Purpose: Crouzon syndrome results from dominant gain-of-function mutations in fibroblast growth factor receptor 2 (FGFR2^C342Y) and is associated with an increased incidence of cleft palate, yet the molecular mechanisms contributing to this phenotype are unknown.  Interestingly, both gain-of-function and loss-of-function genetic mutations have been described in the development of syndromic and nonsyndromic cleft palate.  Evidence of increased FGFR signaling as well as decreased FGFR signaling has been noted by our lab in the mouse model of Crouzon syndrome.  Because cleft palate is a common birth anomaly resulting in substantial morbidity and healthcare costs, the molecular signaling mechanisms involved in the Crouzon model may be fruitful to understanding the pathogenesis of this anomaly.  We have completed expression studies of the FGFR signaling cascade during different stages of palate development in the murine model of Crouzon syndrome to investigate the molecular mechanisms that result in the abnormal palatal phenotype.  We propose that the FGFR2 mutation in Crouzon syndrome results in an increase in mesenchymal FGFR signaling that persists throughout palate development.

 Methods: Fgfr2^C342Y/+ mice were intercrossed to generate litters with homozygous, heterozygous, and wild type (WT) embryos.  In vitro studies from E12.5-15.5 were performed using a dynamic whole-organ palatal culture system to assess the effects of FGF-FGFR signaling augmentation with FGF2 and signaling inhibition with PD176074, a tyrosine kinase inhibitor.  Mutant embryos were compared to WT embryos at embryonic days (E) 13.5-15.5.  Developing palate shelves were dissected from the maxilla and divided into anterior and posterior halves.  Palatal shelf RNA was extracted in Trizol, and 500ng of RNA was used as the starting quantity for cDNA synthesis.  cDNA was made using the SuperScriptIII First Strand kit (Invitrogen) according to the manufacturer’s instructions.  Quantitative RT-PCR was performed using TaqMan probes.    

 Results: Both augmentation and inhibition of FGFR signaling resulted in clefts of WT palates in vitro, while untreated WT palates demonstrated normal palatal shelf elevation and fusion in vitro.  Quantitative RT-PCR analysis demonstrated increased expression of mediators of the mesenchymal FGFR signaling pathway in the posterior palates of the homozygous Crouzon embryos compared to WT throughout palate development (E13.5-15.5).  Just prior to palatal shelf elevation (E14.25), Pea3 expression is nearly doubled and Barx1 expression is increased greater than 3-fold.  In contrast, expression of Sprouty2 and Sprouty4 were down-regulated in the homozygous mutant embryos compared to WT, with significantly decreased expression after palatal shelf elevation at E15.5 (0.29+/-0.09, p<0.05 and 0.21+/-0.07, p<0.02, respectively). 

 Conclusions: Both augmentation and inhibition of the FGF-FGFR signaling cascade result in palatal clefting.  In the case of Crouzon syndrome, the genetic mutation results in increased expression of mediators of the FGFR signaling cascade that persists throughout palate development.  In addition, expression of inhibitors of the FGFR signaling cascade is down-regulated throughout palatogenesis.  These defects foster abnormal palatal outgrowth and elevation resulting in cleft palate.  Knowledge of the mechanistic processes of this disease may contribute to improved therapeutic interventions for clinical application.