Two Distinct Aquaporin 0s Required for Development and Transparency of the Zebrafish Lens

Abstract

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.