title: Overexpression of Pax6 in mouse cornea directly alters corneal epithelial cells: changes in immune function, vascularization, and differentiation.
abstract: OBJECTIVE
  To assess whether Pax6 functions directly in the cornea, a corneal-preferred promoter was used to overexpress Pax6 specifically in the cornea.
  
  
  METHODS
  Transgenic mice harboring a construct containing mouse Pax6 coding sequences fused downstream of the aldehyde dehydrogenase 3a1 (Aldh3a1) promoter were generated (Pax6 Tg). Pax6 expression was analyzed by Western blot and immunohistochemistry. Eye sections were stained with hematoxylin and eosin, Schiff reagent, and fluorescein, to assess morphologic changes, the presence of goblet cells, and barrier integrity, respectively. Gene expression changes in mildly affected Pax6 Tg corneas were compared to age-matched, wild-type (WT) corneas by microarray analysis and quantitative PCR. Promoter regulation of several differentially expressed genes was examined by monitoring luciferase activity of reporter constructs after cotransfection with Pax6 in COS7 cells.
  
  
  RESULTS
  Corneal overexpression of Pax6 produces an abnormal cornea with altered epithelial cell morphology, neovascularization, immune cell invasion, and a compromised barrier; the lens appeared normal. Major changes in expression of genes involved in immune function, vascularization, and epithelial differentiation occurred in corneas from Pax6 Tg versus WT mice. The keratin (K) profile was dramatically altered in the Pax6 Tg corneas, as were several components of the Wnt signaling pathway. In severely affected Pax6 Tg corneas, K12 was reduced, and Pax6 was redistributed into the cytoplasm. Promoters from the chitinase 3-like 3, Wnt inhibitory factor 1, and fms-related tyrosine kinase 1/soluble VEGF receptor genes were upregulated five-, seven-, and threefold, respectively, by Pax6 in transfected COS7 cells.
  
  
  CONCLUSIONS
  Pax6 functions directly to maintain normal, corneal epithelial cells.
authors: Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA. davisj@nei.nih.gov
authors: Janine
authors: J
authors: Davis
authors: Joram
authors: J
authors: Piatigorsky
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002454:Cell Differentiation / Q000235:genetics; D002688:Chitinases / Q000235:genetics; D002522:Chlorocebus aethiops; D003315:Cornea / Q000098:blood supply / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D003318:Corneal Opacity / Q000209:etiology / Q000473:pathology; D004847:Epithelial Cells / Q000473:pathology; D019573:Epithelium, Corneal / Q000276:immunology / Q000473:pathology*; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D053551:Keratin-12 / Q000235:genetics / Q000378:metabolism; D007633:Keratins / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009389:Neovascularization, Pathologic; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014018:Tissue Distribution; D015854:Up-Regulation; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics; D051153:Wnt Proteins / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; D053551:Keratin-12; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012333:RNA, Messenger; D012097:Repressor Proteins; C118544:Wif1 protein, mouse; D051153:Wnt Proteins; D007633:Keratins; C501163:Flt1 protein, mouse; D040281:Vascular Endothelial Growth Factor Receptor-1; D002688:Chitinases; C525260:Ym2 protein, mouse