A GEF activity-independent function for nuclear Net1 in Nodalsignal transduction and mesendoderm formationShi Wei, Guozhu Ning, Linwei Li, Yifang Yan, Shuyan Yang, Yu Cao and Qiang Wang*
ABSTRACTNet1 is a well-characterized oncoprotein with RhoA-specific GEFactivity. Oncogenic Net1, which lacks the first 145 amino acids, ispresent in the cytosol and contributes to the efficient activation ofRhoA and the formation of actin stress fibers in a number of tumor celltypes. Meanwhile, wild-type Net1 is predominantly localized in thenucleus at steady state due to its N-terminal nuclear localizationsequences, where the function of nuclear Net1 has not been fullydetermined. Here, we find that zebrafish net1 is expressedspecifically in mesendoderm precursors during gastrulation.Endogenous Net1 is located in the nucleus during early embryonicdevelopment. Gain- and loss-of-function experiments in zebrafishembryos and mammalian cells demonstrate that, regardless of itsGEF activity, nuclear Net1 is critical for zebrafish mesendodermformation and Nodal signal transduction. Detailed analyses of proteininteractions reveal that Net1 associates with Smad2 in the nucleus ina GEF-independent manner, and then promotes Smad2 activation byenhancing recruitment of p300 (also known as EP300) to thetranscriptional complex. These findings describe a novel geneticmechanism by which nuclear Net1 facilitates Smad2 transcriptionalactivity to guide mesendoderm development.
INTRODUCTIONMembers of the transforming growth factor β (TGF-β) superfamily,including Nodal and bone morphogenetic proteins (BMPs), are keyplayers in embryonic development and homeostasis (Massagué,2008; Wu and Hill, 2009). The Nodal subfamily of these growthfactors [Squint (Sqt) and Cyclops (Cyc) in zebrafish (also known asNdr1 and Ndr2, respectively); Nodal in mammals] are secretedmorphogens that form concentration gradients required formesendoderm induction and patterning (Feldman et al., 1998;Gritsman et al., 1999; Zhou et al., 1993). These ligands bind andactivate heteromeric complexes of type I and II transmembranereceptors, which in turn phosphorylate intracellular mediators, suchas Smad2 and Smad3. Phosphorylated Smad2 and Smad3 can thenform complexes with Smad4 and translocate into the nucleus toregulate transcription of target genes (Schier and Shen, 2000;Schmierer and Hill, 2007).While this signal transduction pathway is
well-characterized and seemingly straightforward, how it is spatio-temporally modulated during embryonic development remainsunclear.
By using ChIP-chip assays, we previously identified severalhundreds of genes directly targeted by Nodal signaling, such as theproto-oncogene neuroepithelioma transforming gene 1 (NET1) (Liuet al., 2011). Net1, the protein product of NET1, was originallyisolated from neuroepithelioma cells and acts as a RhoA-specificguanine nucleotide exchange factor (GEF) to enhance cancer cellmigration and invasion (Alberts and Treisman, 1998; Chan et al.,1996; Murray et al., 2008; Tu et al., 2010). In addition, this gene isexpressed in a dorsoventral-gradient in the blastoderm margin at theonset of gastrulation and has important functions during earlyembryonic development (Liu et al., 2011). Net1-mediated RhoAactivation is required for gastrulation movements during embryonicdevelopment of Xenopus and chicken (Miyakoshi et al., 2004;Nakaya et al., 2008), and, in zebrafish (Danio rerio), Net1 and itsGEF activity are indispensable for dorsal cell fate specificationthrough promotion of maternal β-catenin activation (Wei et al.,2017). TGF-β- and Nodal-regulated genes usually participate infeedback modulation of signal transduction (Liu et al., 2016;Nicklas and Saiz, 2013; Stroschein et al., 1999). The expression ofzebrafish net1 in the blastoderm margin, where mesendodermprecursors are located, is regulated by Nodal signaling (Liu et al.,2011; Wei et al., 2017). However, whether Net1 is involved inNodal signal regulation and mesendoderm induction remainsunknown.
As a typical Dbl family GEF, Net1 has a catalytic Dbl homology(DH) domain and an adjacent pleckstrin homology (PH) domainflanked by N- and C-terminal extensions (Alberts and Treisman,1998). Two nuclear localization sequences (NLS) in the N-terminusfunction to localize Net1 predominantly to the nucleus (Schmidtand Hall, 2002). Deletion of the N-terminal or mutating the NLSresults in translocation of Net1 from the nucleus to the cytosol,which is critical for RhoA activation and filamentous actinformation (Qin et al., 2005; Schmidt and Hall, 2002). Recent datahave demonstrated that enzymatically inactive Net1 can bind tocomponents of the CARD11–BCL10–MALT1 (CBM) complexand modulate nuclear factor (NF)-κB transcriptional activity,suggesting a GEF activity-independent role for Net1 (Vessichelliet al., 2012). Nuclear Net1 also activates RhoA and RhoB inresponse to DNA damage-associated stimulation (Dubash et al.,2011; Srougi and Burridge, 2011). Furthermore, our previous studyuncovered that both nuclear and cytoplasmic Net1 enhanceβ-catenin S675 phosphorylation in a GEF activity-dependentmanner (Wei et al., 2017). These observations raise interestingquestions concerning the developmental functions of nuclear Net1,and suggest the need for further investigation.
In this study, the functions of nuclear Net1 were characterizedusing various gain- and loss-of-function experiments in bothzebrafish embryos and mammalian cells. These studies revealed thatReceived 11 April 2017; Accepted 31 July 2017
State Key Laboratory of Membrane Biology, CASCenter for Excellence in MolecularCell Science, Institute of Zoology, University of Chinese Academy of Sciences,Chinese Academy of Sciences, Beijing 100101, China.
Net1 localizes to the nucleus during gastrulation, where it associateswith Smad2 in a GEF-independent manner, and ultimatelyfacilitates p300 (also known as EP300) recruitment to thetranscriptional complex to promote Smad2 activation andmesendoderm induction.
RESULTSZebrafish net1 is essential for mesendoderm formationIn gastrulating embryos, zebrafish net1 transcripts were present inthe dorsal organizer and lateral marginal region wheremesendoderm progenitors originate, and then became enriched inthe axial mesoderm at the mid-gastrulation stage (75% epiboly)(Fig. 1A) (Wei et al., 2017), suggesting that net1 might take part inmesendoderm formation. We previously generated two zebrafishnull mutant lines named net1Δ20 and net1Δ53, in which anintegrated compensatory network is activated to buffer against theloss of net1 (Wei et al., 2017). Therefore, we disturbed net1expression in embryos by introducing a splice-blocking morpholino(net1MO1) whose efficiency and lack of off-target effects had beenvalidated (Wei et al., 2017). Knockdown of net1 using 4 ng MO1resulted in a notable decrease in the expression of mesoderm markergenes goosecoid (gsc) and no tail a (ntla) at the shield and 75%epiboly stages (Fig. 1B,C). Meanwhile, endoderm marker geneexpression (sox32 and sox17) was also significantly reduced in net1morphants (Fig. 1D). These results suggest that net1 is critical formesendoderm formation in zebrafish embryos.Importantly, the formation of the notochord [as indicated by the
expression of ntl and sonic hedgehog a (shha)], a derivative of theaxial mesoderm, was severely reduced and ruptured in net1morphants (Fig. 1E). Likewise, the expression of marker genes inendodermal derivatives, including liver bud, pancreas rudiment andpharyngeal pouches, was almost abolished in net1-depletedembryos (Fig. 1F). These data indicate that the mesendodermformation defects in net1 morphants were not caused by adevelopmental delay.Shortly after the mid-blastula transition, maternal β-catenin
activates the transcription of the Nodal family member sqt toinduce mesendodermal fate at the margin of embryos (Bellipanniet al., 2006; Zorn and Wells, 2009). We previously published thatzebrafish Net1 is essential for dorsal axis specification throughpromotion of maternal β-catenin activation (Wei et al., 2017). Inaddition, net1 morphants exhibit a much smaller organizer at shieldstage and ventralized phenotypes at later stages, including a variablyreduced head size and shortened body axis, which partiallyresemble Nodal mutants (Wei et al., 2017). To confirm thatmesendoderm defects in net1 morphants are not secondary effectsof Wnt/β-catenin signaling inhibition, we examined the role of net1in Wnt signaling-deficient embryos generated by injecting one-cell-stage embryos with 50 pg ΔN-tcf3 mRNA, which encodes adominant negative form of Tcf3 (Molenaar et al., 1996). Asexpected, introducing the ΔN-tcf3 mRNA significantly decreasedmesoderm and endoderm formation, which was restored uponreintroduction of the Nodal signal by co-injecting 3 pg tar* mRNAthat encodes a constitutively active form of the Nodal type I receptor(Fig. 1G). Meanwhile, embryonic knockdown of net1 decreasedTar*-induced expression of mesendoderm marker genes (Fig. 1G).Therefore, net1 is required for mesendodermal cell fate specificationin a Wnt/β-catenin signaling-independent manner.Next, we sought to exclude possible off-target effects of net1MO
injection during mesendoderm formation. Because injection of net1mRNA leads to very early embryonic lethality (Wei et al., 2017), anantisense photo-cleavable morpholino targeting the N-terminal Flag
sequence of Flag-net1 mRNA (AS-Flag-photo-MO) was used toinhibit translation in the very early embryo. To examine theexpression of mesendoderm marker genes, one-cell-stage embryoswere co-injected with a mixture of net1 MO1, AS-Flag-photo-MOand Flag-net1mRNA (denoted net1 3Mix) and exposed to UV lightto turn on net1 expression at the sphere stage. As shown in Fig. 1H,reintroduction of net1 expression in morphants was sufficient toreverse the mesendoderm defects, suggesting the specificity of net1MO1. Taken together, these results demonstrate an indispensablerole for net1 in mesendoderm development.
Net1 promotes Nodal signaling during mesendodermformationBecause depletion of Net1 suppressed Nodal receptor-inducedmesendoderm induction (Fig. 1G), we expanded our analysis toembryos overexpressing Nodal ligand. As expected, injection of sqtmRNA significantly enlarged the regions of mesoderm andendoderm marker gene expression. However, upon co-injection ofthe net1 MO1, this Nodal ligand-induced mesendoderm expansionwas notably reduced (Fig. 2A,B). These results suggest that net1 isessential for mesendoderm induction through promoting Nodalsignal activity. Furthermore, embryos overexpressing net1 exhibitedectopic expression of mesoderm and endoderm markers comparedto what was seen in the wild-type control, which was eliminated bytreating with the Nodal signal-specific inhibitor SB431542(Fig. 2C). In addition, the expression of fscn1a and cyclops (cyc),the genes directly targeted by the Nodal signal (Liu et al., 2016),was reduced in shield-stage net1 morphants (Fig. 2D). Therefore,Net1 is potentially a positive regulator of Nodal signal transductionduring mesendoderm formation.
To confirm the role of Net1 in Nodal signal transduction, theeffects of Net1 on the expression of ARE-luciferase, a TGF-β/Nodal-responsive reporter, were investigated in mammalian cellsand zebrafish embryos. In HeLa cells, Net1 overexpressionupregulated the luciferase activity of this reporter in a dose-dependent manner (Fig. 2E). In addition, the luciferase activity ofthe reporter was also significantly increased in zebrafish embryosoverexpressing Net1 (Fig. 2F). Conversely, Net1-deficient embryosdisplayed a greater reduction in reporter gene expression (Fig. 2G).Taken together, these results suggest that Net1 promotesmesendoderm formation through upregulating the Nodal signal.
Nuclear Net1 potentiates the Nodal signal independently ofits GEF activityPrevious work has revealed that epitope-tagged Net1 localizesmainly in the nucleus of Xenopus embryonic cells (Miyakoshiet al., 2004). To examine the subcellular localization ofendogenous Net1 during embryonic development, nuclear andcytoplasmic proteins were extracted from zebrafish embryos andexamined by western blotting. It was observed that endogenousNet1 was primarily in the nucleus during different developmentalstages of zebrafish embryogenesis (Fig. 3A). It has been shownthat TGF-β stimulates the transfer of Net1 from the nucleus to thecytoplasm in human retinal pigment epithelial cells (Lee et al.,2010). Therefore, we determined whether Nodal signalingsimilarly influences Net1 translocation in zebrafish embryos.Surprisingly, despite a moderate increase in Net1 expression insqt mRNA-injected embryos, the subcellular localization of Net1did not change upon Nodal signal activation (Fig. 3B). The Nodalsignal is specifically activated in the blastoderm margin (Bennettet al., 2007), suggesting that the other cells in zebrafish gastrulasare deficient in this signal. Since the cytoplasmic Net1 was almost
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undetectable in wild-type embryos (Fig. 3A,B), Net1 israrely present in the cytoplasm in the absence of Nodal signal.Thus, the increased nuclear Net1 proteins in sqt mRNA-injectedembryos might be the result of the upregulated expression of net1,
as it is a direct Nodal-targeted gene (Liu et al., 2011; Wei et al.,2017), and not the nuclear translocation of cytoplasmic Net1induced by Nodal signal. Taken together, these data reveal apredominant nuclear localization of Net1 during early embryonic
Fig. 1. net1 is crucial for mesendoderm formation in zebrafish embryos. (A) Visualization of net1 expression in zebrafish embryos at shield and 75% epiboly(ep) stages using whole-mount in situ hybridization. Shield stage, lateral views with dorsal to the right; 75% epiboly stage, dorsal views with animal pole to the top.(B–F) The expression of mesendoderm marker genes and mesendoderm derivative marker genes in cMO- and net1 MO1-injected embryos at the indicatedstages. (B–D) Shield stage, animal views with dorsal to the right; 75% epiboly stage, dorsal views with animal pole to the top. (E,F) Bud stage, and 24 and 36 hpf.All images are shown in dorsal views with anterior to the top. (G) Wild-type embryos were injected with 50 pg ΔN-tcf3 mRNA alone or co-injected with 3 pg tar*mRNA and 4 ng net1 MO1, and then collected at the shield and 75% epiboly stages for whole-mount in situ hybridization with a gsc or sox17 probe.(H) Overexpression of net1 rescues mesendoderm induction defects in net1morphants. Wild-type embryos were injected with cMO or net1 3Mix (4 ng net1MO1,200 pg Flag-net1 mRNA and 1 ng AS-Flag-photo-MO), and then a subset of net1 3Mix-injected embryos were exposed to UV light at the sphere stage. Theexpression of gsc and sox17 was examined by in situ hybridization at shield and 75% epiboly stages, respectively. In B–H, the percentages (mean±s.d.) of theaffected embryos are shown as calculated from three independent biological repeats with ∼15–30 embryos in each group. UIC, uninjected control.
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development, which is not changed in response to Nodal signalstimulation.Next, it was determined whether nuclear Net1 is responsible
for Nodal signal transduction and mesendoderm induction.First, the effects of nuclear and cytoplasmic Net1 on theactivity of ARE-luciferase reporter were examined in HeLacells upon expression of Net1-NLS and Net1-ΔN NESconstructs, which are restricted to the nucleus and cytoplasm,respectively (Wei et al., 2017). Interestingly, Net1-NLS
potentiated luciferase activity equally to or better than thewild-type protein, while Net1-ΔN NES did not affect reportergene expression (Fig. 3C), suggesting nuclear Net1, rather thancytosolic Net1, enhances the TGF-β signal in mammalian cells.Next, we tested the influence of Net1 subcellular localizationon mesendoderm development in zebrafish embryos. As shownin Fig. 3D, nuclear, but not cytosolic, Net1 clearly promotedmesendoderm formation. Furthermore, the mesendodermdefects in net1 morphants were almost totally eliminated by
Fig. 2. Net1 upregulates Nodal signal transduction. (A,B) Knockdown of net1 decreases Nodal ligand-induced mesendoderm expansion. Wild-type embryoswere injected with 1 pg sqtmRNA alone or concurrently with 4 ng net1MO1 at the one-cell stage, harvested at the shield and 75% epiboly stages, and analyzedfor the expression of indicated marker genes by using whole-mount in situ hybridization. (C) Zebrafish embryos were injected with net1 2Mix(300 pg Flag-net1mRNA and 1 ng AS-Flag-photo-MO) at the one-cell stage. A subset of these embryos was treated with 25 μMSB431542 (SB) from the 64-cellstage, of which a portion was exposed to UV light at the sphere stage. The expression of gsc and sox17 was examined by in situ hybridization at the shield and75% epiboly stages, respectively. (D) The expression of Nodal target genes cyc and fscn1a in cMO- and net1 MO1-injected embryos at shield stage. Lateralview with animal pole to the top. In A–D, the percentages (mean±s.d.) of the affected embryos are shown as calculated from three independent biologicalrepeats with ∼15–25 embryos in each group. UIC, uninjected control. (E,F) Overexpression of Net1 enhanced ARE-luciferase expression. (E) HeLa cells weretransfected with the ARE-luciferase reporter concurrently with increasing amounts of Net1. At 36 h post transfection, the cells were treated with TGF-β1 or leftuntreated overnight, and then harvested for luciferase assays. *P<0.05; NS, not significant (Student’s t-test). (F) At the one-cell stage, zebrafish embryoswere injected with the ARE-luciferase reporter alone or together with net1 2Mix. At the sphere stage, half of the net1 2Mix-injected embryos were exposed to UVlight, and then all embryos were harvested at the shield stage for luciferase assays. *P<0.05 (Student’s t-test). (G) Knockdown of net1 inhibits ARE-luciferaseexpression. At the one-cell stage, zebrafish embryos were co-injected with the ARE-luciferase reporter and cMO or net1 MO1, and then harvested at theshield stage for luciferase assays. **P<0.01 (Student’s t-test). Results in E–G are mean±s.d. (n=3).
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overexpression of the nuclear but not cytosolic Net1 (Fig. 3E,F), indicating a critical requirement of the nuclear Net1 duringzebrafish mesendoderm formation.
To investigate whether Net1 GEF activity is necessary for Nodalsignal transduction, we examined two GEF-deficient zebrafish Net1mutants named as Net1-L266E and Net1-W437L, which contain
Fig. 3. See next page for legend.
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point mutations in the DH and PH domains, respectively (Wei et al.,2017). HeLa cells were co-transfected with the ARE-luciferasereporter and either wild-type Net1 or one of the Net1 mutants. Uponexamination of the lysates from these transfected cells, the cellsexpressing Net1-L266E or Net1-W437L displayed similar TGF-β1-induced levels of ARE-luciferase expression to the wild-type Net1(Fig. 3G). Moreover, nuclear Net1 (Net1-NLS) and its GEFmutants(Net1-NLS L266E and Net1-NLS W437L) contributed equally tothe expression of the reporter gene in HeLa cells and mesendodermformation in zebrafish embryos (Fig. 3H,I). This clearly indicatesthat nuclear Net1 enhances Nodal signaling and mesendodermformation independently of its GEF activity.
Net1 upregulates the Nodal signal in parallel with ordownstream of Smad2Next, we determined which step of Nodal signal transduction isregulated by Net1. Because Net1 overexpression increased TGF-β1-induced ARE-luciferase expression and net1 knockdown inembryos decreased sqt-activated mesendoderm formation(Fig. 2A,B,E), the regulation of Nodal signal by Net1 is unlikelyto occur at the ligand level. Next, it was tested whether Net1 affectsNodal signaling at the receptor level. A constitutively active form ofTGF-β type I receptor ALK5 (CA-ALK5; ALK5 is also known asTGFBR1) was used to activate ARE-luciferase expression in HeLacells. Net1 overexpression augmented this activation (Fig. 4A).Meanwhile, the enhanced expression of gsc and sox17 in zebrafishembryos mediated by constitutively activated Nodal type I receptortar* was eliminated by the introduction of net1 MO1 (Fig. 4B).These results rule out the possibility that Net1-mediated regulationof TGF-β/Nodal signal acts at the receptor level.
By using the same strategy as above, we found that constitutivelyactive Smad2 (CA-Smad2)-induced expression of the reporter geneand mesoderm and endoderm markers were notably changed byNet1 overexpression or depletion (Fig. 4C,D). In addition, neitherectopic wild-type Net1 nor its GEF-deficient mutants affectedSmad2 nuclear translocation and phosphorylation in response toTGF-β1 stimulation in HeLa cells (Fig. 4E,F). Taken together, theseresults suggest that Net1 acts in parallel with or downstream ofSmad2.
Net1 interacts with nuclear Smad2 in a GEF-activity-independent mannerSince Net1 acts in parallel with or downstream of Smad2, it washypothesized that Net1 interacts with Smad2. To test this, epitope-tagged Net1 and Smad2 were overexpressed in HEK293T cells andco-immunoprecipitations were performed, revealing a physicalinteraction between Net1 and Smad2 (Fig. 5A,B). As HeLa cells aremore responsive to TGF-β stimulation than HEK293T cells, weexamined the association between Net1 and Smad2 in HeLa cells inthe absence or presence of TGF-β1.We found that overexpressedNet1was able to interact with endogenous Smad2 (Fig. 5C). Interestingly,this Net1–Smad2 association was enhanced by TGF-β1 treatment(Fig. 5D). Consistent with this, endogenous Net1 in zebrafish gastrulastage embryos could be co-immunoprecipitated with an anti-Smad2antibody, indicating that Net1 and Smad2 form a complex in vivo, andthe endogenous Net1–Smad2 interaction was obviously strengthenedin sqt mRNA-injected embryos (Fig. 5E). Since Net1 is mainlylocated in the nucleus (Figs 4E and 5F) and has a higher affinity foractivated Smad2 (Fig. 5C,E), we speculate that the Net1–Smad2binding occurs in the nucleus. To address this hypothesis, bimolecularfluorescence complementation (BiFC) assay was performed in HeLacells, and the reconstituted fluorescent YFP from YC-Smad2 (fusionof Smad2 to C-terminal half of YFP) and YN-Net1 (fusion of N-terminal half of YFP toNet1) was observed in the nuclei but not in thecytosol (Fig. 5G). These observations strongly support the idea thatNet1 associates with nuclear Smad2.
To determine which domain of Smad2 is responsible for bindingwith Net1, truncated Smad2 mutants expressing the MH1 domain,MH2 domain, MH1 domain and linker region (MH1-L), and linkerregion and MH2 domain (L-MH2), respectively, were constructed.Domain mapping revealed that the linker region and MH2 domainof Smad2 were both required for the interaction between Smad2 andNet1 (Fig. 5H). In addition, in agreement with the GEF-independentfunction of Net1 in Nodal signaling, wild-type Net1 and its GEF-deficient mutants bound to Smad2 with a similar affinity (Fig. 5I).Thus, Net1 interacts with Smad2 in a GEF-activity-independentmanner.
The interaction of Net1 and Smad2 facilitates p300recruitmentNet1 promotes Nodal signaling without affecting the nucleartranslocation or phosphorylation of Smad2 (Fig. 4E,F). Therefore, itwas hypothesized that Net1 enhances Smad2 transcriptionalactivity. Histone acetylation is essential for the regulation oftranscription (Struhl, 1998). The histone acetyl transferase p300 is ageneral transcriptional coactivator, which functions by looseningthe chromatin in an acetylation-dependent manner, while histonedeacetylases (HDACs) act as transcriptional repressors (Ogryzkoet al., 1996; Taunton et al., 1996; Wolffe, 1996). Interactions of theSmad complex with p300 or HDACs represent transcriptionalactivation or repression, respectively (Feng et al., 1998; Janknechtet al., 1998; Pouponnot et al., 1998; Wotton et al., 1999). Therefore,
Fig. 3. Nuclear Net1 enhances Nodal signaling and mesendodermformation in a GEF-independent manner. (A,B) Endogenous Net1 primarilylocalizes in the zebrafish embryonic cell nucleus. Nuclear and cytosolicfractions were extracted from wild-type embryos at indicated stages (A) or fromsqt mRNA-injected embryos at the shield stage (B). Then these resultingsamples were assessed by western blotting with the indicated antibodies.(C) Net1 promotes Nodal signaling in the nucleus. HeLa cells were transfectedwith the ARE-luciferase reporter together with Net1, Net1-NLS or Net1-ΔN-NES. After 36 h of transfection, the cells were treated with or without TGF-β1overnight, then harvested for luciferase assays. **P<0.01; ***P<0.001; NS, notsignificant (Student’s t-test). (D) Nuclear Net1 facilitates mesendoderminduction. At the sphere stage, zebrafish embryos injected with net1 2Mix ornet1-NLS 2Mix or net1-ΔN NES 2Mix were exposed to UV light, and thenharvested for whole-mount in situ hybridization to detect the expression of gscand sox17 at the shield and 75% epiboly stages. (E,F) Overexpression of thenuclear, but not cytosolic, Net1 rescues the mesendoderm defects in net1morphants. Embryos were injected with cMO, net1 MO1, net1-NLS 3Mix ornet1-ΔN NES 3Mix. Embryos injected with net1-NLS 3Mix or net1-ΔN NES3Mix were exposed to UV light at the sphere stage. The expression of gsc (E)and sox17 (F) was examined by in situ hybridization at the shield and 75%epiboly stages, respectively. (G,H) Net1 promotes Nodal signalingindependently of its GEFactivity. HeLa cells were co-transfected with the ARE-luciferase reporter and wild-type Net1 and its GEF-deficient mutants (G) ornuclear Net1 and its GEF-deficient mutants (H). After 36 h of transfection, thecells were treated with or without TGF-β1 overnight, then harvested forluciferase assays. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test). (I) Theinfluence of nuclear Net1 and its GEF-deficient mutants in mesendodermformation. Zebrafish embryos were injected with net1-NLS 2Mix, NLS-L266E2Mix andNLS-W437L 2Mix, respectively. These embryos were exposed to UVat the sphere stage, then harvested for whole-mount in situ hybridization. In D,E,F and I, the percentages (mean±s.d.) of the affected embryos are shown ascalculated from three independent biological repeats with ∼20–30 embryos ineach group. Results in C,G and H are mean±s.d. (n=3). UIC, uninjectedcontrol.
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it was examined whether Net1 affects the association of Smad2with p300 or HDAC1 by performing co-immunoprecipitationexperiments. As shown in Fig. 6A, Smad2 could bind to HDAC1,and this binding was dramatically decreased upon Net1overexpression. By contrast, overexpression of Net1 resulted in aclear promotion of Smad2–p300 interactions (Fig. 6B), indicatingthat Net1 promotes Smad2 transcriptional activity by enhancingp300 recruitment. Interestingly, compared to wild-type Net1, Net1GEF mutants had similar effects on the associations of Smad2 withHDAC1 or p300 (Fig. 6A,B), suggesting that the contribution of theGEF activity of Net1 was negligible in the regulation of Smad2transcriptional activity. This may explain why Net1 promotes Nodal
signaling and mesendoderm induction independently of its GEFactivity. Taken together, these results suggest that nuclear Net1interacts with Smad2 in a GEF-activity-independent manner. Net1–Smad2 interaction induces the dissociation of the corepressorHDAC1 from and recruitment of the coactivator p300 to Smadtranscriptional complexes, thereby activating Nodal target genetranscription during mesendoderm development (Fig. 6C).
DISCUSSIONNodal ligands emerged as endogenous mesendoderm inducers indifferent vertebrate embryos (Feldman et al., 1998; Gritsman et al.,1999; Zhou et al., 1993). Interestingly, loss of Smad2 disrupts the
Fig. 4. Net1 promotes Nodal signal downstreamof or parallel to Smad2. (A) Overexpression of Net1 promotes CA-ALK5-induced ARE-luciferase expression.At 36 h after transfecting with ARE-luciferase reporter together with increasing amounts of Net1 expression plasmids, HeLa cells were re-transfected with orwithout CA-ALK5 for 24 h, and then harvested for luciferase assays. *P<0.05; **P<0.01 (Student’s t-test). (B) One-cell-stage zebrafish embryos were injectedwith 3 pg tar*mRNA alone or together with 4 ng net1MO1, and then harvested for whole-mount in situ hybridization. (C) HeLa cells were co-transfected with theARE-luciferase reporter and indicated plasmids. At 36 h after transfection, the cells were harvested for luciferase assays. **P<0.01 (Student’s t-test). (D) One-cell-stage zebrafish embryos were injected with 25 pg ca-smad2 mRNA alone or together with 4 ng net1 MO1, and then harvested for whole-mount in situhybridization. (E) Overexpression of Net1 does not influence the subcellular localization of Smad2. HeLa cells transfected with Flag–Net1 were treated with orwithout TGF-β1 for 1 h, and then immunostained with anti-Flag (green) and anti-Smad2 (red) antibodies. Nuclei were counterstained with DAPI and areshown in blue. Scale bar: 10 μm. (F) Overexpression of Net1 does not affect the phosphorylation of Smad2. HeLa cells were transfected with Flag-tagged wild-type (WT) Net1 or the Net1-L266E mutant. After 36 h of transfection, these cells were treated with TGF-β1 or left untreated for 4 h, and then harvested forwestern blotting using the indicated antibodies. Results in A and C are mean±s.d. (n=3). In B and D, the percentages (mean±s.d.) of the affected embryos areshown as calculated from three independent biological repeats with ∼20–25 embryos in each group. UIC, uninjected control.
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development of the mesoderm and endoderm, while Smad3inactivation does not result in early embryonic defects. Thissuggests that Smad2 is the major downstream mediator of Nodalsignaling during mesendoderm formation (Nomura and Li, 1998;Weinstein et al., 1998; Yang et al., 1999; Zhu et al., 1998). Wepreviously demonstrated that zebrafish net1 is a direct target of theNodal pathway during gastrulation (Liu et al., 2011). Many Nodaltarget genes have been identified as important feedback regulatoryfactors involved in the control of Nodal-mediated embryonicdevelopment (Liu et al., 2016; Shen, 2007). In light of these reports,we set out to determine whether Net1 functions in the formation of
mesoderm and endoderm by regulating Nodal signal transduction.Our data suggest that Net1 is expressed in mesendoderm progenitorsand required for the specification of mesendodermal cell fatesthrough promoting Smad2 transcriptional activation. However,zebrafish net1mutants exhibit no detectable developmental defects,and a set of genes encoding GEFs, GTPases and GTPase effectorsare upregulated and suspected to compensate for the loss of net1(Wei et al., 2017). It will be interesting to further investigate thecompensatory mechanisms activated in the mutants.
Full-length mammalian Net1, a RhoA guanine nucleotideexchange factor, mainly localizes in the nucleus in quiescent
Fig. 5. Net1 interacts with Smad2. (A,B) Flag-tagged Net1 interacts with overexpressed Smad2. HEK293T cells were transfected with Flag–Net1 and Myc–Smad2 expression plasmids, then harvested for immunoprecipitation with anti-Flag M2 agarose beads (A) or anti-Myc agarose beads (B). Total lysates andimmunoprecipitation (IP) samples were analyzed by western blotting with the indicated antibodies. (C,D) Overexpressed Net1 interacts with endogenous Smad2.HeLa cells transfected with Flag-tagged Net1 were treated with or without TGF-β1 for 4 h, and then harvested for immunoprecipitation with anti-Flag M2 agarosebeads. Note that TGF-β1 treatment enhanced the interaction between Flag–Net1 and endogenous Smad2 (D). (E) The physical interaction of endogenous Net1and Smad2. Whole wild-type embryos were lysed with TNE at the shield stage for immunoprecipitation with anti-Net1 antibody. sqt mRNA was injected whenindicated. (F,G) Detection of the Net1–Smad2 interaction in living cells by means of a BiFC assays. YC-Smad2 and YN-Net1 were transfected alone orsimultaneously into HeLa cells. The expression of YN-Net1 or YC-Smad2 was detected by immunostaining with an anti-GFP antibody and DyLight 549-conjugated secondary antibody (F). The reconstituted YFP fluorescence was detected by confocal microscopy at 488 nm (G). Scale bars: 10 μm. (H) Smad2binds to Net1 through its L-MH2 domain. HEK293T cells were co-transfected with Flag–Net1 and Myc-tagged Smad2 deletion mutants, and then harvested forimmunoprecipitation. (I) Net1 interacts with Smad2 independently of its GEF activity. HEK293T cells were co-transfected with Myc–Smad2 and Flag-tagged wild-type (WT) Net1 or Net1 GEF-deficient mutants, and then harvested for immunoprecipitation.
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cells. Meanwhile, the oncogenic form of this protein is found in thecytoplasm and enhances cellular proliferation and tumorigenesis byactivating RhoA (Alberts and Treisman, 1998; Qin et al., 2005;Schmidt and Hall, 2002). Likewise, Xenopus Net1 is in the nucleusand the activation of the non-canonical Wnt signaling pathway doesnot alter its localization in animal cap cells (Miyakoshi et al.,2004). Interestingly, our study shows that zebrafish Net1 alsopredominantly localizes to the nucleus, and its location does notchange in response to Nodal signal stimulation. However, TGF-βsignaling has been reported to induce redistribution of Net1 to thecytoplasm in human retinal pigment epithelial cells, which isrequired for TGF-β-induced epithelial–mesenchymal transition(EMT) (Lee et al., 2010). Therefore, it is possible that thedifferent effects of TGF-β signaling on Net1 subcellulartranslocation may be dependent on the context.Thus far, the prevailing model is that Net1 export from the
nucleus to the cytoplasm leads to RhoA activation through its GEFactivity. However, recent studies have suggested that Net1 isrequired for the activation of RhoA and RhoB in the nucleus inresponse to DNA damage (Dubash et al., 2011; Srougi andBurridge, 2011). In zebrafish, based on the nuclear localization ofNet1 and the severe defects in mesendoderm formation noted innet1morphants, we hypothesized that Net1 might have a previouslyunidentified function in the nucleus. Indeed, we found that nuclearNet1 plays a role in Nodal signal transduction and mesendodermdevelopment. We further demonstrated that Net1 interacts withSmad2 in the nucleus and promotes Smad2 activation through p300recruitment in a GEF-independent manner. Importantly, to further
support our hypothesis, the overexpression of net1 had didnot promote mesendoderm formation in embryos treated withSB431542, as Smad2 could not be phosphorylated and translocatedinto the nucleus when Nodal signal was inhibited. Thus, nuclearNet1 has at least two functions: (1) activating nuclear Rho GTPasesas a GEF in response to DNA damage, and (2) regulating geneexpression as a scaffold protein in Smad2-containing transcriptionalcomplexes that direct cell fate during early embryogenesis.Identification of other nuclear binding partners of Net1 will beimportant for further understanding of its functions within thenucleus.
Several previous studies have revealed a link between thematernal Wnt/β-catenin pathway and early mesendoderm induction.It has been well established that maternal β-catenin is required forthe expression of dorsal genes, such as Nodal ligands, and for theactivation of MAPK and the mesodermal markers Xbra andeomesodermin (Bellipanni et al., 2006; Schohl and Fagotto, 2003;Zorn andWells, 2009). We previously reported that Net1, as well asits GEF activity, is critical for maternal β-catenin activation (Weiet al., 2017). In this study, extensive experimental evidence andanalysis established that Net1 promotes mesendoderm formationvia activation of Smad2 transcriptional activity. Several lines ofevidence suggest that the mesendoderm defects in net1-depletedembryos, rather than the secondary effects of the suppression ofWnt/β-catenin signal, are the direct results of the reduction of Nodalactivity. First, when Wnt/β-catenin activity is blocked by ΔN-tcf3expression, Net1 depletion significantly decreases Nodal receptor-induced mesendoderm formation. Second, both cytosolic and
Fig. 6. Net1 increases the affinity ofSmad2 for p300. (A,B) Net1 inhibitsthe interactions between Smad2 andHDAC1 (A), but enhances theassociation of Smad2 with p300 (B).HEK293T cells were transfected withindicated plasmids, and then harvestedfor immunoprecipitation with anti-FlagM2 agarose beads. Total lysates andimmunoprecipitation (IP) samples wereanalyzed by western blotting using theindicated antibodies. (C) Proposedmodel of the activation of the Nodalsignal by Net1. When Net1 is absent,Smad2 interacts with the corepressorHDAC1, which results in transcriptionalinhibition. When Net1 is present, Net1–Smad2 interaction induces therecruitment of the coactivator p300,which ultimately leads to the expressionof Nodal target genes duringmesendoderm formation.
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nuclear Net1 promote β-catenin activation (Wei et al., 2017), butonly nuclear Net1 is responsible for promoting Smad2transcriptional activity. Third, Net1 GEF activity is indispensablefor β-catenin activation, but is unnecessary for Nodal signaltransduction. Therefore, Net1 integrates Wnt/β-catenin and Nodalsignals by shifting from its role as a GEF to that of a scaffold proteinduring embryonic development.
MATERIALS AND METHODSZebrafish maintenanceWild-type embryos were obtained from zebrafish (Tuebingen strain)matings. Adult zebrafish and embryos were raised and maintained understandard laboratory conditions. Embryo stages were determined bymorphology as previously described (Kimmel et al., 1995). All zebrafishexperiments were in strict accordance with the Regulations for the Care andUse of Laboratory Animals as published by the Ministry of Science andTechnology of China, and the Institute of Zoology’s Guidelines for the Careand Use of Laboratory Animals.
mRNAs, morpholinos and microinjectionCapped mRNAs of zebrafish net1, tar*, sqt, ca-smad2 and ΔN-tcf3 weresynthesized in vitro from the corresponding linearized plasmids byusing mMessage mMachine kits (Ambion). The morpholinos weredesigned and synthesized by Gene Tools. net1 MO1 was 5′-CTTGCTCCGGCTGTACTCACCTCTT-3′, control MO (mis-MO1) was5′-CTTCCTGCGCCTGTAGTCACGTCTT-3′, and AS-Flag-photo-MOwas 5′-TCATCGTCGTpCTTGTAGTCCAT-3′. To overexpress net1 inzebrafish, Flag-net1 mRNA and AS-Flag-photo-MO were premixed thenco-injected into one-cell-stage embryos. Once at the sphere stage, theseembryos were exposed to 365 nm UV light for 10 min using a Lightbox(Gene Tools, LLC) and then harvested at the shield or 75% epiboly stages.
Whole-mount in situ hybridizationDigoxigenin-UTP-labeled antisense RNA probes were synthesized in vitroby using a MEGAscript® Kit (Ambion) according to the manufacturer’sinstructions. Whole-mount in situ hybridizations were performed aspreviously reported (Liu et al., 2011).
Cell lines and transfectionsHEK293T (CRL-3216, ATCC) and HeLa cell lines (CCL-2, ATCC) werecultured in Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°Cin a humidified incubator with 5% CO2. Cell transfections were performedby using Lipofectamine 2000 (11668019, Invitrogen) according to themanufacturer’s instructions.
Dual-luciferase reporter assaysFor the luciferase reporter assays in mammalian cells, HeLa cells weretransfected with ARE-luciferase (provided by Prof. Yeguang Chen atTsinghua University, China) and the indicated plasmids together with aRenilla luciferase reporter, which serves as an internal control. At 36 h posttransfection, cells were incubated in DMEM supplemented with 2% FBSand 5 ng/ml TGF-β1 (240-B, R&D Systems) overnight, and then harvestedin order to determine luciferase activity assays.
For the luciferase reporter assays in zebrafish embryos, ARE and Renillaluciferase plasmids and the indicated mRNAs or morpholinos were co-injected into one-cell-stage embryos, and then the embryos were harvestedat the shield stage for the luciferase activity assays. Each luciferase reporterassay was performed in triplicate and the data represent the mean±s.d. ofthree independent biological repeats after normalization to Renilla activity.
Immunoprecipitation and western blot analysisHEK293T or HeLa cells were transfected with the indicated plasmids. At 48h post transfection, the cells were harvested and lysed with TNE lysis buffer(10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 0.5% NonidetP-40) containing protease inhibitors. Immunoprecipitation and western
blots were then performed on the resulting cell lysates as previouslydescribed (Liu et al., 2013).
For the immunoprecipitations, anti-Flag M2 (1:100, A2220, Sigma) oranti-Myc agarose beads (1:100, A7470, Sigma) were used. For westernblots, affinity-purified anti-Flag (1:5000, F2555, Sigma), anti-HA (1:3000,CW0092A, CW), anti-Myc (1:3000, M047-3, MBL), anti-Smad2 (1:1000,3122, Cell Signaling Technology) and anti-phospho-Smad2 (Ser245/250/255) (1:500; 3104, Cell Signaling Technology) antibodies were used. Therabbit polyclonal anti-Net1 antibody was generated by our laboratory. Anepitope corresponding to residues SRGEQDLIEDLKLARKAC ofzebrafish Net1 was chosen for immunization. This polyclonal antibodywas affinity purified and validated for specificity by peptide competitionassays. The purified antibody (concentration, 200 μg/ml) was usedat a dilution of 1:1000 for western blots and 1:100 for proteincoimmunoprecipitations in our previous study and current work(Wei et al., 2017).
Immunofluorescence analysisHeLa cells were cultured on coverslips, fixed with 4% paraformaldehyde inPBS for 15 min at room temperature, and then permeabilized with 0.2%Triton X-100 in PBS for 10 min. After blocking for 1 h in 5% FBS in PBS,cells were incubated with anti-Flag M2 (1:1000, A2220, Sigma), anti-Smad2(1:100, 5339, Cell Signaling Technology) or anti-GFP (1:1000, A11122,Invitrogen) antibodies for 4 h, followed by fluorescently conjugated Alexasecondary antibodies for 2 h. Cells were counterstained with DAPI(10236276001, Sigma) to visualize nuclei. All immunofluorescent imageswere captured with a Zeiss LSM780 inverted confocal microscope using thesame settings for all experiments.
BiFC assayFor BiFC assay, Net1 was fused to the N-terminal half of YFP (YN-Net1)and Smad2 was fused to the C-terminal half of YFP (YC-Smad2). YN-Net1and YC-Smad2 were individually or together transfected into HeLa cells.YFP fluorescence was detected 48 h after transfection with a Zeiss LSM780inverted confocal microscope.
AcknowledgementsWe are grateful to members of the Qiang Wang laboratory for assistance anddiscussion.
Competing interestsThe authors declare no competing or financial interests.
FundingThis work was supported by grants from the Ministry of Science and Technology ofthe People’s Republic of China (National Key Research and Development Programof China) (2016YFA0100503) and the National Natural Science Foundation of China(31271532, 31571501).
ReferencesAlberts, A. S. and Treisman, R. (1998). Activation of RhoA and SAPK/JNK
signalling pathways by the RhoA-specific exchange factor mNET1. EMBO J. 17,4075-4085.
Bellipanni, G., Varga, M., Maegawa, S., Imai, Y., Kelly, C., Myers, A. P., Chu, F.,Talbot, W. S. and Weinberg, E. S. (2006). Essential and opposing roles ofzebrafish beta-catenins in the formation of dorsal axial structures andneurectoderm. Development 133, 1299-1309.
Bennett, J. T., Joubin, K., Cheng, S., Aanstad, P., Herwig, R., Clark, M., Lehrach,H. and Schier, A. F. (2007). Nodal signaling activates differentiation genes duringzebrafish gastrulation. Dev. Biol. 304, 525-540.
Chan, A. M., Takai, S., Yamada, K. and Miki, T. (1996). Isolation of a noveloncogene, NET1, from neuroepithelioma cells by expression cDNA cloning.Oncogene 12, 1259-1266.
Dubash, A. D., Guilluy, C., Srougi, M. C., Boulter, E., Burridge, K. andGarcıa-Mata, R. (2011). The small GTPase RhoA localizes to the nucleus and isactivated by Net1 and DNA damage signals. PLoS ONE 6, e17380.
3081
RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3072-3082 doi:10.1242/jcs.204917
Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T., Rennebeck, G., Sirotkin,H. I., Schier, A. F. and Talbot, W. S. (1998). Zebrafish organizer developmentand germ-layer formation require nodal-related signals. Nature 395, 181-185.
Feng, X.-H., Zhang, Y., Wu, R.-Y. and Derynck, R. (1998). The tumor suppressorSmad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for smad3 inTGF-beta-induced transcriptional activation. Genes Dev. 12, 2153-2163.
Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S. and Schier,A. F. (1999). The EGF-CFC protein one-eyed pinhead is essential for nodalsignaling. Cell 97, 121-132.
Janknecht, R., Wells, N. J. and Hunter, T. (1998). TGF-beta-stimulatedcooperation of smad proteins with the coactivators CBP/p300. Genes Dev. 12,2114-2119.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F.(1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253-310.
Lee, J., Moon, H.-J., Lee, J.-M. and Joo, C.-K. (2010). Smad3 regulates Rhosignaling via NET1 in the transforming growth factor-beta-induced epithelial-mesenchymal transition of human retinal pigment epithelial cells. J. Biol. Chem.285, 26618-26627.
Liu, Z., Lin, X., Cai, Z., Zhang, Z., Han, C., Jia, S., Meng, A. andWang, Q. (2011).Global identification of SMAD2 target genes reveals a role for multiple co-regulatory factors in zebrafish early gastrulas. J. Biol. Chem. 286, 28520-28532.
Liu, X., Xiong, C., Jia, S., Zhang, Y., Chen, Y.-G., Wang, Q. and Meng, A. (2013).Araf kinase antagonizes Nodal-Smad2 activity in mesendoderm development bydirectly phosphorylating the Smad2 linker region. Nat. Commun. 4, 1728.
Liu, Z., Ning, G., Xu, R., Cao, Y., Meng, A. andWang, Q. (2016). Fscn1 is requiredfor the trafficking of TGF-beta family type I receptors during endoderm formation.Nat. Commun. 7, 12603.
Massague, J. (2008). TGFbeta in Cancer. Cell 134, 215-230.Miyakoshi, A., Ueno, N. and Kinoshita, N. (2004). Rho guanine nucleotideexchange factor xNET1 implicated in gastrulation movements during Xenopusdevelopment. Differentiation 72, 48-55.
Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J.,Godsave, S., Korinek, V., Roose, J., Destree, O. andClevers, H. (1996). XTcf-3transcription factor mediates beta-catenin-induced axis formation in Xenopusembryos. Cell 86, 391-399.
Murray, D., Horgan, G., MacMathuna, P. and Doran, P. (2008). NET1-mediatedRhoA activation facilitates lysophosphatidic acid-induced cell migration andinvasion in gastric cancer. Br. J. Cancer 99, 1322-1329.
Nakaya, Y., Sukowati, E. W., Wu, Y. and Sheng, G. (2008). RhoA and microtubuledynamics control cell-basementmembrane interaction in EMT during gastrulation.Nat. Cell Biol. 10, 765-775.
Nicklas, D. and Saiz, L. (2013). Characterization of negative feedback networkmotifs in the TGF-beta signaling pathway. PLoS ONE 8, e83531.
Nomura, M. and Li, E. (1998). Smad2 role in mesoderm formation, left-rightpatterning and craniofacial development. Nature 393, 786-790.
Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H. and Nakatani, Y.(1996). The transcriptional coactivators p300 and CBP are histoneacetyltransferases. Cell 87, 953-959.
Pouponnot, C., Jayaraman, L. and Massague, J. (1998). Physical and functionalinteraction of SMADs and p300/CBP. J. Biol. Chem. 273, 22865-22868.
Qin, H., Carr, H. S., Wu, X., Muallem, D., Tran, N. H. and Frost, J. A. (2005).Characterization of the biochemical and transforming properties of theneuroepithelial transforming protein 1. J. Biol. Chem. 280, 7603-7613.
Schier, A. F. and Shen, M. M. (2000). Nodal signalling in vertebrate development.Nature 403, 385-389.
Schmidt, A. and Hall, A. (2002). The Rho exchange factor Net1 is regulated bynuclear sequestration. J. Biol. Chem. 277, 14581-14588.
Schmierer, B. and Hill, C. S. (2007). TGFbeta-SMAD signal transduction:molecular specificity and functional flexibility. Nat. Rev. Mol. Cell Biol. 8, 970-982.
Schohl, A. and Fagotto, F. (2003). A role for maternal beta-catenin in earlymesoderm induction in Xenopus. EMBO J. 22, 3303-3313.
Shen, M. M. (2007). Nodal signaling: developmental roles and regulation.Development 134, 1023-1034.
Srougi, M. C. and Burridge, K. (2011). The nuclear guanine nucleotide exchangefactors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.PLoS ONE 6, e17108.
Stroschein, S. L., Wang, W., Zhou, S., Zhou, Q. and Luo, K. (1999). Negativefeedback regulation of TGF-beta signaling by the SnoN oncoprotein.Science 286,771-774.
Struhl, K. (1998). Histone acetylation and transcriptional regulatory mechanisms.Genes Dev. 12, 599-606.
Taunton, J., Hassig, C. A. and Schreiber, S. L. (1996). A mammalian histonedeacetylase related to the yeast transcriptional regulator Rpd3p. Science 272,408-411.
Tu, Y., Lu, J., Fu, J., Cao, Y., Fu, G., Kang, R., Tian, X. andWang, B. (2010). Over-expression of neuroepithelial-transforming protein 1 confers poor prognosis ofpatients with gliomas. Jpn. J. Clin. Oncol. 40, 388-394.
Vessichelli, M., Ferravante, A., Zotti, T., Reale, C., Scudiero, I., Picariello, G.,Vito, P. and Stilo, R. (2012). Neuroepithelial transforming gene 1 (Net1) binds tocaspase activation and recruitment domain (CARD)- and membrane-associatedguanylate kinase-like domain-containing (CARMA) proteins and regulatesnuclear factor kappaB activation. J. Biol. Chem. 287, 13722-13730.
Wei, S., Dai, M., Liu, Z., Ma, Y., Shang, H., Cao, Y. and Wang, Q. (2017). Theguanine nucleotide exchange factor Net1 facilitates the specification of dorsal cellfates in zebrafish embryos by promoting maternal beta-catenin activation. CellRes. 27, 202-225.
Weinstein, M., Yang, X., Li, C., Xu, X., Gotay, J. and Deng, C.-X. (1998). Failure ofegg cylinder elongation and mesoderm induction in mouse embryos lacking thetumor suppressor smad2. Proc. Natl. Acad. Sci. USA 95, 9378-9383.
Wolffe, A. P. (1996). Histone deacetylase: a regulator of transcription. Science 272,371-372.
Wotton, D., Lo, R. S., Lee, S. and Massague, J. (1999). A Smad transcriptionalcorepressor. Cell 97, 29-39.
Wu, M. Y. and Hill, C. S. (2009). Tgf-beta superfamily signaling in embryonicdevelopment and homeostasis. Dev. Cell 16, 329-343.
Yang, X., Letterio, J. J., Lechleider, R. J., Chen, L., Hayman, R., Gu, H., Roberts,A. B. and Deng, C. (1999). Targeted disruption of SMAD3 results in impairedmucosal immunity and diminished T cell responsiveness to TGF-beta. EMBO J.18, 1280-1291.
Zhou, X., Sasaki, H., Lowe, L., Hogan, B. L. M. and Kuehn, M. R. (1993). Nodal isa novel TGF-beta-like gene expressed in the mouse node during gastrulation.Nature 361, 543-547.
Zhu, Y., Richardson, J. A., Parada, L. F. and Graff, J. M. (1998). Smad3 mutantmice develop metastatic colorectal cancer. Cell 94, 703-714.
Zorn, A. M. and Wells, J. M. (2009). Vertebrate endoderm development and organformation. Annu. Rev. Cell Dev. Biol. 25, 221-251.
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