Down-regulation of the αA, αB, β and γ-crystallins caused by the <i>miak</i> mutation of <i>Pitx3</i>.

<p>Confocal images show the double-labeled crystallin proteins (red) and DAPI (blue) in the lenses of wild-type and <i>miak</i>/<i>miak</i> mice at E12.5. Scale bar <i> = </i>100 µm. <b>A.</b> The αA-crystallins labeling of the lens. The expression patterns of αA-crystallins were similar in the lens epithelium (le) and lens fiber (lf) of the wild-type and <i>miak</i> mice; however, the αA-crystallins signals may be slightly reduced in the <i>miak</i> mice. <b>B.</b> The αB-crystallins labeling of the lens. The αB-crystallin signals were not detected in the <i>miak</i>/<i>miak</i>. <b>C.</b> The β-crystallin labeling of the lens. The β-crystallin signals were barely detected in the <i>miak</i>/<i>miak</i> lens. <b>D.</b> The γ-crystallin labeling of the lens. Immunohistochemistry reveals the dramatically reduced γ-crystallin signals in the <i>miak</i> mice as well as ectopic expression in the anterior region of the lens.</p

Expression analyses of <i>Pitx3</i> transcripts and PITX3 protein in wild-type and <i>miak</i> mice.

<p><b>A.</b> Whole-mount <i>in situ</i> hybridization analysis in <i>miak</i>/+ and <i>miak</i>/<i>miak</i> in E11.5 embryos. The bottom panels show a magnified image of the eyes compared to the top panels. The arrows indicate the expression of <i>Pitx3</i>, detected as blue signals. Scale bar  = 500 µm. <b>B.</b> The relative levels of <i>Pitx3</i> mRNA in the eyes of wild-type (+/+) and <i>miak</i>/<i>miak</i> mice at E11.5, E12.5 and E14.5. <i>Pitx3</i> mRNA expression was measured by real-time RT-PCR analysis using the Mm_Pitx3_1 primer set (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432.s004" target="_blank">Table S1</a>). The values shown in the graph indicate the mean relative expression levels and the SDs of triplicate eye mRNAs. The expression levels in wild-type mice at E11.5 were assigned an arbitrary value of 1 for comparative purposes. ns, no significant differences; *<i>P<</i>0.05; ***<i>P<</i>0.001. <b>C.</b> Over-expression of PITX3 protein during lens development in <i>miak</i> mice. Confocal images show the lenses double-labeled with PITX3 antibody (red) and DAPI (blue) in wild-type and <i>miak</i>/<i>miak</i> mice at E10.5, E11.5 and E12.5. Strong PITX3 signals were observed in the <i>miak</i>/<i>miak</i> mice at E11.5 and E12.5 compared to the wild-type mice. Scale bar <i> = </i>100 µm.</p

mRNA reduction of the downstream targets of PITX3 in <i>miak</i> mice at embryonic stages.

<p>The relative levels of <i>Prox1</i>, <i>Foxe3</i>, and <i>Mip</i> mRNA in the eye of wild-type (+/+) and <i>miak</i>/<i>miak</i> mice at E11.5, E12.5 and E14.5. The mRNA expression levels were measured by real-time RT-PCR analysis using specific primer sets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432.s004" target="_blank">Table S1</a>) for each gene. The values shown in each graph indicate the mean relative expression levels and the SDs of triplicate eye mRNAs. The expression levels in wild-type mice at E11.5 were assigned an arbitrary value of 1 for comparative purposes. **<i>P<</i>0.01; ***<i>P<</i>0.001.</p

Evaluation of the interaction between <i>Foxe3-</i> (A) and <i>Mip-</i> (B) <i>bicoid</i> sites and proteins in wild-type and –<i>miak</i> mice by electrophoretic mobility assay (EMSA).

<p>EMSA performed with <i>Foxe3</i>- <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432-Ahmad1" target="_blank">[16]</a> and <i>Mip-</i> <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432-Sorokina1" target="_blank">[21]</a> <i>bicoid</i> oligonucleotides (oligo probe) and nuclear extracts (NE) from wild-type and <i>miak</i> eyes at E17.5. Although the formation of the specific EMSA complex occurred by combining oligo probes and NEs from wild-type and <i>miak</i> mice, the binding ability was increased with <i>miak</i>-NE and both <i>Foxe3</i>- and <i>Mip-bicoid</i> oligo probes. The binding ability of both oligo probes was inhibited by 10-fold excess unlabeled competitive probes (competitor).</p

Localization and expression of the PROX1 (A) and FOXE3 (B) proteins during lens development in wild-type and <i>miak</i> mice at embryonic stages.

<p>Confocal images show the lenses double-labeled for protein (red) and DAPI (blue) in the wild-type (+/+) and <i>miak</i>/<i>miak</i> mice. Scale bar <i> = </i>100 µm. <b>A.</b> PROX1 labeling of the lens at E11.5 and E12.5. The delay and slight reduction of PROX1 signals was observed in <i>miak</i>/<i>miak</i> mice at both stages. <b>B.</b> FOXE3 labeling of the lens at E11.5 and E12.5. Immunohistochemistry reveals the dramatically reduced signals of FOXE3 in <i>miak</i>/<i>miak</i> mice.</p

Positional cloning of the <i>miak</i> mutation.

<p><b>A.</b> Genetic maps obtained by genotyping and phenotyping of the progeny from the intercross between (C57BL/6J-<i>miak</i>/<i>miak</i> congenic mice × C57BL/6J) F₂. The blue markers <i>D19Mit 112</i> and <i>D19Mit74</i> define the non-recombinant interval containing <i>miak</i> mutation and <i>Pitx3</i> that is responsible gene for the mouse <i>Pitx3^ak</i> and <i>Pitx3^eyl</i> mutation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432-Semina2" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432-Rosemann1" target="_blank">[9]</a>. Distances on chromosome 19 are according to the mouse mm 10 (Genome Reference Consortium GRCm38) genomic sequence. <b>B.</b> Mutation analysis of <i>Pitx3</i> in the <i>miak</i> mouse. <i>miak</i> mice have a c.444C>A nonsense mutation in <i>Pitx3</i>. <b>C.</b> Schematic diagram of the domain structure of the PITX3 protein in the +/+ and <i>miak</i> mice. The domain structures were predicted by the SMART program, and the numbering of the amino acids (aa) is according to the PITX3 aa sequence of the wild-type and <i>miak</i> mice (NP_032878 and AB971349). PITX3 possesses homeodomain (HD, black box) and otp, aristaless, and rax (OAR, light gray box) as major functional domains near the N- and C-termini, respectively. The nonsense mutation in the <i>miak</i> mutants cause truncations of the PITX3 protein that result in a missing C-terminal OAR domain. <b>D.</b> The <i>miak</i> mutation disrupts a <i>Sma</i>I restriction site (CCCGGG) in <i>Pitx3</i>. The digestion of amplicons from wild-type mice produces bands at 136 and 132 bp. However, <i>miak/miak</i> mice are homozygous for the disruption of the <i>Sma</i>I site and yield only a single 268 bp band, whereas the <i>miak</i>/+ mice are heterozygous for the mutation as shown by the two banding patterns superimposed on one another. The top and bottom panels show the RFLP patterns of (C57BL/6J-<i>miak</i>/<i>miak</i> congenic mice × C57BL/6J) F₂ progeny and wild-type inbred strains, respectively. M, marker (100 bp ladder); N, negative control (DDW); CIS, common inbred strain; <i>Dom</i>, <i>domesticus</i>; <i>Mol</i>, <i>molossinus</i>; <i>Mus</i>, <i>musculus</i>; <i>Cas</i>, <i>castaneus</i>.</p

mRNA reduction of the crystallins in <i>miak</i> mice at embryonic stages.

<p>The relative levels of <i>Cryaa</i>, <i>Cryab</i>, <i>Cryb1</i> and <i>Cryga</i> mRNA in the eye of wild-type (+/+) and <i>miak</i>/<i>miak</i> mice at E11.5, E12.5 and E14.5. The mRNA expression levels were measured by real-time RT-PCR analysis using specific primer sets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432.s004" target="_blank">Table S1</a>) for each gene. The values shown in each graph indicate the mean relative expression levels and the SDs of triplicate eye mRNAs. The expression levels in wild-type mice at E11.5 were assigned an arbitrary value of 1 for comparative purposes. *<i>P<</i>0.05; **<i>P<</i>0.01; ***<i>P<</i>0.001.</p