Evolutionary Transition From Water to Land in Vertebrates Illuminated by Basal Ray-Finned Fish Vomeronasal Type 2 Receptor (OlfC) Genes

The vomeronasal type 2 receptor (V2R, also called OlfC) multigene family is found in a broad range of jawed vertebrates from cartilaginous fish to tetrapods. V2Rs encode receptors for food-related amino acids in teleost fish, whereas for peptide pheromones in mammals. In addition, V2Rs of teleost fish are phylogenetically distinct from those of tetrapods, implying a drastic change in the V2Rrepertoire during terrestrial adaptation. To understand the process of diversification of V2Rs in vertebrates from “fish-type” to “tetrapod-type”, we conducted an exhaustive search for V2Rs in cartilaginous fish (chimeras, sharks, and skates) and basal ray-finned fish (reedfish, sterlet, and spotted gar), and compared them with those of teleost, coelacanth, and tetrapods. Phylogenetic and synteny analyses on 1897V2Rs revealed that basal ray-finned fish possess unexpectedly higher number of V2Rs compared with cartilaginous fish, implying that V2Rgene repertoires expanded in the common ancestor of Osteichthyes. Furthermore, reedfish and sterlet possessed various V2Rs that belonged to both “fish-type” and “tetrapod-type”, suggesting that the common ancestor of Osteichthyes possess “tetrapod-type” V2Rs although they inhabited underwater environments. Thus, the unexpected diversity of V2Rs in basal ray-finned fish illuminates the process of how the osteichthyan ancestors adapt from water to land.

Evolution of hes gene family in vertebrates: the hes5 cluster genes have specifically increased in frogs

Background hes genes are chordate homologs of Drosophila genes, hairy and enhancer of split, which encode a basic helix-loop-helix (bHLH) transcriptional repressor with a WRPW motif. Various developmental functions of hes genes, including early embryogenesis and neurogenesis, have been elucidated in vertebrates. However, their orthologous relationships remain unclear partly because of less conservation of relatively short amino acid sequences, the fact that the genome was not analyzed as it is today, and species-specific genome duplication. This results in complicated gene names in vertebrates, which are not consistent in orthologs. We previously revealed that Xenopus frogs have two clusters of hes5, named “the hes5.1 cluster” and “the hes5.3 cluster”, but the origin and the conservation have not yet been revealed. Results Here, we elucidated the orthologous and paralogous relationships of all hes genes of human, mouse, chicken, gecko, zebrafish, medaka, coelacanth, spotted gar, elephant shark and three species of frogs, Xenopus tropicalis (X. tropicalis), X. laevis, Nanorana parkeri, by phylogenetic and synteny analyses. Any duplicated hes5 were not found in mammals, whereas hes5 clusters in teleost were conserved although not as many genes as the three frog species. In addition, hes5 cluster-like structure was found in the elephant shark genome, but not found in cyclostomata. Conclusion These data suggest that the hes5 cluster existed in the gnathostome ancestor but became a single gene in mammals. The number of hes5 cluster genes were specifically large in frogs.

Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development

Nitric oxide (NO) is an ancestral key signaling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission, and immunity. Although our knowledge of nitric oxide synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologs in fish with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in the ray-finned fish lineage, changed this perspective. This prompted us to explore nos gene evolution and expression in depth, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralog in the gills of developing shark, bichir, sturgeon, and gar but not in arctic lamprey, suggest that nos expression in this organ likely arose in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

Genomic Analysis of Hypoxia Inducible Factor Alpha Evolution in Ray-finned Fishes (Actinopterygii)

Two rounds of genome duplication (GD) in the ancestor of vertebrates, followed by additional GD during the evolution of ray-finned fishes (Actinopterygii), expanded certain gene families, including those encoding the hypoxia inducible transcription factor (HIF). The present study analyzed Actinopterygian genomes for duplicates of HIFα, the subunit that confers oxygen-dependent gene regulation. In contrast to tetrapod vertebrates that retain three HIFα genes from the ancestral vertebrate GD, four HIFα forms were found in the genomes of primitive Actinopterygians (spotted gar and Asian arowana). All four forms have been retained in zebrafish and related species (Otocephala) and salmonids and their sister taxa (northern pike) but one of them (HIF4α) was lost during the evolution of more derived fishes (Neoteleostei). In addition, the current analyses confirm that Otocephala retain duplicates of HIF1α and HIF2α from the teleost-specific GD, provide new evidence of salmonid-specific duplicates of HIF1α, HIF2α, and HIF3α, and reveal a broad distribution of a truncated form of HIF2α in salmonids and Neoteleostei. This study delivers a comprehensive view of HIFα evolution in the ray-finned fishes, highlights the need for a consistent nomenclature, and suggests avenues for future research on this critical transcription factor.

Evolution of hes Gene Family in Vertebrates: hes5 Cluster Genes Were Specifically Increased in Xenopus

Background hes genes are chordate homologs of Drosophila genes, hairy and enhancer of split, which encode a basic helix-loop-helix (bHLH) transcriptional repressor with a WRPW motif. Various developmental functions of hes genes, including early embryogenesis and neurogenesis, have been elucidated in vertebrates. However, their orthologous relationships remain unclear partly because of less conservation of relatively short amino acid sequences, less conserved synteny, and species-specific gene duplication. This results in complicated gene names in vertebrates, which are not consistent in orthologs. In a previous study, we revealed that Xenopus frogs have two clusters of hes5, named “the hes5.1 cluster” and “the hes5.3 cluster.” The origin has not yet been revealed. Results Here, we elucidated the orthologous and paralogous relationships of all hes genes of human, mouse, chicken, gecko, zebrafish, medaka, coelacanth, spotted gar, elephant shark, and Xenopus frogs (X. tropicalis and X. laevis) by phylogenic and synteny analysis. Any clusters of hes5 were not found in amniotes, whereas duplicated hes5 clusters in teleost were found although not as many genes as Xenopus. In addition, hes5 cluster-like structure was found in the elephant shark genome, but not found in cyclostomata. Conclusion These data suggest that the hes5 cluster existed in the gnathostome ancestor, but was lost in amniotes.

Cellular and molecular mechanisms of frontal bone development in spotted gar (Lepisosteus oculatus)

BackgroundThe molecular mechanisms initiating vertebrate cranial dermal bone formation is a conundrum in evolutionary and developmental biology. Decades of studies have determined the developmental processes of cranial dermal bones in various vertebrate species, finding possible inducers of dermal bone. However, the evolutionarily derived characters of current experimental model organisms hinder investigations of the ancestral and conserved mechanisms of vertebrate cranial dermal bone induction. Thus, investigating such mechanisms with animals diverging at evolutionarily crucial phylogenetic nodes is imperative.ResultsWe investigated the cellular and molecular foundations of skull frontal bone formation in the spotted gar Lepisosteus oculatus, a basally branching actinopterygian. Whole-mount bone and cartilage stainings and hematoxylin-eosin section stainings revealed that mesenchymal cell condensations in the frontal bone of spotted gar develop in close association with the underlying cartilage. We also identified novel aspects of frontal bone formation: Upregulation of F-actin and plasma membrane in condensing cells, and extension of podia from osteoblasts to the frontal bone, which may be responsible for bone mineral transport.ConclusionThis study highlights the process of frontal bone formation with dynamic architectural changes of mesenchymal cells in spotted gar, illuminating supposedly ancestral and likely conserved developmental mechanisms of skull bone formation among vertebrates.

Conserved Keratin Gene Clusters in Ancient Fish: an Evolutionary Seed for Terrestrial Adaptation

Type I and type II keratins are subgroups of intermediate filament proteins that provide toughness to the epidermis and protect it from water loss. In terrestrial vertebrates, the keratin genes form two major clusters, clusters 1 and 2, each of which is dominated by type I and II keratin genes. By contrast, such clusters are not observed in teleost fish. Although the diversification of keratins is believed to have made a substantial contribution to terrestrial adaptation, its evolutionary process has not been clarified. Here, we performed a comprehensive genomic survey of the keratin genes of a broad range of vertebrates. As a result, we found that ancient fish lineages such as elephant shark, reedfish, spotted gar, and coelacanth share both keratin gene clusters. We also discovered an expansion of keratin genes that form a novel subcluster in reedfish. Syntenic and phylogenetic analyses revealed that two pairs of krt18/krt8 keratin genes were shared among all vertebrates, thus implying that they encode ancestral type I and II keratin protein sets. We further revealed that distinct keratin gene subclusters, which show specific expressions in the epidermis of adult amphibians, stemmed from canonical keratin genes in non-terrestrial ancestors. Molecular evolutionary analyses suggested that the selective constraints were relaxed in the adult epidermal subclusters of amphibians as well as the novel subcluster of reedfish. The results of the present study represent the process of diversification of keratins through a series of gene duplications that could have facilitated the terrestrial adaptation of vertebrates.HighlightsTwo major keratin clusters are conserved from sharks to terrestrial vertebrates.Adult epidermis-specific keratins in amphibians stem from the two major clusters.A novel keratin gene subcluster was found in reedfish.Ancestral krt18/krt8 gene sets were found in all vertebrates.Functional diversification signatures were found in reedfish and amphibian keratins.

Major histocompatibility complex in Osteichthyes

Based on analysis of available genome sequences, five gene lineages of MHC class I molecules (MHC I-U, -Z, -S, -L and -P) and one gene lineage of MHC class II molecules (MHC II-D) have been identified in Osteichthyes. In the latter lineage, three MHC II molecule sublineages have been identified (MHC II-A, -B and -E). As regards MHC class I molecules in Osteichthyes, it is important to take note of the fact that the lineages U and Z in MHC I genes have been identified in almost all fish species examined so far. Phylogenetic studies into MHC II molecule genes of sublineages A and B suggest that they may be descended from the genes of the sublineage named A/B that have been identified in spotted gar (Lepisosteus oculatus). The sublineage E genes of MHC II molecules, which represent the group of non-polymorphic genes with poor expression in the tissues connected with the immune system, are present in primitive fish, i.e. in paddlefish, sturgeons and spotted gar (Lepisosteus oculatus), as well as in cyprinids (Cyprinidae), Atlantic salmon (Salmo salar), and rainbow trout (Oncorhynchus mykiss). Full elucidation of the details relating to the organisation and functioning of the particular components of the major histocompatibility complex in Osteichthyes can advance the understanding of the evolution of the MHC molecule genes and the immune mechanism.