Antiviral Drug Ganciclovir Is a Potent Inhibitor of the Proliferation of Müller Glia–Derived Progenitors During Zebrafish Retinal Regeneration

Abstract

PurposeThe purpose of this study was to investigate the effect of the antiviral drug ganciclovir (GCV) on Müller glia dedifferentiation and proliferation and the underlying cellular and molecular mechanisms in adult zebrafish.MethodsA Tg(1016tuba1a:GFP) transgenic line was generated to identify injury-induced dedifferentiation of Müller 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.ResultsGanciclovir treatment significantly reduced the number of BrdU+ Müller glia–derived progenitor cells (MGPCs) at 4 days post injury. Further analysis showed that GCV had no impact on Müller 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 Müller glia cells were “locked” in a prolonged dedifferentiated state.ConclusionsOur 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.

Opposing Actions of Fgf8a on Notch Signaling Distinguish Two Muller Glial Cell Populations that Contribute to Retina Growth and Regeneration

“…At 2 dpi MG are beginning to proliferate (Fausett and Goldman, 2006). qPCR revealed that forced Fgf8a expression suppressed ascl1a , ccnd1 and cdk2 whose products stimulate MG proliferation, and induced dkk1b and p21 whose products inhibit MG proliferation (Figure 2C) (Fausett et al, 2008; Ramachandran et al, 2010a; Ramachandran et al, 2011, 2012; Zhang et al, 2016). Similar results were seen at 6 hpi and 4 dpi (Figure S2E).…”

Prospective purification and characterization of Müller glia in the mouse retina regeneration assay

“…Of note, we observed strong upregulation of Cdkn1a in MG with increasing explant culture time and animal age. Studies of regenerating zebrafish retina suggest that Cdkn1a upregulation stalls MG in a dedifferentiated state, and limits MG proliferation (Zhang et al, ). Furthermore, Cdkn1a might limit Sox2‐mediated reprogramming in glia‐derived neuronal regeneration of the mammalian spinal cord (Wang et al, ).…”

Inflammation‐induced mammalian target of rapamycin signaling is essential for retina regeneration

“…Adult wild‐type (AB), Tg ( 1016tuba1a:GFP ) (Fausett & Goldman, ), Tg ( mpeg1:nsfB‐mCherry ), Tg ( mpeg1:EGFP ) (Ellett, Pase, Hayman, Andrianopoulos, & Lieschke, ), and Tg ( gfap:GFP ) (Bernardos, Barthel, Meyers, & Raymond, ) transgenic fish were maintained at 28°C on a 14:10‐hr light/dark cycle. The mechanical retinal injury was described previously (Mu et al, ; Zhang et al, ). Briefly, zebrafish were anesthetized in 0.02% Tricaine (Sigma‐Aldrich, St. Louis, MO, USA) in system water.…”

“…An In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science, Penzberg, Upper Bavaria, Germany) was used to detect cell apoptosis in the retina as described previously (Zhang et al, ). DNAse I treated retinal section were used as a positive control.…”

“…Fluorescence images of retinal section or flat mount were captured with a Zeiss Imager M2 upright microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with an Axiocam 506 mono camera. For most cell counting, images were captured with a 10× or 20× objective and counted using the Cell Counter plugin of the ImageJ software as described previously (Zhang et al, ).…”