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The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from several growth factor receptors to the mitogen-activated protein (MAP) kinase signaling cascade, but its biological function during development is not well characterized. Here, we show that the Xenopus homolog of mammalian SNT1/FRS-2 (XSNT1) plays a critical role in the appropriate formation of mesoderm-derived tissue during embryogenesis. XSNT1 has an expression pattern that is quite similar to the fibroblast growth factor receptor-1 (FGFR1) during Xenopus development. Ectopic expression of XSNT1 markedly enhanced the embryonic defects induced by an activated FGF receptor, and increased the MAP kinase activity as well as the expression of a mesodermal marker in response to FGF receptor signaling. A loss-of-function study using antisense XSNT1 morpholino oligonucleotides (XSNT-AS) shows severe malformation of trunk and posterior structures. Moreover, XSNT-AS disrupts muscle and notochord formation, and inhibits FGFR-induced MAP kinase activation. In ectodermal explants, XSNT-AS blocks FGFR-mediated induction of mesoderm and the accompanying elongation movements. Our results indicate that XSNT1 is a critical mediator of FGF signaling and is required for early Xenopus development.
Figure 2. Whole-mount in situ hybridization analysis of XSNT1 and XFGFR1 during embryogenesis. A: Animal pole view of XSNT1 expression in a stage 12 embryo (gastrulation). B: Dorsal view of XSNT1 expression in a stage 19 embryo (neurulation). C: Dorsal view of XFGFR1 expression in a stage 19 embryo. D: Lateral view of XSNT1 expression in stage 26 embryo. E: Lateral view of XSNT1 expression in stage 30 embryo. F: Lateral view of XFGFR1 expression in stage 30 embryo. Nf, neural folds; Ba, branchial arches; So, somites; Br, fore-, mid-, and hindbrain; Pn, pronephros; Tb, tail bud
Figure 7. XSNT-AS disrupts neural and mesoderm-derived tissues. In situ hybridization of Nrp-1 (pan-neural) transcripts in an uninjected embryo (un; A) and in a XSNT1-AS (80 ng) -injected embryo (AS; B). Immunostaining of muscle (12/101 antibody) in an uninjected embryo (un; C) and an XSNT-AS (80 ng) -injected embryo (AS: D). Immunostaining of notochord (Tor-70 antibody) in an uninjected embryo (un; E) and XSNT-AS (80 ng) -injected embryos (AS; F-H). N, neural tissue; Mu, muscle; Nt, notochord.
Figure 4. XSNT1 enhances XFGFR1-induced phenotypic effects. Embryos were left uninjected (un; A) or were injected with RNAs encoding XSNT1 (0.5 ng; SNT; B), FGFRKE (10 pg; KE; C), XSNT1 (0.5 ng) plus FGFRKE (10 pg; KE+SNT; D), FGFRKD (10 pg; KD; E), or XSNT1 (0.5 ng) plus FGFRKD (10 pg; KD+SNT; F). Photographs were taken at stage 29/31.Download figure to PowerPoint
Figure 6. XSNT-AS disrupts posterior structures. A: Uninjected embryos (un). B: Control morpholino oligonucleotide (80 ng) -injected embryos (Cont). C: XSNT1-AS (80 ng) -injected embryos (AS). D: Dorsal view of severely affected XSNT-AS (80 ng) -injected embryo (AS). E: XSNT-AS (80 ng) plus XSNT four-point mutant RNA (0.1 ng) -injected embryo (AS+SNT 4pt. mut). F: Embryos injected with indicated RNAs and/or oligonucleotides were lysed and immunoblotted with anti-XSNT1 antibody. un, uninjected; SNT, XSNT1 RNA (0.1 ng); SNT+Cont, XSNT1 RNA (0.1 ng) plus control morpholino oligonucleotide (80 ng); SNT+AS, XSNT1 RNA (0.1 ng) plus XSNT-AS (80 ng); SNT 4 pt. mut+AS, XSNT1 RNA with four-point mutations (0.1 ng) plus XSNT-AS (80 ng); SNT 4 pt. mut, XSNT1 RNA with four-point mutations (0.1 ng).Download figure to PowerPoint
