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Sonic hedgehog (Shh) protein is a signalling molecule that is important for defining patterning in the development of vertebrates. Shh has been shown to be involved in the morphogenesis of many organ systems such as notochord, floor plate and limbs and in the development of the left-right axis in vertebrates. In this study we show that high levels of protein occur during the initial period of foregut differentiation into trachea and oesophagus, and that low levels of Shh protein are seen during early development of the foregut where oesophageal atresia and/or tracheo-oesophageal fistula ensue. These studies show that Shh protein is expressed in the rat foregut during embryogenesis, and its level declines as the embryo approaches birth. In adriamycin-treated rats the level of Shh protein expression is very low without any time-dependent changes. These results are consistent with the hypothesis that adriamycin influences the Shh signalling pathway, resulting in disruption of normal development of the foregut.

Background/Purpose: The process of tracheoesophageal separation during early development of the foregut has been disputed and has led to difficulties explaining how congenital abnormalities of the trachea and esophagus might occur. This study clarifies the embryogenesis of tracheoesophageal separation by using sequential 3-dimensional imaging at crucial stages of foregut development. Methods: Timed pregnant Sprague Dawley rats were killed at days 11, 11.5, 12, 12.5, and 13. The embryos were harvested, histologically sectioned, and stained with H&E. Digitized photographs were taken of sequential serial transverse sections and their tracings layered in a 3-dimensional rendering program before being "skinned" to produce a 3-dimensional object. Results: The first respiratory structures to develop are the bronchi on day 11.5. They are evident first as bulges on the ventrolateral wall of the foregut approximately two thirds of the way between the lowest pharyngeal pouch and the level of the hepatic diverticulum and pancreatic buds. Lateral grooves dorsal to the respiratory bud on the lateral walls extend cranially. On day 12 the lateral bulges have developed into the 2 main bronchi, although the trachea is yet to separate from the foregut. On days 12.5 to 13 the trachea progressively elongates, and by day 13 tracheoesophageal separation is complete. Conclusions: After the main bronchi have developed, the trachea forms when the ventral component of the foregut is "cut" away from the dorsal component. There is an area of apoptosis at the point of tracheoesophageal separation, and, as the embryo grows, this causes the separation point to stay at a constant distance from the pharynx. Meanwhile, the trachea and esophagus distal to it increase dramatically in length. The area immediately caudal to the initial point of tracheoesophageal separation ultimately forms the stomach. J Pediatr Surg 38:173-177. Copyright 2003, Elsevier Science (USA). All rights reserved.

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). Material and 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. Conclusion 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.

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. Conclusion 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.

Background The hedgehog signalling pathway appears to have a crucial role in the embryogenesis of the foregut in vertebrates. Sonic hedgehog (Shh) protein and gene are involved in the differentiation of many organ systems such as notochord, floor plate, and limbs; development of the left-right axis in vertebrates; and differentiation of trachea and esophagus from the primitive foregut. Methods The prenatal exposure of Sprague-Dawley fetal rats to Adriamycin between days 6 and 9 of gestation was used to induce esophageal atresia and tracheoesophageal fistula. Embryos were harvested by cesarean section on gestational days 11 to 15. Embryos were examined by microscopy, dissection, and serial histology. In situ hybridization was used to study Shh gene expression in the rat embryos. Results In situ hybridization showed that the pattern and level of Shh gene expression are affected by Adriamycin. Adriamycin-treated rats have deformed notochord and undivided foregut, and some of the embryos had a lack of the dorso-ventral patterning of Shh expression seen in control embryos. Conclusions These results are consistent with the hypothesis that Adriamycin influences the Shh signalling pathway, resulting in disruption of normal development of the foregut.