Two Distinct Aquaporin 0s Required for Development and Transparency of the Zebrafish Lens
Two Distinct Aquaporin 0s Required for Development and Transparency of the Zebrafish Lens
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DOI：
10.1167/iovs.10-5626
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发表时间：
2010-12-01
影响因子：
4.4
通讯作者：
Hall, James E.
中科院分区：
文献类型：
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作者：
Froger, Alexandrine;Clemens, Daniel;Hall, James E.
目的. AQP0，以前称为MIP 26，可能在哺乳动物透镜中具有多个独立的功能，包括水运输、薄连接的形成以及与其他透镜组分的相互作用。虽然哺乳动物基因组只含有一个Aqp0基因，但斑马鱼基因组含有两个，Aqp0a和Aqp0b，并且单一哺乳动物蛋白质的推定的多种功能可以在这两个基因之间划分。本研究的目的是利用这种基因的重复和分歧，阐明水通道蛋白0在晶状体中的多种功能。采用全细胞原位杂交和Western blot分析Aqp0a和Aqp0b的表达模式。这两种蛋白质的作用进行了研究，在体内通过显微注射的反义吗啉代寡核苷酸在斑马鱼。两种蛋白质的水渗透性使用爪蟾卵母细胞肿胀测定和酵母收缩测定进行了测试。这两个基因，就像它们在哺乳动物中的对应物一样，都在透镜中表达。单独敲除任一基因都会导致白内障的形成，这表明这两个基因对于正常的透镜发育和透明度都是必需的。当在非洲爪蟾卵母细胞和酵母中表达时，全长Aqp0a是功能性水通道，而Aqp0b不是。然而，在其N末端添加HA标签将Aqp0b转化为非洲爪蟾卵母细胞中的水通道。这些结果表明，Aqp 0 a是透镜的主要水通道，而Aqp 0 b虽然可能是次要水通道，但在透镜中具有未识别的功能。（Invest Ophthalmol维斯科学。2010; 51：6582 - 6592）DOI：10.1167/iovs.10 - 5626
PURPOSE. AQP0, formerly known as MIP26, likely has multiple separate functions in the mammalian lens, including water transport, formation of thin junctions, and interactions with other lens components. Although mammalian genomes contain only one Aqp0 gene, the zebrafish genome contains two, Aqp0a and Aqp0b, and the putative multiple functions of the single mammalian protein may be divided between these two genes. The purpose of this study was to exploit this gene duplication and divergence to illuminate the multiple functions of AQP0 in the lens.METHODS. Wholemount in situ hybridization and Western blot analyses were used to determine the expression pattern of Aqp0a and Aqp0b. The role of both proteins was studied in vivo by microinjection of antisense morpholino oligonucleotides in zebrafish. The water permeability of both proteins was tested using the Xenopus oocyte swelling assay and a yeast shrinkage assay.RESULTS. Both genes, like their mammalian counterpart, are expressed in the lens. Morpholino knock-down of either gene alone led to cataract formation, indicating that both genes are necessary for normal lens development and transparency. Full-length Aqp0a is a functional water channel when expressed in Xenopus oocytes and in yeast, whereas Aqp0b was not. However, the addition of an HA-tag at its N terminus converted Aqp0b to a water channel in Xenopus oocytes.CONCLUSIONS. These results suggest that Aqp0a is the primary water channel of the lens and that Aqp0b, though possibly a secondary water channel, has an unidentified function in the lens. (Invest Ophthalmol Vis Sci. 2010; 51: 6582-6592) DOI:10.1167/iovs.10-5626