Inhibition of Müller glial cell division blocks regeneration of the light-damaged zebrafish retina

Thummel, R., Kassen, S.C., Montgomery, J.E., Enright, J.M., and Hyde, D.R.
Developmental Neurobiology   68(3): 392-408 (Journal)
Registered Authors
Hyde, David R., Kassen, Sean, Montgomery, Jacob, Thummel, Ryan
retinal regeneration, Müller glia, zebrafish, morpholino electroporation, stem cell
MeSH Terms
  • Albinism/genetics
  • Animals
  • Animals, Genetically Modified
  • Cell Death/drug effects
  • Cell Death/radiation effects
  • Disease Models, Animal
  • Embryo, Nonmammalian
  • Eye Proteins/metabolism
  • Gene Expression Regulation/drug effects
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • Homeodomain Proteins/metabolism
  • Light/adverse effects*
  • Microinjections
  • Neuroglia/drug effects
  • Neuroglia/physiology*
  • Oligonucleotides/pharmacology
  • Paired Box Transcription Factors/metabolism
  • Proliferating Cell Nuclear Antigen/genetics
  • Proliferating Cell Nuclear Antigen/metabolism
  • Regeneration/drug effects
  • Regeneration/physiology*
  • Repressor Proteins/metabolism
  • Retinal Degeneration/etiology*
  • Retinal Degeneration/pathology
  • Retinal Degeneration/physiopathology*
  • Rhodopsin/metabolism
  • Time Factors
  • Zebrafish
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/metabolism
18161852 Full text @ Dev. Neurobiol.
The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas.
Genes / Markers
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Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes