The contribution of the sonic hedgehog cascade in the development of the enteric nervous system in fetal rats with anorectal malformations.
OBJECTIVE
This study was designed to determine the expression of Sonic hedgehog (Shh) and its downstream genes during development of the enteric nervous system (ENS) in ethylenethiourea (ETU)-exposed fetal rats with anorectal malformations (ARMs).


METHODS
Anorectal malformations were induced by 1% ETU (125 mg/kg) given on gestational day 10, and the litter was harvested at term. The fetal anorectum and rectosigmoid region, including any communication with the urinary tract, were collected for gene expression studies and immunofluorescence study of the ENS. Gene expression of Shh cascade was performed using reverse transcription and real-time polymerase chain reaction (PCR). The myenteric plexuses of the ENS in normal and ARM rats were visualized with fluorescent antibodies.


RESULTS
Reverse transcription-PCR confirmed expression of Shh and its target genes in all parts of the ARMs. Quantitative PCR demonstrated that levels of expression of the genes of the Shh cascade were low in the ARMs. The immunoreactivity of neuromarkers was markedly reduced in high ARMs and slightly reduced in low ARMs.


CONCLUSIONS
This study demonstrates reduced expression of Shh and its target genes in ARMs in ETU-exposed fetal rats. Neurons in the myenteric plexus were decreased in high and low types of ARMs. Our results support a role for the Shh cascade during development of the ENS during hindgut development.
Mandhan
Parkash
P
Department of Paediatric Surgery, Children's Developmental Genetic Research Group, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand.
Sullivan
Michael
M
Quan
Qi Bao
QB
Beasley
Spencer
S
D001003:Anal Canal / Q000002:abnormalities / Q000196:embryology*; D000818:Animals; D001483:Base Sequence; D004065:Digestive System Abnormalities / Q000196:embryology / Q000235:genetics; D004195:Disease Models, Animal; D017615:Enteric Nervous System / Q000196:embryology*; D005031:Ethylenethiourea; D005260:Female; D005315:Fetal Diseases / Q000175:diagnosis / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental; D053823:Hedgehog Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011247:Pregnancy; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012007:Rectum / Q000002:abnormalities; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D053823:Hedgehog Proteins; D005031:Ethylenethiourea