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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

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.

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.

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.

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.

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 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.

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.

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.

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.

A DNA fragment containing short tandem repeat sequences (approximately 86-bp repeat) was isolated from a Xenopus laevis cDNA library. Southern blot and in situ hybridization analyses revealed that the repeat was highly dispersed in the genome and was present at approximately 1 million copies per haploid genome. We named this element Xstir (Xenopus short tandemly and invertedly repeating element) after its arrangement in the genome. The majority of the genomic Xstir sequences were digested to monomer and dimer sizes with several restriction enzymes. Their sequences were found to be highly homogeneous and organized into tandem arrays in the genome. Alignment analyses of several known sequences showed that some of the Xstir-like sequences were also organized into interspersed inverted repeats. The inverted repeats consisted of an inverted pair of two differently modified Xstirs separated by a short insert. In addition, these were framed by another novel inverted repeat (Xstir-TIR). The Xstir-TIR sequence was also found at the ends of tandem Xstir arrays. Furthermore, we found that Xstir-TIR was linked to a motif characterizing the T2 family which belonged to a vertebrate MITE (miniature inverted-repeat transposable element) family, suggesting the importance of Xstir-TIR for their amplification and transposition. The present study of 11 anuran and 2 urodele species revealed that Xstir or Xstir-like sequences were extensively amplified in the three Xenopus species. Genomic Xstir populations of X. borealis and X. laevis were mutually indistinguishable but significantly different from that of X. tropicalis.

During embryogenesis of the ascidian Halocynthia roretzi, 42 unicellular striated muscle cells are formed in the tail of the tadpole larva. Isolation of cDNA clones demonstrated that multiple genes for larval muscle actin are expressed in this process. Among them, at least five muscle actin genes (HrMA2, HrMA4a, HrMA4b, HrMA5, and HrMA6) form a cluster (HrMA2/4 cluster) within about 30 kb of the genome. The 5′ flanking sequences of the five actin genes resemble each other. When constructs in which 184 bp of the 5′ flanking region of each of these genes fused with lacZ were introduced into fertilized eggs, the reporter gene was expressed in muscle cells of the tailbud embryo, suggesting that the 5′ flanking region of each cluster gene has promoter activity. In addition, a pair of muscle actin genes, HrMA1a and HrMA1b (HrMA1 pair), was isolated from a genomic region different from that of the HrMA2/4 cluster. The HrMA1a and HrMA1b are linked in a head-to-head arrangement on opposite strands and share a 340-bp 5′ flanking sequence containing two symmetrically located TATA boxes. HrMA1a showed basically the same expression pattern as that of HrMA4a. When constructs in which the shared upstream region of HrMA1 pair fused with lacZ in either direction were microinjected into eggs, the reporter gene was expressed in muscle cells of the larval tail, suggesting a bidirectional promoter that regulates muscle-specific transcription of the HrMA1 pair. The tandem cluster of HrMA2/4 genes and the bidirectional promoter of the HrMA1 pair could expedite utilization of muscle-specific trans -acting factors. The organization of genes in the genome may play an important role in the synthesis of a large amount of actins during the process of rapid differentiation.

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 β-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.

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.

SummaryTol1is a DNA-based transposable element identified in the medaka fishOryzias latipesand a member of thehAT(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 theTol1element can undergo excision in the African clawed frogXenopus laevis, a major model organism for vertebrate genetics and developmental biology. An indicator plasmid carrying aTol1element was injected into 2- or 4-cell-stage embryos together with either a helper plasmid coding for the full-lengthTol1transposase or a modified helper plasmid yielding a truncated protein, and recovered from tailbud-stage embryos. Deletion of theTol1region 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 theTol1element was excised from the indicator plasmid by catalysis of the transposase, and suggest that theTol1element is mobile in this frog species.

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.

To elucidate the role of type III iodothyronine 5-deiodinase (5-D) in the temporal regulation of amphibian metamorphosis, the regulation of gene expression of 5-D and thyroid hormone receptor beta (TRbeta) in organs of Xenopus laevis was investigated. High levels of TRbeta mRNA in the respective organs were observed at the times of their major morphological changes. Expression of the 5-D gene was highly regulated among the organs during metamorphosis, including up-regulation in the tail and down-regulation in the liver. The tail and liver expressed 5-D gene before their metamorphic changes. These precocious expressions correlated with the lower responsiveness to exogenously added triiodo-L-thyronine (T3) for inducing a high level of TRbeta mRNA expression. However, the same organs responded to lower doses of T3 to regulate 5-D gene expression as seen in spontaneous metamorphosis. The induction of 5-D gene expression was considerably delayed in the intestine, even at an excess dose of T3. Thus, the two genes in a given organ appeared to respond to T3 either with different dose dependencies or with different timetables. The results obtained are also discussed in respect to recent findings in Rana catesbeiana.

The HrMA4 alpha gene for an embryonic muscle actin of the ascidian Halocynthia roretzi is activated at the gastrula stage in differentiating muscle cells exclusively. The 5' upstream region close to the transcription start site of HrMA4 alpha contains several consensus sequences, which include a TATA box at -30, and E-box at -71, a CArG box at -116, and a cluster of three E-boxes between -150 and -190. When deletion constructs of this region, fused with the bacterial gene for beta-galactosidase (lac-Z), were microinjected into fertilized eggs, the reporter gene was expressed in muscle cells of tailbud embryos. Analyses of the deletion constructs suggested that the 103-bp upstream region is sufficient for the appropriate expression of the gene. However, beta-gal activity was very rarely detectable in the case of 82-bp upstream region and no activity was detected in the case of 72-bp upstream region. Mutations in the proximal E-box sequence did not disturb the muscle-specific expression of the reporter gene. These results suggest that rather short sequences between nucleotides -103 and -72 from the transcription start site are associated with the specific expression of HrMA4 alpha.

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 β-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.