Fig 1.
TGF-β pathway down-regulation from early phases of medaka fish embryo development determines alteration of programmed cell death programs in the retina.
(a) MO-tgfβr1 is designed to sterically block the fourth intron-exon splice donor site of the tgfβr1 transcript, causing a partial retention of the intronic sequence as shown by PCR analysis. (b-g) Control (b), MO-tgfβr1 (c), SB43152 (d) and TGF-β- (e) treated control, MO-miR-181a/b (f) and TGF-β treated MO-miR-181a/b (g) medaka fish embryos at stage 32. The MO-tgfβr1 injected embryos showed a phenotype characterized by abnormal body and head structures, including microphthalmia. (h-m) Alteration of the TGF-β pathway from the early stages of development caused an increase of retina cell death as shown by TUNEL assay (h, i). Administration of drugs that lead to a TGF-β pathway down-regulation (j, SB43152) or increase (k, TGF-β) from stage 30 onwards did not cause cell death alteration in control medaka retina. Similarly, no significant alterations were found in MO-miR-181a/b- (l) and TGF-β-treated MO-miR-181a/b (m) retinas. (n) Quantification of TUNEL positive cells; N = 12 eyes were analyzed for each treatment.
Fig 2.
TGF-β signaling regulates mature miR-181a/b expression levels.
(a-b) TGF-β pathway inhibition leads to a decrease in the expression levels of mature miR-181a and miR-181b. (a) Administration of SB432542, a TGF-β receptor inhibitor, induced a decrease of miR-181a and miR-181b mature forms in St32 eyes with respect to DMSO treatment, as detected by Taqman assays. The miR-181a and miR-181b reduction was comparable with that observed in the morpholino-mediated inhibition of the TGF-β pathway (MO-tgfβr1) at St32 (b). Data are means +SEM. ***, P <0.001 (t-tests). (c-d) TGF-β treatment (10ng/ml) leads to increased levels of miR-181a and miR-181b mature forms in medaka fish St32 eyes in a transcription-independent manner. (c) Administration of TGF-β (10ng/ml) for 24 h (from St30 to St32) led to the increase of mature miR-181a and miR-181b in St32 eyes, as assessed by Taqman assays. Co-treatment with TFG-β and actinomycin D for 24 h (from St30 to St32) did not alter the TGF-β effect on mature miR-181a/b levels. These results indicate that the TGF-β effect on miR-181a/b expression is not transcription-dependent. Data are means +SEM. ***, P <0.001 (two-way ANOVA). (d) qRT-PCR on RNA extracted from DMSO- and TGF-β-treated St32 medaka fish eyes for all the pri-miR-181a and pri-miR-181b transcripts derived from the different genomic loci present in the medaka fish genome. After 24 h (from St30 to St32) of TGF-β treatment (10ng/ml), there were no significant changes in pri-miR-181a/b levels with respect to DMSO treatment. (e) qRT-PCR on RNA extracted from DMSO-, SB432542- and TGF-β-treated St32 medaka fish eyes. In the SB432542-treated eyes the decrease of miR-181a/b levels led to increased prox1 and erk2 transcript levels, whereas in TGF-β-treated eyes the miR-181a/b increase was accompanied by reduced transcript levels of both prox1 and erk2. Data are means +SEM. **P <0.01; ***, P <0.001 (Two-way ANOVA). (f-g) Representative Western blotting (f) and corresponding quantification (g), showing a decrease of total-, phospho-Erk2 proteins and of its downstream target RhoA in TGF-β-treated St32 medaka fish eyes. Data are means +SEM. **P <0.01 (t-tests).
Fig 3.
TGF-β signaling modulates miR-181a/b action in the assembly of retinal circuitry.
(a-e) Retinal frontal sections of St40 DMSO-treated (a), TGF-β-treated (b) control-MO medaka fish embryos, miR-181a/b morphant embryos (c), SB432542-treated embryos (d) and TGF-β-treated (e) miR-181a/b morphant medaka fish embryos processed for Richardson-Romeis staining. Red bars, IPL thickness. Scale bars: 20μm. (f) Quantitative analysis of IPL thickness indicated as the ratio in the central retina between the IPL area and total retinal area. Data are means ± SEM. ***, P <0.001 (one-way ANOVA). (g-j) Representative images of amacrine cells from St40 retinal sections of control-MOs (g), miR-181a/b morphant (h), SB432542-treated control (i) and miR-181a/b morphant treated with TGF-β (j) Six3:eGFP transgenic medaka fish embryos. Cell nuclei are stained with DAPI (blue). GFP (green signal) stains amacrine cell soma and neurites; red arrows, Six3 axon-like structure of amacrine cells; red bars, thickness of the IPL. SB432542 treatment (i) phenocopied the amacrine cell neuritogenesis defects observed in miR-181a/b morphants (h). Addition of TGF-β (from St30 to St40) to miR-181a/b morphants (j) was sufficient to rescue neuritogenesis defects of miR-181a/b morphant transgenic embryos. INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 20μm. (k-n) Representative 2-D reconstruction of confocal images of St32 control-MOs (k), miR-181a/b morphant (l), SB432542-treated (m) and TGF-β-treated miR-181a/b morphant (n) Ath5:eGFP transgenic whole-heads. Dotted white lines mark optic nerve routes. Treatment of control-MOs embryos with 80μM SB432542 (m) phenocopied the miR-181a/b-morphant optic nerve length decrease (l). Addition of TGF-β for 24 h (from St30 to St32) to miR-181a/b morphants (n) was sufficient to rescue correct optic nerve growth in Ath5:eGFP morphant embryos. Scale bars: 50μm. OT, optic tectum.
Fig 4.
TGF-β administration rescues axon defects in miR-181a/b depleted RGCs.
(a-c) Representative images from primary RGC cultures from St30 control-MOs (a), miR-181a/b morphant (b) and TGF-β-treated miR-181a/b morphant medaka fish embryos (c). The RGC axon length defect was rescued by treatment with TGF-β (c). Scale bars: 10μm. (d) Quantification of RGC axonal length. Data are means +SEM (n = 100) from three independent cell culture experiments. ***P <<0.001 (one-way ANOVA).
Fig 5.
TGF-β signaling regulates RhoA levels via two independent and synergistic cascades.
(a) qRT-PCR analysis of erk2 transcripts in total eye RNA derived from St32 control-MOs, miR-181a/b morphants and TGF-β-treated miR-181a/b morphants. The TGF-β-mediated increase of miR-181a/b caused a rescue of miR-181a/b target transcripts, such as prox1 and erk2, in miR-181a/b morphants. (b, c) Representative Western blotting on protein from St32 eyes (b) and corresponding quantification (c) show that administration of TGF-β to MO-miR-181a/b embryos leads to restoration of total-, phospho-Erk2 and RhoA protein levels. When MO-miR-181a/b embryos were treated with both TGF-β and the proteasomal inhibitor MG132, total- and phospho-Erk2 protein levels were still rescued, whereas RhoA levels were only partially rescued. Data are means +SEM.* P <0.05; **P <0.01; *** P <0.001 (two-way ANOVA).
Fig 6.
TGF-β signaling regulates erk2 expression by modulating miR-181a/b levels.
(a) qRT-PCR analysis of prox1 and erk2 transcripts in RNA derived from St32 control-MOs, MO-protector-erk2–injected and TGF-β-treated MO-protector-erk2 medaka fish eyes. The TGF-β rescue on the transcript levels of miR-181a/b targets was mediated by the miR-181a/b increase. Indeed this effect on erk2 was completely abolished in the MO-protector-erk2 embryos (a), while other miR-181a/b targets, such as prox1, whose miR-181 binding sites are unaffected by the MO-protector, were still sensitive to TGF-β action. Data are means ± SEM. * P <0.05; ** P <0.01 (two-way ANOVA). (b-d) Retinal frontal sections of St40 Control (b), MO-protector-erk2 (c) and TGF-β-treated MO-protector-erk2 medaka fish embryos (d) processed for Richardson-Romeis staining. Red bars, IPL thickness. Scale bars: 20μm. (e) Quantitative analysis of IPL thickness, indicated as the ratio in the central retina between the IPL area and total retinal area. Data are means ± SEM. *** P <0.0001 (one-way ANOVA). (f-g) Representative Western blotting on protein from St32 eyes (f) and corresponding quantification (g) show that administration of TGF-β to MO-protector-erk2 embryos leads to partial rescue of RhoA protein to levels. When MO-protector-erk2 embryos were treated with both TGF-β and the proteasomal inhibitor MG132, RhoA levels were not rescued anymore. Data are means +SEM. **P <0.05 (one-way ANOVA).
Fig 7.
Model of TGF-β cascades in retinal axon specification and growth.
The TGF-β pathway regulates axon growth in the retina via two independent and synergistic pathways: the Par6/Smurf1 and the miR-181/ERK pathways. TGF-β–mediated activation of the Par6/Smurf1 cascade leads to ubiquitination and degradation of RhoA. On the other side, TGF-β also generates increased miR-181a/b levels, enhancing the process of miRNA maturation via activation of the SMAD2/3 protein. In turn, by fine modulation of the MAPK/ERK signaling pathway, miR-181a/b has an inhibitory effect on cofilin and RhoA production.