Dalgin G , Goldman DC , Donley N , Ahmed R , Eide CA , Christian JL .

R03 HD050242 NICHD NIH HHS , R03 HD050242-01 NICHD NIH HHS , R03 HD050242-02 NICHD NIH HHS , T32 GM071338 NIGMS NIH HHS

             GATA2-TAL1-TCF3-Lmo2 complex

	Fig. 1. GATA-2 functions non-cell-autonomously to regulate the differentiation of erythrocytes in Xenopus. (A) Anti-HA immunoblot showing that GATA-2 MOs can block the translation of GATA-2 HA mRNA in vivo but have no effect on translation of rGATA-2 HA RNA that has been modified to prevent the MOs from annealing. Three embryo equivalents were loaded in each lane. (B) Two dorsal or ventral animal pole blastomeres of eight-cell embryos were injected with RNA encoding β-galactosidase together with GATA-2 or control MOs as illustrated. Embryos were stained for β-galactosidase activity at stage 32 (red stain) followed by in situ hybridization analysis of Globin expression (purple stain). (C) Two dorsal or ventral animal pole blastomeres of eight-cell embryos were injected with GATA-2 or control MOs alone or together with rGATA-2 HA RNA and expression of Globin was analyzed by Northern blot at stage 32. Levels of Globin transcripts, normalized relative to levels of Odc transcripts, are expressed as a percentage of control below each lane. Similar results were obtained in at least three experiments.
	Fig. 2. Ectodermal GATA-2 is not required for specification of hematopoietic mesoderm. GATA-2 morpholinos were injected into dorsal animal pole cells of eight-cell embryos and expression of Scl (A, B) or Gata-1 (C, D) was analyzed by RT–PCR (A, C) or by in situ hybridization (B, D) at neurula and/or tail bud stages as indicated. Reverse transcriptase was omitted from one set of samples (–RT) as a control for genomic contamination. Levels of Scl or Gata-1 transcripts, normalized relative to levels of Odc transcripts, are expressed as a percentage of control below each lane. Similar results were obtained in at least three experiments.
	Fig. 3. Ectodermally derived GATA-2 is required for normal erythropoiesis. (A) Experimental design is illustrated schematically. (B) Ectoderm (ecto) explanted from embryos injected with either control or GATA-2 MOs was co-cultured with explanted ventral mesoderm (VM) until siblings reached the tailbud stage at which time expression of Globin, Odc and Xpox2 was analyzed by Northern hybridization. Similar results were obtained in at least three experiments.
	Fig. 4. Hematopoietic defects in GATA-2 morphants are not caused by a loss of Bmp expression or activity. (A) Both blastomeres of two-cell embryos were injected with GATA-2 MOs (+), or not (−), ectodermal explants were isolated at stage 9, and cultured until stage 13. Expression of Bmp-2, -4, -7 and Odc was analyzed in whole embryos and ectodermal explants (Ecto) by Northern hybridization. (B, C) Ventral mesoderm (VM) explanted from control embryos, or embryos made to express a constitutively active BMP receptor (caALK) was cultured in isolation (C) or together with ectoderm (ecto) explanted from embryos injected with either control or GATA-2 MOs (B) until siblings reached the tailbud stage. Expression of Globin and Odc was analyzed by Northern hybridization. Levels of Globin transcripts, normalized relative to levels of Odc transcripts, are expressed as a percentage of control below each lane in B. Similar results were obtained in a duplicate experiment.
	Fig. 5. Downregulation of ectodermal GATA-2 leads to a decrease in RBC number. (A) Wright–Giemsa-stained blood collected from control or GATA-2 morphant tadpoles. Arrows indicate RBCs, arrowheads indicate WBCs. (B) Mean number (± S.E.M.) of RBCs (red bars) and WBCs (white bars) present in tadpoles derived from embryos in which control or GATA-2 MOs had been injected into dorsal (dor) or ventral (vent) animal pole blastomeres at the eight-cell stage, expressed as a percentage of cell number in uninjected control embryos. Results are pooled from three independent experiments.
	Fig. 6. GATA-2 functions downstream of BMP and CaM KIV signaling in ectodermal cells. (A–D) Mean number (± S.E.M.) of RBCs present in tadpoles made to express GATA-2 MOs, a dominant mutant form of the BMP receptor (tBR), constitutively active CaM KIV (KIVc) and/or GATA-2 HA RNA as indicated, expressed as a percentage of cell number in control embryos. (E) Experimental design is shown schematically. Mean number (± S.E.M.) of RBCs, expressed as a percentage of cell number in control embryos, present in tadpoles in which CaM KIV signaling was upregulated in dorsal cells followed by injection of GATA-2 RNA into either the same cells, or cells on the opposite side of the embryo as indicated. All results are pooled from a minimum of three independent experiments.
	Fig. 7. Mutation of a putative CaM KIV phosphorylataion site in GATA-2 does not affect nuclear localization or function. (A) Alignment of sequence of human (hGATA-2) and Xenopus Gata-2 (xGATA-2) surrounding the putative phosphorylated serine which is underlined. (B) Immunolocalization of GATA-2 HA in stage 7 or stage 12 embryos that had been injected with the RNA(s) indicated above each panel. (C, D) Northern blot analysis of expression of Globin in stage 32 control embryos or in embryos that had been injected with wild type (WT) or mutant GATA-2 RNA, constitutively active forms of CaM KIV and its upstream kinase (KIVc) and/or MOs as indicated above each lane. Results were reproduced in two independent experiments.
	Fig. 8. Mutation of putative acetylated lysines inactivates GATA-2 in vivo. (A) Alignment of sequence surrounding the N- and C-terminal zinc fingers (NF; N-Finger, CF; C-Finger) of mouse (mGATA-2) and Xenopus Gata-2 (xGATA-2). Acetylated lysine residues that were mutated in the studies of Hayakawa et al. (2004) or in the current studies are underlined. (B, C) Mean number (± S.E.M.) of RBCs present in tadpoles derived from embryos that had been injected with the RNAs and/or MOs indicated below each bar, expressed as a percentage of cell number in control embryos. Results are pooled from a minimum of 4 independent experiments. (D) Two dorsal animal pole blastomeres of eight-cell embryos were injected with varying amounts of RNA encoding CBP alone (control) or together with constitutively active CaM KIV (KIVc) and expression of Globin was analyzed by Northern blot at stage 32. Levels of Globin transcripts, normalized relative to levels of Odc transcripts, are expressed as a percentage of control below each lane. Similar results were obtained in a duplicate experiment.
	Fig. 9. Model for regulation of ectodermal GATA-2 by BMPs and CaM KIV during primitive hematopoiesis. Activation of the BMP signal transduction cascade in ectodermal cells is required to activate transcription of GATA-2. Under normal conditions, GATA-2 is acetylated by CBP and this enables it to regulate transcription of target genes, including those that are required for normal primitive hematopoiesis (left panel). When CaM KIV is hyperactivated, it phosphorylates excessive amounts of a substrate which can then recruit limiting amounts of CBP away from GATA-2. Under these conditions, GATA-2 is hypoacetylated and can no longer activate transcription of target genes (right panel).

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