title: AP-2β Is a Downstream Effector of PITX2 Required to Specify Endothelium and Establish Angiogenic Privilege During Corneal Development.
abstract: OBJECTIVE
  The homeodomain transcription factor, PITX2, is at the apex of a genetic pathway required for corneal development, but the critical effector genes regulated by the PITX2 remain unknown. The purpose of this study was to discover and validate PITX2-dependent mechanisms required for specifying cell lineages and establishing angiogenic privilege within the developing cornea.
  
  
  METHODS
  Microarrays were used to compare gene expression in corneas isolated from temporal Pitx2 knockout embryos and control littermates. Quantitative RT-PCR and immunohistochemistry was used to further validate Tfap2b expression differences in Pitx2 knockout versus control corneas. In situ hybridization and protein immunohistochemistry were used to assay eyes of a Tfap2b allelic series of embryos to identify differentiated cellular lineages in the cornea, blood vessel endothelium, or lymphatic vessel endothelium.
  
  
  RESULTS
  We show that PITX2 is required for the expression of Tfap2b, encoding the AP-2β transcription factor, in the neural crest during corneal development. Markers of differentiated corneal epithelium and stroma are expressed in the absence of AP-2β. In contrast, markers of differentiated corneal endothelium are not expressed in the absence of AP-2β. Endomucin+ blood vessels are present throughout the developing corneal stroma in the absence of AP-2β, whereas LYVE1+ lymphatic vessels are not found.
  
  
  CONCLUSIONS
  The AP-2β transcription factor is an important effector of PITX2 function during corneal development, required for differentiation of corneal endothelium and establishment of angiogenic privilege. Unlike PITX2, AP-2β is not required for the early expression of available lineage specific markers for the corneal epithelium and stroma during embryogenesis, nor establishment of lymphangiogenic privilege. Therefore, additional PITX2-dependent factors likely regulate these latter processes during embryonic development. These results extend our understanding of the genetic mechanisms regulating cornea development.
authors.affiliation: Department of Ophthalmology & Visual Sciences, University of Michigan, Ann Arbor, Michigan, United States.
authors.forename: Lisheng
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Martino
authors.affiliation: Department of Pediatrics, Wayne State University, Detroit, Michigan, United States.
authors.forename: Alan
authors.initials: A
authors.lastname: Dombkowski
authors.affiliation: Department of Craniofacial Biology and Department of Cell and Developmental Biology, University of Colorado-Denver, Denver, Colorado, United States.
authors.forename: Trevor
authors.initials: T
authors.lastname: Williams
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Judith
authors.initials: J
authors.lastname: West-Mays
authors.affiliation: Department of Ophthalmology & Visual Sciences, University of Michigan, Ann Arbor, Michigan, United States 5Department of Cell & Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Gage
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D003315:Cornea / Q000196:embryology / Q000378:metabolism; D004728:Endothelium, Corneal / Q000196:embryology* / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D018345:Mice, Knockout; D009024:Morphogenesis / Q000235:genetics*; D018919:Neovascularization, Physiologic / Q000235:genetics*; D011247:Pregnancy; D011270:Pregnancy, Animal; D050656:Transcription Factor AP-2 / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000097577:Homeobox Protein PITX2
chemical_list: D018398:Homeodomain Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2