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Blood, 1 January 2004, Vol. 103, No. 1, pp. 100-109. Prepublished online as a Blood First Edition Paper on September 4, 2003; DOI 10.1182/blood-2003-04-1063.
HEMATOPOIESIS Ephrin receptor, EphB4, regulates ES cell differentiation of primitive mammalian hemangioblasts, blood, cardiomyocytes, and blood vesselsFrom the Center for Regenerative Medicine and Technology, Massachusetts General Hospital, Boston, MA; and Program in Immunology, Harvard Medical School, Boston, MA.
Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development, however, must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor, EphB4, known to be spatially restricted in expression and critical for organized vessel formation, modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast, blood cell, cardiomyocyte, and vascular differentiation was impaired in EphB4–/– ES cells in conjunction with decreased expression of mesoderm-associated, but not neuroectoderm-associated, genes. Therefore, EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization.
Embryonic stem (ES) cells are pluripotent and their in vitro differentiation provides an experimental window to define the molecular mechanisms controlling germ layer fate determination and tissue formation. Mesoderm induction of ES cells is particularly amenable to in vitro study, and differentiation into blood and vasculature is generally accepted as recapitulating aspects of normal mammalian development. The hematopoietic and vascular systems of the mouse develop from aggregates of extraembryonic mesoderm cells, which colonize the yolk sac at approximately 7.5 days of gestation1,2 and establish blood islands. Blood islands are thought to derive from hemangioblasts,3-5 a common precursor of hematopoietic and endothelial cells. They are organized into an outer layer of endothelial precursors or angioblasts, surrounding an inner cluster of primitive erythroblasts producing embryonic hemoglobin.6-8 The existence of hemangioblasts has been supported by the observation that the hematopoietic and endothelial lineages express a number of genes in common including receptor tyrosine kinases (RTKs), Flk-1 (VEGF-R2) and Tie-2; transcription factors, GATA-2, Runx1, and Scl; and cell surface markers, Sca-1 and CD34.9-14 Recent studies have demonstrated that Flk-1 can serve as a molecular marker for hemangioblasts during early development since Flk-1+ populations can give rise to both hematopoietic and endothelial cells.15-17 Further, Flk-1 is required for hematopoietic and vascular endothelial development within blood islands of the yolk sac.18,19 Molecular regulators of the formation of hemangioblasts are unknown and of considerable interest given the developmental position of the hemangioblast between earliest germ layer formation and tissue-specific stem cell types. Over the past decade, essential roles for Eph receptor tyrosine kinases and their ligands, the ephrins, have emerged from studies of embryonic development.20 Fourteen different Eph receptors have been catalogued into EphA or EphB subclasses based on their affinity for ligands.21 All 8 identified ephrins are membrane proteins of either glycerophosphatidylinositol (GPI)–linked (ephrinA) or transmembrane (ephrinB) proteins.21 Rather than long-range communication, signaling between Eph receptors and their ligands is restricted to sites of direct cell-cell contact and is capable of inducing reciprocal bidirectional events between interacting cells.22-24 Localized expression of ephrins and their receptors are thought to play critical roles in defining tissue patterning and the organizing of highly spatially restricted cell locations. In particular, they participate in modifying cell migration, adhesion, and somite formation.25 EphB4 (or HTK) and its cognate ligand ephrinB2 (or HTKL) play critical roles in determining vascular networks. EphB4 is specifically expressed at the venous endothelium, while ephrinB2 is specifically and reciprocally expressed on arterial endothelial cells at the early stages of vascular development.26 Mice lacking either EphB4 or ephrinB2 are embryonic lethal and display identical defects in forming capillary connections between arterial and venous networks of the head and yolk sac.26-28 EphB4 and ephrinB2 are coexpressed in the yolk sac,27 the first site of hematopoiesis and vascular development during embryogenesis. Our previous studies indicated that EphB4 participates in adult hematopoiesis with primitive human CD34+ hematopoietic cells undergoing accelerated differentiation in the context of activated EphB4.29 We therefore examined EphB4 effects on the hemangioblast, where the processes of hematopoiesis and vasculogenesis intersect. Identification and characterization of hemangioblasts have been hampered by difficulties in accessing the embryo prior to the establishment of the blood islands. In vitro ES differentiation is an alternative experimental system that has provided compelling, direct evidence for the presence of the hemangioblast.30 ES cells differentiate efficiently in vitro and give rise to a differentiated cell mass called embryoid bodies (EBs) upon removal of leukemia inhibitory factor (LIF).31,32 Although the embryoid body is far less organized than the actual embryo, it can partially mimic the spatial organization in the embryo. ES cells can be used to study the differentiation of the mammalian embryo into its earliest recognizable tissue lineages. Many different lineages have been reported within developing EBs including epidermis,33 neuronal and glial cells,34,35 muscle cells,36,37 endothelial cells,38,39 and hematopoietic cells.40,41 It has been reported that the developmental mechanisms of vascular and hematopoietic systems in EBs are similar to that in the yolk sac.16,30,42,43 Recent studies have shown that blast colony–forming cells (BL-CFCs), generated from EBs, represent the long sought common progenitors of hematopoietic and endothelial cells, the hemangioblast. BL-CFCs give rise to primitive, definitive hematopoietic and endothelial cells when plated in medium with hematopoietic and endothelial cell growth factors.30,44 In this study, we report that BL-CFCs and Flk-1+ cells in early-stage EBs were significantly reduced in EphB4-deficient ES cells. Moreover, EphB4 appears to be required for ES cell differentiation into spontaneously beating myocytes and for the induction of alphacardiac mycosin heavy chain (MHC). Analysis of EphB4-deficient ES cells indicated that EphB4 is essential for proper hematopoietic, endothelial, hemangioblast, and primitive mesoderm development. EphB4 affects the rate and magnitude of differentiation suggesting it participates in modifying developmental signals and may be important in coordinating integrated tissue formation.
ES cell culture EphB4-deficient ES cells were kindly provided by Dr David Anderson (California Institute of Technology, Pasadena, CA). EphB4–/– ES cells were generated by a high concentration of G418 selection from EphB4+/– cells that gave germ line transmission for EphB4+/– mice.28 Two clones of undifferentiated EphB4–/– ES cell lines (no. 49 and no. 71 as EphB4–/–1 and EphB4–/–2, respectively) were maintained on a mouse feeder cell line, SNL, in ES medium containing Dulbecco modified Eagle medium (DMEM), 10 ng/mL murine leukemia inhibitory factor (mLIF; Chemicon International, Temecula, CA), 15% fetal calf serum (FCS; HyClone, Logan, UT), 1 mM sodium pyruvate, 2 mM glutamine, 0.1 mM nonessential amino acid, 100 µM monothioglycerol (MTG; Sigma, St Louis, MO), 50 U/mL penicillin, and 50 µg/mL streptomycin. ES cells were cultured on gelatincoated plates for one week prior to EB induction. ES cell differentiation and BL-CFC assay ES cell differentiation into EBs were carried out as described.30,40,44 Briefly, EBs were generated either in liquid or 1% methylcellulose cultures (1 x 104 ES cells per 35-mm Petri dish) in ES differentiation medium containing Iscove modified Dulbecco medium (IMDM), 15% FCS (StemCell Technologies, Vancouver, BC, Canada), 2 mM glutamine, 450 µM MTG, 50 µg/mL ascorbic acid, and 20% BIT (1% bovine serum albumin [BSA], 10 µg/mL insulin, and 200 µg/mL transferrin; StemCell Technologies). After 6 days of differentiation, 50 ng/mL murine stem cell factor (mSCF), 1 ng/mL interleukin 3 (IL-3), 5 ng/mL IL-11, and 2 U/mL human erythropoietin (hEPO; Amgen, Thousand Oaks, CA) were added to cultures to promote hematopoietic differentiation. To assess BL-CFCs, day-3.5 to -4.0 EBs were dissociated by trypsin treatment and by passing through 20-gauge needle to generate single cells. To generate blast colonies from hemangioblasts, 1 x 104/mL EB cells were replated in 1% methylcellulose in the presence of IMDM, 10% PDS (bovine platelet-poor plasma-derived serum; Biomedical Technologies, Stoughton, MA), 2 mM glutamine, 450 µM MTG, 25 µg/mL ascorbic acid, 20% BIT, 5 ng/mL human vascular endothelial growth factor (hVEGF), 50 ng/mL SCF, 10 ng/mL human fibroblast growth factor 2 (hFGF-2), and 2 U/mL hEPO. BL-CFCs can be recognized as loose clusters of cells after 4 days of culture. To analyze the hematopoietic and endothelial developmental potential of the BL-CFC, blast colonies from either EphB4+/– or EphB4–/– were transferred into matrigel-coated plates and cultured in the presence of growth factors known to support both hematopoietic and endothelial cells.30 Following 3 to 4 days in culture, nonadherent hematopoietic and adherent endothelial cells were harvested for reverse transcriptase–polymerase chain reaction (RT-PCR) analysis. Primitive erythroid progenitors (EryP) were analyzed from day-6 EBs in 1% methylcellulose cultures containing IMDM, 10% PDS, 2 mM glutamine, 450 µM MTG, 5% PFHM-II (protein-free hybridoma media II; Gibco-BRL, Carlsbad, CA), and 4 U/mL hEPO. EryP colonies can be identified as small brilliant red colonies and are scored after 5 to 7 days replating.44 Other definitive myeloid progenitors were analyzed from day-10 to -12 EBs in 1% methylcellulose cultures containing IMDM, 10% PDS, 2 mM glutamine, 450 µM MTG, 5% PFHM-II, 4 U/mL hEPO, 50 ng/mL SCF, 1 ng/mL IL-3, 5 ng/mL macrophage colony-stimulating factor (M-CSF), 5 ng/mL thrombopoietin (TPO), 5 ng/mL IL-11, 30 ng/mL granulocyte-CSF (G-CSF), 3 ng/mL GM-CSF, and 5 ng/mL IL-6. Hematopoietic colonies were counted 7 to 10 days after replating. SCF, IL-3, M-CSF, TPO, IL-11, G-CSF, GM-CSF, IL-6, hVEGF, and hFGF-2 were purchased from R&D Systems (Minneapolis, MN). Sprouting EB induction ES cell differentiation into endothelial cells was performed as described.43,45 EBs were initially generated in 1% methylcellulose cultures (1 x 103 ES cells/mL in 2 mL in 35-mm Petri dish) in IMDM containing 15% FCS, 2 mM glutamine, 450 µM MTG, 20% BIT, 50 ng/mL hVEGF, 100 ng/mL hFGF-2, 2 U/mL hEPO, and 10 ng/mL mIL-6. After 11 days of EB differentiation, EBs were harvested and resuspended at 50 EBs/mL in collage medium containing rat-tail collagen type I (Becton Dickinson, San Jose, CA) or Vitrogen (Cohesion Technologies, Vancouver, BC, Canada), 15% FCS, 450 µM MTG, 10 µg/mL insulin, 50 ng/mL hVEGF, 100 ng/mL hFGF-2, and 10 ng/mL mIL-6. After gently mixing EBs into collagen medium, 1 mL was dispensed into 35-mm Petri dishes or double-chamber slides and incubated at 37°C without CO2 for 30 minutes to form a stable collagen gel matrix. The cultures were then incubated for 3 to 6 days at 37°C and 5% CO2. The vascular spindlelike EBs were scored at day 3 on collagen matrix. Flow cytometric analysis and immunostaining
EBs were trypsinized for 2 minutes and passed through a 20-gauge needle to produce a single cell suspension. Cells (1 x 106) were stained for EB cultures in collagen in chamber slides were dehydrated and fixed in methanol-acetone (3:1) solution for 20 minutes. After air drying and rehydrating with PBS, capillary-sprouting EBs were incubated in 5% rat serum for 20 minutes to block nonspecific binding and stained with 1 µg/mL PE-conjugated antimouse platelet endothelial cell adhesion molecule 1 (PECAM1, CD31; Pharmingen) for 1 hour. Gene expression analysis Total RNA was isolated from EBs using Trizol (Invitrogen, Carlsbad, CA) according to manufacturer's instructions and then cleaned by RNeasy kit (Qiagen, Valencia, CA). One microgram RNA was used for reverse transcription using 200 units SuperScript II RNase H– reverse transcriptase (Invitrogen) in the presence of 50 mM Tris (tris(hydroxymethyl)aminomethane; pH 8.3), 75 mM KCl, 3 mM MgCl2, 5 mM dithiothreitol (DTT), 40 units ribonuclease inhibitor, 0.5 mM deoxynucleoside triphosphates (dNTPs), and 100 ng random primers in 20-µL volume. Specific primers used for PCR are listed in Table 1. All the genes were analyzed on more than one occasion using RNA from independently EB samples.
DNA microarrays cDNA synthesis, cRNA synthesis, and labeling were performed as described in the Affymetrix user's manual (Santa Clara, CA). Affymetrix mouse U74Av2 chips (about 12 000 genes) were hybridized on a GeneChip system (Affymetrix) at the Center for Genomics Research of Harvard University and the Massachusetts General Hospital (MGH) Cancer Center according to the manufacturer's instruction. All arrays were globally scaled to a target value of 150 using the average signal from all gene features. Scanned chip images were analyzed using the Microarray Suite Software version 5.0 (Affymetrix). Microsoft Excel was used for further analyses. Four experiments from independent EB samples were used at the Center for Genomics Research of Harvard University and the MGH Cancer Center. A transcript was considered "changed" when the fold change was at least 2.
EphB4–/– ES cells organize into embryoid bodies with altered composition EphB4-null ES cells (EphB4–/–) were generated from EphB4+/– cells by culturing in high concentrations of G418. Two individual clones of both EphB4+/– (EphB4+/–1 and EphB4+/–2) and EphB4–/– (EphB4–/–1 and EphB4–/–2) were originally derived and used in our experiments. However, because identical results were obtained from the 2 clones, subsequent experiments used only one of the clones (EphB4+/–1 and EphB4–/–1), and one clone for each genotype is reported here. The doubling time of undifferentiated ES cells from wild-type, EphB4+/–, and EphB4–/– remained the same (about 18 hours). Gene expression profiles and flow cytometric analyses of wild-type ES cells were very similar to EphB4+/– ES cells (data not shown). Because the wild-type cells had not gone through the transduction and selection processes of the genetically modified cells and we were concerned about alterations induced by these processes, we focused on comparing the EphB4+/– and EphB4–/– cells.
To induce spontaneous differentiation of ES cells into cystic EBs, ES cells were cultured in suspension in the absence of LIF for 4 days. RT-PCR was used to confirm that EphB4–/– EBs did not express EphB4 transcripts, excluding possible contamination by EphB4+/– or EphB4+/+ ES cells (Figure 1A). We initially examined the expression of a hematopoietic gene,
EphB4-deficient ES cells display a defect in hemangioblast development To evaluate the effects of EphB4 on the formation of the early mesoderm-associated hemangioblast, day-3 EBs of EphB4+/– and EphB4–/– ES cells were assayed for their potential to generate hemangioblast-containing BL-CFCs. Previous studies demonstrated that BL-CFCs could be detected as early as 2.5 days of EB differentiation. The maximum numbers of BL-CFCs could be reached from 3.25 to 4.25 days of EB differentiation.44 When cells from EBs are dissociated and replated on methylcellulose, BL-CFCs can be readily recognized as loose clusters compared with the compact secondary embryoid bodies (Figure 2A). BL-CFCs represent entry into a differentiation program where secondary EBs reflect cells retaining a primitive status. Relative to EphB4+/– ES cells, generation of BL-CFCs from EphB4–/– cells was significantly impaired (Figure 2B). EphB4-deficient cells gave rise to more secondary EBs, indicating that more EphB4-deficient cells remain undifferentiated (Figure 2B). To determine whether this decreased production was due to a delay in hemangioblast formation, a more detailed kinetic analysis of the formation of BL-CFCs from EphB4+/– and EphB4–/– ES cells was performed (Figure 2C). EphB4+/– and EphB4–/– ES cells demonstrated similar kinetics as previously described.44 However, the magnitude of BL-CFC formation was significantly lower in EphB4–/– ES cells. Therefore, the absence of EphB4 impairs hemangioblast differentiation.
To determine if the hematopoietic and endothelial developmental potential of EphB4-deficient BL-CFCs was also impaired, blast colonies of day-3 EB cells from either EphB4+/– or EphB4–/– were transferred into matrigel-coated plates and cultured in the presence of growth factors known to support both hematopoietic and endothelial cells. Following 3 to 4 days in culture, nonadherent hematopoietic and adherent endothelial cells were harvested for RT-PCR analysis (Figure 2D). The hematopoietic gene,
Several studies have demonstrated that Flk-1 is a molecular marker for hemangioblasts during early development since Flk-1+ populations can give rise to both hematopoietic and endothelial cells.15-17 To determine whether the EphB4+ and Flk-1+ cells represent overlapping populations in early ES cell differentiation, cells from day-4 EBs were analyzed by flow cytometry. As shown in Figure 3A, half of the cells strongly positive for
EphB4 deficiency affects the developmental potential of both primitive and definitive hematopoiesis
Previous studies demonstrated that BL-CFCs give rise to primitive and definitive hematopoietic cells as well as endothelial cells when plated in medium with hematopoietic and endothelial cell growth factors.30,44 To assess whether EphB4 signals also contribute to the differentiation of hematopoietic and endothelial cells, we used the in vitro ES cell system, which provides easy access to study both primitive and definitive hematopoiesis. At day 4 of EB induction, the number of cells staining for a hematopoietic stem cell immunophenotype (Scal-1+/c-kit+) was decreased in EphB4–/– ES cells and remained low at day 9 (Figure 3C). Decreases in Scal-1+/c-kit+ cells and down-regulation of
EphB4 affects EB endothelial differentiation In vitro differentiation of ES cells has been used to investigate molecular mechanisms of vasculogenesis.38,43,47,48 To assess the role of EphB4 in vasculogenesis and angiogenesis, we used a model of in vitro differentiation using ES cells on a collagen I matrix to form a primitive vascular network. In this model, EBs develop endothelial sprouting in the presence of angiogenic growth factors.45 EphB4+/– and EphB4–/– ES cells were cultured on methylcellulose for 11 days in the presence of VEGF and basic FGF (bFGF) to induce a primitive vascular plexus.43 At day 6, smaller EBs were observed in the EphB4–/– ES cell culture (Figure 5A). After 10 to 11 days of culture, EphB4–/– EBs were of similar size as EphB4+/– EBs, but EphB4–/– EBs displayed a less well-defined morphology (Figure 5A). Subculture of these EBs on a collagen I matrix was used to assess sprouting angiogenesis as described by Feraud et al.45 After the standard 3-day culture on the collagen I matrix, the ability of EphB4–/– EBs to form microtubule outgrowths was significantly reduced (Figure 5). With additional culture, sprouts from individual EBs were able to connect and generate vascular network–like structures that were CD31+ by immunohistochemistry (Figure 5B). These data indicate a regulatory role for EphB4 in primitive vascular development.
Differentiation of ES cells into beating cardiac myocytes is impaired
Hemangioblasts, blood cells, and vascular endothelial cells are of mesodermal origin. Since cardiomyocytes are also developed from mesoderm, we investigated whether EphB4 had any effect on cardiomyocyte formation by an in vitro differentiation assay. After 5 days in methylcellulose culture, individual EBs were transferred into separate wells of a 48-well plate coated with gelatin. After 24 hours, EphB4+/– EBs were attached to the surface of the wells and had a spread-out morphology. In contrast, EphB4–/– EBs had a smaller and more compact morphology (Figure 6A). Further, the expression of the muscle-specific gene,
EphB4 deficiency alters expression time course of mesoderm-specific gene products
To examine the possible molecular mechanisms of EphB4 on ES cell differentiation, we analyzed gene expression of EBs by concentrating on several ES, hematopoietic, endothelial, and mesodermal-associated genes. EBs obtained from different time points were subjected to gene expression analysis using RT-PCR. Consistent with the results previously described by others,49 Rex1 and Oct-4, which are expressed in the inner cell mass and in undifferentiated ES cells, were down-regulated in EphB4+/– and EphB4–/– ES cells during EB differentiation over the course of 5 days (Figure 7). Both EphB4 and ephrinB2 gene expression was detectable in undifferentiated ES cells and in early EB development. The expression of EphB4 and ephrinB2 appeared earlier than the expression of Flk-1, raising the possibility that EphB4 and ephrinB2 may play a role in early EB differentiation. The absence of EphB4 did not effect gene expression of either EphB2 or EphB3, nor did it affect expression of the ligand, ephrinB2. However, expression of both the embryonic hemoglobin gene (
To broadly assess gene expression influenced by EphB4, we used DNA microarray (Affymetrix U74Av2 chips) to compare day-4 EBs from EphB4+/– ES cells with those from EphB4–/– ES cells. We found that 571 genes were down-regulated and 873 genes were up-regulated in EBs on day 4 of EphB4–/–. Among genes down-regulated in EphB4–/– ES cells, there were genes involved in mesoderm formation (such as BMP-4, T-box2, TGF-
The data presented here indicate that EphB4 deficiency results in an alteration in the mesodermal differentiation outcome of ES cells. The effects observed were in the timing of expression of specific gene products, and these were validated as reflecting a substantial functional change by in vitro differentiation systems. EphB4 was not essential for completion of any of the differentiation programs assessed; rather, it modulated the rate and magnitude by which they were accomplished. The systems most well defined were those of very early transition into a multipotent hemangioblast and its descendents, resulting in fewer differentiated cells. These data indicate that EphB4 is an early-acting molecular regulator of hemangioblast formation, perhaps functioning temporally similar to Indian hedgehog or Scl.50,51 Its combined effect on the timing of expression of certain lineage-defining genes and the magnitude of specific phenotypically defined cells suggest that there is a critical window for coordinated gene expression that EphB4 may help orchestrate to achieve maximal mesodermal cell output. Absent signaling through this receptor, mesoderm differentiation programs can proceed but with much diminished efficiency in cell production. EphB4 appears to exert its effects prior to Flk-1 expression. Flk-1 is required for vascular and blood cell differentiation from hemangioblasts, and both EphB4 and Flk-1 appear to regulate this transition, but EphB4 appears to also act at an earlier step in differentiation. EphB4 affects the expression of the most primitive mesodermal gene products involved in hemangioblast and myocyte formation (Figure 8). These are the first data showing a role for EphB4 in myocyte development and place EphB4 uniquely early in mesoderm tissue development.
EphB4's ability to modify differentiation kinetics and outcome poses an interesting set of potential roles. In the simple differentiation systems tested, the time to achieve certain outcomes was delayed, accompanied by a change in the magnitude of cells bearing the more mature phenotype. Rate of differentiation was clearly associated with number of mature cells in settings such as that of embryoid body formation where a reciprocal relationship between mature and immature cells was apparent. The relative balance of mature to immature cells is an expected effect of a change in differentiation kinetics and may be of particular relevance for development. Ephrins and their receptors are often viewed from their association with guidance cues for cell migration, important for establishing organizational patterns of tissues. The data presented here of a differentiation-controlling effect of EphB4 suggests another possible means by which ephrins and Eph receptors may influence tissue pattern formation. While experiments with deficient cells cannot define the physiologic role of receptor activation, the data indicate that EphB4 receptor signaling can modulate the relative abundance of primitive and more differentiated cells. Since ephrins act locally, it may be a mechanism by which tissue boundaries achieve graduated differentiation subsets and perhaps geographically restrict stem cell populations. We previously noted that overexpression (activation) of EphB4 led to accelerated exit of primitive adult hematopoietic cells from a functional stem cell population.29 In the setting of the EphB4-deficient ES model used here, inhibition of differentiation and preservation of a primitive cell type was observed. Therefore the relative gradient of ligand/receptor expression and interaction may dictate the relative abundance of cells from specific differentiation stages. Transition from a stem cell pool may be of particular interest in development at boundaries where ongoing stem cell function would have the potential to be particularly disruptive. Indeed, ectopic expression of EphB4 in mammary tissue resulted in disordered architecture, abnormal tissue function, and a predisposition to malignancy.52 Absence of EphB3 was associated with altered intestinal crypt architecture.53 These effects may be due to cell migratory effects, but containing primitive cells within borders of more differentiated populations may also reflect a graduated differentiation imposed by ephrin signaling. We propose a model by which ephrin expression is a means of creating a boundary of differentiated cells, effectively restricting primitive cells to locations where ephrin receptor activation is minimized. Data from Durbin et al25 in the zebrafish provide support for this possibility. They noted that altered expression of Ephrin A-L1, Ephrin B2, or EphA4 disrupted normal somite formation in conjunction with reduced expression of the muscle differentiation marker, MyoD.25 The data presented here provide further rationale for a link of differentiation to patterning, but other experimental systems are clearly required to definitively assess it in mammals. Since the absence of EphB4 did result in an overabundance of more primitive cells, it raises the additional hypothesis that manipulation of the EphB4/ephrinB2 axis may provide a means of altering cell differentiation kinetics ex vivo. In particular, stem cell expansion has been an important but elusive goal for hematopoietic stem cells due to the propensity of the cells to differentiate when cultured. Whether inhibition of EphB4 signaling can favorably affect adult stem cell kinetics to preserve a stemlike pool is now reasonable to test. In sum, the data presented here indicate that EphB4 is a new molecular modulator of mesodermal differentiation rate and for the first time define it as a regulator of hemangioblast and myocyte formation. The effect of EphB4 on differentiation kinetics, and thereby the relative abundance of mature to immature cells, may be hypothesized to participate in the known role of ephrins and their ligands in defining tissue organization during organogenesis.
We thank Dr David Anderson for the generous gift of EphB4-deficient ES cell lines.
Submitted April 8, 2003; accepted August 13, 2003.
Prepublished online as Blood First Edition Paper, September 4, 2003; DOI 10.1182/blood-2003-04-1063.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David T. Scadden, Massachusetts General Hospital, Harvard Medical School, 149 13th St, Room 5212, Charlestown, MA 02129; e-mail: scadden.david{at}mgh.harvard.edu.
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Abstract
Introduction
-galactosidase (
-cardiac myosin heavy chain, was detected by RT-PCR on day 7 and day 10 in EphB4+/– EBs by RT-PCR, but only detectable in EphB4–/– day-10 EBs (
