Das S , Chang C .

R01 GM083029 NIGMS NIH HHS

Xla Wt + Hsa.smurf2 (fig.6.f)
Xla Wt + smurf1 (fig.2)
Xla Wt + smurf1 (fig.3.b)
Xla Wt + smurf1 (fig.4.a)
Xla Wt + smurf1 (fig.4.b)
Xla Wt + smurf1 (fig.5.b)
Xla Wt + smurf1 + smurf2 (fig.2)
Xla Wt + smurf1 + smurf2 (fig.3.b)
Xla Wt + smurf1 + smurf2 (fig.5.b)
Xla Wt + smurf1 MO (fig.10.b)
Xla Wt + smurf1 MO (fig.7)
Xla Wt + smurf1 MO (fig.8.b)
Xla Wt + smurf1 MO (fig.9.a)
Xla Wt + smurf1 MO (fig.9.b)
Xla Wt + smurf1 MO + smurf2 MO (fig.10.b)
Xla Wt + smurf1 MO + smurf2 MO (fig.10.b)
Xla Wt + smurf1 MO + smurf2 MO (fig.7)
Xla Wt + smurf1 MO + smurf2 MO (fig.7)
Xla Wt + smurf2 (fig.2)
Xla Wt + smurf2 (fig.3.a)
Xla Wt + smurf2 (fig.3.b)
Xla Wt + smurf2 (fig.4.a)
Xla Wt + smurf2 (fig.4.b)
Xla Wt + smurf2 (fig.5.a)
Xla Wt + smurf2 (fig.5.b)
Xla Wt + smurf2 (fig.6.c)
Xla Wt + smurf2 MO (fig.10.a)
Xla Wt + smurf2 MO (fig.10.b)
Xla Wt + smurf2 MO (fig.6.f)
Xla Wt + smurf2 MO (fig.7)
Xla Wt + smurf2 MO (fig.8.a)
Xla Wt + smurf2 MO (fig.8.b)
Xla Wt + smurf2 MO (fig.8.b)
Xla Wt + smurf2 MO (fig.8.b)
Xla Wt + smurf2 MO (fig.9.a)
Xla Wt + smurf2 MO (fig.9.a)
Xla Wt + smurf2 MO (fig.9.a)
Xla Wt + smurf2 MO (fig.9.b)
Xla Wt + smurf2 MO (fig.9.b)
Xla Wt + smurf2 MO (fig.9.b)

	Fig. 1. Expression pattern of Smurf2 during early Xenopus development. Whole mount in situ hybridization shows that Smurf2 transcripts are uniformly localized to the ectodermal and mesodermal regions in blastula (a,b, stage-9 embryos with animal and vegetal views, respectively) and early gastrula (c, stage-10 vegetal view with the arrow pointing to the blastopore lip) embryos. At mid to late gastrula stages, its expression is enhanced in the dorsal region (d, e, stage-11 and -12 embryos, posterior view with dorsal side on top; and f, stage-12 embryo lateral view; D: dorsal; V, ventral). During neurulation, Smurf2 is seen in the neural plate and the migration neural crest cells (g, h, dorsal and anterior views respectively; NC, neural crest). At the tailbud to tadpole stages, Smurf2 transcripts are detected in the neural tissues, the olfactory and otic vesicles, eyes, migrating neural crest, notochord, and tail (i, lateral view with the head on the left side). Br, brain; Nt, notochord; Ol, Olfactory placode; Ot, otic vesicle.
	Fig. 2. Overexpression of Smurfs in the ectoderm induces enlarged or ectopic cement gland and enlarged head. RNAs encoding Smurf1 or 2 were injected into the animal region of early frog embryos and the resulting tailbud embryos are shown here. The numbers indicate the embryos displaying the phenotype.
	Fig. 3. Elevation of Smurf levels affects neural and neural crest development. A: Increased levels of Smurf2 led to expansion or ectopic expression of the neural genes Sox2, NRP1, and Pax6 and reduction of the epidermal marker XK70, but could both up- and down-regulate the neural plate border genes Slug and Pax3. B: Smurf1 and Smurf2 function similarly to control neural and neural crest development. Together they facilitated expansion of the neural genes but reduction of the neural crest marker Slug. The numbers of the embryos displaying the shown changes in marker expression are indicated.
	Fig. 4. Smurf1 and Smurf2 play different roles in mesodermal development. A: Increased Smurf1 expression in dorsal mesoderm results in dorsal-anteriorization of the embryos (46/72 embryos), which often also show anterior neural tube closure defects (in 18 embryos; arrow points to the open brain). In contrast, increased Smurf2 expression in the dorsal mesoderm leads to disruption of axial structures, with embryos frequently displaying gastrulation defects (73/85 embryos with gastrulation defects; among these, 32 embryos had split dorsal structures characterized as the open back phenotype). B: Ectopic expression of Smurf1 in the VMZ induces a partial secondary dorsal axis with the trunk tissues (40/59 embryos; yellow arrowheads); whereas ectopic expression of Smurf2 in the VMZ results in induction of a partial secondary axis that often contains cement gland and hatching gland (36/44 embryos with an ectopic axis, and 25 of these contained cement gland; red arrowheads).
	Fig. 5. Smurf1 and Smurf2 differentially regulate mesodermal markers at gastrula stages. A: Overexpression of Smurf2 in either DMZ or VMZ disrupts expression of the pan-mesodermal marker Brachyury (XBra). Dorsal expression of Smurf2 slightly reduces the dorsal mesoderm marker chordin, whereas ventral expression of Smurf2 inhibits the ventral-lateral mesodermal marker wnt8. B: Unlike Smurf2, Smurf1 does not block, but instead mildly expands, the expression of XBra or chordin, though like Smurf2, it also inhibits wnt8 expression. Co-expression of Smurf1 and 2 leads to reduction of all three mesodermal markers. The numbers indicate the embryos displaying the shown changes in marker expression.
	Fig. 6. Smurf2 antisense morpholino oligonucleotides (Smurf2-MO) acts specifically to block translation and function of Xenopus Smurf2. A: Smurf2 antibody recognizes Xenopus Smurf2, but not Smurf1. B: Smurf2-MO inhibits translation from the RNA encoding Xenopus Smurf2 (XSmurf2), but has no effect on translation of human Smurf2 (hSmurf2). C: Smurf2-MO blocks induction of a partial secondary axis by XSmurf2, but not by hSmurf2. D: Smurf2-MO reduces endogenous Smurf2 protein levels at the gastrula stages. E: Smurf1-MO does not regulate the protein level of Smurf2. F: Ectodermal expression of Smurf2-MO induces head defects, which are partially rescued by low doses of co-expressed hSmurf2.
	Fig. 7. Smurf1 and Smurf2 are both required for normal head development in early Xenopus embryos. Knockdown of either Smurf led to malformation of the head in the resulting tadpoles, with Smurf1 morphants displaying a more severe phenotype. Depletion of both Smurfs exacerbated the head defects, suggesting that the two Smurfs have redundant activities in regulation of head development. The numbers indicate the embryos showing the phenotype.
	Fig. 8. Smurfs regulate ectodermal marker expression. A: Knockdown of Smurf2 slightly reduced the neural marker expression and expanded the epidermal marker XK70, but the neural plate border genes Slug and Pax3 could be both up- and down-regulated in the morphants. B: Knockdown of Smurf1 resulted in slightly reduced Sox2 in a wider domain and decreased Pax6 expression; Slug was also predominantly reduced (10/54 and 35/54 embryos with enhanced and decreased Slug expression, respectively). Depletion of both Smurf1 and Smurf2 caused reduction and disorganization of the Sox2 domain, and decreased expression of both Pax6 and Slug (8/50 and 36/50 embryos, respectively, with enhanced and reduced Slug in 25ng combination of both MOs, and no embryos showed higher expression of Slug in double morphants with 50 ng combination of the MOs).
	Fig. 9. Smurf1 and Smurf2 control mesendodermal development in early Xenopus embryos. A: Knockdown of Smurf1 in dorsal tissues resulted in reduction of axial structures and malformation of the head, which was often accompanied by a delay or failure in neural tube closure (32 out of the 58 embryos with the shown phenotype had neural tube closure defects, arrow). In contrast, knockdown of Smurf2 led to enlargement of the endodermal mass in the embryos. When both Smurfs were knocked down, the embryos developed with severe axial defects, microcephaly, and failure in gastrulation. B: Knockdown of either Smurf1 or Smurf2 in the VMZ region induced tail truncation, though the phenotype was more pronounced in the Smurf1 morphants. Double knockdown of both Smurfs aggravated the tail defects. The numbers indicate the embryos showing the depicted phenotype.
	Fig. 10. Smurf1 and Smurf2 differentially regulate mesodermal marker expression. A: Knockdown of Smurf2 in the marginal zone region caused expansion of the pan-mesodermal marker XBra towards the ectodermal region. Dorsal knockdown of Smurf2 reduced expression of chordin,whereas ventral knockdown of Smurf2 led to expansion of wnt8 into the ectodermal domain. B: Unlike Smurf2 knockdown, Smurf1 knockdown reduced the expression of both XBra and chordin, and double knockdown of both Smurfs resulted in more efficient reduction of these markers. The numbers indicate the embryos displaying the shown changes of marker expression.

Alexandrova, Smurf1 regulates neural patterning and folding in Xenopus embryos by antagonizing the BMP/Smad1 pathway. 2006, Pubmed, Xenbase

Alexandrova, Smurf1 regulates neural patterning and folding in Xenopus embryos by antagonizing the BMP/Smad1 pathway. 2006, Pubmed , Xenbase
Amaravadi, Autonomous neural axis formation by ectopic expression of the protooncogene c-ski. 1997, Pubmed , Xenbase
Aybar, Early induction of neural crest cells: lessons learned from frog, fish and chick. 2002, Pubmed , Xenbase
Baker, The origins of the neural crest. Part I: embryonic induction. 1997, Pubmed , Xenbase
Barembaum, Early steps in neural crest specification. 2005, Pubmed , Xenbase
Bhushan, Smad7 inhibits mesoderm formation and promotes neural cell fate in Xenopus embryos. 1998, Pubmed , Xenbase
Bonni, TGF-beta induces assembly of a Smad2-Smurf2 ubiquitin ligase complex that targets SnoN for degradation. 2001, Pubmed
Burn, Regulation of early Xenopus development by the PIAS genes. 2011, Pubmed , Xenbase
Casellas, Xenopus Smad7 inhibits both the activin and BMP pathways and acts as a neural inducer. 1998, Pubmed , Xenbase
Chang, Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation. 2007, Pubmed , Xenbase
Chang, Neural crest induction by Xwnt7B in Xenopus. 1998, Pubmed , Xenbase
Chang, Cell fate determination in embryonic ectoderm. 1998, Pubmed , Xenbase
Cheng, Phosphorylation of E3 ligase Smurf1 switches its substrate preference in support of axon development. 2011, Pubmed
De Robertis, Dorsal-ventral patterning and neural induction in Xenopus embryos. 2004, Pubmed , Xenbase
Ebisawa, Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. 2001, Pubmed
Fukunaga, Smurf2 induces ubiquitin-dependent degradation of Smurf1 to prevent migration of breast cancer cells. 2008, Pubmed
Harland, Neural induction. 2000, Pubmed , Xenbase
Harland, In situ hybridization: an improved whole-mount method for Xenopus embryos. 1991, Pubmed , Xenbase
Harland, Formation and function of Spemann's organizer. 1997, Pubmed
Hata, Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor. 1998, Pubmed , Xenbase
Hayashi, The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. 1997, Pubmed
Imamura, Smad6 inhibits signalling by the TGF-beta superfamily. 1997, Pubmed
Inoue, Regulation of TGF-beta family signaling by E3 ubiquitin ligases. 2008, Pubmed
Kalkan, Tumor necrosis factor-receptor-associated factor-4 is a positive regulator of transforming growth factor-beta signaling that affects neural crest formation. 2009, Pubmed , Xenbase
Kavsak, Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. 2000, Pubmed , Xenbase
Kong, Smurf2-mediated ubiquitination and degradation of Id1 regulates p16 expression during senescence. 2011, Pubmed
LaBonne, Neural crest induction in Xenopus: evidence for a two-signal model. 1998, Pubmed , Xenbase
Lin, Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-beta signaling. 2000, Pubmed
Liu, Ski/Sno and TGF-beta signaling. 2001, Pubmed
Luo, Ski and SnoN: negative regulators of TGF-beta signaling. 2004, Pubmed
Massagué, Smad transcription factors. 2005, Pubmed
Massagué, Controlling TGF-beta signaling. 2000, Pubmed
Massagué, TGF-beta signal transduction. 1998, Pubmed
Morén, Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases. 2005, Pubmed
Murakami, Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads. 2003, Pubmed , Xenbase
Nakao, Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. 1997, Pubmed , Xenbase
Nakayama, Xenopus Smad8 acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning. 1998, Pubmed , Xenbase
Narimatsu, Regulation of planar cell polarity by Smurf ubiquitin ligases. 2009, Pubmed
Nie, Regulation of Xenopus gastrulation by ErbB signaling. 2007, Pubmed , Xenbase
Piccolo, The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. 1999, Pubmed , Xenbase
Sahai, Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility. 2007, Pubmed
Saint-Jeannet, Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a. 1997, Pubmed , Xenbase
Schier, Nodal signaling in vertebrate development. 2003, Pubmed
Schwamborn, Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity. 2007, Pubmed
Seufert, Expression of a novel Ski-like gene in Xenopus development. 2005, Pubmed , Xenbase
Shi, Mechanisms of TGF-beta signaling from cell membrane to the nucleus. 2003, Pubmed
Soond, How ubiquitination regulates the TGF-β signalling pathway: new insights and new players: new isoforms of ubiquitin-activating enzymes in the E1-E3 families join the game. 2011, Pubmed
Tan, Smad ubiquitination regulatory factor-2 in the fibrotic kidney: regulation, target specificity, and functional implication. 2008, Pubmed
Tang, Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. 2011, Pubmed
Tian, Binding of RhoA by the C2 domain of E3 ligase Smurf1 is essential for Smurf1-regulated RhoA ubiquitination and cell protrusive activity. 2011, Pubmed
Vonica, An obligatory caravanserai stop on the silk road to neural induction: inhibition of BMP/GDF signaling. 2006, Pubmed , Xenbase
Whitman, Nodal signaling in early vertebrate embryos: themes and variations. 2001, Pubmed , Xenbase
Wu, Overexpression of Smurf2 stimulates endochondral ossification through upregulation of beta-catenin. 2008, Pubmed
Wu, Smurf2 induces degradation of GSK-3beta and upregulates beta-catenin in chondrocytes: a potential mechanism for Smurf2-induced degeneration of articular cartilage. 2009, Pubmed
Xia, The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response. 2010, Pubmed
Yamashita, Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation. 2005, Pubmed
Zhang, Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase. 2001, Pubmed , Xenbase
Zhao, E3 ubiquitin ligase Smurf1 mediates core-binding factor alpha1/Runx2 degradation and plays a specific role in osteoblast differentiation. 2003, Pubmed
Zhu, A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. 1999, Pubmed , Xenbase