Alexandrova EM , Thomsen GH .

R01 HD032429-09 NICHD NIH HHS , R01HD032429 NICHD NIH HHS , R01 HD032429 NICHD NIH HHS

	Fig. 1. Smurf1 transcripts are enriched in the dorsal tissues. (A, A′) Lateral view and sagittal section of early gastrula, stage10 (animal pole up, dorsal side at the right, asterisks mark the dorsal blastopore lip). (B, B′) Early neurula, stage 14. (C, C′) Late neurula, stage 20. In panels B, B′ and C, anterior is at the right, (B′, C) sagittal sections, (C′) transversal section. S—somites, sm—somitogenic mesoderm, black arrowheads—neural folds, black arrow—notochord, white arrow—prechordal plate.
	Fig. 2. Characterization of Smurf1 MO and Smurf1CA effectiveness. (A) Alignment of Smurf1 MO sequence with Xenopus laevis (X.l.) Smurf1, zebrafish Danio rerio Smurf1 (zSmurf1) and two versions of X. laevis Smurf2. The translation start site is in bold. (B, C) The amount of Smurf1 protein produced by in vitro translation (B) or in the embryos injected with Smurf1 mRNA (C) is reduced in the presence of Smurf1 MO, but not control MO. (D–G) Smurf1 MO (F) and Smurf1CA (G) block secondary axis induction by the wild-type Smurf1 (E).
	Fig. 3. Knockdown of endogenous Smurf1 results in neural defects. (A–D) Smurf1 MO (B) and Smurf1CA (D), but not control MO (A) or control β-gal mRNA (C) cause neural tube closure defects at neurula stages (top row) and microcephaly and microphtalmy at tailbud stages (bottom row). This phenotype is mimicked by 1–6 ng Smad1 mRNA overexpression (E). (F–I) F-actin accumulation at the neural fold hinge points at stage 18, revealed by phalloidin staining. Control embryos (F, H) have normal loop-shaped pattern. In Smurf1MO (G) or Smurf1CA embryos (I), f-actin staining is weak and the distance between the lateral hinge points is increased. Scale bar = 0.1 mm.
	Fig. 4. Rescue of Smurf1 MO and Smurf1CA embryos by wild-type Smurf1. (A–C, G) Neural folding defect (B) and overall phenotype of tadpoles caused by Smurf1MO is rescued by 0.1 ng of zSmurf1 mRNA (C, G). (D–F) Smurf1CA embryos are similarly rescued by X. laevis Smurf1.
	Fig. 5. Smurf1 knockdown disrupts neural gene expression. Anterior neural marker Otx2 (A–D), prospective eye marker Pax6 (E–H), rhombomere 3 and 5 marker Krox20 and the marker of mid-hindbrain boundary En2 (I–L) at late neurula stage 18. Pan-neural marker N-CAM at stage 18 (M–P) and stage 14 (Q, R). (S–U) Shh target gene in the neural plate, Nkx2.2, at stage 17. All are anterior views.
	Fig. 6. BMP-responsive genes are up-regulated by blocking Smurf1. (A–D) The zone of Msx1, a marker of the neural plate border, is wider in Smurf1 MO and Smurf1CA embryos (B, D, arrowheads), than in control embryos (A, C). (E–G) Expression pattern of the epidermal marker, Xep, encroaches into the placodal zone (F, arrow), but loses its neural domain (asterisk) in Smurf1CA embryos. Its normal pattern is rescued by wt Smurf1 (G). Pink color demarcates lineage tracing by β-gal. (H–L) Double staining of control, Smurf1 MO and Smurf1CA embryos with Xep (purple, arrows) and Sox2 (cyan) and rescue of the Xep pattern by wt Smurf1 (M) and Smad6 (N). All are anterior views.
	Fig. 7. Mesodermal markers are not affected by blocking Smurf1. General mesodermal marker XBra (A, B), the marker of the prospective head mesoderm Frzb (C, D) and the marker of the Organizer and notochord Chd (E–H) are expressed normally in Smurf1CA embryos. (A–D) Vegetal view, dorsal side up. (E–H) Dorsal view, animal pole up. Representative embryos are shown.
	Fig. 8. A low dose of Smad1 overexpression mimics Smurf1 knockdown. N-CAM staining is slightly reduced (A, B), while Xep (D, arrow) and Msx1 (G, arrowheads) are up-regulated at the periphery of the neural plate in embryos injected with 1 ng Smad1 mRNA. As a positive control, 6 ng Smad1 mRNA strongly induces Xep (E, arrow) and Msx1 (H, arrowheads) within the neural plate. Notice the wider neural plate in all Smad1-overexpressing embryos (B, D, E, G, H). All are anterior views at stage 14 (A, B) or 17 (C–H).
	Fig. 9. A Smad1/5 antagonist, Smad6, rescues the Smurf1 knockdown phenotype. (A, B) Representative stage 18 (A) and stage 17 (B) embryos. (C, D) Summary of Smurf1 MO (C) and Smurf1CA (D) rescue by Smad6 (n—number of embryos).
	Fig. 10. Blocking Smurf1 inhibits response to Chordin and enhances response to BMP. (A, B) Smurf1 MO, but not control MO, blocks neural induction by Chordin in animal caps, as scored by in situ hybridization with N-CAM probe. (A) Combined data from two experiments (n—number of animal caps), (B) representative animal caps from each treatment. (C) Similarly, Smurf1CA suppresses expression of N-CAM and two cement gland markers, XAG1 and Xagr2, in Chordin mRNA-injected animal caps, as measured by quantitative RT-PCR at stage 25. (D) Smurf1CA also inhibits secondary axis induction by Chordin mRNA injected in the ventral marginal zone (n—number of embryos). (E) Likewise, embryos injected with 1 ng Chordin mRNA in the animal pole are strongly dorsalized, which is reversed by co-injection of Smurf1CA mRNA. (F) Quantitative RT-PCR on animal caps injected with BMP4 mRNA, with or without Smurf1CA. Expression of both early (Vent1 and Wnt8, scored at stage 10.5) and late BMP4 target genes (GATA6 and αT4-globin, scored at stage 25) is enhanced by Smurf1CA. Panels C and F were repeated twice on independent cDNA with similar results.
	Fig. 11. Phospho-Smad1 levels are elevated in Smurf1CA embryonic neuroectoderm. Dorsal (prospective neural) ectoderm was cut from stage 10.25 embryos injected with indicated mRNAs and analyzed by Western blotting (A). (B) Quantitation of relative P-Smad1/5 levels summarized from four experiments (n—number of experiments, error bars—standard deviation).

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