title: Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium.
abstract: OBJECTIVE
  To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing.
  
  
  METHODS
  The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6).  Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway.  Reporter mice, mosaic for expression of the gene encoding β-galactosidase (LacZ), were crossed to Pax6(+/-) mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6(+/-)Gli3(+/-) double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo.
  
  
  RESULTS
  Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage.  Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation.  Pax6(+/-) corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh.  Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6(+/-) corneal epithelia. Pax6(+/-)Gli3(+/-) double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium.
  
  
  CONCLUSIONS
  The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6(+/-) corneal disease that cannot be remedied pharmacologically by addition of the ligands.
authors.affiliation: School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, UK.
authors.forename: Romana
authors.initials: R
authors.lastname: Kucerova
authors.forename: Natalie
authors.initials: N
authors.lastname: Dorà
authors.forename: Richard L
authors.initials: RL
authors.lastname: Mort
authors.forename: Karen
authors.initials: K
authors.lastname: Wallace
authors.forename: Lucy J
authors.initials: LJ
authors.lastname: Leiper
authors.forename: Christina
authors.initials: C
authors.lastname: Lowes
authors.forename: Carlos
authors.initials: C
authors.lastname: Neves
authors.forename: Petr
authors.initials: P
authors.lastname: Walczysko
authors.forename: Freyja
authors.initials: F
authors.lastname: Bruce
authors.forename: Paul A
authors.initials: PA
authors.lastname: Fowler
authors.forename: Ann M
authors.initials: AM
authors.lastname: Rajnicek
authors.forename: Colin D
authors.initials: CD
authors.lastname: McCaig
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
authors.forename: John D
authors.initials: JD
authors.lastname: West
authors.forename: J Martin
authors.initials: JM
authors.lastname: Collinson
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002999:Clone Cells; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics*; D018628:Gene Dosage; D005786:Gene Expression Regulation / Q000187:drug effects; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D006579:Heterozygote; D018398:Homeodomain Proteins / Q000235:genetics*; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D010455:Peptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D012038:Regeneration / Q000187:drug effects / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014704:Veratrum Alkaloids / Q000494:pharmacology; D014945:Wound Healing / Q000187:drug effects / Q000235:genetics; D000074290:Zinc Finger Protein Gli3
chemical_list: D005136:Eye Proteins; C465571:Gli3 protein, mouse; D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D010455:Peptides; D012097:Repressor Proteins; D014704:Veratrum Alkaloids; D000074290:Zinc Finger Protein Gli3; D019869:Phosphatidylinositol 3-Kinases; C000541:cyclopamine