December 2006
doi: 10.1242/dev.02731

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GATA2 functions at multiple steps in hemangioblast development and differentiation

¹ Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA.
² Molecular Cell Biology Program, Washington University School of Medicine, St Louis, MO 63110, USA.
³ Department of Pharmacology, University of Wisconsin Medical School, Madison, WI, USA.
⁴ Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

View larger version (50K): [in this window] [in a new window]   Fig. 1. Molecular profile of the hemangioblast and relationship to other stem cells. (A) The 37 genes representing the GO term `regulation of transcription, DNA-dependent' were extracted from the list of 667 genes that were differentially expressed between BL-CFC and Blast and used to cluster the duplicate Blast and BL-CFC chips. The scale for relative enrichment of each transcript is depicted at the bottom: 3-fold or greater enrichment for a transcript on one chip relative to the other chips is depicted in dark red, whereas 3-fold less expression of that transcript is in dark blue. (B) The five GO terms with the highest relative representation in the BL-CFC profile are graphed with respect to their fractional representation among the genes of hematopoietic stem cells and their progeny, mature bone marrow cells, and also relative to the Blast profile. The mostrepresented GO terms in the progenitor populations relate to nuclear functions, including synthesis of and binding to nucleic acids. (C) Expression profiles (i.e. lists of genes enriched in a given cell population) from three previous analyses of progenitor and progeny gene expression were used to analyze the progenitor-progeny relationship between BL-CFC and Blast cells. Expression profiles were clustered based on differences between the overall representation of GO terms within each profile. Differences between each profile are represented by Pearson correlation, where 0.0 indicates identical GO term distribution between two profiles, 1.0 indicates no correlation, and 2.0 indicates negative correlation. Cell profiles are as follows: HSC (1), Marrow (1), Neural_SC (1), and HSC_EST(1) indicate Hematopoietic Stem Cell, Bone Marrow Cell, Neural Stem Cell, and Expressed Sequence Tag in Hematopoietic Stem Cells respectively, from Ivanova et al. (Ivanova et al., 2002) and HSC_EST from Phillips et al. (Phillips et al., 2000). HSC (2), Marrow (2), Neural_SC (2), and Brain (2) indicate Hematopoietic Stem Cell, Bone Marrow Cell, Neural Stem Cell, and Brain, respectively, from Ramalho-Santos et al. (Ramalho-Santos et al., 2002). Gastric SC (3) and Chief Cell (3) indicate Gastric Stem Cell and Chief Cells, respectively, from Mills et al. (Mills et al., 2002; Mills et al., 2003).

View larger version (33K): [in this window] [in a new window]   Fig. 2. GATA2 in differentiating ES cells and the generation of inducible ES cells. (A) Rapid induction of Gata2 upon short-term treatment with BMP4. ES cells were differentiated for 2 days in the absence of serum, collected and treated with BMP4, or 10 µg/ml cycloheximide (CHX), or DMSO for 1 hour. Gata2 expression was analyzed and normalized to Gapdh. The quantity of Gata2 expressed in untreated cells was normalized as 1 and used to determine the Gata2 quantity in BMP4-treated samples. Values indicate mean±s.e.m.; *, P<0.01 versus control. (B) Expression kinetics of candidate genes in A2Lox ES cells. RNA from EBs differentiated in serum was utilized in qRT-PCR assays. Candidate genes were first normalized against Gapdh and then the maximal expression for each gene was assigned 100% and the according value for each EB day determined against this 100% value. (C) Schematic of the iGATA2 ES line used, with indicated loci carrying alterations allowing for production of the rtTA, expression of the Gata2 cDNA and generation of hCD4 as a surrogate marker for Scl. (D) Western blot of the iGATA2 ES line. Cells were differentiated in serum for two days, treated with the indicated concentrations of Dox and differentiated for two additional days. EB lysates were subjected to SDS-PAGE followed by blotting with GATA2 and ß-actin antibodies.

View larger version (62K): [in this window] [in a new window]   Fig. 3. Enforced GATA2 results in an increase in mesodermal gene expression. (A) iGATA2 ES cells were differentiated for 2 days in SR and treated with 0.3 µg/ml Dox for an additional 2 days. RNA was then utilized for qRT-PCR to examine the genes indicated. Genes were normalized against Gapdh and then the ratio of the gene quantity (+Dox) to gene quantity (-Dox) was determined to yield normalized fold change. (B) Upper portion depicts the mouse Bmp4 locus. Gray boxes are non-translated exons and the open box indicates the translated exon. Coordinates and arrows indicate conserved, consensus GATA-factor-binding sites. Enlarged below is a section containing the TGE of a 5' to 3' alignment of highly conserved, intronic regions of homologs of the Bmp4 gene. GATA sites 1 and 2 are indicated by brackets; arrows above the alignment indicate GATA site orientation. (C) ChIP analysis of GATA2 occupancy of the Bmp4 locus in D4 EBs. A2Lox ES Cells were differentiated for 4 days in serum, crosslinked with 1% formaldehyde and processed to examine GATA2 occupancy at the three conserved consensus GATA-factor-binding sites. (D) Bmp4 enrichment in Gata2-expressing cells. iGATA2 ES cells were differentiated in SR medium for 4 days and Dox added on D2. At D4, cells were stained for hCD4 expression and sorted into hCD4- and hCD4+ and then RNA generated for qRT-PCR to examine co-enrichment of Gata2 and Bmp4 in hCD4+ cells. (E) FACS analyses of Flk-1+ cell generation by GATA2 and BMP4. Cells were differentiated in SR medium for 2 days and then treated with combinations of Dox (0.3 µg/ml), BMP4 (5 ng/ml) and noggin (10 ng/ml). Cells were utilized for FACS analyses on D3. Numbers indicate percentage of Flk-1+ cells generated. (E') Summary data of four independent sets of FACS experiments showing the percentage of Flk-1+ cells generated in each condition. Values indicate mean±s.e.m.; *, P<0.01 versus untreated.

View larger version (49K): [in this window] [in a new window]   Fig. 4. GATA2 regulates Scl expression. (A) FACS data of hCD4+ (Scl+) cell generation. Cells were grown in serum, SR, or SR+BMP4 as indicated, were treated with or without Dox at D2, and FACS performed on D5 for Flk-1+ and hCD4+ cells. Numbers indicate total percentage of hCD4+ cells (Flk-1+hCD4+ and Flk-1-hCD4+). (A') Summary data of five independent sets of FACS experiments showing the percentage of hCD4+ cells generated in each condition. Values indicate mean±s.e.m.; *, P<0.01 versus untreated. (B) Comparison of human SCL and mouse Scl loci. P indicates alternate promoters, and numbers indicate DNase I hypersensitive sites (Gottgens et al., 2002). Black boxes and white boxes indicate translated and untranslated exons, respectively. Vertical dashes indicate consensus (WGATAR) GATA-factor-binding sites. The bottom line indicates general locus coordinates, where 1 is the start of transcription. (C) Quantitative ChIP assays for GATA-factor occupancy of the Scl loci. Upper panel shows GATA2 occupancy, measured against a standard curve of input dilutions. Individual measurement parameters (EB day, Scl locus site) are the same as on the bottom panel. Bottom panel shows GATA-1 occupancy. Values are mean±s.e.m. for three independent experiments.

View larger version (36K): [in this window] [in a new window]   Fig. 5. GATA2 specifies hemangioblast development. (A) Blast colony formation in wild-type (WT) (J1) or Gata2-/- ES cells. Values are mean±s.e.m. for three independent experiments; *, P<0.05 versus WT. (B) qRT-PCR of Scl (left panel) and Gata1 (right panel) in WT and Gata2-/- ES cells. RNA was from D5 EBs and normalized against Gapdh. Values are mean±s.e.m., from qRT-PCR reactions perfomed in triplicate using two independent RNA samples; *, P<0.05 versus WT. (C) GATA2 induction scheme in differentiating EBs and formation of Blast colonies. Above is the scheme used to generate Blast colonies. Uppermost numbers indicate EB day. Lower gray bars indicate duration of Dox treatment and subsequent treatment of replated EBs. Below is a bar chart displaying Blast colony formation analyses of iGATA2 cells. The x-axis shows the day of EB harvest and replating to assay for Blast colony analysis. The key indicates whether or not Dox was added (1) at D2, during EB formation conditions (before slash); and (2) during growth in semi-solid replating media (after slash). Values are mean±s.e.m., average of three to five individual experiments; *, P<0.05; **, P<0.005. (D) EryP Potential of D2.75 iGATA2 EBs. Cells were grown in serum and treated with or without Dox at D2 and harvested and replated for EryP colonies on D2.75. Values are mean±s.e.m. from two to four independent experiments; *, P<0.05 versus untreated.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Augmented EryP generation and cell proliferation by GATA2. (A) GATA2 induction scheme in differentiating EBs and formation of EryP colonies. Above is the scheme used to generate Blast colonies. Uppermost numbers indicate EB day. Lower gray bars indicate duration of Dox treatment and subsequent treatment of replated EBs. (B) EryP potential of D4 iGATA2 EBs treated with or without Dox at D2 or D3. Values are mean±s.e.m., from four independent experiments; *, P<0.05 versus untreated. (C) Representative bright field images (100x) of EryP colonies from D4 EBs replated in the absence (left) or presence (right) of Dox. (D) BrdU incorporation in EryP cells treated with or without Dox during replating. Values are normalized by subtracting background absorbance and then the BrdU incorporation in untreated EryP cells is normalized to 1. Values are mean±s.e.m. from six independent experiments; *, P<0.005 versus untreated. (E) Cell cycle gene expression profile of iGATA2 EryPs with or without Dox during EryP colony formation. EryP cells from day 2 or 3 of EryP colony formation were harvested and RNA utilized for qRT-PCR assays. Genes were normalized against Gapdh and then the ratio of +Dox to -Dox determined, where values <1 were assigned their inverse to give a negative fold change. Values are mean±s.e.m. from four independent RNA sets.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Augmented endothelial generation by GATA2. (A) GATA2 induction scheme in differentiating EBs and formation of endothelial cells. Uppermost numbers indicate EB day. Gray bar indicates duration of Dox treatment and subsequent analyses of D6 EBs. (B) Generation of Tie2+ endothelial cells by GATA2 expression in serum and SR conditions. Numbers in boxes indicate percentage of Tie2+ cells. (C) Generation of CD31+ and CD31+VE-Cadherin+ endothelial cells in SR media. Numbers in upper left and right quadrants indicate percentage of CD31+ and CD31+VE-Cadherin+ cells, respectively. (C') Quantification of CD31+ and CD31+VE-Cadherin+ cells generated with or without Dox. Values are mean±s.e.m. from four independent experiments; *, P<0.001 versus untreated. (D-E') Representative sprouting EB images. D and E show 100x magnification of brightfield images of EBs generated in the absence (D) and presence (E) of Dox; D' and E' are 800x magnifications of D and E, respectively, showing the tendril-like (D') and endothelial (E') cells generated in the two conditions. (F,F') Brightfield (F) and fluorescence (F') images of 800x magnification of sprouting iGATA2 EBs demonstrating uptake of DiL-Ac-LDL by endothelial cells. (G) Fractional quantification of types of sprouting structures grown by 44 (-Dox) and 46 (+Dox) EBs grown in sprouting media.