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

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