Spatiotemporal Expression of Cdx4 in the Developing Anorectum of Rat Embryos with Ethylenethiourea-Induced Anorectal Malformations

Abstract

Purpose: The aim of this study was to determine the expression of Cdx4 (caudal-type homeobox gene-4) during anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformation (ARM) embryos with a view to establishing the possible role of Cdx4 in ARM pathogenesis. Materials and Methods: ARM was induced by ETU on the 10th gestational day (GD10) in rat embryos. Cesarean deliveries were then performed to harvest the embryos. Spatiotemporal expression of Cdx4 was evaluated in normal rat embryos (n = 354) and ARM embryos (n = 378) from GD13 to GD16. Results: Immunohistochemical staining and immunofluorescence revealed that, in normal embryos, Cdx4 expression was extensively detected on the epithelium of the cloaca on GD13. On GD14, the Cdx4-positive cells were intensively detected on the hindgut. On GD15, the anal membrane was constantly immunoreactive to Cdx4. On GD16, Cdx4-labeled cells were observed on the epithelium of the anus. In the ARM embryos, the epithelium of the cloaca, urorectal septum (URS) and anorectum was negative or faint for Cdx4. In the normal embryo group, Cdx4 protein and mRNA expression showed time-dependent changes in the developing hindgut from GD13 to GD16 on Western blot and real-time reverse transcription plus polymerase chain reaction. Once the URS divided the cloaca into the primitive rectum and urogenital sinus (UGS) on GD15, Cdx4 expression began to decrease. In addition, the expression level of Cdx4 in the ARM group from GD13 to GD15 was significantly lower than that in the normal group (p < 0.05). Conclusions: In ARM embryos, an imbalance in the spatiotemporal expression of Cdx4 was noted during anorectal morphogenesis from GD13 to GD16. This suggests that ETU may cause downregulation of Cdx4 expression. Downregulation of Cdx4 at the time of cloacal separation into the primitive rectum and UGS might thus be related to the development of ARM.