Flrt2 Interacts with Fgfr2 During Murine Craniofacial Development

Background: Craniofacial birth disorders, among the most common of all human congenital anomalies, are often the result of defective patterning during embryonic development. In the midfacial region, aberrant growth of the cartilaginous nasal capsule from neural crest-derived mesenchyme of the frontonasal region is observed in craniofacial birth defects such as Crouzon syndrome where several mutations in the Fibroblast Growth Factor-Receptor (Fgfr) family of genes have been identified. Mutations in the same Fgfr gene yield different clinical presentations, suggesting the additional importance of the Fgf ligands and other receptor-associated proteins in the etiology of the specific craniofacial defects. We have preliminary evidence to suggest that Flrt2, one of the three members of the mouse Fibronectin Leucine Rich Transmembrane gene family, is a putative Fgfr interacting partner during growth and chondrogenesis of the midfacial region. Flrt2 has been shown to be highly expressed in the developing midfacial region (Chung & Gong, unpublished). Previous in vitro studies indicated that Flrt2 binds to members of the Fgfr family and is a modular of Fgf signalling. Objective: To characterize Flrt2/Fgfr2 interactions during midfacial development. Methods: Protein interactions were performed by co-immunoprecipitation and GST pull-down experiments. Lysates prepared from the craniofacial regon were either immunoprecipitated with anti-FLRT2 monoclonal antibody in CO-IP or pulled down with GST-FLRT2 in GST-pull down assay and blotted with FGFR2 antibody. Results: Co-immunoprecipitation experiment indicated mouse Flrt2 interacted with FGF receptor 2 (FGFR2) in vivo and in GST-pull down assays in vitro. Conclusion: The results indicate that Flrt2 interacts with Fgfr2 during midfacial development and is very likely a transmembrane modulator of FGF signalling in the midface. Further experiments will involve the mapping of the domains in Flrt2 involved in these interactions.

Funded by CRC, CFI, CIHR International Opportunities Program and Rosenstadt Funds.