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

• Published in: Molecular vision 2012, Vol.18, p.139-150
• Main Authors: Kucerova, Romana, Dorà, Natalie, Mort, Richard L, Wallace, Karen, Leiper, Lucy J, Lowes, Christina, Neves, Carlos, Walczysko, Petr, Bruce, Freyja, Fowler, Paul A, Rajnicek, Ann M, McCaig, Colin D, Zhao, Min, West, John D, Collinson, J Martin
• Format: Article
• Language: English
• Subjects: Animals; beta -Galactosidase; Cell migration; Cell Movement - drug effects; Cell Proliferation - drug effects; Clone Cells; Cornea; Data processing; Epithelial cells; Epithelial Cells - cytology; Epithelial Cells - drug effects; Epithelial Cells - enzymology; Epithelium; Epithelium, Corneal - cytology; Epithelium, Corneal - drug effects; Epithelium, Corneal - metabolism; Eye Proteins - genetics; Gene Dosage; Gene Expression Regulation - drug effects; Hedgehog protein; Hedgehog Proteins - genetics; Hedgehog Proteins - metabolism; Heterozygote; Heterozygotes; Homeodomain Proteins - genetics; Immunohistochemistry; Intracellular signalling; Kruppel-Like Transcription Factors - metabolism; Mice; Mosaics; Nerve Tissue Proteins - metabolism; Paired Box Transcription Factors - genetics; Pax6 protein; PAX6 Transcription Factor; Peptides - pharmacology; Phosphatidylinositol 3-Kinases - metabolism; Polymerase chain reaction; Regeneration - drug effects; Regeneration - genetics; Repressor Proteins - genetics; Signal transduction; Signal Transduction - drug effects; Signal Transduction - genetics; Transcription factors; Veratrum Alkaloids - pharmacology; Vision; Western blotting; Wound healing; Wound Healing - drug effects; Wound Healing - genetics; Zinc Finger Protein Gli3
• ISSN: 1090-0535; 1090-0535

To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing. 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. 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. 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.