A chordate species lacking Nodal utilizes calcium oscillation and Bmp for left-right patterning.

Larvaceans are chordates with a tadpole-like morphology. In contrast to most chordates of which early embryonic morphology is bilaterally symmetric and the left-right (L-R) axis is specified by the Nodal pathway later on, invariant L-R asymmetry emerges in four-cell embryos of larvaceans. The asymmetric cell arrangements exist through development of the tailbud. The tail thus twists 90° in a counterclockwise direction relative to the trunk, and the tail nerve cord localizes on the left side. Here, we demonstrate that larvacean embryos have nonconventional L-R asymmetries: 1) L- and R-cells of the two-cell embryo had remarkably asymmetric cell fates; 2) Ca2+ oscillation occurred through embryogenesis; 3) Nodal, an evolutionarily conserved left-determining gene, was absent in the genome; and 4) bone morphogenetic protein gene (Bmp) homolog Bmp.a showed right-sided expression in the tailbud and larvae. We also showed that Ca2+ oscillation is required for Bmp.a expression, and that BMP signaling suppresses ectopic expression of neural genes. These results indicate that there is a chordate species lacking Nodal that utilizes Ca2+ oscillation and Bmp.a for embryonic L-R patterning. The right-side Bmp.a expression may have arisen via cooption of conventional BMP signaling in order to restrict neural gene expression on the left side.

Basic helix-loop-helix transcription factors in evolution: Roles in development of mesoderm and neural tissues.

Basic helix-loop-helix (bHLH) transcription factors have attracted the attention of developmental and evolutionary biologists for decades because of their conserved functions in mesodermal and neural tissue formation in both vertebrates and fruit flies. Their evolutionary history is of special interest because it will likely provide insights into developmental processes and refinement of metazoan-specific traits. This review briefly considers advances in developmental biological studies on bHLHs/HLHs. I also discuss recent genome-wide surveys and molecular phylogenetic analyses of these factors in a wide range of metazoans. I hypothesize that interactions between metazoan-specific Group A, D, and E bHLH/HLH factors enabled a sophisticated transition system from cell proliferation to differentiation in multicellular development. This control mechanism probably emerged initially to organize a multicellular animal body and was subsequently recruited to form evolutionarily novel tissues, which differentiated during a later ontogenetic phase.

Redundant mechanisms are involved in suppression of default cell fates during embryonic mesenchyme and notochord induction in ascidians.

During embryonic induction, the responding cells invoke an induced developmental program, whereas in the absence of an inducing signal, they assume a default uninduced cell fate. Suppression of the default fate during the inductive event is crucial for choice of the binary cell fate. In contrast to the mechanisms that promote an induced cell fate, those that suppress the default fate have been overlooked. Upon induction, intracellular signal transduction results in activation of genes encoding key transcription factors for induced tissue differentiation. It is elusive whether an induced key transcription factor has dual functions involving suppression of the default fates and promotion of the induced fate, or whether suppression of the default fate is independently regulated by other factors that are also downstream of the signaling cascade. We show that during ascidian embryonic induction, default fates were suppressed by multifold redundant mechanisms. The key transcription factor, Twist-related.a, which is required for mesenchyme differentiation, and another independent transcription factor, Lhx3, which is dispensable for mesenchyme differentiation, sequentially and redundantly suppress the default muscle fate in induced mesenchyme cells. Similarly in notochord induction, Brachyury, which is required for notochord differentiation, and other factors, Lhx3 and Mnx, are likely to suppress the default nerve cord fate redundantly. Lhx3 commonly suppresses the default fates in two kinds of induction. Mis-activation of the autonomously executed default program in induced cells is detrimental to choice of the binary cell fate. Multifold redundant mechanisms would be required for suppression of the default fate to be secure.

Bivalve-specific gene expansion in the pearl oyster genome: implications of adaptation to a sessile lifestyle.

INTRODUCTION: Bivalve molluscs have flourished in marine environments, and many species constitute important aquatic resources. Recently, whole genome sequences from two bivalves, the pearl oyster, Pinctada fucata, and the Pacific oyster, Crassostrea gigas, have been decoded, making it possible to compare genomic sequences among molluscs, and to explore general and lineage-specific genetic features and trends in bivalves. In order to improve the quality of sequence data for these purposes, we have updated the entire P. fucata genome assembly. RESULTS: We present a new genome assembly of the pearl oyster, Pinctada fucata (version 2.0). To update the assembly, we conducted additional sequencing, obtaining accumulated sequence data amounting to 193× the P. fucata genome. Sequence redundancy in contigs that was caused by heterozygosity was removed in silico, which significantly improved subsequent scaffolding. Gene model version 2.0 was generated with the aid of manual gene annotations supplied by the P. fucata research community. Comparison of mollusc and other bilaterian genomes shows that gene arrangements of Hox, ParaHox, and Wnt clusters in the P. fucata genome are similar to those of other molluscs. Like the Pacific oyster, P. fucata possesses many genes involved in environmental responses and in immune defense. Phylogenetic analyses of heat shock protein70 and C1q domain-containing protein families indicate that extensive expansion of genes occurred independently in each lineage. Several gene duplication events prior to the split between the pearl oyster and the Pacific oyster are also evident. In addition, a number of tandem duplications of genes that encode shell matrix proteins are also well characterized in the P. fucata genome. CONCLUSIONS: Both the Pinctada and Crassostrea lineages have expanded specific gene families in a lineage-specific manner. Frequent duplication of genes responsible for shell formation in the P. fucata genome explains the diversity of mollusc shell structures. These duplications reveal dynamic genome evolution to forge the complex physiology that enables bivalves to employ a sessile lifestyle in the intertidal zone.

Hemichordate genomes and deuterostome origins.

Acorn worms, also known as enteropneust (literally, 'gut-breathing') hemichordates, are marine invertebrates that share features with echinoderms and chordates. Together, these three phyla comprise the deuterostomes. Here we report the draft genome sequences of two acorn worms, Saccoglossus kowalevskii and Ptychodera flava. By comparing them with diverse bilaterian genomes, we identify shared traits that were probably inherited from the last common deuterostome ancestor, and then explore evolutionary trajectories leading from this ancestor to hemichordates, echinoderms and chordates. The hemichordate genomes exhibit extensive conserved synteny with amphioxus and other bilaterians, and deeply conserved non-coding sequences that are candidates for conserved gene-regulatory elements. Notably, hemichordates possess a deuterostome-specific genomic cluster of four ordered transcription factor genes, the expression of which is associated with the development of pharyngeal 'gill' slits, the foremost morphological innovation of early deuterostomes, and is probably central to their filter-feeding lifestyle. Comparative analysis reveals numerous deuterostome-specific gene novelties, including genes found in deuterostomes and marine microbes, but not other animals. The putative functions of these genes can be linked to physiological, metabolic and developmental specializations of the filter-feeding ancestor.

A genome-wide survey of bHLH transcription factors in the Placozoan Trichoplax adhaerens reveals the ancient repertoire of this gene family in metazoan.

Basic helix-loop-helix (bHLH) transcription factors play significant roles in multiple biological processes in metazoan cells. To address the evolutionary history of this gene family, comprehensive and detailed characterization in basal metazoans is essential. Here I report a genome-wide survey of bHLH genes in the Placozoan, Trichoplax adhaerens. The present survey revealed ancient origins of two orthologous families, 48-related-1/Fer1 and ASCb, which both belong to high-order Group A. Group A factors are mainly involved in neural and mesodermal differentiation. I also identified novel members of a Group E orthologous family previously thought to be unique to Homo sapiens. These were discovered in Trichoplax, Saccoglossus kowalevskii, Euperipatoides kanangrensis, and Crassostrea gigas, but apparently are not found in Drosophila melanogaster, Caenorhabditis elegans, or Nematostella vectensis. Furthermore, as reported previously, many unclassified Group A members were observed in Trichoplax. The present study provides important information to infer the ancestral state of bHLH components in the Metazoa.

MarinegenomicsDB: an integrated genome viewer for community-based annotation of genomes.

We constructed a web-based genome annotation platform, MarinegenomicsDB, to integrate genome data from various marine organisms including the pearl oyster Pinctada fucata and the coral Acropora digitifera. This newly developed viewer application provides open access to published data and a user-friendly environment for community-based manual gene annotation. Development on a flexible framework enables easy expansion of the website on demand. To date, more than 2000 genes have been annotated using this system. In the future, the website will be expanded to host a wider variety of data, more species, and different types of genome-wide analyses. The website is available at the following URL: http://marinegenomics.oist.jp.

Evolutionary aspects of variability in bHLH orthologous families: insights from the pearl oyster, Pinctada fucata.

Basic helix-loop-helix (bHLH) transcription factors play significant roles in multiple biological processes in metazoan cells. In recent work, we showed that three orthologous HLH families, pearl, amber, and peridot, have apparently been lost in the Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens lineages. To further address the gain and loss of bHLH proteins during bilaterian evolution, we examined the genome of the pearl oyster, Pinctada fucata, which has recently been sequenced. We characterized the putative full set 65 bHLH genes and showed that genes previously categorized into the orthologous family PTFb, actually fall into two distinct orthologous families, 48-related-1 and 48-related-2. We also identified a novel orthologous family, clockwork orange. Based on these newly identified orthologous family members and on orphan bHLH factors, we propose that genes encoding bHLH factors in bilaterians are not as evolutionarily stable as previously thought.

Draft assembly of the Symbiodinium minutum nuclear genome reveals dinoflagellate gene structure.

BACKGROUND: Dinoflagellates are known for their capacity to form harmful blooms (e.g., "red tides") and as symbiotic, photosynthetic partners for corals. These unicellular eukaryotes have permanently condensed, liquid-crystalline chromosomes and immense nuclear genome sizes, often several times the size of the human genome. Here we describe the first draft assembly of a dinoflagellate nuclear genome, providing insights into its genome organization and gene inventory. RESULTS: Sequencing reads from Symbiodinium minutum were assembled into 616 Mbp gene-rich DNA regions that represented roughly half of the estimated 1,500 Mbp genome of this species. The assembly encoded ∼42,000 protein-coding genes, consistent with previous dinoflagellate gene number estimates using transcriptomic data. The Symbiodinium genome contains duplicated genes for regulator of chromosome condensation proteins, nearly one-third of which have eukaryotic orthologs, whereas the remainder have most likely been acquired through bacterial horizontal gene transfers. Symbiodinium genes are enriched in spliceosomal introns (mean = 18.6 introns/gene). Donor and acceptor splice sites are unique, with 5' sites utilizing not only GT but also GC and GA, whereas at 3' sites, a conserved G is present after AG. All spliceosomal snRNA genes (U1-U6) are clustered in the genome. Surprisingly, the Symbiodinium genome displays unidirectionally aligned genes throughout the genome, forming a cluster-like gene arrangement. CONCLUSIONS: We show here that a dinoflagellate genome exhibits unique and divergent characteristics when compared to those of other eukaryotes. Our data elucidate the organization and gene inventory of dinoflagellates and lay the foundation for future studies of this remarkable group of eukaryotes.

A genomewide survey of bHLH transcription factors in the coral Acropora digitifera identifies three novel orthologous families, pearl, amber, and peridot.

Decoding the genome of the coral, Acropora digitifera, enabled us to characterize a nearly full set of 70 basic helix-loop-helix (bHLH) transcription factors in this organism. This number is comparable to 68 bHLH genes in the sea anemone, Nematostella vectensis, and larger than those in most other invertebrate metazoans. The 70 bHLH genes were assigned to 29 orthologous families previously reported. In addition, we identified three novel HLH orthologous families, which we designated pearl, amber, and peridot, increasing the number of orthologous families to 32. Pearl and amber orthologues were found in genomes and expressed sequenced tags (ESTs) of Mollusca and Annelida in addition to Cnidaria. Peridot orthologues were found in genomes and ESTs of Cephalochordata and Hemichordata in addition to Cnidaria. These three genes were likely lost in the clades of Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens during animal evolution.

Draft genome of the pearl oyster Pinctada fucata: a platform for understanding bivalve biology.

The study of the pearl oyster Pinctada fucata is key to increasing our understanding of the molecular mechanisms involved in pearl biosynthesis and biology of bivalve molluscs. We sequenced ~1150-Mb genome at ~40-fold coverage using the Roche 454 GS-FLX and Illumina GAIIx sequencers. The sequences were assembled into contigs with N50 = 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds with N50 = 14.5 kb. The pearl oyster genome is AT-rich, with a GC content of 34%. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). Version 1.0 of the P. fucata draft genome contains 23 257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. The genes include those reported to have an association with bio-mineralization. Genes encoding transcription factors and signal transduction molecules are present in numbers comparable with genomes of other metazoans. Genome-wide molecular phylogeny suggests that the lophotrochozoan represents a distinct clade from ecdysozoans. Our draft genome of the pearl oyster thus provides a platform for the identification of selection markers and genes for calcification, knowledge of which will be important in the pearl industry.

Analysis of large scale expression sequenced tags (ESTs) from the anural ascidian, Molgula tectiformis.

Anural ascidians show embryogenesis during which tail formation does not take place. This mode of development is a derived character acquired several times independently in ascidian evolution. We identified approximately 20,000 each ESTs (i. e. 10,000 clones each were sequenced from both 5' and 3' ends) of adult gonads, cleaving-embryos, gastrulae/neurulae, embryos before hatching, and hatched larvae of the anural ascidian Molgula tectiformis, in order to comprehensively investigate the molecular mechanism of tailless evolution. Analyses of these ESTs showed that in this species, (1) the expression of embryonic/larval muscle structural genes which are expressed abundantly during embryogenesis of the urodele ascidian Ciona intestinalis, is suppressed; (2) genes that encode proteins with no similarity to known proteins of other organisms are abundantly expressed; (3) genes that show similarity with those up-regulated at metamorphosis in urodele ascidians are up-regulated within several hours after hatching; and (4) 15 of 35 putative orthologues of the downstream components of Brachyury, a key transcription factor for ascidian notochord formation, were found in the ESTs, even though differentiation of notochord is suppressed in this species. We discuss these remarkable results that allow insight into the molecular mechanism(s) responsible for the anural mode of ascidian development.

Expression of a muscle determinant gene, macho-1, in the anural ascidian Molgula tectiformis.

In anural (tailless) ascidian species, functional embryonic muscle is not formed. In urodele (tailed) ascidians, macho-1 functions as a maternally supplied factor for embryonic muscle formation. The failure of embryonic muscle development in anural ascidians may be due to the suppression of macho-1 expression. In this paper, however, we report the expression of macho-1 in embryos of an anural ascidian, Molgula tectiformis. Although M. tectiformis has lost the developmental potential to form functional embryonic muscle, macho-1 was expressed in a very similar manner as in urodele ascidians. This result, together with those of previous studies, strongly suggests that in M. tectiformis the upstream genetic cascade responsible for muscle formation is intact, while the downstream cascade including the expression of muscle structural genes is severely affected.