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Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium

Research output: Contribution to journalJournal article

Published
  • Romana Kucerova
  • Natalie Dorà
  • Richard L. Mort
  • Karen Wallace
  • Lucy J. Leiper
  • Christina Lowes
  • Carlos Neves
  • Petr Walczysko
  • Freyja Bruce
  • Paul A. Fowler
  • Ann M. Rajnicek
  • Colin D. McCaig
  • Min Zhao
  • John D. West
  • J. Martin Collinson
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<mark>Journal publication date</mark>18/01/2012
<mark>Journal</mark>Molecular Vision
Volume18
Number of pages12
Pages (from-to)139-150
Publication StatusPublished
<mark>Original language</mark>English

Abstract

PURPOSE: 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.