Tbx22 expressions during palatal development in fetuses with glucocorticoid-/alcohol-induced C57BL/6N cleft palates.

T-box transcription factor 22 (Tbx22) belongs to the T-box family of transcription factors and was originally found using an in silico approach to identify new genes in the human Xq12-Xq21 region. Mutations in Tbx22 have been reported in families with X-linked cleft palate and ankyloglossia, but the underlying pathogenetic mechanism remains unknown. The aim of this study was to evaluate the expression of Tbx22 messenger RNA (mRNA) during palatogenesis in glucocorticoid-/alcohol-induced cleft palate in a C57BL/6N mouse model. Palatal development was monitored by histomorphologic and immunohistochemical studies and by in situ hybridization. Thirty pregnant C57BL/6N mice at 8 weeks of age, weighing 20 to 25 g, were used in this study. In the experimental group, 12 mice were exposed to alcohol for 7 days before mating, and 12 mice in the control group were not exposed. Six mice in a sham group were exposed to neither alcohol nor glucocorticoids. A total of 18 fetuses with induced cleft palates each from 102 fetuses in the experimental group, 109 in the control group, and 58 in the sham group were used. In both the experimental and the control groups, glucocorticoids were injected subcutaneously on gestational days (GD) 9.5, 10.5, and 11.5, and each mouse was killed on GDs 10.5 to 15.5. Histomorphologic findings were studied using hematoxylin and eosin staining, and antibodies against proliferation cell nuclear antigen, matrix metallopeptidase 9, zinc finger protein 422 (Krox25) heat shock protein 70, and Tbx22 were used in immunohistochemical analysis. Mouse Tbx22 mRNA was identified, and its expression was analyzed during embryogenesis by polymerase chain reaction and in situ hybridization. Coronal sections of the cleft maxilla of the embryos with induced cleft palates had a gap between the palatal shelves, where 2 palatal shelves had fused as in normal development but failed to meet and fuse to each other. By in situ hybridization, Tbx22 mRNA was found to be expressed in distinct areas of the head, such as the mesenchyme of the inferior nasal septum, the posterior palatal shelf before fusion, and the attachment of the tongue during normal development of the palate and maxilla from GD 11.5. Localization in the tongue frenum correlated with the ankyloglossia phenotype in the induced cleft palate animal model.