Abstract
We sought to characterize the regenerated cells, if any, when photoreceptor ablation was mostly limited to a particular cone subtype. This allowed us to uniquely assess whether the remaining cells influence specification of regenerating photoreceptors. The ability to replace lost photoreceptors via stem cell therapy holds promise for treating many retinal degenerative diseases. Zebrafish are potent for modelling this because they have robust regenerative capacity emanating from endogenous stem cells, and abundant cone photoreceptors including multiple spectral subtypes similar to human fovea. We ablated the homolog of the human S-cones, the ultraviolet-sensitive (UV) cones, and tested the hypothesis that the photoreceptors regenerating in their place take on identities matching those expected from normal cone mosaic development. We created transgenic fish wherein UV cones can be ablated by addition of a prodrug. Thus photoreceptors developed normally and only the UV cones expressed nitroreductase; the latter converts the prodrug metronidazole to a cell-autonomous neurotoxin. A significant increase in proliferation of progenitor cell populations (p<0.01) was observed when cell ablation was primarily limited to UV cones. In control fish, we found that BrdU primarily incorporated into rod photoreceptors, as expected. However the majority of regenerating photoreceptors became cones when retinal cell ablation was predominantly restricted to UV cones: a 2-fold increase in the relative abundance of cones (p = 0.008) was mirrored by a 35% decrease in rods. By primarily ablating only a single photoreceptor type, we show that the subsequent regeneration is biased towards restoring the cognate photoreceptor type. We discuss the hypothesis that, after cone death, the microenvironment formed by the remaining retinal cells may be influential in determining the identity of regenerating photoreceptors, though other interpretations are plausible. Our novel animal model provides control of ablation that will assist in identifying mechanisms required to replace cone photoreceptors clinically to restore daytime vision.
Highlights
The adult fish retina possesses a robust innate capacity to regenerate neurons from retinal stem cells [1,2,3], making it an attractive model for stem cell therapies of retinal degenerations
We note that crossing the above lines to an independent 4XUAS line (creating Tg(SWS1:Gal4VP16)ua3016;Tg(UAS-E1b:NfsB-mCherry)c264;Tg(4xUAS:GFP)hzm3) suggested that Gal4 is not expressed in all UV cones (Fig. S1), so this may be a limitation imposed by our Tg(SWS1:Gal4VP16)ua3016 driver line
BrdU-containing cells in the larval remnant were classified into six categories based on opsin in situ hybridization, confocal microscopy and nuclear position by an observer blinded to the treatments. We focused on those cells we could unambiguously categorize and consider three categories of regenerated (BrdU-positive) cells: i) UV cones; ii) BGR cones; and iii) rod photoreceptors
Summary
The adult fish retina possesses a robust innate capacity to regenerate neurons from retinal stem cells [1,2,3], making it an attractive model for stem cell therapies of retinal degenerations. An intriguing alternative is hormonal induction of UV cone loss that parallels normal development in salmonid fish [8,9]; UV cones are normally lost during an ontogenetic shift associated with these fish migrating to deeper waters [9,10,11,12] Perhaps excluding the latter, available retinal cell ablation methods indiscriminately and inconsistently ablate various photoreceptor subtypes (rods and multiple cone subtypes), along with other cells [13]. Available retinal cell ablation methods indiscriminately and inconsistently ablate various photoreceptor subtypes (rods and multiple cone subtypes), along with other cells [13] It appears that all of the ablated cell types are typically replaced during regeneration. The complexity of this suite of regenerating cells has been a roadblock to deciphering the biochemical signalling pathways involved in specifying cell fates during the replacement and rewiring of damaged retina [13]
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