Transcriptomic evidence for Brachyury expression in the caudal tip region of adult Ptychodera flava (Hemichordata).

Most metazoans have a single copy of the T-box transcription factor gene Brachyury. This gene is expressed in cells of the blastopore of late blastulae and the archenteron invagination region of gastrulae. It appears to be crucial for gastrulation and mesoderm differentiation of embryos. Although this expression pattern is shared by most deuterostomes, Brachyury expression has not been reported in adult stages. Here we show that Brachyury of an indirect developer, the hemichordate acorn worm Ptychodera flava, is expressed not only in embryonic cells, but also in cells of the caudal tip (anus) region of adults. This spatially restricted expression, shown by whole-mount in situ hybridization, was confirmed by Iso-Seq RNA sequencing and single-cell RNA-seq (scRNA-seq) analysis. Iso-Seq analysis showed that gene expression occurs only in the caudal region of adults, but not in anterior regions, including the stomochord. scRNA-seq analysis showed a cluster that contained Brachyury-expressing cells comprising epidermis- and mesoderm-related cells, but which is unlikely to be associated with the nervous system or muscle. Although further investigation is required to examine the roles of Brachyury in adults, this study provides important clues for extending studies on Brachyury expression involved in development of the most posterior region of deuterostomes.

Studying Hemichordata WBR Using Ptychodera flava.

Hemichordates are benthic marine invertebrates closely related to chordates. Several species, including Ptychodera flava in the phylum Hemichordates, can undergo whole body regeneration from a small fragment. P. flava is widely distributed in the warm Indo-Pacific region and is easily collected in the lower tidal zone of a shallow beach with a coral reef. Here, we describe the methods for animal collection and preparation of regenerating tissues. The prepared tissues can be used for various molecular and/or histological experiments. We also demonstrate how to examine gene expression patterns in the tissues using whole mount in situ hybridization.

Ancestral Stem Cell Reprogramming Genes Active in Hemichordate Regeneration.

Hemichordate enteropneust worms regenerate extensively in a manner that resembles the regeneration for which planaria and hydra are well known. Although hemichordates are often classified as an extant phylogenetic group that may hold ancestral deuterostome body plans at the base of the deuterostome evolutionary line leading to chordates, mammals, and humans, extensive regeneration is not known in any of these more advanced groups. Here we investigated whether hemichordates deploy functional homologs of canonical Yamanaka stem cell reprogramming factors, Oct4, Sox2, Nanog, and Klf4, as they regenerate. These reprogramming factors are not expressed during regeneration of limbs, fins, eyes or other structures that represent the best examples of regeneration in chordates. We first examined Ptychodera flava EST libraries and identified Pf-Pou3, Pf-SoxB1, Pf-Msxlx, and Pf-Klf1/2/4 as most closely related to the Yamanaka factors, respectively. In situ hybridization analyses revealed that all these homologs are expressed in a distinct manner during head regeneration. Furthermore, Pf-Pou3 partially rescued the loss of endogenous Oct4 in mouse embryonic stem cells in maintaining the pluripotency gene expression program. Based on these results, we propose that hemichordates may have co-opted these reprogramming factors for their extensive regeneration or that chordates may have lost the ability to mobilize these factors in response to damage. The robustness of these pluripotency gene circuits in the inner cell mass and in formation of induced pluripotent stem cells from mammalian somatic cells shows that these programs are intact in humans and other mammals and that these circuits may respond to as yet unknown gene regulatory signals, mobilizing full regeneration in hemichordates.

Genome and transcriptome assemblies of the kuruma shrimp, Marsupenaeus japonicus.

The kuruma shrimp Marsupenaeus japonicus (order Decapoda, family Penaeidae) is an economically important crustacean that occurs in shallow, warm seas across the Indo-Pacific. Here, using a combination of Illumina and Oxford Nanopore Technologies platforms, we produced a draft genome assembly of M. japonicus (1.70 Gbp; 18,210 scaffolds; scaffold N50 = 234.9 kbp; 34.38% GC, 93.4% BUSCO completeness) and a complete mitochondrial genome sequence (15,969 bp). As with other penaeid shrimp genomes, the M. japonicus genome is extremely rich in simple repeats, which occupies 27.4% of the assembly. A total of 26,381 protein-coding gene models (94.7% BUSCO completeness) were predicted, of which 18,005 genes (68.2%) were assigned functional description by at least one method. We also produced an Illumina-based transcriptome shotgun assembly (40,991 entries; 93.0% BUSCO completeness) and a PacBio Iso-Seq transcriptome assembly (25,415 entries; 67.5% BUSCO completeness). We envision that the M. japonicus genome and transcriptome assemblies will serve as useful resources for the basic research, fisheries management, and breeding programs of M. japonicus.

Chloroplast acquisition without the gene transfer in kleptoplastic sea slugs, Plakobranchus ocellatus.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

Integrated omics unveil the secondary metabolic landscape of a basal dinoflagellate.

BACKGROUND: Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Our understanding of dinoflagellate toxin biosynthesis has been hampered by their unusually large genomes. To overcome this challenge, for the first time, we sequenced the genome, microRNAs, and mRNA isoforms of a basal dinoflagellate, Amphidinium gibbosum, and employed an integrated omics approach to understand its secondary metabolite biosynthesis. RESULTS: We assembled the ~ 6.4-Gb A. gibbosum genome, and by probing decoded dinoflagellate genomes and transcriptomes, we identified the non-ribosomal peptide synthetase adenylation domain as essential for generation of specialized metabolites. Upon starving the cells of phosphate and nitrogen, we observed pronounced shifts in metabolite biosynthesis, suggestive of post-transcriptional regulation by microRNAs. Using Iso-Seq and RNA-seq data, we found that alternative splicing and polycistronic expression generate different transcripts for secondary metabolism. CONCLUSIONS: Our genomic findings suggest intricate integration of various metabolic enzymes that function iteratively to synthesize metabolites, providing mechanistic insights into how dinoflagellates synthesize secondary metabolites, depending upon nutrient availability. This study provides insights into toxin production associated with dinoflagellate blooms. The genome of this basal dinoflagellate provides important clues about dinoflagellate evolution and overcomes the large genome size, which has been a challenge previously.

Comparative genomics of four strains of the edible brown alga, Cladosiphon okamuranus.

BACKGROUND: The brown alga, Cladosiphon okamuranus (Okinawa mozuku), is one of the most important edible seaweeds, and it is cultivated for market primarily in Okinawa, Japan. Four strains, denominated S, K, O, and C, with distinctively different morphologies, have been cultivated commercially since the early 2000s. We previously reported a draft genome of the S-strain. To facilitate studies of seaweed biology for future aquaculture, we here decoded and analyzed genomes of the other three strains (K, O, and C). RESULTS: Here we improved the genome of the S-strain (ver. 2, 130 Mbp, 12,999 genes), and decoded the K-strain (135 Mbp, 12,511 genes), the O-strain (140 Mbp, 12,548 genes), and the C-strain (143 Mbp, 12,182 genes). Molecular phylogenies, using mitochondrial and nuclear genes, showed that the S-strain diverged first, followed by the K-strain, and most recently the C- and O-strains. Comparisons of genome architecture among the four strains document the frequent occurrence of inversions. In addition to gene acquisitions and losses, the S-, K-, O-, and C-strains possess 457, 344, 367, and 262 gene families unique to each strain, respectively. Comprehensive Blast searches showed that most genes have no sequence similarity to any entries in the non-redundant protein sequence database, although GO annotation suggested that they likely function in relation to molecular and biological processes and cellular components. CONCLUSIONS: Our study compares the genomes of four strains of C. okamuranus and examines their phylogenetic relationships. Due to global environmental changes, including temperature increases, acidification, and pollution, brown algal aquaculture is facing critical challenges. Genomic and phylogenetic information reported by the present research provides useful tools for isolation of novel strains.

Correlation between Organelle Genetic Variation and RNA Editing in Dinoflagellates Associated with the Coral Acropora digitifera.

In order to develop successful strategies for coral reef preservation, it is critical that the biology of both host corals and symbiotic algae are investigated. In the Ryukyu Archipelago, which encompasses many islands spread over ∼500 km of the Pacific Ocean, four major populations of the coral Acropora digitifera have been studied using whole-genome shotgun (WGS) sequence analysis (Shinzato C, Mungpakdee S, Arakaki N, Satoh N. 2015. Genome-wide single-nucleotide polymorphism (SNP) analysis explains coral diversity and recovery in the Ryukyu Archipelago. Sci Rep. 5:18211.). In contrast, the diversity of the symbiotic dinoflagellates associated with these A. digitifera populations is unknown. It is therefore unclear if these two core components of the coral holobiont share a common evolutionary history. This issue can be addressed for the symbiotic algal populations by studying the organelle genomes of their mitochondria and plastids. Here, we analyzed WGS data from ∼150 adult A. digitifera, and by mapping reads to the available reference genome sequences, we extracted 2,250 sequences representing 15 organelle genes of Symbiodiniaceae. Molecular phylogenetic analyses of these mitochondrial and plastid gene sets revealed that A. digitifera from the southern Yaeyama islands harbor a different Symbiodiniaceae population than the islands of Okinawa and Kerama in the north, indicating that the distribution of symbiont populations partially matches that of the four host populations. Interestingly, we found that numerous SNPs correspond to known RNA-edited sites in 14 of the Symbiodiniaceae organelle genes, with mitochondrial genes showing a stronger correspondence than plastid genes. These results suggest a possible correlation between RNA editing and SNPs in the two organelle genomes of symbiotic dinoflagellates.

Organelle inheritance and genome architecture variation in isogamous brown algae.

Among the brown algal lineages, Ectocarpales species have isogamous fertilization in which male and female gametes are morphologically similar. In contrast, female gametes are much larger than male gametes in the oogamous species found in many other brown algal lineages. It has been reported that the plastids of isogamous species are biparentally inherited whereas the plastids of oogamous species are maternally inherited. In contrast, in both isogamous and oogamous species, the mitochondria are usually inherited maternally. To investigate whether there is any relationship between the modes of inheritance and organellar genome architecture, we sequenced six plastid genomes (ptDNA) and two mitochondrial genomes (mtDNA) of isogamous species from the Ectocarpales and compared them with previously sequenced organellar genomes. We found that the biparentally inherited ptDNAs of isogamous species presented distinctive structural rearrangements whereas maternally inherited ptDNAs of oogamous species showed no rearrangements. Our analysis permits the hypothesis that structural rearrangements in ptDNAs may be a consequence of the mode of inheritance.

Differential gene expression in fronds and stolons of the siphonous macroalga, Caulerpa lentillifera.

The green alga, Caulerpa lentillifera, is composed of a single cell with multiple nuclei, but it possesses structures analogous to leaves or fronds, stems or stolons, and roots or rhizoids. To understand molecular mechanisms involved in formation and function of these structures, we carried out RNA-seq analysis of fronds and stolons (including rhizoids). Taking advantage of the decoded genome of C. lentillifera, the present RNA-seq analysis addressed transcripts corresponding to 9,311 genes identified in the genome. RNA-seq data suggested that 8,734 genes are expressed in sporophytes. Despite the siphonous body of the alga, differential gene expression was evident in the two structures. 1,027 (11.8%) and 1,129 (12.9%) genes were preferentially expressed in fronds and stolons, respectively, while the remaining 6,578 (75.3%) genes were expressed at the same level in both. Most genes preferentially expressed in fronds are associated with photosynthesis and plant hormone pathways, including abscisic acid signaling. In contrast, those preferentially expressed in stolons are associated with translation and DNA replication. These results indicate that gene expression is regulated differently between fronds and stolons, which probably governs the function of each structure. Together with genomic information, the present transcriptomic data provide genic information about development and physiology of this unique, siphonous organism.

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.

Draft genome of the brown alga, Nemacystus decipiens, Onna-1 strain: Fusion of genes involved in the sulfated fucan biosynthesis pathway.

The brown alga, Nemacystus decipiens ("ito-mozuku" in Japanese), is one of the major edible seaweeds, cultivated principally in Okinawa, Japan. N. decipiens is also a significant source of fucoidan, which has various physiological activities. To facilitate brown algal studies, we decoded the ~154 Mbp draft genome of N. decipiens Onna-1 strain. The genome is estimated to contain 15,156 protein-coding genes, ~78% of which are substantiated by corresponding mRNAs. Mitochondrial genes analysis showed a close relationship between N. decipiens and Cladosiphon okamuranus. Comparisons with the C. okamuranus and Ectocarpus siliculosus genomes identified a set of N. decipiens-specific genes. Gene ontology annotation showed more than half of these are classified as molecular function, enzymatic activity, and/or biological process. Extracellular matrix analysis revealed domains shared among three brown algae. Characterization of genes that encode enzymes involved in the biosynthetic pathway for sulfated fucan showed two sets of genes fused in the genome. One is a fusion of L-fucokinase and GDP-fucose pyrophosphorylase genes, a feature shared with C. okamuranus. Another fusion is between an ST-domain-containing gene and an alpha/beta hydrolase gene. Although the function of fused genes should be examined in future, these results suggest that N. decipiens is another promising source of fucoidan.

A siphonous macroalgal genome suggests convergent functions of homeobox genes in algae and land plants.

Genome evolution and development of unicellular, multinucleate macroalgae (siphonous algae) are poorly known, although various multicellular organisms have been studied extensively. To understand macroalgal developmental evolution, we assembled the ∼26 Mb genome of a siphonous green alga, Caulerpa lentillifera, with high contiguity, containing 9,311 protein-coding genes. Molecular phylogeny using 107 nuclear genes indicates that the diversification of the class Ulvophyceae, including C. lentillifera, occurred before the split of the Chlorophyceae and Trebouxiophyceae. Compared with other green algae, the TALE superclass of homeobox genes, which expanded in land plants, shows a series of lineage-specific duplications in this siphonous macroalga. Plant hormone signalling components were also expanded in a lineage-specific manner. Expanded transport regulators, which show spatially different expression, suggest that the structural patterning strategy of a multinucleate cell depends on diversification of nuclear pore proteins. These results not only imply functional convergence of duplicated genes among green plants, but also provide insight into evolutionary roots of green plants. Based on the present results, we propose cellular and molecular mechanisms involved in the structural differentiation in the siphonous alga.

Xenacoelomorph-Specific Hox Peptides: Insights into the Phylogeny of Acoels, Nemertodermatids, and Xenoturbellids.

Xenacoelomorpha has recently been proposed as an animal taxon that includes acoels, nemertodermatids, and xenoturbellids. Their flattened bodies are very simple and lack discrete organs. The Acoela and Nemertodermatida (which comprise Acoelomorpha) were traditionally regarded as early-diverged extant orders of the class Turbellaria of the phylum Platyhelminthes. Recent anatomical studies and molecular phylogenetic studies demonstrate that the two groups belong to the phylum Xenacoelomorpha together with Xenoturbellida. However, debate remains in regard to whether Xenacoelomorpha is monophyletic, and whether xenacoelomorphs are sisters to all other bilaterians or have close affinity to ambulacrarians. The present study addresses the first question by examining the presence or absence of diagnostic peptide sequences shared by the three taxa. Hox genes have been used to investigate the phylogenetic relationships of metazoans. It has been shown that lophotrochozoans, rotifers, and chaetognaths share diagnostic peptide sequences in the C-terminal region of the Lox5 (Hox5/6/7) homeodomain proteins, which supports the clustering of these taxa. Examination of the decoded genome of the acoel Praesagittifera naikaiensis and reported xenacoelomorph Hox genes revealed that acoels share a peptide NLK(S/T)MSQ(V/I)D, which starts immediately after the homeodomain sequence of the central Hox4/5/6. In addition, we found another diagnostic peptide, KEGKL, in the C-terminal region of the anterior Hox1, which is shared by all the three groups of xenacoelomorphs, but not other bilaterians. Furthermore, two acoels, Praesagittifera naikaiensis and Symsagittifera roscoffensis, share another peptide SG(A/P)PGM in the posterior Hox9/11/13. These results support the designation of the phylum Xenacoelomorpha, in which Acoela is a discrete group.

Regeneration in the enteropneust hemichordate, Ptychodera flava, and its evolutionary implications.

Hemichordates are marine invertebrates that are closely related to chordates, but while their body plans are comparable to those of chordates, they possess a remarkable capacity for regeneration, even as adults. A small fragment is sufficient to form a complete individual. Unlike echinoderms, their larvae transform directly into adults; therefore, hemichordate systems offer clear morphological and molecular parallels between regeneration and development. Morphological events in regeneration are generally similar to organogenesis in juveniles. Nonetheless, comparative analysis of gene expression in these two morphological phenomena suggests that hemichordate regeneration is regulated by regeneration-specific mechanisms, as well as by developmental mechanisms. Dependency upon resident pluripotent/multipotent stem cells is a significant difference in metazoan regeneration, and such stem cells are essential for regeneration in many lineages. Based on the present gene expression study, regeneration in acorn worms is more closely related to that in vertebrates, because it employs endogenous stem cell-independent transdifferentiation.

A draft genome of the brown alga, Cladosiphon okamuranus, S-strain: a platform for future studies of 'mozuku' biology.

The brown alga, Cladosiphon okamuranus (Okinawa mozuku), is economically one of the most important edible seaweeds, and is cultivated for market primarily in Okinawa, Japan. C. okamuranus constitutes a significant source of fucoidan, which has various physiological and biological activities. To facilitate studies of seaweed biology, we decoded the draft genome of C. okamuranus S-strain. The genome size of C. okamuranus was estimated as ∼140 Mbp, smaller than genomes of two other brown algae, Ectocarpus siliculosus and Saccharina japonica Sequencing with ∼100× coverage yielded an assembly of 541 scaffolds with N50 = 416 kbp. Together with transcriptomic data, we estimated that the C. okamuranus genome contains 13,640 protein-coding genes, approximately 94% of which have been confirmed with corresponding mRNAs. Comparisons with the E. siliculosus genome identified a set of C. okamuranus genes that encode enzymes involved in biosynthetic pathways for sulfated fucans and alginate biosynthesis. In addition, we identified C. okamuranus genes for enzymes involved in phlorotannin biosynthesis. The present decoding of the Cladosiphon okamuranus genome provides a platform for future studies of mozuku biology.

Hedgehog Expression During Development and Regeneration in the Hemichordate, Ptychodera flava.

Hedgehog is a toolkit gene conserved in metazoans. However, its function differs among taxa, and it shows versatile expression patterns in morphogenesis. We analyzed the expression pattern of hedgehog in the indirect development of the hemichordate, Ptychodera flava, during development and regeneration. Pf-Hh showed distinct enteropneust-specific expression at the anterior tip of the larvae, as well as deuterostome-conserved expression in the pharyngeal endoderm. In contrast, the gene is expressed only in the pharyngeal region during anterior regeneration, but not in the anterior tip of the proboscis. These data suggest that anterior regeneration is driven not only by conserved developmental mechanisms, but also by some regeneration-specific mechanism(s).

A cDNA resource for gene expression studies of a hemichordate, Ptychodera flava.

Recent investigations into the evolution of deuterostomes and the origin of chordates have paid considerable attention to hemichordates (acorn worms), as hemichordates and echinoderms are the closest chordate relatives. The present study prepared cDNA libraries from Ptychodera flava, to study expression and function of genes involved in development of the hemichordate body plan. Expressed sequence tag (EST) analyses of nine cDNA libraries yielded 18,832 cloned genes expressed in eggs, 18,739 in blastulae, 18,539 in gastrulae, 18,811 in larvae, 18,978 in juveniles, 11,802 in adult proboscis, 17,259 in stomochord, 11,886 in gills, and 11,580 in liver, respectively. A set of 34,159 uni-gene clones of P. flava was obtained. This cDNA resource will be valuable for studying temporal and spatial expression of acorn worm genes during development.

Regeneration in the hemichordate Ptychodera flava.

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial regions are severed from the body of the worm. After transection, the body wall contracts and heals closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blastema grows rapidly and begins the process of differentiating into a head with a proboscis and collar. At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the forming proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encircle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to fit the worm's body, has grown attached to the severed site. At about this time the animal regains apparently normal burrowing behavior. After the head is formed, a second blastema-like area appears between the new head and the old body and a new branchial region is inserted by regeneration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented and whitish such that in-situ hybridization can be used to study the expression of genes during the formation of new tissues.