PAX6 Regulates Melanogenesis in the Retinal Pigmented Epithelium through Feed-Forward Regulatory Interactions with MITF

<div><p>During organogenesis, PAX6 is required for establishment of various progenitor subtypes within the central nervous system, eye and pancreas. PAX6 expression is maintained in a variety of cell types within each organ, although its role in each lineage and how it acquires cell-specific activity remain elusive. Herein, we aimed to determine the roles and the hierarchical organization of the PAX6-dependent gene regulatory network during the differentiation of the retinal pigmented epithelium (RPE). Somatic mutagenesis of <i>Pax6</i> in the differentiating RPE revealed that PAX6 functions in a feed-forward regulatory loop with MITF during onset of melanogenesis. PAX6 both controls the expression of an RPE isoform of <i>Mitf</i> and synergizes with MITF to activate expression of genes involved in pigment biogenesis. This study exemplifies how one kernel gene pivotal in organ formation accomplishes a lineage-specific role during terminal differentiation of a single lineage.</p></div

Melanogenic genes down-regulated in the <i>Pax6</i>-deficient RPE.

<p>Melanogenic genes down-regulated in the <i>Pax6</i>-deficient RPE.</p

Model of PAX6 control of melanogenesis in the RPE through a positive feed forward loop with MITF.

<p>PAX6 positively regulates the expression of the <i>D</i>-<i>Mitf</i> isoform. There is a compensation mechanism that maintains pan-<i>Mitf</i> levels in the RPE. PAX6 cooperates with MITF to trans-activate several pigmentation genes.</p

PAX6 is required for the expression of the <i>D</i>-<i>Mitf</i> isoform in the developing RPE.

<p>(A-D) Expression of MITF (red) and CHX10 (green) proteins detected by antibody labeling in the RPE of <i>Pax6^loxP/loxP</i> control and <i>Pax6^loxP/loxP;DctCre</i> mutant E12.5 and E15.5 eyes. Scale bar is 25 µm. (A'-D' insets) Higher magnifications of indicated regions and nuclear staining with DAPI. (E) Relative transcript levels of pan<i>-Mitf</i> and <i>M-</i>, <i>D-</i>, <i>H-</i> and <i>A-Mitf</i> isoforms in RPE fractions using QRT-PCR, *<i>p</i><0.05, ***<i>p</i><0.0005, (n = 5). (F) A scheme of the <i>D-Mitf</i> upstream region showing the putative E-boxes (green rectangles) and PAX6 PD binding sites (light blue rectangles). Red arrows indicate the borders of deletion constructs used for luciferase assay. (G) EMSA examining the binding of PAX6 to the putative PAX6 PD binding sites upstream of the <i>D-Mitf</i> TSS (sites 1-3). The binding of PAX6 to probes 1 and 3 was inhibited using unlabeled probe containing the PAX6 consensus binding site (PAX6CON). (H) Activity of luciferase under the regulation of wild-type or truncated <i>D-Mitf</i> promoter co-transfected into HeLa cells along with different combinations of expression vectors and/or their backbones lacking the ORF (n = 3).</p

PAX6 is required for the expression of several melanogenesis genes.

<p>(A) Relative levels of <i>Tyr</i>, <i>Tyrp1</i>, <i>Si</i>, <i>Mlana</i>, <i>Dct</i> and <i>Myo7a</i> transcripts in RPE of control <i>Pax6^loxP/loxP</i> and mutant <i>Pax6^loxP/loxP;DctCre</i> E15.5 mice determined using QRT-PCR. *<i>p</i><0.05, **<i>p</i><0.005, ***<i>p</i><0.0005, (n = 5). (B-G) Control and mutant RPE (B,E) cryo-sections showing the distal OC subjected to <i>in situ</i> hybridization for <i>Si</i> and (C,D,F,G) paraffin sections labeled with antibodies against TYR and TYRP1. Scale bar is 50 µm in B and E and 25 µm in C,D,F,G.</p

PAX6 trans-activates the promoters of <i>mTyrp1</i> and <i>hTyr</i> in the presence of MITF.

<p>(A,B) Activity of luciferase under the regulation of wild-type or mutated (A) <i>mTyrp1</i> or (B) <i>hTyr</i> promoters co-transfected into HeLa cells along with different combinations of expression vectors and/or their backbones lacking the ORF, as indicated (n = 3). The positions of binding sites for MITF (E/M-box, green rectangle) and potential binding sites for PAX6 (light blue rectangle) are indicated relative to the TSS of each promoter in schematics above each graph. (C) Activity of luciferase under the regulation of four consecutive M-boxes and a basic SV40 promoter co-transfected into HeLa cells along with different combinations of expression vectors and/or their backbones lacking the ORF, as indicated (n = 3). (D) Reciprocal co-immunoprecipitation assay of PAX6 and MITF using protein extracts of ARPE19 cells. Samples were precipitated using anti-PAX6 (lanes 4,7), anti-MITF (lane 3) or IgG (lanes 2,6). Anti-Pax6 (lanes 1-4) or anti-MITF (lanes 5-7) were used for Western blot.</p

<i>D-Mitf</i> is dispensable for melanogenesis in the RPE.

<p>(A-C) Whole eye images of (A) <i>Pax6^loxP/loxP</i>, (B) <i>Mitf^ΔD/ΔD</i> and (C) <i>Pax6^loxP/loxP;DctCre</i> mice. (D) A distal OC view of paraffin section of a <i>Mitf^ΔD/ΔD</i> eye labeled with antibody against MITF. Arrows point at the RPE. (E) Relative transcript levels of pan<i>-Mitf</i> and <i>M-</i>, <i>D-</i>, <i>A-</i> and <i>H-Mitf</i> isoforms in RPE fractions determined using QRT-PCR. (F) Relative transcript levels of <i>Tyr</i>, <i>Tyrp1</i>, <i>Si</i>, <i>Mlana</i>, <i>Dct</i> and <i>Myo7a</i> in RPE fractions determined using QRT-PCR. *<i>p</i><0.05, **<i>p</i><0.005, (n = 5).</p

PAX6 expression is essential for proper pigment accumulation in the RPE but dispensable for RPE polygonal and single layer morphology.

<p>(A-N) RPE of (A-E,K,L) <i>Pax6^loxP/loxP</i> and (F-J,M,N) <i>Pax6^loxP/loxP;DctCre</i> mice analyzed for (A,F) PAX6 expression, (B,C,E,G,H,J,K,M) pigment accumulation and (D,L,I,N,O) morphology and specification. (A,F) Paraffin sections of E12.5 eyes were stained for PAX6 N-terminus and (B,G) viewed by differential interference contrast imaging. Scale bar is 100 µm. (C,H) Whole eye images of E19.5 mice. (D,E,I,J) Transmission electron microscope images of E15.5 eyes. Dashed lines mark the apical and basal membranes of the cells; arrowheads indicate melanosomes. Scale bar is 2 µm. (K-N) RPE flat-mount views of E19.5 eyes (K,M) using bright field or (L,N) stained for actin. Scale bar is 100 µm. (O) Relative transcript levels of <i>connexin-43</i> (a gap junction marker), <i>P-cadherin</i> (an adherens junction marker) and <i>ZO-1</i> (a tight junction marker) from control and <i>Pax6</i>-deficient E15.5 RPE fractions determined using QRT-PCR (n = 6). Abbreviations: CB, ciliary body; CC, choriocapilaris; N, nucleus; PR, photoreceptors.</p