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efficiency for FGFR-4, all four members of the FGFR family can cause ... Signaling specificities of fibroblast growth factor receptors in early Xenopus embryo.
Journal of Cell Science 113, 2865-2875 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1302

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Signaling specificities of fibroblast growth factor receptors in early Xenopus embryo Muriel Umbhauer*, Alfredo Penzo-Méndez*, Léa Clavilier, Jean-Claude Boucaut and Jean-François Riou‡ Laboratoire de Biologie Moléculaire et Cellulaire du Développement, groupe Biologie Expérimentale, UMR CNRS 7622, Université Paris VI, 9 quai Saint-Bernard, 75005 Paris, France *Each author contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 26 May; published on WWW 20 July 2000

SUMMARY Formation of mesoderm and posterior structures in early Xenopus embryos is dependent on fibroblast growth factor (FGF) signaling. Although several FGF receptors (FGFRs) are expressed in the early embryo, their respective role in these processes remains poorly understood. We provide evidence that FGFR-1 and FGFR-4 signals elicit distinct responses both in naive and neuralized ectodermal cells. We show that naive ectodermal cells expressing a constitutively active chimeric torso-FGFR-1 (t-R1) are converted into mesoderm in a Ras-dependent manner, while those expressing torso-FGFR-4 (t-R4) differentiate into epidermis without significant activation of Erk-1. In neuralized ectoderm, expression of t-R4 causes the upregulation of the midbrain markers En-2 and Wnt-1, but not of the hindbrain nor the spinal cord markers Krox20 and Hoxb9. Mutation of tyr776 in the phospholipase C-γ

INTRODUCTION Signals mediated by fibroblast growth factor (FGF) family members are transduced into target cells by tyrosine kinase transmembrane receptors (FGFRs). The FGFR family includes four distinct receptors, FGFR-1, −2, −3 and -4 which share a common general structure. The extracellular domain contains three immunoglobulin-like domains. The first and the second domains are separated by a stretch of acidic amino acids, the acid box. A single transmembrane region joins the extracellular domain to the cytoplasmic domain, which consists of a tyrosine kinase domain and a short C-terminal tail (Green et al., 1996). Activation of FGFR results from ligand induced dimerization and subsequent autophosphorylation, allowing the recruitment of intracellular signaling molecules. Two major signaling pathways downstream of the FGFR have been identified. One involves the recruitment of phospholipase C-γ (PLC-γ) on a single docking phosphotyrosine located in the C-terminal tail (Mohammadi et al., 1992; Peters et al., 1992). Although with a weaker efficiency for FGFR-4, all four members of the FGFR family can cause PLC-γ phosphorylation (Kanai et al., 1997; Shaoul

binding consensus sequence YLDL of t-R4 completely abolishes En-2 and Wnt-1 induction. In contrast to t-R4, platelet derived growth factor (PDGF)-dependent FGFR-1 activation in neuralized ectodermal cells expressing a chimeric PDGFR-FGFR-1 receptor results in the expression of Krox20 and Hoxb9. A similar effect is observed when an inducible form of oncogenic Raf is expressed, therefore implicating FGFR-1 and Raf in the transduction of FGF-caudalizing signals in neural tissue. Our results suggest that FGFR-1 and FGFR-4 transduce distinct signals in embryonic cells, and mainly differ in their ability to activate the Ras/MAPK pathway.

Key words: Xenopus development, Fibroblast growth factor receptor, Signaling specificity

et al., 1995; Vainikka et al., 1994; Wang et al., 1994). A second transduction pathway leads to the activation of mitogenactivated protein kinase (MAPK) via Ras and Raf. Ras activation results from the recruitment of the Grb2/Sos complex to the intracellular domain of the FGFR via a lipidanchored protein, FRS2/SNT-1 which binds to and is phosphorylated by the activated FGFR (Kouhara et al., 1997; Wang et al., 1996). Grb2 recruitment on the FGFR can also be mediated by the adaptor protein Shc (Kanai et al., 1997; Klint et al., 1995; Vainikka et al., 1994). The signals transduced by the different FGFR share some common characteristics but are not identical. Among the four FGFR, FGFR-4 appears to elicit the most divergent responses. For example, FGFR-1 expression promotes FGF1-dependent growth of BaF3 pro-B cells, but not FGFR-4 expression. These differences are correlated with the observation that FGFR-1 strongly induces MAPK phosphorylation in these cells, whereas FGFR-4 only causes a weak activation of MAPK (Wang et al., 1994). In L6E9 rat myoblasts, stimulation of FGFR-1 or FGFR-2 elicits a strong proliferative response with cells becoming rounded. On the contrary, FGFR-4 signals only poorly enhance proliferation while cells remain flattened. Nevertheless,

2866 M. Umbhauer and others FGFR-4 activation in L6E9 cells suppresses myogenin expression and myogenic differentiation, suggesting a function in the maintenance of a non-differentiated state of muscle precursors (Shaoul et al., 1995). FGF signaling plays a major role in the formation of mesoderm and posterior structures of the vertebrate embryo. Results obtained in Xenopus embryos have shown that FGF is involved downstream of early mesoderm inductive signals, in the maintenance of the Xenopus brachyury gene (Xbra) expression in mesoderm precursors (Smith et al., 1997). Ectopic expression of Xbra causes mesoderm formation, but this process requires FGF signaling. Xbra and eFGF, which encodes a Xenopus FGF related to FGF4, have actually been shown to be the components of an autoregulatory loop in which Xbra induces eFGF expression which in turn maintains Xbra expression (Isaacs et al., 1994; Schulte-Merker and Smith, 1995). Transduction of FGF signals leading to Xbra maintenance involves Ras, Raf, the MAPK cascade (Gotoh et al., 1995; LaBonne et al., 1995; MacNicol et al., 1993; Umbhauer et al., 1995), and downstream the heterodimeric AP-1 transcription factor (Kim et al., 1998). AP-1 binding to the Xbra promoter has not been demonstrated, although the Xbra promoter sequence responsible for Xbra activation by FGF has been characterized (Latinkic et al., 1997). FGF signals also regulate the expression of the Xenopus caudal gene family member Xcad3 (Northrop and Kimelman, 1994; Pownall et al., 1996) which has a critical function in the regulation of posterior hox genes (Isaacs et al., 1998). The establishment of the anteroposterior polarity of the Xenopus nervous system is thought to be dependent on FGFcaudalizing activity. Ectodermal explants can be converted into neural tissue by several molecules inhibiting BMP-4 signaling, such as noggin, chordin, follistatin or dominantnegative forms of BMP receptors (Hemmati-Brivanlou and Melton, 1997). These explants only express markers of the anterior neural tube. FGF treatment causes the up-regulation of genes normally expressed in the developing hindbrain and spinal cord. FGF induces caudalization of neuralized ectoderm at gastrula stages. Neuroectodermal competence for caudalization by FGF is lost after the early neurula stage (Cox and Hemmati-Brivanlou, 1995; Kengaku and Okamoto, 1995). cDNA encoding Xenopus homologs of FGFR-1, 2 and 4 have been cloned (Friesel and Brown, 1992; Friesel and Dawid, 1991; Musci et al., 1990; Riou et al., 1996; Shiozaki et al., 1995). Their respective role in the maintenance of Xbra, or in the transduction of caudalization signals is not known. Nonetheless, the developmental expression of Xenopus FGFR-1, 2 or 4 genes is differently regulated, indicating that each FGFR may have a distinct function during development. Such a functional specificity might rely on distinct signals activated downstream of each FGFR. Here, we report that activation of FGFR-1 or FGFR-4 in naive or neuralized Xenopus ectodermal cells elicits distinct responses. Ectodermal cells expressing a constitutive form of FGFR-1 are converted into mesoderm, while those expressing a constitutive FGFR-4 differentiate into epidermis. FGFR-1dependent mesoderm induction is inhibited by a dominantnegative form of Ras and correlates with the activation of Erk-1. In neuralized ectoderm, activation of FGFR-4 causes the up-regulation of the Wnt-1 and En-2 genes, but not that

of the more posterior neural genes Krox20 or Hoxb9. Mutation of the tyrosine residue in the PLC-γ binding consensus sequence YLDL of FGFR-4 completely abolishes Wnt-1 and En-2 up-regulation. Using a chimeric PDGFR/FGFR-1 receptor (P-R1) allowing PDGF-dependent FGFR-1 signaling, we further show that Krox20 and Hoxb9 expression is induced in neuralized ectoderm as a response to FGFR-1 activation. The same response is observed in neuralized ectoderm expressing an inducible form of oncogenic Raf, thus implicating Raf downstream of FGFR-1. MATERIALS AND METHODS Embryos Xenopus lævis embryos were obtained by artificial fertilization as previously described (Umbhauer et al., 1992), and cultured in 10% modified Barth’s solution (MBS). Stages were determined according to Nieuwkoop and Faber (1967). Plasmid contructs and RNA transcription. The different plasmids encoding the torso-FGFR were constructed as follows. DNA sequence encoding the intracellular domains of Xenopus FGFR-1 and FGFR-4 were amplified by PCR using the following primers: 5′-TAATGAATTCACCCGTCGAAGAAG-3′ (FGFR-1 upstream), 5′-ATTATCTAGATCAGCGTTTTTTAAG-3′ (FGFR-1 downstream), 5′-TAATGAATTCAGACACCCCACAGC3′ (FGFR-4 upstream) and 5′-ATTAGCGGCCGCTCAAGTCCCAAGTTG-3′ (FGFR-4 downstream). They represent the sequences from position 1190 to position 2439 of FGFR-1 cDNA (Musci et al., 1990), preceded by an EcoRI linker (GAATTC) and followed by an XbaI linker (TCTAGA), and position 1247 to position 2487 of FGFR-4 cDNA (Riou et al., 1996) preceded by an EcoRI linker (GAATTC) followed by a NotI linker (GCGGCCGC). PCR products were inserted into pBS torso4021-sev (Dickson et al., 1992), after excision of the sevenless cDNA with EcoRI/XbaI or EcoRI/NotI. The torso4021-FGFR sequences were subcloned into the SP64T plasmid containing the 5′ and 3′ untranslated sequences of the Xenopus β-globin gene (Krieg and Melton, 1984). The sequence encoding six Myc tags was amplified by PCR from pCS2+MT plasmid (Rupp et al., 1994), using the primers 5′TAATGGGCGCCGATTTAAAGCTATGGAG-3′ (upstream) and 5′-ATTAGGCGCCCCCTGAATTCAAGTC-3′ (downstream). The Myc tag sequence was inserted as NarI-NarI fragment into a NarI site, immediately after the signal peptide-encoding sequence of torso. The encoded chimeric proteins contain 950 (t-R1) and 948 (tR4) amino acids, respectively. The fusion site of t-R1 encodes the CRIH sequence where C is the cys420 of torso, RI an additional sequence encoded by the EcoRI linker and H the his397 of FGFR-1. The fusion site of t-R4 encodes the CRIQ sequence where Q is the gln415 of FGFR-4. To produce t-R4∆, the sequence encoding the Cterminal region of t-R4 was excised at the level of a BglII site. The truncated protein ends at the level of the leu540 of FGFR-4. Mutation of tyr776 to Phe was produced by sequential PCR using the primers 5′-GAAAACTAGTAACGGCCGACT-3′ (upstream 1), 5′AGATAAGTCCAGAAACTCTTCAGAAACAGC-3′ (downstream 1), 5′-GTCGTTTCTGAAGAGTTTCTGGACTTATCT-3′ (upstream 2) 5′CCGGGGATCCTCTAGAGTCGA-3′ (downstream 2). The wild-type fragment was excised from SP64T-t-R4 with SpeI and SalI and replaced by the amplified mutated fragment. All sequences of amplified DNA were checked. Otherwise, DNA encoding the chimeric receptor containing extracellular human PDGFR, transmembrane and intracellular rat FGFR-4 sequences (P-R4) (Raffioni et al., 1999), or the oncogenic form of human Raf fused to the hormone-binding domain of estrogen receptor (∆Raf:ER; Samuels et al., 1993) have been subcloned in pCS2+ (Rupp et al.,

FGFR signaling specificities in Xenopus 2867 1994) or SP64T, respectively. Construction of the plasmids encoding N17 Ras, P-R1 and ∆1XAR1 has been described elsewhere (Hemmati-Brivanlou and Melton, 1994; Muslin et al., 1994; Whitman and Melton, 1992). For RNA transcription, plasmids were linearized with SacI (t-R1, t-R4, tR4YF, t-R4∆), EcoRI (N17 Ras, P-R1), NotI (P-R4) or BamHI (∆Raf:ER), and were transcribed with SP6 RNA polymerase (Roche Diagnostics) according to previously published procedures (Krieg and Melton, 1987). Microinjection, animal cap assays Microinjection of mRNA was carried out as previously described (Umbhauer et al., 1994). Injected embryos were cultured in 10% MBS. Animal caps were dissected at the midblastula stage (stage 8) and cultured in 100% MBS. Treatment with bovine FGF2 (R&D Systems) was carried out as previously reported (Umbhauer et al., 1992). Activation of P-R1 in neuralized animal caps was performed as described by Cox and Hemmati-Brivanlou (1995) except that ‘aged’ gastrula stage caps were incubated with 2 nM human PDGF BB (Sigma) instead of FGF2. Animal caps expressing ∆Raf:ER were dissected at blastula stage and incubated with 10−6 M estradiol (Sigma) in 1× MBS immediatly after dissection or after culture until the early gastrula stage (stage 10.5).

RESULTS Expression of functional chimeric torso-FGFR receptors in Xenopus embryos To analyse the responses elicited by FGFR-1 or FGFR-4 signals in Xenopus embryos, we have constructed constitutively active chimeric receptors containing the extracellular and transmembrane domains of the Drosophila mutant torso protein (encoded by the torso4021 allele) and the intracellular domain of Xenopus FGFR-1 or FGFR-4. c-Myc tags have been included at the N terminus to allow immunodetection of the chimeric proteins (Fig. 1A). The constitutive activity of the mutant torso protein results from a mutation (Y323C) in the extracellular domain causing spontaneous dimerization (Sprenger and Nüsslein-Volhardt, 1992). This property has been used to express constitutive

Antibodies, protein analysis Proteins were extracted on ice in NP40 buffer (MacNicol et al., 1993) containing 1 mM sodium orthovanadate, except for the analysis of Erk-1 shifts where explants were lysed in β-glycerophosphate buffer (80 mM sodium β-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 1 mM DTT, 2 mM PMSF, 25 µg/ml leupeptin, 0.2 U/ml aprotinin, 1 mM sodium orthovanadate). Whole embryos were lysed in 20 µl extraction buffer, and each explant in 10 µl. Yolk platelets and lipids were eliminated from the extracts by centrifugation at 11000 g for 10 minutes at 4°C. Extracts were subjected to immunoprecipitation with the monoclonal anti-cMyc antibody 9E10 (Santa Cruz Biotechnology), or directly processed for SDS-PAGE. Lysates were run on 7.5% polyacrylamide gels for torso-FGFR protein analyses, or on 15% gels for Erk-1 shifts analysis as described (Rossomando et al., 1989). Proteins were transfered onto nitrocellulose filters (Amersham). After blocking with 5% BSA, filters were incubated with 9E10, monoclonal anti-phosphotyrosine py20, or goat polyclonal anti-rat Erk-1 antibody (Santa Cruz Biotechnology). Bound antibodies were detected with alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories), and were visualized with color developing reagent (BM purple, Roche Diagnostics). RT-PCR analysis of mRNA Total RNA extraction was carried out as previously described (Umbhauer et al., 1992), except that extracts were treated with 0.1 U of DNase I for 30 minutes at 37°C after the LiCl precipitation step. First strand cDNA was prepared from total RNA using the Superscript™ kit (Life Technologies) and random primers. PCR analysis of cDNA was performed according to the method of Wilson and Melton (1994). Primer sequences for EF-1α, N-CAM, Otx-2, Krox20, Hoxb9 (Xlhbox6) and muscle actin (ms act) have been described by Hemmati-Brivanlou and Melton (1994), En-2 and Wnt-1 by Riou et al. (1998), Xbra by Henry et al. (1996) and ODC by Bouwmeester et al. (1996). Primers for Otx-2 were: 5′GGATGGATTTGTTGCACCAGT-3′ (upstream) and 5′CACTCTCCGAGCTCACTTCTC-3′ (downstream) (Pannese et al., 1995). PCR amplification was carried out for a total of 26 cycles except for EF-1α, and ODC which were amplified for a total of 22 cycles. In situ hybridization and histology In situ hybridization and histological analyses were performed as described previously (Riou et al., 1998).

Fig. 1. Functional properties of constitutive chimeric torso-FGFR in Xenopus embryos. (A) Schematic representation of a chimeric torsoFGFR. The chimeric protein is formed of the extracellular and transmembrane domains of the Drosophila protein encoded by the torso4021 allele, and the intracellular domain of Xenopus FGFR-1 (tR1) or FGFR-4 (t-R4). The torso4021 allele contains a mutation (Y327C) in the extracellular domain causing spontaneous dimerization. Myc Tags are included at the N terminus to allow immunodetection. (B) Western blotting analysis of t-R1 and t-R4 expression in embryos. Embryos injected at the 2-cell stage with tR1 or t-R4 mRNA express 140 kDa (t-R1) or 150 kDa (t-R4) proteins from the 32-cell stage until the early tadpole. (C) Analysis of t-R1 and t-R4 phosphorylation in blastula stage animal caps. Animal caps were prepared from embryos injected with t-R1 or t-R4 mRNA at the 2-cell stage. Lysates were subjected to immunoprecipitation with the anti-cMyc antibody 9E10. Immunoprecipitates were analysed on western blots with the antiphosphotyrosine antibody py20 (left panel) or 9E10 (right panel).

2868 M. Umbhauer and others chimeric tyrosine kinase receptor in Drosophila embryos (Dickson et al., 1992; Reichman-Fried et al., 1994). RNA encoding torso-FGFR-1 (t-R1) or torso-FGFR-4 (t-R4) has been microinjected into 2-cell stage embryos. Expression of the chimeric proteins has been analysed by western blotting after various culture periods (Fig. 1B). Both receptors can be detected from the 32-cell stage until the early tadpole (st.35). Strongest protein expression can be observed from blastula to neurula stages, when mesoderm induction and neuralization are taking place. In order to control that torso-FGFRs are functional, we have performed immunoprecipitation with the anti-cMyc antibody, followed by Western blotting with an antiphosphotyrosine antibody. Embryos microinjected with t-R1 or t-R4 mRNA have been cultured until blastula stage and immunoprecipitation has been carried out on animal pole ectodermal tissue (‘animal caps’) extracts. Both receptors react in a similar way with the anti-phosphotyrosine antibody (Fig. 1C), indicating that torso-FGFR are able to activate downstream signals in Xenopus animal cap cells. t-R1 and t-R4 differ in their ability to activate the Ras-MAPK pathway and to induce mesoderm in animal caps In a first attempt to provide data about the signals activated dowstream of FGFR-1 and FGFR-4 in Xenopus embryos, we have compared the ability of t-R1 and t-R4 to cause mesoderm induction in animal caps. RNA encoding t-R1 (1 ng) or t-R4 (1 ng) was microinjected at the 2-cell stage and animal caps dissected as described above. Expression of the mesodermal markers Xbra and ms act were analyzed by RT-PCR at gastrula (st. 11) and tailbud stages (st. 28), respectively (Fig. 2A). Both Xbra and ms act transcripts were detected in response to t-R1 signals. They were never detected in explants expressing t-R4. Histological analysis of differentiated tissues in explants cultured until the tadpole stage (st. 40) confirmed that animal caps expressing t-R1 contain differentiated muscle, while those expressing t-R4 differentiate into epidermis (Fig. 2B). Induction of mesoderm was correlated with the detection of the shifted band of Erk-1 on western blots of t-R1-expressing Fig. 2. Analysis of mesoderm induction in response to t-R1 or t-R4 signals. (A) RT-PCR analysis of mesodermal marker gene expression. Animal caps were dissected from embryos injected with t-R1 or t-R4 mRNA at the 2-cell stage and were cultured until midgastrula stage (st. 11) or tailbud stage (st. 28). Expression of the mesodermal markers Xbra and ms act was detected in the explants expressing t-R1. Only the epidermal marker keratin was detected in t-R4-expressing explants. N-CAM mRNA was detected neither in tR1 nor in t-R4 explants, showing that neuralization did not occur. EF-1α expression was used as loading control. (B) Histological analysis of differentiated tissues. Animal cap explants expressing tR1 (left panel) or t-R4 (right panel) were cultured until tadpole stage (st. 40). Blocks of muscle tissue (ms) were observed in response to tR1 expression. Explants expressing t-R4 only contained atypical epidermis. Bar, 100 µm. (C) Western blotting analysis of Erk-1 activation. Animal caps expressing t-R1 or t-R4 were cultured until midblastula stage (st.8), midgastrula stage (st.11) or midneurula stage (st.17). Lysates were subjected to immunoblotting with antiErk-1 (upper panel) or 9E10 (lower panel). The 44 kDa band corresponding to the activated form of Erk-1 was detected in t-R1expressing explants at stages 8 and 11. It was not detected in t-R4expressing animal caps.

explants. No shift was detected for animal caps expressing t-R4 although both receptors were expressed at a similar level (Fig. 2C). Since mesoderm induction by FGF requires Erk-1 activation downstream of Ras (Gotoh et al., 1995; LaBonne et al., 1995; Umbhauer et al., 1995), we have tested the effect of the dominant-negative form of Ras, N17-Ras (Whitman and Melton, 1992), on Xbra induction in response to t-R1. RT-PCR analysis showed that Xbra transcripts cannot be detected in explants expressing t-R1 and N17 Ras. Suppression of Xbra induction was similar in animal cap explants expressing N17Ras alone and treated with FGF2 (Fig. 3A). In both cases, N17Ras blocked Erk-1 activation as shown by the absence of band shift on western blots (Fig. 3B). These results show that t-R1 expressed in animal caps is able to activate the Ras/MAPK

FGFR signaling specificities in Xenopus 2869 pathway and cause mesoderm induction. This is not the case of t-R4, suggesting that FGFR-4 uses distinct transduction pathways. Expression of t-R4 in neuralized ectoderm results in the up-regulation of Wnt-1 and En-2 In a further attempt to characterize signals activated by t-R4, we tested the effect of t-R4 in neuralized ectodermal explants. Neuralization of animal cap ectoderm can be obtained by the inhibition of BMP-4 signaling using ∆1XAR1, a dominantnegative form of the Xenopus type II activin/BMP-4 receptor ActRIIB (Chang et al., 1997; Hemmati-Brivanlou and Melton, 1994). RNA encoding ∆1XAR1 alone (1 ng) or mixed with the t-R4 RNA (1 ng) was injected into 2-cell stage embryos. Animal caps explants were dissected at the blastula stage and cultured until sibling embryos reached the tailbud stage (st.25-28). Expression of several neural genes including the pan-neural marker N-CAM and the regional markers Otx-2 (forebrain and midbrain), Wnt-1 and En-2 (midbrain), Krox20 (hindbrain) and Hoxb9 (spinal cord) were studied by RT-PCR. ms act transcripts were traced in order to check for the absence of contaminating mesoderm in the explants which otherwise could cause neuroectoderm caudalization independently of t-R4. Only N-

Fig. 3. t-R1 signals cause Xbra up-regulation in a Ras-dependent manner. Embryos were microinjected at the 2-cell stage with RNA encoding the dominant-negative form of Ras (N17 Ras), t-R1 or a mix of t-R1 and N17 Ras. Caps were prepared at the midblastula stage. Explants from uninjected embryos, or from embryos injected with N17 Ras alone were treated with 50 ng/ml FGF2 at the blastula stage. All explants were cultured until midgastrula stage (st. 11). (A) RT-PCR analysis of Xbra expression. N17 Ras abolishes Xbra expression induced in response to t-R1, in the same way as in response to FGF2. ODC was used as loading control. (B) Western blotting analysis of Erk-1 activation. The 44 kDa band corresponding to the activated form of Erk-1 is detected in animal caps expressing tR1 or treated with FGF2. Only the 42 kDa band was detected when explants express N17 Ras.

CAM and Otx-2 mRNA were detected in explants expressing ∆1XAR1 alone, thus attesting of anterior neural ectoderm induction. In response to t-R4, up-regulation of Wnt-1 and En-2 was observed. Expression of other markers was unchanged

Fig. 4. Up-regulation of Wnt-1 and En-2 in neuralized ectoderm expressing t-R4. (A) RT-PCR analysis of neural markers gene expression. RNA encoding ∆1XAR1, or a mix of ∆1XAR1 and t-R4 were microinjected at the 2-cell stage and caps prepared at the midblastula stage. Expression of N-CAM and of regional neural markers Otx-2 (forebrain and midbrain), Wnt-1 (midbrain), En-2 (midbrain), Krox20 (hidbrain) and Hoxb9 (spinal cord) was analyzed in explants cultured until the tailbud stage (St.28). ms act was used to check the absence of contaminating mesoderm and EF-1α as loading control. Wnt-1 and En-2 transcripts are detected in response to t-R4 signaling. (B) Western blotting analysis of Erk-1 activation. Animal caps expressing ∆1XAR1 and t-R4 were cultured until midblastula stage (st.8), midgastrula stage (st.11) or midneurula stage (st.17). Lysates were subjected to immunoblotting with anti-Erk-1 (upper panel) or 9E10 to control the presence of t-R4 (lower panel). The 44 kDa band corresponding to the activated form of Erk-1 was never detected, indicating that Erk-1 activation is not involved in t-R4 signaling

2870 M. Umbhauer and others (Fig. 4A). The shifted form of Erk-1 was not detected in ectodermal explants expressing ∆1XAR1 and t-R4, therefore implying that up-regulation of Wnt-1 and En-2 involves distinct transduction pathways downstream of t-R4 (Fig. 4B). Mutation of Y(776)LDL abolishes t-R4-mediated upregulation of Wnt-1 and En-2 Xenopus FGFR-4 contains the consensus sequence Y(776)LDL for phosphotyrosyl-mediated binding of PLC-γ. This corresponds to the Y(754)LDL site of human FGFR-4. Mutation of Tyr754 into a phenylalanine completely abolishes PLC-γ phosphorylation by human FGFR-4 (Vainikka et al., 1994). In order to test the potential role of PLC-γ downstream of t-R4, we have constructed a mutant t-R4 in which the Tyr776 residue of the catalytic domain of Xenopus FGFR-4 is replaced by a Phe residue. Wild-type t-R4, the Y776F mutant, or a control t-R4 lacking most of the tyrosine kinase domain (tR4∆), have been expressed in animal caps neuralized with ∆1XAR1 (Fig. 5A). Expression of N-CAM, Wnt-1 and En-2 has been analyzed by RT-PCR (Fig. 5B). Wnt-1 and En-2 transcripts could not be detected in neuralized animal caps expressing t-R4 Y776F, whereas they were present when explants expressed the wild-type form of t-R4. This does not result from a lower expression of the mutant chimeric receptor. For each experiment, a subset of the explants was subjected to Western blotting analysis to control the expression of each kind

Fig. 5. The t-R4F776 mutant fails to induce Wnt-1 and En-2 expression in neuralized ectoderm. (A) Schematic representation of the different forms of t-R4 expressed in neuralized animal caps. Tyr776 of the YLDL consensus sequence for phosphotyrosyl-mediated binding of PLC-γ is mutated to a phe residue in the tR4F776 mutant. The truncated t-R4∆ form lacks the activation loop and the catalytic domain including the key asp640 residue of the tyrosine kinase. t-R4∆ is used as negative control. (B) RT-PCR analysis of Wnt-1 and En2 expression. Embryos were microinjected with RNA encoding ∆1XAR1 mixed with RNA encoding t-R4∆, t-R4F776 or wild-type tR4. Animal caps were cultured until tailbud stage (st. 28). N-CAM expression was analyzed to check for neuralization by ∆1XAR1 and ms act for the absence of contaminating mesoderm. EF-1α is used as loading control. Wnt-1 and En-2 expression is completely abolished when tyr776 is mutated into a phe residue. (C) Presence of each form of t-R4 was controled in subsets of explants processed for western blotting with the 9E10 antibody.

of torso-FGFR (Fig. 5C). These results show that the Tyr776 is necessary for signal transduction leading to the up-regulation of Wnt-1 and En-2 in neuralized ectoderm. They strongly suggest that these pathways involve direct activation of PLC-γ by t-R4. Activation of FGFR-1 in neuralized animal caps results in the up-regulation of Krox20 and Hoxb9 Incubation of neuralized animal cap explants with FGF-2 causes the up-regulation of Krox20 and Hoxb9 which are normally expressed in the posterior neuroectoderm (Cox and Hemmati-Brivanlou, 1995; Lamb and Harland, 1995). The results described above show that the activation of FGFR-4 transduction pathways in neuralized ectoderm does not mimick FGF2 treatment. We thus tested the potential role of FGFR-1 in neuroectoderm caudalization. It was not possible to use tR1, since t-R1 expression would cause mesoderm induction in neuralized animal caps. The induced mesoderm might indirectly cause expression of posterior neural markers. We therefore expressed a chimeric PDGFR/FGFR-1 receptor (PR1) allowing PDGF-induced FGFR-1 signaling (Muslin et al., 1994) in gastrula stage explants, when ectodermal cells have lost competence for mesoderm induction. In addition, we analyzed the effect of a PDGF/FGFR-4 receptor (P-R4) activated at the early gastrula stage, to control that the response elicited by t-R4 does not result from its early activation.

FGFR signaling specificities in Xenopus 2871

Fig. 6. PDGF-dependent caudalization of neuralized ectodermal explants expressing a chimeric PDGF-FGFR-1 receptor (P-R1). RT-PCR analysis of neural markers gene expression. RNA encoding P-R1 and ∆1XAR1 (A) or P-R4 and ∆1XAR1 (B) were microinjected at the 2-cell stage. Animal caps were dissected at the early gastrula stage (st. 10) and aged until stage 10.5, when PDGF BB was added. PDGF-treated and untreated explants were cultured until tailbud stage (st. 28). Neural markers N-CAM, Otx-2, Wnt-1, En2, Krox20 and Hoxb9 were analyzed as described in Fig. 4. ms act was analyzed to check for contaminating mesoderm. EF-1α was used as a loading control. The hindbrain and spinal cord markers Krox20 and Hoxb9 were detected in P-R1expressing explants activated with PDGF, showing that FGFR-1 signals can caudalize neuralized ectoderm. Wnt-1 and En-2 were detected in PDGF-treated caps expressing P-R4 in a same way as in response to the constitutive t-R4 (see Fig. 4).

P-R1 mRNA (0.2-0.5 ng) was microinjected at the 2-cell stage in combination with ∆1XAR1 mRNA (1 ng) and animal caps were dissected at the early gastrula stage (st.10). They were incubated with PDGF BB at stage 10.5 and then cultured until tailbud stage (st. 25-28). N-CAM and regional neural marker gene expression were analyzed by RT-PCR. ms act expression was used to control the absence of mesoderm. As shown in Fig. 6A, Wnt-1, En-2, Krox20 and Hoxb9 were expressed in explants treated with PDGF while ms act mRNA was not detected. Otx-2 expression was identical in PDGFtreated and untreated animal caps. Wnt-1 and En-2 transcripts were also detected in untreated explants. This probably indicates that even at a dose which does not cause mesoderm induction, residual P-R1 activity occurs in the animal cap. However, the most posterior markers Krox20 and Hoxb9 were induced in response to PDGF treatment, showing that activation of FGFR-1 transduction pathways is sufficient to elicit neuroectoderm caudalization. Wnt-1 and En-2 upregulation was induced by PDGF in explants expressing P-R4, showing that this process can take place at gastrula stage (Fig. 6B). Finally, we investigated whether up-regulation of neural markers in response to P-R1 activation occured in the entire neural tissue, or whether some regionalization can be observed. In situ hybridization was performed with Wnt-1, En-2, Krox20 and Hoxb9 riboprobes. En-2 transcripts were mostly detected as a small patch in the explant, indicating that the neuralized ectoderm was regionalized. Staining for other markers

generally appeared diffuse although some explants contained patches with higher levels of expression (Fig. 7). Expression of an inducible form of oncogenic Raf causes up-regulation of Krox20 and Hoxb9 in neuralized ectoderm In order to provide further information about the intracellular signals activated during FGF-induced caudalization of neuroectoderm, we have tested the effect of oncogenic Raf, on the expression of neuroectodermal markers in neuralized animal caps. Since mesoderm induction in animal caps by FGF involves the downstream activation of Raf (MacNicol et al., 1993; Umbhauer et al., 1995), it is necessary to activate Raf signaling only at gastrula stage, when ectoderm has lost competence for mesoderm induction. We have therefore expressed an inducible form of oncogenic Raf, ∆Raf:ER. This molecule is formed of a truncated region of human Raf fused to the hormone-binding domain of the estrogen receptor which allows hormone-inducible activation (Samuels et al., 1993). The inhibition of ∆Raf by the fused ER sequence results from the binding of hsp90 which is released upon estrogen treatment. This strategy has been already used to obtain the inducible expression of MyoD or Xbra in Xenopus embryo cells (Kolm and Sive, 1995; Tada et al., 1997). The estrogen-inducible activity of ∆Raf:ER in animal cap cells was first tested according to its ability to induce Xbra expression. Increasing amounts of ∆Raf:ER mRNA were microinjected into 2-cell stage embryos. Xbra expression was

2872 M. Umbhauer and others

Fig. 8. Estrogen-induced expression of Xbra in animal caps expressing ∆Raf:ER. RT-PCR analysis of Xbra expression. Increasing doses of ∆Raf:ER RNA were microinjected at the 2-cell stage and animal caps incubated with 10−6 M β-estradiol at the midblastula stage (st. 8). The highest inducible Xbra expression was detected in caps prepared from embryos injected with the 0.2 ng dose. ODC was used as loading control. Fig. 7. In situ hybridization analysis of neural markers expression in neuralized ectodermal explants expressing P-R1. Animal caps expressing ∆1XAR1 and P-R1 were treated with PDGF BB at gastrula stage (st. 10.5) and were cultured until the tailbud stage (st. 28). Expression of Wnt-1, En-2, Krox20 and Hoxb9 was analyzed by in situ hybridization. En-2 transcripts are detected as patches indicating that some regionalization is taking place in the induced neural tissue. Other markers are generally detected as a diffuse staining, although some explants exhibit patches with higher levels of expression (arrows).

analyzed in animal caps incubated with 10−6 M estradiol at blastula stage. Using 0.2 ng and 0.1 ng RNA doses, Xbra expression was only detected in estradiol-treated caps (Fig. 8). Xbra expression was always correlated with the detection of the activated form of Erk-1 (data not shown). ∆Raf:ER mRNA (0.2 ng) and ∆1XAR1 mRNA (1 ng) were co-injected in 2-cell stage embryos. Animal caps were dissected at the late blastula stage, and were then incubated with 10−6 M estradiol at the early gastrula stage (st. 10.5) when ectoderm has lost competence for mesodermal induction. Caps were cultured until the tailbud stage (stages 28-30), and expression of the neuroectodermal markers was analyzed (Fig. 9). Upon induction of the oncogenic form of Raf, the neuralized ectoderm expressed the most posterior markers Krox20 and Hoxb9. Wnt-1 and En-2 were not significantly detected. Otx-2 expression remained unchanged. These results show that activation of transduction pathways downstream of Raf causes the caudalization of neuralized animal cap cells. They suggest that Raf is a key component of the tranduction machinery activated by FGF in the early steps of posterior nervous system development. DISCUSSION FGF signaling plays an important function in mesoderm formation and development of the posterior structures in Xenopus embryos. Although several FGFR are expressed in the

embryo, their respective roles in these processes remain unclear. In order to test whether intracellular signaling differs from one FGFR to another, we have compared the responses produced in naive or neuralized ectodermal cells, after FGFR1 or FGFR-4 activation. Our results strongly suggest that signals transduced by FGFR-1 and FGFR-4 are different, indicating that FGFR-1 and FGFR-4 may have distinct functions during early embryogenesis. We provide evidence that only FGFR-1 is able to activate the Ras-MAPK pathway to cause mesoderm induction in naive ectoderm. In gastrula neuralized ectoderm, activation of FGFR-1 signaling or expression of an oncogenic form of Raf induce the upregulation of Krox20 and Hoxb9. This implicates FGFR-1 and Raf in the transduction of caudalizing signals. In contrast to FGFR-1, activation of FGFR-4 never induces mesoderm in naive ectodermal cells nor causes Krox20 or Hoxb9 upregulation in neuralized cells. Nonetheless, FGFR-4 activation in neuralized ectoderm induces the expression of Wnt-1 and En-2. This effect is totally abolished when the tyrosine residue in the PLC-γ binding consensus sequence YLDL of FGFR-4 is mutated. It is not possible to specifically activate a defined FGFR in Xenopus embryonic cells since these cells already express several endogenous FGFR. We have used constitutive chimeric torso-FGFRs in order to activate FGFR-1 or FGFR-4 intracellular domains in an identical manner. Since it is very unlikely that the Drosophila torso extracellular domain interferes with any endogenous ligand, we assume that the responses observed in animal caps expressing t-R1 or t-R4 directly reflect the ability of FGFR-1 or FGFR-4 intracellular domains to activate different downstream signaling components. It is unclear however, whether the differences between t-R1 and t-R4 result from a weaker kinase activity of t-R4, or from signaling specificities. Indeed, previous reports indicate that this point is highly dependent on the cellular context wherein FGFR-1 and FGFR-4 are activated. In fibroblast and myoblast cells, FGFR-1 and FGFR-4 autophosphorylation levels are similar (Shaoul et al., 1995;

FGFR signaling specificities in Xenopus 2873

Fig. 9. Raf-dependent caudalization of neuralized ectoderm expressing ∆Raf:ER. RT-PCR analysis of neural markers gene expression. RNA encoding ∆1XAR1 alone or in combination with ∆Raf:ER RNA were microinjected at the 2-cell stage. Animal caps were dissected at the blastula stage and aged until stage 10.5, when β-estradiol was added. Estradiol-treated and untreated explants were cultured until tailbud stage (st. 28). Neural markers N-CAM, Otx-2, Wnt-1, En-2, Krox20 and Hoxb9 were analyzed as described in Fig. 4. ms act was analyzed to check for contaminating mesoderm. EF-1α was used as a loading control. Up-regulation of the posterior neural markers Krox20 and Hoxb9 was only detected in response to the expression of the oncogenic form of Raf. Otx-2 expression is not affected in response to Raf activation.

Vainikka et al., 1994; Wang et al., 1994). In BaF3 and PC12 cells, FGFR-4 phosphorylation is weaker than that of FGFR-1 (Raffioni et al., 1999; Wang and Goldfarb, 1997). Several aminoacid residues of the kinase domain of FGFR-1 which are not conserved in FGFR-4 appear to play a critical role in the mitogenic response of BaF3 cells, and levels of receptor autophosphorylation (Wang and Goldfarb, 1997). Although these amino acid substitutions are all conserved between Xenopus FGFR-1 and FGFR-4 intracellular domains, the absence of any obvious difference between t-R1 and t-R4 phosphorylation in our experiments (Fig. 1C) argues against a decreased level of t-R4 activity. We therefore privilege the interpretation that the absence of mesoderm induction in animal caps expressing t-R4 results rather from a failure to

significantly activate the Ras/MAPK pathway, than from a weaker activity of the intracellular domain of FGFR-4. Our results show that activating FGFR-1 is sufficient to cause Xbra induction in blastula stage ectoderm, as well as Krox20 and Hoxb9 up-regulation in gastrula stage neuralized ectoderm. As expected from previous studies (Gotoh et al., 1995; LaBonne et al., 1995; LaBonne and Whitman, 1997; Umbhauer et al., 1995), FGFR-1-mediated Xbra induction necessitates functional Ras, and is correlated with MAPK activation. Signaling events occuring downstream FGF during neuroectoderm caudalization are less understood. Here, we show that an oncogenic form of Raf expressed in neuralized ectoderm at gastrula stage causes the up-regulation of posterior neural genes. This correlates well with the results showing that the endogenous Raf protein levels peak at the late gastrula/early neurula stages (MacNicol et al., 1993), at the time when the activated form of MAPK is most strongly detected in the posterior region of the embryo (Christen and Slack, 1999). Together with the observations that FGFR-1 gene expression is detected caudally while FGFR-2 and FGFR-4 are not (Friesel and Brown, 1992; Riou et al., 1996), our data suggest that Raf is implicated in a transduction cascade activated downstream of FGFR-1 during the formation of posterior neural tissue. The analysis of the response produced by a constitutive FGFR-4 in ectodermal tissues indicates that FGFR-4 does not activate the Ras/MAPK transduction pathway. Nevertheless, we show that Wnt-1 and En-2 expressions are induced in neuralized ectodermal tissue expressing t-R4. This process is dependent on the recruitment of PLC-γ. PLC-γ catalyzes the hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2), which releases inositol 1,4,5-triphosphate (IP3) and diacylglycerol. These second messengers control transient mobilization of intracellular-free Ca++ and activation of protein kinase C (Carpenter and Ji, 1999). However, downstream signaling events leading to the up-regulation of Wnt-1 and En-2 in Xenopus ectoderm remain to be identified. Several lines of evidence show that FGF signaling plays a pivotal role in the development of the midbrain in vertebrate embryos. The importance of FGF8 signaling in this process has been recently established by the observation that FGF8 mutants in mouse and fish lack posterior midbrain and cerebellar tissue (Meyers et al., 1998; Reifers et al., 1998). In chick (Crossley et al., 1996) and Xenopus (Riou et al., 1998), FGF8-conditioned beads implanted in the caudal forebrain cause the ectopic expression of En-2 and Wnt-1 in the anterior midbrain. In mouse embryos, expression of FGF8 from a transgene in the dorsal mesencephalon and caudal diencephalon induces the ectopic expression of En-2 (Lee et al., 1997). Our results raise the question of the role of FGFR4 in the temporal and spatial regulation of Wnt-1 and En-2 in the Xenopus neural plate. FGFR-4 gene expression is abundantly detected in the anterior neural plate (Riou et al., 1996), and is therefore likely to transduce FGF8 signals. Besides, it is noteworthy that both in Xenopus and in mouse embryos, overexpression of FGF8 always leads to an overgrowth of mesencephalon and diencephalon. In mouse, this effect has been shown to be the consequence of an enhancement of neural precursor cell proliferation at the expense of differentiated cell types (Lee et al., 1997). Although the mitogenic potentials of Xenopus FGFR-1 and

2874 M. Umbhauer and others FGFR-4 were not adressed in this paper, the absence of response involving the Ras/MAPK pathway downstream of FGFR-4 suggest that this receptor might only poorly transduce mitogenic signals in neural cells, in a similar way as in L6E9 myoblasts or BaF3 cells (Shaoul et al., 1995; Wang et al., 1994). It is therefore tempting to propose that FGFR-4 might transduce FGF8 signals affecting Wnt-1 and En-2 expression in a way that does not influence proliferation. We are grateful to Dr B. Dickson for providing the constitutive torso-sev construct, Dr M.Mc Mahon for the ∆Raf:ER construct, Drs D. Tyson and R. A. Bradshaw for the P-R4 construct and Dr L. T. Williams for the P-R1 expression plasmid. We thank Dr Shi De Li for critical reading of the manuscript and helpful discussions, Audrey Bourdelas and Aude Pascal for excellent technical assistance as well as Jean Desrosiers and Philippe Nguyen for illustrations. This work was supported by funds from CNRS, MESR, ARC and AFM

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