April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
DNA methylation activity of Dnmt3a and Dnmt3b is not essential for normal mouse lens development
Author Affiliations & Notes
  • Thanh Hoang
    Department of Biology, Miami University, Oxford, OH
  • Evan K Horowitz
    Department of Biology, Miami University, Oxford, OH
  • Blake R Chaffee
    Department of Biology, Miami University, Oxford, OH
  • Devin G Bruney
    Department of Biology, Miami University, Oxford, OH
  • Savana E Rosalez
    Department of Biology, Miami University, Oxford, OH
  • Brad D Wagner
    Department of Biology, Miami University, Oxford, OH
  • Michael L Robinson
    Department of Biology, Miami University, Oxford, OH
  • Footnotes
    Commercial Relationships Thanh Hoang, None; Evan Horowitz, None; Blake Chaffee, None; Devin Bruney, None; Savana Rosalez, None; Brad Wagner, None; Michael Robinson, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 740. doi:
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      Thanh Hoang, Evan K Horowitz, Blake R Chaffee, Devin G Bruney, Savana E Rosalez, Brad D Wagner, Michael L Robinson, ; DNA methylation activity of Dnmt3a and Dnmt3b is not essential for normal mouse lens development. Invest. Ophthalmol. Vis. Sci. 2014;55(13):740.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Lens fiber cell differentiation from epithelial cells involves a coordinated change in gene expression. Despite the wealth of knowledge of transcription factors involved in lens development, very little is known about the epigenetic regulation of lens fiber cell differentiation. In mammals, Dnmt3a and Dnmt3b enzymes are responsible for creating de novo DNA methylation changes during development. Our study sought to determine the role of DNA methylation mediated by Dnmt3a and Dnmt3b activities during lens development in mice.

Methods: To generate Dnmt3 single knockout (KO) mice, mice with a conditional “loxP-flanked” flanked mutation in Dnmt3a (Dnmt3LL) and mice with a conditional “loxP-flanked” flanked mutation in Dnmt3b (Dnmt3bLL) were bred independently with three different Cre-expressing mouse strains: Sox2-Cre, Le-Cre and MLR10-Cre mice. Sox2-Cre mice express Cre recombinase at 2 cell-stage embryos. Le-Cre mice express Cre in the lens placode by embryonic day 9.0 (E9.0). Cre expression in MLR10-Cre mice initiates at lens vesicle stage (E10.5). Dnmt3ab double KO mice were generated by crossing Dnmt3a KO mice with Dnmt3b KO mice. The levels of Dnmt3a and Dnmt3b mRNA transcripts in the lens were quantified by RT-qPCR. Lens morphology and integrity were examined by hematoxylin and eosin staining.

Results: Dnmt3a/Sox-2 KO mice died within 4 weeks after birth with no obvious eye defect. Dnmt3b/Sox-2 KO mice and Dnmt3ab/Sox-2 double KO mice died early during embryonic development, thus effect on eye phenotype was not examined. Conditional Dnmt3a, Dnmt3b, and Dnmt3ab double KO mice with LeCre and MLR10 Cre did not show any obvious lens defects. RT-qPCR confirmed more than 90% loss of Dnmt3a and Dnmt3b mRNAs in the lens.

Conclusions: Mouse lenses lacking DNA methylation activity of Dnmt3a and Dnmt3b appeared morphologically normal well into adulthood. Whether de novo DNA methylation really has any role in lens development and whether de novo DNA methylation in Dnmt3ab double KO mice were compensated by other Dnmt enzymes needs further studies. Current analyses are focused on determining if loss of Dnmt3a or Dnmt3b or both results in reduced levels of DNA methylation in the lens.

Keywords: 514 enzymes/enzyme inhibitors • 522 eye movements • 497 development  
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