The hagfish genome and the evolution of vertebrates.

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution. Here we investigate the complex history, timing and functional role of genome-wide duplications and programmed DNA elimination in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1R) that predates the early Cambrian cyclostome-gnathostome split, followed by a mid-late Cambrian allo-tetraploidization (2R) in gnathostomes and a prolonged Cambrian-Ordovician hexaploidization (2R) in cyclostomes.

The hagfish genome and the evolution of vertebrates.

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a critical window into early vertebrate evolution. Here, we investigate the complex history, timing, and functional role of genome-wide duplications in vertebrates in the light of a chromosome-scale genome of the brown hagfish . Using robust chromosome-scale (paralogon-based) phylogenetic methods, we confirm the monophyly of cyclostomes, document an auto-tetraploidization (1R) that predated the origin of crown group vertebrates ~517 Mya, and establish the timing of subsequent independent duplications in the gnathostome and cyclostome lineages.

Evolution of cis-regulatory modules for the head organizer gene in chordates: comparisons between and .

Background: In cephalochordates (amphioxus), the notochord runs along the dorsal to the anterior tip of the body. In contrast, the vertebrate head is formed anterior to the notochord, as a result of head organizer formation in anterior mesoderm during early development. A key gene for the vertebrate head organizer, , is broadly expressed in the dorsal mesoderm of amphioxus gastrula.

Platanus-allee is a de novo haplotype assembler enabling a comprehensive access to divergent heterozygous regions.

The ultimate goal for diploid genome determination is to completely decode homologous chromosomes independently, and several phasing programs from consensus sequences have been developed. These methods work well for lowly heterozygous genomes, but the manifold species have high heterozygosity. Additionally, there are highly divergent regions (HDRs), where the haplotype sequences differ considerably.

Actin capping proteins, CapZ (β-actinin) and tropomodulin in amphioxus striated muscle.

CapZ (β-actinin) and tropomodulin (Tmod) are capping proteins involved in the maintenance of thin filaments in vertebrate skeletal muscles. In this study, we focused on amphioxus, the most primitive chordate. We searched for CapZ and Tmod genes in the amphioxus genome and determined their primary structures.

Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution.

Sialic acid, a common terminal substitution of glycoconjugates, has been so far consistently identified in all vertebrates as well as in a growing number of bacterial species. It is assumed to be widely distributed among animal species of the deuterostome phylum, based on its identification in few echinoderm and all vertebrate species. However, whole sections of deuterostome, especially those intermediate species between invertebrates and vertebrates including cephalochordates, urochordates and hemichordates, are still unexplored in term of sialylation capacities.

Evolution of the reproductive endocrine system in chordates.

The cephalochordate, amphioxus, is phylogenetically placed at the most primitive position in the chordate clade. Despite many studies on the endocrine system of amphioxus, definitive evidence has not been reported for the presence an endocrine system comparable to the pituitary-gonadal axis, which is important in the regulation of reproduction in vertebrates. Recent genome analyses in the amphioxus, Branchiostoma floridae, showed that it does not have any pituitary hormone genes except the thyrostimulin gene.

Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region.

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate.

Functional characteristics of amphioxus troponin in regulation of muscle contraction.

Troponin regulates contraction of vertebrate striated muscle in a Ca(2+)-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca(2+) during contraction. In vertebrates, this regulatory mechanism is unlike that in some less highly derived taxa.

Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors.

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends.

Estrogen-dependent transactivation of amphioxus steroid hormone receptor via both estrogen and androgen response elements.

Estrogens are necessary for ovarian differentiation during critical developmental windows in most vertebrates and promote the growth and differentiation of the adult female reproductive system. Estrogen actions are largely mediated through the estrogen receptors (ERs), which are ligand-activated transcription factors. To understand the molecular evolution of sex steroid hormone receptors, we isolated cDNAs encoding two steroid receptors from Japanese amphioxus, Branchiostoma belcheri: an ER ortholog and a ketosteroid receptor (SR) ortholog.

A homolog of the vertebrate thyrostimulin glycoprotein hormone alpha subunit (GPA2) is expressed in Amphioxus neurons.

The cystine-knot glycoprotein hormone alpha (GPA) family regulates gonadal and thyroid functions in vertebrates. Little is known concerning GPA family members in primitive chordates. A previous genomic analysis revealed the presence of two genes homologous to the thyrostimulin alpha subunit (GPA2) in an amphioxus (Branchiostoma florideae); however only one GPA2 homolog contained both the cystine-knot structure and N-glycosylation site characteristic of family members.

Structure of the amphioxus nebulin gene and evolution of the nebulin family genes.

Nebulin family genes are believed to have diverged from a single gene during the evolution of vertebrates. We determined the structure of the amphioxus nebulin gene and showed that in addition to the features of the human nebulin gene, this gene had a LIM domain, secondary super repeats and a giant exon with 98 nebulin repeats containing unique sequences. A transcript of this gene amplified by reverse transcriptase-polymerase chain reaction had a LIM domain, three nebulin repeats and an SH3 domain.

Expression of the gene for ancestral glycoprotein hormone beta subunit in the nerve cord of amphioxus.

Amphioxus belongs to the subphylum cephalochordata, a clade of chordates phylogenetically placed at the most basal position. Despite many studies on the endocrine system of amphioxus, there were no confident lines of evidence on the presence of pituitary hormones, whereas recent amphioxus genome analysis reported that amphioxus has no pituitary hormone except for thyrostimulin, which is a glycoprotein hormone in the pituitary, brain, and other organs of vertebrates. In the present study, we cloned cDNA for one glycoprotein hormone beta subunit (GPB) from amphioxus, AmpGPB5, and phylogenetically indicated that AmpGPB5 is the ancestral molecule of glycoprotein hormone beta subunits of vertebrates including pituitary glycoprotein hormones.

Characterization of amphioxus nebulin and its similarity to human nebulin.

Identification of a large molecule in muscle is important but difficult to approach by protein chemistry. In this study we isolated nebulin cDNA from the striated muscle of amphioxus, and characterized the C-terminal regions of nebulins from other chordates. Although the sequence homology with that of human is only 26%, the C-terminal region of amphioxus nebulin has similar structural motifs of 35 amino acid nebulin repeats and an SH3 domain.

Amphritea japonica sp. nov. and Amphritea balenae sp. nov., isolated from the sediment adjacent to sperm whale carcasses off Kagoshima, Japan.

Two novel species were isolated from the sediment adjacent to sperm whale carcasses off Kagoshima, Japan, at a depth of about 230 m. The isolated strains, JAMM 1866(T), JAMM 1548 and JAMM 1525(T), were Gram-negative, rod-shaped, non-spore-forming and motile by means of a single polar or bipolar flagellum. Phylogenetic analysis based on 16S rRNA gene sequences of strains JAMM 1866(T) and JAMM 1548 indicated a relationship to the symbiotic bacterial clone R21 of Osedax japonicus (100 % sequence similarity) and all three isolates were closely related to Amphritea atlantica (97.

The amphioxus genome illuminates vertebrate origins and cephalochordate biology.

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers.

Neptunomonas japonica sp. nov., an Osedax japonicus symbiont-like bacterium isolated from sediment adjacent to sperm whale carcasses off Kagoshima, Japan.

Novel bacterial species were isolated from sediments adjacent to sperm whale carcasses off Kagoshima, Japan, at a depth of 226-246 m. The isolated strains, JAMM 0745T, JAMM 1380, JAMM 1475 and JAMM 1610, were Gram-negative, rod-shaped, non-spore-forming and motile by means of a single polar or subterminal flagellum. Phylogenetic analysis based on 16S rRNA gene sequences of the novel isolates indicated a relationship to a symbiotic bacterial clone of the polychaete Osedax japonicus (99.

In vitro conversion of sex steroids and expression of sex steroidogenic enzyme genes in amphioxus ovary.

Sex steroids are essential hormones for reproduction in vertebrates. The existence of a sex steroidogenic pathway in invertebrates is controversial because cytochrome P450 (CYP) genes have not been detected in the genomes of an echinoderm and a urochordate. However, cloning and gene expressions of sex steroid-metabolizing enzymes have been reported in a cephalochordate.

Presence of sex steroids and cytochrome P450 genes in amphioxus.

The presence of sex steroids and their receptors has been demonstrated in all vertebrate groups from Agnatha to Mammalia but not in invertebrates. In genomic analyses of urochordates, cytochrome P450 (CYP) genes important for biosynthesis of sex steroids are absent. In the present study, we confirmed the presence of estrogen, androgen, and progesterone by using radioimmunoassay in gonads of amphioxus, Branchiostoma belcheri, which is considered to be evolutionarily closer to vertebrates than other invertebrates.

Sperm-derived sperm motility-initiating substance from amphioxus Branchiostoma belcheri.

The sperm of amphioxus, Branchiostoma belcheri, were immotile when excised from the testis and suspended in seawater. The sperm became motile upon spawning in natural seawater, suggesting mechanisms that triggered sperm motility during spawning. When a male amphioxus that underwent spawning was transferred to a cup containing a small amount of natural seawater, and then the seawater containing the spawned sperm was centrifuged, the supernatant caused motility initiation in the immotile sperm from the testis.

Cephalochordate melanopsin: evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells.

Animal photoreceptor cells can be classified into two distinct types, depending on whether the photopigment is borne on the membrane of a modified cilium (ciliary type) or apical microvilli (rhabdomeric type) [1]. Ciliary photoreceptors are well known as vertebrate rods and cones and are also found in several invertebrates. The rhabdomeric photoreceptor, in contrast, is a predominant type of invertebrate visual cell, but morphologically identifiable rhabdomeric photoreceptors have never been found in vertebrates.

Acrosome reaction in spermatozoa from the amphioxus Branchiostoma belcheri (Cephalochordata, Chordata).

The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected.

Gonadal state of wild amphioxus populations and spawning success in captive conditions during the breeding period in Japan.

Adult individuals of amphioxus (Branchiostoma belcheri) were collected by dredging from a research vessel at selected stations in two areas off the coast of Japan in July 2000: Deyama and Takamatsu so named by local fishermen in the Enshu Nada Sea. The number of males collected exceeded that of females at all the stations in Takamatsu and at four of five stations in Deyama. The over all sex ratio (males : females) of the collected animals was 1.