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Manuela Lahne, David R Hyde; Rock-mediated INM is required for sufficient regeneration of the adult zebrafish retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5838.
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© ARVO (1962-2015); The Authors (2016-present)
In the damaged zebrafish retina, Müller glia reenter the cell cycle and produce neuronal progenitor cells (NPCs) that replace lost retinal neurons. Recently, Müller glia nuclei were shown to migrate between the basal and apical surface of the retina in phase with the cell cycle similar to NPCs in retinal development. We investigated the mechanism governing this interkinetic nuclear migration (INM) and its function in regenerating the light-damaged retina.
Dark-adapted albino or albino Tg[gfap:EGFP]nt11 zebrafish that express EGFP in Müller glia, were exposed to constant intense light for 28 hours (h) and then treated with Rockout, an inhibitor of Rho-associated coiled-coil kinases (Rocks) for up to 44 h. Some zebrafish were subsequently allowed to recover in system water for 8 days. EdU and/or BrdU were intraperitoneally injected to label proliferating cells. Light-damaged retinal sections were immunolabeled with markers for proliferation (PCNA, proliferating cellular nuclear antigen and BrdU), mitosis (pH3, phospho-histone 3), ganglion/amacrine cells (HuCD), blue and red cones (opsins) and GFP.
Inhibition of Rocks, kinases that mediate actin cytoskeletal reorganization, disrupt INM resulting in increased numbers of pH3-positive Müller glia in the basal INL accompanied by a reduction in the numbers of pH3-positive Müller glia in the ONL at 35 h. Proliferation initiation was not affected by inhibiting Rocks as evidenced by similar numbers of PCNA-positive Müller glia. However, reduced numbers of proliferating NPCs were observed at 45 and 72 h of light damage, which was not caused by mitotic defects, as similar numbers of pH3-positive cells were present in Rock-inhibited retinas. EdU and BrdU pulse-chase experiments that labeled activated Müller glia and dividing NPCs, respectively, revealed fewer EdU- and BrdU-double-positive cells at 45 and 50 h of light-damage in Rockout-exposed retinas, suggesting premature cell cycle exit. Moreover, significantly fewer blue and red cones were regenerated at 8 days of recovery (post light damage) in Rock-inhibited retinas, while significantly increased numbers of BrdU and HuCD-positive amacrine/ganglion cells were generated.
Disruption of INM in light-damaged zebrafish reduced NPC proliferation and increased the production of amacrine/ganglion cells at the expense of cone photoreceptors.
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