Wada Kenta, Matsushima Yoshibumi, Tada Tomoki, Hasegawa Sayaka, Obara Yo, Yoshizawa Yasuhiro, Takahashi Gou, Hiai Hiroshi, Shimanuki Midori, Suzuki Sari, Saitou Junichi, Yamamoto Naoki, Ichikawa Masumi, Watanabe Kei, Kikkawa Yoshiaki
Department of Bioproduction, Tokyo University of Agriculture, Abashiri, Japan; Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama, Japan.
PLoS One. 2014 Oct 27;9(10):e111432. doi: 10.1371/journal.pone.0111432. eCollection 2014.
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.
小眼症是一种严重的眼部疾病，这种情况通常是由参与眼睛发育的转录因子突变引起的。携带这些转录因子突变的小鼠将成为确定发育性眼病潜在机制的有用工具。我们在日本野生来源的近交系KOR1/Stm中发现了一种新的自发性隐性小眼症小鼠突变体。纯合突变小鼠在组织学上的特征是晶状体中缺乏晶状体蛋白，这种情况称为无晶状体。通过定位克隆，我们确定了配对样同源结构域转录因子3基因（Pitx3）编码同源结构域的基因组区域外的无义突变c.444C>A是导致小眼症和无晶状体的突变。我们使用RT-PCR检测了突变小鼠胚胎期的Pitx3 mRNA表达，发现其表达水平高于野生型小鼠。通过免疫组织化学分析在小眼症突变体的蛋白质水平上也证实了发育阶段晶状体中Pitx3的过表达。虽然在突变体中未观察到晶状体纤维分化，但在突变晶状体的晶状体泡中观察到强烈的PITX3蛋白信号。因此，我们推测由于无义突变而缺乏C末端（包括OAR结构域）的异常PITX3在突变晶状体中表达。我们发现由于Pitx3突变，下游基因Foxe3、Prox1和Mip的表达发生了改变，突变体的晶状体泡中大幅减少。对这些蛋白质的免疫组织化学分析也观察到了类似的情况。突变体中晶状体蛋白的表达谱也发生了改变。因此，我们推测该突变体中的小眼症/无晶状体是由截短的PITX3表达引起的，导致下游靶点和晶状体纤维蛋白的异常表达。
