Inadequate Cell Proliferation during Palatogenesis of GSK-3β Null Mice

Objectives: GSK-3β is essential in midline development. GSK-3β null mice have been found to exhibit midline defects including incomplete fusion of the ribs at the midline, bifid sternum, delayed sternal ossification as well as complete cleft of the secondary palate. E13.5 to E15.0 has been shown to be the critical period for GSK-3β in mouse palatogenesis. The palatal shelves in these mutant mice undergo normal elevation process but fail to approximate and fuse in the midline. Abnormalities in cell proliferation and/or cell apoptosis in the developing secondary palate may have caused the cleft palate formation. To quantitatively assess cell proliferation and apoptosis in the developing palate of GSK-3β null mice at E13.5 and to compare with that of wildtype and heterozygous mice. Methods: Terminal deoxynucleotidyl transferase-mediated-deoxyuridinetriphosphate nick-end labelling (TUNEL) assay was carried out to detect apoptotic cells within the palatal shelves of wildtype, heterozygous and GSK-3β null mice. The data from these three groups were then analysed using chi-squared tests. Phospho-Histone H3 (pHH3) immunohistochemistry was performed to identify proliferating cells within the palatal shelves of these three groups. The mean numbers of proliferating cells were then compared and statistically analysed using ANOVA followed by Bonferroni post-hoc test. To confirm the results from pHH3 experiment, BrDU assay was carried out. The results were analysed using Student’s t-test. Results:  Histological analyses revealed that the palatal shelves of GSK-3β null mice were consistently smaller with poorly defined palatal bend when compared to that of wildtype littermates. PHH3 and BrDU assays demonstrated a statistically significant reduction (p<0.05) in the number of proliferating cells, while TUNEL assay found unaltered cell apoptosis (p>0.05) in mutant palatal shelves. Conclusion:  Cleft palate in GSK-3β null mice is associated with reduced cell proliferation that results in morphologically smaller palatal shelves, which fail to extend and fuse in the midline.