Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats.
BACKGROUND
shh signaling pathway has been shown to be involved in the morphogenesis of many organ systems. In this study, we investigated the expression of shh and its targets, BMP4 and Hox genes, in the development of anorectal malformations in Ethylenethiourea (ETU)-exposed embryos.


METHODS
We used ETU murine model of the vertebral, anal, cardiac, tracheoesophageal, renal, and limb association. Ethylenethiourea 1% (125 mg/kg) was given to the pregnant females via gavage feeding on gestational day (gD) 10 and saline to control animals. Embryos were collected at gD12 to gD16 and gD21; hindguts were dissected and snap frozen. Highly purified RNA was isolated, and expression of shh, BMP4, Hoxa13, and Hoxd13 genes was confirmed with RT-PCR. Relative quantitative expression of shh and target genes at each time point was done with SYBR Green I qPCR. Normalized gene of interest expression was calculated by geNorm, and data analysis was done with 2-tail Student t test.


RESULTS
shh, BMP4, Hoxa13, and Hoxd13 transcripts were detected in all samples, confirming that shh cascade is active during the process of hindgut development in fetal rats. Relative quantitation demonstrated that shh cascade expression shows time-dependent changes in the developing hindgut.


CONCLUSIONS
This study shows that ETU disturbs the expression of shh signaling pathway during the development of hindgut. We provide evidence that shh plays a pivotal role in the hindgut morphogenesis, and its misexpression affect the expression of targets, BMP4 and Hox genes.
Mandhan
Parkash
P
Department of Paediatric and Surgery, Children's Cancer and Developmental Genetic Research Group, Christchurch School of Medicine & Health Sciences, Christchurch, New Zealand.
Quan
Qi Bao
QB
Beasley
Spencer
S
Sullivan
Michael
M
D001003:Anal Canal / Q000002:abnormalities; D000818:Animals; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D004195:Disease Models, Animal; D005031:Ethylenethiourea / Q000009:adverse effects*; D005260:Female; D005801:Genes, Homeobox / Q000235:genetics; D053823:Hedgehog Proteins / Q000235:genetics; D011247:Pregnancy; D011297:Prenatal Exposure Delayed Effects / Q000139:chemically induced*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012002:Rectal Diseases / Q000139:chemically induced* / Q000151:congenital / Q000235:genetics*; D012007:Rectum / Q000002:abnormalities; D015398:Signal Transduction
C525811:Bmp4 protein, rat; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D053823:Hedgehog Proteins; D005031:Ethylenethiourea