Background: Aquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown. Results: The screening and isolation of transcripts from the zebrafish (Danio rerio) genome revealed eighteen sequences structurally related to the four subfamilies of tetrapod aquaporins, i.e., aquaporins (AQP0, -1 and -4), water and glycerol transporters or aquaglyceroporins (Glps; AQP3 and AQP7-10), a water and urea transporter (AQP8), and two unorthodox aquaporins (AQP11 and -12). Phylogenetic analyses of nucleotide and deduced amino acid sequences demonstrated dual paralogy between teleost and human aquaporins. Three of the duplicated zebrafish isoforms have unlinked loci, two have linked loci, while DrAqp8 was found in triplicate across two chromosomes. Genomic sequencing, structural analysis, and maximum likelihood reconstruction, further revealed the presence of a putative pseudogene that displays hybrid exons similar to tetrapod AQP5 and -1. Ectopic expression of the cloned transcripts in Xenopus laevis oocytes demonstrated that zebrafish aquaporins and Glps transport water or water, glycerol and urea, respectively, whereas DrAqp11b and -12 were not functional in oocytes. Contrary to humans and some rodents, intrachromosomal duplicates of zebrafish AQP8 were water and urea permeable, while the genomic duplicate only transported water. All aquaporin transcripts were expressed in adult tissues and found to have divergent expression patterns. In some tissues, however, redundant expression of transcripts encoding two duplicated paralogs seems to occur. Conclusion: The zebrafish genome encodes the largest repertoire of functional vertebrate aquaporins with dual paralogy to human isoforms. Our data reveal an early and specific diversification of these integral membrane proteins at the root of the crown-clade of Teleostei. Despite the increase in gene copy number, zebrafish aquaporins mostly retain the substrate specificity characteristic of the tetrapod counterparts. Based upon the integration of phylogenetic, genomic and functional data we propose a new classification for the piscine aquaporin superfamily.

In this review, we provide a brief synopsis of the evolution and functional diversity of the aquaporin gene superfamily in prokaryotic and eukaryotic organisms. Based upon the latest data, we discuss the expanding list of molecules shown to permeate the central pore of aquaporins, and the unexpected diversity of water channel genes in Archaea and Bacteria. We further provide new insight into the origin by horizontal gene transfer of plant glycerol-transporting aquaporins (NIPs), and the functional co-option and gene replacement of insect glycerol transporters. Finally, we discuss the origins of four major grades of aquaporins in Eukaryota, together with the increasing repertoires of aquaporins in vertebrates.

A major physiological barrier for aquatic organisms adapting to terrestrial life is dessication in the aerial environment. This barrier was nevertheless overcome by the Devonian ancestors of extant Tetrapoda, but the origin of specific molecular mechanisms that solved this water problem remains largely unknown. Here we show that an ancient aquaporin gene cluster evolved specifically in the sarcopterygian lineage, and subsequently diverged into paralogous forms of AQP2, -5, or -6 to mediate water conservation in extant Tetrapoda. To determine the origin of these apomorphic genomic traits, we combined aquaporin sequencing from jawless and jawed vertebrates with broad taxon assembly of >2,000 transcripts amongst 131 deuterostome genomes and developed a model based upon Bayesian inference that traces their convergent roots to stem subfamilies in basal Metazoa and Prokaryota. This approach uncovered an unexpected diversity of aquaporins in every lineage investigated, and revealed that the vertebrate superfamily consists of 17 classes of aquaporins (Aqp0 - Aqp16). The oldest orthologs associated with water conservation in modern Tetrapoda are traced to a cluster of three aqp2-like genes in Actinistia that likely arose >500 Ma through duplication of an aqp0-like gene present in a jawless ancestor. In sea lamprey, we show that aqp0 first arose in a protocluster comprised of a novel aqp14 paralog and a fused aqp01 gene. To corroborate these findings, we conducted phylogenetic analyses of five syntenic nuclear receptor subfamilies, which, together with observations of extensive genome rearrangements, support the coincident loss of ancestral aqp2-like orthologs in Actinopterygii. We thus conclude that the divergence of sarcopterygian-specific aquaporin gene clusters was permissive for the evolution of water conservation mechanisms that facilitated tetrapod terrestrial adaptation.

The positive buoyancy of marine fish eggs in sea water, allowed by hydration of the oocyte, is critical for their survival and dispersion in the ocean. We isolated an aquaporin, SaAQP1o, that belongs to a unique subfamily of aquaporin-1—like channels specifically evolved in teleosts and mainly expressed in the ovary. We further show that hormone-induced fish oocyte hydration is a highly controlled process based on the interplay between protein hydrolysis and the translocation of SaAQP1o to the plasma membrane, indicating a specialized physiological role for this aquaporin.