Temporal and spatial expression of caudal-type homeobox gene-2 during hindgut development in rat embryos with ethylenethiourea-induced anorectal malformations.

Abstract

The main aim of this study was to determine Cdx2 expression patterns during anorectal development in normal and anorectal malformation (ARM) embryos with a view to establishing the possible role of Cdx2 in ARM pathogenesis. ARM was induced with ethylenethiourea on the 10th gestational day (GD10) in rat embryos, and Cesarean deliveries were performed to harvest the embryos. The temporal and spatial expression of Cdx2 was evaluated in normal rat embryos (n = 303) and ARM embryos (n = 321) from GD13 to GD16. Immunohistochemical staining revealed that, in normal embryos, Cdx2 was mainly expressed on the epithelium of the urorectal septum (URS) and the hindgut on GD13. On GD14, Cdx2-immunopositive cells were extensively detected on the URS, hindgut, and cloacal membrane. On GD15, increased immunopositive tissue staining on the anal membrane was evident. In ARM embryos, the epithelium of the cloaca, URS, and anorectum were negative or faintly immunostaining for Cdx2. Analyses by Western blot and real-time reverse transcription plus the polymerase chain reaction revealed that, in the normal group, Cdx2 protein and mRNA expression showed time-dependent changes in the developing hindgut from GD13 to GD16. Upon the URS division of the cloaca into the primitive rectum and urogenital sinus (UGS) on GD15, Cdx2 expression began to decrease. Moreover, the Cdx2 expression level in the ARM group from GD13 to GD14 was significantly lower than that in the normal group (P < 0.05). In ARM embryos, an imbalance in the spatiotemporal expression of Cdx2 was noted during anorectal morphogenesis from GD13 to GD16. Downregulation of Cdx2 at the time of cloacal separation into the primitive rectum and UGS might thus be related to the development of ARM.