Expression of EphB2 in the development of anorectal malformations in fetal rats

Abstract

Purpose The receptor tyrosine kinase of the Eph family is a large group of highly conserved molecules that function in diverse intercellular recognition events. It has been reported that EphB2 is related to caudal remodeling events. The aim of this study is to investigate EphB2 expression in anorectal development in normal and rat embryos with anorectal malformations (ARMs) and attempt to define its role in anorectal morphogenesis. Methods The ethylenethiourea (ETU) rat model of the ARMs was used in this study. Immunohistochemical analyses and real time quantitative polymerase chain reaction (PCR) were carried out to investigate EphB2 protein localizations and messenger RNA (mRNA) expression levels. (1) Rat embryos with ARMs were obtained by treating pregnant rats (n = 24) with administration of ETU on gestation day (Gd) 10. Normal rat embryos (n = 111) and embryos treated by ETU without ARMs (n = 90) were the control groups, and embryos with ARMs (n = 108) from Gd13 to Gd16 were divided according to the sections taken from specimens. (2) Embryos were sequentially sectioned in the sagittal and transversal planes before staining with a specific antibody to EphB2. Spatiotemporal study was carried out on EphB2 expression. (3) Individual frozen sections were used to manually microdissect the cloaca and anorectal specimens for total RNA extraction. EphB2 expression was evaluated by real time quantitative PCR. Results On the immunologic labeling study, EphB2 expression was confined to the cloaca in control groups, whereas EphB2 expression was mainly located at the urorectal septum (URS) and cloacal membrane on Gd13 and Gd14. The increased positive expression was observed in the fused tissue of the URS and cloacal membrane on Gd15. On Gd16, the anal membrane broke down, and the rectum was able to be in contact with the anus, and EphB2 expression was then noted in mucous membrane of rectum. EphB2 expression was seen in the cloacal and anorectal tissues of embryos with ARMs. By integrated optical density (IOD) measurement, IOD value of EphB2 protein was significantly lower in the ARM group than that in the control groups on Gd13 to Gd16 ( P < .05), respectively. As shown by real time quantitative PCR, EphB2 expression was detected in 3 groups. EphB2 mRNA level increased on Gd13 to Gd16 but gradually decreased after Gd16. The expression level of EphB2 mRNA in the ARM embryos was lower on Gd13 to Gd16 than that in control groups ( P < .05). Conclusions EphB2 expression decreased in the ARM embryos and was confined to URS and cloaca, whereas it was higher in control group. Our data thus indicated that EphB2 molecules possibly contributed to the anorectal morphogenesis and the decreased expression of EphB2 might be related to the development of ARMs.