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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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Requirements for Wnt signaling in craniofacial cartilage development.(A–C, F, G) Alcian blue stained cartilage at 96 hpf, dissected and flat mounted, ventral views, anterior to the left. (A) Wild-type control, (B) dkk1+/− heterozygote, (C) dkk1+/+ homozygote, (F) dntcf3+/− heterozygote, (G) dntcf3+/+ homozygote, heat shocked as described (see Results). Arrowheads indicate clefting of Meckel's cartilage (Mc). (D) Histogram quantifying the frequency of Mc defects in dkk1+ embryos heat shocked at different stages from 16–36 hpf. (E) Histogram quantifying average Mc length, p<0.01. (H) Histogram quantifying the frequency of cartilage defects in arches 1 and 2 in dntcf3+ embryos heat shocked at different stages from 16–30 hpf. Abbreviations: Ch, ceratohyal; Hm, hyomandibular; Mc, Meckels; Pq, palatoquadrate; Sy, symplectic. Scale bar: 100 µm.
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pgen-1004479-g002: Requirements for Wnt signaling in craniofacial cartilage development.(A–C, F, G) Alcian blue stained cartilage at 96 hpf, dissected and flat mounted, ventral views, anterior to the left. (A) Wild-type control, (B) dkk1+/− heterozygote, (C) dkk1+/+ homozygote, (F) dntcf3+/− heterozygote, (G) dntcf3+/+ homozygote, heat shocked as described (see Results). Arrowheads indicate clefting of Meckel's cartilage (Mc). (D) Histogram quantifying the frequency of Mc defects in dkk1+ embryos heat shocked at different stages from 16–36 hpf. (E) Histogram quantifying average Mc length, p<0.01. (H) Histogram quantifying the frequency of cartilage defects in arches 1 and 2 in dntcf3+ embryos heat shocked at different stages from 16–30 hpf. Abbreviations: Ch, ceratohyal; Hm, hyomandibular; Mc, Meckels; Pq, palatoquadrate; Sy, symplectic. Scale bar: 100 µm.

Mentions: Alcian blue staining of cartilage in larvae at 96 hpf revealed that, compared with controls, hs-dkk1+/− heterozygotes heat shocked for 30 min at 16–28 hpf developed mandibular clefting and reduced Meckel's cartilages (Mc), as well as mild reductions in other craniofacial cartilages (Fig. 2A, B, D). Homozygous hs-dkk1+/+ larvae displayed dramatic shortening of Mc ventrally in the first arch, as well as the symplectic (Sy), a more intermediate/dorsal element of the second arch (Fig. 2C, E; [19], [20]). Thus, hereafter “dkk1+” refers to homozygous hs-dkk1 embryos/larvae heat shocked for 30 min between 20–22 hpf.


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)

Requirements for Wnt signaling in craniofacial cartilage development.(A–C, F, G) Alcian blue stained cartilage at 96 hpf, dissected and flat mounted, ventral views, anterior to the left. (A) Wild-type control, (B) dkk1+/− heterozygote, (C) dkk1+/+ homozygote, (F) dntcf3+/− heterozygote, (G) dntcf3+/+ homozygote, heat shocked as described (see Results). Arrowheads indicate clefting of Meckel's cartilage (Mc). (D) Histogram quantifying the frequency of Mc defects in dkk1+ embryos heat shocked at different stages from 16–36 hpf. (E) Histogram quantifying average Mc length, p<0.01. (H) Histogram quantifying the frequency of cartilage defects in arches 1 and 2 in dntcf3+ embryos heat shocked at different stages from 16–30 hpf. Abbreviations: Ch, ceratohyal; Hm, hyomandibular; Mc, Meckels; Pq, palatoquadrate; Sy, symplectic. Scale bar: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004479-g002: Requirements for Wnt signaling in craniofacial cartilage development.(A–C, F, G) Alcian blue stained cartilage at 96 hpf, dissected and flat mounted, ventral views, anterior to the left. (A) Wild-type control, (B) dkk1+/− heterozygote, (C) dkk1+/+ homozygote, (F) dntcf3+/− heterozygote, (G) dntcf3+/+ homozygote, heat shocked as described (see Results). Arrowheads indicate clefting of Meckel's cartilage (Mc). (D) Histogram quantifying the frequency of Mc defects in dkk1+ embryos heat shocked at different stages from 16–36 hpf. (E) Histogram quantifying average Mc length, p<0.01. (H) Histogram quantifying the frequency of cartilage defects in arches 1 and 2 in dntcf3+ embryos heat shocked at different stages from 16–30 hpf. Abbreviations: Ch, ceratohyal; Hm, hyomandibular; Mc, Meckels; Pq, palatoquadrate; Sy, symplectic. Scale bar: 100 µm.
Mentions: Alcian blue staining of cartilage in larvae at 96 hpf revealed that, compared with controls, hs-dkk1+/− heterozygotes heat shocked for 30 min at 16–28 hpf developed mandibular clefting and reduced Meckel's cartilages (Mc), as well as mild reductions in other craniofacial cartilages (Fig. 2A, B, D). Homozygous hs-dkk1+/+ larvae displayed dramatic shortening of Mc ventrally in the first arch, as well as the symplectic (Sy), a more intermediate/dorsal element of the second arch (Fig. 2C, E; [19], [20]). Thus, hereafter “dkk1+” refers to homozygous hs-dkk1 embryos/larvae heat shocked for 30 min between 20–22 hpf.

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