Expression of truncated PITX3 in the developing lens leads to microphthalmia and aphakia in mice.

Microphthalmia is a severe ocular disorder, and this condition is typically caused by mutations in transcription factors that are involved in eye development. Mice carrying mutations in these transcription factors would be useful tools for defining the mechanisms underlying developmental eye disorders. We discovered a new spontaneous recessive microphthalmos mouse mutant in the Japanese wild-derived inbred strain KOR1/Stm. The homozygous mutant mice were histologically characterized as microphthalmic by the absence of crystallin in the lens, a condition referred to as aphakia. By positional cloning, we identified the nonsense mutation c.444C>A outside the genomic region that encodes the homeodomain of the paired-like homeodomain transcription factor 3 gene (Pitx3) as the mutation responsible for the microphthalmia and aphakia. We examined Pitx3 mRNA expression of mutant mice during embryonic stages using RT-PCR and found that the expression levels are higher than in wild-type mice. Pitx3 over-expression in the lens during developmental stages was also confirmed at the protein level in the microphthalmos mutants via immunohistochemical analyses. Although lens fiber differentiation was not observed in the mutants, strong PITX3 protein signals were observed in the lens vesicles of the mutant lens. Thus, we speculated that abnormal PITX3, which lacks the C-terminus (including the OAR domain) as a result of the nonsense mutation, is expressed in mutant lenses. We showed that the expression of the downstream genes Foxe3, Prox1, and Mip was altered because of the Pitx3 mutation, with large reductions in the lens vesicles in the mutants. Similar profiles were observed by immunohistochemical analysis of these proteins. The expression profiles of crystallins were also altered in the mutants. Therefore, we speculated that the microphthalmos/aphakia in this mutant is caused by the expression of truncated PITX3, resulting in the abnormal expression of downstream targets and lens fiber proteins

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

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

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

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

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

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