Pax6 Regulates Gene Expression in the Vertebrate Lens through miR-204

<div><p>During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene <i>miR-204</i> are co-regulated by Pax6 during eye development. <i>miR-204</i> is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of <i>miR-204</i> targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of <i>miR-204</i> were revealed, among them <i>Sox11</i>, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of <i>Trpm/miR-204</i> and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of <i>miR-204</i>.</p> </div

<i>Trpm3/miR-204</i> expression is dependent on Pax6 activity during eye development.

<p>Paraffin sections of control (A–E,G,I,K), <i>Pax6^lacZ/lacZ</i> (F,J) and <i>Pax6^loxP/loxP;Mlr10-cre</i> (H,L) stained for <i>Trpm3</i> mRNA (B,D,E–H), Pax6 protein (A,C, red) and <i>miR-204</i> (I–L, green). <i>Trpm3</i> 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). <i>Trpm3</i> and <i>miR-204</i> are lost from the optic rudiment of <i>Pax6^lacZ/lacZ</i> embryos (F,J; optic cup rudiment is traced with a line) and in the lens of <i>Pax6^loxP/loxP;Mlr10-cre</i> 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.</p

<i>miR-204</i> affects expression of multiple genes and mediates Pax6 suppression of gene expression.

<p>(A) H36CE cells transfected with <i>hsa-miR-204 mimic</i> or control miRNA (<i>cel-miR-67 mimic</i>) or <i>hsa-miR-204 inhibitor</i> or control miRNA inhibitor (<i>cel-miR-67 inhibitor</i>). Significant reduction following over-expression of <i>hsa-miR-204 mimic</i> and significant elevation following transfection with <i>hsa-miR-204 inhibitor</i> was detected by qPCR (expressed as 2-ΔΔCt values) for transcripts of <i>Sox11</i>, <i>Cpn8</i>, <i>Nfia</i>, <i>Myo10</i> and <i>Fbn2</i>. 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 <i>hsa-miR-204 mimic</i> compared to control <i>cel-miR-67mimic</i>, morpholino against miR-204 (<i>Mo-miR-204</i>) and mismatched morpholino (<i>mm-Mo-miR-204</i>). 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 <i>Pax6</i>, <i>Pax6/Mo-miR-204</i> and pGFP-expressing plasmid as a control. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003357#s2" target="_blank">Results</a> are shown as means ± SD, 250 embryos pooled for each assay. Technical triplicate experiments were independently executed at least three times, n = 3, **<i>P</i><0.001, ***<i>P</i><0.0001.</p

Model of Pax6 genetic regulation of <i>Sox11</i> during ocular development.

<p>Pax6 directly regulates <i>miR-204</i> by binding and activating expression of the host genes <i>Trpm3</i> in mammals and <i>ol-Trpm1</i> in fish. During later stages of lens development, <i>miR-204</i> reinforces inhibition of <i>Sox11</i>. <i>miR-204</i> is upstream of several genes involved in neurogenesis and cell motility. The findings in medaka suggest that during the early stages of LP formation, <i>ol-miR-204</i> and <i>Pax6</i> may co-regulate each other via a negative feedback loop through the established <i>Meis2</i>-Pax6 pathway. Red arrows indicate new data presented here.</p

Characterization of the medaka <i>ol-Trpm1/miR-204</i> regulatory region.

<p>(A) RNA <i>in-situ</i> hybridization on frontal eye sections of E11.5 wild-type mouse embryos with the mouse <i>mm-Trpm3</i> probe. (B–D) Bright-field dorsal views of embryos at stage 24 of development; whole-mount <i>in-situ</i> hybridization with (B) ol-Trpm3 or (C) ol-Trpm1 probes and (D) epifluorescence of cI-transgenic embryos for EGF expression from <i>p-ol-Trpm1-GFP</i> transgene. (E) The <i>p-ol-Trpm1-GFP</i> construct includes 1.5 kb upstream of the coding region of <i>ol-Trpm1</i>. 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 <i>p-ol-Trpm1-GFP</i>-transgenic embryos recapitulates endogenous <i>ol-Trpm1</i> expression pattern (C,F). (H,I) <i>Pax6</i> mRNA over-expression activates both <i>ol-Trpm1</i> and <i>EGFP</i> expression in the ventral retina (red arrow) of the OC. (J) Fold-changes (expressed as 2-ΔΔCt values) in <i>miR-204</i> and <i>ol-Trpm1</i> 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 <i>ol-Trpm1</i> promoter sequences. **<i>P</i><0.001; ***<i>P</i><0.0001. Abbreviations: L, Lens; OC, optic cup; OE, olfactory epithelium; NC neural crest melanocytes. Scale bar in C: 20 µm.</p

Pax6 directly binds a <i>Trpm3</i> enhancer sequence <i>in vitro</i> and <i>in vivo.</i>

<p>(A) Schematic diagram of the murine <i>Trpm3</i> locus. Three transcription start sites (TSS, black arrows) are distributed across 600 kb. The black rectangles represent <i>Trpm3</i> exons. The red rectangle represents the <i>miR-204</i>-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) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003357#s2" target="_blank">Results</a> 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. <i>P</i>-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.</p

<i>miR-204</i> down-regulates the expression of <i>Sox11</i>.

<p>(A–B) Sox11 immunofluorescence (A, red) and EGFP (B, green) in Neu-2a cells transfected with <i>miR-204-miRVec</i> 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 <i>scramble-miRVec</i> plasmid or a <i>miR-204</i>-<i>miRVec</i> 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 (<i>P</i> = 0.0104, n = 163;150 cells). (D) Illustration of <i>Sox11</i> cDNA including the untranslated and coding regions drawn in relative proportion. The three predicted <i>miR-204</i> 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 <i>Sox11</i> 3′ UTR, wild-type 3′ UTR and <i>miR-204</i>, or mutated 3′ UTR and <i>miR-204</i>. Error bars represent SEM (<i>P</i> = 0.0011, n = 3). Below are alignments of <i>miR-204</i> RNA sequence, with wild-type and mutated <i>Sox11</i> 3′ UTR regions used in transfections. (E–L) Cryosections of control (E–H), <i>Pax6^loxP/loxP;Mlr10-cre</i> (I) and <i>Pax6^loxP/loxP;a-Cre</i> distal optic cup (J–L) stained with riboprobe against <i>Sox11</i> (E,I,H,L) or <i>Trpm3</i> (G,K) and Pax6 immunofluorescence (F,J, red). Arrowheads in (I) indicate up-regulation in the enlarged transition zone of the <i>Pax6^loxP/loxP;Mlr10-cre</i> lens. (F–H) Adjacent sections of the same control animal: asterisk in (H) marks intermediate level of <i>Sox11</i> staining in outer retina. (J–L) Adjacent sections of the same <i>Pax6^loxP/loxP;a-Cre</i> distal retina: dotted lines demarcate area of <i>Pax6</i> deletion, asterisks mark outer retina with elevated levels of Sox11 (H). LE, lens epithelium; LFC, lens fiber cell; Re, retina. Scale bar = 100 µm.</p

Putative miR-204 targets found to be upregulated in Pax6 CKO lenses.

<p>Putative miR-204 targets found to be upregulated in Pax6 CKO lenses.</p

Pax6 targets that are reduced in developing lens (E14.5) and forebrain (E12.5).

<p>Pax6 targets that are reduced in developing lens (E14.5) and forebrain (E12.5).</p