title: Time-dependent effects of endothelin-3 on enteric nervous system development in an organ culture model of Hirschsprung's disease.
abstract: OBJECTIVE
  Terminal colonic aganglionosis (Hirschsprung disease) results from incomplete rostrocaudal colonisation of the embryonic gut by neural crest cells (NCC). Mutations in the genes encoding endothelin-3 (EDN3) or its receptor (EDNRB) have been shown to result in a similar aganglionosis. This article describes the development of an organ culture model using embryonic murine gut to determine how endothelin-3 regulates development of the enteric nervous system.
  
  
  METHODS
  Gut explants from mice of different gestational ages were cultured for up to 3 days in the presence or absence of 5 micromol/L of the specific endothelin-B receptor antagonist BQ788. EDN3 and EDNRB mRNA expression were analysed by reverse-transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridisation. NCC were localised using immunoreactivity for PGP 9.5, a specific neuronal marker.
  
  
  RESULTS
  EDN3 mRNA continued to be expressed by caecal mesenchymal cells and EDNRB mRNA by the migrating NCC in culture. Embryonic day (E)11.5 explants were already colonised by NCC up to the terminal ileum. Complete colonisation occurred in organ culture over the next 72 hours (equivalent to E 14.5). Explants of E 12.5 and E 13.5 showed complete colonisation after 48 and 24 hours culture, respectively. Terminal aganglionosis resulted from treatment of E 11.5 and E 12.5 gut explants with 5 micromol/L BQ788, whereas there was no inhibitory effect on E 13.5 explants.
  
  
  CONCLUSIONS
  An organ culture model has been developed in which NCC colonisation of embryonic gut mirrors that described in vivo. Blockade of the EDN3/EDNRB receptor pathway shows that the interaction of endothelin-3 with its receptor is only necessary for NCC colonisation at early time-points, despite the continued expression of endothelin-3 mRNA in the gut.
authors.affiliation: Department of Paediatric Surgery, Alder Hey Children's Hospital, University of Liverpool, England.
authors.forename: M N
authors.initials: MN
authors.lastname: Woodward
authors.forename: S E
authors.initials: SE
authors.lastname: Kenny
authors.forename: C
authors.initials: C
authors.lastname: Vaillant
authors.forename: D A
authors.initials: DA
authors.lastname: Lloyd
authors.forename: D H
authors.initials: DH
authors.lastname: Edgar
mesh_terms: D000818:Animals; D002465:Cell Movement; D004064:Digestive System / Q000294:innervation; D065128:Endothelin Receptor Antagonists; D019334:Endothelin-3 / Q000502:physiology*; D017615:Enteric Nervous System / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D006627:Hirschsprung Disease / Q000196:embryology* / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D008815:Mice, Inbred Strains; D009432:Neural Crest / Q000166:cytology / Q000196:embryology; D009842:Oligopeptides / Q000494:pharmacology; D009924:Organ Culture Techniques; D010880:Piperidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
chemical_list: D065128:Endothelin Receptor Antagonists; D019334:Endothelin-3; D009842:Oligopeptides; D010880:Piperidines; D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; C086539:BQ 788