Figure 1.
Trpm3/miR-204 expression is dependent on Pax6 activity during eye development.
Paraffin sections of control (A–E,G,I,K), Pax6lacZ/lacZ (F,J) and Pax6loxP/loxP;Mlr10-cre (H,L) stained for Trpm3 mRNA (B,D,E–H), Pax6 protein (A,C, red) and miR-204 (I–L, green). Trpm3 expression begins after E9.5 in the developing eye (B,D), although it is already active in the otic vesicle on E9.5 (B′ inset). Trpm3 and miR-204 are lost from the optic rudiment of Pax6lacZ/lacZ embryos (F,J; optic cup rudiment is traced with a line) and in the lens of Pax6loxP/loxP;Mlr10-cre mutants (H,L). Di, diencephalon; LP, lens placode; LV, lens vesicle; OV, optic vesicle; NT, neural tube; OC, optic cup; OtV, otic vesicle; RPE, retinal pigmented epithelium; Re, developing retina. Scale bar = 100 µM.
Table 1.
Pax6 targets that are reduced in developing lens (E14.5) and forebrain (E12.5).
Figure 2.
Pax6 directly binds a Trpm3 enhancer sequence in vitro and in vivo.
(A) Schematic diagram of the murine Trpm3 locus. Three transcription start sites (TSS, black arrows) are distributed across 600 kb. The black rectangles represent Trpm3 exons. The red rectangle represents the miR-204-encoding sequence in intron 6. (B) Area downstream of TSS2. Ellipses represent real-time qRT-PCR amplicons. Gray circle represents the location of EMSA probe Trpm3.4. (C) Results of real-time qRT-PCR on DNA from newborn lens ChIP experiment. Y-axis represents relative quantity of template DNA divided by the amount of template of the same reaction in 1% of input DNA. P-values for A1 = 0.0043, A2 = 0.0098, error bars indicate SD. (D) Acrylamide gel of radioactive EMSA probe Trpm3.4. Pax6CON is an oligonucleotide with the consensus binding site of Pax6.. For the Trpm3.4 probe, the first lane is Trpm3.4 probe only, lane 2 is probe+1∶10 flag-Pax6, lane 3 is probe+Pax6-flag, lane 4 is probe with Pax6-flag and competition by cold probe.
Table 2.
Putative miR-204 targets found to be upregulated in Pax6 CKO lenses.
Figure 3.
miR-204 down-regulates the expression of Sox11.
(A–B) Sox11 immunofluorescence (A, red) and EGFP (B, green) in Neu-2a cells transfected with miR-204-miRVec plasmid and pCAG-GFP. White arrows indicate GFP-positive transfected cells with visibly reduced Sox11 immunofluorescence. (C) Quantification of Sox11 immunofluorescence in cultures transfected with either scramble-miRVec plasmid or a miR-204-miRVec control plasmid. Y-axis is Sox11 fluorescence of transfected cells divided by that of non-transfected cells in the same image field. Error bars are SEM (P = 0.0104, n = 163;150 cells). (D) Illustration of Sox11 cDNA including the untranslated and coding regions drawn in relative proportion. The three predicted miR-204 binding sites in the 3′ UTR are indicated as rectangles. Black-labeled rectangles were tested in the luciferase reporter assay. The mutated site is marked with a black arrow. Graph presents the relative luciferase luminescence in cells transfected with wild-type Sox11 3′ UTR, wild-type 3′ UTR and miR-204, or mutated 3′ UTR and miR-204. Error bars represent SEM (P = 0.0011, n = 3). Below are alignments of miR-204 RNA sequence, with wild-type and mutated Sox11 3′ UTR regions used in transfections. (E–L) Cryosections of control (E–H), Pax6loxP/loxP;Mlr10-cre (I) and Pax6loxP/loxP;a-Cre distal optic cup (J–L) stained with riboprobe against Sox11 (E,I,H,L) or Trpm3 (G,K) and Pax6 immunofluorescence (F,J, red). Arrowheads in (I) indicate up-regulation in the enlarged transition zone of the Pax6loxP/loxP;Mlr10-cre lens. (F–H) Adjacent sections of the same control animal: asterisk in (H) marks intermediate level of Sox11 staining in outer retina. (J–L) Adjacent sections of the same Pax6loxP/loxP;a-Cre distal retina: dotted lines demarcate area of Pax6 deletion, asterisks mark outer retina with elevated levels of Sox11 (H). LE, lens epithelium; LFC, lens fiber cell; Re, retina. Scale bar = 100 µm.
Figure 4.
Characterization of the medaka ol-Trpm1/miR-204 regulatory region.
(A) RNA in-situ hybridization on frontal eye sections of E11.5 wild-type mouse embryos with the mouse mm-Trpm3 probe. (B–D) Bright-field dorsal views of embryos at stage 24 of development; whole-mount in-situ hybridization with (B) ol-Trpm3 or (C) ol-Trpm1 probes and (D) epifluorescence of cI-transgenic embryos for EGF expression from p-ol-Trpm1-GFP transgene. (E) The p-ol-Trpm1-GFP construct includes 1.5 kb upstream of the coding region of ol-Trpm1. The red box represents the minimal TK promoter. The sequence with similarty to the Trpm3.4 Pax6-binding site is indicated. The numbers indicate respective genomic locations in the three indicated genomes. Conserved nucleotides between the three species are indicated in red or blue (two out of three analyzed species), and non-conserved nucleotides are in black. (D, G) EGFP expression in the whole (D) or a section (G) of the eye of p-ol-Trpm1-GFP-transgenic embryos recapitulates endogenous ol-Trpm1 expression pattern (C,F). (H,I) Pax6 mRNA over-expression activates both ol-Trpm1 and EGFP expression in the ventral retina (red arrow) of the OC. (J) Fold-changes (expressed as 2-ΔΔCt values) in miR-204 and ol-Trpm1 quantified by qRT-PCR, from Pax6- compared to GFP-injected embryos. (K) Relative luciferase luminescence upon transfection of HeLa cells with Pax6 expression plasmid with or without ol-Trpm1 promoter sequences. **P<0.001; ***P<0.0001. Abbreviations: L, Lens; OC, optic cup; OE, olfactory epithelium; NC neural crest melanocytes. Scale bar in C: 20 µm.
Figure 5.
miR-204 affects expression of multiple genes and mediates Pax6 suppression of gene expression.
(A) H36CE cells transfected with hsa-miR-204 mimic or control miRNA (cel-miR-67 mimic) or hsa-miR-204 inhibitor or control miRNA inhibitor (cel-miR-67 inhibitor). Significant reduction following over-expression of hsa-miR-204 mimic and significant elevation following transfection with hsa-miR-204 inhibitor was detected by qPCR (expressed as 2-ΔΔCt values) for transcripts of Sox11, Cpn8, Nfia, Myo10 and Fbn2. Error bars are SD (**P<0.001 and ***P<0.0001, n = 3). (B) Fold-change (expressed as 2-ΔΔCt values) in mRNA levels of indicated medaka genes quantified by qRT-PCR, from stage 24 embryos injected with hsa-miR-204 mimic compared to control cel-miR-67mimic, morpholino against miR-204 (Mo-miR-204) and mismatched morpholino (mm-Mo-miR-204). 400 embryos were pooled for each assay, and technical triplicate experiments were independently executed at least three times. (C) Fold-change in mRNA levels of the indicated genes quantified by qRT-PCR from embryos injected with Pax6, Pax6/Mo-miR-204 and pGFP-expressing plasmid as a control. Results are shown as means ± SD, 250 embryos pooled for each assay. Technical triplicate experiments were independently executed at least three times, n = 3, **P<0.001, ***P<0.0001.
Figure 6.
Model of Pax6 genetic regulation of Sox11 during ocular development.
Pax6 directly regulates miR-204 by binding and activating expression of the host genes Trpm3 in mammals and ol-Trpm1 in fish. During later stages of lens development, miR-204 reinforces inhibition of Sox11. miR-204 is upstream of several genes involved in neurogenesis and cell motility. The findings in medaka suggest that during the early stages of LP formation, ol-miR-204 and Pax6 may co-regulate each other via a negative feedback loop through the established Meis2-Pax6 pathway. Red arrows indicate new data presented here.