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.
Highlights
A fundamental question in skeletal biology is how cartilages and bones with distinct shapes arise from skeletogenic precursor cells
In this study we examine the role of Wnts in craniofacial patterning using transgenic zebrafish to inhibit downstream Wnt signaling
We show that Wnt signaling deficient embryos have lower jaw specific defects, which strongly resembles loss-of-function phenotypes in both the Bone morphogenetic proteins (Bmps) and Endothelin 1 (Edn1) signaling pathways
Summary
A fundamental question in skeletal biology is how cartilages and bones with distinct shapes arise from skeletogenic precursor cells. Much of the craniofacial skeleton derives from neural crest (NC) cells that migrate in streams into the pharyngeal arches and contain anterior-posterior (A-P) patterning information obtained prior to migration [1,2,3]. These NC cells become intimately associated with epithelia, including surface ectoderm and pharyngeal endoderm, which produce signals important for skeletal patterning. Endoderm-derived Fgf induces cartilage formation [9] and sphingosine phosphate-1 from endoderm modulates Shh signaling to promote mandibular growth and patterning [11,12,13]. Craniofacial skeletal shapes reflect interplay between epithelial signals and intrinsic properties of mesenchyme, but the mechanisms underlying these interactions remain unclear
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