Hoxd-13 Expression In The Anorectal Development Of The Rat Embryos

ObjectiveAnorectal malformations (ARMs) are the most common abnormality of the neonatal digestive system. The pathogenesis of ARMs remains poorly understood. Hoxd-13 gene has been shown to be involved in the morphogenesis of many organ systems. In this study, we investigated Hoxd-13 expression in the development of anorectal malformations in Ethylenethiourea (ETU)-treated embryos to elucidate its role in the anorectal morphogenesis.Materials and Methods1,The ETU murine model of ARMs was employedThe research project was approved by the animal ethics committee of Shengjing hospital. Time-mated pregnant Wistar rats were gavage-fed a single dose of either 125 mg/kg of 1% ETU or an equal dose of saline on gestational day 10 (E0-sperm in vaginal smear after overnight mating). The embryos were harvested via cesarean section on E13 to E21. One half of the clutch was used for immunohistochemistry staining, and the other half one was used for reverse transcription polymerized chain reaction techniques (RT-PCR).2,ImmunohistochemistryThe rat embryos were fixed in 4% paraformaldehyde/0.1 M phosphate buffered saline (PBS) for 12 to 24 hours depending on their size. Then the embryos from each age group were dehydrated, embedded in paraffin and sectioned serially sagittally at 4μm thickness. And then the sections were stained with immunohistochemistry method using specific antibody to Hoxd-13. Temporo-spatial study was carried out on Hoxd-13 expression.3,RT-PCRThe rat embryos were frozen in liquid nitrogen and then dissected under magnification. The anorectal specimens were used for total RNA extraction. Hoxd-13 gene expression was evaluated by RT-PCR.Results1,The morphorlogical resultsSerial histological sections from the control embryos revealed complete descent of the urorectal septum (URS) to the cloacal membrane and division of cloaca into anorectum and urinary bladder. There was no communication between the two structures. It was observed the fusion of URS and the cloacal membrane on the saggital sections of the normal rat embryos on E15 rather than on the sections of ARMs embryos. In ARMs embryos, there was incomplete division of URS with a persistent communication between the rectum and bladder.2,Immunohistochemistry results①Hoxd-13 expression was confined to the mesenchyme of URS and the epithelium of the hindgut, cloaca (fistula) membrane and urogenital sinus in the control group. Hoxd-13 expression was mainly seen in the epithelium of urogenital sinus in the embryos with ARMs.②Hoxd-13 expression levels increased with the gestation day especially after E16. The mean optical density measurements of Hoxd-13 in the mesenchyme of URS and the epithelium of the hindgut, cloaca membrane or fistula membrane were significantly lower in the ARMs group than in the control group on E13-16 (P<0.05), respectively. There was no significant difference between the two groups on E18 and E21.3,RT-PCR resultsRT-PCR amplification of Hoxd-13 gene showed positive bands in the two groups. The mRNA levels of Hoxd-13 gene increased with the gestation day especially after E16. The amount of Hoxd-13 mRNA in the ARMs embryos was lower on E13-16 than in the control group (P<0.05). There was no significant difference between the two groups on E18 and E21. The RT-PCR results were consistent with the immnohistochemistry results.ConclusionsHoxd-13 misexpression in the mesenchyme of URS and the epithelium of the hindgut, cloaca (fistula) membrane may disturb the fusion of URS and the cloacal membrane, which may lead to ARMs. The decreased expression of Hoxd-13 gene may be related to the development of ARMs. Our results imply that Hoxd-13 gene plays a key role in anorectal morphogenesis.