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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 pharyngeal arch cell proliferation.(A–L) Whole mount ISH for pcna in embryos fixed at 3–22 hours post-heat shock (hphs), lateral views, anterior to the left (all heat shocked at 22 hpf). (A–D) pcna is expressed throughout arches, brain and eyes. (E–H) dkk1+ embryos have reduced pcna expression at 3 hphs (E), severe reductions at 6 hphs (F) and virtually no arch expression at 8 hphs (G), before expression returns at 22 hphs (H). (I–L) dntcf3+ embryos show reduced pcna expression at 3 hphs (I), and virtually no expression at 6 hphs (J, K) before expression rebounds between 8–22 hphs (L). (M) Histogram quantifying percentages of dkk1+ and dntcf3+ embryos with moderate versus severe reductions in pcna expression. Scale bar: 100 µm.
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pgen-1004479-g003: Requirements for Wnt signaling in pharyngeal arch cell proliferation.(A–L) Whole mount ISH for pcna in embryos fixed at 3–22 hours post-heat shock (hphs), lateral views, anterior to the left (all heat shocked at 22 hpf). (A–D) pcna is expressed throughout arches, brain and eyes. (E–H) dkk1+ embryos have reduced pcna expression at 3 hphs (E), severe reductions at 6 hphs (F) and virtually no arch expression at 8 hphs (G), before expression returns at 22 hphs (H). (I–L) dntcf3+ embryos show reduced pcna expression at 3 hphs (I), and virtually no expression at 6 hphs (J, K) before expression rebounds between 8–22 hphs (L). (M) Histogram quantifying percentages of dkk1+ and dntcf3+ embryos with moderate versus severe reductions in pcna expression. Scale bar: 100 µm.

Mentions: Cartilages in dntcf3+ and dkk1+ larvae were 30–50% smaller than controls (Fig. 2). This reduced cartilage size was not due to increased cell death as we could detect no differences in the number of acridine orange stained cells in the arches between dntcf3+, dkk1+ and control embryos at 6 hphs (Fig. S5). To examine proliferation in the arches we performed ISH for pcna. Pcna mRNA was detected throughout the pharyngeal arches from 3–22 hphs (25–44 hpf) in controls (Fig. 3A–D), but somewhat reduced at 3 hphs (25 hpf) in both dkk1+ and dntcf3+ embryos (Fig. 3A, E, I, M). By 6 hphs (28 hpf) pcna expression was nearly undetectable in the arches in both dkk1+ (66%, n = 6) and dntcf3+ (60%, n = 10) (Fig. 3B, F, J, M). By 8 hphs (30 hpf), pcna expression had recovered slightly in dntcf3+ embryos (36%, n = 11) (Fig. 3K, M) but not in dkk1+ embryos (80%, n = 5) (Fig. 3G, M). Both recovered completely by 22–28 hphs (44 hpf) (Fig. 3D, H, L, M).


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 pharyngeal arch cell proliferation.(A–L) Whole mount ISH for pcna in embryos fixed at 3–22 hours post-heat shock (hphs), lateral views, anterior to the left (all heat shocked at 22 hpf). (A–D) pcna is expressed throughout arches, brain and eyes. (E–H) dkk1+ embryos have reduced pcna expression at 3 hphs (E), severe reductions at 6 hphs (F) and virtually no arch expression at 8 hphs (G), before expression returns at 22 hphs (H). (I–L) dntcf3+ embryos show reduced pcna expression at 3 hphs (I), and virtually no expression at 6 hphs (J, K) before expression rebounds between 8–22 hphs (L). (M) Histogram quantifying percentages of dkk1+ and dntcf3+ embryos with moderate versus severe reductions in pcna expression. Scale bar: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004479-g003: Requirements for Wnt signaling in pharyngeal arch cell proliferation.(A–L) Whole mount ISH for pcna in embryos fixed at 3–22 hours post-heat shock (hphs), lateral views, anterior to the left (all heat shocked at 22 hpf). (A–D) pcna is expressed throughout arches, brain and eyes. (E–H) dkk1+ embryos have reduced pcna expression at 3 hphs (E), severe reductions at 6 hphs (F) and virtually no arch expression at 8 hphs (G), before expression returns at 22 hphs (H). (I–L) dntcf3+ embryos show reduced pcna expression at 3 hphs (I), and virtually no expression at 6 hphs (J, K) before expression rebounds between 8–22 hphs (L). (M) Histogram quantifying percentages of dkk1+ and dntcf3+ embryos with moderate versus severe reductions in pcna expression. Scale bar: 100 µm.
Mentions: Cartilages in dntcf3+ and dkk1+ larvae were 30–50% smaller than controls (Fig. 2). This reduced cartilage size was not due to increased cell death as we could detect no differences in the number of acridine orange stained cells in the arches between dntcf3+, dkk1+ and control embryos at 6 hphs (Fig. S5). To examine proliferation in the arches we performed ISH for pcna. Pcna mRNA was detected throughout the pharyngeal arches from 3–22 hphs (25–44 hpf) in controls (Fig. 3A–D), but somewhat reduced at 3 hphs (25 hpf) in both dkk1+ and dntcf3+ embryos (Fig. 3A, E, I, M). By 6 hphs (28 hpf) pcna expression was nearly undetectable in the arches in both dkk1+ (66%, n = 6) and dntcf3+ (60%, n = 10) (Fig. 3B, F, J, M). By 8 hphs (30 hpf), pcna expression had recovered slightly in dntcf3+ embryos (36%, n = 11) (Fig. 3K, M) but not in dkk1+ embryos (80%, n = 5) (Fig. 3G, M). Both recovered completely by 22–28 hphs (44 hpf) (Fig. 3D, H, L, M).

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