Sater AK et al. (2003), Evidence for antagonism of BMP-4 signals by MAP...

XB-ART-4706

Differentiation 2003 Sep 01;717:434-44. doi: 10.1046/j.1432-0436.2003.7107006.x.

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Evidence for antagonism of BMP-4 signals by MAP kinase during Xenopus axis determination and neural specification.

Sater AK , El-Hodiri HM , Goswami M , Alexander TB , Al-Sheikh O , Etkin LD , Akif Uzman J .

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We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.

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Species referenced: Xenopus laevis
Genes referenced: acta4 bmp4 hoxb9 mapk1 myc ncam1 otx2 smad1 smad2 smad3 smad4 smad6.2 smad7 tbx2 tbxt tubb2b zic3
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	Fig. 1 Disruption of Smad1 consensus MAP kinase phosphorylation sites by site-directed mutagenesis. (A) Consensus MAP kinase phosphorylation sites within the linker domains of Smad family members. Optimal sites (P-X-S/T-P) are shown in bold; minimal sites (S/T-P) are underlined. Single sites are found in the linker domains of Drosophila MAD, as well as vertebrate Smads 2–4 and 6, although Xenopus and human Smad7 lack consensus MAP kinase sites within the proline-rich linker region. Four sites are present in Smads 1 and 5. (Sequences: Drosophila MAD, Sekelsky et al., 1995; Xenopus Smad1, Thomsen, 1996; human Smad1, Liu et al., 1996; Xenopus Smad2, Graff et al., 1996; human Smad2, Lennon et al., 1996; human Smad3, Zhang et al., 1996; rat Smad4, direct submission, GenBank accession number AF056002; human Smad5, Riggins et al., 1996; Xenopus Smad6, Nakayama et al., 1998a; human Smad6, Hata et al., 1998; Xenopus Smad7, Nakayama et al., 1998b; human Smad7, Hayashi et al., 1997). (B) Each of the four MAP kinase consensus phosphorylation sites was altered by conversion of the phosphoacceptor serine to alanine via two-step site-directed mutagenesis (see Methods).
	Fig. 2 Effects of WT- and M4A-Smad1 on dorso-anterior development. Embryos were injected in two dorsal blastomeres at the four-cell stage with either WT- or M4A-Smad1 mRNA. Embryos were scored for dorso-anterior development using the modified dorso-anterior index (DAI) scale, by which 5 represents normal development and 0 represents a completely ventralized embryo. At all concentrations tested, expression of M4A-Smad1 produces more severe axial defects than does overexpression of WT-Smad1 (n53 independent experiments).
	Fig. 3 Loss of dorsal axial development in embryos expressing WT- or M4A-Smad1. Embryos were injected equatorially in two dorsal blastomeres at the four-cell stage with 1 ng mRNA encoding WT- or M4A-Smad1. When controls reached tailbud stages, embryos were fixed and immunostained with the 12/101 antibody. (A) Uninjected control showing labeled somites. (B) Embryos expressing WT-Smad1. In nearly all cases, small amounts of somitic tissue are present (arrows), even in the most ventralized embryos. (C) Embryos expressing M4ASmad1. 12/101 immunoreactivity is rarely detected in embryos ventralized by overexpression of M4A-Smad1 (n54 independent experiments).
	Fig. 4 Ventral defects in embryos expressing WT- or M4A-Smad1. Embryos were injected in the equatorial region of two ventral blastomeres at the four-cell stage with 1 ng mRNA encoding WTor M4A-Smad1. At tailbud stages, embryos were fixed and immunostained with the Tor70 antibody. Ventral and posterior abnormalities are more severe in embryos expressing M4ASmad1 than in those expressing exogenous WT-Smad1. (A) Uninjected control embryo; (B) embryos overexpressing WTSmad1; (C) embryos expressing M4A-Smad1 (n54 independent experiments).
	Fig. 5 Otx2 expression in embryos overexpressing WT- or M4ASmad1. Embryos were injected with 250 pg/embryo in two cells at the four-cell stage with either WT- or M4A-Smad1. Injected embryos were fixed at st. 21 and hybridized in situ to detect otx2 expression. Embryos overexpressing WT-Smad1 show two domains of strong expression across the anterior neural folds (A). In sibling embryos overexpressing M4A-Smad1, otx2 expression in both domains is greatly reduced, and the two stripes that constitute the more dorsal domain are narrowed (B). In addition, neural tube closure is inhibited or delayed in embryos expressing M4A-Smad1.
	Fig. 6 Stability of WT- or M4A-Smad1 proteins in animal caps. (A) Embryos were injected with 1 ng mRNA encoding myc-tagged WTor M4A-Smad1. Animal caps were isolated at late blastula and collected for immunoblots at intervals between late gastrula (st. 12.5) and early tailbud stages. The myc-tagged proteins are easily detected, indicating that both proteins are translated well in vivo. The WT-Smad1 protein, however, accumulates at earlier stages, and persists at high levels longer than does the M4A-Smad1 protein. This effect was consistent across all experiments and multiple preparations of synthetic mRNA (n56 experiments). (B) Linearity of immunoblot detection. Embryos were injected with 1 ng mRNA encoding myc-tagged WT-Smad1 mRNA. Animal caps were isolated at the late blastula stage and lysed when controls reached st. 10.5. A 2-fold serial dilution was prepared from the lysate, and the dilution series was processed for immunoblotting. Band intensities were quantified and compared with predicted values. A representative experiment and quantification are shown.
	Fig. 7 Overexpression of moderate levels of WT- or M4A-Smad1 in animal caps does not lead to mesoderm formation. Embryos were injected with 500 pg mRNA. Animal caps were isolated at midblastula and collected for RT-PCR at mid-neurula. Ectoderm expressing either WT-Smad1 or M4A-Smad1 shows expression of epidermal keratin. These tissues do not express the mesoderm specific gene Xbra or the posterior gene HoxB9.
	Fig. 8 Overexpression of M4A-Smad1 antagonizes neural specification in response to endogenous signals. (A) Embryos were microinjected with 500 pg/embryo of either WT-Smad1, M4ASmad1, or LacZ mRNA. Ectoderm from injected embryos was isolated at st. 10 and recombined with involuted dorsal mesoderm from mid-gastrula embryos. Recombinates were cultured until controls reached mid-neurula stages (15–16) and were harvested for RT-PCR. (B) Although otx2, NCAM, and N-tubulin are strongly expressed in recombinates of ectoderm expressing LacZ or WTSmad1, both otx2 and NCAM levels are reduced, and N-tubulin is not detected, in recombinates expressing M4A-Smad1 (n58 experiments). we, whole embryo (lane 1); Lac, recombinates of LacZ-expressing ectoderm (lane 2); mes, dorsal mesoderm only (lane 3); WT, recombinates of ectoderm overexpressing WT-Smad1 (lane 4); M4, recombinates of ectoderm expressing M4A-Smad1 (lane 5).