Abstract
Purpose: :
Aquaporin 0 (AQP0, previously MIP) is the primary water channel expressed in lens fiber cells. Mammalian AQP0 functions not only as a water channel but as an adhesion molecule and forms junctions between adjacent fiber cells. AQP0 interacts with the intermediate filament protein filensin, implying it could act as a cytoskeletal anchor. Two isoforms of AQP0, Aqp0a and Aqp0b, are present in zebrafish (Danio rerio). In the Xenopus laevis oocyte and Saccharomyces cerevisiae expression systems Aqp0a functions as a water channel, but Aqp0b does not indicating that a subfunctionalization event has occurred. The purpose of this study is to evaluate the distribution of functions of AQP0 between the two zebrafish isoforms and to determine the role of AQP0 permeability regulation in vivo.
Methods: :
We depleted Aqp0a, Aqp0b, or both, by microinjection of antisense morpholino oligos (MOs) into zebrafish embryos. We tested the ability of DNA constructs encoding the Aqp0a/b genes to rescue the MO-depleted lens defects. By injecting rescue constructs encoding mutant forms of Aqp0a/b or exogenous AQP genes, including mutant varieties of the Fundulus heteroclitus AQP0 (MIPfun), we evaluated the functions of specific residues in AQP0 in the development of the zebrafish lens.
Results: :
MO knockdown of either Aqp0a or Aqp0b caused cataract in the zebrafish lens by 48-72 hours post-fertilization. We obtained statistically significant rescues of each MO knockdown with injection of the corresponding DNA construct. The Aqp0b rescue construct did not rescue the cataracts caused by the Aqp0a-MO and vice-versa. Wild type MIPfun rescued embryos injected with either Aqp0a-MO or Aqp0b-MO, but some mutant variants of MIPfun could rescue only one type of MO knockdown.
Conclusions: :
Zebrafish Aqp0a and Aqp0b are both essential for normal lens development and transparency at very early stages of embryonic lens formation. We conclude that AQP0 has at least two functions required for the normal development of the zebrafish lens, and that these functions have been partitioned into a pair of duplicated AQP0 genes in zebrafish during evolution. This subfunctionalization and our ability to rescue both knock-downs by expression of MIPfun facilitates analyses of the various functions of AQP0 and highlight the advantages of the zebrafish for studies of lens development and physiology.
Keywords: intraocular lens • cataract • transgenics/knock-outs