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Developmental Cell
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Sox5 Is a DNA-Binding Cofactor for BMP R-Smads that Directs Target Specificity during Patterning of the Early Ectoderm

      Highlights

      • Sox5 is expressed in blastula cells and maintained in neural crest cells
      • Sox5 is required for BMP-directed target gene expression in vivo and ex vivo
      • Sox5 is a DNA-binding cofactor for BMP R-Smads required for ectodermal patterning
      • Sox5 recruits Smad complexes to BMP response elements to activate BMP target genes

      Summary

      The SoxD factor, Sox5, is expressed in ectodermal cells at times and places where BMP signaling is active, including the cells of the animal hemisphere at blastula stages and the neural plate border and neural crest at neurula stages. Sox5 is required for proper ectoderm development, and deficient embryos display patterning defects characteristic of perturbations of BMP signaling, including loss of neural crest and epidermis and expansion of the neural plate. We show that Sox5 is essential for activation of BMP target genes in embryos and explants, that it physically interacts with BMP R-Smads, and that it is essential for recruitment of Smad1/4 to BMP regulatory elements. Our findings identify Sox5 as the long-sought DNA-binding partner for BMP R-Smads essential to plasticity and pattern in the early ectoderm.

      Graphical Abstract

      Introduction

      In the bone morphogenetic protein (BMP) branch of transforming growth factor β (TGFβ) signaling, BMP R-Smads (Smad1, Smad5, and Smad8) form a complex with Smad4 following receptor-mediated phosphorylation and translocate to the nucleus (
      • Massagué J.
      • Seoane J.
      • Wotton D.
      Smad transcription factors.
      ). While Smads bind GC-rich DNA motifs in target enhancers, this interaction is of low affinity and selectivity (
      • Blitz I.L.
      • Cho K.W.Y.
      Finding partners: how BMPs select their targets.
      ,
      • Shi Y.
      • Wang Y.-F.
      • Jayaraman L.
      • Yang H.
      • Massagué J.
      • Pavletich N.P.
      Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling.
      ); consequently, the intrinsic DNA-binding activity of this complex is not sufficient to dictate target selectivity. Instead, DNA-binding nuclear partners cooperate with nuclear Smad complexes to recruit them to the context-appropriate target genes (
      • Chen X.
      • Rubock M.J.
      • Whitman M.
      A transcriptional partner for MAD proteins in TGF-beta signalling.
      ,
      • Massagué J.
      • Wotton D.
      Transcriptional control by the TGF-β/Smad signaling system.
      ,
      • Massagué J.
      • Seoane J.
      • Wotton D.
      Smad transcription factors.
      ). For the early developmental roles played by Activin/Nodal R-Smads in establishing the mesendoderm, Fast1 and Mixer provide this specificity (
      • Chen X.
      • Rubock M.J.
      • Whitman M.
      A transcriptional partner for MAD proteins in TGF-beta signalling.
      ,
      • Germain S.
      • Howell M.
      • Esslemont G.M.
      • Hill C.S.
      Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif.
      ,
      • Massagué J.
      • Wotton D.
      Transcriptional control by the TGF-β/Smad signaling system.
      ); however, the analogous partners for the BMP R-Smads in early ectodermal patterning events have remained elusive (
      • Blitz I.L.
      • Cho K.W.Y.
      Finding partners: how BMPs select their targets.
      ).
      BMP signaling plays multiple reiterative roles in the early embryonic ectoderm. This signaling pathway is initially active throughout the germ layer in blastula embryos, when these cells retain the potential to give rise to all embryonic cell types (
      • Hawley S.H.
      • Wünnenberg-Stapleton K.
      • Hashimoto C.
      • Laurent M.N.
      • Watabe T.
      • Blumberg B.W.
      • Cho K.W.
      Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction.
      ,
      • Hemmati-Brivanlou A.
      • Thomsen G.H.
      Ventral mesodermal patterning in Xenopus embryos: expression patterns and activities of BMP-2 and BMP-4.
      ,
      • Knöchel S.
      • Dillinger K.
      • Köster M.
      • Knöchel W.
      Structure and expression of Xenopus tropicalis BMP-2 and BMP-4 genes.
      ). At gastrula stages, cells in the ectoderm respond to BMP antagonists from the organizer by adopting neuronal progenitor fates (
      • Sasai Y.
      • Lu B.
      • Steinbeisser H.
      • De Robertis E.M.
      Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus.
      ,
      • Zimmerman L.B.
      • De Jesús-Escobar J.M.
      • Harland R.M.
      The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4.
      ); continued BMP signaling is essential for epidermis formation (
      • Wilson P.A.
      • Hemmati-Brivanlou A.
      Induction of epidermis and inhibition of neural fate by Bmp-4.
      ). Formation of the neural plate border (NPB) initially requires intermediate levels of BMP signaling as well as Wnt- and fibroblast-growth-factor-derived signals (
      • Milet C.
      • Monsoro-Burq A.H.
      Neural crest induction at the neural plate border in vertebrates.
      ,
      • Prasad M.S.
      • Sauka-Spengler T.
      • LaBonne C.
      Induction of the neural crest state: control of stem cell attributes by gene regulatory, post-transcriptional and epigenetic interactions.
      ). Subsequently, BMP signaling becomes enhanced at the border region (
      • Reichert S.
      • Randall R.A.
      • Hill C.S.
      A BMP regulatory network controls ectodermal cell fate decisions at the neural plate border.
      ,
      • Schohl A.
      • Fagotto F.
      β-catenin, MAPK and Smad signaling during early Xenopus development.
      ), and this is reflected in the expression of a number of direct target genes, including Msx1/2 and Id3 (
      • Karaulanov E.
      • Knöchel W.
      • Niehrs C.
      Transcriptional regulation of BMP4 synexpression in transgenic Xenopus.
      ,
      • Suzuki A.
      • Ueno N.
      • Hemmati-Brivanlou A.
      Xenopus msx1 mediates epidermal induction and neural inhibition by BMP4.
      ), and ultimately leads to neural crest (NC) cell formation (
      • Taylor K.M.
      • LaBonne C.
      Modulating the activity of neural crest regulatory factors.
      ,
      • Tribulo C.
      • Aybar M.J.
      • Nguyen V.H.
      • Mullins M.C.
      • Mayor R.
      Regulation of Msx genes by a Bmp gradient is essential for neural crest specification.
      ). The identity of the DNA-binding cofactor(s) that direct the specificity of the nuclear BMP response to target genes essential for maintaining broad developmental potential at these key embryonic stages is of great importance but has remained unknown.
      Here, we set out to examine a role for the SRY-family factor, Sox5, in NC cells, a multipotent progenitor population unique to vertebrates. We found that Sox5, which is expressed in NC cells and has previously been shown to function in the later development of these cells (
      • Morales A.V.
      • Perez-Alcala S.
      • Barbas J.A.
      Dynamic Sox5 protein expression during cranial ganglia development.
      ,
      • Perez-Alcala S.
      • Nieto M.A.
      • Barbas J.A.
      LSox5 regulates RhoB expression in the neural tube and promotes generation of the neural crest.
      ), is maternally provided (Figure S1;
      • Yanai I.
      • Peshkin L.
      • Jorgensen P.
      • Kirschner M.W.
      Mapping gene expression in two Xenopus species: evolutionary constraints and developmental flexibility.
      ). Like BMP signaling, Sox5 is expressed throughout the pluripotent ectodermal cells of blastula embryos. This factor subsequently becomes enriched at the NPB, and its expression is lost in other ectoderm-derived cells as they become lineage restricted. We find that Sox5 loss of function phenocopies inhibition of BMP signaling, causing ectodermal cells to adopt a pan-neural identity at the expense of epidermis, NC cells, and placodes. Finally, we show that Sox5 physically interacts with the BMP R-Smads in solution and on target promoters and provides critical target specificity. Together, these findings identify Sox5 as a key DNA-binding partner for BMP R-Smads in the early ectoderm.

      Results

      Sox5 Is Required for NC Formation

      Our studies on the roles of Sox proteins in the developing NC (
      • Haldin C.E.
      • LaBonne C.
      SoxE factors as multifunctional neural crest regulatory factors.
      ,
      • Lee P.-C.
      • Taylor-Jaffe K.M.
      • Nordin K.M.
      • Prasad M.S.
      • Lander R.M.
      • LaBonne C.
      SUMOylated SoxE factors recruit Grg4 and function as transcriptional repressors in the neural crest.
      ,
      • Taylor K.M.
      • LaBonne C.
      SoxE factors function equivalently during neural crest and inner ear development and their activity is regulated by SUMOylation.
      ) led us to further examine the role of Sox5, a SoxD family protein, in these cells. Distinct from many NC regulatory factors, ectodermal expression of Sox5 is initially broad and low at early NC stages before becoming strongly enriched at the NPB (
      • Suzuki T.
      • Kusakabe M.
      • Nakayama K.
      • Nishida E.
      The protein kinase MLTK regulates chondrogenesis by inducing the transcription factor Sox6.
      ; Figures S1A–S1F available online). It is also maternally provided and expressed throughout the ectoderm at blastula stages (Figures S1G and S1H). NC cells maintain expression of Sox5 as they begin to migrate, similar to what has been described in chick (
      • Morales A.V.
      • Perez-Alcala S.
      • Barbas J.A.
      Dynamic Sox5 protein expression during cranial ganglia development.
      ,
      • Perez-Alcala S.
      • Nieto M.A.
      • Barbas J.A.
      LSox5 regulates RhoB expression in the neural tube and promotes generation of the neural crest.
      ). The closely related SoxD factor, Sox6, was not detected in early NC cells (
      • Suzuki T.
      • Kusakabe M.
      • Nakayama K.
      • Nishida E.
      The protein kinase MLTK regulates chondrogenesis by inducing the transcription factor Sox6.
      ; Figure S1I).
      To determine if Sox5 is essential for early and/or late aspects of NC cell formation in Xenopus, we generated two morpholinos (MOs) that potently block Sox5 translation (Figures S1J and S1K). These MOs were injected individually into single micromeres at the eight-cell stage targeting the NC. When injected embryos were examined at neurula stages for effects on NC formation, expression of FoxD3, Snail2, Sox9, and Sox10 was found to be significantly reduced (Figure 1A). Both MOs generated this phenotype, and defects could be rescued by a form of Sox5 not targeted by the MOs (Figures S1J and S1K). Loss of NC cells in Sox5 morphant embryos was not due to changes in cell proliferation or cell death (Figures S2A and S2B), and expression of mesodermal markers was unaffected by ectodermally targeted MOs (Figures S2C and S2D).
      Figure thumbnail gr1
      Figure 1Sox5 Is Required for NC and NPB Formation
      (A) In situ hybridization examining FoxD3, Snail2, Sox9, and Sox10 in Xenopus embryos injected with Sox5 MO or Sox5 mRNA. Embryos were collected at midneurula stages (stage 17). Asterisk denotes injected side with β-galactosidase (β-gal) staining (red) as lineage tracer.
      (B) Explant assay examining Snail2 and Sox9 in Wnt8/Chordin-induced explants that were injected with Sox5 MO. Explants were collected alongside sibling embryos cultured to midneurula stages (stage 17).
      (C) In situ hybridization examining Six1 and Foxi1c in embryos injected with Sox5 MO or Sox5 mRNA. Embryos were collected at early neurula stages (stage 13).
      (D) In situ hybridization examining Msx1, Pax3, and Zic1 in embryos injected with Sox5 MO or Sox5 mRNA. Embryos were collected at early neurula stages (stage 13/14).
      (E) Explant assay examining Msx1, Pax3, and Zic1 in Wnt8/Chordin-induced explants that were injected with Sox5 MO. Explants were collected alongside sibling embryos cultured until early neurula stages (stage 14). Asterisk denotes injected side with β-gal staining (red) as lineage tracer.
      See also .
      We further confirmed that loss of NC cells reflected a direct requirement for Sox5 function in the ectoderm by using explants of pluripotent blastula cells programmed to form NC cells by combined Wnt/Chordin treatment (
      • LaBonne C.
      • Bronner-Fraser M.
      Neural crest induction in Xenopus: evidence for a two-signal model.
      ). The robust induction of NC markers seen in Wnt/Chordin-expressing explants was lost if Sox5 was depleted (Figure 1B). Notably, we also found that upregulation of Sox5 resulted in loss FoxD3, Snail2, Sox9, and Sox10 expression (Figure 1A). Sox5 gain of function had previously been reported to enhance NC gene expression in the chick, but this was analyzed at later stages (
      • Perez-Alcala S.
      • Nieto M.A.
      • Barbas J.A.
      LSox5 regulates RhoB expression in the neural tube and promotes generation of the neural crest.
      ). When we examined expression of NC markers at migratory NC stages, we found that expression of some markers, including Sox8, had recovered and were enhanced (Figure S2E). This suggested that Sox5 may have distinct effects early on, at the NPB, and later, in NC cells themselves. Indeed, embryos injected at the eight-cell stage with either Sox5 mRNA or Sox5 MOs show dramatic changes in markers of the NPB (Zic1, Msx1, and Pax3; Figure 1D), as well as in panplacodal markers (Six1 and Foxi1C;
      • Groves A.K.
      • LaBonne C.
      Setting appropriate boundaries: fate, patterning and competence at the neural plate border.
      ) of later NPB derivatives (Figure 1C). Moreover, dependence of NPB gene expression on Sox5 was also observed in Wnt/Chordin-expressing animal explants, indicating a direct requirement for Sox5 in the ectoderm (Figure 1E).

      Sox5 Is Essential for Cell Fate Decisions in the Blastula Ectoderm

      The ectopic Zic1 expression observed in Sox5-depleted embryos extended both medially and laterally to the region of Sox5 enrichment at the NPB (Figure 1D), suggesting that it might be the low panectodermal expression of Sox5 that is essential to establishing proper gene expression in the early ectoderm. As Sox5 is expressed at a time consistent with participation in the earliest steps of ectoderm patterning, we examined whether it was required for establishment of epidermis and neural plate. Depletion of Sox5 resulted in ectopic expression of Sox3, which marks the neural plate, expanding this domain laterally into regions that would normally give rise to epidermis (Figure 2A), while a dramatic loss of Epidermal keratin (EPK), a marker of non-neural ectoderm, was observed in this same region (Figure 2B). Misexpression of Sox5 had similar effects on both Sox3 and EPK (Figures 2A and 2B). EPK expression in blastula animal explants was similarly lost following up or downregulation of Sox5 (Figure 2D). Moreover, these explants were neuralized as a consequence of modulating Sox5 levels, as evidenced by expression of Sox3 (Figure 2C).
      Figure thumbnail gr2
      Figure 2Misregulating Sox5 Phenocopies Loss of BMP Signaling
      (A and B) In situ hybridization examining Sox3 (A) and EPK (B) in embryos injected with Sox5 MO or Sox5 mRNA. Embryos were collected at early neurula stages (stage 13).
      (C and D) Explant assay examining Sox3 (C) and EPK (D) in explants that were either injected with Sox5 MO or Sox5 mRNA. Explants were collected alongside sibling embryos cultured until early neurula stages (stage 13).
      (E and F) In situ hybridization examining Sox3 (E) and EPK (F) in embryos injected with Chordin mRNA showing a similar loss of EPK and expanded expression of Sox3. Embryos were collected at early neurula stages (stage 13/14). Asterisk denotes injected side with β-gal (red) as lineage tracer.
      (G) In situ hybridization examining AP2, Id3, and Vent2 at blastula stages. Embryos were injected with Chordin, Smad7, or Sox5 MO and collected at stage 9. Asterisk denotes injected side with β-gal staining (red) as lineage tracer. See also .

      Activation of Direct Targets of BMP Signaling Requires Sox5

      Strikingly, the consequences of Sox5 depletion in the ectoderm phenocopy the well-characterized effects of BMP inhibition (
      • Piccolo S.
      • Sasai Y.
      • Lu B.
      • De Robertis E.M.
      Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4.
      ,
      • Sasai Y.
      • Lu B.
      • Steinbeisser H.
      • De Robertis E.M.
      Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus.
      ,
      • Wilson P.A.
      • Hemmati-Brivanlou A.
      Induction of epidermis and inhibition of neural fate by Bmp-4.
      ,
      • Zimmerman L.B.
      • De Jesús-Escobar J.M.
      • Harland R.M.
      The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4.
      ), leading to a dramatic expansion of the neural plate at the expense of epidermis (Figures 2E and 2F). Therefore, we hypothesized that Sox5 might function as a BMP effector.
      To test this, we first examined whether BMP signaling was essential for the blastula stage expression of BMP target genes. AP2, Id3, and Vent2 are all are known to be BMP regulated in other systems and/or stages (
      • Henningfeld K.A.
      • Rastegar S.
      • Adler G.
      • Knöchel W.
      Smad1 and Smad4 are components of the bone morphogenetic protein-4 (BMP-4)-induced transcription complex of the Xvent-2B promoter.
      ,
      • Hollnagel A.
      • Oehlmann V.
      • Heymer J.
      • Rüther U.
      • Nordheim A.
      Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells.
      ,
      • Ladher R.
      • Mohun T.J.
      • Smith J.C.
      • Snape A.M.
      Xom: a Xenopus homeobox gene that mediates the early effects of BMP-4.
      ,
      • López-Rovira T.
      • Chalaux E.
      • Massagué J.
      • Rosa J.L.
      • Ventura F.
      Direct binding of Smad1 and Smad4 to two distinct motifs mediates bone morphogenetic protein-specific transcriptional activation of Id1 gene.
      ,
      • Qiao Y.
      • Zhu Y.
      • Sheng N.
      • Chen J.
      • Tao R.
      • Zhu Q.
      • Zhang T.
      • Qian C.
      • Jing N.
      AP2γ regulates neural and epidermal development downstream of the BMP pathway at early stages of ectodermal patterning.
      ). Blocking BMP signaling either with Chordin or with an inhibitory Smad, Smad7 (
      • Casellas R.
      • Brivanlou A.H.
      Xenopus Smad7 inhibits both the activin and BMP pathways and acts as a neural inducer.
      ,
      • Piccolo S.
      • Sasai Y.
      • Lu B.
      • De Robertis E.M.
      Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4.
      ), was found to potently block expression of all three factors (Figure 2G), and depletion of Sox5 led to results indistinguishable from those of blocking BMP signaling.
      Next, we examined the consequences of Sox5 depletion for BMP-mediated induction of direct targets Vent1 and Msx1 (
      • Gawantka V.
      • Delius H.
      • Hirschfeld K.
      • Blumenstock C.
      • Niehrs C.
      Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1.
      ,
      • Rastegar S.
      • Friedle H.
      • Frommer G.
      • Knöchel W.
      Transcriptional regulation of Xvent homeobox genes.
      ,
      • Suzuki A.
      • Ueno N.
      • Hemmati-Brivanlou A.
      Xenopus msx1 mediates epidermal induction and neural inhibition by BMP4.
      ) in blastula explants. BMP4 induced robust expression of both Vent1 and Msx1 in control explants but was unable to do so in the absence of Sox5 (Figures 3A and 3B ), consistent with an essential role for Sox5 in mediating ectodermal responses to BMP signaling. We hypothesized that there were two ways that Sox5 could control BMP responsiveness. First, it could be transcriptionally regulating components of the BMP signal transduction pathway, such as ligands, receptors, or other pathway regulators. Alternatively, Sox5 might function as a DNA-binding cofactor for the BMP R-Smads, functioning to direct target specificity. To distinguish between these possibilities, we first examined whether Sox5 gain or loss of function would affect levels of phosphorylated Smad1/5/8 in the responding cells. We found that neither Sox5 overexpression nor Sox5 depletion altered the levels of either phosphorylated or unphosphorylated Smad1/5/8 (Figure 3C), supporting a model in which Sox5 functions downstream of R-Smad phosphorylation. Similarly, no effects were seen on the levels of phospho-Smad2 (Figure 3D), which functions downstream of the Activin/Nodal branch of TGF-β signaling (
      • Massagué J.
      • Seoane J.
      • Wotton D.
      Smad transcription factors.
      ).
      Figure thumbnail gr3
      Figure 3BMP Signaling Requires Sox5 to Activate Transcriptional Targets
      (A and B) Ectoderm explant assay examining Vent1 (A) and Msx1 (B) in explants injected with BMP4 mRNA or BMP4 mRNA with Sox5 MO. Explants were collected alongside sibling embryos cultured until early neurula stages (stage 14).
      (C) Western blot using lysates from embryos injected either with BMP4, BMP4/Sox5, BMP4/Sox5MO, or Chordin to examine levels of phosphorylated Smad1 and unphosphorylated Smad1.
      (D) Western blot using using lysates from embryos injected with Activin, Activin/Sox5, Activin/Sox5MO, Sox5, or Sox5MO to examine levels of phosphorylated Smad2.
      (E and F) Luciferase assay examining fold activation by the Vent2 promoter, p = 0.0068 (E), or 12XGCCG reporter, p = 0.0227 (F), in the presence of active Smad1/4 complexes or with active Smad1/4 and Sox5 MO.
      (G) Luciferase assay using the Vent2 reporter and the Vent2 3x Mutant reporter (carrying the same mutations as in C). The mutations that reduce Sox5 binding also reduce activation of the reporter by active Smad1/4. Error bars represent SD of three independent experiments.
      See also .

      Sox5 Regulates BMP Target Specificity in the Ectoderm

      We next investigated whether Sox5 was required for activation of two BMP responsive luciferase reporters whose functions are mechanistically distinct. First, we utilized a previously characterized Vent reporter (
      • Henningfeld K.A.
      • Rastegar S.
      • Adler G.
      • Knöchel W.
      Smad1 and Smad4 are components of the bone morphogenetic protein-4 (BMP-4)-induced transcription complex of the Xvent-2B promoter.
      ) that would be expected to require both activated BMP R-Smads and DNA-binding cofactors in order to be activated. We compared activation of this reporter to activation of a minimal “BMP reporter” consisting of 12 multimerized “GCCG” sites (
      • Kusanagi K.
      • Inoue H.
      • Ishidou Y.
      • Mishima H.K.
      • Kawabata M.
      • Miyazono K.
      Characterization of a bone morphogenetic protein-responsive Smad-binding element.
      ). The 12XGCCG reporter responds to activated Smad complexes alone and should not require DNA-binding cofactors. While both reporters are activated in response to BMP/Smads, Sox5 depletion was found to significantly reduce activation of the Vent reporter but not the 12XGCCG reporter (Figures 3E and 3F). This finding is consistent with a model in which Sox5 functions as a DNA-binding cofactor for BMP R-Smads, contributing to target specificity in response to BMP signaling.
      Further supporting this model, we found that BMP-mediated activation of a luciferase reporter driven by Id3 regulatory elements also displayed dependence on Sox5, whereas the Activin/Nodal luciferase reporter, 4XCAGA, did not (Figures S3A and S3B). To ensure that the effects of Sox5 on BMP reporters were direct, we wanted to determine if mutations in Sox5 binding sites would diminish BMP responsiveness. While Sox5 binding sites, which are AT rich, had not previously been examined in Vent2 regulatory regions, we found potential binding sites both within and outside of the best characterized regulatory element, the BRE (
      • von Bubnoff A.
      • Peiffer D.A.
      • Blitz I.L.
      • Hayata T.
      • Ogata S.
      • Zeng Q.
      • Trunnell M.
      • Cho K.W.
      Phylogenetic footprinting and genome scanning identify vertebrate BMP response elements and new target genes.
      ,
      • Henningfeld K.A.
      • Rastegar S.
      • Adler G.
      • Knöchel W.
      Smad1 and Smad4 are components of the bone morphogenetic protein-4 (BMP-4)-induced transcription complex of the Xvent-2B promoter.
      ,
      • Karaulanov E.
      • Knöchel W.
      • Niehrs C.
      Transcriptional regulation of BMP4 synexpression in transgenic Xenopus.
      ). The BRE itself has three potential Sox5 sites and can be bound by both Sox5 and Sox6 (Figures S3C and S3D). Mutation of these sites greatly diminished the ability of both Sox5 and Sox6 to bind the BRE (Figure S3D). Consistent with this, introduction of the same mutations into the much larger regulatory region of the Vent2 reporter significantly diminished BMP responsiveness (Figure 3G).

      Sox5 Physically Interacts with Smad1

      Interestingly, Sox5 depletion was observed to enhance activation of the 12XGCCG reporter (Figure 3F). One interpretation of this finding is that the presence of endogenous Sox5 inhibits activation of this promoter by sequestering BMP R-Smads. Therefore, we examined whether Sox5 could physically bind Smad proteins. In pulldown assays, Sox5 was found to interact with glutathione S-transferase (GST)-Smad1 preferentially to GST-Smad3, and similar results were obtained for Sox6 (Figure 4A). When interactions were assayed by coimmunoprecipitation from embryo lysates, association was also seen with Smad4 (Figure S4A), although this could be due to bridging interactions. Interaction between Sox5 and Smad1 is mediated by the Smad1 MH1 domain (Figure 4B), a domain known to interact with R-Smad DNA-binding cofactors (
      • Massagué J.
      • Seoane J.
      • Wotton D.
      Smad transcription factors.
      ). A panel of Sox5 deletion mutants was generated and tested for interaction with GST-Smad1. Deletion of the LC-domain-containing N terminus or the HMG-domain-containing C terminus did not prevent interaction, whereas a deletion that encompassed the coiled-coil domain region failed to interact (Figures S4B and S4C). This suggests that it is the central coiled-coiled domain region that mediates interaction with Smad1.
      Figure thumbnail gr4
      Figure 4Sox5 Recruits Smad1/4 to Target Promoters through Physical Association
      (A) GST pull-down assay using lysates from uninjected embryos or embryos injected with Sox5 or Sox6 mRNA assaying for binding to GST, Smad1-GST, Smad3-GST, or Smad4-GST. Western blots show expressed levels of Sox5 and Sox6.
      (B) GST pull-down assay using lysates from embryos injected with Sox5 mRNA assaying for binding to GST, Smad1-GST, Smad1-MH1-GST, or Smad1-MH2-GST.
      (C) Schematic representations of Smad1, Smad1-MH1, and Smad1-MH2 constructs used in pull-down assays.
      (D) Coomassie staining of expressed GST fusion proteins used for pull-downs in (A) and (B).
      (E) ChIP-qPCR analysis of Smad1 enrichment on EPK promoter relative to eEF1α promoter. Smad1 enrichment is significantly reduced when Sox5 is depleted. Error bars represent SEM of three independent biological experiments. p = 0.0022.
      (F) ChIP q-PCR analysis of Smad1 enrichment on Vent2 promoter relative to control eEF1α promoter. Smad1 enrichment is significantly reduced when Sox5 is depleted. Error bars represent SEM of three independent biological experiments. p = 0.0472.
      (G) ChIP analysis examining Sox5 enrichment on EPK promoter relative to eEF1α promoter. Smad1 enrichment is significantly reduced in the presence of Chordin. Error bars represent SEM of three independent biological experiments. p = 0.0048.
      (H) ChIP analysis examining Sox5 enrichment on Vent2 promoter relative to eEF1α promoter. Sox5 enrichment is significantly reduced in the presence of Chordin. Error bars represent SEM of three biological experiments. p = 0.0006.
      See also .

      Sox5 Is Essential for Recruitment of Smad1 to BMP Targets

      The aforementioned findings demonstrate that Sox5 is essential for expression of BMP target genes, including EPK, Vent1, and Msx1 in the early ectoderm, and that it is also essential for activating BMP-responsive Vent2 and Id3 reporters. Combined with the physical interaction between Sox5 and Smad1, these results suggest that Sox5 functions as an essential DNA-binding cofactor for BMP R-Smads during patterning of the early ectoderm. To confirm this, we examined Sox5 occupancy on regulatory elements for BMP targets in vivo using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Strong occupancy by both Sox5 and Smad1 was observed on previously characterized regulatory elements for both EPK and Vent2 (Figures 4E–4H) (
      • Henningfeld K.A.
      • Rastegar S.
      • Adler G.
      • Knöchel W.
      Smad1 and Smad4 are components of the bone morphogenetic protein-4 (BMP-4)-induced transcription complex of the Xvent-2B promoter.
      ,
      • Lander R.
      • Nasr T.
      • Ochoa S.D.
      • Nordin K.
      • Prasad M.S.
      • Labonne C.
      Interactions between Twist and other core epithelial-mesenchymal transition factors are controlled by GSK3-mediated phosphorylation.
      ). Notably, Sox5 depletion significantly reduced Smad1 occupancy of these regulatory elements, which is consistent with a role for Sox5 in contributing to Smad1 target specificity (Figures 4E and 4F). We found similar dependence on Sox5 for Smad1 occupancy of regulatory elements for Id3 and Msx1 (Figure S4E). Notably, when Chordin was used to prevent BMP R-Smad activation, Sox5 occupancy of EPK and Vent2 regulatory elements was reduced (Figures 4G and 4H), indicating that, on at least some elements, recruitment of Sox5 is also dependent on Smad1 binding.

      Discussion

      Here, we show that Sox5 is a DNA-binding cofactor that helps recruit BMP R-Smads (Smad1/5/8) to the target genes that are critical for several key aspects of ectodermal development. This role extends from the expression of AP2, Id3, and Vent2 in the early animal cells of blastula embryos, through the formation of the NPB, to the development of NC cells and cranial placodes. Other transcription factors, including znf423/OAZ, Schnurri, and Runx2, have been proposed to partner with Smad 1/5/8 (
      • Alliston T.
      • Choy L.
      • Ducy P.
      • Karsenty G.
      • Derynck R.
      TGF-β-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation.
      ,
      • Blitz I.L.
      • Cho K.W.Y.
      Finding partners: how BMPs select their targets.
      ,
      • Hata A.
      • Seoane J.
      • Lagna G.
      • Montalvo E.
      • Hemmati-Brivanlou A.
      • Massagué J.
      OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways.
      ,
      • Phimphilai M.
      • Zhao Z.
      • Boules H.
      • Roca H.
      • Franceschi R.T.
      BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype.
      ), although they lack the early embryonic expression and function that we show here for Sox5. Furthermore, Runx2 appears to function primarily in osteoblast formation (
      • Franceschi R.T.
      • Xiao G.
      Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways.
      ,
      • Komori T.
      • Yagi H.
      • Nomura S.
      • Yamaguchi A.
      • Sasaki K.
      • Deguchi K.
      • Shimizu Y.
      • Bronson R.T.
      • Gao Y.H.
      • Inada M.
      • et al.
      Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.
      ,
      • Otto F.
      • Thornell A.P.
      • Crompton T.
      • Denzel A.
      • Gilmour K.C.
      • Rosewell I.R.
      • Stamp G.W.
      • Beddington R.S.
      • Mundlos S.
      • Olsen B.R.
      • et al.
      Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.
      ). Znf423/OAZ is essential for central nervous system midline and olfactory neuron development (
      • Cheng L.E.
      • Reed R.R.
      Zfp423/OAZ participates in a developmental switch during olfactory neurogenesis.
      ,
      • Cheng L.E.
      • Zhang J.
      • Reed R.R.
      The transcription factor Zfp423/OAZ is required for cerebellar development and CNS midline patterning.
      ), and in addition to its proposed importance for BMP signaling, it has been shown to bind the Notch intracellular domain to regulate Notch-dependent gene expression (
      • Masserdotti G.
      • Badaloni A.
      • Green Y.S.
      • Croci L.
      • Barili V.
      • Bergamini G.
      • Vetter M.L.
      • Consalez G.G.
      ZFP423 coordinates Notch and bone morphogenetic protein signaling, selectively up-regulating Hes5 gene expression.
      ).
      Sox5, by contrast, meets all criteria for a broadly essential DNA-binding partner for BMP R-Smads. We show here that Sox5 is expressed in pluripotent blastula cells and then, subsequently, in NC cells, both regions where BMPs functions are essential. Intriguingly, Sox5 has long been known to play essential roles in other tissues where BMP signaling is prominent, including NC derivatives such as cartilage and melanoblasts (
      • Dy P.
      • Han Y.
      • Lefebvre V.
      Generation of mice harboring a Sox5 conditional null allele.
      ,
      • Lefebvre V.
      • Li P.
      • de Crombrugghe B.
      A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene.
      ,
      • Smits P.
      • Li P.
      • Mandel J.
      • Zhang Z.
      • Deng J.M.
      • Behringer R.R.
      • de Crombrugghe B.
      • Lefebvre V.
      The transcription factors L-Sox5 and Sox6 are essential for cartilage formation.
      ,
      • Stolt C.C.
      • Lommes P.
      • Hillgärtner S.
      • Wegner M.
      The transcription factor Sox5 modulates Sox10 function during melanocyte development.
      ), as well as oligodendrocytes (
      • Stolt C.C.
      • Schlierf A.
      • Lommes P.
      • Hillgärtner S.
      • Werner T.
      • Kosian T.
      • Sock E.
      • Kessaris N.
      • Richardson W.D.
      • Lefebvre V.
      • Wegner M.
      SoxD proteins influence multiple stages of oligodendrocyte development and modulate SoxE protein function.
      ), myoblasts (
      • Rescan P.-Y.
      • Ralliere C.
      A Sox5 gene is expressed in the myogenic lineage during trout embryonic development.
      ), and germ cells (
      • Denny P.
      • Swift S.
      • Connor F.
      • Ashworth A.
      An SRY-related gene expressed during spermatogenesis in the mouse encodes a sequence-specific DNA-binding protein.
      ), suggesting that it may function as a BMP R-Smad partner in these cell types as well. Notably, we demonstrate here that Sox5 is required for the activation of both endogenous BMP targets and BMP reporters in the early embryonic ectoderm. The requirement for BMP/Sox5 is first observed in the pluripotent cells of the blastula and later is seen in ectodermal cell types that arise from these precursors, particularly those at the NPB. We show that Sox5 physically interacts with BMP R-Smad complexes, as does the closely related protein Sox6, indicating that this interaction is a conserved feature of SoxD family proteins. This finding suggests that Sox6 likely also functions as a Smad1/5 partner and may do so in tissues where Sox5 is not expressed, and this would be an important area of future investigation. In the ectoderm, we find that Sox5 is required for Smad1 recruitment to target regulatory elements and that recruitment of Sox5 to these regulatory elements is attenuated when BMP signaling is blocked. Together, the findings reported here represent a significant advance in our understanding of the earliest events in embryogenesis, as they identify Sox5 as a long-sought DNA-binding partner for BMP R-Smads during regulation of plasticity and pattern in the early ectoderm.

      Experimental Procedures

      Embryological Methods

      Collection, injection, and in situ hybridization of Xenopus embryos were performed as previously described (
      • Bellmeyer A.
      • Krase J.
      • Lindgren J.
      • LaBonne C.
      The protooncogene c-myc is an essential regulator of neural crest formation in xenopus.
      ,
      • LaBonne C.
      • Bronner-Fraser M.
      Neural crest induction in Xenopus: evidence for a two-signal model.
      ). Results shown are representative of at least three independent experiments. Ectodermal explants were manually dissected from the animal pole of blastula (stage 8) embryos previously injected at the two-cell stage with the indicated mRNA or MO and cultured to the indicated stage before being processed for in situ hybridization.

      Luciferase Assays

      Luciferase reporter DNA was coinjected with Renilla luciferase DNA and the indicated mRNA or MO into both cells of two-cell embryos. Embryos were cultured to stage 10, collected as ten-embryo sets in triplicate, and assayed with a Dual-Luciferase Reporter Assay System (Promega) and Promega GloMax Luminometer. Error represents SD between triplicate sets. Unpaired two-tailed t tests were used to determine statistical significance.

      Western Blot Analysis

      For western blot analyses, injected embryos were cultured to stage 10 and lysed as in
      • Lee P.-C.
      • Taylor-Jaffe K.M.
      • Nordin K.M.
      • Prasad M.S.
      • Lander R.M.
      • LaBonne C.
      SUMOylated SoxE factors recruit Grg4 and function as transcriptional repressors in the neural crest.
      study in five embryo sets. Proteins were detected using the following antibodies against the corresponding affinity tag: Myc (9E10, Santa Cruz Biotechnology); Flag (Sigma-Aldrich); hemagglutinin (gift of R. Lamb, Northwestern University) or Actin (Sigma-Aldrich); Smad1 (sc-6031-R, Santa Cruz Biotechnology); phospho-Smad1 (9511, Cell Signaling Technology); phospho-Smad2 (3101, Cell Signaling). Proteins were detected using secondary antibodies conjugated to horseradish peroxidase (HRP) and enhanced chemiluminescence (GE Healthcare).

      Purification of GST Proteins and GST Pull-Down Assays

      GST proteins were expressed in the BL21 strain of E. coli, sonicated, and purified with glutathione-agarose beads (Sigma-Aldrich). Flag- or Myc-epitope-tagged Sox5, Sox5 deletions, and Sox6 constructs expressed Xenopus embryos and lysates prepared at stage 10.5. Bound proteins were released by boiling in SDS, analyzed by SDS-PAGE, and detected using secondary antibodies conjugated to HRP and enhanced chemiluminescence (GE Healthcare).

      ChIP

      ChIP assays were carried out as described elsewhere (
      • Lander R.
      • Nasr T.
      • Ochoa S.D.
      • Nordin K.
      • Prasad M.S.
      • Labonne C.
      Interactions between Twist and other core epithelial-mesenchymal transition factors are controlled by GSK3-mediated phosphorylation.
      ) using 50 stage-13 embryos per immunoprecipitation (IP). IP for myc-tagged proteins was performed using α-Myc (Sigma #C3956) on Protein G magnetic beads (Dynabeads, Invitrogen). Samples were processed as described elsewhere (Supplemental Experimental Procedures). qPCR was performed using SYBR Premix (Clontech) and primers for regulatory elements of epidermal keratin, Vent2, Id3, Msx1, and eEF1α. Primer sequences are provided in the Supplemental Experimental Procedures. Fold enrichment of Smad1 or Sox5 relative to uninjected control was calculated using the ΔΔCT method and represented as a mean from three separate biological replicates, with error bars representing SEM. An unpaired two-tailed t test was used to determine the statistical significance.

      DNA Constructs

      Two forms of the primary “long” isoform of Sox5 have been reported in Xenopus laevis and appear to be splice variants. The first (BC142559) is encoded by an expressed sequence tag obtained from the I.M.A.G.E Consortium and is referred to here as Sox5. A second Sox5 isoform (AB682776) with an extended C terminus was subsequently reported (
      • Suzuki T.
      • Kusakabe M.
      • Nakayama K.
      • Nishida E.
      The protein kinase MLTK regulates chondrogenesis by inducing the transcription factor Sox6.
      ). We confirmed via PCR that both forms are expressed in early Xenopus embryos, and we have found no functional differences between them (Figures 1 and S3; data not shown). Full-length Sox5 is sometimes referred to as L-Sox5 due to the existence of a testes-specific “short” splice variant missing the N-terminal coiled-coil domain; however, it has been determined that “Sox5” is the more appropriate nomenclature (
      • Lefebvre V.
      The SoxD transcription factors—Sox5, Sox6, and Sox13—are key cell fate modulators.
      ). Sox5 is a member of the SoxD SRY family but is distinct from previously characterized Xenopus SoxD (
      • Ito M.
      Function and molecular evolution of mammalian Sox15, a singleton in the SoxG group of transcription factors.
      ,
      • Mizuseki K.
      • Kishi M.
      • Shiota K.
      • Nakanishi S.
      • Sasai Y.
      SoxD: an essential mediator of induction of anterior neural tissues in Xenopus embryos.
      ), which is a member of the SoxG family.

      Acknowledgments

      We thank Joe Nguyen, Maneeshi Prasad, and Caroline Haldin for technical assistance and members of the lab for helpful discussions. We thank Caroline Hill, Gerald Thompsen, and Ken Cho for sharing plasmids. K.N. was supported by National Institute of Dental and Craniofacial Research National Research Service Award F31DE021922 and by NIH grant T32CA009560-24. This work was supported by NIH grant R01GM114058 to C.L.

      Supplemental Information

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