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).

Afouda, GATA4, 5 and 6 mediate TGFbeta maintenance of endodermal gene expression in Xenopus embryos. 2005, Pubmed, Xenbase

Afouda, GATA4, 5 and 6 mediate TGFbeta maintenance of endodermal gene expression in Xenopus embryos. 2005, Pubmed , Xenbase
Ahn, A late phase of cerebellar long-term depression requires activation of CaMKIV and CREB. 1999, Pubmed
Baron, Embryonic origins of mammalian hematopoiesis. 2003, Pubmed
Belaoussoff, Hematopoietic induction and respecification of A-P identity by visceral endoderm signaling in the mouse embryo. 1998, Pubmed
Blobel, CREB-binding protein cooperates with transcription factor GATA-1 and is required for erythroid differentiation. 1998, Pubmed
Brown, The cardiac determination factor, Nkx2-5, is activated by mutual cofactors GATA-4 and Smad1/4 via a novel upstream enhancer. 2004, Pubmed
Cardinaux, Recruitment of CREB binding protein is sufficient for CREB-mediated gene activation. 2000, Pubmed
Chakravarti, Role of CBP/P300 in nuclear receptor signalling. 1996, Pubmed
Christian, Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. 1993, Pubmed , Xenbase
Ciau-Uitz, Distinct origins of adult and embryonic blood in Xenopus. 2000, Pubmed , Xenbase
Cumano, Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. 1996, Pubmed
Davidson, Turning mesoderm into blood: the formation of hematopoietic stem cells during embryogenesis. 2000, Pubmed , Xenbase
de la Calle-Mustienes, Xenopus Xlmo4 is a GATA cofactor during ventral mesoderm formation and regulates Ldb1 availability at the dorsal mesoderm and the neural plate. 2003, Pubmed , Xenbase
de Sousa Lopes, BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. 2004, Pubmed
Friedle, Cooperative interaction of Xvent-2 and GATA-2 in the activation of the ventral homeobox gene Xvent-1B. 2002, Pubmed , Xenbase
Fujiwara, Functional overlap of GATA-1 and GATA-2 in primitive hematopoietic development. 2004, Pubmed
Galloway, Ontogeny of hematopoiesis: examining the emergence of hematopoietic cells in the vertebrate embryo. 2003, Pubmed , Xenbase
Godin, Para-aortic splanchnopleura from early mouse embryos contains B1a cell progenitors. 1993, Pubmed
Goldman, Ectodermally derived steel/stem cell factor functions non-cell autonomously during primitive erythropoiesis in Xenopus. 2006, Pubmed , Xenbase
Gu, The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo. 1999, Pubmed
Gupta, BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis. 2006, Pubmed
Hayakawa, Functional regulation of GATA-2 by acetylation. 2004, Pubmed
Hogan, Specification of the primitive myeloid precursor pool requires signaling through Alk8 in zebrafish. 2006, Pubmed
Horton, Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. 1990, Pubmed
Huo, Important roles of reversible acetylation in the function of hematopoietic transcription factors. 2005, Pubmed
Kamei, A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. 1996, Pubmed
Kato, Neuralization of the Xenopus embryo by inhibition of p300/ CREB-binding protein function. 1999, Pubmed , Xenbase
Kau, Dual contribution of embryonic ventral blood island and dorsal lateral plate mesoderm during ontogeny of hemopoietic cells in Xenopus laevis. 1983, Pubmed , Xenbase
Kelley, Ventral expression of GATA-1 and GATA-2 in the Xenopus embryo defines induction of hematopoietic mesoderm. 1994, Pubmed , Xenbase
Kikkawa, Two-step induction of primitive erythrocytes in Xenopus laevis embryos: signals from the vegetal endoderm and the overlying ectoderm. 2001, Pubmed , Xenbase
Kishimoto, The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning. 1997, Pubmed
Kumano, Spatial and temporal properties of ventral blood island induction in Xenopus laevis. 1999, Pubmed , Xenbase
Lane, The origins of primitive blood in Xenopus: implications for axial patterning. 1999, Pubmed , Xenbase
Lane, Primitive and definitive blood share a common origin in Xenopus: a comparison of lineage techniques used to construct fate maps. 2002, Pubmed , Xenbase
Lugus, GATA2 functions at multiple steps in hemangioblast development and differentiation. 2007, Pubmed
Maeno, The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis. 1996, Pubmed , Xenbase
Maéno, Positive and Negative Regulation of the Differentiation of Ventral Mesoderm for Erythrocytes in Xenopus laevis: (Xenopus laevis/erythropoiesis/embryonic blood island/explant/regulation). 1992, Pubmed , Xenbase
Maéno, Differential participation of ventral and dorsolateral mesoderms in the hemopoiesis of Xenopus, as revealed in diploid-triploid or interspecific chimeras. 1985, Pubmed , Xenbase
Maéno, Regulation of primary erythropoiesis in the ventral mesoderm of Xenopus gastrula embryo: evidence for the expression of a stimulatory factor(s) in animal pole tissue. 1994, Pubmed , Xenbase
Maéno, A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: roles of animal pole tissue in the development of ventral mesoderm. 1994, Pubmed , Xenbase
Maéno, Regulatory signals and tissue interactions in the early hematopoietic cell differentiation in Xenopus laevis embryo. 2003, Pubmed , Xenbase
Massagué, Transcriptional control by the TGF-beta/Smad signaling system. 2000, Pubmed
Mead, SCL specifies hematopoietic mesoderm in Xenopus embryos. 1998, Pubmed , Xenbase
Medvinsky, Definitive hematopoiesis is autonomously initiated by the AGM region. 1996, Pubmed
Medvinsky, An early pre-liver intraembryonic source of CFU-S in the developing mouse. 1993, Pubmed
Menghini, Phosphorylation of GATA2 by Akt increases adipose tissue differentiation and reduces adipose tissue-related inflammation: a novel pathway linking obesity to atherosclerosis. 2005, Pubmed
Mills, Elucidating the origins of the vascular system: a fate map of the vascular endothelial and red blood cell lineages in Xenopus laevis. 1999, Pubmed , Xenbase
Minegishi, The mouse GATA-2 gene is expressed in the para-aortic splanchnopleura and aorta-gonads and mesonephros region. 1999, Pubmed
Molkentin, The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissue-specific gene expression. 2000, Pubmed
Mullins, Genes establishing dorsoventral pattern formation in the zebrafish embryo: the ventral specifying genes. 1996, Pubmed
Nakayama, Xenopus Smad8 acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning. 1998, Pubmed , Xenbase
Narita, Wild-type endoderm abrogates the ventral developmental defects associated with GATA-4 deficiency in the mouse. 1997, Pubmed
Nguyen, Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes. 1998, Pubmed , Xenbase
Nieto, Reorganizing the organizer 75 years on. 1999, Pubmed , Xenbase
Nishinakamura, Activation of Stat3 by cytokine receptor gp130 ventralizes Xenopus embryos independent of BMP-4. 1999, Pubmed , Xenbase
Peterkin, GATA-6 maintains BMP-4 and Nkx2 expression during cardiomyocyte precursor maturation. 2003, Pubmed , Xenbase
Peterkin, The roles of GATA-4, -5 and -6 in vertebrate heart development. 2005, Pubmed
Pyati, Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development. 2005, Pubmed
Rhodes, Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish. 2005, Pubmed
Rooke, Phosphorylation of Gata1 at serine residues 72, 142, and 310 is not essential for hematopoiesis in vivo. 2006, Pubmed
Scarlett, Intact RNA-binding domains are necessary for structure-specific DNA binding and transcription control by CBTF122 during Xenopus development. 2004, Pubmed , Xenbase
Schmid, Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation. 2000, Pubmed
Smith, XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus. 2002, Pubmed , Xenbase
Soderling, The Ca-calmodulin-dependent protein kinase cascade. 1999, Pubmed
Sykes, Suppression of GATA factor activity causes axis duplication in Xenopus. 1998, Pubmed , Xenbase
Takahashi, Inhibitory interaction of c-Myb and GATA-1 via transcriptional co-activator CBP. 2000, Pubmed
Tsai, An early haematopoietic defect in mice lacking the transcription factor GATA-2. 1994, Pubmed
Walmsley, Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands. 1994, Pubmed , Xenbase
Walters, Bone morphogenetic protein function is required for terminal differentiation of the heart but not for early expression of cardiac marker genes. 2001, Pubmed , Xenbase
Walters, Calmodulin-dependent protein kinase IV mediated antagonism of BMP signaling regulates lineage and survival of hematopoietic progenitors. 2002, Pubmed , Xenbase
Wayman, CaM kinase IV regulates lineage commitment and survival of erythroid progenitors in a non-cell-autonomous manner. 2000, Pubmed , Xenbase
Weber, The switch from larval to adult globin gene expression in Xenopus laevis is mediated by erythroid cells from distinct compartments. 1991, Pubmed , Xenbase
Winnier, Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. 1995, Pubmed
Zhang, BMP-like signals are required after the midblastula transition for blood cell development. 1996, Pubmed , Xenbase
Zon, Expression of GATA-binding proteins during embryonic development in Xenopus laevis. 1991, Pubmed , Xenbase