September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Elevated expression of her4.2 and her6 (hairy/Enhancer of split-related) genes facilitates light damage-induced adult zebrafish retinal regeneration
Author Affiliations & Notes
  • Josiah Daniel Nieto
    Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States
    Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana, United States
  • Joshua Hobgood
    Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States
    Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana, United States
  • David R Hyde
    Biological Sciences, University of Notre Dame, Notre Dame, Indiana, United States
    Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana, United States
  • Footnotes
    Commercial Relationships   Josiah Nieto, None; Joshua Hobgood, None; David Hyde, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 2748. doi:
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      Josiah Daniel Nieto, Joshua Hobgood, David R Hyde; Elevated expression of her4.2 and her6 (hairy/Enhancer of split-related) genes facilitates light damage-induced adult zebrafish retinal regeneration. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2748.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Purpose: Adult zebrafish retinal regeneration mechanisms remain poorly understood. Increased expression of her4.2 and her6 (hairy/Enhancer of split-related) genes, observed by microarray analysis of a light damage-induced retinal regeneration paradigm, suggests regeneration-associated roles for these genes. Here, qRT-PCR and morpholino-mediated knockdown were used to test the hypothesis that elevated her4.2 and her6 expression facilitates retinal regeneration following light damage.

Methods : Methods: Zebrafish retinal extraction between 0- and 96-hours of light treatment was executed before total RNA isolation and qRT-PCR. Alongside the her targets, pcna expression confirmed cell proliferation and 18s expression served as a control. The ΔΔCT method allowed comparison of 0-h control and target expression at different timepoints. Morpholino-mediated knockdown of her6 was performed by intravitreal injection and electroporation of the morpholino into the retina. Fish were then placed in constant light until eyes were removed, fixed, and sectioned. After antibody labeling, confocal microscopy and PCNA-positive cell counts were used to compare control and knockdown effects.

Results : Results: Compared to 0-h light treatment, the expression of her4.2 significantly increased at 51-, 72-, and 96-h. Distinctly, her6 expression first increased at 36-h and continued increasing through 72-h. Strikingly, both her4.2 and her6 expression were significantly reduced at 16-h relative to the 0-h control. Relative to the Standard Control morpholino, the her6 morpholino significantly reduced the number of PCNA-positive Müller glia and Müller glia-derived neuronal progenitors at 36-h and 72-h, respectively.

Conclusions : Conclusions: Timing of increased her6 and her4.2 expression corresponds to Müller glia and NPC-related proliferation during regeneration. This suggests that elevated her4.2 and her6 may aid NPC-mediated retinal regeneration. Reduced Müller glia and NPC proliferation after her6 knockdown further supports its role in driving retinal regeneration. Further analysis will define the potential role of these her genes in Müller glia quiescence.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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