Pathogenesis of cleft palate in TGF-beta 3 knockout mice

We previously reported that mutation of the transforming growth factor-beta 3 (TGF-beta 3) gene caused cleft palate in homozygous null (-/-) mice. TGF -beta 3 is normally expressed in the medial edge epithelial (MEE) cells of the palatal shelf. In the present study, we investigated the mechanisms by which TGF-beta 3 deletions caused cleft palate in 129 x CF-1 mice. For orga n culture, palatal shelves were dissected from embryonic day 13.5 (E13.5) m ouse embryos. Palatal shelves were placed singly or in pairs on Millipore f ilters and cultured in DMEM/F12 medium. Shelves were placed in homologous ( +/+ vs +/+, -/- vs -/-, +/- vs +/-) or heterologous (+/+ vs -/-, +/- vs -/- , +/+ vs +/-) paired combinations and examined by macroscopy and histology, Pairs of -/- and -/- shelves failed to fuse over 72 hours of culture where as pairs of +/+ (wild-type) and +/- or +/- (heterozygote) and +/-, as well as +/+ and -/- shelves, fused within the first 48 hour period. Histological examination of the fused +/+ and +/+ shelves showed complete disappearance of the midline epithelial seam whereas -/- and +/- shelves still had some seam remnants. Tn order to investigate the ability of TGF-beta family membe rs to rescue the fusion between -/- and -/- palatal shelves in vitro, eithe r recombinant human (rh) TGF-beta 1, porcine (p) TGF-beta 2, rh TGF-beta 3, rh activin, or p inhibin was added to the medium in different concentratio ns at specific times and for various periods during the culture. In untreat ed organ culture -/- palate pairs completely failed to fuse, treatment with TGF-beta 3 induced complete palatal fusion, TGF-beta 1 or TGF-beta 2 near normal fusion, but activin and inhibin had no effect. We investigated ultra structural features of the surface of the MEE cells using SEM to compare TG F-beta 3-null embryos (E 12.5-E 16.5) with +/- and +/- embryos in vivo and in vitro. Up to E13.5 and after E15.5, structures resembling short rods wer e observed in both +/+ and -/- embryos. Just before fusion, at E14.5, a lot of filopodia-like structures appeared on the surface of the MEE cells in /+ embryos, however, none were observed in -/- embryos, either in vivo or i n vitro. With TEM these filopodia are coated with material resembling prote oglycan. Interestingly, addition of TGF-beta 3 to the culture medium which caused fusion between the -/- palatal shelves also induced the appearance o f these filopodia on their MEE surfaces. TGF-beta 1 and TGF-beta 2 also ind uced filopodia on the -/- MEE but to a lesser extent than TGF-beta 3 and ad ditionally induced lamellipodia on their cell surfaces. These results sugge st that TGF-beta 3 may regulate palatal fusion by inducing filopodia on the outer cell membrane of the palatal medial edge epithelia prior to shelf co ntact. Exogenous recombinant TGF-beta 3 can rescue fusion in -/- palatal sh elves by inducing such filopodia, illustrating that the effects of TGF-beta 3 are transduced by cell surface receptors which raises interesting potent ial therapeutic strategies to prevent and treat embryonic cleft palate.