Cymric, a Maternal and Zygotic HTK-16-Like SHARK Family Tyrosine Kinase Gene, Is Disrupted in Molgula occulta, a Tailless Ascidian.

AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, cymric (Uro-1), a HTK-16-like (HydraTyrosineKinase-16) gene, identified in a subtractive screen for maternal ascidian cDNAs in Molgula oculata, an ascidian species with a tadpole larva. The cymric gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed M. oculata but is not detected in eggs or embryos of the closely related tailless species Molgula occulta. There is a large insertion in cymric in the M. occulta genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The cymric amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the HTK-16-like gene found in other ascidians. Thus, the ascidian cymric genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that cymric is lacking the tyrosine kinase domain in the tailless M. occulta, although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic HTK-16-like tyrosine kinase is another described pseudogene from M. occulta and appears not to be necessary for adult development.

Photocatalytic chemoselective cleavage of C-O bonds under hydrogen gas- and acid-free conditions.

In the presence of a palladium-loaded TiO2 photocatalyst, the cleavage of benzyl phenyl ether in low-molecular-weight alcohol solvents under de-aerated conditions afforded toluene and phenol simultaneously in a 1 : 1 molar ratio.

Cytoplasmic localization and reorganization in ascidian eggs: role of postplasmic/PEM RNAs in axis formation and fate determination.

Localization of maternal molecules in eggs and embryos and cytoplasmic movements to relocalize them are fundamental for the orderly cellular and genetic processes during early embryogenesis. Ascidian embryos have been known as 'mosaic eggs' because of their autonomous differentiation abilities based on localized cell fate determinants. This review gives a historical overview of the concept of cytoplasmic localization, and then explains the key features such as ooplasmic movements and cell lineages that are essential to grasp the process of ascidian development mediated by localized determinant activities. These activities are partly executed by localized molecules named postplasmic/PEM RNAs, originating from approximately 50 genes, of which the muscle determinant, macho-1, is an example. The cortical domain containing these RNAs is relocalized to the posterior-vegetal region of the egg by cytoskeletal movements after fertilization, and plays crucial roles in axis formation and cell fate determination. The cortical domain contains endoplasmic reticulum and characteristic granules, and gives rise to a subcellular structure called the centrosome-attracting body (CAB), in which postplasmic/PEM RNAs are highly concentrated. The CAB is responsible for a series of unequal partitionings of the posterior-vegetal cytoplasmic domain and the postplasmic/PEM RNAs at the posterior pole during cleavage. Some components of this domain, which is rich in granules, are eventually inherited by prospective germline cells with particular postplasmic/PEM RNAs such as vasa. The postplasmic/PEM RNAs are classified into two groups according to their final cellular destinations and localization pathways. Localization of these RNAs is regulated by specific nucleotide sequences in the 3' untranslated regions (3'UTRs).

The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program.

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.

The functional analysis of Type I postplasmic/PEM mRNAs in embryos of the ascidian Halocynthia roretzi.

Maternal factors, such as a muscle determinant macho-1 mRNA that is localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, are crucial for embryonic axis formation and cell fate specification. Maternal mRNAs that show an identical posterior localization pattern to that of macho-1 in eggs and embryos are called Type I postplasmic/PEM mRNAs. We investigated the functions of five of the nine Type I mRNAs so far known in Halocynthia roretzi: Hr-Wnt-5, Hr-GLUT, Hr-PEM3, Hr-PEN1, and Hr-PEN2. Suppression of their functions with specific antisense morpholino oligonucleotides (MOs) had effects on the formation of various tissues: Hr-Wnt-5 on notochord, muscle, and mesenchyme, although zygotic function of Hr-Wnt-5 is responsible for notochord formation; Hr-GLUT on notochord, mesenchyme, and endoderm; and Hr-PEN2 on muscle, mesenchyme, and endoderm. On the other hand, Hr-PEM3 and Hr-PEN1 MOs seemed to have no effect. We conclude that the functions of at least some localized maternal Type I postplasmic/PEM mRNAs are necessary for early embryonic patterning in ascidians.

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo.

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.

Evolutionarily conserved non-AUG translation initiation in NAT1/p97/DAP5 (EIF4G2).

Only a few cases of exclusive translation initiation at non-AUG codons have been reported. We recently demonstrated that mammalian NAT1 mRNA, encoded by EIF4G2, uses GUG as its only translation initiation codon. In this study, we identified NAT1 orthologs from chicken, Xenopus, and zebrafish and found that in all species, the GUG codon also serves as the initiation codon. In all species, the GUG codon fulfilled the reported requirements for non-AUG initiation: an optimal Kozak motif and a downstream hairpin structure. Site-directed mutagenesis showed that nucleotides at positions -3 and +4 are critical for the GUG-mediated translation initiation in vitro. We found that NAT1 orthologs in Drosophila melanogaster and Halocynthia roretzi also use non-AUG start codons, demonstrating evolutionary conservation of the noncanonical translation initiation.

An ascidian homolog of vertebrate iodothyronine deiodinases.

In all classes of vertebrates, the deiodination of the prohormone T(4) to T(3) represents an essential activation step in thyroid hormone action. The possible presence of iodothyronine deiodinase activity in protochordates has been demonstrated in vivo. Recent molecular cloning of the genomes and transcripts of several ascidian species allows further investigation into thyroid-related processes in ascidians. A cDNA clone from Halocynthia roretzi (hrDx) was found to have significant homology (30% amino acid identity) with the iodothyronine deiodinase gene sequences from vertebrates, including the presence of an in-frame UGA codon that might encode a selenocysteine (SeC) in the active site. Because it was not certain that the 3' untranslated region (UTR) contained a SeC insertion sequence (SECIS) element essential for SeC incorporation, a chimeric expression vector of the hrDx coding sequence and the rat deiodinase SECIS element was produced, as well as an expression vector containing the intact hrDx cDNA. COS, CHO, and HEK cells were transfected with these vectors, and deiodinase activity was measured in cell homogenates. Outer-ring deiodinase activity was detected using both T(4) and reverse T(3) as substrates, and activity was enhanced by the presence of the reductive cofactor dithiothreitol. The enzyme activity was optimal during incubation between 20 and 30 C (pH 6-7) and was strongly inhibited by gold-thioglucose. The Halocynthia deiodinase appears to be a high Michaelis-Menten constant (K(m)) enzyme (K(m) reverse T(3), 2 microM; and K(m) T(4), 4 microM). Deiodinase activity was completely lost upon the substitution of the SeC residue in the putative catalytic center by either cysteine or alanine. Transfection of the full-length hrDx cDNA produced deiodinase activity confirming the presence of a SECIS element in the 3'UTR, as revealed by the SECISearch program. In conclusion, our results show, for the first time, the existence of an ascidian iodothyronine outer-ring deiodinase. This raises the hypothesis that, in protochordates, the prohormone T(4) is activated by enzymatic outer-ring deiodination to T(3).

Neural expression of the Huntington's disease gene as a chordate evolutionary novelty.

Huntington's disease is a progressive neuro-degenerative disorder in humans, which is scharacterized by onset of dementia, muscular ataxia, and death. Huntington's disease is caused by the expansion of the polyglutamine (polyQ) tract in the N-terminus of the HD protein (Huntingtin). CAG expansion is a dominant gain of function mutation that affects striated neurons in the brain (Cattaneo, 2003, News Physiol Sci 18:34). The evolutionary origins of the vertebrate Hd gene are not well understood. In order to address the evolutionary history of the Hd gene, we have cloned and characterized the expression of the Hd gene in two invertebrate deuterostomes, an echinoderm and an ascidian, and have examined the expression patterns in a phylogenetic context. Echinoderms are basal deuterostomes and ascidians are basal chordates; both are useful for understanding the origins of and evolutionary trends in genes important in vertebrates such as the Huntigton's disease gene. Expression of Hd RNA is detected at all stages of development in both the echinoderm and ascidian studied. In the echinoderm Heliocidaris erythrogramma, Hd is expressed in coelomic mesodermal tissue derivatives, but not in the central nervous system. In the ascidian Halocynthia roretzi expression is located in both mesoderm and nervous tissue. We suggest that the primitive deuterostome expression pattern is not neural. Thus, neural expression of the Hd gene in deuterostomes may be a novel feature of the chordate lineage, and the original role(s) of HD in deuterostomes may have been non-neural.

Localization and expression pattern of type I postplasmic mRNAs in embryos of the ascidian Halocynthia roretzi.

The posterior-vegetal cytoplasm (PVC) of fertilized ascidian eggs plays important roles in embryo development. It has been reported that some maternal RNAs are localized to the PVC. We identified four novel type I postplasmic mRNAs that are localized to the PVC through the use of data from a cDNA project of maternal mRNAs in the eggs of Halocynthia roretzi (MAGEST database). The mRNAs are HrGLUT, HrPEN-1, and HrPEM-3, which show similarity to a glucose transporter, a g1-related protein, and Ciona pem-3, respectively; and HrPEN-2, with no similarity. Maternal mRNAs of all four genes were identically localized to the PVC after ooplasmic segregation. During cleavage, they were concentrated in the centrosome-attracting body (CAB) and were then segregated into the small blastomeres located at the posterior pole. This localization pattern is common to all known type I postplasmic mRNAs found so far. HrGLUT, HrPEN-1, and HrPEM-3 were expressed zygotically in various tissues later in embryogenesis: HrGLUT and HrPEM-3 in the mesenchyme and nervous system, and HrPEN-1 in the ectodermal cells.

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

Identification of cis elements which direct the localization of maternal mRNAs to the posterior pole of ascidian embryos.

During ascidian embryogenesis, some mRNAs show clear localization at the posterior-most region. These postplasmic mRNAs are divided into two groups (type I and type II) according to their pattern of localization. To elucidate how these localization patterns are achieved, we attempted to identify the localization elements of these mRNAs. When in vitro synthesized postplasmic mRNAs were introduced into eggs, these mRNAs showed posterior localization similar to the endogenous mRNAs. The posterior localization of these mRNAs was mediated by their 3' untranslated regions (3' UTRs), as is the case for several localized Drosophila and Xenopus mRNAs. We identified smaller fragments of the 3' UTRs of HrWnt-5 and HrPOPK-1 mRNAs (type I) and HrPet-3 mRNA (type II) that were sufficient to direct green fluorescent protein mRNA to the posterior pole. For the localization of HrWnt-5 mRNA, two UG dinucleotide repetitive elements were essential. Motifs similar to these small elements also exist within the HrPOPK-1 mRNA localization element and 3' UTR of HrZF-1 mRNA, suggesting the conservation of localization elements among type I mRNAs. In contrast, the smallest sequence that suffices for the posterior localization of HrPet-3 (a type II mRNA) has different features from those of type I mRNAs; indeed, it does not have an identifiable critical element. This difference may distinguish type II mRNAs from type I mRNAs. These findings, especially the identification of the small localization element of HrWnt-5 mRNA, provide new insights into the localization of mRNAs during ascidian embryogenesis.

Neural marker genes differently expressed in subsets of embryonic neural cells of the ascidian Halocynthia roretzi.

Ascidians are lower chordates that possess a possible prototype of the vertebrate nervous system. The central and peripheral nervous systems of ascidian larvae are composed of only a few hundred cells (Nicol and Meinertzhagen, 1991). To investigate how these ascidian nervous systems develop, dissection at the molecular level using subset-specific markers is essential. Here we describe four new genes zygotically expressed in subsets of the ascidian neural cells. The spatial expression domains of these genes overlap in some parts but not in other parts of the nervous systems. Our results suggest that there are functionally different regions in the nervous systems owing to the gene expression differences. Further analyses of these genes will enable us to determine the molecular neuro-developmental characteristics of various clusters of neural cells.

Retinoic acid differently affects the formation of palps and surrounding neurons in the ascidian tadpole.

The anterior-most surface of the ascidian tadpole larvae is composed of specialized complex structures, including adhesive organs (palps) and the surrounding sensory neurons (RTENs) connected to neurons inside the palps. These are derived from a-line blastomeres by inductive effects from A-line blastomeres. The induction is reported to coordinate the expression of homeobox genes in the anterior epidermis, which can be affected by all- trans retinoic acid (RA). RA treatment also results in failure of the morphological formation of palps. Here we first isolated a gene intensely expressed in the cells of the anterior structure from the time of their lineage restriction, and then found that the RA treatment did not affect the specific gene expression in the presumptive palp cells but did affect that in the RTENs. These results suggest that the palp formation involves at least two different processes, a RA-insensitive cell-type specification process and a RA-sensitive morphogenetic process. RA treatment also affects the morphogenetic process of the palp formation and also disturbs the precise patterning of the surrounding epidermis, which may contribute to the regulation of RTEN development.

Update of MAGEST: Maboya Gene Expression patterns and Sequence Tags.

MAGEST is a database for maternal gene expression information for an ascidian, Halocynthia roretzi. The ascidian has become an animal model in developmental biological research because it shows a simple developmental process, and belongs to one of the chordate groups. Various data are deposited into the MAGEST database, e.g. the 3'- and 5'-tag sequences from the fertilized egg cDNA library, the results of similarity searches against GenBank and the expression data from whole mount in situ hybridization. Over the last 2 years, the data retrieval systems have been improved in several aspects, and the tag sequence entries have increased to over 20 000 clones. Additionally, we constructed a database, translated MAGEST, for the amino acid fragment sequences predicted from the EST data sets. Using this information comprehensively, we should obtain new information on gene functions. The MAGEST database is accessible via the Internet at http://www.genome.ad.jp/magest/.

Notch homologue from Halocynthia roretzi is preferentially expressed in the central nervous system during ascidian embryogenesis.

We describe here the primary structure of HrNotch, an ascidian homologue of the Drosophila neurogenic gene Notch. HrNotch transcripts encode a protein of 2352 amino acids and share the principal features of the Notch gene family: extracellular epidermal growth factor (EGF)-like repeats, three Notch/Lin-12 repeats and six intracellular ankyrin repeats. Yet ascidian Notch contains only 33 EGF repeats in the putative extramembrane domain and specifically lacks the three EGF-like repeats. In situ hybridization shows that maternal HrNotch mRNA is distributed uniformly in the cytoplasm of the unfertilized egg. During cleavage, maternal HrNotch transcripts are ubiquitous in the ectoderm cells of the animal hemisphere, which contain less yolk granules. During gastrulation, maternal transcripts persist in most ectoderm lineage cells. Zygotic expression of HrNotch seems to start at the neural plate stage in both a-line cells (descendants of anterior-animal blastomeres) of the dorsal neuroectoderm and b-line cells (descendants of the posterior-animal blastomeres) that comprise the neural fold. Following this stage, transcripts are most evident in the descendants of these cells, that is, the brain lineage cells, precursors of a larval adhesive organ, and dorsal part of the nerve cord (roof plate). Brain lineage cells include the precursors of sensory pigment cells that are known to comprise an equivalence group in ascidian embryos. During tail elongation, transcripts disappear. Predominant expression of HrNotch in epidermal and neural cells is a common feature of chordate Notch genes. Furthermore, the timing of HrNotch expression in sensory pigment cell precursors suggests involvement in the determinative events in the sensory pigment cell equivalence group.

The ascidian embryo: An experimental system for studying genetic circuitry for embryonic cell specification and morphogenesis.

Ascidians have served as an appropriate experimental system in developmental biology for more than a century. The fertilized egg develops quickly into a tadpole larva, which consists of a small number of tissues including the epidermis, central nervous system with two sensory organs, nerve cord, endoderm, mesenchyme, notochord and muscle. Lineage of these embryonic cells is completely described up to the gastrula stage. These features of the ascidian embryo provide an opportunity to study the mechanisms underlying the differentiation of tissues in development. To understand the molecular basis of ascidian embryogenesis, cloning of various genes has been performed, including those that exhibit a lineage-associated expression pattern and those encoding transcription factors, which are expected to be involved in differentiation of tissues, lineage specification, axis formation and regionalization in developmental fields. Here, we present recent advances in the isolation and characterization of these genes. We emphasize the advantages of the ascidian embryo as an experimental system to study genetic circuitries that are required for cellular differentiation and morphogenesis.

Expression of AMD 1, a gene for a MyoD 1-related factor in the ascidian Halocynthia roretzi.

We have isolated and cloned a gene, designated AMD1 (ascidian MyoD-related factor 1), and its cDNAs that encode a member of the family of myogenic basic helix-loop-helix (bHLH) factors from the ascidian Halocynthia roretzi. The AMD1 gene consists of four exons and is transcribed into at least two distinct mRNAs, which differ in their 3' untranslated region. The gene encodes a protein of 435 amino acids, which exhibits homology to the bHLH domain of other myogenic bHLH factors including vertebrate MyoDt. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that transcripts of the gene were not detectable in fertilized eggs or in very early embryos. They were first detected at the 64-cell stage, a few hours prior to the accumulation of mRNAs for embryonic muscle-specific proteins. In addition, expression of the AMD1 gene was evident in adult body-wall muscle but not in heart and other nonmuscle tissues. These results suggest the possibility that, in ascidians as in vertebrates, the myogenic bHLH factor is involved in the specification of embryonic cells as myogenic cells.

Expression of a Gene for Major Mitochondrial Protein, ADP/ATP Translocase, during Embryogenesis in the Ascidian Halocynthia roretzi: (Ascidian embryos/ADP/ATP translocase gene/maternal mRNA/mitochondria).

The ADP/ATP translocase is the most abundant integral protein of the inner mitochondrial membrane and it is encoded by the nuclear DNA. Because mitochondria in the ascidian egg appear to be segregated into blastomeres of muscle lineage, we examined the expression of a gene for ADP/ATP translocase during embryogenesis of the ascidian Halocynthia roretzi. Sequence analysis of a cDNA clone for the ascidian ADP/ATP translocase indicated that it contains a single open reading frame that encodes a polypeptide of 304 amino acids. The polypeptide showed extensive similarity to mammalian ADP/ATP translocases, with as much as 74% identity. The genome of H. roretzi contains a single gene, or two genes at most, for the protein. A large amount of maternal mRNA for ADP/ATP translocase was found in unfertilized eggs and early embryos. The amount of this mRNA decreased after the 64-cell stage, and the mRNA became barely detectable in tailbud embryos, although zygotic transcript for the protein was evident in adult tissues. Both in situ hybridization and Northern blot analyses demonstrated that the mRNA is distributed in the entire cytoplasm of unfertilized eggs. The mRNA is segregated during embryogenesis not only into blastomeres of muscle lineage but also into those of non-muscle lineage.

Regulated spatial expression of fusion gene constructs with the 5' upstream region of Halocynthia roretzi muscle actin gene in Ciona savignyi embryos.

pHrMA4a-Z is a recombinant plasmid in which about 1.4 kb of the 5' flanking region of a gene for muscle actin HrMA4a from the ascidian Halocynthia roretzi is fused with the coding sequence of a bacterial gene for ß-galactosidase (lac-Z). In this study, we examined the expression of the fusion gene construct when it was introduced into eggs of another ascidian, namely Ciona savignyi. When a moderate amount of linearized pHrMA4a-Z was introduced into fertilized Ciona eggs, the expression of the reporter gene was evident in muscle cells of the larvae, suggesting that both species share a common machinery for the expression of muscle actin genes. The 5' upstream region of HrMA4a contains several consensus sequences, including a TATA box at -30, a CArG box at -116 and four E-boxes within a region of 200 bp. A deletion construct, in which only the 216-bp 5' flanking region of HrMA4a was fused with lac-Z, was expressed primarily in larval muscle cells. However, another deletion construct consisting of only the 61-bp upstream region of HrMA4a fused with lac-Z was not expressed at all. When pHrMA4a-Z or ΔpHrMA4a-Z (-216) was injected into each of the muscle-precursor blastomeres of the 8-cell embryo, expression of the reporter gene was observed in larval muscle cells in a lineage-specific fashion. However, expression of the reporter gene was not observed when the plasmid was injected into non-muscle lineage. Therefore, the expression of the reporter gene may depend on some difference in cytoplasmic constituents between blastomeres of muscle and non-muscle lineage in the 8-cell embyo.