Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in β-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context. Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in β-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context. Definitive mesoderm and endoderm both arise from the primitive streak during gastrulation. An entity named mesendoderm representing a transitory state in mesodermal and endodermal development has been identified (1Kimelman D. Griffin K.J. Curr. Opin. Genet. Dev. 2000; 10: 350-356Crossref PubMed Scopus (136) Google Scholar). Several factors have been shown to be involved in the commitment of mesodermal and endodermal lineages during this stage of development (2Vallier L. Reynolds D. Pedersen R.A. Dev. Biol. 2004; 275: 403-421Crossref PubMed Scopus (302) Google Scholar, 3Tada S. Era T. Furusawa C. Sakurai H. Nishikawa S. Kinoshita M. Nakao K. Chiba T. Development (Camb.). 2005; 132: 4363-4374Crossref PubMed Scopus (384) Google Scholar, 4Hallonet M. Kaestner K.H. Martin-Parras L. Sasaki H. Betz U.A. Ang S.L. Dev. Biol. 2002; 243: 20-33Crossref PubMed Scopus (51) Google Scholar, 5Bielinska M. Narita N. Heikinheimo M. Porter S.B. Wilson D.B. Blood. 1996; 88: 3720-3730Crossref PubMed Google Scholar), but the precise mechanism by which they exert their action remains elusive. Differentiation of embryonic stem (ES) 3The abbreviations used are: ES, embryonic stem; EB, embryoid body; VEGFR, vascular endothelial growth factor receptor; FGFR, fibroblast growth factor receptor; TBS, Tris-buffered saline; SH2, Src homology 2; HNF, hepatocyte nuclear factor; RT, reverse transcription. cells to embryoid bodies (EBs) has been a useful tool in understanding early events during development due to the experimental complications of in vivo studies whenever a gene exerts multiple effects. Studies on in vitro differentiation of ES cells have corroborated the existence of mesendoderm (3Tada S. Era T. Furusawa C. Sakurai H. Nishikawa S. Kinoshita M. Nakao K. Chiba T. Development (Camb.). 2005; 132: 4363-4374Crossref PubMed Scopus (384) Google Scholar). Another example of the usefulness of the in vitro EB system relates to studies of endothelial and hematopoietic ontogenesis. Differentiation of these lineages is initiated in specific structures composed of mesodermal cells (called blood islands) present in the yolk sac endoderm (6Palis J. McGrath K.E. Kingsley P.D. Blood. 1995; 86: 156-163Crossref PubMed Google Scholar). Blood islands consist of primitive hematopoietic blood cells surrounded by a layer of endothelial cells and support primitive hematopoiesis between embryonic days 7 and 14.5. Endothelial cells in the yolk sac form the primitive vascular plexus in a process named vasculogenesis, and subsequently a more mature vascular system evolves by angiogenesis. The presence of several factors universal for both endothelial and blood cells, such as VEGFR-2, Tal-1, and CD34 (7Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3358) Google Scholar, 8Kennedy M. Firpo M. Choi K. Wall C. Robertson S. Kabrun N. Keller G. Nature. 1997; 386: 488-493Crossref PubMed Scopus (499) Google Scholar), has suggested a common progenitor for primitive hematopoietic and endothelial cells that has been given the name hemangioblast (9Choi K. Kennedy M. Kazarov A. Papadimitriou J.C. Keller G. Development (Camb.). 1998; 125: 725-732Crossref PubMed Google Scholar). Despite numerous investigations, the mechanisms responsible for differentiation of endothelial and blood cells are still not completely understood. In addition to studies on differentiation of mesodermal lineages, EBs have been employed for studies of endodermal and ectodermal differentiation (10Soria B. Skoudy A. Martin F. Diabetologia. 2001; 44: 407-415Crossref PubMed Scopus (171) Google Scholar, 11Fraichard A. Chassande O. Bilbaut G. Dehay C. Savatier P. Samarut J. J. Cell Sci. 1995; 108: 3181-3188Crossref PubMed Google Scholar, 12Bagutti C. Wobus A.M. Fassler R. Watt F.M. Dev. Biol. 1996; 179: 184-196Crossref PubMed Scopus (133) Google Scholar, 13Abe K. Niwa H. Iwase K. Takiguchi M. Mori M. Abe S.I. Yamamura K.I. Exp. Cell Res. 1996; 229: 27-34Crossref PubMed Scopus (171) Google Scholar). SHB is a ubiquitously expressed adapter protein (14Welsh M. Mares J. Karlsson T. Lavergne C. Breant B. Claesson-Welsh L. Oncogene. 1994; 9: 19-27PubMed Google Scholar). Its N terminus contains proline-rich motifs (15Karlsson T. Songyang Z. Landgren E. Lavergne C. Di Fiore P.P. Anafi M. Pawson T. Cantley L.C. Claesson-Welsh L. Welsh M. Oncogene. 1995; 10: 1475-1483PubMed Google Scholar), the central part includes a phosphotyrosine binding domain (16Welsh M. Songyang Z. Frantz J.D. Trub T. Reedquist K.A. Karlsson T. Miyazaki M. Cantley L.C. Band H. Shoelson S.E. Oncogene. 1998; 16: 891-901Crossref PubMed Scopus (50) Google Scholar, 17Lindholm C.K. Henriksson M.L. Hallberg B. Welsh M. Eur. J. Biochem. 2002; 269: 3279-3288Crossref PubMed Scopus (17) Google Scholar) and several tyrosine phosphorylation sites (17Lindholm C.K. Henriksson M.L. Hallberg B. Welsh M. Eur. J. Biochem. 2002; 269: 3279-3288Crossref PubMed Scopus (17) Google Scholar, 18Lu L. Anneren C. Reedquist K.A. Bos J.L. Welsh M. Exp. Cell Res. 2000; 259: 370-377Crossref PubMed Scopus (24) Google Scholar), and the C terminus contains an Src homology 2 (SH2) domain (15Karlsson T. Songyang Z. Landgren E. Lavergne C. Di Fiore P.P. Anafi M. Pawson T. Cantley L.C. Claesson-Welsh L. Welsh M. Oncogene. 1995; 10: 1475-1483PubMed Google Scholar). The SHB SH2 domain mediates its interactions with several receptors such as the VEGFR-2 (19Holmqvist K. Cross M.J. Rolny C. Hagerkvist R. Rahimi N. Matsumoto T. Claesson-Welsh L. Welsh M. J. Biol. Chem. 2004; 279: 22267-22275Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), FGFR-1 (15Karlsson T. Songyang Z. Landgren E. Lavergne C. Di Fiore P.P. Anafi M. Pawson T. Cantley L.C. Claesson-Welsh L. Welsh M. Oncogene. 1995; 10: 1475-1483PubMed Google Scholar, 20Cross M.J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 13: 2881-2893Crossref PubMed Scopus (75) Google Scholar), T cell receptor (21Lindholm C.K. Gylfe E. Zhang W. Samelson L.E. Welsh M. J. Biol. Chem. 1999; 274: 28050-28057Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and platelet-derived growth factor receptor (15Karlsson T. Songyang Z. Landgren E. Lavergne C. Di Fiore P.P. Anafi M. Pawson T. Cantley L.C. Claesson-Welsh L. Welsh M. Oncogene. 1995; 10: 1475-1483PubMed Google Scholar). The R522K mutation in the SHB SH2 domain renders it inactive. The other SHB domains bind to different cytosolic proteins and thus transduce signals downstream of the receptors. SHB is implicated in many cellular processes such as differentiation of endothelial cells (22Lu L. Holmqvist K. Cross M. Welsh M. Cell Growth & Differ. 2002; 13: 141-148PubMed Google Scholar) and T cell signaling after CD3 stimulation (21Lindholm C.K. Gylfe E. Zhang W. Samelson L.E. Welsh M. J. Biol. Chem. 1999; 274: 28050-28057Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Moreover, SHB is involved in apoptotic signaling in fibroblasts, islets of Langerhans, and endothelial cells (23Welsh M. Christmansson L. Karlsson T. Sandler S. Welsh N. Mol. Med. 1999; 5: 169-180Crossref PubMed Google Scholar, 24Karlsson T. Welsh M. Oncogene. 1996; 13: 955-961PubMed Google Scholar, 25Dixelius J. Larsson H. Sasaki T. Holmqvist K. Lu L. Engstrom A. Timpl R. Welsh M. Claesson-Welsh L. Blood. 2000; 95: 3403-3411Crossref PubMed Google Scholar). EBs expressing the R522K SHB mutant displayed a lower mRNA content of several liver and pancreatic markers (26Kriz V. Anneren C. Lai C. Karlsson J. Mares J. Welsh M. Exp. Cell Res. 2003; 286: 40-56Crossref PubMed Scopus (12) Google Scholar). In addition, SHB was found to be essential for EB blood vessel formation (27Rolny C. Lu L. Agren N. Nilsson I. Roe C. Webb G.C. Welsh M. Exp. Cell Res. 2005; 308: 381-393Crossref PubMed Scopus (15) Google Scholar), as EBs expressing R522K SHB failed to form normal blood vessels. To address the role of the SHB adapter protein in development, we have established knock-out ES cell lines deficient for one or both SHB alleles. These ES cell lines were differentiated in vitro and aimed at understanding the impact of SHB on hemangiogenesis. The data suggest an important involvement of SHB in the development of mesoderm. Gene Targeting—The murine embryonic stem cell line GSI-1 derived from 129SvJ mice was inactivated at both (SHB-/-) or one (SHB+/-) of the SHB alleles. These were inactivated in two subsequent steps. The first targeting vector contained three loxP sites. The first and the second loxP sites flanked the neomycin resistance gene, and the second and third loxP sites bordered the first exon (Fig. 1A). One of the clones in which homologous recombination had not occurred was used as an SHB+/+ control. The clone in which homologous recombination had taken place and all three loxP sites were present (loxPSHB) was treated with Cre recombinase by in vitro transfection. One of the clones, which had lost both the neomycin resistance gene and the first SHB exon, was used as a maternal clone for the second transfection. The second targeting vector contained an insertion of the neomycin gene into the first exon without loxP sites (Fig. 1A). Two sister clones to SHB-/-, in which homologous recombination had not occurred were used as SHB+/-. Clones were screened by PCR with one primer outside the sequence of the construct (supplemental Fig. 1). ES Cell Culture—ES cells were cultured in the presence of murine embryonic fibroblasts in Dulbecco's modified Eagle's medium/glutamax (Invitrogen), penicillin/streptomycin, 15% (Invitrogen), (Invitrogen), (Invitrogen), and factor as previously (26Kriz V. Anneren C. Lai C. Karlsson J. Mares J. Welsh M. Exp. Cell Res. 2003; 286: 40-56Crossref PubMed Scopus (12) Google Scholar). were at in For ES cell differentiation, fibroblasts and factor were ES cells were at and by the of for 2 days A.M. G. J. J. G. B. J. J. Mol. Cell. 1997; Full Text PDF PubMed Scopus Google to form EBs. These were in for one more day and on EBs were and days after the of Alternatively, EBs formation by the for 2 days by 2 days of were as in for an 7 days a differentiation of and stained for or to vascular structures or cells. was the to the Gene expression was by a the PCR were for and for by at for and for at the by at for Alternatively, gene expression was by a reverse was used to of into For PCR the was PCR were for by at for and for at the by at for are shown in The PCR were on a levels were of EBs with were stained to a previously P. Rolny C. L. C. Wu Claesson-Welsh L. J. Cell Sci. 2004; PubMed Scopus Google Scholar). EBs on were with Tris-buffered and with and the EBs were with and treated with in to EBs were with TBS, and interactions were in The was with at with and with and with EBs were with with and and the was Alternatively, EBs were stained for that was to cells. In cells were differentiated to a L. E. A. 2003; PubMed Scopus Google Scholar) from Cell The is in Fig. Two days to differentiation, ES cells were and cultured in ES modified Eagle's with penicillin/streptomycin, 15% (Invitrogen), (Invitrogen), (Invitrogen), and factor 2 days in ES the ES cells had and were and with ES cells were to at a of in differentiation modified Eagle's penicillin/streptomycin, 15% (Invitrogen), and to form EBs. These were at in for 7 the EBs were in and the EBs to were and EBs were at after which the was inactivated by and EBs several by an and a ES cells were and by with and with a at a of The contains modified Dulbecco's with for stem cell and to support hematopoietic Cell colonies were after an days of thus a differentiation of by the were a on or are shown due to the of the In all other a was of the SHB the of we of ES cells the construct (Fig. 1A). The first one clone of in which homologous recombination had occurred with all three loxP sites recombination occurred in clones, but in the two loxP sites the neomycin gene were present and were thus for of the first The second one clone of the insertion of the neomycin gene, in the second of the first Another two clones contained insertion in the first the second was For cells, two sister clones with insertion of the targeting construct were The in of SHB protein expression (Fig. of in SHB-/- studies have suggested an involvement of SHB in vascular formation (27Rolny C. Lu L. Agren N. Nilsson I. Roe C. Webb G.C. Welsh M. Exp. Cell Res. 2005; 308: 381-393Crossref PubMed Scopus (15) Google Scholar). have the expression of two markers for early mesoderm at appropriate of EB The markers were Brachyury and Brachyury and are expressed in and to a in endoderm M. S. S. M. Nishikawa S. Chiba T. Era T. 2005; PubMed Scopus Google Scholar). Brachyury gene expression was in the the SHB-/- clone exhibited a in this (Fig. of delayed down-regulation of gene expression was in the the failed to The expression of mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages was also These were the hematopoietic markers Blood. 1994; PubMed Google Scholar), M.J. M. W. Development (Camb.). 2003; PubMed Scopus Google Scholar, M. I. K. V. F. W. Development (Camb.). 2002; PubMed Google Scholar), and K. J. K. A. E. K. M.L. C. Cell. Full Text PDF PubMed Scopus Google Scholar), the vascular markers Blood. 2000; 95: PubMed Google Scholar, A. C. T. Development (Camb.). 1994; PubMed Google Scholar) and VEGFR-2 (7Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3358) Google Scholar, F. J. Schuh A.C. L. A. Rossant J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and the cardiac marker M. O. J. N. Sci. S. A. 2000; PubMed Scopus Google Scholar). In the all of expression with which in the of and on day 8 (Fig. The SHB-/- clone reduced expression of of these markers on day 6 or 8 of day of differentiation, expression of and in the SHB-/- clone and was than that of the (Fig. the clone with the and the SHB-/- clones on day of differentiation, it was that the of one SHB in a that was to that of the SHB knock-out (Fig. was expression of VEGFR-2, which has been shown to be essential for hematopoietic and vascular development (7Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3358) Google Scholar), reduced in both the and SHB knock-out ES cells (Fig. at this In addition, FGFR-1 expression was SHB with which is a receptor that also a role for vascular and hematopoietic development R. P. P. L. J. A. Claesson-Welsh L. Biol. 2005; PubMed Scopus (33) Google Scholar). The expression of FGFR-1 was at day in the SHB-/- clone (Fig. The data suggest differentiation to mesodermal lineages in SHB-/- EBs as a of delayed down-regulation of markers with early mesodermal of hematopoietic and vascular markers in EBs after days of the of mRNA for of mRNA was by or was of VEGFR-2 in EBs FGFR-1 in SHB-/- marker is expressed in cells to hematopoiesis M.J. M. W. Development (Camb.). 2003; PubMed Scopus Google Scholar), and the delayed of it an content after days of differentiation in the SHB-/- clone in to the SHB+/+ clone (Fig. To this the expression of the protein was also on the protein by of EBs differentiated for cells derived from the SHB-/- and clones were stained more than derived from the SHB+/+ clone (Fig. the that mRNA is on day in SHB-/- EBs and that this also in protein Blood in SHB-/- EBs after in an for the study of and angiogenesis. vasculogenesis, EBs form a primitive vascular the formation of a mature blood vessel with in a subsequent process angiogenesis. of EBs with the of endothelial cells from the EB into the the EB the blood form a vascular plexus P. Rolny C. L. C. Wu Claesson-Welsh L. J. Cell Sci. 2004; PubMed Scopus Google Scholar) (Fig. was to study the of SHB-/- ES cells to form blood SHB previously has been shown to EB blood vessel SHB-/- or EBs not in their with of the SHB+/+ EBs cultured (Fig. These EBs formed a that was to the central of the EB with vascular structures the after in the presence of the SHB-/- and EBs than the SHB+/+ EBs with fewer structures (Fig. were with the other clone not is in the line with the study a role of SHB in the development of in EBs (27Rolny C. Lu L. Agren N. Nilsson I. Roe C. Webb G.C. Welsh M. Exp. Cell Res. 2005; 308: 381-393Crossref PubMed Scopus (15) Google Scholar). SHB-/- ES in data for the hematopoietic markers and suggest a delayed of gene expression but also an of SHB-/- ES cells to into blood cells as their expression was on day of To SHB is important for in vitro EBs from the SHB-/-, and SHB+/+ clones were for their to form blood cell Cell colonies were after days of differentiation in by a EB differentiation a of days of SHB-/- EBs fewer blood cell colonies than EBs. In three of the EBs also formed fewer blood cell colonies than the SHB+/+ clone (Fig. that the of SHB knock-out is delayed expression and differentiation to the hematopoietic In the present we have the of SHB-/- ES cells to in ES cells in vitro in structures as which to the cells from all three and but the for the normal development of the ES cells can but not studies on murine of the first exon in the SHB gene of SHB protein The homologous recombination was not in the SHB The in a SHB-/- clone after the second be due to the that one is more to the than the other Another is that SHB can cell and thus SHB-/- clones not to be is a between endoderm and mesoderm during of these in the primitive streak in a to as mesendoderm (1Kimelman D. Griffin K.J. Curr. Opin. Genet. Dev. 2000; 10: 350-356Crossref PubMed Scopus (136) Google Scholar). during development, mesoderm and endoderm interactions for differentiation M. Kaestner K.H. Martin-Parras L. Sasaki H. Betz U.A. Ang S.L. Dev. Biol. 2002; 243: 20-33Crossref PubMed Scopus (51) Google Scholar, M. N. D. A. Dev. Biol. 2003; 259: PubMed Scopus Google Scholar). the existence of mesendoderm was also in vitro (3Tada S. Era T. Furusawa C. Sakurai H. Nishikawa S. Kinoshita M. Nakao K. Chiba T. Development (Camb.). 2005; 132: 4363-4374Crossref PubMed Scopus (384) Google Scholar). data support the role of SHB in mesodermal of the early mesodermal marker Brachyury was by a delayed in the expression of several mesodermal such as VEGFR-2, and expression of which is a hematopoietic marker expressed subsequently to was not in the SHB knock-out cells. The markers three different mesodermal lineages, and thus the data suggest a in mesodermal differentiation early Brachyury of VEGFR-2 was in both and SHB-/- EBs. VEGFR-2 is as a factor involved in hematopoiesis and in vivo and in vitro P. Rolny C. L. C. Wu Claesson-Welsh L. J. Cell Sci. 2004; PubMed Scopus Google Scholar, F. J. Schuh A.C. L. A. Rossant J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Choi K. A. W. Keller G. Development (Camb.). 1997; PubMed Google Scholar). the and SHB-/- EBs for their to form blood it was that both clones were to form blood These are in with study the SH2 domain mutant of (27Rolny C. Lu L. Agren N. Nilsson I. Roe C. Webb G.C. Welsh M. Exp. Cell Res. 2005; 308: 381-393Crossref PubMed Scopus (15) Google Scholar). ES cells failed to form blood of growth factor was The more of the ES cells may have several not with the SHB protein but also with other of this protein such as and in the SHB-/- cells, these other may for SHB may with in which the SHB protein is not and SHB-/- EBs form vascular after stimulation. SHB is involved in the differentiation of endothelial endothelial cells (22Lu L. Holmqvist K. Cross M. Welsh M. Cell Growth & Differ. 2002; 13: 141-148PubMed Google Scholar) and in of endothelial cells by binding to tyrosine in in the VEGFR-2 (19Holmqvist K. Cross M.J. Rolny C. Hagerkvist R. Rahimi N. Matsumoto T. Claesson-Welsh L. Welsh M. J. Biol. Chem. 2004; 279: 22267-22275Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The data thus suggest impaired signaling downstream of VEGFR-2 in cells during blood vessel is as a marker for hematopoietic M. I. K. V. F. W. Development (Camb.). 2002; PubMed Google Scholar, J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The present that EBs a delayed in expression that levels of expression with at day suggest an to to blood cells. was not a formation the is the delayed in expression that further expression without differentiation has previously been and was by the existence of two of cells, and T. T. K. S. R. K. Exp. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were shown to have the to into hematopoietic to cells, which a to It is that the cells in the SHB-/- EBs the of cells that to of hematopoietic the marker was in EBs. N. C. K. S. J. J. and M. the formation of blood from the to in cultured EBs P. Rolny C. L. C. Wu Claesson-Welsh L. J. Cell Sci. 2004; PubMed Scopus Google Scholar). FGFR-1 knock-out EBs also vascular structures R. P. P. L. J. A. Claesson-Welsh L. Biol. 2005; PubMed Scopus (33) Google Scholar). have not in SHB-/- EBs after the addition of not The that both addition and FGFR-1 knock-out vessel formation in EBs the of of SHB-/- in this context, SHB may of FGFR-1 signaling M.J. Lu L. Magnusson P. Nyqvist D. Holmqvist K. Welsh M. Claesson-Welsh L. Mol. Biol. Cell. 2002; 13: 2881-2893Crossref PubMed Scopus (75) Google Scholar). In the present data suggest that the SHB adapter protein is important for the formation of vascular structures and hematopoietic at a stage than mesodermal SHB a role in the of ES cells. are to the and for and the ES cell The was by also by with
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