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Members of the fibroblast growth factor (FGF) ligand family play a critical role in mesoderm formation in the frog Xenopus laevis. While many components of the signaling cascade triggered by FGF receptor activation have been identified, links between these intracellular factors and the receptor itself have been difficult to establish. We report here the characterization of Xenopus SNT-1 (FRS2alpha), a scaffolding protein previously identified as a mediator of FGF activity in other biological contexts. SNT-1 is widely expressed during early Xenopus development, consistent with a role for this protein in mesoderm formation. Ectopic SNT-1 induces mesoderm in Xenopus ectodermal explants, synergizes with low levels of FGF, and is blocked by inhibition of Ras activity, suggesting that SNT-1 functions to transmit signals from the FGF receptor during mesoderm formation. Furthermore, dominant-inhibitory SNT-1 mutants inhibit mesoderm induction by FGF, suggesting that SNT-1 is required for this process. Expression of dominant-negative SNT-1 in intact embryos blocks mesoderm formation and dramatically disrupts trunk and tail development, indicating a requirement for SNT-1, or a related factor inhibited by the mutant construct, during axis formation in vivo. Finally, we demonstrate that SNT-1 physically associates with the Src-like kinase Laloo, and that SNT-1 activity is required for mesoderm induction by Laloo, suggesting that SNT-1 and Laloo function as components of a signaling complex during mesoderm formation in the vertebrate.
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Fig. 1. Sequence and expression of Xenopus SNT-1. (A) Alignment of the putative human and Xenopus SNT-1 amino acid sequences. Identical amino acids are shown in filled boxes. The conserved phosphotyrosine binding (PTB) domains are indicated. The location of the six tyrosine residues mutated in 6YF are highlighted (see below). (B) RT–PCR analysis of XSNT-1 expression during development. (C) RT–PCR analysis of XSNT-1 expression in gastrula stage explants (stage 10.5). The ‘-RT’ lane contains all reagents except reverse transcriptase and was used as a negative control. Ornithine decarboxylase (ODC) is used as a loading control (Bassez et al., 1990). Xbrachyury (Xbra) is a panmesodermal marker at this stage (Smith et al., 1991); Chordin is a dorsal mesodermal marker (Sasai et al., 1994); Xwnt8 is a ventrolateral mesodermal marker ( Smith and Harland, 1991 and Christian et al., 1991).
Fig. 2. Effects of SNT-1 misexpression. (A) Top: lateral view of stage 35 embryo injected with 1 ng human SNT-1 RNA in the animal pole. Bottom: lateral view of uninjected sibling embryo. Anterior is to left. SNT-1 injection results in a loss of anterior structures, including eyes, the generation of small, ectopic tail-like structures (arrowheads), and occasional enlargement of the proctodeum. (B) Human SNT-1 induces mesoderm in animal caps. RT–PCR analysis of animal caps dissected at late blastula stages and cultured until midgastrula stages. Animal caps were isolated from embryos injected with SNT-1 RNA at the 2-cell stage, as listed. (C) Xenopus SNT-1 (XSNT-1) induces mesoderm in animal caps. RT–PCR analysis of animal caps dissected at late blastula stages and cultured until midgastrula stages. Animal caps were isolated from embryos injected at the 2-cell stage with 1 ng XSNT-1 RNA.
Fig. 3. SNT-1 functions within a signaling cascade that includes FGF and Ras. (A) SNT-1 activity is inhibited by expression of a dominant inhibitory Ras construct. 1 ng SNT-1 and 1ng dominant inhibitory Ras (dnRas) RNA were injected, as listed. (B) SNT-1 and basic FGF (bFGF) protein synergize in the mesoderm induction assay. 50 pg SNT-1 and 1 ng/ml bFGF were used, as listed. RT–PCR analysis of animal caps cultured until midgastrula stages.
Fig. 4. SNT-1 mutants function as putative dominant-negative reagents. (A) Inhibition of FGF-mediated mesoderm induction by δMT, and subsequent rescue with full-length SNT-1. (B) Inhibition of FGF-mediated mesoderm induction by 6YF, and subsequent rescue with full-length SNT-1. RT–PCR analysis of animal caps cultured until midgastrula stages. 2 ng δMT, 1 ng 6YF, and 1 ng SNT-1 RNA were injected, as listed. 25 ng/ml bFGF was added, as listed.
Fig. 5. The SNT-1 mutant 6YF inhibits mesoderm induction and normal development in the Xenopus embryo. (A) Stage 32 embryos, injected with 500 pg 6YF RNA alone (top panel), co-injected with 500 pg 6YF and 40 pg activated Ras (const. active Ras) RNA, or uninjected. Anterior is to left. Bottom two embryos in top panel are dorsal views, showing the open blastopore remnant; all other views are lateral. (B) β-Gal staining and whole-mount in situ hybridization of midgastrula stage albino embryos using an antisense Xbrachyury probe; vegetal-posterior view. The embryo on the left was injected with 100 pg β-galactosidase RNA in the equatorial region of one blastomere at the 2-cell stage; note overlap between Xbrachyury expression (blue) and the red β-gal product (arrow). The embryo on the right was injected with both 100 pg β-galactosidase RNA and 500 pg 6YF RNA in the same region; note the presence of β-gal product in the gap in Xbrachyury expression (arrow).
Fig. 6. Physical association between SNT-1 and Laloo. (A) Western blot analysis of SNT-1/Laloo co-immunoprecipitation. (B) Yeast 2-hybrid analysis demonstrating interaction between the SH4/SH3 domains of Laloo and SNT-1. Expression vectors for Gal4 DNA binding domain fusion proteins (Laloo or FGFR1 segments) and Gal4 activation domain fusion proteins (SNT-1 or PLCγ-SH2 domain) were co-transformed in yeast and plated onto synthetic medium without leucine/tryptophan to measure co-transformation efficiency or onto medium without leucine/tryptophan/histidine+25 mM 3-amino-1,2,4-triazole (AT) to select clones displaying fusion protein interaction. No proteins tested interacted with the negative control PLCγ fusion protein (not shown). Bottom panel: LalooSH4/3, LalooSH4, and LalooSH3–30 protein are expressed in yeast. Yeast protein extracts were immunoprecipitated with anti-Myc monoclonal antibody and analyzed by Western blot with a monoclonal antibody against the GAL4 DNA binding domain.
Fig. 7. Expression of dominant-negative SNT-1 inhibits mesoderm induction by the activated Laloo mutant Y492F. RT–PCR analysis of animal caps cultured until midgastrula stages. 100 pg Y492F RNA and 1 ng 6YF RNA were injected, as listed.
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