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. Author manuscript; available in PMC: 2011 Dec 22.
Published in final edited form as: Mech Dev. 2001 Dec;109(2):195–204. doi: 10.1016/s0925-4773(01)00524-x

SNT-1/FRS2α physically interacts with Laloo and mediates mesoderm induction by Fibroblast Growth Factor

Joanne Hama 1, Hong Xu 2,#, Mitchell Goldfarb 2, Daniel C Weinstein 1,*
PMCID: PMC3244880  NIHMSID: NIHMS334182  PMID: 11731233

Abstract

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 (FRS2α), 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.

Keywords: Mesoderm, Xenopus, SNT-1, FRS2α, Laloo, FGF

1. Introduction

Understanding the processes that lead from a fertilized egg to the formation of the germ layers, and ultimately to the adult body plan, is a central goal of embryology. All tissues in the animal derive from the three primary germ layers: ectoderm, endoderm, and mesoderm. Endodermal derivatives contribute to the organs of the gut, while ectodermal derivatives form the epidermis and central nervous system. The mesodermal germ layer plays a pivotal role in organizing the vertebrate body axes, and itself gives rise to the muscular, skeletal, and circulatory systems.

In the vertebrate embryo, mesoderm is first observed at the initiation of gastrulation, a process by which the mesodermal germ layer spreads between the outer ectodermal and inner endodermal layers. In the frog, Xenopus laevis, factors secreted from the vegetal pole induce mesoderm in the cells of the adjacent marginal zone. Members of both the Transforming Growth Factor-β (TGF-β) and Fibroblast Growth Factor (FGF) ligand families play critical roles in this process (Klein and Melton, 1994). Addition of FGF or members of the Activin/Nodal-related class of TGF-βs will induce mesoderm in explants of competent ectoderm, while inhibition of the signaling cascades downstream of these factors blocks mesoderm formation in vivo (Slack, 1994; Harland and Gerhart, 1997). The primary mesoderm-inducing signal secreted by the vegetal pole is thought to be a TGF-β ligand, the zygotic expression of which is initiated by the actions of the transcription factor VegT (Clements et al., 1999; Kofron et al., 1999). Although FGF has also been proposed as a primary inducer, the expression patterns and activities of Xenopus FGFs are more consistent with a model in which FGF pathway activation is required for the proper response to and maintenance of mesoderm induction. It is clear, however, that FGF signaling is essential for the proper formation and patterning of trunk and tail mesoderm in Xenopus.

Signaling downstream of the TGF-β receptors is mediated by the intracellular Smad proteins, which provide a relatively direct route from the cell surface to the nucleus, involving few intermediates (Massague, 1998). Signaling through the FGF receptor tyrosine kinase, however, is mediated by a complex transduction cascade involving a number of diverse molecules, including SHP2, PI3K, Grb2, Laloo, and components of the Ras/Raf/MAPK cascade (Whitman and Melton, 1992; MacNicol et al., 1993; LaBonne et al., 1995; Gotoh et al., 1995; Umbhauer et al., 1995; Northrup et al., 1995; Tang et al., 1995; Weinstein et al,. 1998; Gupta and Mayer, 1998; Carballada et al., 2000). Although studies have demonstrated a requirement for all of these factors in FGF-mediated mesoderm induction, the molecular interactions underlying this requirement remain, in several cases, poorly defined. While studies in other biological systems can be used to infer functional relationships between these proteins during development, several components of this pathway have not been well-characterized in other contexts; a notable example of such a molecule is the Src-like kinase Laloo (Weinstein et al., 1998).

A physical link between FGF receptor and the intracellular transduction machinery has only recently been identified. Suc1-associated Neurotrophic Target-1 (SNT-1, also termed FRS2α) interacts with the juxtamembrane domain of FGF receptor-1 (FGFR1) via a phosphotyrosine-binding (PTB) domain on SNT-1 (Xu et al., 1998; Ong et al., 2000; Dhalluin et al., 2000). This interaction allows for FGF-induced, receptor-mediated SNT tyrosine phosphorylation, which in turn enables SNT-1 to bind downstream targets, including the Grb2 adaptor in association with the Ras activator Sos (Wang et al., 1996; Ong et al., 1996; Kouhara et al., 1997) and the SHP2 tyrosine phosphatase (Ong et al., 1997; Hadari et al., 1998). Certain FGF-induced biological responses in cultured cells have been shown to be mediated or potentiated by SNT-1 (Kouhara et al., 1997, Hadari et al., 1998; Xu and Goldfarb, 2001). However, no function for SNT-1 or the related SNT-2 during development has been demonstrated to date.

We describe here the characterization of SNT-1 function during early vertebrate embryogenesis. We first show that ectopic SNT-1 induces mesoderm in Xenopus animal cap explants, and that SNT-1 strongly synergizes with FGF in this context. We then demonstrate that dominant inhibitory SNT-1 constructs inhibit mesoderm induction by FGF, and block trunk and tail formation in intact embryos, indicating that SNT-1 is required for FGF-mediated mesoderm induction, and for normal development, in vivo. Finally, we demonstrate that SNT-1 physically interacts with Laloo through the latter’s SH3 and SH4 domains, and show that inhibition of SNT-1 activity blocks Laloo function. These results provide important insights into Laloo’s role within the signaling cascade triggered by FGF receptor activation.

2. Results

2.1 Isolation of Xenopus SNT-1

In order to identify Xenopus SNT-1, we performed a database search with the full-length human SNT-1 cDNA. Several expressed sequence tags were identified in Xenopus with high homology to the amino and carboxy-termini of human SNT-1. Polymerase Chain Reaction (PCR) strategies were then used to isolate a full-length clone from a gastrula stage cDNA library (Weinstein et al., 1998). This cDNA encodes a putative open reading frame of 509 amino acids, and shares high identity (80%) with mammalian SNT-1; homologous regions include a phosphotyrosine binding domain (PTB), as well as six conserved tyrosine residues that function as putative docking sites for Grb2 and SHP2 (Kouhara et al., 1997; Hadari et al., 1998; Xu and Goldfarb, 2001); these residues are equivalently spaced in the Xenopus clone and in mammalian SNT-1 (Fig. 1A). Because of the high degree of conservation between the Xenopus cDNA and SNT-1, and because this clone shares significantly less identity (42%) with the related SNT-2, we believe that this cDNA encodes the Xenopus homolog of SNT-1 (XSNT-1).

Fig. 1.

Fig. 1

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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; Christian et al., 1991).

2.2 Distribution of SNT-1 in Xenopus embryos

In order to determine the temporal range of XSNT-1 expression during early development, we performed reverse transcription polymerase chain reaction (RT-PCR) on RNA from early embryos. Mesoderm induction in Xenopus occurs between cleavage and early gastrula stages; XSNT-1 is expressed throughout this period (Figure 1B). XSNT-1 is expressed maternally, as transcripts are present prior to the initiation of zygotic transcription at stage 8.5. Strong expression of XSNT-1 is maintained throughout neurula and tailbud stages, and persists through early tadpole stages (stage 35). Whole-mount in situ hybridization studies did not reveal tissue-restricted localization of XSNT-1 through tailbud stages (data not shown). The ubiquitous early expression of intracellular components of both TGF-β– and FGF-stimulated mesoderm induction pathways have been described previously (Lagna et al., 1996; Weinstein et al., 1998; Song et al., 2001). To confirm that XSNT-1 is expressed in the cells of the presumptive mesoderm, we performed RT-PCR analysis on explants from gastrula stage embryos. As shown in Figure 1C, XSNT-1 transcripts are present throughout the gastrula embryo, including in the cells of the dorsal and ventral marginal zone, the regions from which the mesoderm is derived. Thus, XSNT-1 is expressed in a manner consistent with a role for this factor in mesoderm formation.

2.3 SNT-1 activity in embryos and explants

As an initial assay for the function of SNT-1 in Xenopus, we injected synthetic SNT-1 RNA into the animal pole of early cleavage stage embryos. Due to initial difficulties in isolating a full-length Xenopus SNT-1 clone, human SNT-1 cDNA was used for these and most of the subsequent functional studies. Misexpression of SNT-1 throughout the ectoderm results in a range of abnormalities, including a reduction of anterior structures, and the generation of ectopic tail-like structures (Fig. 2A). Dorsal misexpression of SNT-1 results in a loss of anterior structures, with no ectopic tails, in 75% of injected embryos (N=28). Ventral misexpression of SNT-1 results in ectopic tail formation in 22% of injected embryos that survive to tailbud stages (N=23); no loss of anterior structures are observed in these embryos. The high lethality of ventral SNT-1 injection (approximately 50% at 1ng) may account for the low penetrance of the tail duplication phenotype. These anterior truncations and posterior duplications resemble those seen following misexpression of several FGF pathway components, including eFGF, Xbrachyury, Laloo, and Gli2, although SNT-1 RNA appears to generate a higher number of smaller ectopic tails per embryo than do these other reagents (Pownall et al., 1996; Tada et al., 1997; Weinstein et al.,1998; Brewster et al., 2000; JH and DCW, unpublished observations).

Fig. 2.

Fig. 2

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Effects of SNT-1 misexpression. (A) top: Lateral view of stage 35 embryo injected with 1ng 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.

To better define the function of SNT-1 during embryogenesis, we tested its activity in an ectodermal explant (animal cap) assay. At midgastrula stages, RT-PCR analysis revealed that SNT-1-injected caps express both the panmesodermal marker Xbrachyury (Xbra) and the ventrolateral mesodermal marker Xwnt8 in a dose-dependent fashion (Fig 2B; Smith et al., 1991; Smith and Harland, 1991; Christian et al., 1991). Subsequent studies revealed that misexpression of Xenopus SNT-1 (XSNT-1) results in a similar induction of Xbra and Xwnt8 in animal caps (Fig. 2C). These data indicate that misexpression of SNT-1 induces ventrolateral mesoderm in competent ectoderm; a similar mesodermal fate is induced following addition of FGF protein (Klein and Melton, 1994; Fig. 4).

Fig. 4.

Fig. 4

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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.

2.4 SNT-1 functions as a component of an FGF-receptor activated pathway

Studies in mammalian cells have shown that SNT-1 functions to mediate signaling by the FGF receptor. To determine whether SNT-1 induces mesoderm by a similar route, we performed epistasis studies using a dominant-inhibitory Ras construct. Ras blockade has been shown to inhibit all mesoderm induction by FGF, but not the induction of Xwnt8 by TGF-β pathway reagents (Whitman and Melton, 1992; LaBonne and Whitman, 1994; Cornell and Kimelman, 1994). Dominant-inhibitory Ras completely blocks both Xbra and Xwnt8 expression by SNT-1, indicating that SNT-1 induces mesoderm through a Ras-dependent pathway (Fig. 3A, compare lanes 1 and 2).

Fig. 3.

Fig. 3

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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.

As further support for the hypothesis that SNT-1 and FGF induce mesoderm through a single molecular cascade, we assayed for synergy between SNT-1 and basic FGF (bFGF). As shown in Figure 3B, 50 pg of SNT-1 RNA does not induce mesodermal marker gene expression in gastrula stage animal caps (Fig. 3B, lane 1); similarly, 1 ng/mL bFGF protein induces little or no expression of Xwnt8 and Xbra (Fig. 3B, lane 3). bFGF treatment of animal caps derived from embryos injected with 50 pg SNT-1 RNA, however, results in a dramatic induction of ventrolateral mesoderm (Fig. 3B, lane 4). These data suggest that SNT-1 and FGF induce mesoderm through a conserved molecular pathway.

2.5 Inhibition of SNT-1 blocks mesoderm induction by FGF and disrupts axis formation in vivo

As mentioned above, SNT-1 contacts the FGF receptor via a PTB domain that is conserved between Xenopus and mammals. Studies in cell culture suggest that an SNT-1 construct containing only the N-terminal myristoylation site and PTB domain might serve as a dominant-inhibitory reagent, competing with endogenous SNT-1 for binding to the FGF receptor, and thus preventing phosphorylation and activation of SNT-1 (H. Xu, unpublished data). We thus decided to assay the effects of expression of this construct (ΔMT) on mesoderm formation. Expression of ΔMT in animal caps does not induce mesoderm, indicating that regions of SNT-1 3′ of the PTB domain are required for its activity (Fig. 4A, lane 1). More important, expression of ΔMT dramatically inhibits mesoderm induction by bFGF (Fig. 4A, compare lanes 2 and 3). This inhibition is rescued by co-expression of full-length SNT-1, demonstrating the specificity of inhibition by ΔMT (Figure 4A, compare lanes 3 and 4). These results indicate that a C-terminally truncated SNT-1 can function as a dominant-negative molecule, and demonstrate that SNT-1 activity, or that of a similar molecule inhibited by ΔMT, is required for mesoderm induction by FGF.

SNT-1 contains six tyrosine residues that are phosphorylated following treatment with FGF; these residues serve as docking sites for the Grb2 adaptor protein and the SHP2 phosphatase (Kouhara et al., 1997; Hadari et al., 1998, Xu and Goldfarb, 2001). We next examined whether an unphosphorylatable SNT-1 mutant construct might also inhibit signaling through the wild-type protein. To this end, we used 6YF, an SNT-1 construct in which these six tyrosine residues were mutated to phenylalanine (Xu and Goldfarb, 2001). 6YF RNA does not induce mesoderm in animal caps, indicating a requirement for the six tyrosine residues in mesoderm induction by SNT-1 (Fig. 4B, lane 1). As with ΔMT, expression of 6YF inhibits mesoderm induction by bFGF (Fig. 4B, compare lanes 2 and 3); this inhibition is relieved by co-expression of full-length SNT-1, demonstrating the specificity of inhibition by 6YF (Figure 4B, compare lanes 3 and 4). These data provide additional support for a model in which SNT-1 is a required component of the cascade mediating mesoderm induction by FGF.

In order to determine whether SNT-1 plays a role in Xenopus development in vivo, we attempted to inhibit SNT-1 activity in the presumptive mesoderm of intact embryos. Towards this end, we injected 6YF into the equatorial region of early cleavage stage embryos. Marginal zone expression of 500 pg 6YF RNA results in a failure of blastopore closure and a complete loss of tail structures in 49% of injected embryos (Fig. 5A; N=53). Higher doses of 6YF produced greater phenotypic penetrance, but were prohibitively toxic. This phenotype is similar to that seen following injection of other reagents that disrupt signaling downstream of the FGF receptor (Amaya et al., 1991; Whitman nd Melton, 1992; MacNicol et al., 1993; LaBonne et al., 1995; Gotoh et al., 1995; Umbhauer et al., 1995; Amaya and Kroll, 1996; Weinstein et al., 1998; Brewster et al., 2000; Carballada et al., 2001). Co-expression of activated Ras partially or completely rescues axis formation in a fraction of these embryos: 23% of these embryos lack tails and show failure of blastopore closure (Fig. 5A; N=53); rescue could not be achieved by co-expression of full-length SNT-1, due to the toxicity of 6YF and SNT-1 in intact embryos. These results indicate that SNT-1, or a related molecule inhibited by 6YF, is essential for normal development of the body axis.

Fig. 5.

Fig. 5

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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 100pg 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 100pg 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).

To test directly whether SNT-1 is required for mesoderm formation in Xenopus, embryos injected with 6YF RNA were assayed for Xbrachyury expression by whole-mount in situ hybridization. At midgastrula stages, Xbrachyury is normally expressed throughout the marginal zone; equatorial injection of β-galactosidase RNA, used as a lineage trace, has no effect on Xbrachyury expression (Smith et al., 1991; Fig. 5B). Co-injection of β-galactosidase and 6YF RNA in one of two blastomeres at the two-cell stage, however, effectively inhibits Xbrachyury expression at the site of injection (Fig. 5B). These results suggest that SNT-1 activity is required for mesoderm formation, in vivo.

2.6 Physical association between SNT-1 and the Src-like kinase Laloo

SNT-1 contains several docking sites for the SHP2 tyrosine phosphatase, the activity of which is required for mesoderm induction by FGF (Tang et al., 1995); cell culture studies suggest that SHP2 functions upstream of the Ras/MAPK cascade (Shi et al., 2000). An attractive target for this phosphatase is a tyrosine residue found near the C-terminus of the Src-like kinase Laloo (Weinstein et al., 1998). To examine whether SNT-1 might serve as a link between Laloo and SHP2, we assayed for physical association between SNT-1 and Laloo. RNA encoding epitope-tagged SNT-1 and Laloo constructs were expressed in blastula stage embryos; as shown in Figure 6A, immunoprecipitation of epitope-tagged Laloo co-precipitates exogenous SNT-1. While this result does not prove that endogenous Laloo and SNT-1 form a stable complex, it does suggest that these proteins may physically interact in vivo.

Fig. 6.

Fig. 6

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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 Myc-tagged Gal4 DNA binding domain fusion proteins (Laloo or FGFR1 segments) and Gal4 activation domain fusion proteins (SNT-1 or PLCγ-SH2 domain) were cotransformed in yeast and plated onto synthetic medium without leucine/tryptophan to measure cotransformation efficiency or onto medium without leucine/tryptophan/histidine + 25mM 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.

In order to determine which region of Laloo mediates its association with SNT-1, we used a yeast two-hybrid system. For these studies, we co-expressed pACT-SNT-1, encoding full-length SNT-1 fused to the GAL4 activation domain, and pGBKT7 derivative plasmids expressing Laloo segments fused to the GAL4 DNA binding domain. As a positive control, pACT-SNT-1 was co-transfected with pAS-FR1-JM1, expressing the juxtamembrane region of FGF Receptor-1 previously shown to bind SNT-1 (Fig. 6B; Xu et al., 1998). As domain-swapping studies in our lab suggested that the SH3 domain was a critical determinant of specificity among active Src family kinases, we first co-expressed full-length SNT-1 with LalooSH4/3, a construct that contained only the SH4 (“unique”) and SH3 domains of Laloo (aa 1-117). LalooSH4/3 demonstrated a clear interaction with SNT-1 (Fig. 6B); this construct did not form colonies when co-expressed with a control activation domain construct (pGAD-PLCγ-SH2; data not shown). We next co-expressed SNT-1 with either LalooSH4, a construct that contains only the SH4 domain of Laloo, or LalooSH3-30, a construct that differs from LalooSH4/3 only in the removal of amino acids 1-25 of Laloo. Surprisingly, neither LalooSH4 nor LalooSH3-30 interacted with SNT-1 (Fig. 6B). These results suggest that the physical association between Laloo and SNT-1 requires both the SH3 and SH4 domains of Laloo.

2.7 SNT-1 activity is required for Laloo function

To address the functional relevance of the interaction between Laloo and SNT-1, an epistasis study was performed in animal caps. Our previous work has shown that wild-type Laloo activity is dependent on basal signaling through the FGF receptor, and is thus sensitive to inhibition at any point within this auto-activating pathway; the activated Laloo construct Y492F, however, is only sensitive to “downstream” inhibition (Weinstein et al., 1998). Y492F induces the expression of both Xbra and Xwnt8 in gastrula stage animal caps (Fig. 7, lane1; Weinstein et al., 1998). Co-expression of 6YF potently inhibits mesoderm induction by Y492F (Fig. 7, compare lanes 1 and 2). This result indicates that SNT-1 is required for mesoderm induction by the activated Laloo construct, and suggests that SNT-1, or another molecule associated with or downstream of SNT-1, is a target of Laloo kinase activity.

Fig. 7.

Fig. 7

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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.

3. Discussion

FGF receptor-mediated signal transduction plays a critical role during mesoderm formation in the vertebrate, and is required for the correct formation of the trunk and tail in Xenopus embryos. In this paper, we demonstrate that the scaffolding protein SNT-1 is an essential component of this signaling pathway. Gain-of-function studies indicate that SNT-1 activity is sufficient for mesoderm induction and tail formation, as misexpression of SNT-1 induces mesoderm in animal cap explants and generates ectopic tail-like duplications in intact embryos. Furthermore, SNT-1 synergizes with FGF in animal caps, and mesoderm induction by SNT-1 is blocked by inhibition of FGF signaling; these results strongly suggest that SNT-1 and FGF function as components of a linked cascade. Finally, loss-of-function studies indicate that SNT-1 function is necessary both for FGF-mediated mesoderm induction, and for the formation of the trunk and tail. This work provides the first demonstration of an in vivo requirement for SNT-1.

A potential limitation of this study is our use of human, rather than Xenopus, SNT-1 for many of our non-descriptive studies. The high identity between human and Xenopus SNT-1, however, suggests that similar results would be expected with Xenopus SNT-1 (XSNT-1) in gain-of-function or protein interaction studies. In fact, our experiments have confirmed that ectopic XSNT-1 induces mesoderm in animal cap explants (Fig. 2C). Furthermore, we have demonstrated that XSNT-1 is present in the marginal zone of the gastrula stage embryo, consistent with a role for XSNT-1 protein during mesoderm formation. The most compelling validation of our studies with human SNT-1, however, stems from our analysis of the dominant-negative SNT-1 constructs. These reagents, which presumably target endogenous SNT-1, are shown to inhibit the same molecular pathway that is activated by misexpression of wild-type SNT-1. Thus, it is highly likely that our studies reflect the role of endogenous SNT proteins during vertebrate development.

We have demonstrated that FGF-mediated mesoderm formation requires the activity of SNT-1, and that SNT-1 acts in part through a physical and functional interaction with Laloo. Figure 8 presents a revised model of FGF signaling that adds our new findings to the previous body of literature. Studies in mammalian cell culture have shown that SNT-1 undergoes FGF-dependent tyrosine phosphorylation mediated by a direct physical association between the receptor and SNT (Xu et al., 1998; Ong et al., 2000). SNT-1 tyrosine phosphorylation creates docking sites for Grb2/Sos and SHP2 (Kouhara et al., 1997; Hadari et al., 1998; Xu and Goldfarb, 2001), both of which are required for mesoderm induction by FGF through their activation of the Ras/MAPK pathway (Tang et al., 1995; Gupta and Mayer, 1998). Previous studies have shown that Laloo is essential for FGF-induced mesoderm formation and have suggested that activation of Laloo by the FGF receptor is dependent upon the dephosphorylation of Laloo tyrosine 492 (Weinstein et al., 1998). As our epistasis data presented here place SNT-1 downstream of Laloo, we suggest that Laloo kinase activity contributes to the initial or sustained tyrosine phosphorylation of SNT-1. Laloo may be activated for this task by the SHP2 phosphatase recruited to the SNT-1/Laloo complex: although epistasis studies have demonstrated a role for SHP2 downstream of or in parallel to Laloo, they do not preclude additional involvement of SHP2 upstream of Laloo (Weinstein and Hemmati-Brivanlou, 2001). Laloo activation may also require the downregulation of the Laloo inhibitor Csk (Song et al, 2001). The sustained phosphorylation of SNT-1 mediated by FGF receptor and Laloo would, in turn, allow for continued engagement of SHP2 and Grb2/Sos on SNT, resulting in sustained Ras/MAPK activation required to drive mesoderm differentiation.

Fig. 8.

Fig. 8

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Model depicting the role of SNT-1 and Laloo in FGF signaling during early vertebrate development. Arrows do not necessarily indicate direct molecular interactions. See text for details.

4. Experimental procedures

4.1 RNA preparation, explant dissection, and cell culture

RNA was synthesized in vitro in the presence of cap analog using the mMessage mMachine kit (Ambion). Microinjection, explant dissection and dissociation cultures were performed as described (Hemmati-Brivanlou and Melton, 1994; Wilson and Hemmati-Brivanlou, 1995).

4.2 RT-PCR

RT-PCR was performed as described (Wilson and Hemmati-Brivanlou, 1995; Weinstein et al., 1998). Primers designed specifically for this study are as follows: XSNT-1-U: 5′-AGGAGGTTCGTGTTTCCAG XSNT-1-D: 5′-GTTTCTCTGTGTTCATCGC All other primer sequences are as described (Weinstein et al., 1998; Hemmati-Brivanlou and Melton, 1994).

4.3 Preparation of SNT-1 and Laloo constructs

6YF is a Myc-tagged human SNT-1 mutated from tyrosine to phenylalanine at all six phosphorylation sites (Xu and Goldfarb, 2001). ΔMT is a Myc-tagged human SNT-1 with deletion of residues 138-508 (courtesy of Kyung Lee). 6YF, ΔMT, and wild-type human SNT-1 were subcloned into the BamHI and NotI sites of pCS2.

4.4 Whole-mount in situ hybridization and β-gal staining

Automated in situ detection was performed using the InSituPro (ABiMED). Protocols for whole-mount in situ hybridization were derived from Harland (1991), with the following changes: 1) RNase steps were eliminated; 2) BM purple AP substrate (Boehringer Mannheim) replaced BCIP/NBT. The antisense Xbra probe was synthesized in the presence of digoxygenin-11-UTP (Boehringer Mannheim). For β-gal staining, fixed embryos were incubated with magenta-gal substrate (Anatrace) prior to in situ hybridization, as described in Smith and Harland (1991).

4.5 Xenopus co-immunoprecipitation assay

5ng of Laloo-Flag and/or SNT-1-Myc RNA were injected into the animal poles of early cleavage stage embryos. Laloo-Flag includes the sequence DYKDDDDK at the Laloo C-terminus (Song et al., 2001), while SNT-1-Myc includes a triple-Myc epitope tag at the SNT-1 C-terminus (Xu et al., 1998). Embryos were lysed at stage 8 in 500ul MW Lysis Buffer (25mM Tris pH 7.5, 0.1M NaCl, 0.5% Triton X-100), and centrifuged twice at 14,000K; following each centrifugation, only the clear lysate was retained. Lysates were incubated overnight at 40C with 1:500 antibody (Anti-flag M2 monoclonal or anti-Myc-tag monoclonal 9E10 (Sigma)), followed by incubation with Protein A/G-PLUS-Agarose (Santa Cruz Biotechnology) for 1 hour at 40C. After 4 washes in MW Lysis Buffer, protein was eluted in 0.1M Glycine, pH 3.5, neutralized with wash buffer (0.05M Tris, pH 7.4, 0.15M NaCl), and subjected to standard SDS-PAGE and Western Blotting protocols.

4.6 Yeast two hybrid assay and vector construction

LalooSH4/3, LalooSH4, and LalooSH3-30 were constructed by PCR. LalooSH4/3 includes Laloo amino acids 1-117; LalooSH4 includes Laloo amino acids 1-55; LalooSH3-30 includes Laloo amino acids 26-117. PCR products were cloned into the EcoRI and BamHI sites of pGBKT7, which also contains the Gal4 DNA-binding domain (Clontech). pACT-SNT-1, expressing human SNT-1 residues 2-508 fused to the GAL4 activation domain, and pAS-FR1-JM1, expressing the juxtamembrane region of mouse FGFR1 fused to the GAL4 DNA binding domain, were previously described (Xu et al., 1998). The yeast two hybrid assay was performed as previously described (Xu et al., 1998).

Acknowledgements

The authors thank Y. Song for excellent technical assistance, and K.W. Lee for providing the SNT-ΔMT construct. This work was supported by an Irma T. Hirschl Career Scientist Award, by the Speaker’s Fund for Biomedical Research: Toward the Science of Patient Care, awarded by the City of New York, and by the AMDeC Foundation of New York City, through its “Tartikoff/Perelman/EIF Fund for Young Investigators in Women’s Cancers,” and by PHS grant R01-GM61671 (all awarded to D.C.W.) and by PHS grant R01-GM59432 (to M.G.)

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