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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.
Abstract
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, Val(423)-Thr(424), 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.
Figure 1
FGFR-VT− expression is spatially and temporally restricted during blastula stage. A, schematic illustrating the 261-nt probe that corresponds to the sequence of the VT+ isoform as well as the VT+ and VT− protected fragments produced by RNase digestion for the analysis shown inB. Digestion of probe:VT+ hybrids results in a 162-nt protected fragment, while digestion of probe:VT− hybrids results in digestion of the 6-nt single strand loop encoding the VT (black box), producing two protected fragments of 107 and 49 nt. The size in nt is listed below each fragment.B, RNase protection analysis of FGFR-VT+ and FGFR-VT− mRNA in Xenopus blastulae. Stage 8 blastulae were dissected into animal, vegetal, and marginal zone regions (as illustrated in the schematic diagram shown above lanes 5–7), as described (14). Total RNA was isolated from each region, and RNase protection analysis was performed using a 32P-labeled 261-nt probe, as in Ref. 7. A representative experiment is shown. Lane 1, probe;lane 2, digested probe; lanes 3–7, protected fragments from in vitro transcribed FGFR-VT+ cRNA, in vitro transcribed FGFR-VT− cRNA, marginal zone cells (M), animal cells (A), and vegetal cells (V), respectively. The positions of the undigested probe (arrow), the VT+ protected fragments (square brackets), and VT− 107-nt protected fragment (arrow) are indicated; the 49-nt VT− protected fragment is not shown. C, RT-PCR analysis of the VT+ and VT− temporal expression pattern in marginal zone cells. Blastula stage embryos were collected at the following postfertilization times: 4.5 h (stage 7), 5.0 h (stage 8), 5.5 h (stage 8), and 6.0 h (stage 8). Marginal zones were dissected, and total RNA was isolated as inB. RT-PCR was performed as described under “Experimental Procedures,” and the VT+ and VT− products, which differ in size by 6 nt, were analyzed by autoradiography on a 6% polyacrylamide, 6M urea sequencing gel. A representative experiment is shown. Amplification products of VT+ cDNA (lane 1) and VT− cDNA (lane 2) mark the position of the VT+ and VT− products (arrows) representing the two isoforms in the marginal zone cells (lanes 3–6). Each marginal zone sample was also amplified using primers for histone (H4), as an input control, and for elongation factor 1-α, as a measure of zygotic transcription.Dev Time, development time.
Figure 2
FGF can stimulate an increase in VT− expression. Animal cap explants were cultured with (+) or without (−) 100 ng/ml FGF-2, and at the time indicated above each lane, RNA was extracted. RT-PCR analysis was performed as in Fig. 1 C. The experiment was performed on four separate occasions, and the increase in VT− expression ranged from 2- to 3-fold. A representative experiment is shown. The positions of the VT+ and VT− isoforms as well as the H4 input control are indicated.
Figure 3
Overexpression of the FGFR-VT+ but not the VT− isoform leads to abnormal development inXenopus. FGFR-VT+ and VT− cRNA was prepared and microinjected into fertilized eggs as described under “Experimental Procedures.” Control embryos (Con) were injected with the same volume of diethyl pyrocarbonate-treated H2O. A, embryos were left to develop for 72 h at room temperature until they reached tadpole stage and then scored for normal development (n); the percentage is based on the total number injected. A total of 150 embryos were used for each experiment, and the averages and S.D. values of 14 individual experiments are shown.B, total RNA (five embryos per treatment) was extracted at 24 h after injection and analyzed by RT-PCR (as described in the legend to Fig. 1) for VT+, VT−, and histone H4 (input control) expression levels. The positions of the VT+, VT−, and H4 PCR products are indicated. C, embryo proteins were labeled by injection of [35S]methionine, and after 24 h, total protein was extracted (100 embryos per sample), immunoprecipitated, and analyzed as described under “Experimental Procedures.” The position of FGFR1 protein is indicated.
Figure 4
FGFR-VT+ overexpression effects on embryonic development. Embryos from the experiment in Fig. 2 were fixed in 10% formalin and photographed. Examples are shown of embryos injected with diethyl pyrocarbonate-treated H2O (control (Con), A), FGFR-VT+ (B), and FGFR-VT− (C). Scale bar, 1.0 mm.
Figure 5
The VT+ and VT− isoforms have differential effects on mesoderm induction in vitro. A, animal cap explants (30 per sample) from stage 8 blastulae of H2O-injected (○), VT−-injected (▪), or VT+-injected (▴) embryos were cultured in the presence of the indicated concentration of FGF-2 for 72 h. Mesoderm induction was scored by morphological criteria as described in Ref. 2. Values from 10 individual experiments were plotted; the bars represent S.E.B, total RNA was extracted from stage 8 animal cap explants (five per sample) from injected embryos and analyzed by RT-PCR for VT+, VT−, and histone H4 expression levels, as described in the legend to Fig. 1 C. A representative experiment is shown. The positions of the VT+, VT−, and H4 PCR products are indicated. Quantitation by densitometry of the VT+ and VT− expression levels in each sample was performed as described in Ref. 17, and the ratio of VT− to VT+ obtained from these measurements is indicated below the appropriate lane.
Figure 6
Overexpression of the VT+ isoform reduces mesoderm formation in vivo. Fertilized eggs were injected as described in the legend to Fig. 2 and cultured until they reached gastrula stage (stage 10.5). Whole mount in situ hybridization was performed as described under “Experimental Procedures,” using a probe for either Xbra (A) or chordin (B). The white arrows indicate regions of expression in the control embryos. Scale bar, 0.25 mm.
