Characterization of a novel species-specific 51-amino acid peptide, PEP51, as a caspase-3/7 activator in ovarian follicles of the ascidian, Ciona intestinalis Type A.

Invertebrates lack hypothalamic-pituitary-gonadal axis, and have acquired species-specific regulatory systems for ovarian follicle development. Ascidians are marine invertebrates that are the phylogenetically closest living relatives to vertebrates, and we have thus far substantiated the molecular mechanisms underlying neuropeptidergic follicle development of the cosmopolitan species, Ciona intestinalis Type A. However, no ovarian factor has so far been identified in Ciona. In the present study, we identified a novel Ciona-specific peptide, termed PEP51, in the ovary. Immunohistochemical analysis demonstrated the specific expression of PEP51 in oocyte-associated accessory cells, test cells, of post-vitellogenic (stage III) follicles. Immunoelectron microscopy revealed that PEP51 was localized in the cytosol of test cells in early stage III follicles, which lack secretory granules. These results indicate that PEP51 acts as an intracellular factor within test cells rather than as a secretory peptide. Confocal laser microscopy verified that activation of caspase-3/7, the canonical apoptosis marker, was detected in most PEP51-positive test cells of early stage III. This colocalization of PEP51 and the apoptosis marker was consistent with immunoelectron microscopy observations demonstrating that a few normal (PEP51-negative) test cells reside in the aggregates of PEP51-positive apoptotic test cells of early stage III follicles. Furthermore, transfection of the PEP51 gene into COS-7 cells and HEK293MSR cells resulted in activation of caspase-3/7, providing evidence that PEP51 induces apoptotic signaling. Collectively, these results showed the existence of species-specific ovarian peptide-driven cell metabolism in Ciona follicle development. Consistent with the phylogenetic position of Ciona as the closest sister group of vertebrates, the present study sheds new light on the molecular and functional diversity of the regulatory systems of follicle development in the Chordata.

Differentiation of endostyle cells by Nkx2-1 and FoxE in the ascidian Ciona intestinalis type A: insights into shared gene regulation in glandular- and thyroid-equivalent elements of the chordate endostyle.

Due to similarities in iodine concentrations and peroxidase activities, the thyroid in vertebrates is considered to originate from the endostyle of invertebrate chordates even though it is a glandular (mucus-producing) organ for aquatic suspension feeding. Among chordates with an endostyle, urochordates are useful evolutionary research models for the study of vertebrate traits. The ascidian Ciona intestinalis forms an endostyle with specific components of glandular- and thyroid-related elements, and molecular markers have been identified for these components. Since we previously examined a simple endostyle in the larvacean Oikopleura dioica, the expression of the thyroid-related transcription factor genes, Ciona Nkx2-1 and FoxE, was perturbed by TALEN-mediated gene knockout in the present study to elucidate the shared and/or divergent features of a complex ascidian endostyle. The knockout of Ciona Nkx2-1 and FoxE exerted different effects on the morphology of the developing endostyle. The knockout of Nkx2-1 eliminated the expression of both glandular and thyroidal differentiation marker genes, e.g., vWFL1, vWFL2, CiEnds1, TPO, and Duox, while that of FoxE eliminated the expression of the differentiation marker genes, TPO and CiEnds1. The supporting element-related expression of Pax2/5/8a, Pax2/5/8b, FoxQ1, and ß-tubulin persisted in the hypoplastic endostyles of Nkx2-1- and FoxE-knockout juveniles. Although the gene regulation of ascidian-specific CiEnds1 remains unclear, these results provide insights into the evolution of the vertebrate thyroid as well as the urochordate endostyle.

The TRP channel PKD2 is involved in sensing the mechanical stimulus of adhesion for initiating metamorphosis in the chordate Ciona.

Metamorphosis is the dramatic and irreversible reconstruction of animal bodies transitioning from the larval stage. Because of the significant impact of metamorphosis on animal life, its timing is strictly regulated. Invertebrate chordate ascidians are the closest living relatives of vertebrates. Ascidians exhibit metamorphosis that converts their swimming larvae into sessile adults. Ascidian metamorphosis is triggered by a mechanical stimulus generated when adhesive papillae adhere to a substrate. However, it is not well understood how the mechanical stimulus is generated and how ascidian larvae sense the stimulus. In this study, we addressed these issues by a combination of embryological, molecular, and genetic experiments in the model ascidian Ciona intestinalis Type A, also called Ciona robusta. We here showed that the epidermal neuronal network starting from the sensory neurons at the adhesive papillae is responsible for the sensing of adhesion. We also found that the transient receptor potential (TRP) channel PKD2 is involved in sensing the stimulus of adhesion. Our results provide a better understanding of the mechanisms underlying the regulation of the timing of ascidian metamorphosis.

d-Serine controls epidermal vesicle release via NMDA receptor, allowing tissue migration during the metamorphosis of the chordate Ciona.

d-Serine, a free amino acid synthesized by serine racemase, is a coagonist of N-methyl-d-aspartate-type glutamate receptor (NMDAR). d-Serine in the mammalian central nervous system modulates glutamatergic transmission. Functions of d-serine in mammalian peripheral tissues such as skin have also been described. However, d-serine's functions in nonmammals are unclear. Here, we characterized d-serine-dependent vesicle release from the epidermis during metamorphosis of the tunicate Ciona. d-Serine leads to the formation of a pocket that facilitates the arrival of migrating tissue during tail regression. NMDAR is the receptor of d-serine in the formation of the epidermal pocket. The epidermal pocket is formed by the release of epidermal vesicles' content mediated by d-serine/NMDAR. This mechanism is similar to observations of keratinocyte vesicle exocytosis in mammalian skin. Our findings provide a better understanding of the maintenance of epidermal homeostasis in animals and contribute to further evolutionary perspectives of d-amino acid function among metazoans.

Vasopressin Promoter Transgenic and Vasopressin Gene-Edited Ascidian, Ciona intestinalis Type A (Ciona robusta): Innervation, Gene Expression Profiles, and Phenotypes.

Oxytocin (OT) and vasopressin (VP) superfamily neuropeptides are distributed in not only vertebrates but also diverse invertebrates. However, no VPergic innervation of invertebrates has ever been documented. In the ascidian, Ciona intestinalis Type A (Ciona robusta), an OT/VP superfamily peptide was identified, and the Ciona vasopressin (CiVP) induces oocyte maturation and ovulation. In the present study, we characterize the innervation and phenotypes of genetically modified Ciona: CiVP promoter-Venus transgenic and CiVP mutants. CiVP promoter-Venus transgenic Ciona demonstrated that CiVP gene was highly expressed in the cerebral ganglion and several nerves. Fluorescence was also detected in the ovary of young CiVP promoter-Venus transgenic ascidians, suggesting that the CiVP gene is also expressed temporarily in the ovary of young ascidians. Furthermore, a marked decrease of post-vitellogenic (stage III) follicles was observed in the ovary of CiVP mutants, whereas pre-vitellogenic (stage I) and vitellogenic (stage II) follicles were increased in the mutant ovary, compared with that of wildtype Ciona. Gene expression profiles showed that the expression of various genes, including genes related to ovarian follicle growth, was altered in the ovary of CiVP mutants. Altogether, these results indicated that CiVP, mainly as a neuropeptide, plays pivotal roles in diverse biological functions, including growth of early-stage ovarian follicles via regulation of the expression of a wide variety of genes. This is the first report describing a VP gene promoter-transgenic and VP gene-edited invertebrate and also on its gene expression profiles and phenotypes.

Orchestration of the distinct morphogenetic movements in different tissues drives tail regression during ascidian metamorphosis.

Metamorphosis is the dramatic conversion of an animal body from larva to adult. In ascidians, tadpole-shaped, swimming larvae become sessile juveniles by losing their tail during metamorphosis. This study investigated the cellular and molecular mechanisms underlying this metamorphic event called tail regression, in the model ascidian Ciona. The ascidian tail consists of internal organs such as muscle, notochord, nerve cord, and the outer epidermal layer surrounding them. We found that the epidermis and internal organs show different regression strategies. Epidermal cells are shortened along the anterior-posterior axis and gather at the posterior region. The epidermal mass is then invaginated into the trunk by apical constriction. The internal tissues, by contrast, enter into the trunk by forming coils. During coiling, notches are introduced into the muscle cells, which likely reduces their rigidness to promote coiling. Actin filament is the major component necessary for the regression events in both the epidermis and internal tissues. The shortening and invagination of the epidermis depend on the phosphorylation of the myosin regulatory light chain (mrlc) regulated by rho-kinase (ROCK). The coiling of internal tissues does not require ROCK-dependent phosphorylation of mrlc, and they can complete coiling without epidermis, although epidermis can facilitate the coiling of internal tissues. We conclude that tail regression in ascidians consists of active morphogenetic movements in which each tissue's independent mechanism is orchestrated with the others to complete this event within the available time window.

GABA-Induced GnRH Release Triggers Chordate Metamorphosis.

Metamorphosis, a widespread life history strategy in metazoans, allows dispersal and use of different ecological niches through a dramatic body change from a larval stage [1, 2]. Despite its conservation and importance, the molecular mechanisms underlying its initiation and progression have been characterized in only a few animal models. In this study, through pharmacological and gene functional analyses, we identified neurotransmitters responsible for metamorphosis of the ascidian Ciona. Ciona metamorphosis converts swimming tadpole larvae into vase-like, sessile adults. Here, we show that the neurotransmitter GABA is a key regulator of metamorphosis. We found that gonadotropin-releasing hormone (GnRH) is a downstream neuropeptide of GABA. Although GABA is generally thought of as an inhibitory neurotransmitter, we found that it positively regulates secretion of GnRH through the metabotropic GABA receptor during Ciona metamorphosis. GnRH is necessary for reproductive maturation in vertebrates, and GABA is an important excitatory regulator of GnRH in the hypothalamus during puberty [3, 4]. Our findings reveal another role of the GABA-GnRH axis in the regulation of post-embryonic development in chordates.

Hox13 is essential for formation of a sensory organ at the terminal end of the sperm duct in Ciona.

Species-specific traits are thought to have been acquired by natural selection. Transcription factors play central roles in the evolution of species-specific traits. Hox genes encode a set of conserved transcription factors essential for establishing the anterior-posterior body axis of animals. Changes in the expression or function of Hox genes can lead to the diversification of animal-body plans. The tunicate ascidian Ciona intestinalis Type A has an orange-colored structure at the sperm duct terminus. This orange-pigmented organ (OPO) is the characteristic that can distinguish this ascidian from other closely related species. The OPO is formed by the accumulation of orange-pigmented cells (OPCs) that are present throughout the adult body. We show that Hox13 is essential for formation of the OPO. Hox13 is expressed in the epithelium of the sperm duct and neurons surrounding the terminal openings for sperm ejection, while OPCs themselves do not express this gene. OPCs are mobile cells that can move through the body vasculature by pseudopodia, suggesting that the OPO is formed by the accumulation of OPCs guided by Hox13-positive cells. Another ascidian species, Ciona savignyi, does not have an OPO. Like Hox13 of C. intestinalis, Hox13 of C. savignyi is expressed at the terminus of its sperm duct; however, its expression domain is limited to the circular area around the openings. The genetic changes responsible for the acquisition or loss of OPO are likely to occur in the expression pattern of Hox13.

A novel ER membrane protein Ehg1/May24 plays a critical role in maintaining multiple nutrient permeases in yeast under high-pressure perturbation.

Previously, we isolated 84 deletion mutants in Saccharomyces cerevisiae auxotrophic background that exhibited hypersensitive growth under high hydrostatic pressure and/or low temperature. Here, we observed that 24 deletion mutants were rescued by the introduction of four plasmids (LEU2, HIS3, LYS2, and URA3) together to grow at 25 MPa, thereby suggesting close links between the genes and nutrient uptake. Most of the highly ranked genes were poorly characterized, including MAY24/YPR153W. May24 appeared to be localized in the endoplasmic reticulum (ER) membrane. Therefore, we designated this gene as EHG (ER-associated high-pressure growth gene) 1. Deletion of EHG1 led to reduced nutrient transport rates and decreases in the nutrient permease levels at 25 MPa. These results suggest that Ehg1 is required for the stability and functionality of the permeases under high pressure. Ehg1 physically interacted with nutrient permeases Hip1, Bap2, and Fur4; however, alanine substitutions for Pro17, Phe19, and Pro20, which were highly conserved among Ehg1 homologues in various yeast species, eliminated interactions with the permeases as well as the high-pressure growth ability. By functioning as a novel chaperone that facilitated coping with high-pressure-induced perturbations, Ehg1 could exert a stabilizing effect on nutrient permeases when they are present in the ER.

piRNA-like small RNAs are responsible for the maternal-specific knockdown in the ascidian Ciona intestinalis Type A.

The mRNAs stored in eggs are crucial for embryogenesis. To address functions of maternal mRNAs, we recently reported the novel method MASK (maternal mRNA-specific knockdown), which we used to specifically knockdown maternal transcripts in the ascidian Ciona intestinalis Type A. In MASK, the cis element of a maternal gene is fused with eGFP or Kaede reporter gene, and the cassette is introduced into Ciona genome by transposon-mediated transgenesis. In eggs of the transgenic lines, the maternal expression of the gene whose cis element is used for driving the reporter gene is suppressed. The zygotic expression of the gene is not suppressed, suggesting that the MASK method can distinguish between maternal and zygotic functions of a gene. Here we investigated the cis and trans factors responsible for MASK results. In the ovaries in which knockdown of a maternal gene occurs, a number of antisense small RNAs are expressed that are complementary to the sequence of the knocked-down genes. We suspect that these antisense small RNAs are the factor responsible for MASK results. The antisense small RNAs have several features that are seen in PIWI-interacting RNAs (piRNAs), suggesting that MASK is likely to use a piRNA-mediated mechanism to knock down maternal mRNAs.

Formation of adult organs through metamorphosis in ascidians.

The representative characteristic of ascidians is their vertebrate-like, tadpole shape at the larval stage. Ascidians lose the tadpole shape through metamorphosis to become adults with a nonmotile, sessile body and a shape generally considered distinct from that of vertebrates. Solitary ascidians including Ciona species are extensively studied to understand the developmental mechanisms of ascidians, and to compare these mechanisms with their counterparts in vertebrates. In these ascidian species, the digestive and circulatory systems are not well developed in the larval trunk and the larvae do not take food. This is in contrast with the inner conditions of vertebrate tadpoles, which have functional organs comparable to those of adults. The adult organs and tissues of these ascidians become functional during metamorphosis that is completed quickly, suggesting that the ascidian larvae of solitary species are a transient stage of development. We here discuss how the cells and tissues in the ascidian larval body are converted into those of adults. The hearts of ascidians and vertebrates use closely related cellular and molecular mechanisms that suggest their shared origin. Hox genes of ascidians are essential for forming adult endodermal structures. To fully understand the development and evolution of chordates, a complete elucidation of the mechanisms underlying the adult tissue/organ formation of ascidians will be needed. WIREs Dev Biol 2018, 7:e304. doi: 10.1002/wdev.304 This article is categorized under: Comparative Development and Evolution > Body Plan Evolution Early Embryonic Development > Development to the Basic Body Plan.

Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells.

The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona.

Neuronal map reveals the highly regionalized pattern of the juvenile central nervous system of the ascidian Ciona intestinalis.

BACKGROUND: The dorsally located central nervous system (CNS) is an important hallmark of chordates. Among chordates, tunicate ascidians change their CNS remarkably by means of a metamorphosis from a highly regionalized larval CNS to an oval-shaped juvenile CNS without prominent morphological features. The neuronal organization of the CNS of ascidian tadpole larvae has been well described, but that in the CNS of postmetamorphosis juveniles has not been characterized well. RESULTS: We investigated the number of neural cells, the number and position of differentiated neurons, and their axonal trajectories in the juvenile CNS of the ascidian Ciona intestinalis. The cell bodies of cholinergic, glutamatergic, and GABAergic/glycinergic neurons exhibited different localization patterns along the anterior-posterior axis in the juvenile CNS. Cholinergic neurons extended their axons toward the oral, atrial and body wall muscles and pharyngeal gill to regulate muscle contraction and ciliary movement. CONCLUSIONS: Unlike its featureless shape, the juvenile CNS is highly patterned along the anterior-posterior axis. This patterning may be necessary for exerting multiple roles in the regulation of adult tissues distributed throughout the body. This basic information of the juvenile CNS of Ciona will allow in-depth studies of molecular mechanisms underlying the reconstruction of the CNS during ascidian metamorphosis.

CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis.

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site. CRISPR/Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.

Transposon-mediated targeted and specific knockdown of maternally expressed transcripts in the ascidian Ciona intestinalis.

Maternal mRNAs play crucial roles during early embryogenesis of ascidians, but their functions are largely unknown. In this study, we developed a new method to specifically knockdown maternal mRNAs in Ciona intestinalis using transposon-mediated transgenesis. We found that GFP expression is epigenetically silenced in Ciona intestinalis oocytes and eggs, and this epigenetic silencing of GFP was used to develop the knockdown method. When the 5' upstream promoter and 5' untranslated region (UTR) of a maternal gene are used to drive GFP in eggs, the maternal gene is specifically knocked down together with GFP. The 5' UTR of the maternal gene is the major element that determines the target gene silencing. Zygotic transcription of the target gene is unaffected, suggesting that the observed phenotypes specifically reflect the maternal function of the gene. This new method can provide breakthroughs in studying the functions of maternal mRNAs.

Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases.

Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis.

Enhancer activity sensitive to the orientation of the gene it regulates in the chordate genome.

Enhancers are flexible in terms of their location and orientation relative to the genes they regulate. However, little is known about whether the flexibility can be applied in every combination of enhancers and genes. Enhancer detection with transposable elements is a powerful method to identify enhancers in the genome and to create marker lines expressing fluorescent proteins in a tissue-specific manner. In the chordate Ciona intestinalis, this method has been established with a Tc1/mariner superfamily transposon Minos. Previously, we created the enhancer detection line E[MiTSAdTPOG]15 (E15) that specifically expresses green fluorescent protein (GFP) in the central nervous system (CNS) after metamorphosis. In this study, we identified the causal insertion site of the transgenic line. There are two genes flanking the causal insertion of the E15 line, and the genomic region around the insertion site contains the enhancers responsible for the expression in the endostyle and gut in addition to the CNS. We found that the endostyle and gut enhancers show sensitivity to the orientation of the GFP gene for their enhancer activity. Namely, the enhancers cannot enhance the expression of GFP which is inserted at the same orientation as the E15 line, while the enhancers can enhance GFP expression inserted at the opposite orientation. The CNS enhancer can enhance GFP expression in both orientations. The DNA element adjacent to the endostyle enhancer is responsible for the orientation sensitivity of the enhancer. The different sensitivity of the enhancers to the orientation of the transgene is a cause of CNS-specific GFP expression in the E15 line.

Germline transgenesis of the chordate Ciona intestinalis with hyperactive variants of sleeping beauty transposable element.

BACKGROUND: Transposon-mediated transgenesis is an excellent method for creating stable transgenic lines and insertional mutants. In the chordate Ciona intestinalis, Minos is the only transposon that has been used as the tool for germline transformation. Adding another transposon system in this organism enables us to conduct genetic techniques which can only be realized with the use of two transposons. RESULTS: In the present study, we found that another Tc1/mariner superfamily transposon, sleeping beauty (SB), retains sufficient activity for germline transformation of C. intestinalis. SB shows efficiencies of germline transformation, insertion into gene coding regions, and enhancer detection comparable to those of Minos. We have developed a system for the remobilization of SB copies in the C. intestinalis genome by using transgenic lines expressing SB transposase in the germ cells. With this system, we examined the manner of SB mobilization in the C. intestinalis genome. SB shows intrachromosomal transposition more frequently than Minos. CONCLUSIONS: SB-based germline transformation and the establishment of a new method that uses its frequent intrachromosomal transposition will result in breakthroughs in genetic approaches that use C. intestinalis together with Minos.

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis.

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.

Efficient transposition of a single Minos transposon copy in the genome of the ascidian Ciona intestinalis with a transgenic line expressing transposase in eggs.

Transgenesis with transposons is an important technique for studying genetic functions. In the ascidian Ciona intestinalis, methods for germline transformation with the Tc1/mariner transposon Minos have been established. A system to remobilize a single Minos copy in the genome is needed to refine this transgenic technique. In this study, such an experimental system was established with a transgenic line expressing Minos transposase in eggs. In the eggs of a double transgenic animal from a cross between the egg transposase line and a transgenic line having a single Minos insertion, the transposon was transposed into new positions of the Ciona genome, thus creating new insertions. Some of the new insertions caused enhancer detection. The majority of the new insertion sites were mapped on different chromosomes from that of the transposon donor. This characteristic of Minos is in contrast to that of the Sleeping Beauty transposon, which causes frequent intrachromosomal transposition.