Regeneration of inner retinal neurons after intravitreal injection of ouabain in zebrafish.
We examined the regenerative capacity of the adult zebrafish retina by intravitreal injection of a low ouabain concentration to rapidly damage the ganglion cell layer (GCL) and inner nuclear layer (INL) with minimal photoreceptor cell damage. By 24 h after ouabain injection, maximal numbers of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-positive cells were detected in the INL and GCL, with low numbers of TUNEL-positive cells in the outer nuclear layer. Immunolabeling revealed that approximately 85% of the HuC/D-positive amacrine and ganglion cells were lost by 7 d post-ouabain injection (dpi). This ganglion cell loss was consistent with the small, but statistically significant, decrease in the optic nerve diameter. The regeneration response began within 1 dpi with increased proliferating cell nuclear antigen (PCNA) expression in both the INL and GCL. By 3 dpi, PCNA expression is primarily restricted to the Müller glia. By 5 dpi, most of the PCNA expression was localized to neuronal progenitors expressing the olig2:egfp transgene rather than the Müller glia. By 7 dpi, the neuronal progenitors began committing to the ganglion cell fate based on the coexpression of the atoh7:EGFP transgene and the zn5 antigen. The regeneration of ganglion and amacrine cells continued until 60 dpi, when they reached 75% of their uninjected control number. This demonstrates that inner retinal damage, without extensive photoreceptor damage, is sufficient to induce a regeneration response that is marked by the Müller glial cells reentering the cell cycle to produce neuronal progenitor cells that regenerate INL and ganglion cells in the zebrafish retina.
Fimbel
Shane M
SM
Department of Biological Sciences and Center for Zebrafish Research, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Montgomery
Jacob E
JE
Burket
Christopher T
CT
Hyde
David R
DR
D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D015153:Blotting, Western / Q000379:methods; D004305:Dose-Response Relationship, Drug; D051959:ELAV Proteins / Q000378:metabolism; D000067778:ELAV-Like Protein 3; D004791:Enzyme Inhibitors / Q000633:toxicity*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D009419:Nerve Tissue Proteins / Q000235:genetics; D009457:Neuroglia / Q000378:metabolism / Q000473:pathology; D000076104:Oligodendrocyte Transcription Factor 2; D010042:Ouabain / Q000633:toxicity*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012038:Regeneration / Q000502:physiology*; D012160:Retina / Q000473:pathology*; D012165:Retinal Ganglion Cells / Q000187:drug effects*; D013997:Time Factors; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D051959:ELAV Proteins; D000067778:ELAV-Like Protein 3; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D049452:Green Fluorescent Proteins; D009419:Nerve Tissue Proteins; D000076104:Oligodendrocyte Transcription Factor 2; D010042:Ouabain; D018809:Proliferating Cell Nuclear Antigen; D029961:Zebrafish Proteins; C088635:elavl3 protein, zebrafish; C465361:olig2 protein, zebrafish