AP-2β Is a Downstream Effector of PITX2 Required to Specify Endothelium and Establish Angiogenic Privilege During Corneal Development

Invest Ophthalmol Vis Sci. 2016 Mar;57(3):1072-81. doi: 10.1167/iovs.15-18103.


Purpose: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation
  • Cornea / embryology
  • Cornea / metabolism
  • Endothelium, Corneal / embryology*
  • Endothelium, Corneal / metabolism
  • Female
  • Gene Expression Regulation, Developmental*
  • Homeodomain Proteins / biosynthesis
  • Homeodomain Proteins / genetics*
  • Homeodomain Proteins / metabolism
  • Immunohistochemistry
  • In Situ Hybridization
  • Mice
  • Mice, Knockout
  • Morphogenesis / genetics*
  • Neovascularization, Physiologic / genetics*
  • Pregnancy
  • Pregnancy, Animal*
  • Transcription Factor AP-2 / genetics*
  • Transcription Factor AP-2 / metabolism
  • Transcription Factors / biosynthesis
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism


  • Homeodomain Proteins
  • Transcription Factor AP-2
  • Transcription Factors
  • homeobox protein PITX2