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Fig. 7
miR-216a and Dot1l Regulate Retinal Regeneration through the Wnt/β-Catenin Pathway
(A) Control MOs or Dot1l MOs were injected and electroporated into the left eyes of wild-type zebrafish, followed by either 4% DMSO or GSK-3β-inhibitor (1 mM) before intense light exposure (0 h). Eyes were collected after 51 h of intense light lesioning and immunostained using antibodies against PCNA. Nuclei were counterstained with TOPRO (blue).
(B) dot1l MOs significantly decreased the number of PCNA+ cells in the INL, while there was no significant difference between dotl1l MO + GSK-3β-inhibitor co-injected eyes and control eyes.
(C) Quantification of PCNA+ proliferating progenitors in MO-ctl + DMSO, MO-dot1l + DMSO, MO-ctl + GSK-3β-inhibitor, and MO-dot1l + GSK-3β-inhibitor electroporated retinas. Activation of Wnt signaling rescued the decrease in the number of proliferating progenitors upon dot1l knockdown after 51 h of intense light lesioning. Data represent means ± SEMs; n = 9–11 fish. ∗p < 0.05, p = 0.0167 (MO-ctl + DMSO versus MO-dot1l + DMSO) by 1-way ANOVA with Dunnett’s multiple comparisons test.
(D) GSK-3β-inhibitor alone, Dot1l inhibitor (iDot1l) alone, or the combination was injected into the left eyes of Tg(1016tuba1a:GFP)zebrafish; DMSO alone was used as a control. At 51 h post-injection, eyes were collected for PCNA immunostaining. GSK-3β-inhibitor alone induced MG proliferation in undamaged eyes while co-injection of the Dot1l inhibitor led to no significant changes in number of proliferating MG. Data represent means ± SEMs. Each data point represents an individual fish. ∗∗∗∗p < 0.0001, by 1-way ANOVA with Dunnett’s multiple comparisons test.
(E) Control MO or miR-216a MO was injected and electroporated into the left eyes of Tg(1016tuba1a:GFP)zebrafish, followed by either 4% DMSO or XAV939 (10 μM). Eyes were collected at 51 h post-injection and immunostained using antibodies against GFP for dedifferentiated MG and PCNA for proliferating progenitors. Nuclei were counterstained with TOPRO (blue).
(F) Suppression of miR-216a by MO-216a injection stimulates MG proliferation; however, upon co-injection with XAV939, no significant increase in the number of proliferating progenitors was detected.
(G) Quantification of PCNA+ proliferating progenitors in MO-ctl + DMSO, MO-216a + DMSO, MO-ctl + XAV939, and MO-216a + XAV939 electroporated retinas. Inhibition of Wnt signaling reversed the increase in the number of progenitors upon miR-216a knockdown after 51 h of intense light lesioning. Data represent means ± SEMs; n = 18–21 fish. ∗∗p < 0.01, p = 0.0089 (MO-ctl + DMSO versus MO-216 + DMSO) by 1-way ANOVA with Dunnett’s multiple comparisons test.
GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars, 50 μm.
See also Figure S4.
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