Stable aquaculture of the Japanese lancelet Branchiostoma japonicum for 7 years.

Despite advances in the study on animal evolution in the last two decades, paucity of experimental data on cephalochordates comparable to those on the other chordates hinders an integrative understanding of chordate evolutionary history. To obtain lancelet data under well-controlled experiments, laboratory cultures of lancelets have been performed at several institutions. In a mass culture started in 2005, we have obtained up to three consecutive generations of Branchiostoma japonicum. Using sand substratum, survival rates of laboratory lancelets until maturation have improved to greater than 30%, much higher than compared to previously, and for adults the annual average survival rate was 82.3%. The high survival rate allows maintaining animals at least 6 years and potentially longer. Water temperatures lower than 23°C obviously reduced the frequency of spawning even after the onset of spawning period, and 1-2 days after changing the temperature at 25°C animals became spawned well. We also observed obvious sex reversal from male to female in individuals that had been cultured for 3 years or more. Our continuous culture has provided sufficient materials for vital experiments on early development and for studying metamorphosis, as well as for the conservation of wild populations. The subculture of successive laboratory generations will provide a valuable resource for genetic studies.

The Hox8 of the hemichordate Balanoglossus misakiensis.

Deuterostomes comprise a monophyletic group of animals that include chordates, xenoturbellids, and the Ambulacraria, which consists of echinoderms and hemichordates. The ancestral chordate probably had 14 Hox genes aligned linearly along the chromosome, with the posterior six genes showing an independent duplication compared to protostomes. In contrast, ambulacrarians are characterized by a duplication of the posterior Hox genes, resulting in three genes known as Hox11/13a, Hox11/13b, and Hox11/13c. Here, we isolated 12 Hox genes from the hemichordate Balanoglossus misakiensis and found an extra Hox gene that has not been reported in hemichordates. The extra B. misakiensis gene was suggested to be Hox8 from paralog-characteristic residues in its hexapepetide motif and homeodomain and a comparison with Strongylocentrotus purpuratus Hox genes. Our data suggest that the ancestor of echinoderms and hemichordates may have had a full complement of 12 Hox genes.

The lancelet and ammocoete mouths.

The evolutionary history of the vertebrate mouth has long been an intriguing issue in comparative zoology. When the prevertebrate state was considered, the oral structure in adult lancelets (amphioxus) was traditionally referred to because of its general similarity to that of the ammocoete larva of lampreys. The larval mouth in lancelets, however, shows a peculiar developmental mode. Reflecting this, the affinity of the lancelet mouth has long been argued, but is still far from a consensus. The increase in available data from molecular biology, comparative developmental biology, paleontology, and other related fields makes it prudent to discuss morphological homology and homoplasy. Here, we review how the lancelet mouth has been interpreted in the study of evolution of the vertebrate mouth, as well as recent advances in chordate studies. With this background of increased knowledge, our innervation analysis supports the interpretation that the morphological similarity in the oral apparatus between ammocoetes and lancelets is a homoplasy caused by their similar food habits.

Larval development of the oriental lancelet, Branchiostoma belcheri, in laboratory mass culture.

We are successfully maintaining a laboratory colony of the lancelet Branchiostoma belcheri bred in the laboratory. Based on living individuals in this mass culture, morphological characteristics from the seven-day larval to benthic juvenile stages have been studied. Most striking was that later larval development of B. belcheri showed great individual variation even in a rather stable culture environment. Metamorphosis first occurred on 60 days post fertilization (dpf) and was continuously observed throughout the present study up to 100 dpf. Morphological traits such as the number of primary gill slits and body size at the start of metamorphosis are apparently affected by culture condition. Body size measured in the largest individuals showed nearly linear growth at 0.087 mm/day. The variability found in larval development calls for caution when developmental stages and chronological ages are compared between populations. However, the developmental flexibility of this animal also raises the possibility that growth and sexual maturation could be controlled artificially in captivity.

Laboratory culture of the Oriental lancelet Branchiostoma belcheri.

To overcome difficulties in getting research materials of cephalochordate lancelets, which has severely hampered experimental studies of this animal, we have attempted to establish a culture system in the laboratory. Adult animals collected from the wild were maintained in 2.5-L plastic containers filled with natural seawater without sand substratum. They were fed daily with unicellular algae. About 25% of the animals collected in 2003, 2004, and 2005 developed gonads in our culture system. Some of the sexually mature animals collected in the breeding seasons in 2005 and 2006 spawned spontaneously in the plastic containers of this system. Broods obtained in 2005 were maintained longer than a year in a glass tank without sand substratum. The progeny born in the laboratory showed great individual variation in growth but metamorphosed normally, and some of them started to develop gonads around 10 months after fertilization. Our mass culture methods for both adults and their progeny made daily observation possible and allowed the constant spawning of animals collected from the wild, at least in the summer season. Our culture method saves labor in maintenance and is easily set up without any specific demands except for running seawater, though still required to better survival rate and spawning control. Lancelet populations maintained in the laboratory can promote studies on these animals across disciplines and especially contribute to elucidation of the evolutionary history of chordates.

Early development of amphioxus links evolutionary events with vertebrates.

Comparison of early development is a powerful approach to understand how spherical embryos set up the basis for body patterning. Localization of the germ plasm likely couples with the site of gastrulation in many animals including cnidarians. A center of single or complex Wnt signaling pathway(s) is also co-localized with germ plasm and plays a role primarily in antero-posterior patterning in most animals. In addition, a Nodal signaling center appears in deuterostomes perpendicular to Wnt signaling and governs dorso-ventral patterning. Wnt and Nodal signaling pathways function as orthogonal coordinates to pattern embryos in three-dimensions. Amphioxus early embryos establish similar coordinates, but the location of Wnt signaling along the equator might modify the function of the Nodal signaling center to specify the chordate basic body pattern inverting dorso-ventral polarity, which is thought to have occurred during the evolution of deuterostomes. Surprising similarities in early developmental processes found in cnidarians and deuterostomes, and divergences of cnidarians-bilaterians, deuterostomes-protostomes, and ambulacrarians-chordates occurred one after another possibly within a geologically short period may link molecular mechanisms that gave rise to bilaterians, deuterostomes, and chordates.

Evolutionary history of the extant amphioxus lineage with shallow-branching diversification.

Amphioxus or lancelets have been regarded as a key animal in understanding the origin of vertebrates. However, the evolutionary history within this lineage remains unexplored. As the amphioxus lineage has likely been separated from other chordates for a very long time and displays a marked left-right asymmetry, its evolutionary history is potentially helpful in better understanding chordate and vertebrate origins. We studied the phylogenetic relationships within the extant amphioxus lineage based on mitochondrial genomes incorporating new Asymmetron and Epigonichthys populations, and based on previously reported nuclear transcriptomes. The resulting tree patterns are consistent, showing the Asymmetron clade diverging first, followed by the Epigonichthys and Branchiostoma clades splitting. Divergence time estimates based on nuclear transcriptomes with vertebrate calibrations support a shallow diversification of the extant amphioxus lineage in the Tertiary. These estimates fit well with the closure of seaways between oceans by continental drift, ocean currents, and present geographical distributions, and suggest a long cryptic history from the origin of amphioxus to its most recent diversification. Deduced character polarities based on phylogenetic analyses suggest that the common ancestor of the extant amphioxus existed in a tiny epibenthic state with larva-like appearance of extant amphioxus, likely with ciliate epidermis.

Acquisition of the dorsal structures in chordate amphioxus.

Acquisition of dorsal structures, such as notochord and hollow nerve cord, is likely to have had a profound influence upon vertebrate evolution. Dorsal formation in chordate development thus has been intensively studied in vertebrates and ascidians. However, the present understanding does not explain how chordates acquired dorsal structures. Here we show that amphioxus retains a key clue to answer this question. In amphioxus embryos, maternal nodal mRNA distributes asymmetrically in accordance with the remodelling of the cortical cytoskeleton in the fertilized egg, and subsequently lefty is first expressed in a patch of blastomeres across the equator where wnt8 is expressed circularly and which will become the margin of the blastopore. The lefty domain co-expresses zygotic nodal by the initial gastrula stage on the one side of the blastopore margin and induces the expression of goosecoid, not-like, chordin and brachyury1 genes in this region, as in the oral ectoderm of sea urchin embryos, which provides a basis for the formation of the dorsal structures. The striking similarity in the gene regulations and their respective expression domains when comparing dorsal formation in amphioxus and the determination of the oral ectoderm in sea urchin embryos suggests that chordates derived from an ambulacrarian-type blastula with dorsoventral inversion.

Amphioxus mouth after dorso-ventral inversion.

INTRODUCTION: Deuterostomes (animals with 'secondary mouths') are generally accepted to develop the mouth independently of the blastopore. However, it remains largely unknown whether mouths are homologous among all deuterostome groups. Unlike other bilaterians, in amphioxus the mouth initially opens on the left lateral side. This peculiar morphology has not been fully explained in the evolutionary developmental context. We studied the developmental process of the amphioxus mouth to understand whether amphioxus acquired a new mouth, and if so, how it is related to or differs from mouths in other deuterostomes. RESULTS: The left first somite in amphioxus produces a coelomic vesicle between the epidermis and pharynx that plays a crucial role in the mouth opening. The vesicle develops in association with the amphioxus-specific Hatschek nephridium, and first opens into the pharynx and then into the exterior as a mouth. This asymmetrical development of the anterior-most somites depends on the Nodal-Pitx signaling unit, and the perturbation of laterality-determining Nodal signaling led to the disappearance of the vesicle, producing a symmetric pair of anterior-most somites that resulted in larvae lacking orobranchial structures. The vesicle expressed bmp2/4, as seen in ambulacrarian coelomic pore-canals, and the mouth did not open when Bmp2/4 signaling was blocked. CONCLUSIONS: We conclude that the amphioxus mouth, which uniquely involves a mesodermal coelomic vesicle, shares its evolutionary origins with the ambulacrarian coelomic pore-canal. Our observations suggest that there are at least three types of mouths in deuterostomes, and that the new acquisition of chordate mouths was likely related to the dorso-ventral inversion that occurred in the last common ancestor of chordates.

Expression of Bblhx3, a LIM-homeobox gene, in the development of amphioxus Branchiostoma belcheri tsingtauense.

Amphioxus Bblhx3 was identified as a LIM-homeobox gene expressed in gastrulae. Structural analysis suggested that it is a member of lhx3 but not of lhx1 gene group. Whole mount in situ hybridization revealed that expression of Bblhx3 was initiated at the early gastrula stage and continued at least until 10-day larvae. Expression of Bblhx3 first appeared in the vegetal and future dorsal area in initial gastrulae and became restricted to the endoderm during gastrulation. In neurulae and early larvae, Bblhx3 was expressed in the developing neural tube, the notochord and preoral pit lineage. In 10-day larvae, Bblhx3 was expressed only in the preoral pit. This expression pattern is apparently distinct from that of vertebrate lhx3 genes that are not expressed during gastrulation.

Development of oral and branchial muscles in lancelet larvae of Branchiostoma japonicum.

The perforated pharynx has generally been regarded as a shared characteristic of chordates. However, there still remains phylogenetic ambiguity between the cilia-driven system in invertebrate chordates and the muscle-driven system in vertebrates. Giant larvae of the genus Asymmetron were reported to develop an orobranchial musculature similar to that of vertebrates more than 100 years ago. This discovery might represent an evolutionary link for the chordate branchial system, but few investigations of the lancelet orobranchial musculature have been completed since. We studied staged larvae of a Japanese population of Branchiostoma japonicum to characterize the developmental property of the orobranchial musculature. The larval mouth and the unpaired primary gills develop well-organized muscles. These muscles function only as obturators of the openings without antagonistic system. As the larval mouth enlarged posteriorly to the level of the ninth myomere, the oral musculature was fortified accordingly without segmental patterning. In contrast, the iterated branchial muscles coincided with the dorsal myomeric pattern before metamorphosis, but the pharynx was remodeled dynamically irrespective of the myomeric pattern during metamorphosis. The orobranchial musculature disappeared completely during metamorphosis, and adult muscles in the oral hood and velum, as well as on the pterygial coeloms developed independently. The lancelet orobranchial musculature is apparently a larval adaptation to prevent harmful intake. However, vestigial muscles appeared transiently with the secondary gill formation suggest a bilateral ancestral state of muscular gills, and a segmental pattern of developing branchial muscles without neural crest and placodal contributions is suggestive of a precursor of vertebrate branchiomeric pattern.

A diploblastic radiate animal at the dawn of cambrian diversification with a simple body plan: distinct from Cnidaria?

BACKGROUND: Microfossils of the genus Punctatus include developmental stages such as blastula, gastrula, and hatchlings, and represent the most complete developmental sequence of animals available from the earliest Cambrian. Despite the extremely well-preserved specimens, the evolutionary position of Punctatus has relied only on their conical remains and they have been tentatively assigned to cnidarians. We present a new interpretation of the Punctatus body plan based on the developmental reconstruction aided by recent advances in developmental biology. RESULTS: Punctatus developed from a rather large egg, gastrulated in a mode of invagination from a coeloblastura, and then formed a mouth directly from the blastopore. Spiny benthic hatchlings were distinguishable from swimming or crawling ciliate larvae found in cnidarians and sponges. A mouth appeared at the perihatching embryonic stage and was renewed periodically during growth, and old mouths transformed into the body wall, thus elongating the body. Growing animals retained a small blind gut in a large body cavity without partitioning by septa and did not form tentacles, pedal discs or holdfasts externally. A growth center at the oral pole was sufficient for body patterning throughout life, and the body patterning did not show any bias from radial symmetry. CONCLUSIONS: Contrary to proposed cnidarian affinity, the Punctatus body plan has basic differences from that of cnidarians, especially concerning a spacious body cavity separating ectoderm from endoderm. The lack of many basic cnidarian characters in the body patterning of Punctatus leads us to consider its own taxonomic group, potentially outside of Cnidaria.

Development of deciduous and permanent dentitions in the upper jaw of the house shrew (Suncus murinus).

The diphyodont tooth replacement in mammals is characterized by a single replacement of a deciduous dentition by a permanent dentition. Despite its significance in mammalian biology and paleontology, little is known about the developmental mechanisms regulating the diphyodont replacement. Because the mouse never replaces its teeth, this study used the house shrew, Suncus murinus, as a model to investigate the control of the diphyodont replacement of a deciduous dentition by successions and additions of permanent teeth. Using morphological and gene expression analyses of serial sections, we have demonstrated the development of the upper dentition of the house shrew. In this species, the deciduous tooth germs are formed but soon become vestigial, whereas the successional and accessional (molar) germs are subsequently formed and developed. There are distinct Shh expression domains in the deciduous, successional, and accessional tooth germs, and those of the latter two germs are identified from the appearance of their primary enamel knots. The developmental sequence of tooth germs in the house shrew indicates that two adjacent primary enamel knots of the successional and accessional germs do not develop simultaneously, but with a constant time lag. We suggest that this mode of tooth succession and accession can be explained by a sequential inhibitory cascade model in which the timing of initiation and the spacing of tooth development are determined by the inhibition from the primary enamel knots of developmentally preceding adjacent tooth germs.

Tiny sea anemone from the Lower Cambrian of China.

BACKGROUND: Abundant fossils from the Ediacaran and Cambrian showing cnidarian grade grossly suggest that cnidarian diversification occurred earlier than that of other eumetazoans. However, fossils of possible soft-bodied polyps are scanty and modern corals are dated back only to the Middle Triassic, although molecular phylogenetic results support the idea that anthozoans represent the first major branch of the Cnidaria. Because of difficulties in taxonomic assignments owing to imperfect preservation of fossil cnidarian candidates, little is known about forms ancestral to those of living groups. METHODS AND FINDINGS: We have analyzed the soft-bodied polypoid microfossils Eolympia pediculata gen. et sp. nov. from the lowest Cambrian Kuanchuanpu Formation in southern China by scanning electron microscopy and computer-aided microtomography after isolating fossils from sedimentary rocks by acetic acid maceration. The fossils, about a half mm in body size, are preserved with 18 mesenteries including directives bilaterally arranged, 18 tentacles and a stalk-like pedicle. The pedicle suggests a sexual life cycle, while asexual reproduction by transverse fission also is inferred by circumferential grooves on the body column. CONCLUSIONS: The features found in the present fossils fall within the morphological spectrum of modern Hexacorallia excluding Ceriantharia, and thus Eolympia pediculata could be a stem member for this group. The fossils also demonstrate that basic features characterizing modern hexacorallians such as bilateral symmetry and the reproductive system have deep roots in the Early Cambrian.

Dynamic modification of oral innervation during metamorphosis in Branchiostoma belcheri, the oriental lancelet.

The oral apparatus in lancelets undergoes a remarkable modification during larval development, especially during metamorphosis, when the oral innervation is radically altered. The larval mouth opens on the left side at the early larval stage, and a peripheral nerve network, the oral nerve ring (ONR), develops around it. The ONR enlarges as the mouth expands caudally, eventually receiving fibers from nerves as far back as the tenth on the left side. The mouth shrinks during metamorphosis, and with this change the ONR regresses; the posterior sixth to tenth nerves become freed from the connection with the ONR, whereas the fourth and fifth nerves retain their connections. This modification is the basis for the asymmetric innervation to the velum. There is no mesodermal or mesenchymal restriction for guiding nerve patterning as typically found in vertebrate cranial nerves. Rather, it seems to be the ONR, which has no counterpart in vertebrates, that plays pivotal roles for patterning the nervous system in the oral region. The oral innervation pattern in lancelets represents a derived character state that may be related to the asymmetry of the ancestral body and head.

Development of heterodont dentition in house shrew (Suncus murinus).

Mammalian heterodont dentition comprises incisors, canines, premolars, and molars. Although there has been intensive research, the patterning of these specific tooth types has not yet been elucidated. In order for the gene expression data to be linked with tooth type determination, it is first necessary to determine precisely the incisor-, canine-, premolar-, and molar-forming regions in the jaw primordia. To accomplish this, we studied dentition development in the house shrew (Suncus murinus), which has retained all the tooth types, using three-dimensional reconstructions from serial histological sections and the Sonic hedgehog (Shh) expression patterns. Before the appearance of morphological signs of odontogenesis, Shh expression localized to the presumptive tooth-forming regions, in which the mesial and distal expression domains corresponded to the incisor- and premolar-forming regions, respectively. The upper incisor region was found to extend across the boundary between the frontonasal and the maxillary processes. The canine-forming regions later appeared in the intermediate portions of the maxillary and the mandibular processes. The molar-forming regions later appeared distal to the initially demarcated tooth-forming regions by secondary extension of the distal ends. The demarcation visualized by the Shh expression pattern in the jaw primordia of the house shrew probably represents the basic developmental pattern of mammalian heterodont dentition.

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates.

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/FoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage.

Comparison of expression patterns of fibroblast growth factor 8, bone morphogenetic protein 4 and sonic hedgehog in jaw development of the house shrew, Suncus murinus.

To elucidate the mechanism underlying jaw development in mammals, we used a new laboratory animal, Suncus murinus (house shrew, an insectivore) as the subject for the investigation, because Suncus has all types of teeth (incisor, canine, premolar and molar) in its upper and lower jaws and is thought to be a good model animal having a general mammalian tooth pattern. At the start, by use of degenerate primers we cloned Suncus homologues of fibroblast growth factor 8 (sFgf8), bone morphogenetic protein 4 (sBmp4) and sonic hedgehog (sShh) genes from cDNA library derived from whole Suncus embryos at day 12 (E12). Thereafter, we examined the expression patterns of these genes in the jaw development of Suncus E11-16 embryos (for mouse E9.5-12 embryos). sFgf8 and sBmp4 were expressed in E11 but not in E15 and onward during orofacial development. sShh was expressed from E11 onward, and its expression was increased in the orofacial area. The expression pattern of sFgf8 in the maxillary and mandibular arches of E14 coincided with the area of the presumptive tooth arch. However, sShh and sBmp4 were expressed only in the outer area (= buccal/labial side) of presumptive tooth arch. Thus, these 3 genes showed specific expression pattern in jaw development of Suncus, and their distributions did not overlap each other except in a few regions. These findings suggest that sFgf8, sBmp4 and sShh have a specific function respectively during jaw development in Suncus murinus.

beta-Catenin in early development of the lancelet embryo indicates specific determination of embryonic polarity.

The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.

Amphi-Eomes/Tbr1: an amphioxus cognate of vertebrate Eomesodermin and T-Brain1 genes whose expression reveals evolutionarily distinct domain in amphioxus development.

A cDNA for a novel T-box containing gene was isolated from the amphioxus Branchiostoma belcheri. A molecular phylogenetic tree constructed from the deduced amino acid sequence of the isolated cDNA indicates that this gene belongs to the T-Brain subfamily. In situ hybridization reveals that the expression is first detected in the invaginating archenteron at the early gastrula stage and this expression is down-regulated at the neurula stage. In early larvae, the expression appears again and transcripts are detected exclusively in the pre-oral pit (wheel organ-Hatschek's pit of the adult). In contrast to the vertebrate counterparts, no transcripts are detected in the brain vesicle or nerve cord throughout the development. These results are interpreted to mean that a role of T-Brain products in vertebrate forebrain development was acquired after the amphioxus was split from the lineage leading to the vertebrates. On the other hand, comparison of the tissue-specific expression domain of T-Brain genes and other genes between amphioxus and vertebrates revealed that the pre-oral pit of amphioxus has several molecular features which are comparable to those of the vertebrate olfactory and hypophyseal placode.