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© The Rockefeller University Press,
0021-9525/2000//811 $5.00
The Journal of Cell Biology, Volume 151, Number 4,
, 2000 811-824
Original Article |
CAM Kinase IV Regulates Lineage Commitment and Survival of Erythroid Progenitors in a Non-Cell–Autonomous Manner
Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.
Key Words: Xenopus • CaM KIV • hematopoiesis • erythropoiesis • embryogenesis
© 2000 The Rockefeller University Press
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Introduction |
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CaM-dependent kinase IV (CaM KIV) is a member of a recently characterized CaM kinase cascade (for review see Soderling 1999). Binding of Ca2+–CaM to CaM KIV serves two purposes: it relieves autoinhibition, as described above, and it allows a critical threonine residue, located within the activation loop, to be phosphorylated by a CaM-dependent kinase kinase (CaM KK). Phosphorylation of this Thr 196 by CaM KK results in a 10–20-fold activation of CaM KIV. In addition to CaM KIV, CaM KK has two other known substrates: CaM-dependent kinase I (CaM KI) (Haribabu et al. 1995; Soderling 1996) and protein kinase B (Yano et al. 1998).
The substrate specificity of CaM KIV is not well defined, but it has been shown to phosphorylate and regulate various transcription factors (for review see Soderling 1999). Consistent with a role in regulating transcription, CaM KIV and CaM KK show strong nuclear localization, but are also present in the cytosol (Sakagami et al. 1999). By contrast, CaM KI appears to be only cytosolic (Picciotto et al. 1995). In addition to responding to intracellular calcium, the CaM kinase cascade participates in cross talk with other signaling cascades. For example, CaM KIV directly phosphorylates and inhibits the activity of type I adenylyl cyclase (Wayman et al. 1996) and nitric oxide synthase (Hayashi et al. 1999), and it may indirectly activate mitogen-activated protein kinase pathways (Enslen et al. 1996).
In adult mammals, CaM KIV is expressed predominantly in a subset of neurons, in T lymphocytes and in male germ cells (for review see Means et al. 1997). In the immune system, CaM KIV appears to be required to prevent apoptosis during T cell development and may function in the cascade of events that is required for activation of mature T cells (Anderson et al. 1997; for review see Means et al. 1997). CaM KIV also plays a role in learning and memory within the brain (Bito et al. 1996; Ahn et al. 1999).
Little is known about embryonic expression patterns or developmental roles of CaM KIV. To elucidate such function(s), we examined the consequences of inactivating or constitutively activating this signaling pathway in Xenopus embryos. Misregulation of CaM KIV activity led to several developmental defects. In this study, we have focused on the role of CaM KIV in primitive hematopoiesis.
Vertebrate hematopoiesis is a multistep process in which pluripotent, self-renewing stem cells commit to and ultimately differentiate along one of the various mature lineages of the blood (for review see Zon 1995; Evans 1997). In Xenopus, blood cell progenitors can arise from any blastomere of the 16-cell embryo (Mills et al. 1999). These cells will populate the ventral blood island (VBI, which is the functional equivalent of the yolk sac blood island of higher vertebrates), which gives rise to all progenitors of primitive hematopoiesis (Turpen et al. 1997). Primitive hematopoiesis is carried out by a transient population of stem cells and differs significantly from adult, definitive hematopoiesis. Whereas primitive hematopoiesis produces predominantly embryonic erythrocytes (red blood cells [RBCs]), definitive hematopoiesis yields adult erythroid cells, as well as progenitors of the lymphoid (lymphocyte) and myeloid (granulocyte, monocyte, and platelet) lineages. In Xenopus embryos, definitive hematopoietic cells are derived from the VBI and from the mesoderm of the dorsal–lateral plate (Kau and Turpen 1983; Máeno et al. 1985; Weber et al. 1991; Turpen et al. 1997).
The induction, proliferation, and differentiation of blood progenitors is directed by cues from nonhematopoietic tissues and by signals intrinsic to the hematopoietic stem cells (HSCs) themselves. For example, bone morphogenetic protein-4 specifies ventral fate within the mesoderm, enabling blood progenitors to be born (for review see Lemaire and Yasuo 1998), and may subsequently regulate the proliferation and differentiation of primitive hematopoietic cells (Bhatia et al. 1999). Unidentified signals provided by endodermal (Yoder et al. 1994; Belaoussoff et al. 1998) and endothelial cells (Yoder et al. 1994; Fennie et al. 1995; Ohneda et al. 1998) can also influence the proliferation, survival, and/or lineage fate of HSCs. Downstream of these extrinsic signals, a regulatory network of hematopoietic-specific transcription factors functions in the specification and further development of blood progenitors (Huber and Zon 1998; for review see Sieweke and Graf 1998).
In this study, we found that the proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for hematopoietic progenitors to commit to the erythroid lineage and for the survival of erythroid precursors.
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Materials and Methods |
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Generation of Mutant Forms of Xenopus CaM KIV
The constitutively active Xenopus CaM KIV cDNA (CaM KIVc) was generated by introducing point mutations such that the sequence encoding HMDN (amino acids 313–316) (see Fig. 1) within the autoinhibitory domain was changed to DMDD. Introduction of these acidic charged mutations inactivates the autoinhibitory domain, generating a calcium-independent kinase (Tokumitsu et al. 1994). The dominant-negative CaM KIV cDNA (DnCaM KIV) encodes a protein containing the acidic charged mutations present in CaM KIVc, in addition to the following mutations: (a) Lys 79 in the ATP-binding site was mutated to Gln and (b) the activation loop phosphorylation site (Thr 204) was mutated to Ala. Mutations were introduced by PCR-mediated amplification of the Xenopus CaM KIV cDNA using primers that contained appropriate nucleotide substitutions. PCR-generated portions of mutant cDNAs were sequenced and all cDNAs were subcloned into the expression vector pCS2+ (Rupp et al. 1994).
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β-Galactosidase Staining, In Situ Hybridization, and Histological Analysis
Embryos were stained for β-galactosidase activity using Red-gal (Research Organics, Inc.) as a substrate and processed for in situ hybridization, as descibed previously (Nakayama et al. 1998). After in situ hybridization, some embryos were dehydrated in methanol, embedded in paraffin, and sections 14–20-µM thick were cut and counterstained with eosin (Christian and Moon 1993).
Northern Blot and Reverse Transcription–PCR Analysis of Gene Expression
RNA was isolated and reverse transcription(RT)-PCR analysis was performed, as descibed previously (Cui et al. 1996), using the following PCR conditions: one cycle at 95°C for 5 min, followed by cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The cycle number was determined empirically for each primer pair so that PCR products were examined during the exponential phase of amplification. The following primer pairs (in 5' to 3' orientation) were used to amplify CaM KIV: upstream GGG ATG CTG CAG ATG CTG and downstream CAT GTA CTG GTC TCC TCG. For globin, the primers were: upstream TTG CTG TCT CAC ACC ATC CAG G and downstream TCT GTA CTT GGA GGT GAG GAC G. Ornithine decarboxylase (ODC) (Nastos et al. 1998) and GATA-1 (Zon et al. 1991) primers have been published. Northern blot hybridization was performed as described previously (Christian and Moon 1993). Radioactive bands were visualized with a Molecular Dynamics phosphorimager, and the Macintosh IP lab gel program was used to quantitate relative levels of expression of each gene.
Western Blot Analysis and Activity Assays of CaM KK
Proteins were extracted from Xenopus embryos, as descibed previously (Moon and Christian 1989), and analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and probed with anti–rat CaM KK or CaM KIV antibodies (Transduction Labs). Immunocomplexes were visualized using an anti–mouse HRP-conjugated secondary antibody and a chemiluminescent detection system (NEN Life Science Products). CaM KK activity in embryo extracts was measured and normalized relative to the total protein content, as descibed in Wayman et al. 1997.
Collection and Analysis of Peripheral Blood Samples
Tails were severed from tadpole stage embryos and hematopoietic cells were collected into amphibian PBS containing 0.5% BSA and 10 IU/ml of heparin. Cells were concentrated onto slides using a cytocentrifuge. For general morphological examination, blood cells were stained with a Hema 3 stain set (Biochemical Sciences Inc.) and examined by light microscopy. Apoptotic cells in cytospin preparations of peripheral blood were detected by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay using a fluorescein apoptosis detection kit (Promega). Nuclei of cells were counterstained with propidium iodide to determine the total number of cells.
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Results |
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Transcripts encoding CaM KIV are present in Xenopus eggs and persist at a uniform level throughout embryonic development (Fig. 2 A). Analysis of RNA isolated from various dissected regions of embryos revealed that CaM KIV transcripts are distributed fairly uniformly across the dorsal–ventral and anterior–posterior axes and are present in both ectodermal and mesendodermal germ layers (Fig. 2 B).
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68 kD that comigrated with recombinant rat CaM KK
on Western blots of Xenopus egg and embryo extracts. This protein increased in abundance by tadpole stage 42, at which time a second immunoreactive protein of 70 kD was also detected. The size of this higher molecular weight species is consistent with it being the Xenopus ortholog of CaM KKβ. The increase in CaM KK protein levels at the tadpole stage correlated with an approximately threefold increase in CaM KK activity. To determine whether CaM KK activity is spatially restricted, we analyzed protein expression and activity in extracts isolated from dissected dorsal or ventral halves of early (stage 10) gastrulae. CaM KK protein and activity were detected in dorsal and ventral halves of embryos.
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10-fold more RNA (1–2 ng) encoding CaM KIVc alone (data not shown), indicating that the effects were due to activated CaM KIV, rather than another target of CaM KK. Injection of RNA encoding DnCaM KIV or CaM KIVc led to the accumulation of ectopic protein at levels greater than endogenous levels, at least until tailbud stage 28 (Fig. 5 A).
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Northern blot analysis confirmed that constitutive activation or inhibition of CaM KIV activity on either the dorsal or ventral side of embryos led to a dramatic decrease in globin transcripts (Fig. 5 C). Tailbud stage 29 embryos in which CaM KIV activity was misregulated expressed globin at only 16–32% of control levels, and expression remained substantially repressed in stage 39 tadpoles.
CaM KIV Plays a Non-Cell–Autonomous Role in Regulating Hematopoiesis
Both dorsal and ventral blastomeres of cleavage-stage Xenopus embryos contribute to the VBI (Tracey et al. 1998; Mills et al. 1999; Lane and Smith 1999), which might explain why misregulation of CaM KIV activity in dorsal cells disrupts globin expression in a ventral region. However, our finding that expression of globin is almost completely repressed when CaM KIV activity is perturbed in only a subset of embryonic cells (Fig. 5b and Fig. C; Table ) raised the possibility that CaM KIV functions non-cell–autonomously in blood development. Specifically, CaM KIV may be required in nonhematopoietic cells to regulate the generation of extrinsic signals that direct normal erythropoiesis. To test this hypothesis, we targeted RNAs encoding mutant CaM KIV to blastomeres that do not contribute to the RBC lineage and then analyzed expression of globin at the tailbud stage. As illustrated in the diagram above in Fig. 6 A, RNA encoding β-galactosidase (200 pg), which was used as a lineage label, was injected, either alone or together with constitutively active or DnCaM KIV RNAs, into dorsal midline animal pole blastomeres of 32-cell stage embryos (A1 blastomeres by the nomenclature of Nakamura and Kishiyama 1971; D1.1.1 by the nomenclature of Mills et al. 1999). These blastomeres give rise to anterior ectodermal derivatives and do not contribute to erythroid cells in the VBI (Mills et al. 1999). As a control, the same RNAs were injected into the two ventral midline animal pole cells (A4 blastomeres) of sibling 32-cell embryos. These blastomeres give rise to erythroid cells in a minor fraction of embryos (Mills et al. 1999). Half of the injected embryos in each group were stained for β-galactosidase activity at the tailbud stage (to verify the accuracy of injections), followed by in situ hybridization to detect globin transcripts. RNA was extracted from the remaining embryos for quantitative analysis of globin expression by Northern blot hybridization. Misregulation of CaM KIV activity in the progeny of A1 blastomeres (indicated by red β-galactosidase staining, white arrows) led to a notable reduction in globin staining (black arrows) in many embryos (Fig. 6 A; Table ). Northern blot analysis revealed that when CaM KIV activity was misregulated in the progeny of A1 or A4 blastomeres, globin expression was reduced to 45–64% of control levels (Fig. 6 B). These data suggest that CaM KIV functions in nonhematopoietic cells to regulate production of a signal(s) that is required for normal erythropoiesis.
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Misregulation of CaM KIV Does Not Disrupt Hematopoietic Specification
The decrease in globin expression caused by either upregulation or inhibition of CaM KIV activity could be due to defects at any stage in RBC development, including specification of HSCs from ventral mesoderm, commitment of a subset of these pluripotent cells to the erthyroid lineage, and/or proliferation, survival, or differentiation of hematopoietic precursors (Zon 1995; Evans 1997). To ask whether misregulation of CaM KIV interferes with the initial specification of HSCs, we analyzed expression of two early markers of blood development, Xaml (Tracey et al. 1998) and GATA-1 (Kelley et al. 1994). Neurula stage 18 embryos, in which CaM KIV was misregulated in ventral cells, expressed Xaml (Fig. 8 A) and GATA-1 (Fig. 8 B) at levels indistinguishable from control embryos. By contrast, at tailbud stage 28 expression of GATA-1 (Fig. 8 B) and Xaml (data not shown) was repressed in embryos injected with mutant CaM KIV RNAs. Since Xaml is initially expressed in cells derived from the dorsal side of embryos (Tracey et al. 1998), we also examined its expression following dorsal misregulation of CaM KIV and obtained identical results (data not shown). We conclude that misregulation of CaM KIV activity does not disrupt the initial specification of hematopoietic fate.
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To look for specific defects in hematopoietic maturation and differentiation, we examined RBC number and morphology in cytospin preparations of peripheral blood samples isolated from stage 45 tadpoles. At this stage of development, all of the circulating blood is derived from the VBI as a result of primitive hematopoiesis (Turpen et al. 1997). In control embryos, 80% of circulating cells were RBCs and 20% were white blood cells (WBCs) (Fig. 9A and Fig. B) that are predominantly of the monocyte/macrophage lineage (Ohinata et al. 1990). In embryos where CaM KIV activity was inhibited by injection of RNA encoding DnCaM KIV into either ventral (Fig. 9) or dorsal (data not shown) cells, the total number of circulating blood cells was unchanged, but a greater proportion of these were myeloid, as opposed to erythroid cells (Fig. 9A and Fig. B) such that the ratio of WBCs to RBCs was increased approximately eightfold relative to controls (C). Based on morphology (Fig. 9 A) (Hadji-Azimi et al. 1987) and staining with alpha-naphthyl acetate esterase (data not shown), the majority of WBCs in these embryos, as in controls, were of the monocyte/macrophage lineage. In support of this conclusion, expression of myeloperoxidase, a marker of early embryonic macrophages (Smith, S., and T. Mohun, unpublished data) was increased severalfold in CaM KIV–deficient embryos relative to controls (Fig. 9 D). The increase in the ratio of WBCs to RBCs in embryos injected with DnCaM KIV RNA was specifically caused by a blockade of the CaM KIV branch of the CaM KK cascade, since it was rescued by coinjection of 1 ng of RNA encoding CaM KIVc (Fig. 9 C). These results indicate that embryonic CaM KIV activity is required for hematopoietic stem cell commitment to erythroid, as opposed to myeloid, differentiation.
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We used a TUNEL assay to compare apoptosis in blood cells isolated from control embryos and embryos in which CaM KIV activity was misregulated. As shown in Fig. 9E and Fig. F, constitutive activation of CaM KIV not only caused a decrease in total blood cell number, but almost 50% of the cells were apoptotic. In contrast, although inhibition of CaM KIV also led to a decrease in the number of circulating RBCs, <10% of blood cells isolated from these embryos or from controls were apoptotic (Fig. 9 F). We have not definitively identified the apoptotic cells in CaM KIVc–expressing embryos as erythroid precursors, but this is likely the case based on the aberrant morphology and selective decrease in number of RBCs as opposed to WBCs. We conclude that inappropriate activation of CaM KIV during embryonic development leads to anemia secondary to an abnormally high rate of RBC apoptosis.
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Discussion |
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CaM KIV Directs Primitive Blood Progenitors into the Erythroid Lineage
During embryogenesis, primitive blood progenitors are committed almost exclusively to erythroid differentiation (Tavassoli 1991), yet can be induced to adopt a myeloid or lymphoid fate when cultured in the presence of appropriate hematopoietic differentiation factors in vitro (Moore and Metcalf 1970), or when transplanted to a different hematopoietic site in vivo (Huang and Auerbach 1993; Turpen et al. 1997). These studies suggest that primitive blood progenitors are pluripotent, but that factors present in their microenvironment restrict their fate and direct them towards erythroid differentiation. Signals that function non-cell–autonomously to restrict the developmental potential of primitive blood progenitors have not been identified. Our studies involving inhibition of endogenous CaM KIV demonstrate that CaM KIV is required to generate these signals.
The increase in WBC number in CaM KIV–deficient embryos could potentially result from expansion of a population of progenitors that arise independent of the VBI, rather than from commitment of primitive HSCs to myeloid differentiation, as proposed above. Grafting experiments in Xenopus have demonstrated the existence of nonlymphoid leukocytes (primarily macrophages) that are not derived from primitive stem cells of the VBI, but originate entirely from the head region of the embryo (Ohinata et al. 1990). More recent studies in zebrafish have identified a similar population of self-renewing macrophage precursors that are not derived from the blood islands (Herbomel et al. 1999). At present, we cannot rule out the possibility that inactivation of CaM KIV induces the proliferation of this distinct leukocyte population. However, this seems unlikely given that we do not see an increase in myeloperoxidase-positive cells in CaM KIV–deficient embryos before formation of the VBI (data not shown). Furthermore, the observation that the increase in WBCs in CaM KIV-deficient embryos is accompanied by a corresponding decrease in RBCs is more consistent with the hypothesis that VBI-derived stem cells become committed to myeloid differentiation at the expense of the erythroid lineage.
Downregulation of CaM KIV Activity May Be Required for Survival and Maturation of Erythroid Progenitors
Our studies show that though a certain level of CaM KIV activity is required for committment of HSCs to erythroid differentiation, abnormally high levels of CaM KIV result in apoptosis of erythroid progenitors. Programmed cell death is a major component of normal erythropoiesis (Koury and Bondurant 1990) and provides a mechanism to upregulate or downregulate red cell number in response to environmental conditions. This process has been best characterized for erythroid progenitors of definitive hematopoiesis. The survival, proliferation, and differentiation of these cells is highly dependent on the hormone erythropoietin (EPO) (for review see Krantz 1991), which exerts its antiapoptotic effects, at least in part, by inducing expression of Bcl-x (Socolovsky et al. 1999). Programmed cell death most likely also plays an important role in regulating the production of primitive erythrocytes, since mice lacking either EPO or its receptor show partial defects in yolk sac hematopoiesis (Wu et al. 1995). Furthermore, Bcl-x appears to be required for the survival of primitive, as well as definitive, erthyrocytes (Motoyama et al. 1999), though this remains controversial (Gregory et al. 1999). It is possible that inappropriate activation of CaM KIV inhibits expression of EPO or another antiapoptotic hormone, thereby leading to excessive death of primitive RBCs.
A caveat of our analysis of phenotypic defects caused by hyperactivation of the CaM KIV–signaling pathway is that these studies can only demonstrate potential functions for this enzyme. Further studies will be needed to determine whether negative regulation of endogenous CaM KIV signaling is required to prevent apoptosis during the normal program of erythroid maturation.
CaM KIV Regulates Production of Extrinsic Signals That Direct Normal Hematopoiesis
Based on the well-documented role of CaM KIV in modulating transcription factor function, a simple hypothesis is that the hematopoietic defects caused by misregulation of CaM KIV activity are a direct result of changes in transcriptional regulation of the blood development program. Numerous hematopoietic-specific transcription factors that participate in proliferation, lineage commitment, survival, and/or differentiation of HSCs have been identified that might be regulated by CaM KIV. For example, GATA-1, SCL, and LMO-2 have been implicated as transcriptional regulators that are required for commitment of progenitors to the erythroid lineage (for review see Sieweke and Graf 1998), whereas the Ets family member PU.1 can instruct pluripotent hematopoietic progenitors to differentiate along the myeloid lineage (Nerlov and Graf 1998). GATA-1 is also required to block apoptosis and thus permit survival and maturation of erythroid precursors (Weiss and Orkin 1995; De Maria et al. 1999). CaM KIV could potentially function cell autonomously within HSCs to activate or inactivate one or more of these transcriptional regulators, either directly, by phosphorylating the factor itself, or indirectly, by regulating the activity of cofactors such as cAMP-responsive element-binding protein–(CREB) binding protein (Hung et al. 1999).
As described below, our data demonstrate that CaM KIV regulates the production of extrinsic signals that act upstream of blood cell–specific transcription factors to control hematopoiesis. Unfortunately, this non-cell–autonomy precludes the use of the Xenopus embryo system to definitively test whether CaM KIV directly regulates signals intrinsic to blood progenitors. Further analyses of CaM KIV function in purified populations of HSCs, or in transgenic mice in which CaM KIV is specifically misregulated in HSCs, will help to resolve this issue.
The observation that erythropoiesis is defective even when CaM KIV activity is perturbed solely in nonerythroid tissues demonstrates a non-cell–autonomous role for this enzyme. Embryologic studies have shown that signals produced by nonhematopoietic tissues are required for normal blood development. In Xenopus, explant studies have shown that signals from the dorsal lateral plate can stimulate progenitors in the VBI to differentiate as myeloid cells of the monocyte/macrophage lineage (Turpen and Smith 1985). Tissue recombination studies have further shown that signals from ectodermal cells, including those in the dorsal half of the animal pole, are required for erythroid cells to synthesize globin protein (Máeno et al. 1994). Finally, transplantation studies have shown that signals present in ventral regions of the embryo are required for commitment of multipotential progenitors to primitive hematopoiesis during gastrulation, and they are sufficient to induce cells that would normally contribute only to definitive hematopoiesis to, instead, form primitive erythrocytes (Turpen et al. 1997). Extrinsic signals are also required for mammalian hematopoiesis. For example, signals generated by the primitive endoderm are required for the initial induction of mesoderm with hematopoietic potential (Belaoussoff et al. 1998). Furthermore, factors produced by endothelial or endodermal-like cell lines derived from the yolk sac (Yoder et al. 1994; Fennie et al. 1995), or other hematopoietic sites (Ohneda et al. 1998), can stimulate self-renewal and direct the lineage commitment and maturation of primitive and definitive HSCs. One hypothesis that is consistent with our results is that endogenous CaM KIV regulates expression of a gene(s) encoding a cell–cell signaling molecule(s) that normally functions to promote survival/proliferation of primitive erythrocytes, but which can specify HSCs to adopt a myeloid fate when present at abnormal levels. This hypothesis could account for the observation that both upregulation and inhibition of CaM KIV activity cause defects in hematopoiesis, since both would lead to abnormal levels of this gene product.
Despite embryological evidence showing that cell–cell signaling between HSCs and their environment is required for normal blood development, the molecular nature of the extrinsic signals that dictate the fate of blood progenitors is unknown. Mutations in many unidentified genes have been shown to disrupt various aspects of hematopoiesis in zebrafish (for review see Parker et al. 1999), and at least one of these (cloche) functions non-cell–autonomously (Parker and Stainier 1999). Our studies demonstrate a critical role for CaM KIV in the generation of extrinsic signals that regulate several different steps in erythroid development. Further analysis of when and where CaM KIV functions during embryonic hematopoiesis will provide a first step toward the eventual identification of extrinsic signals that control the blood program.
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Acknowledgments |
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This work was supported in part by grants from the National Institutes of Health (HD01167 to J.L. Christian, DK44239 to T.R. Soderling, and CA78593 to K. Kolibaba) and the American Heart Association (9750143N to J.L. Christian). M.J. Walters is the recipient of a Tartar Trust Fellowship and G.A. Wayman is supported by a postdoctoral fellowship from the American Heart Association, Northwest Affiliate (99204092).
Submitted: 31 May 2000
Revised: 11 September 2000
Accepted: 3 October 2000
Abbreviations used in this paper: CaM, calmodulin; CaM KI, CaM-dependent kinase I; CaM KIV, CaM-dependent kinase IV; CaM KIVc, constitutively active CaM KIV; CaM KK, CaM-dependent kinase kinase; DMZ, dorsal marginal zone; DnCaM KIV, dominant-negative CaM KIV; EPO, erythropoietin; GATA, erythroid-specific DNA-binding factor; HSC, hematopoietic stem cell; ODC, ornithine decarboxylase; RT, reverse transcription; TUNEL, terminal deoxynucleotidyl transferase nick end labeling; VBI, ventral blood island; VMZ, ventral marginal zone; WBC, white blood cell.
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