Elsevier

Surgery

Volume 134, Issue 2, August 2003, Pages 345-350
Surgery

Society of University Surgeons
Defective sonic hedgehog signaling in esophageal atresia with tracheoesophageal fistula*,**

Presented at the 64th Annual Meeting of the Society of University Surgeons, Houston, Texas, February 12-15, 2003.
https://doi.org/10.1067/msy.2003.243Get rights and content

Abstract

Background. The pathogenesis of esophageal atresia and tracheoesophageal fistula (EA/TEF) remains unknown. We have found previously that an initial esophageal atresia, followed by an abnormal (absent) branching pattern of the middle branch of a trifurcation of the lung/tracheal bud, leads to the neonatal finding of TEF. Mice null mutant for hedgehog signaling can experience the development of EA/TEF, but the mechanism for this development is also unknown. Given that EA/TEF in humans appears not to be due to genetic defects, a hedgehog mutation cause seems very unlikely. However, defective hedgehog signaling that is caused by environmental effects in the human embryo likely could be implicated. We studied a teratogen-induced model of EA/TEF to determine the mechanism by which defective hedgehog signaling may lead to EA/TEF. Methods. We injected Adriamycin into pregnant rats to induce EA/TEF in rat embryos. We first quantified sonic hedgehog (Shh) signaling pathway molecule expression using real-time, semiquantitative reverse-transcriptase polymerase chain reaction for Shh, Shh receptors (patched and smoothened), and downstream intracellular targets of those receptors (Gli family members). On the basis of these findings, we then developed an in vitro culture system for the day-12 embryonic TEF and manipulated Shh signaling using either exogenous Shh or Shh inhibitors. Results. By reverse transcriptase-polymerase chain reaction, a unique difference between the fistula tract and control tissues was that Gli-2 (downstream signaling molecule of Shh) messenger RNA levels were much lower in the fistula tract than in the adjacent esophagus (P =.002). Surprisingly, in the culture experiments, the fistula tract was induced to branch by exogenous Shh. Such branching of the fistula was unexpected and further supports the presumed respiratory origin of the fistula tract because the normal lung, but not normal esophagus, branched in response to Shh. The Shh inhibitor had no effect, which indicated that defective signaling, rather than hyperfunctioning Shh, is critical to the nonbranching phenotype of the fistula tract in TEF. Conclusion. The recapitulation of respiratory developmental morphogenesis by the fistula tract of TEF in the presence of exogenous Shh, together with the quantitative reduction in normal, endogenous levels of Gli-2, strongly suggests that 1 mechanism for the formation of the fistula tract is the lack of proper Shh signaling because of Gli-2 deficiency, with subsequent straight, nonbranching caudal growth of the fistula tract. This deficiency can be rescued by excess exogenous Shh, thus reestablishing respiratory morphogenesis. (Surgery 2003;134:345-50.)

Section snippets

Adriamycin induction of EA/TEF

After protocol approval was obtained from our Institutional Animal Care and Use Committee, time-dated pregnant Sprague-Dawley rats (day 0.5 corresponding with noon of the day of discovery of a vaginal plug) were obtained from Charles River Laboratories (Wilmington, Mass). On gestational days 6 through 9, the experimental rats were injected intraperitoneally with 2 mg/kg of Adriamycin. Control animals were not injected. Litters of embryos from both groups were harvested on embryonic day 12.5

Semiquantitative RT-PCR

By semiquantitative fluorescence-activated real-time RT-PCR, there were no differences noted among levels of Shh, patched, smoothened, and Gli-1 or Gli-3 at e12.5 (data not shown). However, our data for Gli-2 revealed a statistically significant lower level of expression in the fistula tract when compared with the esophagus (P =.019; Fig 1).

. E12 Gli-2 levels in the fistula tract (Fist) are significantly lower (P =.019) when compared with normal esophagus (esoph) at the same age. This suggests

Discussion

The pathogenesis of congenital EA/TEF remains an enigma. Although there are many theories of its development, no single theory encompasses all of the variations nor the associated variable penetrance of the VACTERL syndrome. Most cases of EA/TEF occur sporadically, so it is highly unlikely that there is a simple, inheritable genetic mechanism, which lends support to the notion that the cause is multifactorial. With the discovery of the teratogen-induced rodent model of EA/TEF, we have been able

Acknowledgements

The monoclonal antibody, 5E1, a sonic hedgehog inhibitor, which was developed by Thomas M. Jessell, was obtained from the Developmental Studies Hybridoma Bank that was developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences.

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*

Reprint requests: George K. Gittes, MD, The Children's Mercy Hospital, Laboratory for Surgical Organogenesis, 2401 Gillham Road, Kansas City MO 64108.

**

0039-6060/2003/$30.00 + 0

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