1 Division of Hematology/Oncology, Department of Pediatrics, Harvard Medical School,
2 Howard Hughes Medical Institute, Childrens Hospital, 300 Longwood Ave, Boston, MA 02115, USA
* Present address: Department of Pathology, D4047C, St. Jude Childrens Research Hospital, Memphis, TN 38103, USA
Author for correspondence (e-mail: zon{at}rascal.med.harvard.edu)
Accepted March 26, 2001
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SUMMARY |
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Key words: Xenopus, LMO-2, SCL, GATA-binding factors, Hematopoiesis
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INTRODUCTION |
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The formation of ventral mesoderm requires the activity of soluble growth factors such as bone morphogenetic proteins (BMPs). Ectopic expression of BMP-4 in the developing Xenopus embryo leads to ventralization that is characterized by lack of notochord, decreased muscle formation and increased blood formation. Animal pole ectoderm explants from BMP-4-injected embryos express globin mRNA, suggesting that BMP-4 can directly induce erythroid differentiation (Dale et al., 1992; Jones et al., 1992). Interference with normal BMP-4 signaling by ectopic expression of a truncated BMP-receptor results in dorsalized embryos with decreased blood formation (Graff et al., 1994; Maeno et al., 1994a; Suzuki et al., 1994). BMP-4 signaling results in expression of a variety of downstream targets including GATA-1, GATA-2 and SCL (Maeno et al., 1996; Zhang and Evans, 1996; Mead et al., 1998).
GATA-1 and GATA-2 are zinc-finger DNA-binding proteins required for normal blood cell development. Targeted disruption of each of these genes in mice results in a failure of hematopoiesis and death due to anemia (Pevny et al., 1991; Tsai et al., 1994). In Xenopus, GATA-1 and GATA-2 are early markers of hematopoietic mesoderm (Kelley et al., 1994). GATA-1 expression is restricted to hematopoietic cells during embryogenesis whereas GATA-2 is more widely expressed (Kelley et al., 1994; Walmsley et al., 1994). GATA-2 is present at a very low level as a maternal RNA and zygotic expression begins during gastrulation (Zon et al., 1991; Kelley et al., 1994). GATA-1 is undetectable by in situ hybridization until late neurula stages when it becomes expressed in the ventral blood island (Kelley et al., 1994).
Stem cell leukemia (SCL) is a basic helix-loop-helix (bHLH) transcription factor and is an early marker of hematopoietic cells in the vertebrate embryo. SCL was originally identified in a chromosomal translocation in T-cell acute lymphoblastic leukemia (Begley et al., 1989; Aplan et al., 1990; Bernard et al., 1990; Begley et al., 1991; Chen et al., 1990a; Chen et al., 1990b; Visvader et al., 1991). Gene targeting experiments have indicated an essential role for SCL in hematopoiesis and vasculogenesis (Shivdasani et al., 1995; Robb et al., 1995; Porcher et al., 1996; Visvader et al., 1998). We have recently reported the cloning and characterization of Xenopus SCL and showed that ectopic expression of SCL was sufficient to direct primitive mesoderm to a hematopoietic fate. SCL expression is induced by BMP-4 and we proposed that SCL lay downstream of BMP in a ventral signaling cascade to specify HSCs from undifferentiated mesoderm (Mead et al., 1998). SCL binds DNA as a heterodimer in complex with the bHLH proteins E12/E47. Gel mobility shift studies have shown that SCL can associate in a high order complex on DNA that includes SCL, E12/E47, GATA-1, Ldb-1 and LMO-2 (Wadman et al., 1997).
Like SCL, LMO-2 (LIM-only protein 2; also known as TTG-2 and Rbtn-2) was first identified as a chromosomal translocation associated with T-cell acute lymphoblastic leukemia (Royer-Pokora et al., 1991). LMO-2 is more broadly expressed than SCL, although high levels of LMO-2 expression are found in the sites of hematopoiesis (Foroni et al., 1992). Targeted gene disruption experiments in mice have shown an essential role for LMO-2 in hematopoiesis (Yamada et al., 1998). Homozygous mice null for LMO-2 die at day 9-10 of severe anemia and LMO-2-negative stem cells fail to contribute to the hematopoietic cells of adult chimeric mice. LMO-2 does not bind DNA, but instead acts as a bridging molecule between DNA-binding molecules such as SCL and GATA-1. Wadman and colleagues have demonstrated that LMO-2 is an integral part of a five-member erythroid transcription-activating complex, which includes SCL, E2A, GATA-1, Ldb1/NL1 and LMO-2 (Wadman et al., 1997). This erythroid complex recognizes a bipartite DNA motif of an E-box (CAGGTG) approximately nine base pairs upstream of a GATA site. DNA-site CASTing experiments in T cells isolated from LMO-2 transgenic mice identified a novel LMO-2-containing complex that forms on two E-box sites separated by approximately ten base pairs (Grutz et al., 1998). This complex is restricted to immature (CD8-/CD4-) T-cells and may indicate a role for this LMO-2-containing complex in the block in differentiation of pre-cancerous thymocytes. The 5' regulatory regions of the gene for GATA-1 contain sites that promote the formation of a multimeric erythroid complex suggesting that GATA-1 itself may be a transcriptional target of the complex (Vyas et al., 1999). Recently, a pentameric protein complex (LMO-2, SCL, E2A, Ldb-1 and pRb), that assembles on a consensus SCL site, has been shown to downregulate the expression of c-kit in maturing erythroblasts (Vitelli et al., 2000). Thus, LMO-2 plays a pivotal role in hematopoiesis by bridging multi-protein DNA-binding complexes to regulate transcription of downstream targets during blood cell formation.
We now report the cloning and functional characterization of Xenopus LMO-2. In situ hybridization studies show that LMO-2 is highly expressed in the ventral region of the early neurula, and as development proceeds, becomes localized to the ventral blood island (VBI). LMO-2 is also highly expressed outside the hematopoietic system with abundant expression in the nascent tailbud region and in the brain. Ectopic expression of LMO-2 in animal pole explants treated with basic fibroblast growth factor (bFGF) results in erythroid differentiation. Like SCL and GATA-1, ectopic expression of LMO-2 on its own has little effect on developing embryos; the embryos develop normally with normal blood island formation. Ectopic expression of both SCL and LMO-2, however, results in embryos developing with a truncated body and an enlarged ventral blood island. This ventral phenotype is exaggerated by ectopic expression of GATA-1 together with SCL and LMO-2. Embryos injected with LMO-2, SCL and GATA-1 mRNAs develop with virtually no dorsal-anterior structures and express globin throughout the body axis. These transcription factors also work synergistically in animal pole explant assays to induce the development of erythroid cells. Our studies strongly suggest that the formation of the LMO-2/SCL/GATA-1 complex in vivo specifies ventral mesoderm to become blood.
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MATERIALS AND METHODS |
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Whole embryo RNA in situ hybridization analysis and immunohistochemistry
RNA in situ hybridization analysis using labeled probes was performed as described (Harland, 1991; Hemmati-Brivanlou et al., 1990a). Antisense XLMO-2 RNA probes were in vitro transcribed in the presence of digoxigenin-labeled rUTP (Boehringer Mannheim Biochemicals). Whole embryo immunohistochemistry was performed, using a monoclonal antibody (L4-27) directed against tadpole -globin at 1/40 (v/v), as described (Hemmati-Brivanlou et al., 1990b). Embryos were staged according to Nieuwkoops normal table of development (Nieuwkoop and Faber, 1967).
RT-PCR assay
RNA extractions, first strand cDNA synthesis and PCR analysis were performed as previously described (Kelley et al., 1994). PCR primer sets and conditions for EF-1 and
T3 globin were as described (Kelley et al., 1994). PCR conditions were determined for each primer set to ensure that amplification was in the exponential range. PCR primers for XLMO-2: forward 5'-GGG AAG TCG GAA GGA GAC-3'; and reverse, 5'-CGG TCA CCC ACG CAG AAG-3'. These primers amplify a 208 bp fragment with an optimal annealing temperature of 55°C.
In vitro transcription and micro-injection
Synthetic mRNA was prepared from linearized plasmid DNA using mMessage mMachine in vitro transcription kits (Ambion, TX). RNA yield was quantitated spectrophotometrically. The integrity of the in vitro transcribed RNA was determined by agarose gel electrophoresis in the presence of formaldehyde. Injection of Xenopus embryos was as previously described (Smith and Harland, 1992). o-dianisidine staining of isolated animal cap cells was as described (Huber et al., 1998). Injection experiments were repeated at least three times with similar results.
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RESULTS |
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In an attempt to quantitate the synergy between these hematopoietic transcription factors, we used an animal pole explant assay. Embryos were injected with CMV-driven plasmids encoding LMO-2, SCL and GATA-1, either alone, in pairs, or all three together. Animal pole explants were dissected from the embryos at stage 8 and treated with activin (100 pM). The explants were cultured to swimming tadpole stage (Stage 33) and then dissociated with collagenase. Animal cap cell suspensions were stained with o-dianisidine to identify hemoglobin-containing erythrocytes. The cell suspensions were transferred to glass slides and the hemoglobin-positive cells counted under a microscope (Fig. 8). Activin treatment alone does not induce erythrocyte development: activin induces dorsal mesoderm such as muscle and notochord and not ventral mesoderm (blood). Expression of LMO-2, SCL or GATA-1 alone results in the formation of a small number of red blood cells in the animal pole explants (100 hemoglobin-positive cells). Injection of the transcription factors in pairs results in a moderate increase in the number of red blood cells. Combination of LMO-2, SCL and GATA-1 in the injection cocktail results in a massive increase in the numbers of hemoglobinized cells in the animal pole explants. The number of erythrocytes produced by the combination of LMO-2, SCL and GATA-1 is higher than the additive value for each transcription factor alone, indicating that these transcription factors synergize in nascent mesoderm to promote primitive hematopoiesis. The total number of red blood cells is approximately tenfold higher in the triple-injected embryos compared with the single transcription factors alone. Similar results were obtained when basic FGF was used as the mesoderm-inducing agent on the dissected animal pole explants instead of activin (data not shown).
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DISCUSSION |
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Using animal cap explant assays we have shown that LMO-2 expression is upregulated by BMP-4 activity. This is consistent with a role for LMO-2 in the regulation of primitive hematopoiesis. XLMO-2, which does not bind DNA itself, can induce the expression of -globin in FGF-treated animal pole explants. Treatment of animal caps with FGF, however, results in a low level of SCL expression (Fig. 6; Mead et al., 1998). In the presence of a small amount of SCL, ectopic LMO-2 expression may result in the stabilization of an erythroid-promoting transcription complex and thus upregulate
-globin expression. The GATA-binding factors, GATA-1 and GATA-2, are normally present in animal pole ectoderm and are thus likely to be able to participate in the multimeric transcription complex (Kelley et al., 1994). In support of this hypothesis, SCL expression is also upregulated in the presence of XLMO-2, perhaps indicating a positive feedback mechanism promoting erythroid differentiation (Fig. 6).
LMO-2, SCL and GATA-1 act synergistically to promote red blood cell formation
Overexpression of SCL, LMO-2 and GATA-1 in activin-treated animal pole explants leads to the production of a large numbers of hemoglobinized cells. Expression of these transcription factors alone, or in pairs, leads to a mild increase in erythrocyte production. The synergy of these transcription factors was also noted in the phenotypes resulting from embryos injected with LMO-2, SCL and GATA-1. The phenotype of the "triple-injected" embryos is reminiscent of those injected with BMP-4, where ventralization results in expansion of ventral mesoderm derivatives (such as blood) at the expense of dorsal structures (such as muscle and notochord). These data, taken together with the overlapping expression patterns of these transcription factors and the fact that these proteins interact in vitro, strongly suggest that LMO-2, SCL and GATA-1 act synergistically in a multi-protein complex to promote red blood cell formation. It is important to note, however, that the activity of this transcription complex in explanted embryonic tissue may not necessarily reflect the response seen in the context of the whole embryo.
Two models, which do not preclude each other, can be proposed to explain the phenotypic effects of the injecting these erythroid transcription factors. The first model predicts the cell autonomous activation of the blood program by the formation of the hematopoietic complex containing SCL, LMO-2 and GATA-1. In this manner, the complex provides a molecular address for hematopoietic mesoderm formation within the embryo and overrides endogenous signals that specify other tissues. For instance, when overexpressed, the erythroid complex (SCL, LMO-2 and GATA-1) may compete with a myogenic complex that specifies muscle. The second model posits that the overexpression of SCL, LMO-2 and GATA-1 acts, in part, in a non-cell autonomous manner. Non-neuronal ectoderm has been shown to participate in hematopoiesis in a non-cell autonomous manner, in part, through the action of bone morphogenetic proteins (BMPs). When ectodermal cells, a source of BMP signaling, are co-cultured with ventral mesoderm, there is an enhancement of blood formation (Maeno et al., 1994b). In support of this model, we have demonstrated that a cocktail of SCL, LMO-2 and GATA-1 can activate a BMP promoter reporter construct in Xenopus embryos (data not shown). Indeed, the BMP-4 promoter region contains multiple GATA and E-box binding sites and may be directly induced by factors that bind these sites (Kim et al., 1998). BMP-4, secreted from cells expressing the LMO-2/SCL/GATA-1 transcription complex, could influence neighboring cells to adopt a ventral (hematopoietic) fate. An auto-activation loop of BMP signaling may then enhance the initial BMP induction (Metz et al., 1998). Such sequential activation of BMP may normally occur during embryonic development. Our data do not rule out either model, and it is likely that the observed effects are the result of both cell autonomous and cell non-autonomous effects.
Control of hematopoietic transcription in the developing embryo
Our data show that the co-expression of LMO-2, SCL and GATA-1 can drive ectopic erythropoiesis in the Xenopus embryo. A model for primitive erthryopoiesis emerges in which the overlapping expression patterns of LMO-2, SCL and GATA-1 allow the formation of a transcription activating complex that drives the early blood program. The composition of these transcription complexes may differ in a cell type- and cis-element-specific context. Furthermore, the composition of the complex formed on discrete DNA binding sites may lead to either induction or repression of target genes (Visvader et al., 1997; Anderson et al., 1998; Ono et al., 1998; Vyas et al., 1999; Vitelli et al., 2000; Anderson et al., 2000). The interaction of proteins in these multimeric complexes allows exquisite control of gene regulation at the transcriptional level. Our experiments have indicated an inductive role for the combination of LMO-2, SCL and GATA-1 in developing Xenopus embryos. It will be of interest to include other potential members of the transcription complex in this type of mRNA cocktail injection experiments. For example, the LIM-binding protein Ldb1, and LMO-2 itself, have been shown to be negative regulators of the erythroid differentiation in a proerythroblast cell line (Visvader et al., 1997). Inhibitory factors, such as Ldb1, FOG and pRb (Visvader et al., 1997; Deconinck et al., 2000; Vitelli et al., 2000), may counteract the blood inducing activity of LMO-2, SCL and GATA-1 and may serve to limit erythroid differentiation. Future studies will help dissect how complex transcription factor interactions regulate the hematopoietic program in the developing vertebrate embryo.
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ACKNOWLEDGMENTS |
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