Antiviral drug ganciclovir is a potent inhibitor of the proliferation of Müller glia-derived progenitors during zebrafish retinal regeneration

Abstract

PURPOSE. The purpose of this study was to investigate the effect of the antiviral drug ganciclovir (GCV) on M¨uller glia dedifferentiation and proliferation and the underlying cellular and molecular mechanisms in adult zebrafish. METHODS. A Tg(1016tuba1a:GFP) transgenic line was generated to identify injury-induced dedifferentiation of M¨uller glia. Mechanical retinal damage was induced by a needle-poke injury on the back of the eyes in adult zebrafish. Phosphate-buffered saline or GCV was injected into the vitreous of the eye at the time of injury or through the cornea. The GCV clearance rate from the eye was determined by a reversed-phase HPLC method. Green fluorescent protein (GFP) and bromodeoxyuridine (BrdU) immunofluorescence were used to determine the effect of GCV on retinal regeneration. Cell apoptosis was evaluated by TUNEL staining. Microglia were labeled by vitreous injection of isolectin IB4 conjugates. Quantitative (q)PCR and Western blot analysis were used to determine gene expression in the retina. RESULTS. Ganciclovir treatment significantly reduced the number of BrdUþ M¨uller glia-derived progenitor cells (MGPCs) at 4 days post injury. Further analysis showed that GCV had no impact on M¨uller glia dedifferentiation and the initial formation of MGPCs. Our data indicate that GCV irreversibly inhibited MGPC proliferation likely through a p53-p21cip1-dependent pathway. Interestingly, unlike control cells, GCV-treated M¨uller glia cells were ‘‘locked’’ in a prolonged dedifferentiated state. CONCLUSIONS. Our study uncovered a novel inhibitory effect of GCV on MGPC proliferation and suggests its potential use as a tool to uncover molecular mechanisms underlying retinal regeneration in zebrafish.