166. Modulation of Wnt Pathway Signaling Determines Ectopic Craniofacial Fusion and Orofacial Clefting

Abstract

Introduction: The etiology of facial clefting is a complex synthesis of multiple environmental and genetic factors. Therefore few inroads have been made into the design or development of targeted preventions or treatments for these birth defects. The identification of a developmental signaling pathway that definitively regulates facial fusion would be a crucial first step. Previous gene expression studies in mice have demonstrated that Wnt genes are expressed in the right time and place to be critical for facial fusion. Studies of mouse strains prone to facial clefting have identified causative mutations in the Wnt9b locus. This study examines for the first time how the experimental manipulation of Wnt signaling at the pathway level influences the normal events of craniofacial morphogenesis. Here we use two animal models in which the early events of facial development are remarkably conserved, combining mouse transgenetics with in ovo viral-mediated gene transfer in developing chick embryos. Methods: Wild type (CD1) or Wnt reporter (BATGAL) mouse embryos and normal chicken embryos representing the normal course of craniofacial outgrowth and fusion were collected at E10.5-E13.5 (mouse) and St. 25-28 (E5-E7; chick). Gene expression was detected by non-radioactive in situ hybridization or X-gal detection of beta-galactosidase transgene activity in either whole-mount or sectioned embryos. To achieve viral-mediated gene transfer in chick embryos, adenovirus encoding the soluble Wnt inhibitor Dkk1 or retrovirus encoding Wnt3a ligand were delivered in ovo to the maxillary prominence (MXP) by microinjection. Results: in mouse and chick embryos, Wnt7b and Wnt9b ligand gene expression was found to localize to the surface ectoderm of fusing facial prominences. In BATGAL mice, Wnt-responsive cells were found in the surface ectoderm of the fusing lateral and frontonasal promences, as well as in the underlying mesenchyme of the developing facial prominences. To determine whether repression of Wnt pathway activity prevented normal facial fusion, Ad-Dkk1 or Ad-Fc (as a control) was injected into the developing maxillary prominence at St. 25 (E5.5). After 36 hours of incubation, facial clefts were visible in embryos treated with Ad-Dkk1, whereas the face had fused normally in controls. To assess whether exogenous Wnt pathway activation might induce ectopic fusion of craniofacial tissues, RCAS-Wnt3a or RCAS-AP (alkaline phosphatase) was injected into the developing maxillary facial prominence at St. 23 (E4). After 36 hours of incubation, the injected maxillary prominences of RCAS-Wnt3a treated embryos had grown out and fused to the frontonasal prominence, whereas the contralateral, uninjected maxillary facial prominence and the RCAS-AP injected MXP had not yet fused. Conclusions: Our gain- and loss-of-function data provide strong evidence that normal Wnt pathway activity is necessary for physiologic facial fusion, and that exogenous activation of the Wnt pathway by administration of the ligand is sufficient to induce ectopic fusion events within the craniofacial complex. Further studies using this model will determine how regulation of the Wnt pathway influence epithelial seam cell fate decisions during facial fusion, such as proliferation rates, apoptosis, migration, and epithelial-mesenchymal transformations. Finally, our model suggests that therapeutic modulation of a cell signaling pathway may be a viable strategy for in utero treatment of facial clefts.