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Wnt signaling interacts with bmp and edn1 to regulate dorsal-ventral patterning and growth of the craniofacial skeleton.

Alexander C, Piloto S, Le Pabic P, Schilling TF - PLoS Genet. (2014)

Bottom Line: These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches.Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression.Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.

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Wnt signaling in the pharyngeal arches.(A–H) In situ hybridization (ISH) and (I) quantitative, real-time PCR (qPCR) analysis of Wnt target gene expression; (A, C, E–H) lateral views, anterior to the left; (B, D) transverse sections through arch 2. (A, B) mycn mRNA is ventrally restricted (black arrowheads) in wild type (WT) embryos. Arches 1 and 2 are outlined by dotted lines. (C, D) GFP mRNA is ventrally restricted (arrowheads) in Tg(7xTCF-Xla.Siam:GFP)ia4 transgenics. (B, D) Both mycn (B) and 7xTCF;GFP (D) are expressed in ventral nc cells and pharyngeal endoderm, and excluded from pharyngeal ectoderm. (E–H) mycn and axin2 expression in controls (E, G), and dntcf3+ embryos at 26 hpf (F, H; heat shocked at 22 hpf). (I) qPCR analysis of axin2, lef1 and mycn expression in dntcf3+ embryos, normalized to nontransgenic, heat-shocked controls, with ef1alpha as an internal control. * P<0.05, ** P<0.001. Abbreviations: e, eye; D, dorsal arch; end, pharyngeal endoderm; hb, hindbrain; I, intermediate arch; mhb, mid-hindbrain boundary; nc, neural crest; pe, pharyngeal ectoderm; V, ventral arch. Scale bars: 100 µm.
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pgen-1004479-g001: Wnt signaling in the pharyngeal arches.(A–H) In situ hybridization (ISH) and (I) quantitative, real-time PCR (qPCR) analysis of Wnt target gene expression; (A, C, E–H) lateral views, anterior to the left; (B, D) transverse sections through arch 2. (A, B) mycn mRNA is ventrally restricted (black arrowheads) in wild type (WT) embryos. Arches 1 and 2 are outlined by dotted lines. (C, D) GFP mRNA is ventrally restricted (arrowheads) in Tg(7xTCF-Xla.Siam:GFP)ia4 transgenics. (B, D) Both mycn (B) and 7xTCF;GFP (D) are expressed in ventral nc cells and pharyngeal endoderm, and excluded from pharyngeal ectoderm. (E–H) mycn and axin2 expression in controls (E, G), and dntcf3+ embryos at 26 hpf (F, H; heat shocked at 22 hpf). (I) qPCR analysis of axin2, lef1 and mycn expression in dntcf3+ embryos, normalized to nontransgenic, heat-shocked controls, with ef1alpha as an internal control. * P<0.05, ** P<0.001. Abbreviations: e, eye; D, dorsal arch; end, pharyngeal endoderm; hb, hindbrain; I, intermediate arch; mhb, mid-hindbrain boundary; nc, neural crest; pe, pharyngeal ectoderm; V, ventral arch. Scale bars: 100 µm.

Mentions: Numerous Wnt ligands (Wnt2, Wnt4, Wnt5a/b, Wnt 6, and Wnt7a/b) and receptors (Fzd1, Fzd3, Fzd4, Fzd6, Fzd7, Fzd8, and Fzd10) are expressed broadly in the pharyngeal ectoderm, endoderm, neural crest (NC), and mesoderm [29], [32], [34], [35]. To determine which regions of the pharyngeal arches respond directly to Wnt signaling we used in situ hybridization (ISH) to examine expression of the direct downstream Wnt target mycn (Fig. 1A,B), an oncogene with roles in regulating Wnt-dependent morphogenesis and proliferation [47], [48]. mycn mRNA was detected throughout the arches but at higher levels in the ventral domain, primarily within the NC mesenchyme (arrowheads in Fig. 1A,B). To further address which pharyngeal tissues respond directly, we examined expression of a transgenic Wnt reporter zebrafish Tg(7xTCF-Xla.Siam:GFP)ia4 (7xTCF:GFP) [49], which contains seven TCF response elements driving expression of GFP, thus acting as a live reporter in cells where stabilized β-catenin (βcat) interacts with Tcf transcription factors. ISH for GFP mRNA at 28 hours postfertilization (hpf) revealed regions of 7xTCF:GFP expression in the ventral first and second arches (Fig. 1C), which in transverse sections appeared localized both to arch NC cells and pharyngeal endoderm, but not pharyngeal ectoderm (Fig. 1D).


Wnt signaling interacts with bmp and edn1 to regulate dorsal-ventral patterning and growth of the craniofacial skeleton.

Alexander C, Piloto S, Le Pabic P, Schilling TF - PLoS Genet. (2014)

Wnt signaling in the pharyngeal arches.(A–H) In situ hybridization (ISH) and (I) quantitative, real-time PCR (qPCR) analysis of Wnt target gene expression; (A, C, E–H) lateral views, anterior to the left; (B, D) transverse sections through arch 2. (A, B) mycn mRNA is ventrally restricted (black arrowheads) in wild type (WT) embryos. Arches 1 and 2 are outlined by dotted lines. (C, D) GFP mRNA is ventrally restricted (arrowheads) in Tg(7xTCF-Xla.Siam:GFP)ia4 transgenics. (B, D) Both mycn (B) and 7xTCF;GFP (D) are expressed in ventral nc cells and pharyngeal endoderm, and excluded from pharyngeal ectoderm. (E–H) mycn and axin2 expression in controls (E, G), and dntcf3+ embryos at 26 hpf (F, H; heat shocked at 22 hpf). (I) qPCR analysis of axin2, lef1 and mycn expression in dntcf3+ embryos, normalized to nontransgenic, heat-shocked controls, with ef1alpha as an internal control. * P<0.05, ** P<0.001. Abbreviations: e, eye; D, dorsal arch; end, pharyngeal endoderm; hb, hindbrain; I, intermediate arch; mhb, mid-hindbrain boundary; nc, neural crest; pe, pharyngeal ectoderm; V, ventral arch. Scale bars: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4109847&req=5

pgen-1004479-g001: Wnt signaling in the pharyngeal arches.(A–H) In situ hybridization (ISH) and (I) quantitative, real-time PCR (qPCR) analysis of Wnt target gene expression; (A, C, E–H) lateral views, anterior to the left; (B, D) transverse sections through arch 2. (A, B) mycn mRNA is ventrally restricted (black arrowheads) in wild type (WT) embryos. Arches 1 and 2 are outlined by dotted lines. (C, D) GFP mRNA is ventrally restricted (arrowheads) in Tg(7xTCF-Xla.Siam:GFP)ia4 transgenics. (B, D) Both mycn (B) and 7xTCF;GFP (D) are expressed in ventral nc cells and pharyngeal endoderm, and excluded from pharyngeal ectoderm. (E–H) mycn and axin2 expression in controls (E, G), and dntcf3+ embryos at 26 hpf (F, H; heat shocked at 22 hpf). (I) qPCR analysis of axin2, lef1 and mycn expression in dntcf3+ embryos, normalized to nontransgenic, heat-shocked controls, with ef1alpha as an internal control. * P<0.05, ** P<0.001. Abbreviations: e, eye; D, dorsal arch; end, pharyngeal endoderm; hb, hindbrain; I, intermediate arch; mhb, mid-hindbrain boundary; nc, neural crest; pe, pharyngeal ectoderm; V, ventral arch. Scale bars: 100 µm.
Mentions: Numerous Wnt ligands (Wnt2, Wnt4, Wnt5a/b, Wnt 6, and Wnt7a/b) and receptors (Fzd1, Fzd3, Fzd4, Fzd6, Fzd7, Fzd8, and Fzd10) are expressed broadly in the pharyngeal ectoderm, endoderm, neural crest (NC), and mesoderm [29], [32], [34], [35]. To determine which regions of the pharyngeal arches respond directly to Wnt signaling we used in situ hybridization (ISH) to examine expression of the direct downstream Wnt target mycn (Fig. 1A,B), an oncogene with roles in regulating Wnt-dependent morphogenesis and proliferation [47], [48]. mycn mRNA was detected throughout the arches but at higher levels in the ventral domain, primarily within the NC mesenchyme (arrowheads in Fig. 1A,B). To further address which pharyngeal tissues respond directly, we examined expression of a transgenic Wnt reporter zebrafish Tg(7xTCF-Xla.Siam:GFP)ia4 (7xTCF:GFP) [49], which contains seven TCF response elements driving expression of GFP, thus acting as a live reporter in cells where stabilized β-catenin (βcat) interacts with Tcf transcription factors. ISH for GFP mRNA at 28 hours postfertilization (hpf) revealed regions of 7xTCF:GFP expression in the ventral first and second arches (Fig. 1C), which in transverse sections appeared localized both to arch NC cells and pharyngeal endoderm, but not pharyngeal ectoderm (Fig. 1D).

Bottom Line: These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches.Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression.Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.

Show MeSH
Related in: MedlinePlus