Investigation of hoxa2 gene function in palate development using a retroviral gene delivery system

Abstract

Cleft palate is a common human birth defect caused by any process which interferes with palatogenesis. Studies in Hoxa2 mutant (Hoxa2-/-) mice which exhibit a secondary cleft palate were reported to be due to an abnormal positioning of the tongue which prevents normal palatal shelf fusion to occur. To obtain direct evidence for the importance of Hoxa2 in murine palate development, an in vitro whole organ palatal culture model was developed, eliminating any influences from the tongue. A retroviral gene delivery system was employed, containing either Hoxa2 sense or Hoxa2 antisense cDNA, to respectively enhance or knockdown the expression of Hoxa2 mRNA in the developing palate. Our results show that palatal cultures infected with the lowest titer of Hoxa2 sense virus induce a fusion rate of 72.7%, which is similar to palatal cultures treated with the control virus (81.8%), although fusion rates of 41.2% to 50.0% were observed in palates infected with higher titers. With the antisense virus treated group, a more profound inhibition of the fusion rate was observed (27.7% - 46.1%), which is comparable with the frequency of palatal fusion in Hoxa2-/- mice (44.4%). Additionally, the palatal shelves in both sense and antisense virus treated groups appear to be relatively shorter in length, than those measured in the control group. Interestingly, in the antisense virus treated group, the ratio of the length of the fused portion to the length of palatal shelves appears to be relatively large compared to the control group. Verification and quantification of Hoxa2 mRNA in the developing palate between E12.5 and E15.5 was performed by real-time RT-PCR. Hoxa2 gene expression was observed at all stages studied, with expression being the highest at E12.5 and declining from E13.5. The expression level remained constant from E13.5 through E15.5. These findings demonstrate for the first time that Hoxa2 may play a direct role in murine palate development. Results suggest that both factors (the absence of Hoxa2 gene in the palate causing delayed palatal development, as well as the position of the tongue) appear to act in unison to produce cleft palate in Hoxa2 knockout mice.