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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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Bmp receptor expression in dntcf3+ embryos.(A–F) Whole mount ISH for bmpr1ab (A, B), bmpr1ba (C, D), and bmpr1bb (E, F) expression, lateral views, anterior to the left. (G) Histogram quantifying the number of dntcf3+ embryos with reduced expression. (H,I) qPCR analysis of Bmp receptor expression levels in dntcf3+ embryos at different times post heat shock, normalized to nontransgenic, heat-shocked controls, with ef1alpha as an internal control. * P<0.05, ** P<0.001. Abbreviations: e, eye; nc, neural crest. Scale bar: 100 µm.
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pgen-1004479-g008: Bmp receptor expression in dntcf3+ embryos.(A–F) Whole mount ISH for bmpr1ab (A, B), bmpr1ba (C, D), and bmpr1bb (E, F) expression, lateral views, anterior to the left. (G) Histogram quantifying the number of dntcf3+ embryos with reduced expression. (H,I) qPCR analysis of Bmp receptor expression levels in dntcf3+ embryos at different times post heat shock, normalized to nontransgenic, heat-shocked controls, with ef1alpha as an internal control. * P<0.05, ** P<0.001. Abbreviations: e, eye; nc, neural crest. Scale bar: 100 µm.

Mentions: To further investigate how Wnts might regulate the ability of NC cells to respond to Bmp signaling, we examined whether or not dntcf3+ embryos show any changes in expression of Bmp receptors. Zebrafish have four type 1 receptors (Bmpr1aa, ab, ba, bb) and two type II receptors (Bmpr2a and b). Whole mount ISH for all six receptors revealed that only bmpr1ab, bmpr1ba, and bmpr1bb are expressed strongly in the arches at 24 hpf (Fig. S9C–F, I–J, M–R). bmpr1aa, bmpr2a, and bmpr2b were detected much more broadly throughout the embryo at this stage (Fig. S9A–B, G–H, K–L). Bmpr1ab expression extended throughout arches 1 and 2, while bmpr1ba and bmpr1bb expression was restricted to more intermediate and ventral domains (Fig. S9M–O). Transverse sections additionally showed that bmpr1ab and bmpr1ba expression is limited to arch NC cells and not surrounding epithelia (Fig. S9P–R). In dntcf3+ embryos bmpr1ab was severely reduced (57% n = 35) (Fig. 8A–B, G), while bmpr1ba was slightly reduced (bmpr1ab: 46% n = 57) and bmpr1bb expression was largely unaffected (bmpr1bb: 31% n = 29) (Fig. 8C–F, G).


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)

Bmp receptor expression in dntcf3+ embryos.(A–F) Whole mount ISH for bmpr1ab (A, B), bmpr1ba (C, D), and bmpr1bb (E, F) expression, lateral views, anterior to the left. (G) Histogram quantifying the number of dntcf3+ embryos with reduced expression. (H,I) qPCR analysis of Bmp receptor expression levels in dntcf3+ embryos at different times post heat shock, normalized to nontransgenic, heat-shocked controls, with ef1alpha as an internal control. * P<0.05, ** P<0.001. Abbreviations: e, eye; nc, neural crest. Scale bar: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004479-g008: Bmp receptor expression in dntcf3+ embryos.(A–F) Whole mount ISH for bmpr1ab (A, B), bmpr1ba (C, D), and bmpr1bb (E, F) expression, lateral views, anterior to the left. (G) Histogram quantifying the number of dntcf3+ embryos with reduced expression. (H,I) qPCR analysis of Bmp receptor expression levels in dntcf3+ embryos at different times post heat shock, normalized to nontransgenic, heat-shocked controls, with ef1alpha as an internal control. * P<0.05, ** P<0.001. Abbreviations: e, eye; nc, neural crest. Scale bar: 100 µm.
Mentions: To further investigate how Wnts might regulate the ability of NC cells to respond to Bmp signaling, we examined whether or not dntcf3+ embryos show any changes in expression of Bmp receptors. Zebrafish have four type 1 receptors (Bmpr1aa, ab, ba, bb) and two type II receptors (Bmpr2a and b). Whole mount ISH for all six receptors revealed that only bmpr1ab, bmpr1ba, and bmpr1bb are expressed strongly in the arches at 24 hpf (Fig. S9C–F, I–J, M–R). bmpr1aa, bmpr2a, and bmpr2b were detected much more broadly throughout the embryo at this stage (Fig. S9A–B, G–H, K–L). Bmpr1ab expression extended throughout arches 1 and 2, while bmpr1ba and bmpr1bb expression was restricted to more intermediate and ventral domains (Fig. S9M–O). Transverse sections additionally showed that bmpr1ab and bmpr1ba expression is limited to arch NC cells and not surrounding epithelia (Fig. S9P–R). In dntcf3+ embryos bmpr1ab was severely reduced (57% n = 35) (Fig. 8A–B, G), while bmpr1ba was slightly reduced (bmpr1ab: 46% n = 57) and bmpr1bb expression was largely unaffected (bmpr1bb: 31% n = 29) (Fig. 8C–F, G).

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