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Daniel M. Clemens, Tailin Zhang, Katalin Kalman, Karin L. Németh-Cahalan, Thomas F. Schilling, James E. Hall; Assigning Functions To The Two Isoforms Of Aqp0 In Zebrafish With A Novel Knock-down/rescue System. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3412.
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© ARVO (1962-2015); The Authors (2016-present)
Aquaporin 0 (AQP0, previously MIP) is the primary water channel expressed in lens fiber cells. In addition to being a water channel, mammalian AQP0 functions as an adhesion molecule and participates in forming junctions between adjacent fiber cells. Functions beyond water permeability and adhesion have also been proposed for AQP0. Two isoforms of AQP0 are present in zebrafish (Danio rerio), Aqp0a and Aqp0b.In the Xenopus laevis oocyte and Saccharomyces cerevisiae expression systems, Aqp0a functions as a water channel but Aqp0b does not. The purpose of this study is to evaluate the importance of AQP0 water permeability in the development and transparency of the zebrafish lens and to explore the adhesive properties of zebrafish Aqp0a and Aqp0b.
We depleted Aqp0a, Aqp0b, or both, by microinjection of antisense morpholino oligos (MOs). We tested the ability of DNA constructs encoding the Aqp0a/b genes to rescue the MO-depleted lens defects by co-injection. By injecting rescue constructs encoding mutant forms of Aqp0a/b or misexpressing exogenous AQP genes, we evaluated the functions of specific residues in AQP0 in the development of the zebrafish lens. The adhesive properties of Aqp0a and Aqp0b are being evaluated using a mouse fibroblast cell adhesion assay.
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. Preliminary results suggested that injection of exogenous MIPfun, a killifish (Fundulus heteroclitus) AQP0 protein, rescued embryos injected with Aqp0a-MO. We are currently testing the ability of bovine AQP0 (possessing all necessary AQP0 functions) to rescue Aqp0a/b deficient zebrafish.
Zebrafish Aqp0a and Aqp0b are both essential for normal lens development and transparency at very early stages of embryonic lens formation. We conclude that at least two functions of AQP0 are 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 facilitates genetic analyses and highlights the advantages of the zebrafish for studies of lens development and potential causes and treatments of cataract.Grant: NIH EY05661
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