Geographical Distribution and Genetic Diversity of Praesagittifera naikaiensis (Acoelomorpha) in the Seto Inland Sea, Japan.

Acoel flatworms are simple bilaterians that lack digestive lumens and coelomic cavities. Although they are a significant taxon for evaluating the evolution of metazoans, suitable species for biological experiments are not available in Japan. We recently focused on Praesagittifera naikaiensis, which inhabits the sandy shores of intertidal zones in the Seto Inland Sea in Japan, as a candidate for a representative acoel species to be used in experiments. However, reports on its distribution range remain limited. Here, we surveyed the habitats of P. naikaiensis on 108 beaches along the Seto Inland Sea. Praesagittifera naikaiensis is reported here from 37 sites (six previously known and 31 newly discovered sites) spread over a wide area of the Seto Inland Sea, from Awaji Island in Hyogo Prefecture to Fukuoka Prefecture (364 km direct distance). Based on the mitochondrial cytochrome oxidase subunit I (COI) gene haplotypes, we evaluated the genetic diversity of 145 individuals collected from 33 sites. Out of 42 COI haplotypes, 13 haplotypes were shared by multiple individuals. The most frequent haplotype was observed in 67 individuals collected from 31 sites. Eight other haplotypes were detected at geographically distant locations (maximum of 299 km direct distance). Multiple haplotypes were found at 32 sites. These results demonstrate that sufficient genetic flow exists among P. naikaiensis populations throughout the Seto Inland Sea. Molecular phylogenetic analysis of the COI haplotypes of P. naikaiensis revealed that all specimens were grouped into one clade. The genetic homogeneity of the animals in this area favors their use as an experimental animal.

A draft nuclear-genome assembly of the acoel flatworm Praesagittifera naikaiensis.

BACKGROUND: Acoels are primitive bilaterians with very simple soft bodies, in which many organs, including the gut, are not developed. They provide platforms for studying molecular and developmental mechanisms involved in the formation of the basic bilaterian body plan, whole-body regeneration, and symbiosis with photosynthetic microalgae. Because genomic information is essential for future research on acoel biology, we sequenced and assembled the nuclear genome of an acoel, Praesagittifera naikaiensis. FINDINGS: To avoid sequence contamination derived from symbiotic microalgae, DNA was extracted from embryos that were free of algae. More than 290x sequencing coverage was achieved using a combination of Illumina (paired-end and mate-pair libraries) and PacBio sequencing. RNA sequencing and Iso-Seq data from embryos, larvae, and adults were also obtained. First, a preliminary ∼17-kilobase pair (kb) mitochondrial genome was assembled, which was deleted from the nuclear sequence assembly. As a result, a draft nuclear genome assembly was ∼656 Mb in length, with a scaffold N50 of 117 kb and a contig N50 of 57 kb. Although ∼70% of the assembled sequences were likely composed of repetitive sequences that include DNA transposons and retrotransposons, the draft genome was estimated to contain 22,143 protein-coding genes, ∼99% of which were substantiated by corresponding transcripts. We could not find horizontally transferred microalgal genes in the acoel genome. Benchmarking Universal Single-Copy Orthologs analyses indicated that 77% of the conserved single-copy genes were complete. Pfam domain analyses provided a basic set of gene families for transcription factors and signaling molecules. CONCLUSIONS: Our present sequencing and assembly of the P. naikaiensis nuclear genome are comparable to those of other metazoan genomes, providing basic information for future studies of genic and genomic attributes of this animal group. Such studies may shed light on the origins and evolution of simple bilaterians.

Multiple massive domestication and recent amplification of Kolobok superfamily transposons in the clawed frog Xenopus.

BACKGROUND: DNA transposons are generally destroyed by mutations and have short lifespans in hosts, as they are neutral or harmful to the host and therefore not conserved by natural selection. The clawed frog Xenopus harbors many DNA transposons and certain families, such as T2-MITE, have extremely long lives. These have ancient origins, but have shown recent transposition activity. In addition, certain transposase genes may have been "domesticated" by Xenopus and conserved over long time periods by natural selection. The aim of this study was to elucidate the evolutionary interactions between the host and the long-lived DNA transposon family it contains. Here, we investigated the molecular evolution of the Kolobok DNA transposon superfamily. Kolobok is thought to contribute to T2-MITE transposition. RESULTS: In the diploid western clawed frog Xenopus tropicalis and the allotetraploid African clawed frog Xenopus laevis, we searched for transposase genes homologous to those in the Kolobok superfamily. To determine the amplification and domestication of these genes, we used molecular phylogenetics and analyses of copy numbers, conserved motifs, orthologous gene synteny, and coding sequence divergence between the orthologs of X. laevis and X. tropicalis, or between those of two distant X. tropicalis lineages. Among 38 X. tropicalis and 24 X. laevis prospective transposase genes, 10 or more in X. tropicalis and 14 or more in X. laevis were apparently domesticated. These genes may have undergone multiple independent domestications from before the divergence of X. laevis and X. tropicalis. In contrast, certain other transposases may have retained catalytic activity required for transposition and could therefore have been recently amplified. CONCLUSION: Multiple domestication of certain transposases and prolonged conservation of the catalytic activity in others suggest that Kolobok superfamily transposons were involved in complex, mutually beneficial relationships with their Xenopus hosts. Some transposases may serve to activate long-lived T2-MITE subfamilies.

Genome evolution in the allotetraploid frog Xenopus laevis.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Distribution of the T2-MITE Family Transposons in the Xenopus (Silurana) tropicalis Genome.

The T2 family of miniature inverted-repeat transposable elements (T2-MITE) is a prevalent MITE family found in both Xenopus(Silurana) tropicalis and X. laevis. Some subfamilies, particularly T2-A1 and T2-C, may have originated prior to the diversification of the 2 Xenopus lineages and currently include active members in X. tropicalis, whereas another subfamily, T2-E, may have lost its transposition activity even earlier. The distribution of each T2-MITE subfamily in X. tropicalis was investigated and compared to evaluate the evolutionary dynamics of the T2-MITE subfamilies. The subfamilies showed differences in chromosomal distribution, uniformity of insertion density on scaffolds, ratios of upstream to downstream insertions with respect to genes, and their distance from genes. Among these, the T2-C subfamily was interesting because it was frequently inserted upstream and close to genes and because genes with close insertions of this subfamily showed high correlations in spatial expression patterns. This unique distribution and long-lived transposition activity may reflect a mutual relationship evolved between this subfamily and the host.

Mechanisms of maternal inheritance of dinoflagellate symbionts in the acoelomorph worm Waminoa litus.

Waminoa litus is a zooxanthella-bearing acoel worm that infests corals. It is unique to Bilateria in that it transmits its algal symbionts vertically via eggs irrespective of the heterogeneity of the symbionts. It simultaneously harbors two dinoflagellate genera: Symbiodinium and Amphidinium. In this study, we examined the timing and vertical transmission pathway of algal symbionts in W. litus using light and electron microscopy. The oogenesis of the worm can be divided into three stages: stage I, in which the ovary is absent; stage II, the early vitellogenic zone containing immature oocytes formed in the ovary; and stage III, with both early and late vitellogenic zones in the body. In the early vitellogenic zone at stage II, oocytes are surrounded by accessory-follicle cells (AFCs). Both Symbiodinium and Amphidinium symbionts are not initially observed in the oocytes, but are observed in the AFCs. In the late vitellogenic zone at stage III, oocytes are enveloped by a complete sheath of AFCs; the algal symbionts are taken up by the late vitellogenic oocytes. These observations suggest that AFCs mediate the transfer of the algae from the parent to the oocytes. Ribotype analyses of the Symbiodinium symbionts revealed that they differ from those harbored by coral in the same experimental aquarium. These results indicate that W. litus has an active algal transport pathway and maintains a specific lineage of Symbiodinium via vertical transmission.

Recent transposition activity of Xenopus T2 family miniature inverted-repeat transposable elements.

To investigate the recent transposition activity of T2 family miniature inverted-repeat transposable elements (MITEs) in Xenopus tropicalis (Western clawed frog), we analyzed the intraspecific polymorphisms associated with MITE insertion in X. tropicalis for three subfamilies of the T2 family (T2-A1, T2-C, and T2-E). A high frequency of MITE-insertion polymorphisms was observed at the T2-A1 (50%) and T2-C insertion loci (60%), but none were noted at the T2-E insertion locus (0%). Analyses of the collected data indicated that members of the T2-A1 and T2-C subfamilies may be currently active in the host species. Identification of these active transpositions will help us in understanding the mechanisms underlying the long-term survival (over several tens of millions of years) of the T2-A1 and T2-C subfamilies.

A systematic search and classification of T2 family miniature inverted-repeat transposable elements (MITEs) in Xenopus tropicalis suggests the existence of recently active MITE subfamilies.

To reveal the genome-wide aspects of Xenopus T2 family miniature inverted-repeat transposable elements (MITEs), we performed a systematic search and classification of MITEs by a newly developed procedure. A terminal sequence motif (T2-motif: TTAAAGGRR) was retrieved from the Xenopus tropicalis genome database. We then selected 51- to 1,000-bp MITE candidates framed by an inverted pair of 2 T2-motifs. The 34,398 candidates were classified into possible clusters by a novel terminal sequence (TS)-clustering method on the basis of differences in their short terminal sequences. Finally, 19,242 MITEs were classified into 16 major MITE subfamilies (TS subfamilies), 10 of which showed apparent homologies to known T2 MITE subfamilies, and the rest were novel TS subfamilies. Intra- and inter-subfamily similarities or differences were investigated by analyses of diversity in GC content, total length, and sequence alignments. Furthermore, genome-wide conservation of the inverted pair structure of subfamily-specific TS stretches and their target site sequence (TTAA) were analyzed. The results suggested that some TS subfamilies might include active or at least recently active MITEs for transposition and/or amplification, but some others might have lost such activities a long time ago. The present methodology was efficient in identifying and classifying MITEs, thereby providing information on the evolutionary dynamics of MITEs.

Unique form and osmoregulatory function of a neurohypophysial hormone in a urochordate.

The cyclic nonapeptides, oxytocin and vasopressin, are neurohypophysial hormones that regulate many significant physiological processes related especially to reproduction and osmoregulation. In this study, we characterized an oxytocin-related peptide cDNA from a urochordate, Styela plicata, thought to be a sister group to vertebrates. Sequence analysis of the deduced precursor polypeptide revealed that the precursor is composed of three segments: a signal peptide, an oxytocin-like sequence flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic processing site, and a neurophysin domain, similar to other oxytocin/vasopressin family precursors. However, unlike other members of this family, the tunicate oxytocin-like peptide (CYISDCPNSRFWST-NH2) is a tetradecapeptide. We termed this peptide Styela oxytocin-related peptide (SOP). Furthermore, analyses of mass spectrometry, in situ hybridization, and immunohistochemistry demonstrated production of mature SOP in the cerebral ganglion. To elucidate the physiological action of SOP, we kept the tunicate for 2 d under the three different concentrations of seawater, 60, 100, and 130%, and measured the expression levels of SOP mRNA in the cerebral ganglion. The greatest expression of SOP mRNA was observed in the 60% seawater. In 60% seawater, but not in 100 or 130%, the tunicate mostly closed the atrial and branchial siphons. Therefore, we investigated the contractile effects of SOP on the siphons in vitro. SOP caused contractions in both siphons in a dose-dependent manner. Taken together, these results suggest that SOP acts to prevent the influx of a low concentration of seawater into the body and thus play an important role in osmoregulation.

Evolution of the Xenopus piggyBac transposon family TxpB: domesticated and untamed strategies of transposon subfamilies.

A new family, termed TxpB, of DNA transposons belonging to the piggyBac superfamily was found in 3 Xenopus species (Xenopus tropicalis, Xenopus laevis, and Xenopus borealis). Two TxpB subfamilies of Kobuta and Uribo1 were found in all the 3 species, and another subfamily termed Uribo2 was found in X. tropicalis. Molecular phylogenetic analyses of their open reading frames (ORFs) revealed that TxpB transposons have been maintained for over 100 Myr. Both the Uribo1 and the Uribo2 ORFs were present as multiple copies in each genome, and some of them were framed by terminal inverted repeat sequences. In contrast, all the Kobuta ORFs were present as a single copy in each genome and exhibited high evolutionary conservation, suggesting domestication of Kobuta genes by the host. Genomic insertion polymorphisms of the Uribo1 and Uribo2 transposons (nonautonomous type) were observed in a single species of X. tropicalis, indicating recent transposition events. Transfection experiments in cell culture revealed that an expression vector construct for the intact Uribo2 ORF caused precise excision of a nonautonomous Uribo2 element from the target vector construct but that for the Kobuta ORF did not. The present results support our viewpoint that some Uribo2 members are naturally active autonomous transposons, whereas Kobuta members may be domesticated by hosts.

Identification and expression-profiling of Xenopus tropicalis miRNAs including plant miRNA-like RNAs at metamorphosis.

Small RNAs (miR159-like RNAs) identical to some plant miR159s were found in Xenopus tropicalis miRNA cDNA libraries (30 miRNA families consisting of 75 unique sequences). Preferential expression of this RNA species was found in neural tissues during development. A sequence matching to this RNA species was not found in the 21 available animal's genome databases, but its resembling sequences associated with transposons were found in the X. tropicalis database. A possibility of horizontal transfer of the miR159 genes from plants will be discussed. Expression profiles of other miRNA species at metamorphosis were shown by DNA array and/or Northern hybridization.

PCR detection of excision suggests mobility of the medaka fish Tol1 transposable element in the frog Xenopus laevis.

Tol1 is a DNA-based transposable element identified in the medaka fish Oryzias latipes and a member of the hAT (hobo/Activator/Tam3) transposable element family. Its mobility has already been demonstrated in the human and mouse, in addition to its original host species. This element is thus expected to be useful in a wide range of vertebrates as a genomic manipulation tool. Herein, we show that the Tol1 element can undergo excision in the African clawed frog Xenopus laevis, a major model organism for vertebrate genetics and developmental biology. An indicator plasmid carrying a Tol1 element was injected into 2- or 4-cell-stage embryos together with either a helper plasmid coding for the full-length Tol1 transposase or a modified helper plasmid yielding a truncated protein, and recovered from tailbud-stage embryos. Deletion of the Tol1 region of the indicator plasmid was observed in the experiment with the full-length transposase, and not in the other case. The deletion was associated with various footprint sequences at breakpoints, as frequently observed with many DNA-based transposable elements. These results indicate that the Tol1 element was excised from the indicator plasmid by catalysis of the transposase, and suggest that the Tol1 element is mobile in this frog species.

Lineage-specific tandem repeats riding on a transposable element of MITE in Xenopus evolution: a new mechanism for creating simple sequence repeats.

Xstir is a repetitive DNA sequence element that is extremely amplified as a common component of two different structures: a tandem repeat (Xstir array) and a MITE (miniature inverted-repeat transposable element) in the genome of Xenopus laevis. To elucidate the origin and evolutionary history of Xstir-related sequences, we investigated their species specificity among three Xenopus species (X. laevis, X. borealis, and X. tropicalis). Analyses by sequence alignment and digestion with restriction enzymes of genomic Xstir-related sequences revealed that the MITE (Xmix MITE) was well conserved among the three Xenopus species, with small lineage-specific differences. On the other hand, the tandem repeat element (tropXstir) in X. tropicalis was different from the Xstir that X. laevis and X. borealis have in common. Both sequences of Xstir and tropXstir were, however, different segments of the Xmix MITE. The results suggest that these tandem repeats were formed by partial tandem duplication of the MITE internal sequence in each lineage of X. tropicalis and of X. borealis/X. laevis after their branching. A molecular mechanism for creating and elongating the tandem repeats from the MITE is proposed.

Tissue-specific regulation of type III iodothyronine 5-deiodinase gene expression mediates the effects of prolactin and growth hormone in Xenopus metamorphosis.

Prolactin (PRL) and growth hormone (GH) are known to be able to act as antimetamorphic hormones. From investigations of how PRL inhibits Xenopus tail regression in vitro, it was found that the both hormones could, in addition to their known antimetamorphic actions, upregulate mRNA expression of type III iodothyronine 5-deiodinase (5D), an enzyme that inactivates thyroid hormones (TH). Conversely, both PRL and GH were found to downregulate 5D mRNA expression in the liver. Blockage by PRL of TH-induced tail regression in organ culture was released by treatment with iopanoic acid (IOP, an inhibitor of 5D activity). The IOP-released tail regression displayed a unique morphology of the larger fins retained on the regressing tails, consistent with the finding that mRNA for both PRL receptor and 5D were enriched in the fin. The results suggest that the metamorphosis-modulating actions of PRL and GH are mediated, at least partially, by tissue-specific regulation of 5D mRNA expression.

Thyroid hormone-dependent repression of α1-microglobulin/bikunin precursor (AMBP) gene expression during amphibian metamorphosis.

A Xenopus AMBP (xAMBP) cDNA clone was isolated from a subtracted liver cDNA library by differential hybridization screening. The deduced amino acid sequence shared 50-60% identity with its mammalian counterparts, which are the precursors of the plasma glycoproteins, α1-microglobulin and bikunin. Both peptide structures were well conserved in xAMBP. Northern and in situ hybridization revealed that the xAMBP gene was specifically expressed in liver parenchymal cells. The gene was activated around embryo hatching and repressed at the metamorphic climax stage. During adult life the mRNA level remained low. Treating the tadpoles with thyroid homone prematurely reduced the mRNA level. Furthermore, thyroid hormone acted on larval hepatocytes in primary culture and reduced the mRNA level. Thus, xAMBP gene expression appears to be repressed through the direct action of thyroid hormone on the hepatocytes at the metamorphic climax stage. On the other hand, adult hepatocytes in thyroid hormone-free culture medium expressed mRNA at a low level, which was not reduced in response to thyroid hormone, suggesting that the repressed xAMBP gene expression in adult hepatocytes was maintained in a thyroid hormone-independent manner. The unique expression profile suggested that the xAMBP gene plays a biological role in the progression of amphibian metamorphosis.

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.

Introduction and Expression of Recombinant Genes in Ascidian Embryos.

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocynthia roretzi, respectively. The plasmid pmiwZ contains the coding sequence of bacterial ß-galactosidase gene (lac-Z) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4-kb long 5' flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene (CAT). Injection of approximately 160 pl of 10 µg/ml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 µg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac-Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell-types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocynthia eggs and the appearance of CAT protein was examined later by the anti-CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4-kb long 5' flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.