Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats

Abstract

Sonic hedgehog (Shh) has been shown to be involved in the morphogenesis of many organ systems including the notochord, floor plate and limbs, as well as in the development of the left-right axis in vertebrates. Recent evidence suggests the Shh cascade plays a crucial role in the development of the foregut and hindgut. We have previously shown that prenatal exposure of fetal rats to ethylenethiourea (ETU) induces hindgut malformations and other abnormalities of the VACTERL association. The aim of this study was to determine the pattern of expression of Shh and its downstream genes during hindgut development in ETU-exposed embryos with anorectal malformations (ARMs). Pregnant Sprague-Dawley rats were mated together overnight and a positive vaginal plug was marked as gD0. On gD10, 1% ETU (125 mg/kg) was given to the experimental group and controls received the same volume of saline. Embryos were collected from both groups at gD12-16. The developing hindgut of each embryo was dissected under magnification and snap frozen. Highly purified RNA was isolated from each hindgut and first strand cDNA was prepared with appropriate negative controls. Reverse transcriptase (RT) polymerase chain reaction (PCR) was done to determine the transcripts of Shh in each sample and quantitative real-time PCR was carried out to show relative quantitative expression of Shh at each time point. Shh was detected in all samples confirming that Shh is active during the process of hindgut development in fetal rats. Relative quantitation demonstrated that Shh expression shows time-dependent changes in the developing hindgut of ETU-exposed rat embryos, and when results were compared with control samples, there was significant decrease in expression on gD14 and 15, when the cloaca normally separates into the rectum and urethra occurs in the rat fetus. The misregulated expression of Shh in the hindgut of ETU-exposed rat embryos suggests that ETU may interfere with Shh signalling. Downregulation at the time of cloacal separation into rectum and urethra indicates that Shh plays a crucial role in the development of hindgut.