FIGURE
Fig. 1
- ID
- ZDB-FIG-200513-13
- Publication
- Kara et al., 2019 - The miR-216a-Dot1l Regulatory Axis Is Necessary and Sufficient for Müller Glia Reprogramming during Retina Regeneration
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Fig. 1
Suppression of miR-216a Is Required for MG Dedifferentiation and Proliferation during Retina Regeneration (A) Schematic for post-mitotic and dedifferentiated MG sorting. Adult zebrafish were dark adapted for 2 weeks and then exposed to constant intense light lesioning for 45 h. For post-mitotic MG isolation, GFP+cells were sorted from dark adapted Tg(gfap:gfp)retinas. For dedifferentiated MG isolation, GFP+ cells were isolated from 45 h light lesioned Tg(1016tuba1a:GFP)retinas. (B) Fold changes in miR-216a levels in FACS-purified MG were determined by qPCR. miR-216a is enriched in post-mitotic MG (GFP+) from undamaged retinas in Tg(gfap:gfp) fish. After 45 h of light damage, miR-216ais downregulated ∼5-fold in dedifferentiated MG (GFP+) in Tg(1016tuba1a:gfp) fish. miR-216a expression did not change in non-MG cells (GFP−) during regeneration. Data are from 5 independent experiments with 3 technical replicates of qPCR. MG were purified from 18 and 20 light-damaged fish in each experiment. Error bars represent the SEMs. ∗p < 0.05 and ∗∗p < 0.01 using 2-way ANOVA with Fisher’s least significant difference (LSD) post hoc test. (C) Control miRNA (siRNA against luciferase [siLuc]) or miR-216a was injected and electroporated into the left eyes of Tg(1016tuba1a:gfp) zebrafish before intense light exposure (0 h). After 45 h, retinas were collected, sectioned, and immunostained using antibodies against GFP and PCNA. Nuclei were counterstained with TOPRO (blue). miR-216again of function abolished tuba1a:GFP transgene expression and significantly reduced the number of INL PCNA+ proliferating cells. (D) Quantification of PCNA+ cells in the INL and ONL. Error bars represent the SEMs (n = 5–6 fish); ∗∗p < 0.01 using Student’s t test. (E) Overexpression of miR-216a reduced the number of GFP+/PCNA+proliferating progenitor cells after 60 h of intense light damage using Tg(1016tuba1a:gfp) zebrafish. (F) Quantification of total GFP+ and PCNA+ cells. Error bars represent the SEMs (n = 10 fish); ∗p < 0.03 and ∗∗p < 0.003 using Student’s t test. GCL, ganglion cell layer; INL, inner nuclear layer; ns, not significant; ONL, outer nuclear layer. Scale bars, 50 μm. |
Expression Data
Expression Detail
Antibody Labeling
Phenotype Data
Phenotype Detail
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