The VT+ and VT− Isoforms of the Fibroblast Growth Factor Receptor Type 1 Are Differentially Expressed in the Presumptive Mesoderm of Xenopus Embryos and Differ in Their Ability to Mediate Mesoderm Formation

Previously, we cloned a variant form of the type 1 fibroblast growth factor receptor (FGFR1), FGFR-VT−, from Xenopus embryos (Gillespie, L. L., Chen, G., and Paterno, G. D. (1995) J. Biol. Chem. 270, 22758–22763). This isoform differed from the reported FGFR1 sequence (FGFR-VT+) by a 2-amino acid deletion, Val423-Thr424, in the juxtamembrane region. This deletion arises from the use of an alternate 5′ splice donor site, and the activity of the VT+ and VT− forms of the FGFR1 was regulated by phosphorylation at this site. We have now investigated the expression pattern and function of these two isoforms in mesoderm formation in Xenopus embryos. Cells within the marginal zone are induced to form mesoderm during blastula stages. RNase protection analysis of blastula stage embryos revealed that the VT+ isoform was expressed throughout the embryo but that the VT− isoform was expressed almost exclusively in the marginal zone. The ratio of VT+:VT− transcripts in the marginal zone indicated that the VT+ form was predominant throughout blastula stages except for a brief interval, coinciding with the start of zygotic transcription, when a dramatic increase in VT− expression levels was detected. This increase could be mimicked in part by treatment of animal cap explants with FGF-2. Overexpression of the VT+ isoform in Xenopus embryos resulted in development of tadpoles with severe reductions in trunk and tail structures, while embryos overexpressing the VT− isoform developed normally. A standard mesoderm induction assay revealed that a 10-fold higher concentration of FGF-2 was required to reach 50% induction in VT+-overexpressing animal cap explants compared with those overexpressing the VT− isoform. Furthermore, little or no expression of the panmesodermal marker Brachyury (Xbra) was detected in VT+-overexpressing embryos, while VT−-overexpressing embryos showed normal staining. This demonstrates that VT+ overexpression had a negative effect on mesoderm formation in vivo. These data are consistent with a model in which mesoderm formation in vivo is regulated, at least in part, by the relative expression levels of the VT+ and VT− isoforms. Previously, we cloned a variant form of the type 1 fibroblast growth factor receptor (FGFR1), FGFR-VT−, from Xenopus embryos (Gillespie, L. L., Chen, G., and Paterno, G. D. (1995) J. Biol. Chem. 270, 22758–22763). This isoform differed from the reported FGFR1 sequence (FGFR-VT+) by a 2-amino acid deletion, Val423-Thr424, in the juxtamembrane region. This deletion arises from the use of an alternate 5′ splice donor site, and the activity of the VT+ and VT− forms of the FGFR1 was regulated by phosphorylation at this site. We have now investigated the expression pattern and function of these two isoforms in mesoderm formation in Xenopus embryos. Cells within the marginal zone are induced to form mesoderm during blastula stages. RNase protection analysis of blastula stage embryos revealed that the VT+ isoform was expressed throughout the embryo but that the VT− isoform was expressed almost exclusively in the marginal zone. The ratio of VT+:VT− transcripts in the marginal zone indicated that the VT+ form was predominant throughout blastula stages except for a brief interval, coinciding with the start of zygotic transcription, when a dramatic increase in VT− expression levels was detected. This increase could be mimicked in part by treatment of animal cap explants with FGF-2. Overexpression of the VT+ isoform in Xenopus embryos resulted in development of tadpoles with severe reductions in trunk and tail structures, while embryos overexpressing the VT− isoform developed normally. A standard mesoderm induction assay revealed that a 10-fold higher concentration of FGF-2 was required to reach 50% induction in VT+-overexpressing animal cap explants compared with those overexpressing the VT− isoform. Furthermore, little or no expression of the panmesodermal marker Brachyury (Xbra) was detected in VT+-overexpressing embryos, while VT−-overexpressing embryos showed normal staining. This demonstrates that VT+ overexpression had a negative effect on mesoderm formation in vivo. These data are consistent with a model in which mesoderm formation in vivo is regulated, at least in part, by the relative expression levels of the VT+ and VT− isoforms. fibroblast growth factor FGF receptor polymerase chain reaction RT, reverse transcription-PCR protein kinase C nucleotide(s) Fibroblast growth factors (FGFs)1 represent a family of related polypeptides known to stimulate a variety of cellular activities (reviewed in Ref. 1.Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar), including mesoderm differentiation in the Xenopus embryo (2.Slack J.M.W. Darlington B.G. Heath J.K. Godsave S.F. Nature. 1987; 326: 197-200Crossref PubMed Scopus (652) Google Scholar). Their effects are mediated by high affinity transmembrane FGF receptors (FGFRs) containing intrinsic tyrosine kinase activity (reviewed in Ref. 3.Klint P. Claesson-Welsh L. Front. Biosci. 1999; 4: 165-177Crossref PubMed Google Scholar). Like other receptor tyrosine kinases, FGFR signal transduction is initiated by ligand binding and results in activation of several well characterized intracellular signaling pathways, including the protein kinase C (PKC) and Ras/mitogen-activated protein kinase pathways (4.Gillespie L.L. Paterno G.D. Mahadevan L.C. Slack J.M.W. Mech. Dev. 1992; 38: 99-108Crossref PubMed Scopus (20) Google Scholar, 5.Huang J. Mohammadi M. Rodrigues G.A. Schlessinger J. J. Biol. Chem. 1995; 270: 5065-5072Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Four FGFR genes have been described to date, FGFR1–FGFR4, along with a number of alternately spliced variants (reviewed in Refs. 3.Klint P. Claesson-Welsh L. Front. Biosci. 1999; 4: 165-177Crossref PubMed Google Scholar and 6.Friesel R.E. Maciag T. FASEB J. 1995; 9: 919-925Crossref PubMed Scopus (406) Google Scholar). Previously, we cloned from Xenopus embryos an alternately spliced isoform of the FGFR1 that contains a deletion of Val423-Thr424 in the juxtamembrane region (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). We demonstrated that this site could be phosphorylated by PKC. Furthermore, in a functional assay, activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate significantly reduced the activity of the VT-containing isoform (VT+) in Xenopusoocytes while having little effect on the deletion isoform (VT−). We speculated that differential expression of these two isoforms might represent an important mechanism for regulating FGFR activity in theXenopus embryo (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In the blastula stage Xenopus embryo, cells located in the equatorial region (marginal zone) are induced to differentiate into mesoderm in response to signal(s) from neighboring vegetal cells (reviewed in Refs. 8.Kimelman D. Christian J.L. Moon R.T. Development. 1992; 116: 1-9Crossref PubMed Google Scholar and 9.Isaacs H.V. Cell. Mol. Life Sci. 1997; 53: 350-361Crossref PubMed Scopus (47) Google Scholar). Use of a dominant negative form of the FGFR1 to block FGF activity has provided evidence that FGF/FGFR signaling is required for normal development of the mesoderm (10.Amaya E. Musci T.J. Kirschner M.W. Cell. 1991; 66: 257-270Abstract Full Text PDF PubMed Scopus (927) Google Scholar). The current view is that FGF does not act as the initial inducing signal(s) but rather as a competence factor in the responding cells and that its activity is required for the full range of responses leading to mesoderm formation (9.Isaacs H.V. Cell. Mol. Life Sci. 1997; 53: 350-361Crossref PubMed Scopus (47) Google Scholar, 11.Cornell R.A. Musci T.J. Kimelman D. Development. 1995; 121: 2429-2437Crossref PubMed Google Scholar). In this report, we examine the role of the VT+ and VT− isoforms in mesoderm formation in Xenopus and show that the two isoforms are differentially expressed in the presumptive mesoderm and that they differ in their ability to mediate mesoderm formation in vitro and in vivo. Xenopus laevis were purchased from Nasco. Eggs were artificially inseminated, and embryos were cultured as described under Godsave et al. (12.Godsave S.F. Isaacs H. Slack J.M.W. Development. 1988; 102: 555-566Crossref PubMed Google Scholar); embryonic stages were determined according to Nieuwkoop and Faber (13.Nieuwkoop P.D. Faber J. Normal Table of Xenopus laevis. North Holland Publishing, Amsterdam1967Google Scholar). Stage 8 blastulae were dissected into animal, vegetal, and marginal zone regions as described (14.Gillespie L.L. Paterno G.D. Slack J.M.W. Development. 1989; 106: 203-208Crossref PubMed Google Scholar). cRNA was transcribed from FGFRSP64T constructs (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) using the SP6 Ribomax system (Promega). 4.6 nl containing diethyl pyrocarbonate-treated H2O or 650 pg/nl cRNA was microinjected into stage 1 embryos. Embryos were cultured at room temperature until they reached the stage required for the assays described below. Probe preparation, RNA extraction, and RNase protection analysis were performed as described (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). RT-PCR analysis was performed as described in Ref. 15.Paterno G.D. Li Y. Luchman H.A. Ryan P.J. Gillespie L.L. J. Biol. Chem. 1997; 272: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar using forward (5′-GGGCTGCTTTTGTGTCCGCAAT-3′) and reverse (5′-CATTGATGAGCTGGAGTCCCCTG-3′) primers that bracket the VT region and generate 156- and 162-bp fragments for the FGFR-VT− and FGFR-VT+ gene products, respectively. Histone H4 was used as an input control with forward and reverse primers as described (16.Niehrs C. Steinbeisser H. De Robertis E.M. Science. 1994; 263: 817-820Crossref PubMed Scopus (144) Google Scholar). EF1α was amplified using 5′-CCTGAATCACCCAGGCCAGATTGGTG-3′ and 5′-GAGGGTAGTCTGAGAAGCTCTCCACG-3′, as forward and reverse primers, respectively. The [32P]CTP-labeled PCR products were analyzed in the linear range for amplification, determined empirically (16.Niehrs C. Steinbeisser H. De Robertis E.M. Science. 1994; 263: 817-820Crossref PubMed Scopus (144) Google Scholar) to be 19 cycles for histone H4, 22 cycles for EF1α, and 25 cycles for FGFR, and visualized on a 6% polyacrylamide/6 murea gel by autoradiography. Quantitation by densitometry was performed as in Ref. 17.Paterno G.D. Mercer F.C. Chayter J.J. Yang X. Robb J.D. Gillespie L.L. Gene (Amst.). 1998; 222: 77-82Crossref PubMed Scopus (20) Google Scholar. For expression analysis of injected FGFR cRNA, 0.5 μCi of [35S]methionine was co-injected into each embryo. Protein extraction, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis analysis were performed as in Ref. 18.Ryan P.J. Gillespie L.L. Dev. Biol. 1994; 166: 101-111Crossref PubMed Scopus (32) Google Scholar. The anti-Xenopus FGFR1 used for immunoprecipitation was a polyclonal antibody raised against a synthetic C-terminal peptide (18.Ryan P.J. Gillespie L.L. Dev. Biol. 1994; 166: 101-111Crossref PubMed Scopus (32) Google Scholar). Recombinant XenopusFGF-2 was expressed and purified according to Kimelman et al. (19.Kimelman D. Abraham J.A. Haaparanta T. Palisi T.M. Kirschner M.W. Science. 1988; 242: 1053-1056Crossref PubMed Scopus (272) Google Scholar). Animal cap explants were excised from injected embryos and treated with FGF-2 as described (18.Ryan P.J. Gillespie L.L. Dev. Biol. 1994; 166: 101-111Crossref PubMed Scopus (32) Google Scholar). Explants were cultured for various times then extracted for RNA analysis or scored for mesoderm induction as in Ref 2.Slack J.M.W. Darlington B.G. Heath J.K. Godsave S.F. Nature. 1987; 326: 197-200Crossref PubMed Scopus (652) Google Scholar. Whole mount in situ hybridization with digoxygenin (Roche Molecular Biochemicals)-labeled Xenopus Brachyury (Xbra) orXenopus chordin cRNA was performed as described (20.Yang S. Lockwood A. Hollett P. Ford R. Kao K. J. Biol. Chem. 1998; 273: 13746-13752Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), using maleic acid buffer. The cDNAs for Xenopus Brachyury andchordin were kindly provided by Dr. R. T. Moon (University of Washington). Previously, we demonstrated that the activity of the VT+ and VT− isoforms could be differentially regulated by phosphorylation (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). One obvious question is whether these two isoforms function differently in mesoderm formation in Xenopus embryos. Induction to form mesoderm takes place in the marginal zone cells of the blastula stage embryo, so we began by examining the spatial expression pattern of the VT+ and VT− isoforms during this stage of development. Embryos were dissected into three regions representing the three germ layers: animal (presumptive ectoderm), marginal zone (presumptive mesoderm), and vegetal (presumptive endoderm). RNase protection assays of these three regions revealed that the VT− isoform was expressed predominantly in the marginal zone with very little detectable message in the animal and vegetal regions (Fig.1 B). In contrast, only small differences in VT+ expression were observed in the three regions (Fig.1 B). Although we had previously reported that the VT+ isoform was the major form expressed throughout development and that no change in the ratio of VT+ to VT− isoforms was detected (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), the time intervals used in that study were large in order to cover a broad range of developmental stages. In light of the results in Fig. 1 B, we decided to reexamine the temporal VT+ and VT− expression patterns in the marginal zone, using shorter time intervals and focusing on the stages when mesoderm induction is known to take place. Blastula stage embryos were collected at 0.5-h time intervals, and the VT+ and VT− expression levels in the marginal zone were analyzed by RT-PCR. For this purpose, we employed primers that bracket the VT region and generate VT+ and VT− products that can be distinguished on a sequencing gel. Our analysis revealed that in early blastulae (4.5 h postfertilization; late stage 7), the VT+ isoform was the major form in marginal zone cells (Fig. 1 C, lane 3), consistent with our previous findings (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, 30 min later (stage 8), a dramatic increase in the level of VT− relative to VT+ was observed, such that the VT− isoform became predominant (Fig. 1 C,lane 4). This was quickly followed by a decrease in VT− expression to initial levels, with the VT+ isoform remaining predominant at all subsequent time points examined (Fig. 1 C,lanes 5 and 6). Our previous work demonstrated that these two FGFR1 isoforms arise by alternate use of a 5′ splice donor site during transcription (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). InXenopus embryos, however, zygotic transcription does not begin until midblastula transition (21.Newport J. Kirschner M. Cell. 1982; 30: 675-686Abstract Full Text PDF PubMed Scopus (1190) Google Scholar). This occurs during stage 8, but the precise timing of this developmental event cannot be determined by either the number of cell divisions or the time after fertilization (Ref. 21.Newport J. Kirschner M. Cell. 1982; 30: 675-686Abstract Full Text PDF PubMed Scopus (1190) Google Scholar; reviewed in Ref. 22.Masui Y. Wang P. Biol. Cell. 1998; 90: 537-548Crossref PubMed Scopus (78) Google Scholar). Instead, an increase in elongation factor 1-α expression, one of the earliest transcripts to be expressed by the embryonic genome (23.Krieg P.A. Varnum S.M. Wormington W.M. Melton D.A. Dev. Biol. 1989; 133: 93-100Crossref PubMed Scopus (342) Google Scholar), has been frequently used to indicate that zygotic transcription has begun. We measured elongation factor 1-α levels in our marginal zone samples and determined that expression levels began to increase as early as 5 h (Fig.1 C, lane 4). This demonstrates that the increase in VT− expression takes place concurrent with the onset of zygotic transcription. We investigated the possibility that FGF itself was involved in this switch in expression pattern, since Musci et al. (24.Musci T.J. Amaya E. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8365-8369Crossref PubMed Scopus (113) Google Scholar) and Friesel and Dawid (25.Friesel R. Dawid I. Mol. Cell. Biol. 1991; 11: 2481-2488Crossref PubMed Scopus (68) Google Scholar) have reported that FGFR1 mRNA levels in animal cap explants were regulated by FGF. We cultured blastula stage animal cap explants with FGF-2 in a standard mesoderm induction assay (2.Slack J.M.W. Darlington B.G. Heath J.K. Godsave S.F. Nature. 1987; 326: 197-200Crossref PubMed Scopus (652) Google Scholar) and determined VT+ and VT− expression levels at various times after the addition of FGF-2. VT− expression levels in FGF-2-treated explants increased within 0.5 h, compared with untreated control explants (Fig. 2, 1 and all subsequent time intervals VT− expression levels in explants the as control levels (Fig. 2, VT+ expression levels, on the other These data that a increase in the expression level of the VT− isoform can be by FGF. is however, that FGF induction cannot for the large increase in VT− levels observed in vivo C, lane with Fig. The for the increase in VT− expression in the marginal zone to be The and switch to VT− expression in the presumptive mesoderm that these two FGFR1 isoforms have in early development. We investigated this possibility by examining the effects of overexpressing each isoform. cRNA was microinjected into and embryos were compared with control embryos for their ability to into normal embryos overexpressing the VT− isoform developed of those overexpressing the VT+ isoform developed into normal tadpoles A and B). embryos began to at h was an with embryos to The obvious effect was a in trunk and tail in the tadpoles (Fig. B). This differential effect was not to differential or of the since levels of VT+ and VT− RNA (Fig. and protein (Fig. were detectable at h, the stage when became in the VT+-overexpressing overexpression effects on embryonic development. Embryos from the in Fig. were in and are of embryos injected with diethyl pyrocarbonate-treated H2O FGFR-VT+ and FGFR-VT− The VT+ were to those reported by Amaya et al. (10.Amaya E. Musci T.J. Kirschner M.W. Cell. 1991; 66: 257-270Abstract Full Text PDF PubMed Scopus (927) Google Scholar) in embryos overexpressing a dominant negative FGFR1 These showed that FGFR signaling and that overexpression in embryos severe from a in mesoderm as well as in This that the VT+ from a in mesoderm we investigated the effect of VT+ and VT− overexpression on mesoderm formation in vitro and in vivo. we measured the FGF-2 in explants from embryos microinjected with either VT+ or VT− cRNA and compared with the for explants from embryos. Our results that overexpression of the VT+ isoform reduced the level of mesoderm induction by overexpressing explants required a higher concentration of FGF control explants to 50% induction (Fig. 5 Explants overexpressing the VT− on the other reached 50% induction at a concentration control explants (Fig. 5 overexpression of VT+ to while overexpression of VT− increased of the VT− and VT+ expression levels in these explants revealed that the to FGF was with the relative expression levels of the two isoforms B). The ratio of VT− to VT+ in explants from embryos was that of control explants (Fig. 5 1 and 3), as was the of induced VT+ explants at concentration of FGF (Fig. 5 Explants from embryos, on the other had the ratio of VT− to VT+ (Fig. 5 and the to FGF In of the were induced in the of FGF-2 (Fig. 5 This from of VT− with FGF in animal cap explants (9.Isaacs H.V. Cell. Mol. Life Sci. 1997; 53: 350-361Crossref PubMed Scopus (47) Google Scholar). the results of the one that overexpressing the VT+ isoform in a level of mesoderm formation in vivo. this we examined the expression of an early panmesodermal which is expressed throughout the presumptive mesoderm of the early stage embryo D. B.G. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). Furthermore, FGFR signaling is required for expression (reviewed in S. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). for was not detectable or was very in embryos embryos, on the other expressed levels to those of (Fig. a marker involved in development Y. Steinbeisser H. D. De Robertis E.M. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, Y. Steinbeisser H. De Robertis E.M. Nature. 1995; PubMed Scopus Google Scholar), was in VT+ embryos (Fig. that of expression in embryos was not the of a of transcription. the VT+ isoform can function to mesoderm formation in Xenopus embryos. In this we have that while the VT+ isoform was predominant in the presumptive mesoderm during of blastula a brief but dramatic increase in VT− mRNA expression during this time (Fig. 1 coinciding with mesoderm induction in vivo (9.Isaacs H.V. Cell. Mol. Life Sci. 1997; 53: 350-361Crossref PubMed Scopus (47) Google Scholar). be important to this in VT− expression known FGF signaling pathways in the embryo, such as protein kinase and and C. M. Dev. Biol. 1997; PubMed Scopus Google Scholar) reported that protein kinase activity was detectable during midblastula and by of on the other a of h during early blastula to midblastula stages (18.Ryan P.J. Gillespie L.L. Dev. Biol. 1994; 166: 101-111Crossref PubMed Scopus (32) Google Scholar). activation of these pathways a time that reported in this is that the VT− protein has a its We are to generate that can the VT+ and VT− isoforms and of signaling We have that the VT+ and VT− isoforms differ significantly in their ability to mediate mesoderm This in isoform function could be to PKC PKC is known to be during mesoderm induction by FGF (4.Gillespie L.L. Paterno G.D. Mahadevan L.C. Slack J.M.W. Mech. Dev. 1992; 38: 99-108Crossref PubMed Scopus (20) Google Scholar), and PKC a in FGF signaling the but not the isoform (7.Gillespie L.L. Chen G. Paterno G.D. J. Biol. Chem. 1995; 270: 22758-22763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The differential activity of these two FGFR isoforms that two in the embryo, one predominantly VT− and the other predominantly could be to the concentration of and only the high levels of VT− be Our data then that mesoderm formation in vivo is not only on the concentration of FGF but on the relative expression levels of the VT+ and VT− isoforms in the responding This a mechanism for mesoderm induction to marginal zone all cells in the blastula stage embryo have been to R.A. Musci T.J. Kimelman D. Development. 1995; 121: 2429-2437Crossref PubMed Google Scholar, L.L. Paterno G.D. Slack J.M.W. Development. 1989; 106: 203-208Crossref PubMed Google Scholar). of the work on mesoderm induction has on analysis of such as and (reviewed in Refs. 9.Isaacs H.V. Cell. Mol. Life Sci. 1997; 53: 350-361Crossref PubMed Scopus (47) Google Scholar, Dev. Biol. 1999; PubMed Scopus Google Scholar, and E.M. Melton D.A. Development. 1998; PubMed Google Scholar), our results that regulated expression of receptors and receptor isoforms a We Mercer for

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