A novel model of retinal ablation demonstrates that the extent of rod cell death regulates the origin of the regenerated zebrafish rod photoreceptors

Zebrafish is a popular model to study retinal regeneration, as its retina produces a robust regenerative response following multiple forms of injury (Hitchcock and Raymond, 2004). Conventional models propose that regenerated retinal neurons derive from two sources, depending on the cell class lost (Otteson and Hitchcock, 2003; Morris et al., 2008). Muller glia possess qualities of multipotent stem cells, as they are the source of many regenerated neuronal cell classes in the retina (Fausett and Goldman, 2006; Bernardos et al., 2007; Fimbel et al., 2007; Stenkamp, 2007; Thummel et al., 2008a,b). A variety of damage paradigms induce the Muller glial cells to reenter the cell cycle and divide asymmetrically to produce neuronal progenitor cells, which continue to proliferate and migrate radially to the damaged retinal layer where they differentiate into the neuronal cell classes that are missing (Yurco and Cameron, 2005; Fausett and Goldman, 2006; Raymond et al., 2006; Fimbel et al., 2007; Thummel et al., 2008a,b). New retinal neurons are also produced during persistent neurogenesis, which occurs as the fish eye grows throughout its life and new retinal neurons are produced to accompany this growth. During this process, Muller glia proliferate at a slow rate to produce neuronal progenitor cells that migrate to the outer nuclear layer (ONL). Upon reaching the ONL, they become rod precursor cells that are committed to differentiating into only rod photoreceptors (Otteson and Hitchcock, 2003; Raymond et al., 2006). Thus, the damaged retina possesses two classes of cells with a regenerative capacity, the pluripotent Muller glia and the committed rod precursors. Different damage models helped to elucidate the conditions involved in inducing a regenerative response from either the Muller glia or rod precursors. Surgical lesion of a portion of the retina and retinal puncture both induce Muller glial cell proliferation and formation of neuronal progenitor cell clusters in the inner nuclear layer (INL; Yurco and Cameron, 2005; Fausett and Goldman, 2006). Intravitreal injection of the neurotoxin ouabain, which kills neurons in all retinal layers at high doses and primarily removes INL neurons and ganglion cells at low doses, also produces a regeneration response that originates with Muller glial cell division (Fimbel et al., 2007; Sherpa et al., 2008). Thus, loss of non-photoreceptor neurons requires proliferation of the pluripotent Muller glia. Similarly, light-induced apoptosis of the rod and cone photoreceptors yields a robust regeneration response involving proliferation of the Muller glia (Vihtelic and Hyde, 2000; Bernardos et al., 2007; Kassen et al., 2007; Thummel et al., 2008a,b). In contrast, the light-damaged ventral retina exhibits rod photoreceptor cell death without any apparent cone cell loss, which results in significant ONL rod precursor cell proliferation and minimal Muller glial cell division (Vihtelic et al., 2006). This suggests that if the damage is restricted to rod photoreceptors, regeneration requires only increasing rod precursor cell proliferation. This was further examined in the pde6cw59 mutant, which is unable to maintain cones, and the Tg(Xops:mCFP) transgenic line, which exhibits chronic rod cell death (Morris et al., 2008). Whereas the pde6cw59 mutant possesses Muller glial proliferation, the Tg(Xops:mCFP) fish exhibit increased rod precursor cell division without greater Muller glial cell proliferation (Morris et al., 2008). These studies suggest that all regenerated retinal neurons originate from the Muller glial cells, whereas loss of only rod photoreceptors can be regenerated from the resident rod precursors. However, none of these damage models examined regeneration following acute and specific loss of all mature rod photoreceptors, which may require accelerated production of additional rod precursor cells that would necessitate increased proliferation of the Muller glia. To address this hypothesis, we utilized the bacterial nitroreductase (NTR)/metronidazole cell ablation system to specifically induce death of all rod photoreceptors. The NTR enzyme reduces nitroimidazole prodrugs (Lindmark and Muller, 1976), such as metronidazole, into cytotoxins that generate DNA interstrand crosslinking and cell death (Edwards, 1977; Roberts et al., 1986). Whereas many prodrugs exhibit a bystander effect, in which they cause the death of adjacent cells that do not express the activating enzyme, the NTR/metronidazole system does not display such an effect (Roberts et al., 1986; Edwards, 1993; Bridgewater et al., 1997). It was previously demonstrated that metronidazole treatment of zebrafish expressing the E. coli nitroreductase (nfsB) gene under control of known promoters specifically ablated pancreatic β cells in larval (Curado et al., 2007; Pisharath et al., 2007) and adult (Moss et al., 2009) fish, as well as larval cardiomyocytes, hepatocytes (Curado et al., 2007), and cells of the developing notochord and ventral CNS (Davison et al., 2007). Thus, unlike previous methods used, this system holds great promise for inducing rod cell death without damaging cone photoreceptors. We describe the generation of two transgenic zebrafish lines that express an NTR-EGFP fusion protein from the zebrafish rod opsin promoter (zop). The Tg(zop:nfsB- EGFP)nt19 line expresses NTR-EGFP uniformly in all rod photoreceptors, whereas Tg(zop:nfsB-EGFP)nt20 displays a non-uniform expression of NTR-EGFP in a subset of rods. Treatment of both transgenic lines with metronidazole resulted in the death of all cells expressing the NTR-EGFP fusion protein, followed by regeneration of the lost photoreceptors. In the Tg(zop:nfsB-EGFP)nt19 line, prodrug treatment resulted in the loss of all rods in the retina. In contrast, metronidazole induced death in only a subset of rods in the Tg(zop:nfsB-EGFP)nt20 line. Unexpectedly, metronidazole treatment increased Muller glial cell proliferation in the Tg(zop:nfsB-EGFP)nt19 retina, but only increased rod precursor cell proliferation in the Tg(zop:nfsB-EGFP)nt20 retina. Thus, acute loss of a large number of only rod photoreceptors is sufficient to induce a Muller glial regeneration response, possibly due to an insufficient number of rod precursor cells in the ONL to effectively regenerate the full rod photoreceptor cell population.