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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 dorsal-ventral arch patterning.(A–P) Whole mount ISH for genes involved in dorsal-ventral (D-V) patterning in control (A, D, G, J), dkk1+ embryos heat shocked at 20–22 hpf (B, E, H, K), and dntcf3+ embryos heat shocked at 22–24 hpf (C, F, I, L), lateral views, anterior to the left. hand2 (B, C), dlx3b (E, F), and dlx6a (H, I) expression is reduced, while jag1b (K, L) expression expands ventrally (arrowheads) particularly in dntcf3+ embryos, compared with controls. (M–P) dlx2a expression in control (M,O), dntcf3+ (N), and dkk1+ (P) embryos. The dorsal boundary of dlx2a expression (arrowheads) appears to shift ventrally in some cases, (N,P) compared to the control (M,O). (Q) Histogram quantifying percentages of dkk1+ and dntcf3+ embryos with defects in D-V patterning. Scale bar: 100 µm.
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pgen-1004479-g004: Requirements for Wnt signaling in dorsal-ventral arch patterning.(A–P) Whole mount ISH for genes involved in dorsal-ventral (D-V) patterning in control (A, D, G, J), dkk1+ embryos heat shocked at 20–22 hpf (B, E, H, K), and dntcf3+ embryos heat shocked at 22–24 hpf (C, F, I, L), lateral views, anterior to the left. hand2 (B, C), dlx3b (E, F), and dlx6a (H, I) expression is reduced, while jag1b (K, L) expression expands ventrally (arrowheads) particularly in dntcf3+ embryos, compared with controls. (M–P) dlx2a expression in control (M,O), dntcf3+ (N), and dkk1+ (P) embryos. The dorsal boundary of dlx2a expression (arrowheads) appears to shift ventrally in some cases, (N,P) compared to the control (M,O). (Q) Histogram quantifying percentages of dkk1+ and dntcf3+ embryos with defects in D-V patterning. Scale bar: 100 µm.

Mentions: To investigate roles for Wnt signaling in D-V patterning within the arches, we examined expression of genes that mark distinct ventral, intermediate and dorsal regions of the arch primordia in dntcf3+ and dkk1+ embryos with ISH [19]. hand2 expression in the ventral-most domains of each arch was severely reduced in both dkk1+ (53%, n = 15) and dntcf3+ (59%, n = 17) embryos (Fig. 4A–C, Q), with a small domain of expression remaining at the arch 1–2 boundary in dntcf3+ embryos (Fig. 4C). Similarly, expression of dlx3b and dlx6a in the intermediate domains of each arch were mildly reduced in dkk1+ (44%, n = 18, 23%, n = 21) and severely reduced in dntcf3+ embryos (83%, n = 12; 90%, n = 21) (Fig. 4D–I, Q). Finally, expression of the Notch ligand, jag1b in the dorsal-most domains of each arch [28], was variably expanded ventrally in dkk1+ (10.5%, n = 57) embryos and consistently expanded in dntcf3+ embryos (55.5%, n = 9) as well as chimeras in which dntcf3+ cells were transplanted into the pharyngeal endoderm (Fig. 4J–L, Q; Fig. S2B–C). These gene expression changes were not simply due to an overall loss of arches or NC cells, since dlx2a expression (Fig. 4N, P) as well as sox10:lynTdtom expression throughout the D-V extent of the arch NC were unaffected in the arches of both dkk1+ and dntcf3+ embryos (Fig. S7). Additionally, BIO treatments of wild type embryos caused dorsal expansion of expression of the ventral-intermediate gene msxe, mild expansion of dlx3b and hand2 expression, and reduced jag1b expression in the dorsal domain (Fig. S8). Therefore, Wnt signaling promotes ventral and intermediate-cell fates in the arches. Dntcf3+ embryos in particular, with residual hand2 expression at the arch 1–2 boundary, closely resemble mutants in Bmp and Edn1 signaling [19], [20].


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 dorsal-ventral arch patterning.(A–P) Whole mount ISH for genes involved in dorsal-ventral (D-V) patterning in control (A, D, G, J), dkk1+ embryos heat shocked at 20–22 hpf (B, E, H, K), and dntcf3+ embryos heat shocked at 22–24 hpf (C, F, I, L), lateral views, anterior to the left. hand2 (B, C), dlx3b (E, F), and dlx6a (H, I) expression is reduced, while jag1b (K, L) expression expands ventrally (arrowheads) particularly in dntcf3+ embryos, compared with controls. (M–P) dlx2a expression in control (M,O), dntcf3+ (N), and dkk1+ (P) embryos. The dorsal boundary of dlx2a expression (arrowheads) appears to shift ventrally in some cases, (N,P) compared to the control (M,O). (Q) Histogram quantifying percentages of dkk1+ and dntcf3+ embryos with defects in D-V patterning. Scale bar: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004479-g004: Requirements for Wnt signaling in dorsal-ventral arch patterning.(A–P) Whole mount ISH for genes involved in dorsal-ventral (D-V) patterning in control (A, D, G, J), dkk1+ embryos heat shocked at 20–22 hpf (B, E, H, K), and dntcf3+ embryos heat shocked at 22–24 hpf (C, F, I, L), lateral views, anterior to the left. hand2 (B, C), dlx3b (E, F), and dlx6a (H, I) expression is reduced, while jag1b (K, L) expression expands ventrally (arrowheads) particularly in dntcf3+ embryos, compared with controls. (M–P) dlx2a expression in control (M,O), dntcf3+ (N), and dkk1+ (P) embryos. The dorsal boundary of dlx2a expression (arrowheads) appears to shift ventrally in some cases, (N,P) compared to the control (M,O). (Q) Histogram quantifying percentages of dkk1+ and dntcf3+ embryos with defects in D-V patterning. Scale bar: 100 µm.
Mentions: To investigate roles for Wnt signaling in D-V patterning within the arches, we examined expression of genes that mark distinct ventral, intermediate and dorsal regions of the arch primordia in dntcf3+ and dkk1+ embryos with ISH [19]. hand2 expression in the ventral-most domains of each arch was severely reduced in both dkk1+ (53%, n = 15) and dntcf3+ (59%, n = 17) embryos (Fig. 4A–C, Q), with a small domain of expression remaining at the arch 1–2 boundary in dntcf3+ embryos (Fig. 4C). Similarly, expression of dlx3b and dlx6a in the intermediate domains of each arch were mildly reduced in dkk1+ (44%, n = 18, 23%, n = 21) and severely reduced in dntcf3+ embryos (83%, n = 12; 90%, n = 21) (Fig. 4D–I, Q). Finally, expression of the Notch ligand, jag1b in the dorsal-most domains of each arch [28], was variably expanded ventrally in dkk1+ (10.5%, n = 57) embryos and consistently expanded in dntcf3+ embryos (55.5%, n = 9) as well as chimeras in which dntcf3+ cells were transplanted into the pharyngeal endoderm (Fig. 4J–L, Q; Fig. S2B–C). These gene expression changes were not simply due to an overall loss of arches or NC cells, since dlx2a expression (Fig. 4N, P) as well as sox10:lynTdtom expression throughout the D-V extent of the arch NC were unaffected in the arches of both dkk1+ and dntcf3+ embryos (Fig. S7). Additionally, BIO treatments of wild type embryos caused dorsal expansion of expression of the ventral-intermediate gene msxe, mild expansion of dlx3b and hand2 expression, and reduced jag1b expression in the dorsal domain (Fig. S8). Therefore, Wnt signaling promotes ventral and intermediate-cell fates in the arches. Dntcf3+ embryos in particular, with residual hand2 expression at the arch 1–2 boundary, closely resemble mutants in Bmp and Edn1 signaling [19], [20].

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