Apoptotic and proliferative defects characterize ocular development in a microphthalmic BMP model

Invest Ophthalmol Vis Sci. 2013 Jul 10;54(7):4636-47. doi: 10.1167/iovs.13-11674.


Purpose: Vision is critically dependent on ocular size, which is regulated by environmental and genetic factors. Mutation of human Growth and Differentiation Factor 6 (GDF6) or zebrafish gdf6a results in a spectrum of small eye phenotypes (microphthalmia, anophthalmia, and coloboma). However, current models do not explain their etiology fully. As such, analyses of apoptosis and cell cycle regulation were undertaken in a zebrafish gdf6a mutant.

Methods: Microarray analysis was performed at 2 days after fertilization to uncover novel gdf6a-dependent cell cycle regulators. Altered expression of Gdf6a targets was confirmed by in situ hybridization, and resulting changes in cell proliferation were assessed by phosphohistone H3 immunohistochemistry. Analysis of apoptosis was evaluated through activated Caspase 3 immunohistochemistry and chemical inhibitors of cell death.

Results: Reduced numbers of retinal progenitor cells are observed at 24 hours post fertilization (hpf), resulting in microphthalmic eyes in gdf6a(-/-) embryos. At 28 hpf, a wave of apoptosis occurs; however, apoptosis inhibition does not rescue eye size, indicating a limited contribution. Mutants display altered proliferation and expression levels of cell cycle regulators, including members of the forkhead box i (foxi) transcription factor family expressed in the ciliary marginal zone. Notably, inhibition of foxi2 in gdf6a(-/-) embryos further reduces eye size.

Conclusions: These data support a model whereby the gdf6a(-/-)-induced microphthalmia is based on early regulation of retinal progenitor cell number, and later by regulation of proliferation in the ciliary marginal zone. Foxi genes represent downstream effectors of Gdf6a function in the CMZ required for eye size determination.

Keywords: Gdf6; apoptosis; bone morphogenetic protein; forkhead box; proliferation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / physiology*
  • Bone Morphogenetic Proteins / physiology*
  • Cell Cycle / genetics
  • Cell Proliferation*
  • Disease Models, Animal
  • Embryo, Nonmammalian
  • Eye / embryology
  • Forkhead Transcription Factors / metabolism
  • Gene Expression Regulation
  • Growth Differentiation Factor 6 / metabolism
  • Immunohistochemistry
  • Microarray Analysis
  • Microphthalmos / genetics
  • Microphthalmos / pathology*
  • Microphthalmos / physiopathology
  • Stem Cells / pathology
  • Zebrafish / embryology
  • Zebrafish Proteins / metabolism


  • Bone Morphogenetic Proteins
  • Forkhead Transcription Factors
  • Growth Differentiation Factor 6
  • Zebrafish Proteins
  • gdf6a protein, zebrafish