Final published version
Research output: Contribution to Journal/Magazine › Journal article › peer-review
Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium. / Kucerova, Romana; Dorà, Natalie; Mort, Richard L. et al.
In: Molecular Vision, Vol. 18, 18.01.2012, p. 139-150.Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium
AU - Kucerova, Romana
AU - Dorà, Natalie
AU - Mort, Richard L.
AU - Wallace, Karen
AU - Leiper, Lucy J.
AU - Lowes, Christina
AU - Neves, Carlos
AU - Walczysko, Petr
AU - Bruce, Freyja
AU - Fowler, Paul A.
AU - Rajnicek, Ann M.
AU - McCaig, Colin D.
AU - Zhao, Min
AU - West, John D.
AU - Collinson, J. Martin
PY - 2012/1/18
Y1 - 2012/1/18
N2 - 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.
AB - 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.
KW - Animals
KW - Cell Movement
KW - Cell Proliferation
KW - Clone Cells
KW - Epithelial Cells
KW - Epithelium, Corneal
KW - Eye Proteins
KW - Gene Dosage
KW - Gene Expression Regulation
KW - Hedgehog Proteins
KW - Heterozygote
KW - Homeodomain Proteins
KW - Kruppel-Like Transcription Factors
KW - Mice
KW - Nerve Tissue Proteins
KW - PAX6 Transcription Factor
KW - Paired Box Transcription Factors
KW - Peptides
KW - Phosphatidylinositol 3-Kinases
KW - Regeneration
KW - Repressor Proteins
KW - Signal Transduction
KW - Veratrum Alkaloids
KW - Wound Healing
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
M3 - Journal article
C2 - 22275805
VL - 18
SP - 139
EP - 150
JO - Molecular Vision
JF - Molecular Vision
SN - 1090-0535
ER -