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Caudal-related Homeobox (Cdx) Protein-dependent Integration of Canonical Wnt Signaling on Paired-box 3 (Pax3) Neural Crest Enhancer*

  • Oraly Sanchez- Ferras
    Footnotes
    Affiliations
    Molecular Genetics of Development, Department of Biological Sciences, and BioMed Research Center, Faculty of Sciences, University of Quebec, Montreal, Quebec H2X 3Y7, Canada
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  • Baptiste Coutaud
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    Affiliations
    Molecular Genetics of Development, Department of Biological Sciences, and BioMed Research Center, Faculty of Sciences, University of Quebec, Montreal, Quebec H2X 3Y7, Canada
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  • Taraneh Djavanbakht Samani
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    Affiliations
    Molecular Genetics of Development, Department of Biological Sciences, and BioMed Research Center, Faculty of Sciences, University of Quebec, Montreal, Quebec H2X 3Y7, Canada
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  • Isabelle Tremblay
    Footnotes
    Affiliations
    Molecular Genetics of Development, Department of Biological Sciences, and BioMed Research Center, Faculty of Sciences, University of Quebec, Montreal, Quebec H2X 3Y7, Canada
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  • Ouliana Souchkova
    Affiliations
    Molecular Genetics of Development, Department of Biological Sciences, and BioMed Research Center, Faculty of Sciences, University of Quebec, Montreal, Quebec H2X 3Y7, Canada
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  • Nicolas Pilon
    Correspondence
    To whom correspondence should be addressed: Dept. of Biological Sciences and BioMed Research Center, Faculty of Sciences, University of Quebec at Montreal, 141 President-Kennedy Ave., Montreal, Quebec H2X 3Y7, Canada. Tel.: 514-987-3000 (ext. 3342); Fax: 514-987-4647
    Affiliations
    Molecular Genetics of Development, Department of Biological Sciences, and BioMed Research Center, Faculty of Sciences, University of Quebec, Montreal, Quebec H2X 3Y7, Canada
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  • Author Footnotes
    * This work was supported by Canadian Institutes of Health Research Grants DCO190GP and IHD-94366 as well as a new investigator grant from the Banting Research Foundation.
    This article contains supplemental Table S1 and Figs. S1 and S2.
    1 Recipient of an Alexander-Graham-Bell scholarship from the Natural Science and Engineering Research Council (NSERC) of Canada.
    2 Supported by University of Quebec at Montreal scholarships.
    3 Supported by an NSERC undergraduate research award.
Open AccessPublished:March 28, 2012DOI:https://doi.org/10.1074/jbc.M112.356394
      One of the earliest events in neural crest development takes place at the neural plate border and consists in the induction of Pax3 expression by posteriorizing Wnt·β-catenin signaling. The molecular mechanism of this regulation is not well understood, but several observations suggest a role for posteriorizing Cdx transcription factors (Cdx1/2/4) in this process. Cdx genes are known as integrators of posteriorizing signals from Wnt, retinoic acid, and FGF pathways. In this work, we report that Wnt-mediated regulation of murine Pax3 expression is indirect and involves Cdx proteins as intermediates. We show that Pax3 transcripts co-localize with Cdx proteins in the posterior neurectoderm and that neural Pax3 expression is reduced in Cdx1-null embryos. Using Wnt3a-treated P19 cells and neural crest-derived Neuro2a cells, we demonstrate that Pax3 expression is induced by the Wnt-Cdx pathway. Co-transfection analyses, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transgenic studies further indicate that Cdx proteins operate via direct binding to an evolutionarily conserved neural crest enhancer of the Pax3 proximal promoter. Taken together, these results suggest a novel neural function for Cdx proteins within the gene regulatory network controlling neural crest development.

      Introduction

      Pax3 is a paired-box homeodomain transcription factor essential for normal neural crest (NC),
      The abbreviations used are: NC
      neural crest
      NT
      neural tube
      NCC
      NC cell(s)
      PNP
      posterior neural plate
      NCE
      neural crest enhancer
      RA
      retinoic acid
      CdxBS
      Cdx binding site
      X-gal
      5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside
      CHX
      cycloheximide
      W3a
      Wnt3a-conditioned medium
      Ctl
      control-conditioned medium
      en
      embryonic day n.
      neural tube (NT) and skeletal muscle development. In mice, homozygous Pax3 loss of function, as seen in Splotch (Sp) mutants, leads to early embryonic lethality due to its role in NC cells (NCC). Although Pax3+/Sp mice only display pigmentation anomalies (white belly spot), most Pax3Sp/Sp embryos die around embryonic day 14.0 (e14.0) due to a severe NC defect leading to lack of outflow tract septation and heart failure (
      • Li J.
      • Liu K.C.
      • Jin F.
      • Lu M.M.
      • Epstein J.A.
      Transgenic rescue of congenital heart disease and spina bifida in Splotch mice.
      ). Pax3Sp/Sp mice also display other severe anomalies, including spinal ganglia malformations, intestinal aganglionosis, and posterior NT defects (spina bifida) (
      • Auerbach R.
      Analysis of the developmental effects of a lethal mutation in the house mouse.
      ). At the molecular level, Pax3 plays a critical role in the gene regulatory network controlling NCC development downstream of canonical Wnt signals. Together with members of the Msx, Dlx, and Zic families, Pax3 specifies the neural plate border and promotes induction of NCC (
      • Meulemans D.
      • Bronner-Fraser M.
      Gene-regulatory interactions in neural crest evolution and development.
      ,
      • Betancur P.
      • Bronner-Fraser M.
      • Sauka-Spengler T.
      Assembling neural crest regulatory circuits into a gene regulatory network.
      ,
      • Monsoro-Burq A.H.
      • Wang E.
      • Harland R.
      Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction.
      ). At later stages, Pax3 also controls survival of dorsal NT progenitors through stimulation of p53 degradation (
      • Wang X.D.
      • Morgan S.C.
      • Loeken M.R.
      Pax3 stimulates p53 ubiquitination and degradation independent of transcription.
      ,
      • Pani L.
      • Horal M.
      • Loeken M.R.
      Rescue of neural tube defects in Pax-3-deficient embryos by p53 loss of function. Implications for Pax-3-dependent development and tumorigenesis.
      ). In humans, heterozygous PAX3 mutations have been associated with Waardenburg syndrome, which is characterized by NC defects such as cranio-facial and pigmentary anomalies (
      • Baldwin C.T.
      • Hoth C.F.
      • Amos J.A.
      • da-Silva E.O.
      • Milunsky A.
      An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome.
      ,
      • Baldwin C.T.
      • Hoth C.F.
      • Macina R.A.
      • Milunsky A.
      Mutations in PAX3 that cause Waardenburg syndrome type I. Ten new mutations and review of the literature.
      ,
      • Tassabehji M.
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      • Newton V.E.
      • Harris R.
      • Balling R.
      • Gruss P.
      • Strachan T.
      Waardenburg's syndrome patients have mutations in the human homologue of the Pax-3 paired box gene.
      ).
      Neural Pax3 expression begins at the early somite stage (around e8.25) prior to initiation of NT closure. At this stage, Pax3 transcripts are detected on the lateral borders of both the anterior neural plate and posterior neural plate (PNP) (
      • Goulding M.D.
      • Chalepakis G.
      • Deutsch U.
      • Erselius J.R.
      • Gruss P.
      Pax-3, a novel murine DNA binding protein expressed during early neurogenesis.
      ). Following neural plate bending in a closed NT (from e8.5 onward), Pax3 transcripts are then detected in the dorsal neurectoderm, including pre-migratory NCC, in an almost continuous manner along the anterior-posterior (AP) axis. Indeed, strong Pax3 expression is detected in two large domains extending 1) from the forebrain down to rhombomere 4 and 2) from rhombomere 6 down to the rostral half of the PNP. Although detectable, Pax3 expression is clearly much weaker in rhombomere 5. At later stages, caudal Pax3 expression follows progression of posterior elongation and is maintained in the dorsal half of the closed neural tube until e14.5. Pax3 expression is also detected in a subset of migratory NCC contributing to the cardiac outflow tract, peripheral, and enteric nervous systems as well as in melanocytes but is generally down-regulated as NCC differentiate.
      Regulatory sequences sufficient to mediate induction and dorsal restriction of Pax3 expression in the hindbrain and trunk are contained in the proximal 1.6-kb promoter (
      • Natoli T.A.
      • Ellsworth M.K.
      • Wu C.
      • Gross K.W.
      • Pruitt S.C.
      Positive and negative DNA sequence elements are required to establish the pattern of Pax3 expression.
      ). Deletion analysis of this promoter has revealed that a block of 674 bp containing two evolutionarily conserved regions of ∼250 bp, called neural crest enhancer 1 and 2 (NCE1 and -2), is sufficient to drive expression in the dorsal NT and NCC (
      • Milewski R.C.
      • Chi N.C.
      • Li J.
      • Brown C.
      • Lu M.M.
      • Epstein J.A.
      Identification of minimal enhancer elements sufficient for Pax3 expression in neural crest and implication of Tead2 as a regulator of Pax3.
      ). NCE1 bears Pbx binding sites (activated by Pbx1-containing transcriptional complexes), which appear to be specifically required to control Pax3 expression in the hindbrain (
      • Chang C.P.
      • Stankunas K.
      • Shang C.
      • Kao S.C.
      • Twu K.Y.
      • Cleary M.L.
      Pbx1 functions in distinct regulatory networks to pattern the great arteries and cardiac outflow tract.
      ,
      • Pruitt S.C.
      • Bussman A.
      • Maslov A.Y.
      • Natoli T.A.
      • Heinaman R.
      Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro in vivo.
      ). NCE2 contains a Tead binding site that was shown to be required for the activity of the whole 674-bp NCE in e10.5 transgenic embryos (
      • Milewski R.C.
      • Chi N.C.
      • Li J.
      • Brown C.
      • Lu M.M.
      • Epstein J.A.
      Identification of minimal enhancer elements sufficient for Pax3 expression in neural crest and implication of Tead2 as a regulator of Pax3.
      ). In addition, both NCE1 and NCE2 contain a binding site for Pou class III members, and mutation of these sites leads to reduced NT activity of the 1.6-kb promoter in e9.5 transgenic embryos (
      • Pruitt S.C.
      • Bussman A.
      • Maslov A.Y.
      • Natoli T.A.
      • Heinaman R.
      Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro in vivo.
      ). On the other hand, Pax3 expression is induced by posteriorizing Wnt signals and dorsally restricted in response to dorso-ventral patterning signals, such as Sonic Hedgehog (Shh) (
      • Monsoro-Burq A.H.
      • Wang E.
      • Harland R.
      Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction.
      ,
      • Taneyhill L.A.
      • Bronner-Fraser M.
      Dynamic alterations in gene expression after Wnt-mediated induction of avian neural crest.
      ,
      • Goulding M.D.
      • Lumsden A.
      • Gruss P.
      Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord.
      ,
      • Bang A.G.
      • Papalopulu N.
      • Kintner C.
      • Goulding M.D.
      Expression of Pax-3 is initiated in the early neural plate by posteriorizing signals produced by the organizer and by posterior non-axial mesoderm.
      ,
      • Bang A.G.
      • Papalopulu N.
      • Goulding M.D.
      • Kintner C.
      Expression of Pax-3 in the lateral neural plate is dependent on a Wnt-mediated signal from posterior nonaxial mesoderm.
      ,
      • de Crozé N.
      • Maczkowiak F.
      • Monsoro-Burq A.H.
      Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network.
      ). However, how the canonical Wnt and Shh signals are integrated at the transcriptional level is still unclear.
      The vertebrate Cdx genes (Cdx1, Cdx2, and Cdx4) are related to Drosophila caudal (cad) (
      • Brooke N.M.
      • Garcia-Fernàndez J.
      • Holland P.W.
      The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster.
      ), and their gene products have conserved the ancestral ability to specify the posterior embryo and pattern the AP axis. Murine Cdx genes are sequentially activated in ectodermal and mesodermal cells of the primitive streak around e7.25, with Cdx1 activated first and Cdx4 activated last (
      • Beck F.
      • Erler T.
      • Russell A.
      • James R.
      Expression of Cdx-2 in the mouse embryo and placenta. Possible role in patterning of the extra-embryonic membranes.
      ,
      • Meyer B.I.
      • Gruss P.
      Mouse Cdx-1 expression during gastrulation.
      ,
      • Gamer L.W.
      • Wright C.V.
      Murine Cdx-4 bears striking similarities to the Drosophila caudal gene in its homeodomain sequence and early expression pattern.
      ). At e8.5, all Cdx genes are expressed in the caudal embryo and form a nested set along the AP axis. Whereas Cdx1 and Cdx2 exhibit an almost perfect overlap in expression around the hindbrain/spinal cord boundary, Cdx4 is expressed slightly more posterior at this stage. The most anterior domain of Cdx expression appears to be restricted to the dorsal NT, with Cdx1 protein expressed in NCC emigrating from this domain (
      • Meyer B.I.
      • Gruss P.
      Mouse Cdx-1 expression during gastrulation.
      ,
      • Gaunt S.J.
      • Drage D.
      • Trubshaw R.C.
      Cdx4/LacZ and Cdx2/LacZ protein gradients formed by decay during gastrulation in the mouse.
      ). This anterior limit of expression regresses concomitantly with axial elongation, persisting in the caudal embryo until e10.5 for Cdx1 and Cdx4 and until e12.5 for Cdx2. All three Cdx genes are also expressed in the developing hindgut epithelium with Cdx1 and Cdx2 expression maintained postnatally (
      • Beck F.
      Homeobox genes in gut development.
      ).
      Cdx gene expression is regulated by posteriorizing signals from Wnt, retinoic acid (RA), and fibroblast growth factor (FGF) pathways in multiple species (
      • Prinos P.
      • Joseph S.
      • Oh K.
      • Meyer B.I.
      • Gruss P.
      • Lohnes D.
      Multiple pathways governing Cdx1 expression during murine development.
      ,
      • Pilon N.
      • Oh K.
      • Sylvestre J.R.
      • Savory J.G.
      • Lohnes D.
      Wnt signaling is a key mediator of Cdx1 expression in vivo.
      ,
      • Pilon N.
      • Oh K.
      • Sylvestre J.R.
      • Bouchard N.
      • Savory J.
      • Lohnes D.
      Cdx4 is a direct target of the canonical Wnt pathway.
      ,
      • Lickert H.
      • Domon C.
      • Huls G.
      • Wehrle C.
      • Duluc I.
      • Clevers H.
      • Meyer B.I.
      • Freund J.N.
      • Kemler R.
      Wnt/β-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine.
      ,
      • Lengerke C.
      • Schmitt S.
      • Bowman T.V.
      • Jang I.H.
      • Maouche-Chretien L.
      • McKinney-Freeman S.
      • Davidson A.J.
      • Hammerschmidt M.
      • Rentzsch F.
      • Green J.B.
      • Zon L.I.
      • Daley G.Q.
      BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway.
      ,
      • Keenan I.D.
      • Sharrard R.M.
      • Isaacs H.V.
      FGF signal transduction and the regulation of Cdx gene expression.
      ,
      • Ikeya M.
      • Takada S.
      Wnt-3a is required for somite specification along the anteroposterior axis of the mouse embryo and for regulation of Cdx-1 expression.
      ,
      • Houle M.
      • Sylvestre J.R.
      • Lohnes D.
      Retinoic acid regulates a subset of Cdx1 function in vivo.
      ,
      • Houle M.
      • Prinos P.
      • Iulianella A.
      • Bouchard N.
      • Lohnes D.
      Retinoic acid regulation of Cdx1. An indirect mechanism for retinoids and vertebral specification.
      ,
      • Faas L.
      • Isaacs H.V.
      Overlapping functions of Cdx1, Cdx2, and Cdx4 in the development of the amphibian Xenopus tropicalis.
      ,
      • Bel-Vialar S.
      • Itasaki N.
      • Krumlauf R.
      Initiating Hox gene expression. In the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups.
      ,
      • Shimizu T.
      • Bae Y.K.
      • Muraoka O.
      • Hibi M.
      Interaction of Wnt and caudal-related genes in zebrafish posterior body formation.
      ). Among these posteriorizing signals, the evolutionarily conserved role of the canonical Wnt pathway in Cdx regulation is the best characterized. Indeed, both Cdx1 and Cdx4 have been clearly identified as direct targets of the Wnt·β-catenin pathway (
      • Prinos P.
      • Joseph S.
      • Oh K.
      • Meyer B.I.
      • Gruss P.
      • Lohnes D.
      Multiple pathways governing Cdx1 expression during murine development.
      ,
      • Pilon N.
      • Oh K.
      • Sylvestre J.R.
      • Bouchard N.
      • Savory J.
      • Lohnes D.
      Cdx4 is a direct target of the canonical Wnt pathway.
      ,
      • Lickert H.
      • Domon C.
      • Huls G.
      • Wehrle C.
      • Duluc I.
      • Clevers H.
      • Meyer B.I.
      • Freund J.N.
      • Kemler R.
      Wnt/β-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine.
      ). Moreover, other data suggest that Cdx2 is also responsive to canonical Wnt signals, although evidence for a direct regulation is sparse (
      • Benahmed F.
      • Gross I.
      • Gaunt S.J.
      • Beck F.
      • Jehan F.
      • Domon-Dell C.
      • Martin E.
      • Kedinger M.
      • Freund J.N.
      • Duluc I.
      Multiple regulatory regions control the complex expression pattern of the mouse Cdx2 homeobox gene.
      ,
      • He S.
      • Pant D.
      • Schiffmacher A.
      • Meece A.
      • Keefer C.L.
      Lymphoid enhancer factor 1-mediated Wnt signaling promotes the initiation of trophoblast lineage differentiation in mouse embryonic stem cells.
      ,
      • Joo J.H.
      • Taxter T.J.
      • Munguba G.C.
      • Kim Y.H.
      • Dhaduvai K.
      • Dunn N.W.
      • Degan W.J.
      • Oh S.P.
      • Sugrue S.P.
      Pinin modulates expression of an intestinal homeobox gene, Cdx2, and plays an essential role for small intestinal morphogenesis.
      ,
      • Marikawa Y.
      • Tamashiro D.A.
      • Fujita T.C.
      • Alarcón V.B.
      Aggregated P19 mouse embryonal carcinoma cells as a simple in vitro model to study the molecular regulations of mesoderm formation and axial elongation morphogenesis.
      ,
      • Nordström U.
      • Maier E.
      • Jessell T.M.
      • Edlund T.
      An early role for WNT signaling in specifying neural patterns of Cdx Hox gene expression and motor neuron subtype identity.
      ,
      • Saegusa M.
      • Hashimura M.
      • Kuwata T.
      • Hamano M.
      • Wani Y.
      • Okayasu I.
      A functional role of Cdx2 in β-catenin signaling during transdifferentiation in endometrial carcinomas.
      ,
      • Zhao X.
      • Duester G.
      Effect of retinoic acid signaling on Wnt/β-catenin and FGF signaling during body axis extension.
      ). In addition, Cdx proteins can interact with the Lef1-β-catenin transcriptional effector of the canonical Wnt pathway (
      • Béland M.
      • Pilon N.
      • Houle M.
      • Oh K.
      • Sylvestre J.R.
      • Prinos P.
      • Lohnes D.
      Cdx1 autoregulation is governed by a novel Cdx1-LEF1 transcription complex.
      ).
      Our understanding of Cdx function has long been hampered by the functional redundancy between Cdx members and the vital role of Cdx2 during implantation (
      • Strumpf D.
      • Mao C.A.
      • Yamanaka Y.
      • Ralston A.
      • Chawengsaksophak K.
      • Beck F.
      • Rossant J.
      Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst.
      ,
      • Savory J.G.
      • Pilon N.
      • Grainger S.
      • Sylvestre J.R.
      • Béland M.
      • Houle M.
      • Oh K.
      • Lohnes D.
      Cdx1 and Cdx2 are functionally equivalent in vertebral patterning.
      ). Recent development of a conditional Cdx2 allele (
      • Savory J.G.
      • Bouchard N.
      • Pierre V.
      • Rijli F.M.
      • De Repentigny Y.
      • Kothary R.
      • Lohnes D.
      Cdx2 regulation of posterior development through non-Hox targets.
      ,
      • Gao N.
      • White P.
      • Kaestner K.H.
      Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2.
      ) and analysis of Cdx double mutants (
      • Young T.
      • Rowland J.E.
      • van de Ven C.
      • Bialecka M.
      • Novoa A.
      • Carapuco M.
      • van Nes J.
      • de Graaff W.
      • Duluc I.
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      • Beck F.
      • Mallo M.
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      Cdx Hox genes differentially regulate posterior axial growth in mammalian embryos.
      ,
      • van Nes J.
      • de Graaff W.
      • Lebrin F.
      • Gerhard M.
      • Beck F.
      • Deschamps J.
      The Cdx4 mutation affects axial development and reveals an essential role of Cdx genes in the ontogenesis of the placental labyrinth in mice.
      ,
      • van den Akker E.
      • Forlani S.
      • Chawengsaksophak K.
      • de Graaff W.
      • Beck F.
      • Meyer B.I.
      • Deschamps J.
      Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation.
      ,
      • Savory J.G.
      • Mansfield M.
      • Rijli F.M.
      • Lohnes D.
      Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7.
      ) has demonstrated important regulatory roles for Cdx proteins in several processes during mouse embryogenesis. In the mesoderm, Cdx proteins regulate axial patterning, axial elongation, and somitogenesis in addition to placentogenesis and hematopoiesis. An important part of the Cdx function is executed through direct regulation of diverse Hox genes (
      • Pilon N.
      • Oh K.
      • Sylvestre J.R.
      • Savory J.G.
      • Lohnes D.
      Wnt signaling is a key mediator of Cdx1 expression in vivo.
      ,
      • Young T.
      • Rowland J.E.
      • van de Ven C.
      • Bialecka M.
      • Novoa A.
      • Carapuco M.
      • van Nes J.
      • de Graaff W.
      • Duluc I.
      • Freund J.N.
      • Beck F.
      • Mallo M.
      • Deschamps J.
      Cdx Hox genes differentially regulate posterior axial growth in mammalian embryos.
      ,
      • van Nes J.
      • de Graaff W.
      • Lebrin F.
      • Gerhard M.
      • Beck F.
      • Deschamps J.
      The Cdx4 mutation affects axial development and reveals an essential role of Cdx genes in the ontogenesis of the placental labyrinth in mice.
      ,
      • van den Akker E.
      • Forlani S.
      • Chawengsaksophak K.
      • de Graaff W.
      • Beck F.
      • Meyer B.I.
      • Deschamps J.
      Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation.
      ,
      • Wang Y.
      • Yabuuchi A.
      • McKinney-Freeman S.
      • Ducharme D.M.
      • Ray M.K.
      • Chawengsaksophak K.
      • Archer T.K.
      • Daley G.Q.
      Cdx gene deficiency compromises embryonic hematopoiesis in the mouse.
      ,
      • Subramanian V.
      • Meyer B.I.
      • Gruss P.
      Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of Hox genes.
      ). However, recent work has shown that a significant proportion of the Cdx function is also fulfilled via direct regulation of several non-Hox targets (
      • Savory J.G.
      • Bouchard N.
      • Pierre V.
      • Rijli F.M.
      • De Repentigny Y.
      • Kothary R.
      • Lohnes D.
      Cdx2 regulation of posterior development through non-Hox targets.
      ,
      • Grainger S.
      • Lam J.
      • Savory J.G.
      • Mears A.J.
      • Rijli F.M.
      • Lohnes D.
      Cdx regulates Dll1 in multiple lineages.
      ). In the endoderm, Cdx proteins are involved in intestinal patterning and cell differentiation via Hox-dependent and -independent mechanisms (
      • Gao N.
      • White P.
      • Kaestner K.H.
      Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2.
      ,
      • Verzi M.P.
      • Shin H.
      • Ho L.L.
      • Liu X.S.
      • Shivdasani R.A.
      Essential and redundant functions of caudal family proteins in activating adult intestinal genes.
      ,
      • Suh E.
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      • Taylor J.
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      A homeodomain protein related to caudal regulates intestine-specific gene transcription.
      ,
      • Grainger S.
      • Savory J.G.
      • Lohnes D.
      Cdx2 regulates patterning of the intestinal epithelium.
      ,
      • Crissey M.A.
      • Guo R.J.
      • Funakoshi S.
      • Kong J.
      • Liu J.
      • Lynch J.P.
      Cdx2 levels modulate intestinal epithelium maturity and Paneth cell development.
      ).
      The function of Cdx members in the neurectoderm is less well understood. As in the mesoderm and endoderm, Cdx proteins control neurectoderm AP patterning via Hox-dependent and -independent mechanisms (
      • Bel-Vialar S.
      • Itasaki N.
      • Krumlauf R.
      Initiating Hox gene expression. In the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups.
      ,
      • Nordström U.
      • Maier E.
      • Jessell T.M.
      • Edlund T.
      An early role for WNT signaling in specifying neural patterns of Cdx Hox gene expression and motor neuron subtype identity.
      ,
      • Sturgeon K.
      • Kaneko T.
      • Biemann M.
      • Gauthier A.
      • Chawengsaksophak K.
      • Cordes S.P.
      Cdx1 refines positional identity of the vertebrate hindbrain by directly repressing Mafb expression.
      ,
      • Skromne I.
      • Thorsen D.
      • Hale M.
      • Prince V.E.
      • Ho R.K.
      Repression of the hindbrain developmental program by Cdx factors is required for the specification of the vertebrate spinal cord.
      ,
      • Isaacs H.V.
      • Pownall M.E.
      • Slack J.M.
      Regulation of Hox gene expression and posterior development by the Xenopus caudal homologue Xcad3.
      ). Multiple gain- or loss-of-function mouse models have also demonstrated that Cdx proteins are important for proper formation of the NT and NC-derived spinal ganglia, but whether they do so in a tissue-autonomous or -non-autonomous (via the mesoderm) manner remains an open question (
      • van den Akker E.
      • Forlani S.
      • Chawengsaksophak K.
      • de Graaff W.
      • Beck F.
      • Meyer B.I.
      • Deschamps J.
      Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation.
      ,
      • Savory J.G.
      • Mansfield M.
      • Rijli F.M.
      • Lohnes D.
      Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7.
      ,
      • van de Ven C.
      • Bialecka M.
      • Neijts R.
      • Young T.
      • Rowland J.E.
      • Stringer E.J.
      • Van Rooijen C.
      • Meijlink F.
      • Nóvoa A.
      • Freund J.N.
      • Mallo M.
      • Beck F.
      • Deschamps J.
      Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone.
      ,
      • Gaunt S.J.
      • Drage D.
      • Trubshaw R.C.
      Increased Cdx protein dose effects upon axial patterning in transgenic lines of mice.
      ,
      • Charité J.
      • de Graaff W.
      • Consten D.
      • Reijnen M.J.
      • Korving J.
      • Deschamps J.
      Transducing positional information to the Hox genes. Critical interaction of cdx gene products with position-sensitive regulatory elements.
      ). Nevertheless, analysis of neurectoderm-specific Cdx loss of function in ascidian embryos demonstrates that Cdx proteins may impact NT and NC formation in a tissue-autonomous manner. Indeed, such mutant embryos display NT defects as well as an absence of pigment cells, which are derived from NC-like cells (
      • Mita K.
      • Fujiwara S.
      Nodal regulates neural tube formation in the Ciona intestinalis embryo.
      ,
      • Jeffery W.R.
      • Strickler A.G.
      • Yamamoto Y.
      Migratory neural crest-like cells form body pigmentation in a urochordate embryo.
      ).
      In this study, we investigated the possibility that Cdx proteins might convey the posteriorizing Wnt signals to Pax3. We found that murine Pax3 is in fact an indirect Wnt target and that Cdx proteins can directly activate neural Pax3 expression at least via the previously described NCE2 (
      • Milewski R.C.
      • Chi N.C.
      • Li J.
      • Brown C.
      • Lu M.M.
      • Epstein J.A.
      Identification of minimal enhancer elements sufficient for Pax3 expression in neural crest and implication of Tead2 as a regulator of Pax3.
      ). Altogether, our data strengthen the idea that Cdx members are involved in both NT and NCC development in a tissue-autonomous manner downstream of Wnt signals.

      DISCUSSION

      We presented data indicating that the dorsal NT/NCC marker Pax3 is a direct target of the Cdx proteins downstream of canonical Wnt signals. Cdx proteins convey canonical Wnt signals to the proximal Pax3 promoter through direct binding to Cdx binding sites located in the evolutionarily conserved NCE2. These Cdx binding sites are essential both for Cdx-mediated transactivation of NCE2 in cell culture experiments and for expression of a NCE2 reporter in the dorsal NT and NCC of e9.5 transgenic embryos, supporting the existence of the Wnt-Cdx-Pax3 pathway in vivo.

      Wnt-mediated Induction of Pax3 Expression at Neural Plate Border

      Although several studies have reported that Pax3 is a posterior Wnt-induced gene, very little is known regarding the possible mechanism of this regulation (
      • Monsoro-Burq A.H.
      • Wang E.
      • Harland R.
      Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction.
      ,
      • Taneyhill L.A.
      • Bronner-Fraser M.
      Dynamic alterations in gene expression after Wnt-mediated induction of avian neural crest.
      ,
      • Bang A.G.
      • Papalopulu N.
      • Goulding M.D.
      • Kintner C.
      Expression of Pax-3 in the lateral neural plate is dependent on a Wnt-mediated signal from posterior nonaxial mesoderm.
      ,
      • de Crozé N.
      • Maczkowiak F.
      • Monsoro-Burq A.H.
      Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network.
      ). Our data now indicate that induction of murine Pax3 expression by canonical Wnt signals is indirect, involving Cdx proteins as intermediaries. In this regard, it is interesting to note that all three Cdx genes are direct targets of Wnt·β-catenin signaling in P19 cells. Although this was expected for Cdx1 and Cdx4 (
      • Prinos P.
      • Joseph S.
      • Oh K.
      • Meyer B.I.
      • Gruss P.
      • Lohnes D.
      Multiple pathways governing Cdx1 expression during murine development.
      ,
      • Pilon N.
      • Oh K.
      • Sylvestre J.R.
      • Savory J.G.
      • Lohnes D.
      Wnt signaling is a key mediator of Cdx1 expression in vivo.
      ,
      • Pilon N.
      • Oh K.
      • Sylvestre J.R.
      • Bouchard N.
      • Savory J.
      • Lohnes D.
      Cdx4 is a direct target of the canonical Wnt pathway.
      ,
      • Lickert H.
      • Domon C.
      • Huls G.
      • Wehrle C.
      • Duluc I.
      • Clevers H.
      • Meyer B.I.
      • Freund J.N.
      • Kemler R.
      Wnt/β-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine.
      ), such an outcome was somehow surprising for Cdx2, given a previous report indicating that exogenous Wnt3a can specifically induce Cdx1 but not Cdx2 in embryo culture (
      • Prinos P.
      • Joseph S.
      • Oh K.
      • Meyer B.I.
      • Gruss P.
      • Lohnes D.
      Multiple pathways governing Cdx1 expression during murine development.
      ). However, this result is in agreement with more recent work suggesting that Cdx2 is also a direct target of canonical Wnt signals (
      • Benahmed F.
      • Gross I.
      • Gaunt S.J.
      • Beck F.
      • Jehan F.
      • Domon-Dell C.
      • Martin E.
      • Kedinger M.
      • Freund J.N.
      • Duluc I.
      Multiple regulatory regions control the complex expression pattern of the mouse Cdx2 homeobox gene.
      ,
      • He S.
      • Pant D.
      • Schiffmacher A.
      • Meece A.
      • Keefer C.L.
      Lymphoid enhancer factor 1-mediated Wnt signaling promotes the initiation of trophoblast lineage differentiation in mouse embryonic stem cells.
      ,
      • Joo J.H.
      • Taxter T.J.
      • Munguba G.C.
      • Kim Y.H.
      • Dhaduvai K.
      • Dunn N.W.
      • Degan W.J.
      • Oh S.P.
      • Sugrue S.P.
      Pinin modulates expression of an intestinal homeobox gene, Cdx2, and plays an essential role for small intestinal morphogenesis.
      ,
      • Marikawa Y.
      • Tamashiro D.A.
      • Fujita T.C.
      • Alarcón V.B.
      Aggregated P19 mouse embryonal carcinoma cells as a simple in vitro model to study the molecular regulations of mesoderm formation and axial elongation morphogenesis.
      ,
      • Saegusa M.
      • Hashimura M.
      • Kuwata T.
      • Hamano M.
      • Wani Y.
      • Okayasu I.
      A functional role of Cdx2 in β-catenin signaling during transdifferentiation in endometrial carcinomas.
      ).
      Our data challenge a recent report showing that Pax3 expression in the dorsal NT can also be regulated by another evolutionarily conserved enhancer located in intron 4 (named ECR2) and described as containing multiple putative Lef/Tcf binding sites (
      • Degenhardt K.R.
      • Milewski R.C.
      • Padmanabhan A.
      • Miller M.
      • Singh M.K.
      • Lang D.
      • Engleka K.A.
      • Wu M.
      • Li J.
      • Zhou D.
      • Antonucci N.
      • Li L.
      • Epstein J.A.
      Distinct enhancers at the Pax3 locus can function redundantly to regulate neural tube and neural crest expressions.
      ). Indeed, it was reported that mutation of these putative binding sites abrogates ECR2 activity in transgenic zebrafishes. However, this mutated transgene was not assayed in mice, and these putative binding sites were not shown to be bound by Lef/Tcf proteins. Because the consensus Lef/Tcf binding site exhibits rather low binding specificity for HMG-box proteins, this raises the possibility that putative Lef/Tcf binding sites identified in Pax3 ECR2 are not bound by Lef/Tcf proteins but rather by other HMG-box proteins, such as Sox members (
      • Huang B.L.
      • Brugger S.M.
      • Lyons K.M.
      Stage-specific control of connective tissue growth factor (CTGF/CCN2) expression in chondrocytes by Sox9 and β-catenin.
      ,
      • Kormish J.D.
      • Sinner D.
      • Zorn A.M.
      Interactions between SOX factors and Wnt/β-catenin signaling in development and disease.
      ,
      • Kuwabara T.
      • Hsieh J.
      • Muotri A.
      • Yeo G.
      • Warashina M.
      • Lie D.C.
      • Moore L.
      • Nakashima K.
      • Asashima M.
      • Gage F.H.
      Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis.
      ,
      • Liu X.
      • Luo M.
      • Xie W.
      • Wells J.M.
      • Goodheart M.J.
      • Engelhardt J.F.
      Sox17 modulates Wnt3A/β-catenin-mediated transcriptional activation of the Lef-1 promoter.
      ). Alternatively, it is also possible that Pax3 is a direct Wnt target in zebrafish and not in mice. Regardless of the mechanism operating in other organisms, our CHX experiments indicate that murine Pax3 is an indirect Wnt target. Moreover, we have found that a luciferase reporter construct driven by ECR2 is, like NCE2, very poorly activated by Lef1·β-catenin complexes in transient transfection assays using P19 and N2a cells (data not shown).
      Recent work in Xenopus embryos also suggests that Wnt-mediated induction of Pax3 expression at the neural plate border might be controlled by species-specific mechanisms. Indeed, de Crozé et al. (
      • de Crozé N.
      • Maczkowiak F.
      • Monsoro-Burq A.H.
      Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network.
      ) have reported that Pax3 expression at the neural plate border is regulated by canonical Wnt signals via both direct and indirect means. This work showed that, although Pax3 can be directly induced by canonical Wnt signals, the transcription factor AP2a is required as an intermediate for full activation (
      • de Crozé N.
      • Maczkowiak F.
      • Monsoro-Burq A.H.
      Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network.
      ). The existence of such a mechanism in mice is very unlikely because knock-out of all AP2 isoforms (via deletion of exon 5) has been shown to result in cranial neural crest defects that do not involve reduced Pax3 expression (
      • Schorle H.
      • Meier P.
      • Buchert M.
      • Jaenisch R.
      • Mitchell P.J.
      Transcription factor AP-2 essential for cranial closure and craniofacial development.
      ).
      On the other hand, other studies in Xenopus embryos have revealed that the homeobox gene Gbx2 is a direct downstream target of Wnt·β-catenin signaling acting upstream of Pax3 at the neural plate border (
      • Li B.
      • Kuriyama S.
      • Moreno M.
      • Mayor R.
      The posteriorizing gene Gbx2 is a direct target of Wnt signaling and the earliest factor in neural crest induction.
      ). Similarly to Cdx genes, Gbx2 is a known posteriorizing gene. However, in marked contrast to Cdx proteins, Gbx2 cannot directly activate Pax3 expression because it acts as a repressor (
      • Li B.
      • Kuriyama S.
      • Moreno M.
      • Mayor R.
      The posteriorizing gene Gbx2 is a direct target of Wnt signaling and the earliest factor in neural crest induction.
      ,
      • Heimbucher T.
      • Murko C.
      • Bajoghli B.
      • Aghaallaei N.
      • Huber A.
      • Stebegg R.
      • Eberhard D.
      • Fink M.
      • Simeone A.
      • Czerny T.
      Gbx2 and Otx2 interact with the WD40 domain of Groucho/Tle corepressors.
      ). Therefore, in this case, it appears that Gbx2 is required to repress an unknown repressor of Pax3 at the neural plate border. More work will be required to determine whether this mechanism is conserved in mice.

      Regulation of Pax3 Expression via NCE2

      We have shown for the first time that the Pax3 NCE2 alone is sufficient to recapitulate induction as well as dorsal restriction of Pax3 expression in the caudal NT. Taken together with previous work, this suggests that NCE2 is involved in the posterior whereas NCE1 is rather involved in the anterior expression of Pax3 (
      • Milewski R.C.
      • Chi N.C.
      • Li J.
      • Brown C.
      • Lu M.M.
      • Epstein J.A.
      Identification of minimal enhancer elements sufficient for Pax3 expression in neural crest and implication of Tead2 as a regulator of Pax3.
      ,
      • Chang C.P.
      • Stankunas K.
      • Shang C.
      • Kao S.C.
      • Twu K.Y.
      • Cleary M.L.
      Pbx1 functions in distinct regulatory networks to pattern the great arteries and cardiac outflow tract.
      ,
      • Pruitt S.C.
      • Bussman A.
      • Maslov A.Y.
      • Natoli T.A.
      • Heinaman R.
      Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro in vivo.
      ). As summarized in Fig. 7, our in vitro and in vivo data further indicate that the activity of NCE2 is regulated by the posteriorizing Wnt-Cdx pathway. Given the broad distribution of Cdx proteins in the posterior neurectoderm, it is currently unclear how NCE2 exhibits dorsally restricted activity. As described previously for Pax3 expression, this could first be ensured by a repressive mechanism involving Shh signals emerging from the node, notochord, and floor plate (
      • Goulding M.D.
      • Lumsden A.
      • Gruss P.
      Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord.
      ), implying that Shh-responsive regulatory sequences are contained within NCE2. Such restricted activity of NCE2 might also be due to an interaction between Cdx proteins and a neural plate border co-factor. In this regard, our transfection assays in N2a cells have suggested that Cdx-mediated transactivation of NCE2 might rely in part on the presence of a neural factor. The identity of such co-factor is currently unknown, and it is most likely not a transcription factor previously reported to act on NCE2 (Tead2 and Brn1/2) (
      • Milewski R.C.
      • Chi N.C.
      • Li J.
      • Brown C.
      • Lu M.M.
      • Epstein J.A.
      Identification of minimal enhancer elements sufficient for Pax3 expression in neural crest and implication of Tead2 as a regulator of Pax3.
      ,
      • Pruitt S.C.
      • Bussman A.
      • Maslov A.Y.
      • Natoli T.A.
      • Heinaman R.
      Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro in vivo.
      ). Indeed, although both Tead2 and Brn1/2 have been implicated in the regulation of Pax3 expression, their expression pattern is not consistent with a role in the induction of Pax3 expression in vivo. On one hand, Tead2 and its co-factor YAP65 are almost ubiquitously expressed at e8.5–e9.5, becoming restricted to neural tissues only after e10.0 (
      • Yasunami M.
      • Suzuki K.
      • Houtani T.
      • Sugimoto T.
      • Ohkubo H.
      Molecular characterization of cDNA encoding a novel protein related to transcriptional enhancer factor-1 from neural precursor cells.
      ,
      • Sawada A.
      • Nishizaki Y.
      • Sato H.
      • Yada Y.
      • Nakayama R.
      • Yamamoto S.
      • Nishioka N.
      • Kondoh H.
      • Sasaki H.
      Tead proteins activate the Foxa2 enhancer in the node in cooperation with a second factor.
      ). On the other hand, the proneural factors Brn1 and Brn2, as well as other Pou class III members Brn4 and Tst1/Oct6, are not expressed in the PNP (
      • Bouchard M.
      • Grote D.
      • Craven S.E.
      • Sun Q.
      • Steinlein P.
      • Busslinger M.
      Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region.
      ,
      • He X.
      • Treacy M.N.
      • Simmons D.M.
      • Ingraham H.A.
      • Swanson L.W.
      • Rosenfeld M.G.
      Expression of a large family of POU-domain regulatory genes in mammalian brain development.
      ,
      • Heydemann A.
      • Nguyen L.C.
      • Crenshaw 3rd, E.B.
      Regulatory regions from the Brn4 promoter direct LACZ expression to the developing forebrain and neural tube.
      ,
      • Sugitani Y.
      • Nakai S.
      • Minowa O.
      • Nishi M.
      • Jishage K.
      • Kawano H.
      • Mori K.
      • Ogawa M.
      • Noda T.
      Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons.
      ,
      • Mathis J.M.
      • Simmons D.M.
      • He X.
      • Swanson L.W.
      • Rosenfeld M.G.
      Brain 4. A novel mammalian POU domain transcription factor exhibiting restricted brain-specific expression.
      ,
      • Monuki E.S.
      • Kuhn R.
      • Weinmaster G.
      • Trapp B.D.
      • Lemke G.
      Expression and activity of the POU transcription factor SCIP.
      ). Thus, these observations strongly suggest that Tead2 and Brn1/2 are involved in the maintenance rather than induction of Pax3 expression. More work will obviously be required to better understand the regulatory mechanisms involved in the dorsal restriction of Pax3 expression, and our data indicate that at least some of them are operating via NCE2.
      Figure thumbnail gr7
      FIGURE 7Control of Pax3 expression in the caudal neurectoderm via NCE2. Induction of Pax3 expression in the posterior neural plate is controlled by the Wnt-Cdx pathway. Expression in the closed neural tube is later maintained by the activity of Tead2 as well as Brn1/2 transcription factors. Restriction of Pax3 expression at the lateral neural plate and dorsal neural tube is ensured by repressive Shh signals emerging from the node, notochord, and floor plate. An unknown neuron-specific Cdx co-factor might also be involved in the spatial restriction of NCE2 activity.

      Novel Function for Cdx Proteins in Caudal Neurectoderm Development

      Strong Cdx expression in the caudal neurectoderm is highly conserved across chordates. However, Cdx function in this lineage is poorly understood because of functional redundancy. Until recently, Cdx proteins were mostly known for their evolutionarily conserved role in the control of neural AP patterning via Hox-dependent mechanisms (
      • Bel-Vialar S.
      • Itasaki N.
      • Krumlauf R.
      Initiating Hox gene expression. In the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups.
      ,
      • Isaacs H.V.
      • Pownall M.E.
      • Slack J.M.
      Regulation of Hox gene expression and posterior development by the Xenopus caudal homologue Xcad3.
      ,
      • Charité J.
      • de Graaff W.
      • Consten D.
      • Reijnen M.J.
      • Korving J.
      • Deschamps J.
      Transducing positional information to the Hox genes. Critical interaction of cdx gene products with position-sensitive regulatory elements.
      ,
      • Shimizu T.
      • Bae Y.K.
      • Hibi M.
      Cdx-Hox code controls competence for responding to Fgfs and retinoic acid in zebrafish neural tissue.
      ). Cdx proteins are now also known to regulate neural AP patterning via Hox-independent mechanisms in different species (
      • Sturgeon K.
      • Kaneko T.
      • Biemann M.
      • Gauthier A.
      • Chawengsaksophak K.
      • Cordes S.P.
      Cdx1 refines positional identity of the vertebrate hindbrain by directly repressing Mafb expression.
      ,
      • Skromne I.
      • Thorsen D.
      • Hale M.
      • Prince V.E.
      • Ho R.K.
      Repression of the hindbrain developmental program by Cdx factors is required for the specification of the vertebrate spinal cord.
      ). More recently, Cdx1-Cdx2 double knock-out mice were generated and revealed a novel redundant role for Cdx members in the control of NT closure (
      • Savory J.G.
      • Mansfield M.
      • Rijli F.M.
      • Lohnes D.
      Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7.
      ). This work showed that Cdx proteins regulate the planar cell polarity gene Ptk7 and further suggested that Cdx members are involved in the regulation of convergent extension movements in the caudal embryo. This analysis involved a conditional mutagenesis approach to circumvent the peri-implantation lethality associated with the Cdx2 null mutation, via a CMV-β-actin-Cre-ERT2 transgene and a floxed allele of Cdx2. Thus, the Cdx function was lost in all three germ layers, and it is uncertain whether this novel Cdx role in neurulation is tissue-autonomous, -non-autonomous, or a combination of both. Ptk7 loss of function in Xenopus appears to affect convergent extension of the neurectoderm (
      • Wehner P.
      • Shnitsar I.
      • Urlaub H.
      • Borchers A.
      RACK1 is a novel interaction partner of PTK7 that is required for neural tube closure.
      ), but this has not been reported in mice. Indeed, although Ptk7−/− mouse mutants have been shown to have defective convergent extension movements in the mesoderm, an analysis of the neurectoderm has not been reported (
      • Yen W.W.
      • Williams M.
      • Periasamy A.
      • Conaway M.
      • Burdsal C.
      • Keller R.
      • Lu X.
      • Sutherland A.
      PTK7 is essential for polarized cell motility and convergent extension during mouse gastrulation.
      ). Therefore, more detailed analysis of Cdx1-Cdx2 double knock-out animals and conditional approaches involving tissue-specific loss of function will be required to clarify the Cdx-dependent processes involved in NT formation.
      On the other hand, our work now suggests that Cdx proteins may impact caudal neurectoderm development in a tissue-autonomous manner, at least via the regulation of Pax3 expression. As evidenced by the severe NC and NT defects observed in Pax3Sp/Sp mutants, Pax3 plays a crucial role in the neurectoderm (
      • Li J.
      • Liu K.C.
      • Jin F.
      • Lu M.M.
      • Epstein J.A.
      Transgenic rescue of congenital heart disease and spina bifida in Splotch mice.
      ,
      • Auerbach R.
      Analysis of the developmental effects of a lethal mutation in the house mouse.
      ). Pax3 is important for NCC induction, and analysis of Pax3-deficient embryos has indicated that NC defects are due to a marked reduction in the number of NCC that emigrate from the NT at cranial levels and a progressive complete loss of NCC at more caudal levels (
      • Conway S.J.
      • Bundy J.
      • Chen J.
      • Dickman E.
      • Rogers R.
      • Will B.M.
      Decreased neural crest stem cell expansion is responsible for the conotruncal heart defects within the splotch (Sp(2H))/Pax3 mouse mutant.
      ,
      • Epstein J.A.
      • Li J.
      • Lang D.
      • Chen F.
      • Brown C.B.
      • Jin F.
      • Lu M.M.
      • Thomas M.
      • Liu E.
      • Wessels A.
      • Lo C.W.
      Migration of cardiac neural crest cells in Splotch embryos.
      ,
      • Olaopa M.
      • Zhou H.M.
      • Snider P.
      • Wang J.
      • Schwartz R.J.
      • Moon A.M.
      • Conway S.J.
      Pax3 is essential for normal cardiac neural crest morphogenesis but is not required during migration nor outflow tract septation.
      ). This progressive increase in the severity of NC defects along the AP axis reflects the cranial co-expression of the functionally redundant Pax7 (
      • Mansouri A.
      • Stoykova A.
      • Torres M.
      • Gruss P.
      Dysgenesis of cephalic neural crest derivatives in Pax7−/− mutant mice.
      ). Pax3 is also important for NT closure, being required for the survival of dorsal progenitors via down-regulation of p53 activity (
      • Wang X.D.
      • Morgan S.C.
      • Loeken M.R.
      Pax3 stimulates p53 ubiquitination and degradation independent of transcription.
      ,
      • Pani L.
      • Horal M.
      • Loeken M.R.
      Rescue of neural tube defects in Pax-3-deficient embryos by p53 loss of function. Implications for Pax-3-dependent development and tumorigenesis.
      ). Thus, by acting upstream of Pax3, the canonical Wnt-Cdx pathway might control cell specification and maintenance of progenitor populations required for proper NC and NT development.
      A role for Cdx proteins in NC development has not been formally reported in any species. This is most likely because such a role is masked by functional redundancy and/or the presence of very severe posterior truncation phenotypes in Cdx compound mutants. However, several observations are in agreement with a role for Cdx proteins in NC development as well as the conservation of this role through evolution. In mice, an analysis of NCC in Cdx1-Cdx2 double knockouts has not been reported, but Cdx1−/−Cdx2+/− mutants are known to display abnormal and fused dorsal root ganglia (
      • van den Akker E.
      • Forlani S.
      • Chawengsaksophak K.
      • de Graaff W.
      • Beck F.
      • Meyer B.I.
      • Deschamps J.
      Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation.
      ). In zebrafish, Cdx loss of function leads to a reduced number of Rohon-Beard cells (which share a common precursor with NCC) and the absence of NC-derived spinal nerve roots (
      • Skromne I.
      • Thorsen D.
      • Hale M.
      • Prince V.E.
      • Ho R.K.
      Repression of the hindbrain developmental program by Cdx factors is required for the specification of the vertebrate spinal cord.
      ,
      • Epperlein H.H.
      • Selleck M.A.
      • Meulemans D.
      • McHedlishvili L.
      • Cerny R.
      • Sobkow L.
      • Bronner-Fraser M.
      Migratory patterns and developmental potential of trunk neural crest cells in the axolotl embryo.
      ). In ascidian, Cdx loss of function results in the absence of pigment cells, which are derived from NC-like cells (
      • Mita K.
      • Fujiwara S.
      Nodal regulates neural tube formation in the Ciona intestinalis embryo.
      ,
      • Jeffery W.R.
      • Strickler A.G.
      • Yamamoto Y.
      Migratory neural crest-like cells form body pigmentation in a urochordate embryo.
      ). Future work focusing on a more detailed characterization of compound mutants or tissue-specific loss-of-function studies should help validate these observations and confirm a role for Cdx proteins in NC development.
      In conclusion, our work suggests that Cdx proteins occupy a strategic position between canonical Wnt signals and Pax3 at the beginning of the gene regulatory cascade controlling NCC development. Because Cdx genes are not expressed in the anterior neurectoderm, the Wnt-Cdx pathway cannot impact cranial NC induction. Therefore, our data are in accordance with the general idea that NCC are intrinsically different along the AP axis (
      • Abzhanov A.
      • Tzahor E.
      • Lassar A.B.
      • Tabin C.J.
      Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro.
      ,
      • Le Douarin N.M.
      • Creuzet S.
      • Couly G.
      • Dupin E.
      Neural crest cell plasticity and its limits.
      ,
      • Lwigale P.Y.
      • Conrad G.W.
      • Bronner-Fraser M.
      Graded potential of neural crest to form cornea, sensory neurons, and cartilage along the rostrocaudal axis.
      ,
      • Thibaudeau G.
      • Holder S.
      • Gerard P.
      Anterior/posterior influences on neural crest-derived pigment cell differentiation.
      ) and strongly suggest that these differences are already in place during induction of NCC.

      Acknowledgments

      We thank Denis Flipo (University of Quebec at Montreal) for the FACS analyses as well as Qinzhang Zhu and Li Lian (Institut de Recherches Cliniques de Montréal) for microinjections. We thank Jonathan Epstein for the Pax3 probe, James B. Jaynes for the Engrailed cDNA, and David Lohnes for the Cdx antibodies as well as Cdx1-null mice.

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