ascl1a and lin-28 mRNAs are rapidly induced in dedifferentiating MG following retinal injury. (a) RT-PCR shows induction of pluripotency factor mRNAs following retinal injury. (b) Real-time PCR quantification of pluripotency factor mRNA levels during retina regeneration. Data represent means ± s.d. (n=3 individual fish; compared to uninjured retina, P=0.0001 or less for lin28, c-myc_b, and ascl1a expression at all time points post retinal injury; P=0.0001 for klf4 at 15 hpi, and 2 and 4 dpi; P=0.02 for klf4 at 7 dpi; P=0.0001 or less for sox2, c-myc_a and nanog at 2 and 4 dpi; P= 0.0425 for c-myc_a at 7 dpi; P=0.0178 and 0.0069 for oct4 at 2 and 4 dpi, respectively). Note Y-axis is fold induction in log scale and normalized to 0 hr time point that is assigned a value of 1. (c) RT-PCR shows ascl1a and lin-28 are coordinately induced following retinal injury. (d) In situ hybridization and immunofluorescence shows lin-28 RNA co-localizes with 1016 tuba1a:gfp transgene expression in glutamine synthetase (GS)⁺ MG at 4 dpi. Scale bar is 10 microns. (e) lin-28 and ascl1a double fluorescence in situ hybridization and BrdU immunofluorescence show lin-28 and ascl1a are co-expressed in proliferating MG-derived progenitors at 4 dpi. Three hrs prior to sacrifice, adult fish, whose retina was injured 4 days earlier, received an intraperitoneal injection of BrdU. Scale bar is 10 microns. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; BrdU, bromodeoxyuridine; GS, glutamine synthetase; GFP, green fluorescent protein.

Ascl1a regulates lin-28 expression. (a, b) Lissamine-labeled control, ascl1a or lin-28-targeting MOs were electroporated into injured retinas of 1016 tuba1a:gfp transgenic zebrafish. At 4 dpi GFP, and ascl1a and lin-28 mRNA expression was detected by immunofluorescence and in situ hybridization, respectively. Control MO-treated retinas retain GFP, ascl1a and lin-28 expression (arrows), while Lin-28 knockdown suppresses GFP expression and knockdown of Ascl1a suppresses both GFP and lin-28 expression. Scale bar is 10 microns. (c,d) RT-PCR shows Lin-28 knockdown has no effect on injury-dependent induction of ascl1a mRNA at 2 dpi (c), while Ascl1a knockdown blocks injury-dependent induction of lin-28 mRNA at 2 dpi (d). Lane C is control uninjured retina. (e,f) Real-time PCR quantification of the effects of Lin-28 (e) or Ascl1a (f) knockdown on lin-28 and ascl1a mRNA levels at 2 dpi. Data are normalized to uninjured retinas and represent means ± s.d. from 3 replicas of a single experiment. (g) Ascl1a regulates lin-28 promoter activity. HEK293 cells were transfected with lin-28:luciferase and the indicated amounts of cmv:ascl1a along with SV40:Renilla luciferase for normalization. Normalized promoter activity is reported as means ± s.d. (n=3; compared to control, P=0.0123 for 50 ng Ascl1a expression vector and P=0.0001 for 200 ng of Ascl1a expression vector). (h) Ascl1a binds to regions of the lin-28 promoter that harbor multiple E-boxes. ChIP analysis of zebrafish embryos (single cell stage) injected with either myc-RNA or myc-ascl1a mRNA. Immunoprecipitated chromatin was assayed by PCR using primers (arrows) flanking putative Ascl1a binding sites 1 and 2 (ethidium bromide stained gel shown). The predicted fragments sizes of 287 bp and 323 bp were amplified. The 3.1 kb lin-28 promoter diagramed below the gel shows consensus E-box binding sites (ovals). Orange ovals are putative Ascl1 binding sites.

let-7 miRNAs suppress expression of regeneration and pluripotency-associated genes. (a) hspd1 in situ hybridization and glutamine synthetase (GS) immunofluorescence shows hspd1 RNA co-localizes (arrows) with GS-expressing MG in the uninjured retina. (b) RT-PCR shows hspd1 mRNA expression in uninjured retina that is increased following retinal injury, while pax6_b expression is undetectable in the uninjured retina and dramatically induced following retinal injury. (c) Real-time PCR quantification of hspd1, pax6_a and pax6_b mRNA levels in uninjured and 4 day post injured retina (injured). Data represent means ± s.d. (n=3 fish; compared to uninjured retina, P=0.0001 or less for hspd1, pax6_a and pax6_b, respectively, at 4 dpi. (d) let-7-dependent suppression of zebrafish proteins that are necessary for retina regeneration or associated with pluripotency. HEK 293 cells were transfected with flag or myc-tagged regeneration or pluripotency-associated gene expression vectors (50 ng) with increasing concentrations (0, 50, 200, 500 ng) ubC:let-7a,let-7f along with pCS2:mCherry for normalization. (−) lane indicates untransfected cells. Forty-eight hrs later cells were harvested and proteins resolved by denaturing SDS-PAGE. Western blots were probed with anti-mCherry, anti-Flag or anti-Myc antibodies. let-7-dependent regulation of protein expression was quantified by densitometry (Supplementary Information, Fig. S7). Experiments were repeated 3 times with similar results. See Fig. S9 for full scans. (e) Constructs used to overexpress let-7 and investigate the function of putative let-7 miRNA binding sites in the hspd1 3’ UTR. Mutations in the let-7 binding site seed sequence are indicated. (f) The hspd1 3’ UTR let-7 binding site at position 1970 confers let-7-dependent regulation on luciferase expression. Luciferase activity from 24 hpf zebrafish embryos that were co-injected at the single cell stage with the luciferase reporter and the let-7 miRNA expression vector described in (e) along with capped Renilla luciferase mRNA for normalization. Data represent means ± s.d. (n=3 fish; compared to wt 3’UTR construct in absence of pri-let-7 miRNA or mut 3’ UTR construct with pri-let-7 miRNA, P=0.0001 or less for luciferase-wt 3’ UTR activity treated with 1, 5 and 20 ng of ubC:let-7a,let- 7f construct.

Ascl1a and Lin-28 knockdown inhibit 1016 tuba1a:gfp transgene expression and MG-derived progenitor proliferation. (a) Control, ascl1a or lin-28 lissamine-tagged MOs were electroporated into the retina of 1016 tuba1a:gfp transgenic fish at the time of retinal injury and 3 hrs prior to sacrifice, at 4 dpi, fish received an intraperitoneal injection of BrdU. Arrows point to cells harboring lissamine-tagged MO. GFP and BrdU immunofluorescence show Ascl1a and Lin-28 knockdown suppress 1016 tuba1a:gfp expression and proliferation of MG-derived progenitors. (b) Quantification of the total number of MO⁺ cells that are in S phase as indicated by BrdU uptake. Data represent means ± s.d. (n=3 individual fish; compared to control MO, lin-28 MO and ascl1a MO P=0.000178 or less). (*) identifies autofluorescence in ONL. Scale bar is 10 microns and applies to all photomicrographs.

Lin-28 regulates let-7 miRNA levels in MG-derived progenitors. (a) RT-PCR shows let-7 miRNA expression is high and lin-28 expression is low in differentiated MG (lane C), while let-7 miRNA expression is suppressed and lin-28 expression is highly induced in MG-derived progenitors at 4 dpi. (b) Real-time PCR quantification of lin-28 mRNA and TaqMan PCR quantification of let-7a and let-7f miRNA levels in purified MG and MG-derived progenitors at 4 dpi. Data are normalized to uninjured retina and represent means ± s.d. A single sample, consisting of MG purified from 2 uninjured fish and MG-derived progenitors purified from 10 injured fish, was assayed in triplicate. (c) let-7a in situ hybridization (LNA probe) and BrdU immunofluorescence shows a dearth of let-7a miRNA in BrdU⁺ MG-derived progenitors of the injured retina at 4dpi. Scale bar is 20 microns. (d) TaqMan PCR quantification of let-7 miRNA levels in uninjured and injured retinas at different times after injury. Data represent means ± s.d. (n=3 fish; compared to control uninjured retina, P=0.0001 or less for let-7a and let-7f at all time points). (e) RT-PCR shows Lin-28 knockdown with 2 different MOs relieves injury-dependent let-7a and let-7f miRNA suppression. Lane C is uninjured retina. (f) Lin-28 or Ascl1a knockdown relieves injury-dependent let-7 miRNA suppression. TaqMan PCR was used to quantify let-7a and let-7f miRNA levels. Data represent means ± s.d. from 3 replicas of a single experiment.