Defective sonic hedgehog signaling in esophageal atresia with tracheoesophageal fistula

Surgery. 2003 Aug;134(2):345-50. doi: 10.1067/msy.2003.243.


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.

Conclusions: 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.

MeSH terms

  • Animals
  • Doxorubicin
  • Embryo, Mammalian / metabolism
  • Embryonic and Fetal Development / drug effects
  • Esophageal Atresia / chemically induced
  • Esophageal Atresia / embryology*
  • Esophageal Atresia / etiology*
  • Female
  • Hedgehog Proteins
  • Kruppel-Like Transcription Factors
  • Organ Culture Techniques
  • Pregnancy
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction*
  • Tracheoesophageal Fistula / chemically induced
  • Tracheoesophageal Fistula / embryology*
  • Tracheoesophageal Fistula / etiology*
  • Trans-Activators / metabolism*
  • Trans-Activators / pharmacology
  • Transcription Factors / genetics
  • Zinc Finger Protein Gli2


  • Gli2 protein, mouse
  • Hedgehog Proteins
  • Kruppel-Like Transcription Factors
  • RNA, Messenger
  • SHH protein, human
  • Trans-Activators
  • Transcription Factors
  • Zinc Finger Protein Gli2
  • Doxorubicin