Roderick Nigel Finn

Adaptive evolution associated with dispensable gene loss is widespread in the animal kingdom. However, loss of genes essential for embryonic survival and the mechanisms that evolved to bypass negative selection and extinction are less well known. Here, we show that all Gadiformes, an ecologically dominant order of codfishes, lost the aqp1ab -type genes that are essential for the formation of hydrated pelagic eggs and embryonic survival of marine teleosts in oceanic environments. Despite such gene loss, codfishes produce some of the most highly hydrated pelagic eggs of all marine teleosts. To resolve this apparent paradox, we initially confirmed that the mechanisms of osmotic drive are conserved with other pelagic-egg-producing marine teleosts, and then conducted comparative transcript analyses of aquaporin water channels in distantly-related freshwater and marine teleosts. This uncovered Aqp4-type channels that have been chromosomally linked to the Aqp1-type channels in jawed vertebrate genomes for >450 million years as the primary candidates. Using immunolocalization, functional assays, and inhibition experiments with zebrafish and Atlantic cod as model organisms, we show that short isoforms of Aqp4 are required for egg ovulation in more ancestral lineages of freshwater teleosts and were recruited to the oocyte hydration pathway of codfishes. By contrast, the oldest and most modern lineages of marine teleosts producing pelagic eggs still utilize the aqp1ab genes for egg hydration. Our data thus reveal that codfishes survived essential gene loss by functionally co-opting freshwater ovulatory pathways to marine egg hydration.

In marine teleosts, the neuroendocrine regulation of aquaporin intracellular trafficking in osmoregulatory organs such as the intestinal rectum remains underexplored. By using paralog-specific antibodies, here we determined the subcellular localization of seven aquaporin and aquaglyceroporin paralogs in the rectal enterocytes of the marine teleost gilthead seabream (Sparus aurata) and investigated the neuropeptinergic effects of arginine vasopressin (Avp) and oxytocin (Oxt) on channel trafficking. The data reveal that Aqp1aa, -1ab2 and -3a are mainly localized in the cytoplasm and basolateral membranes of the enterocytes, while Aqp1ab1, -10bb and -14 are cytoplasmatic and also present in the apical plasma membrane microvilli. In contrast, Aqp0a localizes in the enterocyte subapical cytoplasm. Treatment of rectal explants with Avp in vitro increases the targeting of Aqp1ab1, -1ab2, -10bb and -14, but not of Aqp0a, -1aa and -3a, to the enterocyte apical microvilli, which involves protein kinase A (PKA)-mediated phosphorylation of the channel subdomains. In addition, we find that the differential interaction of the Aqp1ab-type paralogs with teleost-specific 14-3-3ζ-like binding proteins is associated with the trafficking of the channels to the apical microvilli membranes. Conversely, Oxt treatment does not affect the subcellular distribution of any of the investigated aquaporins, except Aqp14, which is internalized in response to the neuropeptide by a partially independent protein kinase C pathway. These data suggest that Avp-PKA signaling controls plasma membrane trafficking of several seabream rectal aquaporins, possibly to facilitate water and solute fluxes across the enterocyte membrane.

The intracellular unorthodox aquaporins, AQP11 and AQP12, remain the least characterized members of the aquaporin superfamily. Mammalian and teleost AQP12 channels have recently been established as polytransporters in native membranes. They are localized intracellularly in pancreatic acinar cells and considered to play roles in zymogen granule maturation and exocytosis. In contrast with the vertebrate channels, the permeability properties and physiological roles of invertebrate AQP12-like (Aqp12L) orthologs are completely unknown. Here, we report that the Aqp12L channel from the extremophile crustacean Artemia franciscana is specifically targeted to the membrane of the yolk platelets (YPs) when heterologously expressed in frog (Xenopus laevis) oocytes, as recently reported for the vertebrate orthologs. Using YPs isolated from A. franciscana Aqp12L-expressing frog oocytes, we further show that this channel can transport water, H2O2, ammonia, and urea but not glycerol. Immunostaining of the A. franciscana ovary using a specific Aqp12L antibody revealed that the in vivo localization of the channel also occurs in the YP membranes of vitellogenic oocytes. RNAi-mediated silencing of Aqp12L substantially reduces the offspring output of both ovoviviparous and oviparous females through the impairment of oocyte vitellogenesis (oocyte growth) and the premature activation of ovarian apoptotic and atretic pathways. These findings provide the first direct evidence demonstrating that invertebrate Aqp12L is a functional polytransporter with an essential role in crustacean oogenesis.

Unorthodox AQP12-type channels are poorly understood intracellular aquaporins localized in the endoplasmic reticulum and zymogen granules (ZGs) of pancreatic acinar cells. Despite connections to major diseases, their biophysical properties and intracellular trafficking regulation remain largely unknown. Here, we show that heterologously expressed plant and metazoan AQP12-related channels specifically localize to the intracellular yolk platelet (YP) membrane of frog oocytes, a feature that is recapitulated in vivo for invertebrate and vertebrate orthologs. Using native YP membranes, we show that the vertebrate channels are mercury-sensitive polytransporters with intracellular trafficking regulated by Ca2+ and cAMP signaling pathways. We identify a novel pan-vertebrate C-terminal YP-targeting domain (YPD) in AQP12, which also drives orthodox aquaporin chimeras and truncated channels to YPs and ZGs. In cultured pancreatic cells, the YPD and Ca2+-induced AQP12 N-terminal phosphorylation coregulate secretagogue-triggered channel transport to the ZGs for enzyme secretion. These findings uncover conserved signaling mechanisms for AQP12 trafficking to intracellular protein storage vesicles, and open unexpected avenues for targeted delivery systems.

AQP8-type water channels are expressed superficially in the plasma membrane or intracellularly in the inner mitochondrial membrane, where they respectively function in osmohomeostasis or as peroxiporins to alleviate oxidative stress. To date only single-copy AQP8 or AQP16 genes are known in tetrapods and two binary gene clusters composed of aqp8aa-aqp8ab and aqp8ba-aqp8bb in teleost fishes. Here, using phylogenomic and synteny analyses, we revise this view and show that bony fish aqp8aa, -ab, -ba and -bb genes are non-canonical co-orthologs that independently arose at chromosomal breakpoints. Conversely, canonical orthologs of tetrapod AQP8 are now detected in all vertebrate classes except hagfishes. In cartilaginous fishes, intact aqp8 orthologs and linked pseudogenes exist in squalomorph sharks and only fractionated aqp8-like pseudogenes in galeomorph sharks. Some isolated aqp8-like exons are detected in batoid ray genomes, while no aqp8-type coding sequences are currently found in holocephalan genomes. In the actinopterygian (ray-finned fish) lineage, the canonical ortholog of tetrapod AQP8 is estimated to have undergone gene translocation in their common ancestor ~400 million years ago but was subsequently inactivated or lost in many descendant lineages. In close temporal proximity to this gene translocation event, the actinopterygian aqp8aa-aqp8ab binary gene cluster was generated in the original syntenic locus, potentially as a result of meiotic recombination. Our data support a model of total chromosomal replication for the generation of AQP16 genes and the teleost aqp8ba-aqp8bb gene cluster. We further uncover additional duplicates in Strepsirrhini primates that provide an eminent example of the stochastic nature of neofunctionalization. The present data thus suggest that that common ancestral genome duplications combined with lineage-level chromosomal translocation, recombination and replication events contributed to the diversification of vertebrate AQP8-type genes.

AQP8-type water channels are expressed superficially in the plasma membrane or intracellularly in the inner mitochondrial membrane where they respectively function in osmohomeostasis or as peroxiporins to alleviate oxidative stress. To date only single-copy AQP8 or AQP16 genes are known in tetrapods and two binary gene clusters composed of aqp8aa-aqp8ab and aqp8ba-aqp8bb in teleost fishes. Here using phylogenomic and synteny analyses we revise this view and show that bony fish aqp8aa, -ab, -ba and -bb genes are non-canonical co-orthologs that independently arose at chromosomal breakpoints. Conversely, canonical orthologs of tetrapod AQP8 are now detected in all vertebrate classes except hagfishes. In cartilaginous fishes, intact aqp8 orthologs and linked pseudogenes exist in squalomorph sharks, but only fractionated aqp8-like pseudogenes in galeomorph sharks. Some isolated aqp8-like exons are detected batoid ray genomes, while no aqp8-type coding sequences are currently found in holocephalan genomes. In ray-finned fishes, the canonical ortholog of tetrapod AQP8 underwent gene translocation in their common ancestor ~400 million years ago, but was subsequently inactivated or lost in many descendant lineages. In close temporal proximity to this gene translocation event, the actinopterygian aqp8aa-aqp8ab binary gene cluster was generated in the original syntenic locus potentially as a result of meiotic recombination. Our data support a model of total chromosmal replication for the generation of tetropod AQP16 genes and the teleost aqp8ba-aqp8bb gene cluster. We further uncover additional duplicates in Strepsirrhini primates that provide an eminent example of the stochastic nature of neofunctionalization. The present data thus suggest that chromosomal translocation, recombination and replication events contributed to the diversification of vertebrate AQP8-type genes.

Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca²⁺ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon.

The term peroxiporin has been created to define a transmembrane water channel protein (aquaporin) that also conducts hydrogen peroxide (H₂O₂). Such channels facilitate cell signaling and redox balance. Recent studies have shown that multiple differentially localized aquaporins play important roles in the activation, detoxification, maintenance of motility and swimming performance of piscine spermatozoa. For the first time, the peroxiporin function of one of these channels has been identified as a critical player in the mitochondrial elimination of H₂O₂, with important implications for sperm competition in fishes. This chapter provides an up-to-date overview of the phylogenetic interrelationships of the gnathostome aquaporin superfamily and the duplicates in teleosts, together with information on which of these channels are expressed in spermatozoa and which retain the biophysical property of H₂O₂ conductance. We discuss the site-specific functions of the aquaporins for piscine sperm cell physiology and summarize how the signaling pathways governing the intracellular trafficking and function of mitochondrial peroxiporins evolved in their spermatozoa.

Aquaporin-mediated oocyte hydration is considered important for the evolution of pelagic eggs and the radiative success of marine teleosts. However, the molecular regulatory mechanisms controlling this vital process are not fully understood. Here, we analyzed >400 piscine genomes to uncover a previously unknown teleost-specific aquaporin-1 cluster (TSA1C) comprised of tandemly arranged aqp1aa-aqp1ab2-aqp1ab1 genes. Functional evolutionary analysis of the TSA1C reveals a ∼300-million-year history of downstream aqp1ab-type gene loss, neofunctionalization, and subfunctionalization, but with marine species that spawn highly hydrated pelagic eggs almost exclusively retaining at least one of the downstream paralogs. Unexpectedly, one-third of the modern marine euacanthomorph teleosts selectively retain both aqp1ab-type channels and co-evolved protein kinase-mediated phosphorylation sites in the intracellular subdomains together with teleost-specific Ywhaz-like (14-3-3ζ-like) binding proteins for co-operative membrane trafficking regulation. To understand the selective evolutionary advantages of these mechanisms, we show that a two-step regulated channel shunt avoids competitive occupancy of the same plasma membrane space in the oocyte and accelerates hydration. These data suggest that the evolution of the adaptive molecular regulatory features of the TSA1C facilitated the rise of pelagic eggs and their subsequent geodispersal in the oceanic currents.