Essential Role of the Transcription Factor Ets-2 inXenopus Early Development

Abstract

The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate thatXenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription. The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate thatXenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription. During Xenopus early embryonic development, mesoderm arises from ectoderm by induction that requires signals from the vegetal hemisphere of the embryo (1Sive H.L. Genes Dev. 1993; 1: 1-12Crossref Scopus (149) Google Scholar, 2Slack J.M. Curr. Biol. 1994; 4: 116-126Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The FGF 1The abbreviations used are: FGF, fibroblast growth factor; bFGF, basic fibroblast growth factor; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; Xbra, Xenopus brachyury; En-R, repressor domain ofDrosophila Engrailed; XFD, Xenopusdominant-negative form of FGF receptor; RT-PCR, reverse transcription-coupled polymerase chain reaction; GST, glutathioneS-transferase; MO, morpholino oligonucleotides /MAP kinase (MAPK) pathway has been shown to be involved in early mesodermal patterning (3Whitman M. Melton D.A. Nature. 1992; 357: 252-254Crossref PubMed Scopus (183) Google Scholar, 4MacNicol A.M. Muslin A.J. Williams L.T. Cell. 1993; 73: 571-583Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 5Gotoh Y. Masuyama N. Suzuki A. Ueno N. Nishida E. EMBO J. 1995; 14: 2491-2498Crossref PubMed Scopus (134) Google Scholar, 6LaBonne C. Burke B. Whitman M. Development. 1995; 121: 1475-1486PubMed Google Scholar, 7Umbhauer M. Marshall C.J. Mason C.S. Old R.W. Smith J.C. Nature. 1995; 376: 58-62Crossref PubMed Scopus (204) Google Scholar, 8Kusakabe M. Masuyama N. Hanafusa H. Nishida E. EMBO Rep. 2001; 2: 727-735Crossref PubMed Scopus (24) Google Scholar). One of the genes that are thought to be regulated directly by FGF via the MAPK signal transduction pathway is XenopusBrachyury (Xbra). Brachyury is an important regulatory gene in early vertebrate development (9Herrmann B.G. Labeit S. Poustka A. King T.R. Lehrach H. 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Ho R.K. Walker C. Kimmel C.B. Cell. 1993; 75: 99-111Abstract Full Text PDF PubMed Scopus (472) Google Scholar, 16Schulte-Merker S. van Eeden F.J. Halpern M.E. Kimmel C.B. Nusslein-Volhard C. Development. 1994; 120: 1009-1015Crossref PubMed Google Scholar, 17Conlon F.L. Sedgwick S.G. Weston K.M. Smith J.C. Development. 1996; 122: 2427-2435PubMed Google Scholar). However, the link between the FGF/MAPK pathway and the induction of Xbra expression has not been fully defined. The ETS family of transcription factors, comprising more than 30 different members, has been found to play a crucial role in controlling transcription of a variety of genes involved in important cellular processes, such as proliferation and differentiation (19Yordy J.S. Muise-Helmericks R.C. Oncogene. 2000; 55: 6503-6513Crossref Scopus (272) Google Scholar, 20Sharrocks A.D. Nat. Rev. Mol. Cell Biol. 2001; 11: 827-837Crossref Scopus (839) Google Scholar). They share a unique DNA binding domain, the ETS domain, which interacts specifically with GGA(A/T)-based recognition sites (18Wasylyk B. Hagman J. Gutierrez-Hartmann A. Trends Biochem. Sci. 1998; 23: 213-216Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). As targets of the Ras-MAPK signaling pathway, Ets transcription factors are phosphorylated by MAPK and function as critical nuclear integrators of ubiquitous signaling cascades. The ETS family is divided into subfamilies by sequence similarity based on the ETS domain or additional sequence motifs. Ets-2 is a member of the ETS subfamily, which consists of three members: Ets-1, Ets-2, andDrosophila Pointed (21Yang B.S. Hauser C.A. Henkel G. Colman M.S. Van Beveren C. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (319) Google Scholar, 22Wasylyk C. Bradford A.P. Gutierrez-Hartmann A. Wasylyk B. Oncogene. 1997; 14: 899-913Crossref PubMed Scopus (93) Google Scholar). Studies with mammalian cultured cells have shown that Ets-2 is activated by MAPK-dependent phosphorylation of threonine 72 in an N-terminal regulatory domain (the Pointed domain) (21Yang B.S. Hauser C.A. Henkel G. Colman M.S. Van Beveren C. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (319) Google Scholar, 23McCarthy S.A. Chen D. Yang B.S. Garcia Ramirez J.J. Cherwinski H. Chen X.R. Klagsbrun M. Hauser C.A. Ostrowski M.C. McMahon M. Mol. Cell. Biol. 1997; 17: 2401-2412Crossref PubMed Scopus (151) Google Scholar). Xenopus Ets-2 is maternally expressed in both the animal pole and the intermediate zone (24Wolff C.M. Stiegler P. Baltzinger M. Meyer D. Ghysdael J. Stehelin D. Befort N. Remy P. Cell Growth & Differ. 1991; 9: 447-456Google Scholar, 25Meyer D. Durliat M. Senan F. Wolff M. Andre M. Hourdry J. Remy P. Int. J. Dev. Biol. 1997; 41: 607-620PubMed Google Scholar). On the basis of these results, we hypothesized that Ets-2 relays the FGF/MAPK signaling and induces mesoderm by inducing Xbra gene transcription. In this study, we have presented several lines of evidence indicating that Ets-2 plays an essential role in mesodermal patterning, lying between the FGF/MAPK pathway and the Xbra transcription. Xenopus Ets-2 cDNA was obtained by screening a λZAP II cDNA library made from stage 10.5 embryos. To construct CS2-En-N, the 888-bp fragment coding for amino acids 1–294 of Drosophila engrailed protein was inserted into the StuI site of CS2+. To generate EtsΔN En-R (the repressor domain of DrosophilaEngrailed), the DNA binding domain of Ets-2 (amino acid residues 310–468) was subcloned into CS2-En-N. In vitro synthesis of capped mRNA was performed using mMESSAGE mMACHINE kit (Ambion) according to the manufacturer's instructions. Embryos werein vitro fertilized, dejellied, and cultured in 0.1× modified Barth's saline (1.5 mm HEPES, pH 7.4, 8.8 mm NaCl, 0.1 mm KCl, 0.24 mmNaHCO3, 0.082 mm MgSO4, 0.03 mm Ca(NO3)2, and 0.041 mm CaCl2). Embryos were staged according to Nieuwkoop and Faber (26Nieuwkoop P.D. Faber J. Normal Table of Xenopus laevis (Daudin). North Holland Publishing Co., Amsterdam1967Google Scholar). Embryos at the four-cell stage were injected with mRNA as described in the text and figure legends. Animal caps were dissected from the injected embryos at stages 8–8.5 and cultured in 1× Steinberg solution containing 0.1% bovine serum albumin to various stages for further analysis as described in the figure legends. RT-PCR experiments were performed according to standard protocol. The primer pairs used here for RT-PCR have been described elsewhere (27Masuyama N. Hanafusa H. Kusakabe M. Shibuya H. Nishida E. J. Biol. Chem. 1999; 274: 12163-12170Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,28Hanafusa H. Masuyama N. Kusakabe M. Shibuya H. Nishida E. EMBO Rep. 2000; 1: 32-39Crossref PubMed Scopus (22) Google Scholar). For morpholino oligonucleotide injections, an Ets-2 antisense oligonucleotide with the sequence 5′-AGCTGAGGGAGGGTATGTCCTTCC-3′ was obtained from Gene Tools, LLC. Oligonucleotides were resuspended in sterile, filtered water and injected into four-cell stage embryos with the indicated amounts (29Yamanaka H. Moriguchi T. Masuyama N. Kusakabe M. Hanafusa H. Takada R. Takada S. Nishida E. EMBO Rep. 2002; 3: 69-75Crossref PubMed Scopus (364) Google Scholar). Embryos were injected with 200 pg of Xbra-pOLUC (provided by Dr. K. W. Y. Cho) and 100 pg of pCMV-β-galactosidase together with 1 ng of Ets-2 mRNA into animal poles at the two-cell stage. Dissected animal caps were assayed for luciferase and β-galactosidase activities at stage 11 (13, 14). The ratio of luciferase to β-galactosidase activity provides a normalized measure of luciferase expression. In all cases, fold activation was calculated using the results from only Xbra pOLUC- and CMV-β-galactosidase-injected embryos as background. Each experiment was performed three times to ensure reproducibility of results. To obtain the recombinant GST-Ets-2 protein, the entire coding region of Ets-2 was subcloned into pGEX-6P. Production of GST-Ets-2 and GST protein was performed as described (30Basuyaux J.P. Ferreira E. Stehelin D. Buttice G. J. Biol. Chem. 1997; 272: 26188-26195Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). A DNA sequence for oligonucleotides of the Ets binding site was 5′-CAGGTGTCAGTTCTTACTGGATGTAAGTTTATTGAAGGCA-3′. A gel mobility shift assay was carried out as described (31Ma X. Neurath M. Gri G. Trinchieri G. J. Biol. Chem. 1997; 272: 10389-10395Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). For the competition assay, the Ets binding site was mutated using a site-directed mutagenesis kit (QuikChangeTM; Stratagene. The Ets binding site was mutated using forward primer 5′-CAGGTGTCAGTTCTTACTGGCCGTAAGTTTATTGAAGGC-3′ and reverse primer 5′-TGCCTTCAATAAAC TTACGGCCAGTAAGAACTGACACCTG-3′. We first examined the effect of overexpression of wild typeXenopus Ets-2 mRNA on early embryonic development. Ets-2 mRNA was injected into the dorsal or ventral marginal zones of four-cell stage Xenopus embryos. When compared with normal uninjected control embryos at tadpole stage 35, embryos with dorsal injections of Ets-2 showed anterior truncation and shortened body axis. The embryos injected with relatively high doses of Ets-2 mRNA (1–2 ng) lacked eyes and cement glands (Fig. 1 A). About 70% of embryos injected with 2 ng of Ets-2 mRNA showed the severe anterior defects (n = 34). A typical image is shown in Fig. 1 A. About 40% of embryos injected with 1 ng of Ets-2 mRNA showed similar severe defects (n = 45). A typical phenotype is shown (Fig. 1 A, Ets-2 1 ng DMZ (dorsal marginal zone)). Moreover, injections with high doses of mRNA sometimes resulted in embryos with tail-like protrusions (data not shown). The ventral injections had little or no effect on the development of the embryos (Fig. 1 A, Ets-2 1 ng VMZ (ventral marginal zone)). To investigate these embryos in more detail, we examined the wide range of molecular markers in Ets-2 mRNA-injected embryos. The results of the RT-PCR analysis carried out on stages 26 and 37 whole embryos injected with Ets-2 mRNA of 1 ng are shown (Fig. 1 B). Sibling embryos were used as control. Overexpression of Ets-2 mRNA resulted in remarkable reduction of expression of a forebrain marker Otx2 and a cement gland marker XAG. Moreover, expression of a pan-neural marker neural cell adhesion molecule was also reduced by injection of Ets-2 mRNA. Thus, Ets-2 overexpression suppressed expression of anterior markers. Moreover, Ets-2 overexpression increased expression of the posterior markers such as Xbra and Xcad3 (Fig. 1 B). A transverse section through the anterior of the posteriorized embryo showed no development of brain ventricles (data not shown). These data suggest that overexpression of Ets-2 causes anterior truncation and induction of posterior mesoderm. Next, to examine whether Ets-2 functions downstream of FGF signaling, we tested whether expression of Ets-2 could rescue the defects caused by inhibition of FGF signal inXenopus embryos. Dominant-negative FGF receptor (XFD) mRNA with or without Ets-2 mRNA was injected into dorsal marginal zones of the four-cell stage. Overexpression of XFD caused a severe posterior defect (18 out of 18, Fig. 2). This defect is thought to result from inhibition of both gastrulation movement and posterior mesoderm formation (32Amaya E. Musci T.J. Kirschner M.W. Cell. 1991; 66: 257-270Abstract Full Text PDF PubMed Scopus (930) Google Scholar). Co-injection of Ets-2 rescued this morphological defect, and the injected embryos developed almost normally out of (Fig. 2). This rescue of the phenotype by Ets-2 that Ets-2 functions in mesoderm patterning downstream of the defects by Embryos were injected into the dorsal marginal zones at four-cell stage and cultured stage ng of XFD mRNA was injected with or without ng of Ets-2 To the of Ets-2, we various markers. Animal caps were dissected from Ets-2 mRNA-injected embryos and cultured to stage 10.5 or In stage 10.5 animal Ets-2 the expression of the marker Xbra in a (Fig. A). The dorsal mesoderm marker and the ventral mesoderm marker were not by Ets-2 overexpression (data not shown). In stage animal Ets-2 the expression of the posterior markers Xcad3 and (Fig. B). The expression of which is expressed in mesoderm and was little by the injection of Ets-2 mRNA. To examine whether Ets-2 is for mesoderm patterning, we a dominant-negative form of The of the construct used here was shown (Fig. A). Ets-2 a N-terminal regulatory domain (the Pointed domain) and a that the DNA binding domain (the ETS EtsΔN En-R was made of the region of Ets-2, which the DNA binding domain the activation to the transcription We examined the effect of overexpression of EtsΔN En-R (Fig. B). EtsΔN En-R mRNA was injected into dorsal marginal zones of four-cell stage and these embryos were cultured stage A typical image is shown in Fig. B. EtsΔN embryos showed the severe posterior defect out of truncation and dorsal of embryos were also These of the injected embryos were similar to of the embryos (Fig. 2). Embryos injected developed almost normally (Fig. B). We examined whether EtsΔN En-R is to FGF- or constitutively active expression of Xbra (Fig. is a of MAPK J. 1991; PubMed Scopus Google Scholar, E. Y. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, J.M. J. Rev. 2001; PubMed Scopus Google Scholar, J. V. P. Biochem. 2002; PubMed Scopus Google Scholar). uninjected animal caps with FGF expressed a high of Xbra, EtsΔN animal caps with FGF not Xbra expression. injection of active Xbra expression of EtsΔN En-R resulted in reduction of Xbra expression. These results suggest that the FGF/MAPK pathway induces Xbra expression via To the of the EtsΔN En-R EtsΔN En-R mRNA was injected with wild Ets-2 and expression of Xbra was by RT-PCR with whole embryos (Fig. EtsΔN whole embryos showed reduced expression of Xbra compared with uninjected whole embryos. The wild Ets-2 embryos showed expression of Xbra, indicating that wild Ets-2 rescued the effect by EtsΔN Next, we tested the effect of expression of antisense morpholino oligonucleotides against We injected the Ets-2 into dorsal marginal zones in four-cell stage embryos. About of embryos injected with the Ets-2 in dorsal showed a severe posterior defect (n = A typical phenotype is shown in Fig. Expression of control in dorsal of embryos had no effect (Fig. Embryos injected with the Ets-2 in ventral were almost normal (data not shown). To that the Ets-2 phenotype is specifically caused by the Ets-2 we Ets-2 mRNA with Ets-2 Co-injection of Ets-2 mRNA rescued of embryos from the defects in posterior by Ets-2 (n = A typical image is shown (Fig. these results suggest that Ets-2 is for patterning of posterior mesoderm. RT-PCR analysis showed that expression of Ets-2 is to expression of We that Ets-2 regulates the Xbra transcription. To the role of Ets-2 in of Xbra we performed a reporter assay using the luciferase reporter containing the fragment of the Xbra promoter region B.V. Umbhauer M. Neal K.A. Lerchner W. Smith J.C. Cunliffe V. Genes Dev. 1997; 23: 3265-3276Crossref Scopus (166) Google Scholar, 14Artinger M. Blitz I. Inoue K. Tran U. Cho K.W. Mech. Dev. 1997; 65: 187-196Crossref PubMed Scopus (92) Google Scholar). The Xbra reporter construct was with Ets-2 mRNA and the control β-galactosidase into animal poles of two-cell stage embryos. Animal caps were dissected at stage and assayed for luciferase and β-galactosidase activities at stage The ratio of luciferase to β-galactosidase activity provides a normalized measure of luciferase expression. Ets-2 activated luciferase expression to control (Fig. A). FGF the luciferase activity by Ets-2 was On the EtsΔN En-R the luciferase activity EtsΔN En-R inhibited the luciferase activity (Fig. A). These results are in with that Ets-2 regulates the transcription of we whether Ets-2 protein to the promoter region of A has shown that a region of the Xbra promoter is for expression W. B.V. D. Smith J.C. Development. 2000; Google Scholar). We for the Ets binding sites this region of Xbra We found Ets binding in the Xbra promoter and tested to to Ets-2 in the gel mobility shift The gel mobility shift assay was performed on to and to of the Xbra promoter using the expressed protein or GST The fragment bound only to GST-Ets-2 (data not the region of the Xbra promoter bound to Ets-2 (Fig. to the wild and the mutated region were for the gel mobility shift assay (Fig. B). The GST-Ets-2 protein bound to the wild oligonucleotide GST not to the (Fig. The binding was inhibited by with an of oligonucleotide (Fig. However, the binding with an of oligonucleotide (Fig. Thus, Ets-2 is in a with a Xbra promoter is that mesoderm via the pathway in early Xenopus development (3Whitman M. Melton D.A. Nature. 1992; 357: 252-254Crossref PubMed Scopus (183) Google Scholar, 4MacNicol A.M. Muslin A.J. Williams L.T. Cell. 1993; 73: 571-583Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 5Gotoh Y. Masuyama N. Suzuki A. Ueno N. Nishida E. EMBO J. 1995; 14: 2491-2498Crossref PubMed Scopus (134) Google Scholar, 6LaBonne C. Burke B. Whitman M. Development. 1995; 121: 1475-1486PubMed Google Scholar, 7Umbhauer M. Marshall C.J. Mason C.S. Old R.W. Smith J.C. Nature. 1995; 376: 58-62Crossref PubMed Scopus (204) Google Scholar, 8Kusakabe M. Masuyama N. Hanafusa H. Nishida E. EMBO Rep. 2001; 2: 727-735Crossref PubMed Scopus (24) Google Scholar). have shown that of at subfamilies of ETS are nuclear targets of the is a member of the factor subfamily, and is that phosphorylation of by MAPK activities of However, the Xbra expression was not reduced by overexpression of dominant-negative in animal assay F. B. T. A. T. EMBO J. 1998; 17: PubMed Scopus Google Scholar). The member of ETS which to the subfamily, was in has been to be a of FGF signaling, not Xbra expression in animal Y. T. H. T. Mech. Dev. 1999; PubMed Scopus Google Scholar). Moreover, overexpression of not the expression of Xbra H. Mech. Dev. 1999; PubMed Scopus Google Scholar). results here have that Ets-2 induces Xbra expression and plays an essential role in mesoderm patterning downstream of the FGF/MAPK overexpression of Ets-2 caused posteriorized embryos. were to be by Xcad3 that is for posterior development downstream of FGF signaling M.E. J.M. EMBO J. 1998; 17: PubMed Scopus Google Scholar). Ets-2 and Xcad3 to anterior neural Ets-2 with Xcad3 and the gene pathway in posterior development to be M.E. J.M. Development. 1996; 122: Google Scholar). In Xenopus mesoderm patterning, no transcription factor that the FGF signal at the Xbra promoter has been M. Smith J.C. Development. 1997; Google Scholar). results suggest that Ets-2 regulates the transcription of Xbra downstream of the FGF/MAPK J. G. G. V. V. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google have shown that Ets-2 protein to a site of the They on the role of Ets sites in transcription through with transcription J. G. G. V. V. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the Ets sites are on the Xbra is that with several sites Xbra transcription. transcription factors, such as in of Xbra J. J.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus (24) Google Scholar). is that transcription factors in with Ets-2 Xbra transcription. of the of Ets-2 with transcription factors be a the of results here suggest that Ets-2 plays an essential role in mesoderm patterning. has been as a of Overexpression of in embryos caused cells to cellular adhesion and to D. T. K. S. H. K. Mech. Dev. 1999; PubMed Scopus Google Scholar, D. T. K. S. H. K. 2000; PubMed Google Scholar). In is that Ets-2 is also involved in cellular adhesion and cell We are of Ets-2 in Xenopus developmental We Dr. W. Y. Cho for the We also H. Hanafusa and M. Kusakabe in for and