Division of Reproductive and Developmental Sciences, University of Edinburgh, Edinburgh, United Kingdom.
PLoS One. 2011;6(12):e28895. doi: 10.1371/journal.pone.0028895. Epub 2011 Dec 29.
Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6⁺/⁻ heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6⁺/⁻) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication.
METHODOLOGY/PRINCIPAL FINDINGS: We used electron microscopy to investigate ocular defects in Pax6⁺/⁻ heterozygotes (low Pax6 levels) and PAX77(Tg/-) transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZ(Tg/-)) mice to investigate corneal epithelial maintenance by LESC clones in Pax6⁺/⁻ and PAX77(Tg/-) mosaic mice. PAX77(Tg/-) mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6⁺/⁻ mosaics were corrected by introducing the PAX77 transgene (in Pax6⁺/⁻, PAX77(Tg/-) mosaics). Pax6(Leca4/+), XLacZ(Tg/-) mosaic mice (heterozygous for the Pax6(Leca4) missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZ(Tg/-) mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6⁺/⁻ and PAX77(Tg/-) mosaic corneas, suggesting Pax6 under- and over-expression both affect LESC clones.
CONCLUSIONS/SIGNIFICANCE: Pax6⁺/⁻ and PAX77(Tg/-) genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK.
转录因子 PAX6 的剂量改变会导致多种人类眼部病理生理学改变。PAX6⁺/⁻杂合子患有无虹膜症和无虹膜相关角膜病（ARK），这是一种角膜恶化，可能涉及到角膜缘上皮干细胞（LESC）的缺乏。杂合 Pax6(+/Sey-Neu)（Pax6⁺/⁻）小鼠重现了人类疾病，是 ARK 的良好模型。其他小鼠 Pax6 突变体和 PAX77(Tg/-)转基因也存在角膜病变，这些转基因过度表达 Pax6 并模拟人类 PAX6 重复。
方法/主要发现：我们使用电子显微镜研究 Pax6⁺/⁻杂合子（低 Pax6 水平）和 PAX77(Tg/-)转基因（高 Pax6 水平）的眼部缺陷。除了众所周知的上皮缺陷外，异常的 Pax6 剂量对两种基因型的角膜基质和内皮都有深远的影响，包括细胞空泡化，类似于人类黄斑角膜营养不良的报道。我们使用 X 连锁 LacZ 转基因在 X 染色体失活镶嵌雌性（XLacZ(Tg/-)）小鼠中的镶嵌表达，以研究 Pax6⁺/⁻和 PAX77(Tg/-)镶嵌小鼠中 LESC 克隆对角膜上皮的维持作用。过度表达 Pax6 的 PAX77(Tg/-)镶嵌体产生了正常的角膜上皮放射状条纹模式（尽管存在其他角膜缺陷），表明细胞向心性运动不受影响。Pax6⁺/⁻镶嵌体中中度紊乱的模式通过引入 PAX77 转基因（在 Pax6⁺/⁻，PAX77(Tg/-)镶嵌体中）得到纠正。Pax6(Leca4/+)，XLacZ(Tg/-)镶嵌小鼠（杂合 Pax6(Leca4)错义突变）显示出更严重的镶嵌模式紊乱。在野生型 XLacZ(Tg/-)镶嵌体中，随着年龄（15 至 30 周）的增长，校正后的角膜上皮条纹数量（活跃的 LESC 克隆数量的间接估计）减少。相比之下，在 Pax6⁺/⁻和 PAX77(Tg/-)镶嵌角膜中，校正后的条纹数量在 15 周时已经较低，这表明 Pax6 的过表达和低表达都会影响 LESC 克隆。
结论/意义：PAX6⁺/⁻和 PAX77(Tg/-)基因型对 LESC 克隆数量只有相对较小的影响，但会导致更严重的角膜内皮和基质缺陷。这应该促使进一步研究人类无虹膜症和 ARK 的病理生理学。
