Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina

Abstract

Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the residentMüller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage.

mTor-signaling is required for the formation of proliferating Müller glia-derived progenitor cells in the chick retina

“…6A,B). Nuclear migration may be required for the reprogramming of Müller glia to a more progenitor-like state (Lahne et al, 2015). We found significantly lower levels of Pax6/cell in the few Sox9-positive glia that migrated into the ONL in rapamycin-treated retinas compared with controls, whereas levels of Pax6 in the Sox9-positive glia that were in the INL were not significantly different from controls (Fig.…”

Retina regeneration in zebrafish

“…The reprogrammed nuclei of injury-responsive Müller glia migrate from the INL to the outer nuclear layer (ONL) where they divide asymmetrically and then return to the INL (Fig. 2), a process referred to as interkinetic nuclear migration 13,14 . This asymmetric division results in the generation of a retinal progenitor that also exhibits interkinetic nuclear migration as it amplifies in a Pax6-dependent manner 14,15 , which results in a small population of progenitors capable of regenerating all major retinal neuron types (Fig.…”

“…2), a process referred to as interkinetic nuclear migration 13,14 . This asymmetric division results in the generation of a retinal progenitor that also exhibits interkinetic nuclear migration as it amplifies in a Pax6-dependent manner 14,15 , which results in a small population of progenitors capable of regenerating all major retinal neuron types (Fig. 2) 16,17 .…”

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

“…Tumor necrosis factor α, a signaling molecule that is released from dying retinal neurons induces Müller glia residing in the basal inner nuclear layer (INL) of the retina, to proliferate 5 and produce neuronal progenitor cells that continue to proliferate before differentiating into the neuronal cell types that died 2–4 . During the proliferative phase of the regeneration response, the nuclei of Müller glia and their derived neuronal progenitor cells undergo a repetitive migratory pattern in phase with the cell cycle (interkinetic nuclear migration, INM) 6, 7 . Nuclei positioned in the basal INL replicate their DNA before migrating to the outer nuclear layer (ONL) where they divide before the arising nuclei return basally to the INL.…”

“…9, 11–13 However, the mechanisms governing INM in the adult regenerating retina have not been studied in much detail. 6, 7 Live-cell imaging will be an invaluable approach to advance our knowledge of the signaling pathways that control INM in the adult regenerating retina.…”