Interference with miR-204 expression modifies lens cell differentiation via Meis2 targeting. Frontal sections of St24 control (A–C), Mo-miR-204 (D–F), miR-204 (G–I), Mo-Meis2/Mo-miR-204 (J–L), Meis2-TPmiR-204 (M–O), and Meis2-TPmiR-204/miR-204 (P–R)–injected medaka embryos processed for whole-mount RNA ISH with probes specific for olMeis2 (A, D, G, J, M, and P), olPax6 (B, E, H, K, N, and Q), and olα-ACrystallin (C, F, I, L, O, and R). Expression of olMeis2 (D and M) and olPax6 (E and N) is up-regulated in lens of miR-204 and Meis2-TPmiR-204 morphant embryos, whereas that of olα-ACrystallin is increased in the lens placode and ectopically expressed in the epithelial lens monolayer (F and O, yellow arrowheads). Lens epithelial (D–F and J–L, red arrowheads) and primary fiber (D–F and J–L, black arrowheads) cells are displaced. (J) MOs act at the translational level; thus, Mo-Meis2/Mo-miR-204 coinjection does not rescue olMeis2 mRNA expression. miR-204 gain-of-function has opposite effects in lens gene expression, without affecting lens epithelial monolayer (G–I, red arrowheads) and the primary fibers (G–I, black arrowheads). Mo-Meis2/Mo-miR-204 and Meis2-TPmiR-204/miR-204 coinjections restore correct expression of lens differentiation markers (J–L and P–R). Mo-Meis2/Mo-miR-204 coinjections do not rescue cell displacement (J–L, red and black arrowheads). Broken lines mark boundaries between the lens epithelial monolayer and the primary fiber cells. (Scale bars: 20 μm.)

Model for function of miR-204 in lens and retina development. miR-204 controls expression of the Meis2 gene, contributing to regulation of the Meis2/Pax6 pathway in both lens and retina development.

miR-204 directly targets Meis2. (A–C′) Frontal sections of St24 WT medaka embryos hybridized in both single (A–B′) and double (C and C′) whole-mount RNA ISH with probes against olMeis2 (red) and miR-204 (blue). miR-204 (A and A′) and olMeis2 (blue) (B–B′) colocalize in the lens placode and ciliary marginal zone (C–C′). Boxed areas are magnified in A′–C′. (D) Predicted target site of miR-204 within the 3′-UTR of the Meis2 gene in different species, showing conserved nucleotides (red) and nonconserved nucleotides (black). The blue line represents the sequence against which Meis2-TPmiR-204 Mo was designed. (E) Relative Luc activities in H36CE cells as fold differences in the Luc/Renilla ratios normalized to the value of Luc reporter constructs. miR-204 addition significantly decreases Luc activity of the construct containing 3′-UTR of MEIS2 when compared with controls. ***P < 0.0001 (t tests). Three point mutations in the predicted miR-204 target site in Meis2 inhibit this effect (no significant variation when compared with the thymidine kinase (TK)-Luc control). Densitometric analysis (F) of Western blotting (G) shows reduction of Meis2 protein levels in the presence of miR-204 duplexes and increase after miR-204 depletion when compared with cel-miR-67 control transfections. **P < 0.001; ***P < 0.0001 (t tests). 1, inhibitor hsa-miR-204; 2, inhibitor cel-miR-67; 3, mimic hsa-miR-204; 4, mimic cel-miR-67. Relative levels of the Meis2 protein measured 48 h after transfection of H36CE cells. (H–M) Frontal sections of St24 control (H and K), miR-204–overexpressing (I and L), and Mo-miR-204–injected (J and M) embryos treated for whole-mount RNA ISH with an olMeis2 probe (H–J) or immunostained with an anti-Meis2 antibody (green) (K–M). Sections are counterstained with propidium iodide (PI, red). Both olMeis2 mRNA and protein are down-regulated in lens placode and retina of miR-204–overexpressing embryos (I and L) but up-regulated in miR-204 morphants (J and M). (Scale bars: 20 μm.).

miR-204 is required for establishment of D-V polarity of the optic cup. Frontal vibratome sections of control (A–C), Mo-miR-204 (D–F), miR-204 (G–I), Mo-Meis2/Mo-miR-204 (J–L), Meis2-TPmiR-204 (M–O), and Meis2-TPmiR-204/miR-204 (P–R)–injected embryos processed for whole-mount RNA ISH with probes specific for olPax6 (A, D, G, J, M, and P), olVax2 (B, E, H, K, N, and Q), and olTbx5 (C, F, I, L, O, and R). olPax6 is up-regulated and ectopically expressed in ventral retina of morphant embryos (D and M, asterisk). Expression of ventral gene olVax2 is reduced, whereas that of dorsal marker olTbx5 is expanded ventrally in morphant embryos (E, F, N, and O) when compared with control embryos (B and C). miR-204–overexpressing embryos show reciprocal alterations in Pax6 and D-V marker expression (G–I). Defects in D-V optic cup polarity are completely rescued by coinjection of Mo-miR-204 with Mo-Meis2 (J–L) and Meis2-TPmiR-204 with miR-204 (P–R). (Scale bars: 20 μm.)