The single amphioxus Mox gene: insights into the functional evolution of Mox genes,somites, and the asymmetry of amphioxus somitogenesis

Mox genes are members of the "extended" Hox-cluster group of Antennapedia-like homeobox genes. Homologues have been cloned from both invertebrate and vertebrate species, and are expressed in mesodermal tissues. In vertebrates, Mox1 and Mox2 are distinctly expressed during the formation of somites and differentiation of their derivatives. Somites are a distinguishing feature uniquely shared by cephalochordates and vertebrates. Here, we report the cloning and expression of the single amphioxus Mox gene. AmphiMox is expressed in the presomitic mesoderm (PSM) during early amphioxus somitogenesis and in nascent somites from the tail bud during the late phase. Once a somite is completely formed, AmphiMox is rapidly downregulated. We discuss the presence and extent of the PSM in both phases of amphioxus somitogenesis. We also propose a scenario for the functional evolution of Mox genes within chordates, in which Mox was co-opted for somite formation before the cephalochordate-vertebrate split. Novel expression sites found in vertebrates after somite formation postdated Mox duplication in the vertebrate stem lineage, and may be linked to the increase in complexity of vertebrate somites and their derivatives, e.g., the vertebrae. Furthermore, AmphiMox expression adds new data into a long-standing debate on the extent of the asymmetry of amphioxus somitogenesis.     

Phylogenetic dating and characterization of gene duplications in vertebrates: the cartilaginous fish reference

Vertebrates originated in the lower Cambrian. Their diversification and morphological innovations have been attributed to large-scale gene or genome duplications at the origin of the group. These duplications are predicted to have occurred in two rounds, the "2R" hypothesis, or they may have occurred in one genome duplication plus many segmental duplications, although these hypotheses are disputed. Under such models, most genes that are duplicated in all vertebrates should have originated during the same period. Previous work has shown that indeed duplications started after the speciation between vertebrates and the closest invertebrate, amphioxus, but have not set a clear ending. Consideration of chordate phylogeny immediately shows the key position of cartilaginous vertebrates (Chondrichthyes) to answer this question. Did gene duplications occur as frequently during the 45 Myr between the cartilaginous/bony vertebrate split and the fish/tetrapode split as in the previous approximately 100 Myr? Although the time interval is relatively short, it is crucial to understanding the events at the origin of vertebrates. By a systematic appraisal of gene phylogenies, we show that significantly more duplications occurred before than after the cartilaginous/bony vertebrate split. Our results support rounds of gene or genome duplications during a limited period of early vertebrate evolution and allow a better characterization of these events.     

Loss/retention and evolution of NBS-encoding genes upon whole genome triplication of Brassica rapa

A genome triplication took place in the ancestor of Brassiceae species after the split of the Arabidopsis lineage. The postfragmentation and shuffling of the genome turned the ancestral hexaploid back to diploids and caused the radiation of Brassiceae species. The course of speciation was accompanied by the loss of duplicate genes and also influenced the evolution of retained genes. Of all the genes, those encoding NBS domains are typical R genes that confer resistance to invading pathogens. In this study, using the genome of Arabidopsis thaliana as a reference, we examined the loss/retention of orthologous NBS-encoding loci in the tripled Brassica rapa genome and discovered differential loss/retention frequencies. Further analysis indicated that loci of different retention ratios showed different evolutionary patterns. The loci of classesII and III (maintaining two and three syntenic loci, respectively, multi-loci) show sharper expansions by tandem duplications, have faster evolutionary rates and have more potential to be associated with novel gene functions. On the other hand, the loci that are retained at the minimal rate (keeping only one locus, class I, single locus) showed opposite patterns. Phylogenetic analysis indicated that recombination and translocation events were common among multi-loci in B. rapa, and differential evolutionary patterns between multi- and single-loci are likely the consequence of recombination. Investigations towards other gene families demonstrated different evolutionary characteristics between different gene families. The evolution of genes is more likely determined by the property of each gene family, and the whole genome triplication provided only a specific condition.     

Fungal regulatory evolution: cis and trans in the balance

Regulatory divergence is likely a major driving force in evolution. Comparative genomics is being increasingly used to infer the evolution of gene regulation. Ascomycota fungi are uniquely suited among eukaryotes for regulatory evolution studies, due to broad phylogenetic scope, many sequenced genomes, and tractability of genomic analysis. Here we review recent advances in the identification of the contribution of cis- and trans-factors to expression divergence. Whereas current strategies have led to the discovery of surprising signatures and mechanisms, we still understand very little about the adaptive role of regulatory evolution. Empirical studies including experimental evolution, comparative functional genomics and hybrid and engineered strains are showing early promise toward deciphering the contribution of regulatory divergence to adaptation.     

Isolation and mapping the chicken zona pellucida genes: an insight into the evolution of orthologous genes in different species

The avian oocyte is surrounded by a specialized extracellular glycoproteinaceous matrix, the perivitelline membrane, which is equivalent to the zona pellucida (ZP) in mammals and the chorion in teleosts. A number of related ZP genes encode the proteins that make up this matrix. These proteins play an important role in the sperm/egg interaction and may be involved in speciation. The human genome is known to contain ZP1, ZP2, ZP3, and ZPB genes, while a ZPAX gene has also been identified in Xenopus. The rapid evolution of these genes has confused the nomenclature and thus orthologous relationships across species. In order to clarify these homologies, we have identified ZP1, ZP2, ZPC, ZPB, and ZPAX genes in the chicken and mapped them to chromosomes 5, 14, 10, 6, and 3, respectively, establishing conserved synteny with human and mouse. The amino acid sequences of these genes were compared to the orthologous genes in human, mouse, and Xenopus, and have given us an insight into the evolution of these genes in a variety of different species. The presence of the ZPAX gene in the chicken has highlighted a pattern of probable gene loss by deletion in mouse and gene inactivation by deletion, and base substitution in human.     

Tangled up in two: a burst of genome duplications at the end of the Cretaceous and the consequences for plant evolution

Genome sequencing has demonstrated that besides frequent small-scale duplications, large-scale duplication events such as whole genome duplications (WGDs) are found on many branches of the evolutionary tree of life. Especially in the plant lineage, there is evidence for recurrent WGDs, and the ancestor of all angiosperms was in fact most likely a polyploid species. The number of WGDs found in sequenced plant genomes allows us to investigate questions about the roles of WGDs that were hitherto impossible to address. An intriguing observation is that many plant WGDs seem associated with periods of increased environmental stress and/or fluctuations, a trend that is evident for both present-day polyploids and palaeopolyploids formed around the Cretaceous–Palaeogene (K–Pg) extinction at 66 Ma. Here, we revisit the WGDs in plants that mark the K–Pg boundary, and discuss some specific examples of biological innovations and/or diversifications that may be linked to these WGDs. We review evidence for the processes that could have contributed to increased polyploid establishment at the K–Pg boundary, and discuss the implications on subsequent plant evolution in the Cenozoic.     

New insight into the evolution of aquaporins from flowering plants and vertebrates: orthologous identification and functional transfer is possible

Aquaporins (AQPs) represent a family of channel proteins that transport water and/or small solutes across cell membranes in the three domains of life. In all previous phylogenetic analysis of aquaporin, trees constructed using proteins with very low amino acid identity (<15%) were incongruent with rRNA data. In this work, restricting the evolutionary study of aquaporins to proteins with high amino acid identity (>25%), we showed congruence between AQPs and organismal trees. On the basis of this analysis, we defined 19 orthologous gene clusters in flowering plant species (3 PIP-like, 7 TIP-like, 6 NIP-like and 3 SIP-like). We described specific conserved motifs for each subfamily and each cluster, which were used to develop a method for automatic classification. Analysis of amino acid identity between orthologous monocotyledon and dicotyledon AQPs from each cluster, suggested that PIPs are under high evolutionary constraint. The phylogenetic analysis allowed us the assignment of orthologous aquaporins for very distant animal lineages (tetrapods-fishes). We also demonstrated that the location of all vertebrate AQPs in the ortholog clusters could be predicted by comparing their amino acid identity with human AQPs. We defined four AQP subfamilies in animals: AQP1-like, AQP8-like, AQP3-like and AQP11-like. Phylogenetic analysis showed that the four animal AQPs subfamilies are related with PIP-like, TIP-like, NIP-like and SIP-like subfamilies, respectively. Thus, this analysis would allow the prediction of individual AQPs function on the basis of orthologous genes from Arabidopsis thaliana and Homo sapiens.     

Gene and genome duplications in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions

One important mechanism for functional innovation during evolution is the duplication of genes and entire genomes. Evidence is accumulating that during the evolution of vertebrates from early deuterostome ancestors entire genomes were duplicated through two rounds of duplications (the 'one-to-two-to-four' rule). The first genome duplication in chordate evolution might predate the Cambrian explosion. The second genome duplication possibly dates back to the early Devonian. Recent data suggest that later in the Devonian, the fish genome was duplicated for a third time to produce up to eight copies of the original deuterostome genome. This last duplication took place after the two major radiations of jawed vertebrate life, the ray-finned fish (Actinopterygia) and the sarcopterygian lineage, diverged. Therefore the sarcopterygian fish, which includes the coelacanth, lungfish and all land vertebrates such as amphibians, reptiles, birds and mammals, tend to have only half the number of genes compared with actinopterygian fish. Although many duplicated genes turned into pseudogenes, or even 'junk' DNA, many others evolved new functions particularly during development. The increased genetic complexity of fish might reflect their evolutionary success and diversity.     

Identification,expression and bioactivity of hexokinase in amphioxus: Insights into evolution of vertebrate hexokinase genes

Hexokinase family includes hexokinases I, II, III and IV, that catalyze the phosphorylation of glucose to produce glucose 6-phosphate. Hexokinase IV, also known as glucokinase, is only half size of the other types of hexokinases that contain two hexokinase domains. Despite the enormous progress in the study of hexokinases, the evolutionary relationship between glucokinase and other hexokinases is still uncertain, and the molecular processes leading to the emergence of hexokinases in vertebrates remain controversial. Here we clearly demonstrated the presence of a single hexokinase-like gene in the amphioxus Branchiostoma japonicum, Bjhk, which shows a tissue-specific expression pattern, with the most abundant expression in the hepatic caecum, testis and ovary. The phylogenetic and synteny analyses both reveal that BjHK is the archetype of vertebrate hexokinases IV, i.e. glucokinases. We also found for the first time that recombinant BjHK showed functional enzyme activity resembling vertebrate hexokinases I, II, III and IV. In addition, a native glucokinase activity was detected in the hepatic caecum. Finally, glucokinase activity in the hepatic caecum was markedly reduced by fasting, whereas it was considerably increased by feeding. Altogether, these suggest that Bjhk represents the archetype of glucokinases, from which vertebrate hexokinase gene family was evolved by gene duplication, and that the hepatic caecum plays a role in the control of glucose homeostasis in amphioxus, in favor of the notion that the hepatic caecum is a tissue homologous to liver.     

Pigmentary function and evolution of tyrp1 gene duplicates in fish

The function of the tyrosinase‐related protein 1 (Tyrp1) has not yet been investigated in vertebrates basal to tetrapods. Teleost fishes have two duplicates of the tyrp1 gene. Here, we show that the teleost tyrp1 duplicates have distributed the ancestral gene expression in the retinal pigment epithelium (RPE) and melanophores in a species‐specific manner. In medaka embryos, tyrp1a expression is found in the RPE and in melanophores while tyrp1b is only expressed in melanophores. In zebrafish embryos, expression of tyrp1 paralogs overlaps in the RPE and in melanophores. Knockdown of each zebrafish tyrp1 duplicate alone does not show pigmentary defects, but simultaneous knockdown of both tyrp1 genes results in the formation of brown instead of black eumelanin accompanied by severe melanosome defects. Our study suggests that the brown melanosome color in Tyrp1‐deficient vertebrates is an effect of altered eumelanin synthesis. Black eumelanin formation essentially relies on the presence of Tyrp1 and some of its function is most likely conserved from the common ancestor of bony vertebrates.     

Molecular cloning and sequencing of metallothionein in squamates: New insights into the evolution of the metallothionein genes in vertebrates

Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian metallothioneins, we have cloned and sequenced the cDNAs encoding for metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of metallothioneins in reptiles. Phylogenetic analysis shows that reptilian metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of metallothionein genes along the major vertebrate lineages.     

Characterization of Brachyury genes in the dogfish S. canicula and the lamprey L. fluviatilis. Insights into gastrulation in a chondrichthyan

In order to gain insights into the evolution of gastrulation mechanisms among vertebrates, we have characterized a Brachyury-related gene in a lamprey, Lampetra fluviatilis, and in a chondrichthyan, Scyliorhinus canicula. These two genes, respectively termed LfT and ScT, share with their osteichthyan counterparts prominent expression sites in the developing notochord, the tailbud, but also a transient expression in the prechordal plate, which is likely to be ancestral among vertebrates. In addition, the lamprey LfT gene is transcribed in the endoderm of the pharyngeal arches and the epiphysis, two expression sites that have not been reported thus far in gnathostomes, and, as in the chick, in the differentiating nephrotomes. Since Brachyury expression in nascent mesoderm and endoderm is highly conserved among vertebrates as well as cephalochordates, we have used this marker to identify these cell populations during gastrulation in the dogfish. The results suggest that these cells are initially present over the whole margin of the blastoderm and are displaced during gastrulation to its posterior part, which may correspond to the site of mesoderm and endoderm internalization. These data provide the first molecular characterization of gastrulation in a chondrichthyan. They indicate that gastrulation in the dogfish and in some amniotes shares striking similarities despite the phylogenetic distance between these species. This supports the hypothesis that the extensively divergent morphologies of gastrulae among vertebrates largely result from adaptations to the presence of yolk.     

Evolution and functional divergence of monocarboxylate transporter genes in vertebrates

Monocarboxylate transporters (MCTs) form a gene family with an ancient past. The identification of MCTs (MCHs) from bacteria, protozoa, fungi, invertebrates, as well as vertebrates, but not from plants and virus, allowed illuminating the phylogenetic and evolutionary history of this gene family. The significant expansion of vertebrate MCT genes should have primarily occurred after the divergence of vertebrates and invertebrates, but before the divergence time between ray-finned fish and mammals. The divergence of insect MCTs should have at least occurred in the common ancestor of fruit fly, beetle, and honeybee. Fungi monocarboxylate transporter homologues (MCHs) might evolve independently from an ancient ancestor. The results of functional divergence analysis provided statistical evidences for shifted evolutionary rate and/or changes of amino acid property after gene duplication. The sliding window analysis of the d(N)/d(S) ratio values showed that strong functional constraints must impose on the N- and C-terminal domains of vertebrate MCTs. These corresponding regions may play crucial roles for functionality of MCT proteins.     

Evolution of trace amine associated receptor (TAAR) gene family in vertebrates: lineage-specific expansions and degradations of a second class of vertebrate chemosensory receptors expressed in the olfactory epithelium

The trace amine-associated receptors (TAARs) form a specific family of G protein-coupled receptors in vertebrates. TAARs were initially considered neurotransmitter receptors, but recent study showed that mouse TAARs function as chemosensory receptors in the olfactory epithelium. To clarify the evolutionary dynamics of the TAAR gene family in vertebrates, near-complete repertoires of TAAR genes and pseudogenes were identified from the genomic assemblies of 4 teleost fishes (zebrafish, fugu, stickleback, and medaka), western clawed frogs, chickens, 3 mammals (humans, mice, and opossum), and sea lampreys. Database searches revealed that fishes had many putatively functional TAAR genes (13-109 genes), whereas relatively small numbers of TAAR genes (3-22 genes) were identified in tetrapods. Phylogenetic analysis of these genes indicated that the TAAR gene family was subdivided into 5 subfamilies that diverged before the divergence of ray-finned fishes and tetrapods. In tetrapods, virtually all TAAR genes were located in 1 specific region of their genomes as a gene cluster; however, in fishes, TAAR genes were scattered throughout more than 2 genomic locations. This possibly reflects a whole-genome duplication that occurred in the common ancestor of ray-finned fishes. Expression analysis of zebrafish and stickleback TAAR genes revealed that many TAARs in these fishes were expressed in the olfactory organ, suggesting the relatively high importance of TAARs as chemosensory receptors in fishes. A possible evolutionary history of the vertebrate TAAR gene family was inferred from the phylogenetic and comparative genomic analyses.     

Five Nkx5 genes show differential expression patterns in anlagen of sensory organs in medaka: insight into the evolution of the gene family

We report the identification and characterisation of five different Nkx5-related genes in medaka fish (Oryzias latipes). They constitute homologues of genes previously isolated in higher vertebrates, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3 and SOHo-1, and were named accordingly: OlNkx5--1.1, OlNkx5--2, OlNkx5--3 and OlSOHo. For the Nkx5--1 gene a new, second homologue, OlNkx5--1.2, was isolated. In medaka, Nkx5 and SOHo genes are differentially expressed in three developing sensory organs: eye, ear and lateral line and later in defined brain regions. Phylogenetic analyses of the entire Nkx5 family revealed that four paralogous Nkx5 groups, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3/GH6 and SOHo, are present in vertebrates. Only some of the Nkx5 family members have been identified in singular vertebrate species so far. Here we present, for the first time, the isolation of representatives of each Nkx5 subgroup in one species, the medaka fish. Based on similarities in sequence and expression patterns, and genomic organisation we propose a model of the evolutionary history of the Nkx5 family. The model predicts that the four vertebrate Nkx5 genes arose by a tandem duplication, followed by chromosomal duplication. The two Nkx5--1 genes identified so far exclusively in medaka most probably result from an additional genome duplication in the fish lineage.     

Ossification of the posterior longitudinal ligament related genes identification using microarray gene expression profiling and bioinformatics analysis

Ossification of the posterior longitudinal ligament (OPLL) is a kind of disease with physical barriers and neurological disorders. The objective of this study was to explore the differentially expressed genes (DEGs) in OPLL patient ligament cells and identify the target sites for the prevention and treatment of OPLL in clinic. Gene expression data GSE5464 was downloaded from Gene Expression Omnibus; then DEGs were screened by limma package in R language, and changed functions and pathways of OPLL cells compared to normal cells were identified by DAVID (The Database for Annotation, Visualization and Integrated Discovery); finally, an interaction network of DEGs was constructed by string. A total of 1536 DEGs were screened, with 31 down-regulated and 1505 up-regulated genes. Response to wounding function and Toll-like receptor signaling pathway may involve in the development of OPLL. Genes, such as PDGFB, PRDX2 may involve in OPLL through response to wounding function. Toll-like receptor signaling pathway enriched genes such as TLR1, TLR5, and TLR7 may involve in spine cord injury in OPLL. PIK3R1 was the hub gene in the network of DEGs with the highest degree; INSR was one of the most closely related genes of it. OPLL related genes screened by microarray gene expression profiling and bioinformatics analysis may be helpful for elucidating the mechanism of OPLL.