Repressing Notch Signaling and Expressing TNFα Are Sufficient to Mimic Retinal Regeneration by Inducing Müller Glial Proliferation to Generate Committed Progenitor Cells

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

“…To maintain Rockout activity, system water containing the drug was exchanged every 24 h and zebrafish were maintained in a dark incubator set at 30°C until eyes were enucleated at 35, 45, 50, 55, or 72 h after beginning the light treatment. Having previously established that exposure to constant intense light for 24 h was sufficient to induce maximal photoreceptor cell death by 16 h of light damage and a complete regeneration response Conner et al, 2014), we chose to apply Rockout from 28 h of light treatment to prevent any effect it may exert during the period of cell death (Bermel et al, 2009;Koch et al, 2014;Yamamoto et al, 2014). To study the effect of Rockout on retinal regeneration, zebrafish were exposed to constant intense light for 28 h, treated with 25 M Rockout for 44 h (corresponding to 72 h after beginning the constant light treatment), and subsequently transferred into system water lacking drugs to recover for either 8 or 15 d. In a subset of experiments, the ␥-secretase inhibitor R04929097 (750 M; SelleckBio; Conner et al, 2014) or its vehicle control, DMSO (10%), were intraperitoneally injected into albino; Tg[gfap:EGFP] nt11 zebrafish using a 32 gauge syringe immediately before exposing them to either 25 M Rockout or DMSO (1:2000) in system water from 28 h of light treatment.…”

“…To maintain R04929097 concentrations at a constant level, intraperitoneal injections of either R04929097 or DMSO were performed every 8 h. The treatment groups were the following: (1) DMSO (1:2000)/DMSO (10%), (2) Rockout/ DMSO (10%), (3) DMSO (1:2000)/R04929097, or (4) Rockout/ R04929097. In addition, 25 M cytochalasin D (Tocris Bioscience; Carlier et al, 1986;Becker and Hart, 1999) or its vehicle control, DMSO (1:1000), were injected intraperitoneally into anesthetized zebrafish at 28 h of light treatment using a 32 gauge syringe and maintained in system water in a dark incubator set at 30°C until 35 h of light treatment (Conner et al, 2014). Because the Rho activator CN03 is conjugated to a cellpenetrating moiety, either CN03 (5 g/ml; Cytoskeleton) or water was intravitreally injected into healthy undamaged albino; Tg[gfap:EGFP] nt11 or albino zebrafish eyes using a Hamilton syringe as described previously (Thummel et al, 2008a).…”

“…Eyes were collected for analysis at times corresponding to 45 and 50 h after the start of the light treatment. EdU detection was performed on frozen retinal sections according to manufacturer's guidelines, followed by immunocytochemistry for BrdU (Conner et al, 2014).…”

“…As a result of this process, termed interkinetic nuclear migration (INM), NPC nuclei undergo DNA replication (S-phase) in a basal region and perform mitosis at the apical surface of the neuroepithelium (Pearson et al, 2005;Baye and Link, 2007;Xie et al, 2007;Del Bene et al, 2008;Norden et al, 2009;Tsuda et al, 2010;Kosodo et al, 2011;Leung et al, 2011;Lee and Norden, 2013). Disruption of INM is thought to cause premature cell cycle exit of NPCs due to their exposure to different spatial cues along apicobasal Notch-Delta gradients (Murciano et al, 2002;Del Bene et al, 2008). Furthermore, the degree of basal migration during the G 1 and S phases was correlated with the decision to either remain in or exit the cell cycle after the next cell division such that more basally located NPC nuclei are more likely to exit the cell cycle than those positioned further medially (Baye and Link, 2007).…”

“…Furthermore, the degree of basal migration during the G 1 and S phases was correlated with the decision to either remain in or exit the cell cycle after the next cell division such that more basally located NPC nuclei are more likely to exit the cell cycle than those positioned further medially (Baye and Link, 2007). During INM, nuclei are driven apically in the G 2 phase of the cell cycle mainly by actin-myosin-mediated contraction, although microtubules also exert some effect (Del Bene et al, 2008;Norden et al, 2009;Yu et al, 2011). Actin-myosin-mediated contraction requires the phosphorylation of the myosin light chain (MLC), which is mediated by a variety of kinases, including Rhoassociated coiled-coil kinases (Rocks) (Amano et al, 1996;Vicente-Manzanares et al, 2009).…”