Abnormal expression of TBX4 during anorectal development in rat embryos with ethylenethiourea-induced anorectal malformations

ABSTRACT

BACKGROUND: To assess the expression of T-box transcription factor 4 (TBX4) during the anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformations (ARM) rat embryos. METHODS: Anorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatiotemporal expression of TBX4 was evaluated in normal (n = 490) and ETU-induced ARM rat embryos (n = 455) from E13 to E16 by immunohistochemical staining, Western blot analysis and real-time RT-PCR. RESULTS: In the normal embryos, immunohistochemical staining revealed that TBX4 expression was detected in the epithelium of hindgut and urorectal septum (URS) on E13. TBX4-immunopositive cells were increased significantly in the epithelium of hindgut and URS, the future anal orifice part of cloacal membrane on E14. On E15, abundant stained cells were observed in the rectum, URS and dorsal cloacal membrane and the expression of positive cells reached its peak. On E16, only sporadic positive cells were distributed in the epithelium of the distal rectum. In the ARM embryos, the hindgut/rectum, URS and dorsal cloacal membrane were faint for TBX4 immunohistochemical staining. In the normal group, TBX4 protein and mRNA expression showed time-dependent changes in the hindgut/rectum from E13 to E16 on Western blot and real-time RT-PCR. On E13 and E15, the expression level of TBX4 mRNA in the ARM group was significantly lower than that in the normal group (P < 0.05). On E15, the expression level of TBX4 protein in the ARM group was significantly lower than that in the normal group (P < 0.05). CONCLUSIONS: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.