Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and cleavage stage embryos.     

Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

An enzyme capture assay for analysis of active hyaluronan synthases

We describe a sensitive assay for detection of active hyaluronan synthases (HASs) capable of synthesizing hyaluronan (HA) without use of radioactive uridine 5'-diphosphate sugar precursors. The HAS capture assay is based on the binding of a biotinylated HA binding protein (bHABP) to HA chains that are associated with HAS and the subsequent capture of bHABP-HA-HAS complexes with streptavidin-agarose. Specific HAS proteins (e.g., HAS1, not HAS2 or HAS3) captured in this pull-down approach are readily immunodetected by Western blot analysis using appropriate antibodies. The assay was used to detect active HAS proteins in cell membranes, purified recombinant Streptococcus equisimilis HAS (SeHAS), and in vitro translated human HAS1 or SeHAS. The HAS capture assay was also used to assess the fraction of HAS molecules that were active, which cannot be done using standard assays for synthase activity. Assay sensitivity for detection of purified SeHAS is <1 pmol.     

Functional molecular mass of a vertebrate hyaluronan synthase as determined by radiation inactivation analysis.

Hyaluronan (HA), a linear polysaccharide composed of N-acetylglucosamine-glucuronic acid repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. The HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The prototypical vertebrate hyaluronan synthase, xlHAS1 (or DG42) from Xenopus laevis, is a 588-residue membrane protein. Recently, the streptococcal enzyme was found to function as a monomer of protein with approximately 16 lipid molecules. The vertebrate enzymes are larger than the streptococcal enzymes; based on the vertebrate HAS deduced amino acid sequence, two additional membrane-associated regions at the carboxyl terminus are predicted. We have utilized radiation inactivation to measure the target size of yeast-derived recombinant xlHAS1. The target size of HAS activity was confirmed using two internal standards. First, samples were spiked with glucose-6-phosphate dehydrogenase, an enzyme of known molecular weight. Second, parallel samples of native xlHAS1 and a xlHAS1-green fluorescent protein fusion (833 residues) were compared; substantial confidence was gained by using this novel internal standard. Our test also corroborated the basic tenets of radiation inactivation theory. We found that the vertebrate HAS protein functions catalytically as a monomer.     

The human hyaluronan synthase genes: genomic structures,proximal promoters and polymorphic microsatellite markers

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.     

Evolution of Chordate Actin Genes: Evidence from Genomic Organization and Amino Acid Sequences

The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing two new ascidian cytoplasmic and muscle actin genes. The exon–intron organization and sequences of these genes were compared with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome. Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates. Received: 20 June 1996 / Accepted: 16 October 1996     

EST analysis of gene expression in testis of the ascidian Ciona intestinalis

To explore the gene expression underlying spermatogenesis, a large-scale analysis has been done on the cDNAs from testis of the ascidian, Ciona intestinalis. A set of 5,461 expressed sequence tags was analyzed and grouped into 2,806 independent clusters. Approximately 30% of the clusters showed significant sequence matches to the proteins reported in DDBJ/GenBank/EMBL database including a set of proteins closely related to the gene regulation during spermatogenesis, functional and morphological changes of spermatogenic cells during spermiogenesis, and physiological functions of sperm, as well as those with housekeeping functions commonly expressed in other cells. Some clones show similarities to the proteins present in vertebrate lymphocytes, suggesting a primitive immune system in ascidians. We have also found some genes that are known to participate in hormonal regulation of spermatogenesis in vertebrates. The large majority of the genes expressed in Ciona testis show no significant matches to known proteins and the further analysis of these genes may shed new light on the molecular mechanism of spermatogenesis and sperm functions.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

A genome-wide survey of the genes for planar polarity signaling or convergent extension-related genes in Ciona intestinalis and phylogenetic comparisons of evolutionary conserved signaling components

Non-canonical Wnt signals similar to planar cell polarity (PCP) signaling in the fly control convergent extension (CE) of the dorsal mesoderm during gastrulation in vertebrates. Using the Ciona complete genome sequence and EST sequence data, we present here an initial and exhaustive search in non-vertebrate chordates, Ciona intestinalis for the family members as well as homologs or orthologs that are involved in PCP/CE signaling cascades. We clarified 7 cardinal gene families, including the MAPK, STE20 group kinase, Rho small GTPase, STAT, Glypican, Fz and Wnt gene families, as well as gene homologs or orthologs for known PCP/CE signaling components with their phylogenetic nature. As a result, we characterized 62 Ciona component genes. Among them, 59 genes were novel and functional genes which were supported by EST expressions and 15 genes belonged to PCP/CE component orthologs of other organisms or common ancestor genes. Moreover, from the phylogenetic point of view, we compared these components genome-widely with the PCP signaling components of fly and the CE signaling components of vertebrates. We then discovered not only that ascidians contain the basic ancestral signaling pathway components in chordates but also that several signaling components have not found in ascidian, indicating that ascidian CE pathway might have several gaps from vertebrate CE pathway. The present study provides an initial step for the subsequent analysis of CE in the non-vertebrate chordates, ascidians. In addition, this phylogenetic approach will help to facilitate understanding of the relationship between fly PCP signaling and the vertebrate CE pathway.     

An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli

The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.     

Complete nucleotide sequence of the mitochondrial genome of Doliolum nationalis with implications for evolution of urochordates

The evolutionary history of the diverse lifestyles adopted by urochordates has attracted intense interest because it may effect the evolutionary history of vertebrates. Here, we report the complete mitochondrial (mt) DNA sequence of the pelagic thaliacean doliolid Doliolum nationalis. The doliolid mt genome shares the unusual tRNAs of trnM(uau) and trnG(ucu) with other ascidians, such as Halocynthia and Ciona. On the other hand, the gene order of the doliolid mt genome is significantly different from that of any ascidian species or vertebrate reported to date. Phylogenetic analyses of the amino acid sequences of 12 protein-coding genes strongly support the sister-grouping of doliolids and the Phlebobranch ascidian Ciona, with the Stolidobranch ascidian alocynthia as the outgroup, thereby providing strong support for the paraphyly of ascidians, as has been suggested by 18S rDNA studies. Given the paraphyletic nature of ascidians, it seems likely that the common ancestor of ascidians and thaliaceans was sessile, as are the present-day ascidians, and that the thaliaceans subsequently evolved a pelagic lifestyle.     

Region specific gene expressions in the central nervous system of the ascidian embryo

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. Fibroblast growth factor 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/16/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.     

Hremx, the ascidian homologue of ems/emx, is expressed in the anterior and posterior-lateral epidermis but not in the central nervous system during embryogenesis

Recent comparative studies on expression patterns of homeobox genes in the development between ascidians and vertebrates have come to suggest a possibility that a common basic mechanism may exist in the patterning of the central nervous system (CNS). The ems/emx genes have been demonstrated to be involved in the formation and patterning of the anterior CNS in Drosophila and vertebrate embryos. In the present study, we have isolated and analyzed expression of Hremx, the ascidian homologue of ems/emx with particular attention to whether it is expressed in the larval ascidian CNS. Expression of Hremx was detected in the anterior trunk and lateral tail epidermis but not in the anterior CNS. The two expression domains of the epidermis responded in different ways upon treatment with retinoic acid: the anterior expression domain was unaltered, while the posterior expression domain extended to the anterior. The present result suggests that Hremx may have a function in anterior patterning but not in the patterning of the CNS in the ascidian embryo. We suggest the possibility that the function of ems/emx genes in the patterning of the anterior CNS in Drosophila and vertebrate embryos might have been acquired independently in the lineages to Drosophila and vertebrates.     

Decoding cis-regulatory systems in ascidians

Ascidians, or sea squirts, are lower chordates, and share basic gene repertoires and many characteristics, both developmental and physiological, with vertebrates. Therefore, decoding cis-regulatory systems in ascidians will contribute toward elucidating the genetic regulatory systems underlying the developmental and physiological processes of vertebrates. cis-Regulatory DNAs can also be used for tissue-specific genetic manipulation, a powerful tool for studying ascidian development and physiology. Because the ascidian genome is compact compared with vertebrate genomes, both intergenic regions and introns are relatively small in ascidians. Short upstream intergenic regions contain a complete set of cis-regulatory elements for spatially regulated expression of a majority of ascidian genes. These features of the ascidian genome are a great advantage in identifying cis-regulatory sequences and in analyzing their functions. Function of cis-regulatory DNAs has been analyzed for a number of tissue-specific and developmentally regulated genes of ascidians by introducing promoter-reporter fusion constructs into ascidian embryos. The availability of the whole genome sequences of the two Ciona species, Ciona intestinalis and Ciona savignyi, facilitates comparative genomics approaches to identify cis-regulatory DNAs. Recent studies demonstrate that computational methods can help identify cis-regulatory elements in the ascidian genome. This review presents a comprehensive list of ascidian genes whose cis-regulatory regions have been subjected to functional analysis, and highlights the recent advances in bioinformatics and comparative genomics approaches to cis-regulatory systems in ascidians.     

Analysis of ascidian <Emphasis Type="Italic">Not</Emphasis> genes highlights their evolutionarily conserved and derived features of structure and expression in development

The ascidian larva is often regarded as an organism close to the ancestral form of chordates, while it is generally accepted that the Spemanns organizer is absent from ascidian embryos. Not is one of the genes expressed in the organizer to execute functions in vertebrate embryos. To address the extent of conservation of Not gene expression among ascidians and vertebrates, we examined the structure and developmental expression of Not of the two distantly related ascidian species, Halocynthia and Ciona. Putative ascidian Not proteins were noted by the absence of one of the two motifs conserved among Not proteins of sea urchin and vertebrates. Analysis by in situ hybridization revealed that Not gene expression of ascidians could be categorized into three types: expression likely to be conserved between ascidians and vertebrates, that probably unique to ascidians, and that specific to ascidian species. Expression of ascidian Not in the posterior end of the tail as well as the notochord and a small part of the anterior neural tube at the tailbud stage is reminiscent of the expression of the vertebrate counterparts in the tailbud, which is regarded as a continuation of the organizer and the pineal gland, respectively. The expression of Not in the epidermis precursors during cleavage stage may be unique to ascidians. In the light of the present findings, evolutionary aspects of Not genes are discussed.Electronic Supplementary Material Supplementary material is available for this article at Edited by N. Satoh     

Cross-phylum regulatory potential of the ascidian Otx gene in brain development in Drosophila melanogaster

The origin of molecular mechanisms of cephalic development is an intriguing question in evolutionary and developmental biology. Ascidians, positioned near the origin of the phylum Chordata, share a conserved set of anteroposterior patterning genes with vertebrates. Here we report the cross-phylum regulatory potential of the ascidian Otx gene in the development of the Drosophila brain and the head vertex structures. The ascidian Otx gene rescued the embryonic brain defect caused by a null mutation of the Drosophila orthodenticle (otd) gene and enhanced rostral brain development while it suppressed trunk nerve cord formation. Furthermore, the ascidian Otx gene restored the head vertex defects caused by a viable otd mutation, ocelliless, via specific activation and repression of downstream regulatory genes. These cross-phylum regulatory potentials of the ascidian Otx gene are equivalent to the activities of the Drosophila and human otd/Otx genes in these developmental processes. These results support the notion that basal chordates such as ascidians have the same molecular patterning mechanism for the anterior structures found in higher chordates, and suggest a common genetic program of cephalic development in invertebrate, protochordate and vertebrate.