Teleost Radiation Research Articles

Haemoglobin Gene Repertoire in Teleost and Cichlid Fishes Shaped by Gene Duplications and Genome Rearrangements.

Haemoglobin is a key molecule for oxygen transport in vertebrates. It exhibits remarkable gene diversity in teleost fishes, reflecting adaptation to various aquatic environments. In this study, we present the dynamic evolution of haemoglobin subunit genes based on a comparison of high-quality genome assemblies of 24 vertebrate species, including 17 teleosts (of which six are cichlids). Our findings indicate that teleost genomes contain a range of haemoglobin genes, from as few as five in fugu to as many as 43 in salmon, with the latter being the largest repertoire found in vertebrates. We find evidence that the teleost ancestor had at least four Hbα and three or four Hbβ subunit genes, and that the current gene diversity emerged during teleost radiation, driven primarily by (tandem) gene duplications, genome compaction, and rearrangement dynamics. We provide insights into the genomic organisation of haemoglobin clusters in different teleost species. We further show that the evolution of paralogous rhbdf1 genes flanking both teleost clusters (LA and MN) supports the hypothesis for the origin of the LA cluster by rearrangement within teleosts, rather than by the teleost specific whole-genome duplication. We specifically focus on cichlid fishes, where adaptation to low oxygen environment plays role in species diversification. Our analysis of six cichlid genomes, including Pungu maclareni from the Barombi Mbo crater lake, for which we sequenced a representative genome, reveals 18-32 copies of the Hb genes, and elevated rates of non-synonymous substitutions compared to other teleosts. Overall, this work facilitates a deeper understanding of how haemoglobin genes contribute to the adaptive potential of teleosts.

Early Cretaceous radiation of teleosts recorded by the otolith-based ichthyofauna from the Valanginian of Wąwał, central Poland

ABSTRACT We report a Valanginian (Early Cretaceous) otolith-based ichthyofauna from a section at Wąwał in central Poland. We describe one new genus (Palaeoargentina gen. nov.), six new otolith-based species (Pteralbula polonica sp. nov., Protalbula pentangularis sp. nov., Kokenichthys kuteki sp. nov., Protoelops gracilis sp. nov., Palaeoargentina plicata sp. nov., Archaeotolithus aptychoides sp. nov.) from Wąwał and compare them to species known from similar assemblages elsewhere. The comparison of teleost diversity shows similarity to the Aptian (late Early Cretaceous) and less distinctly to the Maastrichtian (latest Cretaceous) assemblages, rather than to its coeval equivalents from Germany and southern England, and indicates that a considerable teleost diversity already existed before the mid-Cretaceous. The vertical succession of otolith taxa in the Wąwał section is in concordance to the pattern already revealed from the succession of bivalves and other benthic invertebrates and it is attributed to sea level and temperature variations. Previously identified causes of benthic invertebrate succession in the Wąwał section are used to infer paleoenvironmental factors governing fish distribution in the Valanginian marine environment recorded at this site. The new findings suggest that the radiation of teleosts started before the Valanginian, and it was a relatively long and apparently gradual process. This fossil association also reveals a significant shift in the abundance ratio of fish otoliths vs. cephalopod statoliths in fully marine deposits, with otoliths much more abundant than the statoliths in Valanginian and younger sediments while it is otherwise in the Jurassic deposits.

Origin, form and function of extraembryonic structures in teleost fishes.

Teleost eggs have evolved a highly derived early developmental pattern within vertebrates as a result of the meroblastic cleavage pattern, giving rise to a polar stratified architecture containing a large acellular yolk and a small cellular blastoderm on top. Besides the acellular yolk, the teleost-specific yolk syncytial layer (YSL) and the superficial epithelial enveloping layer are recognized as extraembryonic structures that play critical roles throughout embryonic development. They provide enriched microenvironments in which molecular feedback loops, cellular interactions and mechanical signals emerge to sculpt, among other things, embryonic patterning along the dorsoventral and left-right axes, mesendodermal specification and the execution of morphogenetic movements in the early embryo and during organogenesis. An emerging concept points to a critical role of extraembryonic structures in reinforcing early genetic and morphogenetic programmes in reciprocal coordination with the embryonic blastoderm, providing the necessary boundary conditions for development to proceed. In addition, the role of the enveloping cell layer in providing mechanical, osmotic and immunological protection during early stages of development, and the autonomous nutritional support provided by the yolk and YSL, have probably been key aspects that have enabled the massive radiation of teleosts to colonize every ecological niche on the Earth. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Neuroendocrine Control of Reproduction in Teleost Fish: Concepts and Controversies.

During the teleost radiation, extensive development of the direct innervation mode of hypothalamo-pituitary communication was accompanied by loss of the median eminence typical of mammals. Cells secreting follicle-stimulating hormone and luteinizing hormone cells are directly innervated, distinct populations in the anterior pituitary. So far, ∼20 stimulatory and ∼10 inhibitory neuropeptides, 3 amines, and 3 amino acid neurotransmitters are implicated in the control of reproduction. Positive and negative sex steroid feedback loops operate in both sexes. Gene mutation models in zebrafish and medaka now challenge our general understanding of vertebrate neuropeptidergic control. New reproductive neuropeptides are emerging. These include but are not limited to nesfatin 1, neurokinin B, and the secretoneurins. A generalized model for the neuroendocrine control of reproduction is proposed. Hopefully, this will serve as a research framework on diverse species to help explain the evolution of neuroendocrine control and lead to the discovery of new hormones with novel applications.

Revision of the Mesozoic freshwater fish clade Archaeomaenidae

Archaeomaenidae is a clade of Mesozoic freshwater fishes that includes the Australian species Archaeomaene tenuis, Madariscus robustus and Wadeichthys oxyops, the Antarctic Oreochima ellioti, the Chinese Zaxilepis qinglongensis, and the Mongolian Gurvanichthys mongoliensis. A comprehensive redescription of the genus Archaeomaene demonstrates that Madariscus robustus is a junior synonym of Archaeomaene tenuis, and thus constitutes an ontogenetic body-size series. A detailed revision of W. oxyops and O. ellioti also confirms their phylogenetic placements within Archaeomaenidae. Furthermore, the incompletely documented taxa Z. qinglongensis and G. mongoliensis are recognized as archaeomaenids. Archaeomaenidae therefore comprises a geographically widely distributed radiation of stem teleosts or teleosteomorphs that is closely related to the archetypal Eurasian clade Pholidophoridae. Lynne Bean [Lynne.bean@anu.edu.au] Research School of Earth Sciences, Australian National University, 142 Mills Road, Acton, ACT 2601, Australia.

Phylogeny and expression modulation of interleukin 1 receptors in grass carp (Ctenopharyngodon idella)

The interleukin (IL) −1 family members play an important role in regulating inflammatory responses and their functions are mediated by a group of receptors consisting of immunoglobulin and Toll/IL-1 receptor (TIR) domains. In humans, 10 IL-1Rs are found. In this study, 5 IL-1 receptors including IL-1R3/IL-1RAcP, IL-1R8/SIGIRR, IL-1R9a/IL-1RAcPL1a, IL-1R9b/IL-1RAcPL1b and IL-1R10/IL-1RAcPL2 were identified in grass carp (Ctenopharyngodon idella). Phylogenetic analysis reveals that the IL-1R9a/IL-1RAcPL1a and IL-1R9b/IL-1RAcPL1b share significantly high sequence similarity and are believed to have been duplicated from the same gene prior to the radiation of teleosts. Further, these two receptors closely relate to the IL-1R10/IL-1RAcPL2, suggesting that they may have evolved from a common ancestor. The IL-1R3/IL-1RAcP, IL-1R9a/IL-1RAcPL1a, IL-1R9b/IL-1RAcPL1b and IL-1R10/IL-1RAcPL2 are highly expressed in the brain. Stimulation of primary spleen leucocytes by LPS and intraperitoneal injection of fish with poly (I:C) or bacterial infection results in significant increases of IL-1R3/IL-1RAcP expression. Interestingly, the IL-1R8/SIGIRR and IL-1R10/IL-1RAcPL2 showed similar expression patterns.

Genome-wide identification and transcriptome-based expression analysis of sox gene family in the Japanese flounder Paralichthys olivaceus

Sox genes are transcription factors that ubiquitously exist in animal species, and share a conserved high mobility group (HMG) box. They play important biological roles in diverse developmental processes, such as sex determination and differentiation, chondrogenesis, neurogenesis, and early embryonic development. In this study, we identified 25 sox genes from genome and transcriptome of Japanese flounder Paralichthys olivaceus. These sox genes could be mainly classified into seven subfamilies (B1, B2, C, D, E, F, and K), and each subfamily exhibited a relatively conserved gene structure. Besides, subfamilies A and G were found exclusively in human and mouse, and sox 32 in subfamily K only existed in teleosts. Compared with other mammals, some sox genes in teleosts had two duplicates. The loss, duplication, and divergence of sox genes during evolution provided an evidence for whole-genome duplication that occurred in the radiation of teleosts. The expression of Japanese flounder sox genes was also analyzed by FPKM value. Our results showed that certain sox genes exhibited obviously tissue-specific and spatio-temproal expression. Especially, gonal-basied expression analysis uncovered that sox7 and sox2 were ovary-biased, and sox8b was testis-biased. Moreover, sox10a was expressed specifically in ovary, and sox8a in testis. Therefore this study provide a solid foundation for future functional and evolutionary analysis of sox genes in Japanese flounder.

Teleost Fish-Specific Preferential Retention of Pigmentation Gene-Containing Families After Whole Genome Duplications in Vertebrates.

Vertebrate pigmentation is a highly diverse trait mainly determined by neural crest cell derivatives. It has been suggested that two rounds (1R/2R) of whole-genome duplications (WGDs) at the basis of vertebrates allowed changes in gene regulation associated with neural crest evolution. Subsequently, the teleost fish lineage experienced other WGDs, including the teleost-specific Ts3R before teleost radiation and the more recent Ss4R at the basis of salmonids. As the teleost lineage harbors the highest number of pigment cell types and pigmentation diversity in vertebrates, WGDs might have contributed to the evolution and diversification of the pigmentation gene repertoire in teleosts. We have compared the impact of the basal vertebrate 1R/2R duplications with that of the teleost-specific Ts3R and salmonid-specific Ss4R WGDs on 181 gene families containing genes involved in pigmentation. We show that pigmentation genes (PGs) have been globally more frequently retained as duplicates than other genes after Ts3R and Ss4R but not after the early 1R/2R. This is also true for non-pigmentary paralogs of PGs, suggesting that the function in pigmentation is not the sole key driver of gene retention after WGDs. On the long-term, specific categories of PGs have been repeatedly preferentially retained after ancient 1R/2R and Ts3R WGDs, possibly linked to the molecular nature of their proteins (e.g., DNA binding transcriptional regulators) and their central position in protein-protein interaction networks. Taken together, our results support a major role of WGDs in the diversification of the pigmentation gene repertoire in the teleost lineage, with a possible link with the diversity of pigment cell lineages observed in these animals compared to other vertebrates.

Neopterygian phylogeny: the merger assay.

The phylogenetic relationships of the recently described genus †Ticinolepis from the Middle Triassic of the Monte San Giorgio are explored through cladistic analyses of the so far largest morphological dataset for fossil actinopterygians, including representatives of the crown-neopterygian clades Halecomorphi, Ginglymodi and Teleostei, and merging the characters from previously published systematic studies together with newly proposed characters. †Ticinolepis is retrieved as the most basal Ginglymodi and our results support the monophyly of Teleostei and Holostei, as well as Halecomorphi and Ginglymodi within the latter clade. The patterns of relationships within these clades mostly agree with those of previous studies, although a few important differences require future research. According to our results, ionoscopiforms are not monophyletic, caturids are not amiiforms and leptolepids and luisiellids form a monophyletic clade. Our phylogenetic hypothesis confirms the rapid radiation of the holostean clades Halecomorphi and Ginglymodi during the Early and Middle Triassic and the radiation of pholidophoriform teleosts during the Late Triassic. Crown-group Halecomorphi have an enormous ghost lineage throughout half of the Mesozoic, but ginglymodians and teleosts show a second radiation during the Early Jurassic. The crown-groups of Halecomorphi, Ginglymodi and Teleostei originated within parallel events of radiation during the Late Jurassic.

Fatty acid-binding protein genes of the ancient, air-breathing, ray-finned fish, spotted gar (Lepisosteus oculatus)

With the advent of high-throughput DNA sequencing technology, the genomic sequence of many disparate species has led to the relatively new discipline of genomics, the study of genome structure, function and evolution. Much work has been focused on the role of whole genome duplications (WGD) in the architecture of extant vertebrate genomes, particularly those of teleost fishes which underwent a WGD early in the teleost radiation >230 million years ago (mya). Our past work has focused on the fate of duplicated copies of a multigene family coding for the intracellular lipid-binding protein (iLBP) genes in the teleost fishes. To define the evolutionary processes that determined the fate of duplicated genes and generated the structure of extant fish genomes, however, requires comparative genomic analysis with a fish lineage that diverged before the teleost WGD, such as the spotted gar (Lepisosteus oculatus), an ancient, air-breathing, ray-finned fish. Here, we describe the genomic organization, chromosomal location and tissue-specific expression of a subfamily of the iLBP genes that code for fatty acid-binding proteins (Fabps) in spotted gar. Based on this work, we have defined the minimum suite of fabp genes prior to their duplication in the teleost lineages ~230–400 mya. Spotted gar, therefore, serves as an appropriate outgroup, or ancestral/ancient fish, that did not undergo the teleost-specific WGD. As such, analyses of the spatio-temporal regulation of spotted gar genes provides a foundation to determine whether the duplicated fabp genes have been retained in teleost genomes owing to either sub- or neofunctionalization.

Analysis of the spotted gar genome suggests absence of causative link between ancestral genome duplication and transposable element diversification in teleost fish.

Teleost fish have been shown to contain many superfamilies of transposable elements (TEs) that are absent from most tetrapod genomes. Since theories predict an increase in TE activity following polyploidization, such diversity might be linked to the 3R whole-genome duplication that occurred approximately 300 million years ago before the teleost radiation. To test this hypothesis, we have analyzed the genome of the spotted gar Lepisosteus oculatus, which diverged from the teleost lineage before the 3R duplication. Our results indicate that TE diversity and copy numbers are similar in gar and teleost genomes, suggesting that TE diversity was ancestral and not linked to the 3R whole-genome duplication. We propose that about 25 distinct superfamilies of TEs were present in the last ancestor of gars and teleost fish about 300 million years ago in the ray-finned fish lineage.

A stem lepidosireniform lungfish (Sarcopterygia: Dipnoi) from the Upper Eocene of Libya, North Africa and implications for Cenozoic lungfish evolution

Modern lungfish are represented by three families, Neoceratodontidae in Australia, Protopteridae in Africa, and Lepidosirenidae in South America, with the latter two comprising the Lepidosireniformes. However, the group was far more diverse as recently as the Cretaceous. Here, a new lungfish, Xenoceratodus labyrinthus is described on the basis of upper and lower tooth-bearing elements from the Late Eocene of Dur at Talah, eastern Libya. Toothplates are characterized by four denticulations, low crests and ridges, and a well-developed crushing surface. The occlusal surface bears a unique ornament of enamel lines that branch to form a reticulate pattern across the crushing surface of the jaws. The new lungfish is closely related to the Paleocene Lavocatodus giganteus and the Late Cretaceous Lavocatodus? humei, which in turn are interpreted as a clade of stem Lepidosireniformes. Along with the presence of archaic lungfish lineages in the Cenozoic of South America and Australia, Xenoceratodus suggests that a wide range of lungfish lineages crossed the Cretaceous-Paleogene boundary, only to later become extinct; climate change and teleost radiation in the Cenozoic may have ultimately led to their decline.

Demonstration of the Coexistence of Duplicated LH Receptors in Teleosts, and Their Origin in Ancestral Actinopterygians.

Pituitary gonadotropins, FSH and LH, control gonad activity in vertebrates, via binding to their respective receptors, FSHR and LHR, members of GPCR superfamily. Until recently, it was accepted that gnathostomes possess a single FSHR and a single LHR, encoded by fshr and lhcgr genes. We reinvestigated this question, focusing on vertebrate species of key-phylogenetical positions. Genome analyses supported the presence of a single fshr and a single lhcgr in chondrichthyans, and in sarcopterygians including mammals, birds, amphibians and coelacanth. In contrast, we identified a single fshr but two lhgcr in basal teleosts, the eels. We further showed the coexistence of duplicated lhgcr in other actinopterygians, including a non-teleost, the gar, and other teleosts, e.g. Mexican tetra, platyfish, or tilapia. Phylogeny and synteny analyses supported the existence in actinopterygians of two lhgcr paralogs (lhgcr1/ lhgcr2), which do not result from the teleost-specific whole-genome duplication (3R), but likely from a local gene duplication that occurred early in the actinopterygian lineage. Due to gene losses, there was no impact of 3R on the number of gonadotropin receptors in extant teleosts. Additional gene losses during teleost radiation, led to a single lhgcr (lhgcr1 or lhgcr2) in some species, e.g. medaka and zebrafish. Sequence comparison highlighted divergences in the extracellular and intracellular domains of the duplicated lhgcr, suggesting differential properties such as ligand binding and activation mechanisms. Comparison of tissue distribution in the European eel, revealed that fshr and both lhgcr transcripts are expressed in the ovary and testis, but are differentially expressed in non-gonadal tissues such as brain or eye. Differences in structure-activity relationships and tissue expression may have contributed as selective drives in the conservation of the duplicated lhgcr. This study revises the evolutionary scenario and nomenclature of gonadotropin receptors, and opens new research avenues on the roles of duplicated LHR in actinopterygians.

Whole-genome duplication in teleost fishes and its evolutionary consequences.

Whole-genome duplication (WGD) events have shaped the history of many evolutionary lineages. One such duplication has been implicated in the evolution of teleost fishes, by far the most species-rich vertebrate clade. After initial controversy, there is now solid evidence that such event took place in the common ancestor of all extant teleosts. It is termed teleost-specific (TS) WGD. After WGD, duplicate genes have different fates. The most likely outcome is non-functionalization of one duplicate gene due to the lack of selective constraint on preserving both. Mechanisms that act on preservation of duplicates are subfunctionalization (partitioning of ancestral gene functions on the duplicates), neofunctionalization (assigning a novel function to one of the duplicates) and dosage selection (preserving genes to maintain dosage balance between interconnected components). Since the frequency of these mechanisms is influenced by the genes' properties, there are over-retained classes of genes, such as highly expressed ones and genes involved in neural function. The consequences of the TS-WGD, especially its impact on the massive radiation of teleosts, have been matter of controversial debate. It is evident that gene duplications are crucial for generating complexity and that WGDs provide large amounts of raw material for evolutionary adaptation and innovation. However, it is less clear whether the TS-WGD is directly linked to the evolutionary success of teleosts and their radiation. Recent studies let us conclude that TS-WGD has been important in generating teleost complexity, but that more recent ecological adaptations only marginally related to TS-WGD might have even contributed more to diversification. It is likely, however, that TS-WGD provided teleosts with diversification potential that can become effective much later, such as during phases of environmental change.

Enigmatic orthology relationships between Hox clusters of the African butterfly fish and other teleosts following ancient whole-genome duplication.

Numerous ancient whole-genome duplications (WGD) have occurred during eukaryote evolution. In vertebrates, duplicated developmental genes and their functional divergence have had important consequences for morphological evolution. Although two vertebrate WGD events (1R/2R) occurred over 525 Ma, we have focused on the more recent 3R or TGD (teleost genome duplication) event which occurred approximately 350 Ma in a common ancestor of over 26,000 species of teleost fishes. Through a combination of whole genome and bacterial artificial chromosome clone sequencing we characterized all Hox gene clusters of Pantodon buchholzi, a member of the early branching teleost subdivision Osteoglossomorpha. We find 45 Hox genes organized in only five clusters indicating that Pantodon has suffered more Hox cluster loss than other known species. Despite strong evidence for homology of the five Pantodon clusters to the four canonical pre-TGD vertebrate clusters (one HoxA, two HoxB, one HoxC, and one HoxD), we were unable to confidently resolve 1:1 orthology relationships between four of the Pantodon clusters and the eight post-TGD clusters of other teleosts. Phylogenetic analysis revealed that many Pantodon genes segregate outside the conventional “a” and “b” post-TGD orthology groups, that extensive topological incongruence exists between genes physically linked on a single cluster, and that signal divergence causes ambivalence in assigning 1:1 orthology in concatenated Hox cluster analyses. Out of several possible explanations for this phenomenon we favor a model which keeps with the prevailing view of a single TGD prior to teleost radiation, but which also considers the timing of diploidization after duplication, relative to speciation events. We suggest that although the duplicated hoxa clusters diploidized prior to divergence of osteoglossomorphs, the duplicated hoxb, hoxc, and hoxd clusters concluded diploidization independently in osteoglossomorphs and other teleosts. We use the term “tetralogy” to describe the homology relationship which exists between duplicated sequences which originate through a shared WGD, but which diploidize into distinct paralogs from a common allelic pool independently in two lineages following speciation.

Evolutionary Origin of the Scombridae (Tunas and Mackerels): Members of a Paleogene Adaptive Radiation with 14 Other Pelagic Fish Families

Uncertainties surrounding the evolutionary origin of the epipelagic fish family Scombridae (tunas and mackerels) are symptomatic of the difficulties in resolving suprafamilial relationships within Percomorpha, a hyperdiverse teleost radiation that contains approximately 17,000 species placed in 13 ill-defined orders and 269 families. Here we find that scombrids share a common ancestry with 14 families based on (i) bioinformatic analyses using partial mitochondrial and nuclear gene sequences from all percomorphs deposited in GenBank (10,733 sequences) and (ii) subsequent mitogenomic analysis based on 57 species from those targeted 15 families and 67 outgroup taxa. Morphological heterogeneity among these 15 families is so extraordinary that they have been placed in six different perciform suborders. However, members of the 15 families are either coastal or oceanic pelagic in their ecology with diverse modes of life, suggesting that they represent a previously undetected adaptive radiation in the pelagic realm. Time-calibrated phylogenies imply that scombrids originated from a deep-ocean ancestor and began to radiate after the end-Cretaceous when large predatory epipelagic fishes were selective victims of the Cretaceous-Paleogene mass extinction. We name this clade of open-ocean fishes containing Scombridae “Pelagia” in reference to the common habitat preference that links the 15 families.

Fish Lateral Line Innovation: Insights into the Evolutionary Genomic Dynamics of a Unique Mechanosensory Organ

The mechanosensory lateral line, found only in fishes and amphibians, is an important sense organ associated with aquatic life. Lateral line patterns differ among teleost, the most diverse vertebrate taxa, hypothetically in response to selective pressures from different aquatic habitats. In this article, we conduct evolutionary genomic analyses of 34 genes associated with lateral line system development in teleosts to elucidate the significance of contrasting evolutionary rates and changes in the protein coding sequences. We find that duplicated copies of these genes are preferentially retained in the teleost genomes and that episodic events of positive selection have occurred in 22 of the 30 postduplication branches. In general, teleost genes evolved at a faster rate relative to their tetrapod counterparts, and the mutation rates of 26 of the 34 genes differed among teleosts and tetrapods. We conclude that following whole genome duplication, evolutionary rates and episodic events of positive selection on the lateral line system development genes might have been one of the factors favoring the subsequent adaptive radiation of teleosts into diverse habitats. These results provide the foundation for further detailed explorations into lateral line system genes and the evolution of diverse phenotypes and adaptations.

Characterization of the Atlantic salmon (Salmo salar) brain-type fatty acid binding protein (fabp7) genes reveals the fates of teleost fabp7 genes following whole genome duplications

Whole genome duplications (WGDs) are considered to have been a driving force in the generation of evolutionary diversity that is characteristic of higher eukaryotes. The ancestor of salmonids underwent two additional WGDs compared to mammals, one (3R) at the base of the teleost radiation and another (4R) in the common ancestor of extant salmonids. We have chosen the fatty acid binding protein (fabp) gene family as a model to study the fate of duplicated genes in teleosts following WGDs. As previously described for zebrafish, we identified two copies (fabp7a and fabp7b) of the brain-type fabp gene in several fish including rainbow smelt, but there was only a single transcript in northern pike, the closest relative of the salmonids, and two rather than the expected four fabp7 genes in Atlantic salmon, rainbow trout and grayling. A phylogenetic analysis revealed that a loss of the fabp7a gene occurred in the common ancestor of the northern pike and salmonids after it had diverged from the rainbow smelt, and that the 4R WGD then gave rise to the fabp7bI and fabp7bII observed in salmonids. This is supported by genetic mapping that placed the Atlantic salmon duplicated fabp7b genes on homeologous chromosomes. There was no evidence of neo-functionalization in the salmonid fabp7bI and fabp7bII genes based on dN/dS ratios and an examination of amino acid substitutions. Atlantic salmon fabp7bI and fabp7bII genes are both expressed broadly like fabp7b expression in northern pike. However, only Atlantic salmon fabp7bII, like its counterpart in northern pike and zebrafish, was expressed in the liver. A comparison of ~2000bp upstream of Atlantic salmon fabp7b gene duplicates revealed an insertion of 62bp in fabp7bI relative to fabp7bII. The presence of predicted transcription factor binding sites in this insertion sequence may explain the differential expression of the fabp7b gene duplicates in Atlantic salmon liver.

Identification and Biochemical Characterization of Two Functional CMP-Sialic Acid Synthetases in Danio rerio

Sialic acids (Sia) form the nonreducing end of the bulk of cell surface-expressed glycoconjugates. They are, therefore, major elements in intercellular communication processes. The addition of Sia to glycoconjugates requires metabolic activation to CMP-Sia, catalyzed by CMP-Sia synthetase (CMAS). This highly conserved enzyme is located in the cell nucleus in all vertebrates investigated to date, but its nuclear function remains elusive. Here, we describe the identification and characterization of two Cmas enzymes in Danio rerio (dreCmas), one of which is exclusively localized in the cytosol. We show that the two cmas genes most likely originated from the third whole genome duplication, which occurred at the base of teleost radiation. cmas paralogues were maintained in fishes of the Otocephala clade, whereas one copy got subsequently lost in Euteleostei (e.g. rainbow trout). In zebrafish, the two genes exhibited a distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored N-acetylneuraminic acid, whereas the cytosolic dreCmas2 showed highest affinity for 5-deamino-neuraminic acid. The subcellular localization was confirmed for the endogenous enzymes in fractionated zebrafish lysates. Nuclear entry of dreCmas1 was mediated by a bipartite nuclear localization signal, which seemed irrelevant for other enzymatic functions. With the current demonstration that in zebrafish two subfunctionalized cmas paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells.

Nrf2b, Novel Zebrafish Paralog of Oxidant-responsive Transcription Factor NF-E2-related Factor 2 (NRF2)

NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.