Fuentealba LC , Eivers E , Ikeda A , Hurtado C , Kuroda H , Pera EM , De Robertis EM .

HD21502-21 NICHD NIH HHS , R01 HD021502-21A1 NICHD NIH HHS , R01 HD021502 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS

	Figure 1. Smad1 Is Phosphorylated and Inhibited by GSK3(A) Smad1 contains MAPK (blue) and GSK3 (red) phosphorylation sites in its linker region. The PPAY binding site of Smurf1 is boxed and serines 210 and 214 used to raise antibodies indicated by asterisks.(B) Smad1 constructs encoding Smad1 wild-type (SWT) or phosphorylation-resistant mutants for GSK3 (SGM) and MAPK (SMM) sites.(C–F) Injection of SGM or SMM, but not of SWT, mRNA increased expression of the ventral marker sizzled in Xenopus embryos.(G) A BMP-independent phospho-mimetic activated Smad1 (SEVE) in which the SVS terminus was mutated into EVE.(H–J) Activity of SEVE is increased by phosphorylation-resistant linker mutations.(K) GSK3 radioactively phosphorylates Smad1 in vitro, but only when primed by MAPK. The recombinant Smad1 linker substrate was about 90% pure in polyacrylamide gels (data not shown).(L and M) Phospho-specific antibodies for hSmad1 Ser214 (pSmad1MAPK) and Ser210 (pSmad1GSK3 antibody B was used).(N) Phospho-specific antibodies (pSmad1MAPK and pSmad1GSK3-A) demonstrate that GSK3 phosphorylation of recombinant Smad1 requires MAPK priming, in nonradioactive Western blots. Recombinant Smad1 substrate was used in the same amount as in (K).
	Figure 7. Wnt Signaling Induces Epidermis in a Smad1/5/8- and β-Catenin-Dependent, but Tcf3-Independent, Manner(A) LiCl induces epidermis (Cytokeratin) and inhibits neural differentiation (NCAM, Otx2). Radioactive RT-PCR analysis of whole embryos (WE), animal cap (AC) explants, and dissociated animal cap cells at stage 13. MyoD indicates lack of mesoderm induction and Ornithine decarboxylase (ODC) equal loading.(B) Quantitative PCR of dissociated animal caps injected with pCSKA-Wnt8 and pCS2-LRP6 DNA. DN-Smad5 mRNA was coinjected to block Smad1/5/8 activity. Cytokeratin mRNA levels at stage 13 were normalized for ODC mRNA, and the standard deviation from three independent experiments is indicated.(C and C′) Wnt3a protein (60 nl of 16 ng/μl) microinjected into the blastula cavity at stage 9 inhibits anterior neural plate and expands epidermis (n = 40 and 42, respectively).(D and D′) DN-Smad5 converts the entire ectoderm into neural tissue and is epistatic to Wnt3a protein injection (n = 27 and 35). ADMP MO was coinjected to eliminate all traces of epidermis.(E and E′) Dkk1 mRNA expands the neural plate (n = 100 and 53).(F) δN-Tcf3 mRNA eliminates the neural plate; only a ring of Sox2 expression in ventral mesoderm remained (90%, n = 30).(F′) Dkk1 mRNA rescues neural plate in the presence of δN-Tcf3 in 60% of embryos (n = 70).(G and G′) The induction of neural plate by Dkk1 mRNA has a complete requirement for β-Catenin (100%, n = 17 and 55, respectively).(H) Model in which the BMP (D-V) and Wnt (A-P) patterning pathways are integrated at the level of Smad1/5/8 phosphorylations. Black arrows indicate direct protein-protein interactions and blue arrows transcriptional regulation by Smad1/5/8; all interactions are supported by overexpression or morpholino studies in Xenopus (Lee et al., 2006, and data not shown).

Aubert, Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. 2002, Pubmed

Aubert, Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. 2002, Pubmed
Aubin, In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis. 2004, Pubmed
Badano, The centrosome in human genetic disease. 2005, Pubmed
Beck, The role of BMP signaling in outgrowth and patterning of the Xenopus tail bud. 2001, Pubmed , Xenbase
Bilic, Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. 2007, Pubmed
Christian, Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. 1993, Pubmed , Xenbase
Cohen, The renaissance of GSK3. 2001, Pubmed
De Robertis, Dorsal-ventral patterning and neural induction in Xenopus embryos. 2004, Pubmed , Xenbase
De Robertis, Spemann's organizer and self-regulation in amphibian embryos. 2006, Pubmed
Feng, Specificity and versatility in tgf-beta signaling through Smads. 2005, Pubmed
Glinka, Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. 1998, Pubmed , Xenbase
Harland, Neural induction. 2000, Pubmed , Xenbase
Heasman, Patterning the early Xenopus embryo. 2006, Pubmed , Xenbase
Hoppler, BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm. 1998, Pubmed , Xenbase
Huang, A novel role of phospho-beta-catenin in microtubule regrowth at centrosome. 2007, Pubmed
Koay, Genetic disorders of the LRP5-Wnt signalling pathway affecting the skeleton. 2005, Pubmed
Korchynskyi, Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. 2002, Pubmed
Kretzschmar, Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. 1997, Pubmed
Kuroda, Default neural induction: neuralization of dissociated Xenopus cells is mediated by Ras/MAPK activation. 2005, Pubmed , Xenbase
Lee, Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. 2006, Pubmed , Xenbase
Ligon, Dynein binds to beta-catenin and may tether microtubules at adherens junctions. 2001, Pubmed
Liu, Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. 2002, Pubmed , Xenbase
Logan, The Wnt signaling pathway in development and disease. 2004, Pubmed
Marcus, The role of GSK3beta in regulating neuronal differentiation in Xenopus laevis. 1998, Pubmed , Xenbase
Marshall, Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. 1995, Pubmed
Molenaar, XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. 1996, Pubmed , Xenbase
Morvan, Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass. 2006, Pubmed
Niehrs, Regionally specific induction by the Spemann-Mangold organizer. 2004, Pubmed
Nishita, Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer. 2000, Pubmed , Xenbase
Pera, Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. 2003, Pubmed , Xenbase
Reversade, Regulation of ADMP and BMP2/4/7 at opposite embryonic poles generates a self-regulating morphogenetic field. 2005, Pubmed , Xenbase
Sapkota, Balancing BMP signaling through integrated inputs into the Smad1 linker. 2007, Pubmed , Xenbase
Semënov, SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. 2005, Pubmed , Xenbase
Shi, Mechanisms of TGF-beta signaling from cell membrane to the nucleus. 2003, Pubmed
Stern, Neural induction: old problem, new findings, yet more questions. 2005, Pubmed , Xenbase
Tamai, A mechanism for Wnt coreceptor activation. 2004, Pubmed , Xenbase
Wilson, Neural induction: toward a unifying mechanism. 2001, Pubmed
Wilson, Induction of epidermis and inhibition of neural fate by Bmp-4. 1995, Pubmed , Xenbase
Zhu, A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. 1999, Pubmed , Xenbase