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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 regulates Bmp and Edn1 signaling in the arches.(A–F) Anti-pSmad1/5/8 staining in control (A–C) and dntcf3+ (D–F) embryos heat shocked at 22–24 hpf, lateral views, anterior to the left. pSmad1/5/8 expression is localized ventrally in the arches (arrowheads), reduced at 2 hphs (D), severely reduced at 4 hphs (E), and absent (asterisk) by 6 hphs (F). (G) Western blot analysis of anti-pSmad1/5/8 in control and dntcf3+ embryos. Protein was extracted at 2 hphs or 6 hphs and alpha tubulin was used as a loading control. (H) Histogram quantifying pSmad1/5/8 expression at 6 hphs, based on Western blots, in control and dntcf3+ embryos, levels normalized to alpha tubulin. (I) Histogram quantifying Edn1 expression by qPCR, in control and dntcf3+ embryos, p<0.01.
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pgen-1004479-g005: Wnt signaling regulates Bmp and Edn1 signaling in the arches.(A–F) Anti-pSmad1/5/8 staining in control (A–C) and dntcf3+ (D–F) embryos heat shocked at 22–24 hpf, lateral views, anterior to the left. pSmad1/5/8 expression is localized ventrally in the arches (arrowheads), reduced at 2 hphs (D), severely reduced at 4 hphs (E), and absent (asterisk) by 6 hphs (F). (G) Western blot analysis of anti-pSmad1/5/8 in control and dntcf3+ embryos. Protein was extracted at 2 hphs or 6 hphs and alpha tubulin was used as a loading control. (H) Histogram quantifying pSmad1/5/8 expression at 6 hphs, based on Western blots, in control and dntcf3+ embryos, levels normalized to alpha tubulin. (I) Histogram quantifying Edn1 expression by qPCR, in control and dntcf3+ embryos, p<0.01.

Mentions: Because dntcf3+ embryos showed D-V defects in cartilage morphology and gene expression that more closely resembled Bmp- and Edn1-deficient embryos than dkk1+ we focused on dntcf3+. To examine interactions between Wnt and Bmp signaling in the arches we used an antibody that recognizes phosphorylated Smad1/5/8 (pSmad1/5/8) in dntcf3+ embryos. In controls pSmad1/5/8 localized to ventral arches 1 and 2 where levels of Bmp signaling have been shown to be highest at 24 hpf (Fig. 5A–C; [19]). Anti-pSmad1/5/8 staining was slightly reduced in the first arch at 2 hphs (24–26 hpf) in dntcf3+ embryos (Fig. 5D), in both arches by 4 hphs (Fig. 5E), and virtually lost altogether at 6 hphs (Fig. 5F). Western blots confirmed that pSmad1/5/8 levels were much lower than controls at 6 hphs (Fig. 5G, H). To examine potential interactions between Wnt and Edn1 signaling in the arches we performed qPCR for Edn1 in dntcf3+ embryos at 6 hphs. Edn1 expression was significantly reduced relative to control (Fig. 5I). These results reveal an indirect role for Wnts in D-V patterning through regulation of both Bmp and Edn1 signaling.


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 regulates Bmp and Edn1 signaling in the arches.(A–F) Anti-pSmad1/5/8 staining in control (A–C) and dntcf3+ (D–F) embryos heat shocked at 22–24 hpf, lateral views, anterior to the left. pSmad1/5/8 expression is localized ventrally in the arches (arrowheads), reduced at 2 hphs (D), severely reduced at 4 hphs (E), and absent (asterisk) by 6 hphs (F). (G) Western blot analysis of anti-pSmad1/5/8 in control and dntcf3+ embryos. Protein was extracted at 2 hphs or 6 hphs and alpha tubulin was used as a loading control. (H) Histogram quantifying pSmad1/5/8 expression at 6 hphs, based on Western blots, in control and dntcf3+ embryos, levels normalized to alpha tubulin. (I) Histogram quantifying Edn1 expression by qPCR, in control and dntcf3+ embryos, p<0.01.
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pgen-1004479-g005: Wnt signaling regulates Bmp and Edn1 signaling in the arches.(A–F) Anti-pSmad1/5/8 staining in control (A–C) and dntcf3+ (D–F) embryos heat shocked at 22–24 hpf, lateral views, anterior to the left. pSmad1/5/8 expression is localized ventrally in the arches (arrowheads), reduced at 2 hphs (D), severely reduced at 4 hphs (E), and absent (asterisk) by 6 hphs (F). (G) Western blot analysis of anti-pSmad1/5/8 in control and dntcf3+ embryos. Protein was extracted at 2 hphs or 6 hphs and alpha tubulin was used as a loading control. (H) Histogram quantifying pSmad1/5/8 expression at 6 hphs, based on Western blots, in control and dntcf3+ embryos, levels normalized to alpha tubulin. (I) Histogram quantifying Edn1 expression by qPCR, in control and dntcf3+ embryos, p<0.01.
Mentions: Because dntcf3+ embryos showed D-V defects in cartilage morphology and gene expression that more closely resembled Bmp- and Edn1-deficient embryos than dkk1+ we focused on dntcf3+. To examine interactions between Wnt and Bmp signaling in the arches we used an antibody that recognizes phosphorylated Smad1/5/8 (pSmad1/5/8) in dntcf3+ embryos. In controls pSmad1/5/8 localized to ventral arches 1 and 2 where levels of Bmp signaling have been shown to be highest at 24 hpf (Fig. 5A–C; [19]). Anti-pSmad1/5/8 staining was slightly reduced in the first arch at 2 hphs (24–26 hpf) in dntcf3+ embryos (Fig. 5D), in both arches by 4 hphs (Fig. 5E), and virtually lost altogether at 6 hphs (Fig. 5F). Western blots confirmed that pSmad1/5/8 levels were much lower than controls at 6 hphs (Fig. 5G, H). To examine potential interactions between Wnt and Edn1 signaling in the arches we performed qPCR for Edn1 in dntcf3+ embryos at 6 hphs. Edn1 expression was significantly reduced relative to control (Fig. 5I). These results reveal an indirect role for Wnts in D-V patterning through regulation of both Bmp and Edn1 signaling.

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