FGFR1 and the Bloodline of the Vasculature
http://www.100md.com
动脉硬化血栓血管生物学 2005年第5期
From the Laboratory for Physiology (V.W.M.w.H.), Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, and the Department of Nephrology (T.J.R.), Leiden University Medical Center, Leiden, The Netherlands.
Correspondence to Victor W.M. van Hinsbergh, PhD, Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. E-mail v.vanhinsbergh@vumc.nl
Since the recognition of basicfibroblast growth factor (bFGF, FGF-2) and acidic FGF (aFGF, FGF-1) as proliferation and maintenance factors for endothelial cells in culture,1,2 much interest has arisen regarding their role as potential angiogenic factors in tumors and tissue healing in vivo. The FGFs, of which presently at least 22 members have been recognized,3 interact with FGF receptors (FGFRs). After FGF binding, the FGFRs dimerize and become activated displaying protein tyrosine kinase activity and subsequent signaling in the cell.4,5 Four functional FGFRs have been recognized, FGFR1–4, most of which can be encountered in various alternative spliced isoforms. FGFR1 plays an essential role during early stages of development of the mesoderm and at several levels during organogenesis.3,6 FGF/FGFR1 is expressed almost exclusively in the mesenchyme, including endothelial cells, smooth muscle cells, and hemopoietic progenitor cells. FGFR1 is involved in the stimulation and proliferation of endothelial cells and vascularization by FGFs, particularly bFGF and aFGF. FGFs also promote the formation of hematopoietic progenitor cells in early development, which requires the FGFR1 as well.7,8 In line with this, chromosomal translocation involving FGFR1 has been observed in patients with stem-cell myeloproliferative disease, probably caused by constitutive activation of the FGFR1 gene.9
See page 944
It has long been anticipated that hematopoietic progenitor cells and endothelial progenitor cells derive from a common cell type, the hemangioblast.10 Studies on mouse embryos have shown that such hemangioblast cells indeed exist. These cells develop in the primitive streak of the embryo and migrate to the yolk sac, where they form blood islands.11 The outer cells of these blood islands mature into endothelial cells and form a vascular plexus, while the inner cells develop into primitive and definitive hematopoietic progenitor cells and their subsequent lineages of blood cells. In addition, in the para-aortic splanchnopleura, hemangioblasts develop into vascular structures.11 Later in development the primitive and definitive hematopoietic progenitor ("stem") cells migrate to the fetal liver and subsequently to the bone marrow, where they remain life-long active as a source for white and red blood cells, mast cells, and megakaryocytes, from which blood platelets are formed.
One of the issues that remained unresolved was the contribution of FGF signaling to the early development of the vascular tree. Embryos deficient in FGFR1 stop growing at an early stage of gestation because of abnormal mesodermal patterning.12,13 However, when embryonic stem cells were derived from FGFR1-deficient blastocysts and administered subcutaneously in nude mice, these embryonic stem cells were still able to differentiate into different types of mesodermal cells.14 Thus FGFR1 causes inappropriate mesodermal patterning rather than impeding mesodermal differentiation. Because FGFs are required for proper mesodermal pattern development, deficiency of FGFR1 interfered with the early development and viability of embryos in the mouse. To overcome this initial blockade of development, Faloon et al7 and Magnusson et al15,16 used the technique of growing embryoid bodies from embryonic stem cells. Pluripotent embryonic stem cells can be isolated from the inner cell mass of a blastula stage embryo. These cells can be grown in culture and can be induced to aggregate into spheroid bodies, the embryoid bodies, by specific culture conditions, which includes the withdrawal of the factor LIF. The inner mass of these embryoid bodies subsequently develops into mesodermal cells, a part of which further differentiate into hemangioblast cells and subsequently into hematopoietic cells, endothelial cells, and vascular smooth muscle cells (Figure 1). The embryoid bodies appeared helpful in elucidating the contribution of various factors involved in hematopoietic and endothelial development.17–19 Their regulation is highly comparable with that in the yolk sac of murine embryos. It has become clear that the differentiation steps from mesodermal progenitor cells into hemangioblasts and subsequently into primitive and definitive lineages of hematopoietic and endothelial progenitor cells required the expression of growth factors including BMP-4 and VEGF, the growth factor receptors VEGFR-2 (Flk1, KDR) and FGFR1, CD41, and transcription factors, such as SCL/Tal1 and Runx1 (required for hematopoietic cells formation).20–29 Members of the transforming growth factor (TGF)-? signaling family, activin A, TGF-?, and possibly BMP-2, act as positive (Activin A) and negative regulators (TGF-?).30,21 The various steps thought to be involved in the differentiation of mesoderm into hematopoietic and endothelial progenitor cells are summarized in Figure 2.
Figure 1. Development of early vasculature in embryoid bodies. EB indicates embryoid body; pcd, postcoidal day; LIF, leukemia inhibitory factor.
Figure 2. Differentiation of hemangioblasts into hematopoietic cells and endothelial cells.
In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Magnusson et al15 report on the expression of FGFR-1 in embryoid bodies and its requirement for hematopoietic and endothelial cell development and function. They show that FGFR1 is expressed and becomes activated in a subpopulation of endothelial cells during the development of embryoid bodies. Its expression was associated with cell division, suggesting that particularly dividing endothelial cells express FGFR1. Whether this is related to the cell cycle state of the cell or reflects a subpopulation of rapidly dividing endothelial cells remains uncertain. In FGFR1-expressing endothelial cells (FGFR1+/CD31+) bFGF stimulated mitogenesis, both in the absence and presence of VEGF. Addition of bFGF to embryoid bodies particularly stimulates angiogenesis in embryonic bodies by the length growth of vascular structures, while the stimulatory effect of VEGF regards the formation of a complex capillary plexus.16 Although embryoid bodies derived from stem cells that lack one or both alleles of FGFR1 are still able to form endothelial cells and vascular structures, the formation of hematopoietic progenitor cells is impaired. Analysis of the mRNA expression in FGFR1–/– embryoid bodies showed a 50% increase of the mesodermal marker brachyury at day 4, indicating a delayed progression of differentiation as compared with their FGFR1+/– counterparts. After 8 days, when endothelial cells and vascular structures had become developed, a relatively normal development of the endothelial lineage in these embryonic bodies was observed. However, a number of genes involved in hematopoietic differentiation were highly suppressed in the FGFR1–/– embryoid bodies, including SCL/Tal1, CD41, and the ?-H1 and GATA-1, two proteins involved in development of erythrocytes. Indeed, at day 12 day virtually no expression of the leukocyte marker CD45 was seen in FGFR1–/– embryoid bodies, in contrast to the FGFR1+/– ones. This indicates that the differentiation into hematopoietic progenitor cells is severely impaired, and suggests that FGF/FGFR1 signaling is required for this differentiation.
These data are consistent with previous observations of Faloon et al,7 who showed a considerable reduction of VEGFR2, SCL/Tal1, ?-H1, and ?-major production in FGFR–/– embryoid bodies as compared with their FGFR1 wild-type counterparts. SCL/Tal1 is required for the commitment of hemangioblasts into hematopoietic progenitor cell differentiation.21–23,31 Therefore, it is likely that the absence of SCL/Tal1 in FGFR1–/– embryonic bodies is an important contributor to the lack of hematopoietic differentiation. Overexpression of SCL/Tal1 in FGFR1–/– embryonic bodies may resolve whether this is the only factor preventing hematopoiesis or whether other factors also contribute to this phenomenon. Faloon et al7 indicated that in SCL/Tal1–/– embryoid bodies endothelial markers were elevated 2- to 4-fold, suggesting preferential differentiation into the endothelial lineage. This raises two questions. First, are the endothelial cells differentiating directly from the primitive hemangioblast in FGFR1–/– and SCL/Tal1–/– conditions identical in characteristics as those deriving wild-type conditions from the definitive hemangioblasts? Second, does (over)expression of SCL/Tal1 reduce differentiation into the angioblast and endothelial lineage, or is the increase in endothelial differentiation in FGFR1–/– and SCL/Tal1–/– embryonic bodies merely caused by the lack of transition into hematopoietic progenitor cells? Other studies have shown that SCL/Tal1 deficiency causes defects in endothelial growth and yolk sac vascularization, indicating that this factor also contributes to proper endothelial development.31,32 Apparently experimental conditions may influence the degree to which endothelial development occurs in this deficiency. Because replenishment of SCL/Tal1 in 3.5- to 4-day-old SCL/Tal1–/– embryoid cells by Cre-lox–mediated induction or viral transduction showed that the function of hematopoietic and endothelial cells can be normalized,31,33 it is likely that SCL/Tal1 deficiency causes deflection of the hemangioblast differentiation into the direction of the angioblast lineage producing endothelial cells and smooth muscle cells. A similar mechanism may occur in the case of FGFR1 deficiency.
Although the studies of Magnusson et al15 and Faloon et al7 indicate that FGFR1 is dispensable for early vascular development in embryoid bodies, this does not exclude that FGFR1 contributes to vascular development and angiogenesis. Several lines of evidence support such a role. First, part of the endothelial cells in the embryoid bodies and in the new vasculature of teratomas does contain FGFR1. A considerable part of these cells is involved in cell division. Whether this expression is paralleled by effects of FGF/FGFR1 on cell division or migration cannot be deduced from these experiments. An alternative explanation might be that FGF/FGFR1 signaling reduces the vulnerability of endothelial cells for apoptosis, as shown in vitro.34,35 It may be a general strategy in development to enhance apoptosis vulnerability of cells that make important steps in differentiation or morphogenic patterning, to avoid improper growth. If FGF/FGFR1 signaling would be able to overcome this vulnerability, FGF signaling would act as a "permissive factor" in development. Indeed, such a permissive role of FGF has been well documented in neural stem cell differentiation where FGF has a dual action maintaining the size of the progenitor pool by its proliferative effects as well as stimulating proneural differentiation by modulating signaling36 Given the involvement of FGF/FGFR1 signaling in the process of differentiation of many different cells, including cardiomyocytes, adipocytes, bone cells, and hematopoietic cells and in patterning of mesoderm and embryo vessels,7,15,37–39 this is a concept that warrants further investigation. Such a concept may also explain why the subpopulation of circulating progenitor cells that contain FGFR1 may be particularly successful in differentiating into an endothelial phenotype in adult man.40
Interestingly in the FGFR1–/– embryoid bodies the lack of FGFR-1 signaling resulted in an abnormal branching pattern of the vasculature.15,16 Although these in vitro systems do not completely reflect the normal embryonic or organ-specific multicellular environment (embryoid bodies lack the polarity of the embryo) and the precise state of differentiation of endothelial cells, they underpin a potential role of FGF/FGFR1 signaling in patterning of the vascular network. This is in line with studies of Lee et al,29 who used a dominant-negative truncated FGFR1 behind a CMV-promoter, which was injected in the 9-day-old murine embryo, and becomes expressed preferentially in vascular and endocardial endothelial cells. The overexpression resulted in abnormal development including incomplete branching of the vascular network.29
The formation of a vascular network as well as mesodermal development require coordination of cell signaling and stepwise recruitment of cytokines in a temporally and spatially coordinated manner. These processes warrant factors that can change the cellular environment from a nonpermissive one into a permissive milieu for such processes. The current study of Magnusson adds another piece of the puzzle to the concept that FGF/FGFR1 is not a conditional factor but rather one of these permissive factors.
References
Gospodarowicz D, Moran J, Braun D, Birdwell C. Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent. Proc Natl Acad Sci U S A. 1976; 73: 4120–4124.
Maciag T, Cerundolo J, Ilsley S, Kelley PR, Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc Natl Acad Sci U S A. 1979; 76: 5674–5678.
Itoh N, Ornitz DM. Evolution of the Fgf and Fgfr gene families. Trends Genet. 2004; 20: 563–569.
Auguste P, Javerzat S, Bikfalvi A. Regulation of vascular development by fibroblast growth factors. Cell Tissue Res. 2003; 314: 157–166.
Stachowiak MK, Fang X, Myers JM, Dunham SM, Berezney R, Maher PA, Stachowiak EK. Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation. J Cell Biochem. 2003; 90: 662–691.
Coumoul X, Deng CX. Roles of FGF receptors in mammalian development and congenital diseases. Birth Defects Res C Embryo Today. 200; 69: 286–304.
Faloon P, Arentson E, Kazarov A, Deng CX, Porcher C, Orkin S, Choi K. Basic fibroblast growth factor positively regulates hematopoietic development. Development. 2000; 127: 1931–1941.
Poole TJ, Finkelstein EB, Cox CM. The role of FGF and VEGF in angioblast induction and migration during vascular development. Dev Dyn. 2001; 220: 1–17.
Guasch G, Mack GJ, Popovici C, Dastugue N, Birnbaum D, Rattner JB, Pebusque MJ. FGFR1 is fused to the centrosome-associated protein CEP110 in the 8p12 stem cell myeloproliferative disorder with t(8;9)(p12;q33). Blood. 2000; 95: 1788–1796.
Bailey AS, Fleming WH. Converging roads: evidence for an adult hemangioblast. Exp Hematol. 2003; 31: 987–993.
Huber TL, Kouskoff V, Fehling HJ, Palis J, Keller G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature. 2004; 432: 625–630.
Deng CX, Wynshaw-Boris A, Shen MM, Daugherty C, Ornitz DM, Leder P. Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev. 1994; 8: 3045–3057.
Yamaguchi TP, Harpal K, Henkemeyer M, Rossant J. Fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 1994; 8: 3032–3044.
Xu X, Weinstein M, Li C, Deng C. Fibroblast growth factor receptors (FGFRs) and their roles in limb development. Cell Tissue Res. 1999; 296: 33–43.
Magnusson PU, Ronca R, Dell’era P, Carlstedt P, Jakobsson L, Partanen J, Dimberg A, Claesson-Welsh L. Fibroblast Growth Factor Receptor-1 Expression Is Required for Hematopoietic but not Endothelial Cell Development. Arterioscler Thromb Vasc Biol. 2005; 25: 944–949.
Magnusson P, Rolny C, Jakobsson L, Wikner C, Wu Y, Hicklin DJ, Claesson-Welsh L. Deregulation of Flk-1/vascular endothelial growth factor receptor-2 in fibroblast growth factor receptor-1–deficient vascular stem cell development. J Cell Sci. 2004; 117: 1513–1523.
Risau W, Sariola H, Zerwes HG, Sasse J, Ekblom P, Kemler R, Doetschman T. Vasculogenesis and angiogenesis in embryonic-stem-cell–derived embryoid bodies. Development. 1988; 102: 471–478.
Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998; 125: 725–732.
Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development. 1998; 125: 1747–1757.
Huber TL, Zhou Y, Mead PE, Zon LI. Cooperative effects of growth factors involved in the induction of hematopoietic mesoderm. Blood. 1998; 92: 4128–4137.
Park C, Afrikanova I, Chung YS, Zhang WJ, Arentson E, Fong Gh G, Rosendahl A, Choi K. A hierarchical order of factors in the generation of FLK1- and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells. Development. 2004; 131: 2749–2762.
Robb L, Lyons I, Li R, Hartley L, Kontgen F, Harvey RP, Metcalf D, Begley CG. Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc Natl Acad Sci U S A. 1995; 92: 7075–7059.
Ema M, Faloon P, Zhang WJ, Hirashima M, Reid T, Stanford WL, Orkin S, Choi K, Rossant J. Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse. Genes Dev. 2003; 17: 380–393.
Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996; 380: 435–439.
Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996; 380: 439–442.
Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L, Bernstein A, Rossant J. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell. 1997; 89: 981–990.
Ferkowicz MJ, Starr M, Xie X, Li W, Johnson SA, Shelley WC, Morrison PR, Yoder MC. CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo. Development. 2003; 130: 4393–4403.
Fehling HJ, Lacaud G, Kubo A, Kennedy M, Robertson S, Keller G, Kouskoff V. Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development. 2003; 130: 4217–4227.
Lacaud G, Gore L, Kennedy M, Kouskoff V, Kingsley P, Hogan C, Carlsson L, Speck N, Palis J, Keller G. Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood. 2002; 100: 458–466.
Dickson MC, Martin JS, Cousins FM, Kulkarni AB, Karlsson S, Akhurst RJ. Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development. 1995; 121: 1845–1854.
D’Souza SL, Elefanty AG, Keller G. SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast, but not for its development. Blood. 2005Jan 27;.
Visvader JE, Fujiwara Y, Orkin SH. Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development. Genes Dev. 1998; 12: 473–479.
Endoh M, Ogawa M, Orkin S, Nishikawa S. SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation. EMBO J. 2002; 21: 6700–6708.
Lee SH, Schloss DJ, Swain JL. Maintenance of vascular integrity in the embryo requires signaling through the fibroblast growth factor receptor. J Biol Chem. 2000; 275: 33679–33687.
Erez N, Zamir E, Gour BJ, Blaschuk OW, Geiger B. Induction of apoptosis in cultured endothelial cells by a cadherin antagonist peptide: involvement of fibroblast growth factor receptor-mediated signalling. Exp Cell Res. 2004; 294: 366–378.
Israsena N, Hu M, Fu W, Kan L, Kessler JA. The presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells. Dev Biol. 2004; 268: 220–231.
Dell’Era P, Ronca R, Coco L, Nicoli S, Metra M, Presta M. Fibroblast growth factor receptor-1 is essential for in vitro cardiomyocyte development. Circ Res. 2003; 93: 414–420.
Debiais F, Lefevre G, Lemonnier J, Le Mee S, Lasmoles F, Mascarelli F, Marie PJ. Fibroblast growth factor-2 induces osteoblast survival through a phosphatidylinositol 3-kinase-dependent, -beta-catenin-independent signalling pathway. Exp Cell Res. 2004; 297: 235–246.
Patel NG, Kumar S, Eggo MC. Essential role of fibroblast growth factor signaling in preadipoctye differentiation. J Clin Endocrinol Metab. 2005; 90: 1226–1232.
Burger PE, Coetzee S, McKeehan WL, Kan M, Cook P, Fan Y, Suda T, Hebbel RP, Novitzky N, Muller WA, Wilson EL. Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells. Blood. 2002; 100: 3527–3535.(Victor W.M. van Hinsbergh)
Correspondence to Victor W.M. van Hinsbergh, PhD, Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. E-mail v.vanhinsbergh@vumc.nl
Since the recognition of basicfibroblast growth factor (bFGF, FGF-2) and acidic FGF (aFGF, FGF-1) as proliferation and maintenance factors for endothelial cells in culture,1,2 much interest has arisen regarding their role as potential angiogenic factors in tumors and tissue healing in vivo. The FGFs, of which presently at least 22 members have been recognized,3 interact with FGF receptors (FGFRs). After FGF binding, the FGFRs dimerize and become activated displaying protein tyrosine kinase activity and subsequent signaling in the cell.4,5 Four functional FGFRs have been recognized, FGFR1–4, most of which can be encountered in various alternative spliced isoforms. FGFR1 plays an essential role during early stages of development of the mesoderm and at several levels during organogenesis.3,6 FGF/FGFR1 is expressed almost exclusively in the mesenchyme, including endothelial cells, smooth muscle cells, and hemopoietic progenitor cells. FGFR1 is involved in the stimulation and proliferation of endothelial cells and vascularization by FGFs, particularly bFGF and aFGF. FGFs also promote the formation of hematopoietic progenitor cells in early development, which requires the FGFR1 as well.7,8 In line with this, chromosomal translocation involving FGFR1 has been observed in patients with stem-cell myeloproliferative disease, probably caused by constitutive activation of the FGFR1 gene.9
See page 944
It has long been anticipated that hematopoietic progenitor cells and endothelial progenitor cells derive from a common cell type, the hemangioblast.10 Studies on mouse embryos have shown that such hemangioblast cells indeed exist. These cells develop in the primitive streak of the embryo and migrate to the yolk sac, where they form blood islands.11 The outer cells of these blood islands mature into endothelial cells and form a vascular plexus, while the inner cells develop into primitive and definitive hematopoietic progenitor cells and their subsequent lineages of blood cells. In addition, in the para-aortic splanchnopleura, hemangioblasts develop into vascular structures.11 Later in development the primitive and definitive hematopoietic progenitor ("stem") cells migrate to the fetal liver and subsequently to the bone marrow, where they remain life-long active as a source for white and red blood cells, mast cells, and megakaryocytes, from which blood platelets are formed.
One of the issues that remained unresolved was the contribution of FGF signaling to the early development of the vascular tree. Embryos deficient in FGFR1 stop growing at an early stage of gestation because of abnormal mesodermal patterning.12,13 However, when embryonic stem cells were derived from FGFR1-deficient blastocysts and administered subcutaneously in nude mice, these embryonic stem cells were still able to differentiate into different types of mesodermal cells.14 Thus FGFR1 causes inappropriate mesodermal patterning rather than impeding mesodermal differentiation. Because FGFs are required for proper mesodermal pattern development, deficiency of FGFR1 interfered with the early development and viability of embryos in the mouse. To overcome this initial blockade of development, Faloon et al7 and Magnusson et al15,16 used the technique of growing embryoid bodies from embryonic stem cells. Pluripotent embryonic stem cells can be isolated from the inner cell mass of a blastula stage embryo. These cells can be grown in culture and can be induced to aggregate into spheroid bodies, the embryoid bodies, by specific culture conditions, which includes the withdrawal of the factor LIF. The inner mass of these embryoid bodies subsequently develops into mesodermal cells, a part of which further differentiate into hemangioblast cells and subsequently into hematopoietic cells, endothelial cells, and vascular smooth muscle cells (Figure 1). The embryoid bodies appeared helpful in elucidating the contribution of various factors involved in hematopoietic and endothelial development.17–19 Their regulation is highly comparable with that in the yolk sac of murine embryos. It has become clear that the differentiation steps from mesodermal progenitor cells into hemangioblasts and subsequently into primitive and definitive lineages of hematopoietic and endothelial progenitor cells required the expression of growth factors including BMP-4 and VEGF, the growth factor receptors VEGFR-2 (Flk1, KDR) and FGFR1, CD41, and transcription factors, such as SCL/Tal1 and Runx1 (required for hematopoietic cells formation).20–29 Members of the transforming growth factor (TGF)-? signaling family, activin A, TGF-?, and possibly BMP-2, act as positive (Activin A) and negative regulators (TGF-?).30,21 The various steps thought to be involved in the differentiation of mesoderm into hematopoietic and endothelial progenitor cells are summarized in Figure 2.
Figure 1. Development of early vasculature in embryoid bodies. EB indicates embryoid body; pcd, postcoidal day; LIF, leukemia inhibitory factor.
Figure 2. Differentiation of hemangioblasts into hematopoietic cells and endothelial cells.
In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Magnusson et al15 report on the expression of FGFR-1 in embryoid bodies and its requirement for hematopoietic and endothelial cell development and function. They show that FGFR1 is expressed and becomes activated in a subpopulation of endothelial cells during the development of embryoid bodies. Its expression was associated with cell division, suggesting that particularly dividing endothelial cells express FGFR1. Whether this is related to the cell cycle state of the cell or reflects a subpopulation of rapidly dividing endothelial cells remains uncertain. In FGFR1-expressing endothelial cells (FGFR1+/CD31+) bFGF stimulated mitogenesis, both in the absence and presence of VEGF. Addition of bFGF to embryoid bodies particularly stimulates angiogenesis in embryonic bodies by the length growth of vascular structures, while the stimulatory effect of VEGF regards the formation of a complex capillary plexus.16 Although embryoid bodies derived from stem cells that lack one or both alleles of FGFR1 are still able to form endothelial cells and vascular structures, the formation of hematopoietic progenitor cells is impaired. Analysis of the mRNA expression in FGFR1–/– embryoid bodies showed a 50% increase of the mesodermal marker brachyury at day 4, indicating a delayed progression of differentiation as compared with their FGFR1+/– counterparts. After 8 days, when endothelial cells and vascular structures had become developed, a relatively normal development of the endothelial lineage in these embryonic bodies was observed. However, a number of genes involved in hematopoietic differentiation were highly suppressed in the FGFR1–/– embryoid bodies, including SCL/Tal1, CD41, and the ?-H1 and GATA-1, two proteins involved in development of erythrocytes. Indeed, at day 12 day virtually no expression of the leukocyte marker CD45 was seen in FGFR1–/– embryoid bodies, in contrast to the FGFR1+/– ones. This indicates that the differentiation into hematopoietic progenitor cells is severely impaired, and suggests that FGF/FGFR1 signaling is required for this differentiation.
These data are consistent with previous observations of Faloon et al,7 who showed a considerable reduction of VEGFR2, SCL/Tal1, ?-H1, and ?-major production in FGFR–/– embryoid bodies as compared with their FGFR1 wild-type counterparts. SCL/Tal1 is required for the commitment of hemangioblasts into hematopoietic progenitor cell differentiation.21–23,31 Therefore, it is likely that the absence of SCL/Tal1 in FGFR1–/– embryonic bodies is an important contributor to the lack of hematopoietic differentiation. Overexpression of SCL/Tal1 in FGFR1–/– embryonic bodies may resolve whether this is the only factor preventing hematopoiesis or whether other factors also contribute to this phenomenon. Faloon et al7 indicated that in SCL/Tal1–/– embryoid bodies endothelial markers were elevated 2- to 4-fold, suggesting preferential differentiation into the endothelial lineage. This raises two questions. First, are the endothelial cells differentiating directly from the primitive hemangioblast in FGFR1–/– and SCL/Tal1–/– conditions identical in characteristics as those deriving wild-type conditions from the definitive hemangioblasts? Second, does (over)expression of SCL/Tal1 reduce differentiation into the angioblast and endothelial lineage, or is the increase in endothelial differentiation in FGFR1–/– and SCL/Tal1–/– embryonic bodies merely caused by the lack of transition into hematopoietic progenitor cells? Other studies have shown that SCL/Tal1 deficiency causes defects in endothelial growth and yolk sac vascularization, indicating that this factor also contributes to proper endothelial development.31,32 Apparently experimental conditions may influence the degree to which endothelial development occurs in this deficiency. Because replenishment of SCL/Tal1 in 3.5- to 4-day-old SCL/Tal1–/– embryoid cells by Cre-lox–mediated induction or viral transduction showed that the function of hematopoietic and endothelial cells can be normalized,31,33 it is likely that SCL/Tal1 deficiency causes deflection of the hemangioblast differentiation into the direction of the angioblast lineage producing endothelial cells and smooth muscle cells. A similar mechanism may occur in the case of FGFR1 deficiency.
Although the studies of Magnusson et al15 and Faloon et al7 indicate that FGFR1 is dispensable for early vascular development in embryoid bodies, this does not exclude that FGFR1 contributes to vascular development and angiogenesis. Several lines of evidence support such a role. First, part of the endothelial cells in the embryoid bodies and in the new vasculature of teratomas does contain FGFR1. A considerable part of these cells is involved in cell division. Whether this expression is paralleled by effects of FGF/FGFR1 on cell division or migration cannot be deduced from these experiments. An alternative explanation might be that FGF/FGFR1 signaling reduces the vulnerability of endothelial cells for apoptosis, as shown in vitro.34,35 It may be a general strategy in development to enhance apoptosis vulnerability of cells that make important steps in differentiation or morphogenic patterning, to avoid improper growth. If FGF/FGFR1 signaling would be able to overcome this vulnerability, FGF signaling would act as a "permissive factor" in development. Indeed, such a permissive role of FGF has been well documented in neural stem cell differentiation where FGF has a dual action maintaining the size of the progenitor pool by its proliferative effects as well as stimulating proneural differentiation by modulating signaling36 Given the involvement of FGF/FGFR1 signaling in the process of differentiation of many different cells, including cardiomyocytes, adipocytes, bone cells, and hematopoietic cells and in patterning of mesoderm and embryo vessels,7,15,37–39 this is a concept that warrants further investigation. Such a concept may also explain why the subpopulation of circulating progenitor cells that contain FGFR1 may be particularly successful in differentiating into an endothelial phenotype in adult man.40
Interestingly in the FGFR1–/– embryoid bodies the lack of FGFR-1 signaling resulted in an abnormal branching pattern of the vasculature.15,16 Although these in vitro systems do not completely reflect the normal embryonic or organ-specific multicellular environment (embryoid bodies lack the polarity of the embryo) and the precise state of differentiation of endothelial cells, they underpin a potential role of FGF/FGFR1 signaling in patterning of the vascular network. This is in line with studies of Lee et al,29 who used a dominant-negative truncated FGFR1 behind a CMV-promoter, which was injected in the 9-day-old murine embryo, and becomes expressed preferentially in vascular and endocardial endothelial cells. The overexpression resulted in abnormal development including incomplete branching of the vascular network.29
The formation of a vascular network as well as mesodermal development require coordination of cell signaling and stepwise recruitment of cytokines in a temporally and spatially coordinated manner. These processes warrant factors that can change the cellular environment from a nonpermissive one into a permissive milieu for such processes. The current study of Magnusson adds another piece of the puzzle to the concept that FGF/FGFR1 is not a conditional factor but rather one of these permissive factors.
References
Gospodarowicz D, Moran J, Braun D, Birdwell C. Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent. Proc Natl Acad Sci U S A. 1976; 73: 4120–4124.
Maciag T, Cerundolo J, Ilsley S, Kelley PR, Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc Natl Acad Sci U S A. 1979; 76: 5674–5678.
Itoh N, Ornitz DM. Evolution of the Fgf and Fgfr gene families. Trends Genet. 2004; 20: 563–569.
Auguste P, Javerzat S, Bikfalvi A. Regulation of vascular development by fibroblast growth factors. Cell Tissue Res. 2003; 314: 157–166.
Stachowiak MK, Fang X, Myers JM, Dunham SM, Berezney R, Maher PA, Stachowiak EK. Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation. J Cell Biochem. 2003; 90: 662–691.
Coumoul X, Deng CX. Roles of FGF receptors in mammalian development and congenital diseases. Birth Defects Res C Embryo Today. 200; 69: 286–304.
Faloon P, Arentson E, Kazarov A, Deng CX, Porcher C, Orkin S, Choi K. Basic fibroblast growth factor positively regulates hematopoietic development. Development. 2000; 127: 1931–1941.
Poole TJ, Finkelstein EB, Cox CM. The role of FGF and VEGF in angioblast induction and migration during vascular development. Dev Dyn. 2001; 220: 1–17.
Guasch G, Mack GJ, Popovici C, Dastugue N, Birnbaum D, Rattner JB, Pebusque MJ. FGFR1 is fused to the centrosome-associated protein CEP110 in the 8p12 stem cell myeloproliferative disorder with t(8;9)(p12;q33). Blood. 2000; 95: 1788–1796.
Bailey AS, Fleming WH. Converging roads: evidence for an adult hemangioblast. Exp Hematol. 2003; 31: 987–993.
Huber TL, Kouskoff V, Fehling HJ, Palis J, Keller G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature. 2004; 432: 625–630.
Deng CX, Wynshaw-Boris A, Shen MM, Daugherty C, Ornitz DM, Leder P. Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev. 1994; 8: 3045–3057.
Yamaguchi TP, Harpal K, Henkemeyer M, Rossant J. Fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 1994; 8: 3032–3044.
Xu X, Weinstein M, Li C, Deng C. Fibroblast growth factor receptors (FGFRs) and their roles in limb development. Cell Tissue Res. 1999; 296: 33–43.
Magnusson PU, Ronca R, Dell’era P, Carlstedt P, Jakobsson L, Partanen J, Dimberg A, Claesson-Welsh L. Fibroblast Growth Factor Receptor-1 Expression Is Required for Hematopoietic but not Endothelial Cell Development. Arterioscler Thromb Vasc Biol. 2005; 25: 944–949.
Magnusson P, Rolny C, Jakobsson L, Wikner C, Wu Y, Hicklin DJ, Claesson-Welsh L. Deregulation of Flk-1/vascular endothelial growth factor receptor-2 in fibroblast growth factor receptor-1–deficient vascular stem cell development. J Cell Sci. 2004; 117: 1513–1523.
Risau W, Sariola H, Zerwes HG, Sasse J, Ekblom P, Kemler R, Doetschman T. Vasculogenesis and angiogenesis in embryonic-stem-cell–derived embryoid bodies. Development. 1988; 102: 471–478.
Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998; 125: 725–732.
Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development. 1998; 125: 1747–1757.
Huber TL, Zhou Y, Mead PE, Zon LI. Cooperative effects of growth factors involved in the induction of hematopoietic mesoderm. Blood. 1998; 92: 4128–4137.
Park C, Afrikanova I, Chung YS, Zhang WJ, Arentson E, Fong Gh G, Rosendahl A, Choi K. A hierarchical order of factors in the generation of FLK1- and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells. Development. 2004; 131: 2749–2762.
Robb L, Lyons I, Li R, Hartley L, Kontgen F, Harvey RP, Metcalf D, Begley CG. Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc Natl Acad Sci U S A. 1995; 92: 7075–7059.
Ema M, Faloon P, Zhang WJ, Hirashima M, Reid T, Stanford WL, Orkin S, Choi K, Rossant J. Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse. Genes Dev. 2003; 17: 380–393.
Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996; 380: 435–439.
Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996; 380: 439–442.
Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L, Bernstein A, Rossant J. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell. 1997; 89: 981–990.
Ferkowicz MJ, Starr M, Xie X, Li W, Johnson SA, Shelley WC, Morrison PR, Yoder MC. CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo. Development. 2003; 130: 4393–4403.
Fehling HJ, Lacaud G, Kubo A, Kennedy M, Robertson S, Keller G, Kouskoff V. Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development. 2003; 130: 4217–4227.
Lacaud G, Gore L, Kennedy M, Kouskoff V, Kingsley P, Hogan C, Carlsson L, Speck N, Palis J, Keller G. Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood. 2002; 100: 458–466.
Dickson MC, Martin JS, Cousins FM, Kulkarni AB, Karlsson S, Akhurst RJ. Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development. 1995; 121: 1845–1854.
D’Souza SL, Elefanty AG, Keller G. SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast, but not for its development. Blood. 2005Jan 27;.
Visvader JE, Fujiwara Y, Orkin SH. Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development. Genes Dev. 1998; 12: 473–479.
Endoh M, Ogawa M, Orkin S, Nishikawa S. SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation. EMBO J. 2002; 21: 6700–6708.
Lee SH, Schloss DJ, Swain JL. Maintenance of vascular integrity in the embryo requires signaling through the fibroblast growth factor receptor. J Biol Chem. 2000; 275: 33679–33687.
Erez N, Zamir E, Gour BJ, Blaschuk OW, Geiger B. Induction of apoptosis in cultured endothelial cells by a cadherin antagonist peptide: involvement of fibroblast growth factor receptor-mediated signalling. Exp Cell Res. 2004; 294: 366–378.
Israsena N, Hu M, Fu W, Kan L, Kessler JA. The presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells. Dev Biol. 2004; 268: 220–231.
Dell’Era P, Ronca R, Coco L, Nicoli S, Metra M, Presta M. Fibroblast growth factor receptor-1 is essential for in vitro cardiomyocyte development. Circ Res. 2003; 93: 414–420.
Debiais F, Lefevre G, Lemonnier J, Le Mee S, Lasmoles F, Mascarelli F, Marie PJ. Fibroblast growth factor-2 induces osteoblast survival through a phosphatidylinositol 3-kinase-dependent, -beta-catenin-independent signalling pathway. Exp Cell Res. 2004; 297: 235–246.
Patel NG, Kumar S, Eggo MC. Essential role of fibroblast growth factor signaling in preadipoctye differentiation. J Clin Endocrinol Metab. 2005; 90: 1226–1232.
Burger PE, Coetzee S, McKeehan WL, Kan M, Cook P, Fan Y, Suda T, Hebbel RP, Novitzky N, Muller WA, Wilson EL. Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells. Blood. 2002; 100: 3527–3535.(Victor W.M. van Hinsbergh)