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A muscle-specic monoclonal antibody, termed Mu-2, recognizes a single 220-kd polypeptide which first appears in early tailbud-stage embryos of the ascidian Halocynthia roretzi (17). In the present investigation, a cDNA library was prepared from poly(A)+ RNA isolated from tailbud embryos. The resulting cDNAs were inserted into the expression vector γgt11, and the library was screened with the Mu-2. One of positive clones, containing an insert about 1.6-kb in length, was selected and subcloned. Partial sequence analyses at the 5′ terminus and at the 3′ terminus of the 1.6-kb cDNA allowed the amino acid sequence. The deduced sequence showed extensive homology to the rodent myosin heavy chain protein. Using an antisense RNA probe generated from the cDNA, Northern blot hybridization was carried out to measure the level of the myosin heavy chain gene transcripts during normal embryogenesis. These transcripts were not detected at pregastruia stages, after which they accumulated rapidly.

Embryological development takes place in four dimensions and requires the existence of time measuring processes within the embryo. Evidence is accumulating that suggests that the emergence of many events during early embryonic development is controlled by timing mechanisms or developmental clocks. The purpose of this work is to review recent studies on developmental timing with speculations about underlying possible mechanisms. It is an attractive idea that the development of an embryo is timed by a single clock set in motion at fertilization, but this seems not feasible. The clock mechanism which determines the time of initiation of cellular differentiation may be independent of that for the timing of morphogenesis. The clock mechanism for cellular differentiation may be closely associated with the cycles of DNA replication, while the clock which counts the time to onset of early morphogenetic events is found in the cytoplasm. These ideas can provide a framework which may help to organize existing observations and to stimulate new experimental approaches to the problem.

When isolated embryonic cells of the medaka, Oryzias latipes, were brought into physical contact within 30 sec of dissociation, cell fusion could be induced, but fusion could no longer be induced between cells that had been isolated for more than 90 sec. Observations with a scanning electron microscope revealed that the cell surface was smooth immediately after dissociation. Cells examined about 30 sec after dissociation, however, had a great wealth of surface folds. The cell surface observed about 90 sec after dissociation was again smooth. Fusing cells were highly plicated. At the contact sites of the two fusing cells many plicae were intertwined in complex patterns. The transitory existence of cell surface folds is believed to be a prerequisite for this cell fusion.

The ascidian embryo has long provided a model system for ‘mosaic’ development. This article reviews recent advances in the study of ascidian developmental biology. These include: (a) the re-analysis of cell lineages in ascidian embryos with the ascertainment of developmental fates of every blastomere of a 110-cell embryo; (b) the development of several tissue-specific monoclonal antibodies; (c) the investigation and description of cell cycle requirements for differentiation; it has been found that neither cytokinesis nor nuclear division is required for differentiation, but that several rounds of DNA replication are essential for the expression of certain tissue-specific genes; and (d) the demonstration by new descriptive and experimental studies of the presence of cytoplasmic factors or determinants responsible for specification of embryonic cell features; myoplasm which is thought to contain muscle determinants has been isolated, and immunological attempts to elucidate the molecular nature of the factors have begun.

The surface of eggs of the ascidian Halocynthia roretzi, observed with SEM, is essentially smooth until immediately before cell division when numerous microvilli appear and remain during cytokinesis. As the dividing blastomeres become closely adherent, however, the microvilli disappear and the eggs recover their smooth surface. This periodic appearance-disappearance of microvilli is repeated at each cleavage cycle up to at least the 32-cell stage. During blastomere adhesion, microvilli that have appeared near the plane of the first cleavage or of the bilateral symmetry seem to fuse together across the plane to form a zipper-like complex of cytoplasmic processes, which might be responsible for attachment of the two halves of these bilaterally symmetrical embryos via the blastomeres bordering the plane of symmetry.

In order to determine whether or not a crucial number of DNA replications are prerequisite for cellular differentiation, we have studied development of a tissue-specific enzyme, muscle acetylcholinesterase (AChE) in the presumptive muscle cells of cleavage-arrested ascidian embryos. Embryos were cleavage-arrested with cytochalasin B (an inhibitor of cytokinesis) and aphidicolin (an inhibitor of DNA synthesis). The 64-ceIl-stage embryos which had been permanently cleavage-arrested with cytochalasin B developed AChE in all the eight presumptive muscle cells, but the same stage embryos which had been prevented from undergoing further divisions by simultaneous treatment with aphidicolin and cytochalasin did not produce AChE at all. Cytochalasin-arrested 76-cell-stage embryos were able to differentiate AChE in the ten presumptive muscle cells, while aphidicolin-cytochalasin-arrested 76-cell stages in as many as four cells. The early gastrulae which had been arrested with cytochalasin B produced AChE in all the sixteen presumptive muscle cells, while the same stage embryos arrested with aphidicolin and cytochalasin in as many as twelve cells. Cytochalasin-arrested late gastrulae developed AChE in twenty blastomeres, while aphidicolin-cytochalasin-arrested late gastrulae in eighteen cells. The presumptive muscle cells at these four stages consist of those of three different (seventh, eighth, and ninth) generations, and the relative positions of the blastomeres in the cleavage-arrested embryos remained fixed. Judging from the relative positions of the blastomeres, the AChE-producing cells in aphidicolin-cytochalasin-arrested embryos were always at eighth or ninth generation, while those with no AChE activity were certainly at seventh generation. Based on these findings it was supposed that aphidicolin-sensitive cell-cyclic events, presumably DNA replication, are closely associated with AChE development and that the eighth cleavage cycle may be ‘quantal’ for the histospecific enzyme development.

During ascidian embryogenesis a tissue-specific enzyme, muscle acetylcholinesterase (AChE) may first be detected histochemically in the presumptive muscle cells of the neurula. In order to investigate the ‘clock’ or counting mechanism that is determining the time when AChE first appears, Whittaker’s experiment (1973) has been repeated using eggs of the ascidian, Halocynthia roretzi. Embryos that had been permanently cleavage-arrested with cytochalasin B were able to differentiate AChE in their muscle lineage blastomeres. The time of first AChE occurrence in embryos that had been cleavage-arrested in the 32-cell stage with cytochalasin B was about the same as in normal embryos. This result indicates that the clock is not apparently regulated by the events of cytokinesis. The early gastrulae which had been arrested with colchicine or with colcemid could develop AChE activity, although no histochemically detectable AChE activity was observed in the cleavage-stage embryos that had been arrested with either drug. Therefore the clock does not seem to be controlled by the mitotic cycle of the nucleus. It is suggested that the cycle of DNA replication may be related to the regulation of the clock that is determining the time of development of histospecific protein.

Cell lineages during development of ascidian embryos were analyzed by injection of horseradish peroxidase as a tracer enzyme into identified cells at the one-, two-, four-, and eight-cell stages of the ascidians, Halocynthia roretzi, Ciona intestinalis , and Ascidia ahodori . Identical results were obtained with eggs of the three different species examined. The first cleavage furrow coincided with the bilateral symmetry plane of the embryo. The second furrow did not always divide the embryo into anterior and posterior halves as each of the anterior and posterior cell pairs gave rise to different tissues according to their destinies, which became more definitive in the cell pairs at the eight-cell stage. Of the blastomeres constituting the eight-cell stage embryo, the a4.2 pair (the anterior animal blastomeres) differentiated into epidermis, brain, and presumably sense organ and palps. Every descendant cell of the b4.2 pair (the posterior animal blastomeres) has been thought to become epidermis; however, the horseradish peroxidase injection probe revealed that the b4.2 pair gave rise to not only epidermis but also muscle cells at the caudal tip region of the developing tailbud-stage embryos. The A4.1 pair (the anterior vegetal blastomeres) developed into endoderm, notochord, brain stem, spinal cord, and also muscle cells next the caudal tip muscle cells. From the B4.1 pair (the posterior vegetal blastomeres) originated muscle cells of the anterior and middle parts of the tail, mesenchyme, endoderm, endodermal strand, and also notochord at the caudal tip region. These results clearly demonstrate that muscle cells are derived not only from the B4.1 pair, as has hitherto been believed, but also from both the b4.2 and A4.1 pairs.

In order to examine whether or not sex differentiation in the medaka, Oryzias latipes, is modified by the physiological level of sex hormones in the adult fish, trunk regions containing the gonads of newly hatched fry were transplanted unilaterally into the anterior chamber of the eyes of adult fish. The grafts could be classified into two types according to the vascularization. One type of graft developed well ; some of them protruded from the eyes of the host fish. In these grafts the connexion of the blood circulation between the graft and the host was detectable without exception. The other type of graft consisted of those specimens without vascularization. The grafts of this type did not grow in size; this condition seemed to be similar to the so-called in vivo culture in the anterior chamber of the eye in rodents. Most of these grafts, however, degenerated. Judging from the histology of the gonad in the graft, a genetic male graft in the eye of a fish developed into a testis, regardless of the sexuality of the host. This fact was confirmed by two series of experiments. The gonad of a genetic female fry developed into an ovary if the graft was transplanted into a female fish. On the other hand, the gonad of a genetic female graft transplanted into a male fish failed to develop into an ovary, but formed spermatogenetic cells in a gonad of an abnormal structure. Therefore, it is highly probable that the reversal of sex differentiation from genetic oogonia into spermatogenetic cells is accomplished by the physiological level of the sex hormones in male fish. On the contrary, the sex reversal of genetic males to females is not induced by the physiological level of female sex hormones.

Mitotic and meiotic activities of germ cells during early development in the medaka, Oryzias latipes, are dealt with in this report. Primordial germ cells were obviously distinguishable from somatic cells 3 days after fertilization and began to proliferate about 7 days after fertilization. The mean number of primordial germ cells increased during a period of 7–10 days after fertilization, reaching about 90 immediately before hatching. Newly hatched fry could be classified into two types according to the number and the nucleic activity of germ cells in the gonadal rudiment. One type consisted of fry containing about 100 germ cells and no cells in the meiotic prophase. In the other type of fry the number of germ cells increased by mitotic divisions and some of the cells began to enter into the meiotic prophase. During the course of further development the fry of the former type differen tiated into males and the latter into females. Therefore it can be concluded that the morphological sex differentiation of germ cells occurs at the time of hatching. However, no sexual differences in the histological structure of somatic elements in the gonad are observable at that time.

We have investigated the mechanism of an evolutionary change in ascidian muscle cell differentiation. The ascidians Molgula oculata and Molgula occulta are closely related species with different modes of development. M. oculata embryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereas M. occulta embryos develop into tailless larvae with undifferentiated vestigial muscle cells. The muscle actin gene MocuMA1 was isolated from an M. oculata genomic library. MocuMA1 is a single-copy, larval-type muscle actin gene which appears to lack introns. However, the 5' upstream region of MocuMA1 is sufficient to drive expression of a lacZ fusion construct in the larval muscle cells, implying that it is a functional gene. MocuMA1 mRNA first appears in the prospective muscle cells of M. oculata embryos during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected during M. occulta embryogenesis, although the same probe was capable of detecting muscle actin mRNA in more distantly related ascidian species with tail muscle cells. Interspecific hybrids produced by fertilizing M. occulta eggs with M. oculata sperm recover the ability to express muscle actin mRNA in the vestigial muscle cells, suggesting that trans-acting factors responsible for muscle actin gene expression are conserved in M. occulta. The presence of these trans-acting factors was confirmed by showing that the MocuMA1/lacZ fusion construct is expressed in the vestigial muscle cells of M. occulta larvae. The orthologous larval muscle actin genes MoccMA1a and MoccMA1b were isolated from a M. occulta genomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products nonfunctional. Although the 5' upstream regions of the M. occulta muscle actin genes also contain numerous changes, expression of MoccMA1a/lacZ and MoccMA1b/lacZ fusion constructs showed that they both retain specific promoter activity, although it is reduced in MoccMAlb. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression.

Embryonic cell specification and pattern formation in the ascidian embryo are controlled by prelocalized egg cytoplasmic determinants. In previous studies, we showed that overexpression of a maternal gene, posterior end mark (pem), whose transcript localizes to posterior-vegetal cytoplasm of the fertilized egg, causes a loss of the anterior and dorsal structures of the larva (Yoshida et al., Development 122, 2005-2012, 1996). In the present study, first we observed that lithium treatment resulted in reduction of the larval tail. Lineage tracing analyses revealed that descendants of the A4.1 blastomere of the 8-cell-stage embryo (which forms the greater part of notochord and nerve cord) were missing from the tail region, that they were translocated anteriorly into the trunk region, and that the fate of the A4.1-line notochord cells had changed to endoderm. These results suggest that lithium treatment affects the trunk-tail patterning during embryogenesis by changing the cell fate of specific cell lineages. Second, we showed that lithium treatment could rescue the anterior and dorsal structures in pem-overexpressed larvae. This result suggests that pem plays a role in the patterning of the ascidian embryo via a signaling cascade that is affected by lithium. Third, we isolated an ascidian beta-catenin gene and found that overexpression of beta-catenin in the A4.1 blastomere had effects very similar to lithium treatment, such as reduction of the tail and anterior translocation of A4.1 descendants. These results suggest that the target of lithium is, at least in part, the Wnt-signaling cascade and that pem may also function via this cascade.

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stages that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.

Acetylcholinesterase (AChE) is a tissue-specific enzyme of the muscle cells of ascidian embryos and its synthesis begins at the neurula stage. Embryos which had been permanently cleavage-arrested with cytochalasin B could develop AChE activity. The time of first AChE occurrence in embryos which had been arrested in the 32-cell stage with cytochalasin was about the same as in normal embryos. The nucleus in the cell of cytochalasin-arrested embryos divided in good synchrony with that of normal embryos. Embryos which had been continuously arrested with colchicine could also produce AChE activity at nearly the same time as did normal embryos. In the cell of colchicine-arrested embryos normal nuclear divisions did not occur, but the cell showed repeated cycles of nuclear envelope breakdown and nuclear envelope reformation in almost parallel with cell cycles of normal embryos. The cell of colchicine-arrested embryos incorporated [3H]thymidine. Aphidicolin, a specific inhibitor of DNA synthesis, prevented cleavages of ascidian eggs. Embryos which had been permanently arrested with aphidicolin in the cleavage stages up to the 64-cell stage did not develop AChE activity, while embryos which had been treated with it from the 76-cell stage onwards were found to be able to differentiate AChE activity. Based on these findings it was proposed that DNA replication is prerequisite for development of the histospecific protein and that the cycle of DNA replication is closely associated with the clock mechanism which is determining the time of initiation of the enzyme development.

Recent studies on temporal control of early amphibian development are reviewed. It is becoming clear that the development of an embryo is not timed by a single clock set in motion at fertilization, instead each developmental event seems to be timed by its own clock-like mechanism. The timing of developmental events is rigidly determined within embryonic cells, and usually can not be altered experimentally. One exception, however, is the timing of midblastula transition in amphibian embryos; recent studies have shown that its timing is regulated by the nucleocytoplasmic ratio. Several developmental events, particularly those associated with transcriptional activities, require DNA replication prior to their occurrence, suggesting an intimate relationship between DNA replication cycles and their onset. On the other hand, there are many other developmental events where timing is not controlled by the number of cell divisions, DNA replication cycles, or the nucleocytoplasmic ratio. Cytoplasmic machinery with autonomous oscillatory properties is thought to be involved in the timing of these events.

We have produced two monoclonal antibodies (Epi-1 and Epi-2) which specifically recognize epidermal cells and their derivative, the larval tunic, of developing embryos of the ascidian Halocynthia roretzi. The antigens, examined by indirect immunofluorescence staining, first appear at the early tailbud stage and are present until at least the swimming larval stage. There were distinct and separate puromycin and actinomycin D sensitivity periods for each antigen. Aphidicolin, a specific inhibitor of DNA synthesis, prevented the appearance of each antigen when embryos were exposed to the drug continuously from cleavage stages. These results suggest that the antigens are synthesized during embryogenesis by developing epidermal cells and that several rounds of DNA replication are required for the antigen expression. Early cleavage stage embryos, including fertilized but unsegmented eggs, in which cytokinesis had been blocked with cytochalasin B expressed the antigens, and blastomeres exhibiting the antigens were always of the epidermis lineage. In partial embryos produced by four separated blastomere pairs of the 8-cell embryos, the expression of antigens was seen only in those developed from the animal blastomere pairs, which are progenitors of epidermal cells. These observations indicate that differentiation of epidermal cells in ascidian embryos takes place in a typical “mosaic” fashion.

The brain of the ascidian larva comprises two pigment cells, termed the ocellus melanocyte and the otolith melanocyte. Cell lineage analysis has shown that the two bilateral pigment lineage cells (a-line blastomeres) in the animal hemisphere give rise to these melanocytes in a complementary manner. The results of the present investigation suggest that the specification of the fate of pigment cells proceeds in two distinct steps. First, the determination of pigment lineage cells requires an inductive interaction from the vegetal blastomeres of the A-line. Cell dissociation experiments demonstrated that the inductive interaction is completed by the midgastrula stage. However, the two bilaterally positioned cells destined to become the pigment cells in the first step are still equipotent at this stage in that they can give rise to either the ocellus or otolith. Thus, they constitute what is termed an “equivalence group.” In the second step, the individual fates of the two cells that compose the equivalence group are determined. Namely, one cell develops into an ocellus and the other cell develops into an otolith. Photoablation of one of the pigment precursor cells at various stages indicated that the second step of determination occurs at the midtailbud stage. It is suggested that the cue to choose one of the alternative developmental pathways may be positional information that exists along the anteroposterior axis. The second step of determination is thought to be mediated by a hierarchical interaction. In the absence of this interaction, melanocyte specification proceeds along the dominant pathway that results in the differentiation of an ocellus.

Background: The T-box family transcription-factor gene, Brachyury, has two expression domains with discretefunctions during animal embryogenesis. The primary domain, associated with the blastopore, is shared by mostmetazoans, while the secondary domain, involved in the notochord, is specific to chordates. In most animals,Brachyury is present in a single copy, but in cephalochordates, the most basal of the chordates, the gene is presentin two copies, suggesting allotment of the two domains to each of the duplicates.Results: In order to clarify whether Brachyury duplication occurred in the common ancestor of chordates afterwhich one of duplicates was lost in the urochordate and vertebrate lineages, we estimated phylogeneticrelationships of Brachyury genes and examined the synteny of a Brachyury-containing genomic region ofdeuterostomes with decoded genomes. The monophyletic origin of tandemly arranged Brachyury genes ofcephalochordates indicates that the tandem duplication occurred in the cephalochordate lineage, but not in thechordate ancestor.Conclusions: Our results thus suggest that, in the common ancestor of chordates, a single copy of Brachyuryacquired two expression domains and that the duplication was not involved in the acquisition of the notochord.However, in relation to regulatory mechanisms, both possibilities—namely a single copy with two domains andtwo copies with different domains—should be considered in future studies of Brachyury.

Background Dinoflagellates are unicellular marine and freshwater eukaryotes. They possess large nuclear genomes (1.5–245 gigabases) and produce structurally unique and biologically active polyketide secondary metabolites. Although polyketide biosynthesis is well studied in terrestrial and freshwater organisms, only recently have dinoflagellate polyketides been investigated. Transcriptomic analyses have characterized dinoflagellate polyketide synthase genes having single domains. The Genus Symbiodinium, with a comparatively small genome, is a group of major coral symbionts, and the S. minutum nuclear genome has been decoded. Results The present survey investigated the assembled S. minutum genome and identified 25 candidate polyketide synthase (PKS) genes that encode proteins with mono- and multifunctional domains. Predicted proteins retain functionally important amino acids in the catalytic ketosynthase (KS) domain. Molecular phylogenetic analyses of KS domains form a clade in which S. minutum domains cluster within the protist Type I PKS clade with those of other dinoflagellates and other eukaryotes. Single-domain PKS genes are likely expanded in dinoflagellate lineage. Two PKS genes of bacterial origin are found in the S. minutum genome. Interestingly, the largest enzyme is likely expressed as a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly of 10,601 amino acids, containing NRPS and PKS modules and a thioesterase (TE) domain. We also found intron-rich genes with the minimal set of catalytic domains needed to produce polyketides. Ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) along with other optional domains are present. Mapping of transcripts to the genome with the dinoflagellate-specific spliced leader sequence, supports expression of multifunctional PKS genes. Metabolite profiling of cultured S. minutum confirmed production of zooxanthellamide D, a polyhydroxy amide polyketide and other unknown polyketide secondary metabolites. Conclusion This genomic survey demonstrates that S. minutum contains genes with the minimal set of catalytic domains needed to produce polyketides and provides evidence of the modular nature of Type I PKS, unlike monofunctional Type I PKS from other dinoflagellates. In addition, our study suggests that diversification of dinoflagellate PKS genes comprises dinoflagellate-specific PKS genes with single domains, multifunctional PKS genes with KS domains orthologous to those of other protists, and PKS genes of bacterial origin. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2195-8) contains supplementary material, which is available to authorized users.

During ascidian embryogenesis mesenchymal cells proliferate and have no known function until after metamorphosis, when they give rise to various mesodermal tissues of the adult. Despite their simple lineage, specification mechanisms of the embryonic mesenchyme cells remain to be investigated; this is mainly due to lack of specific molecular markers for this cell type. Here we report that, in Halocynthia roretzi, zygotic expression of a cytoskeletal actin gene (HrCA1) begins at the late gastrula stage and that transcripts were predominantly distributed in embryonic mesenchyme cells, although some expression was observed in special cells of the central nervous system as well as in notochord cells. When HrCA1 expression was examined in cleavage-arrested embryos, it was found only in mesenchyme-lineage blastomeres of arrested 8-cell and later stages, consistent with the predominant expression of HrCA1 being in mesenchyme cells. To examine whether cell–cell contact until the 8-cell stage is required for mesenchyme cell specification, blastomeres were continuously dissociated during the period of 2–8-cell stages and division of the blastomeres was then arrested. Results showed that two of eight dissociated and division-arrested blastomeres from a single fertilized egg (presumably those corresponding to B4.1 cells) expressed HrCA1, suggesting that specification of embryonic mesenchyme cells does not require cell–cell contact until the 8-cell stage.

The tadpole larva of an ascidian develops 40 notochord cells in the center of its tail. Most of the notochord cells originate from the A-line precursors, among which inductive interactions are required for the subsequent differentiation of notochord. The presumptive-endoderm blastomeres or presumptive-notochord blastomeres themselves are inducers of notochord formation. Notochord induction takes place during the 32-cell stage. In amphibia, mesoderm induction is thought to be mediated by several growth factors, for example, activins and basic fibroblast growth factor (bFGF). In the ascidian, Halocynthia roretzi, treatment with bFGF of presumptive-notochord blastomeres that had been isolated at the early 32-cell stage promoted the formation of notochord at a low concentration of bFGF (0.02 ng/ml), while activin failed to induce notochord differentiation. The effect of bFGF reached a maximum at the end of the 32-cell stage and rapidly faded at the beginning of the subsequent cleavage, the time for full induction of notochord being at least 20 minutes. The expression of As-T, a previously isolated ascidian homolog of the mouse Brachyury (T) gene, starts at the 64cell stage and is detectable exclusively in the presumptivenotochord blastomeres. The present study showed that presumptive-notochord blastomeres, isolated at the early 32-cell stage, neither differentiated into notochord nor expressed the As-T gene. However, when the presumptivenotochord blastomeres were coisolated or recombined with inducer blastomeres, transcripts of As-T were detected. When presumptive-notochord blastomeres were treated with bFGF, the expression of the As-T gene was also detected. These results suggest that inductive interaction is required for the expression of the As-T gene and that the expression of the As-T gene is closely correlated with the determined state of the notochord-precursor cells.

The complete nucleotide sequence of the mitochondrial genome of the hemichordate Balanoglossus carnosus (acorn worm) was determined. The arrangement of the genes encoding 13 protein, 22 tRNA, and 2 rRNA genes is essentially the same as in vertebrates, indicating that the vertebrate and hemichordate mitochondrial gene arrangement is close to that of their common ancestor, and, thus, that it has been conserved for more than 600 million years, whereas that of echinoderms has been rearranged extensively. The genetic code of hemichordate mitochondria is similar to that of echinoderms in that ATA encodes isoleucine and AGA serine, whereas the codons AAA and AGG, whose amino acid assignments also differ between echinoderms and vertebrates, are absent from the B. carnosus mitochondrial genome. There are three noncoding regions of length 277, 41, and 32 bp: the larger one is likely to be equivalent to the control region of other deuterostomes, while the two others may contain transcriptional promoters for genes encoded on the minor coding strand. Phylogenetic trees estimated from the inferred protein sequences indicate that hemichordates are a sister group of echinoderms.

Background The interaction between biological researchers and the bioinformatics tools they use is still hampered by incomplete interoperability between such tools. To ensure interoperability initiatives are effectively deployed, end-user applications need to be aware of, and support, best practices and standards. Here, we report on an initiative in which software developers and genome biologists came together to explore and raise awareness of these issues: BioHackathon 2009. Results Developers in attendance came from diverse backgrounds, with experts in Web services, workflow tools, text mining and visualization. Genome biologists provided expertise and exemplar data from the domains of sequence and pathway analysis and glyco-informatics. One goal of the meeting was to evaluate the ability to address real world use cases in these domains using the tools that the developers represented. This resulted in i) a workflow to annotate 100,000 sequences from an invertebrate species; ii) an integrated system for analysis of the transcription factor binding sites (TFBSs) enriched based on differential gene expression data obtained from a microarray experiment; iii) a workflow to enumerate putative physical protein interactions among enzymes in a metabolic pathway using protein structure data; iv) a workflow to analyze glyco-gene-related diseases by searching for human homologs of glyco-genes in other species, such as fruit flies, and retrieving their phenotype-annotated SNPs. Conclusions Beyond deriving prototype solutions for each use-case, a second major purpose of the BioHackathon was to highlight areas of insufficiency. We discuss the issues raised by our exploration of the problem/solution space, concluding that there are still problems with the way Web services are modeled and annotated, including: i) the absence of several useful data or analysis functions in the Web service "space"; ii) the lack of documentation of methods; iii) lack of compliance with the SOAP/WSDL specification among and between various programming-language libraries; and iv) incompatibility between various bioinformatics data formats. Although it was still difficult to solve real world problems posed to the developers by the biological researchers in attendance because of these problems, we note the promise of addressing these issues within a semantic framework.