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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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Related in: MedlinePlus

BMP protein rescues craniofacial phenotypes in dntcf+ embryos.(A,B) Whole mount ISH for gfp in Tg(Bre:gfp) embryos, lateral views, anterior to the left. (A) Wildtype and (B) following injection with 25 ng/embryo BMP4/7 into the arch region at 20 hpf. (C) Histogram quantifying Mc length (µM) in controls, dntcf3+ embryos, and dntcf3+ embryos injected with either 50 ng of BMP4/7 or 5 ng EDN1. (D) Histograms quantifying numbers of dntcf3+ embryos, alone or injected with BMP4/7 or EDN1 proteins, with reductions in Ch (left) and joint fusions between Mc and Pq (right). Numbers of embryos with rescue of Ch or Mc-Pq were averaged from three independent experiments (minimum of 10 embryos per protein injection experiment) and samples compared using a paired student t-test. (E, G, I) Whole mounted, alcian-stained 4 dpf larvae; control (E), dntcf3+ (G), and dntcf3+ injected with 25 ug BMP4/7 (I), lateral views, anterior to the left. Arrowhead in G indicates Ch loss. Black lines in G and I indicate Mc length and asterisks indicate Mc-Pq joint fusion. (F, H, J–L) Dissected, flat-mounted alcian-stained cartilages at 4 dpf, ventral views, anterior to the left; control with unilateral injection of 50 ng BMP4/7 (F), dntcf3+ (H), dntcf3+ with 25 ng BMP4/7 (J), control with 5 ng EDN1 (K) and dntcf3+ with 5 ng EDN1 (L). Arrowhead in F indicates duplicate Mc. Black lines in J indicate Mc length. Arrowheads in H indicate Sy and Ch. Asterisks in H, J and L indicate Mc-Pq joint fusions. **P<0.001. Abbreviations: Ch, ceratohyal; mc, Meckel's; mc', duplicated Meckel's; Sy, symplectic. Scale bar: 100 µm.
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pgen-1004479-g006: BMP protein rescues craniofacial phenotypes in dntcf+ embryos.(A,B) Whole mount ISH for gfp in Tg(Bre:gfp) embryos, lateral views, anterior to the left. (A) Wildtype and (B) following injection with 25 ng/embryo BMP4/7 into the arch region at 20 hpf. (C) Histogram quantifying Mc length (µM) in controls, dntcf3+ embryos, and dntcf3+ embryos injected with either 50 ng of BMP4/7 or 5 ng EDN1. (D) Histograms quantifying numbers of dntcf3+ embryos, alone or injected with BMP4/7 or EDN1 proteins, with reductions in Ch (left) and joint fusions between Mc and Pq (right). Numbers of embryos with rescue of Ch or Mc-Pq were averaged from three independent experiments (minimum of 10 embryos per protein injection experiment) and samples compared using a paired student t-test. (E, G, I) Whole mounted, alcian-stained 4 dpf larvae; control (E), dntcf3+ (G), and dntcf3+ injected with 25 ug BMP4/7 (I), lateral views, anterior to the left. Arrowhead in G indicates Ch loss. Black lines in G and I indicate Mc length and asterisks indicate Mc-Pq joint fusion. (F, H, J–L) Dissected, flat-mounted alcian-stained cartilages at 4 dpf, ventral views, anterior to the left; control with unilateral injection of 50 ng BMP4/7 (F), dntcf3+ (H), dntcf3+ with 25 ng BMP4/7 (J), control with 5 ng EDN1 (K) and dntcf3+ with 5 ng EDN1 (L). Arrowhead in F indicates duplicate Mc. Black lines in J indicate Mc length. Arrowheads in H indicate Sy and Ch. Asterisks in H, J and L indicate Mc-Pq joint fusions. **P<0.001. Abbreviations: Ch, ceratohyal; mc, Meckel's; mc', duplicated Meckel's; Sy, symplectic. Scale bar: 100 µm.

Mentions: Bmps act together with Edn1 to promote ventral-intermediate cell fates in the arches [16], [17], [21], [22], [58]. Therefore we examined the ability of Bmp and Edn1 to restore ventral-intermediate gene expression in Wnt signaling-deficient embryos. Beads coated in human recombinant BMP4/7 heterodimers effectively induce Bmp target genes in zebrafish pharyngeal arches [19]. Similarly, microinjection of a 25 ng/nl BMP4/7 solution extracellularly on one side of the head induced Bmp signaling, as measured by expression of the transgenic Bmp-response element reporter (Bre:Gfp; [19]) at 8 hours post injection (hpi) (Fig. 6A, B). Unilateral injections of BMP4/7 protein into dntcf3+ embryos at 4 hphs partially rescued cartilage defects on the injected side (Fig. 6C–J). Typically this restored Mc length and Ch, but not the Mc-Pq joint, and rescue was dose-dependent (Fig. 6C, D). These results suggest that Wnt signaling acts upstream of, or possibly in parallel to, Bmp signaling to promote ventral cartilage cell fates in the arches. EDN1 protein injections have previously been shown to rescue an Edn1 mutant phenotype and partially rescue a Bmp loss of function phenotype [16], [19]. EDN1 injections into dntcf3+ embryos also partially rescued Mc length, but notably were more proficient at rescuing Ch and joint development (Fig. 6C–D, L).


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 protein rescues craniofacial phenotypes in dntcf+ embryos.(A,B) Whole mount ISH for gfp in Tg(Bre:gfp) embryos, lateral views, anterior to the left. (A) Wildtype and (B) following injection with 25 ng/embryo BMP4/7 into the arch region at 20 hpf. (C) Histogram quantifying Mc length (µM) in controls, dntcf3+ embryos, and dntcf3+ embryos injected with either 50 ng of BMP4/7 or 5 ng EDN1. (D) Histograms quantifying numbers of dntcf3+ embryos, alone or injected with BMP4/7 or EDN1 proteins, with reductions in Ch (left) and joint fusions between Mc and Pq (right). Numbers of embryos with rescue of Ch or Mc-Pq were averaged from three independent experiments (minimum of 10 embryos per protein injection experiment) and samples compared using a paired student t-test. (E, G, I) Whole mounted, alcian-stained 4 dpf larvae; control (E), dntcf3+ (G), and dntcf3+ injected with 25 ug BMP4/7 (I), lateral views, anterior to the left. Arrowhead in G indicates Ch loss. Black lines in G and I indicate Mc length and asterisks indicate Mc-Pq joint fusion. (F, H, J–L) Dissected, flat-mounted alcian-stained cartilages at 4 dpf, ventral views, anterior to the left; control with unilateral injection of 50 ng BMP4/7 (F), dntcf3+ (H), dntcf3+ with 25 ng BMP4/7 (J), control with 5 ng EDN1 (K) and dntcf3+ with 5 ng EDN1 (L). Arrowhead in F indicates duplicate Mc. Black lines in J indicate Mc length. Arrowheads in H indicate Sy and Ch. Asterisks in H, J and L indicate Mc-Pq joint fusions. **P<0.001. Abbreviations: Ch, ceratohyal; mc, Meckel's; mc', duplicated Meckel's; Sy, symplectic. Scale bar: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004479-g006: BMP protein rescues craniofacial phenotypes in dntcf+ embryos.(A,B) Whole mount ISH for gfp in Tg(Bre:gfp) embryos, lateral views, anterior to the left. (A) Wildtype and (B) following injection with 25 ng/embryo BMP4/7 into the arch region at 20 hpf. (C) Histogram quantifying Mc length (µM) in controls, dntcf3+ embryos, and dntcf3+ embryos injected with either 50 ng of BMP4/7 or 5 ng EDN1. (D) Histograms quantifying numbers of dntcf3+ embryos, alone or injected with BMP4/7 or EDN1 proteins, with reductions in Ch (left) and joint fusions between Mc and Pq (right). Numbers of embryos with rescue of Ch or Mc-Pq were averaged from three independent experiments (minimum of 10 embryos per protein injection experiment) and samples compared using a paired student t-test. (E, G, I) Whole mounted, alcian-stained 4 dpf larvae; control (E), dntcf3+ (G), and dntcf3+ injected with 25 ug BMP4/7 (I), lateral views, anterior to the left. Arrowhead in G indicates Ch loss. Black lines in G and I indicate Mc length and asterisks indicate Mc-Pq joint fusion. (F, H, J–L) Dissected, flat-mounted alcian-stained cartilages at 4 dpf, ventral views, anterior to the left; control with unilateral injection of 50 ng BMP4/7 (F), dntcf3+ (H), dntcf3+ with 25 ng BMP4/7 (J), control with 5 ng EDN1 (K) and dntcf3+ with 5 ng EDN1 (L). Arrowhead in F indicates duplicate Mc. Black lines in J indicate Mc length. Arrowheads in H indicate Sy and Ch. Asterisks in H, J and L indicate Mc-Pq joint fusions. **P<0.001. Abbreviations: Ch, ceratohyal; mc, Meckel's; mc', duplicated Meckel's; Sy, symplectic. Scale bar: 100 µm.
Mentions: Bmps act together with Edn1 to promote ventral-intermediate cell fates in the arches [16], [17], [21], [22], [58]. Therefore we examined the ability of Bmp and Edn1 to restore ventral-intermediate gene expression in Wnt signaling-deficient embryos. Beads coated in human recombinant BMP4/7 heterodimers effectively induce Bmp target genes in zebrafish pharyngeal arches [19]. Similarly, microinjection of a 25 ng/nl BMP4/7 solution extracellularly on one side of the head induced Bmp signaling, as measured by expression of the transgenic Bmp-response element reporter (Bre:Gfp; [19]) at 8 hours post injection (hpi) (Fig. 6A, B). Unilateral injections of BMP4/7 protein into dntcf3+ embryos at 4 hphs partially rescued cartilage defects on the injected side (Fig. 6C–J). Typically this restored Mc length and Ch, but not the Mc-Pq joint, and rescue was dose-dependent (Fig. 6C, D). These results suggest that Wnt signaling acts upstream of, or possibly in parallel to, Bmp signaling to promote ventral cartilage cell fates in the arches. EDN1 protein injections have previously been shown to rescue an Edn1 mutant phenotype and partially rescue a Bmp loss of function phenotype [16], [19]. EDN1 injections into dntcf3+ embryos also partially rescued Mc length, but notably were more proficient at rescuing Ch and joint development (Fig. 6C–D, L).

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