Regulatory cocktail for dopaminergic neurons in a protovertebrate identified by whole-embryo single-cell transcriptomics.

The CNS of the protovertebrate Ciona intestinalis contains a single cluster of dopaminergic (DA) neurons, the coronet cells, which have been likened to the hypothalamus of vertebrates. Whole-embryo single-cell RNA sequencing (RNA-seq) assays identified Ptf1a as the most strongly expressed cell-specific transcription factor (TF) in DA/coronet cells. Knockdown of Ptf1a activity results in their loss, while misexpression results in the appearance of supernumerary DA/coronet cells. Photoreceptor cells and ependymal cells are the most susceptible to transformation, and both cell types express high levels of Meis Coexpression of both Ptf1a and Meis caused the wholesale transformation of the entire CNS into DA/coronet cells. We therefore suggest that the reiterative use of functional manipulations and single-cell RNA-seq assays is an effective means for the identification of regulatory cocktails underlying the specification of specific cell identities.

Neuronal identities derived by misexpression of the POU IV sensory determinant in a protovertebrate.

The protovertebrate Ciona intestinalis type A (sometimes called Ciona robusta) contains a series of sensory cell types distributed across the head-tail axis of swimming tadpoles. They arise from lateral regions of the neural plate that exhibit properties of vertebrate placodes and neural crest. The sensory determinant POU IV/Brn3 is known to work in concert with regional determinants, such as Foxg and Neurogenin, to produce palp sensory cells (PSCs) and bipolar tail neurons (BTNs), in head and tail regions, respectively. A combination of single-cell RNA-sequencing (scRNA-seq) assays, computational analysis, and experimental manipulations suggests that misexpression of POU IV results in variable transformations of epidermal cells into hybrid sensory cell types, including those exhibiting properties of both PSCs and BTNs. Hybrid properties are due to coexpression of Foxg and Neurogenin that is triggered by an unexpected POU IV feedback loop. Hybrid cells were also found to express a synthetic gene battery that is not coexpressed in any known cell type. We discuss these results with respect to the opportunities and challenges of reprogramming cell types through the targeted misexpression of cellular determinants.

Zic-r.b controls cell numbers in Ciona embryos by activating CDKN1B.

The control of cell numbers and the establishment of cell types are two processes that are essential in early embryonic development. We have a reasonable understanding of how these processes occur individually, but we have considerably less sophisticated understanding of how these processes are linked. Tunicates have fixed cell lineages with predictable cell cycles, making them well suited to investigate these processes. In the ascidian Ciona, we show that the transcription factor Zic-r.b, known to be involved in establishing several cell types in early development also activates the expression of the cell cycle inhibitor CDKN1B. Zic-r.b is a major missing component of the cell division clock establishing specific cell numbers. We also show that a larvacean homolog of Zic-r.b is expressed one cell cycle earlier than its Ciona counterpart. The early expression in larvaceans may explain why they have half as many notochord cells as ascidians and may illustrate a general mechanism to evolve changes in morphology.

Detection of TALEN-mediated genome cleavage during the early embryonic stage of the starfish Patiria pectinifera.

BACKGROUND: Echinoderms have long been utilized as experimental materials to study the genetic control of developmental processes and their evolution. Among echinoderms, the molecular study of starfish embryos has received considerable attention across research topics such as gene regulatory network evolution and larval regeneration. Recently, experimental techniques to manipulate gene functions have been gradually established in starfish as the feasibility of genome editing methods was reported. However, it is still unclear when these techniques cause genome cleavage during the development of starfish, which is critical to understand the timeframe and applicability of the experiment during early development of starfish. RESULTS: We herein reported that gene functions can be analyzed by the genome editing method TALEN in early embryos, such as the blastula of the starfish Patiria pectinifera. We injected the mRNA of TALEN targeting rar, which was previously constructed, into eggs of P. pectinifera and examined the efficiency of genome cleavage through developmental stages from 6 to 48 hours post fertilization. CONCLUSION: The results will be key knowledge not only when designing TALEN-based experiments but also when assessing the results.

Distribution changes of non-self-test cells and self-tunic cells surrounding the outer body during Ciona metamorphosis.

BACKGROUND: Ascidians significantly change their body structure through metamorphosis, but the spatio-temporal cell dynamics in the early metamorphosis stage has not been clarified. A natural Ciona embryo is surrounded by maternally derived non-self-test cells before metamorphosis. However, after metamorphosis, the juvenile is surrounded by self-tunic cells derived from mesenchymal cell lineages. Both test cells and tunic cells are thought to be changed their distributions during metamorphosis, but the precise timing is unknown. RESULTS: Using a metamorphosis induction by mechanical stimulation, we investigated the dynamics of mesenchymal cells during metamorphosis in a precise time course. After the stimulation, two-round Ca2+ transients were observed. Migrating mesenchymal cells came out through the epidermis within 10 min after the second phase. We named this event "cell extravasation." The cell extravasation occurred at the same time as the backward movement of posterior trunk epidermal cells. Timelapse imaging of transgenic-line larva revealed that non-self-test cells and self-tunic cells temporarily coexist outside the body until the test cells are eliminated. At the juvenile stage, only extravasated self-tunic cells remained outside the body. CONCLUSIONS: We found that mesenchymal cells extravasated following two-round Ca2+ transients, and distributions of test cells and tunic cells changed in the outer body after tail regression.

Mechanism underlying retinoic acid-dependent metamorphosis in the starfish.

The evolution of the biphasic life cycle in marine invertebrates has attracted considerable interest in zoology. We recently provided evidence that retinoic acid (RA) is involved in the regulation of metamorphosis in starfish. It also functions in life cycle transitions of jellyfish (cnidaria). Thus, documenting the evolutionarily conserved role of RA in such transitions will help to trace the life cycle evolution of bilaterians and cnidarians. In this study, we examined the molecular mechanisms by which RA signaling is involved in the commencement of metamorphosis in starfish. First, we measured RA levels during the larval and metamorphosis stages by liquid chromatography-tandem mass spectrometry. We found that all-trans RA levels in the larval body are high before larvae acquire competence for metamorphosis, suggesting that the commencement of metamorphosis is not controlled by increased RA synthesis. Furthermore, the suppression of rar gene expression by TALEN-mediated gene knockout revealed that RA receptor (RAR) is essential for metamorphosis. These observations suggest that the initiation of metamorphosis is regulated at the level of synthesized RA to activate RAR. We discuss the divergence of ligand molecules and receptors during the evolution of life cycle regulation.

Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract.

Multi-regional expression of pancreas-related digestive enzyme genes in the intestinal chamber of the ascidian Ciona intestinalis type A.

Bilateria share sequential steps in their digestive systems, and digestion occurs in a pre-absorption step within a chamber-like structure. Previous studies on the ascidian Ciona intestinalis type A, an evolutionary research model of vertebrate organs, revealed that Ciona homologs of pancreas-related exocrine digestive enzymes (XDEs) are exclusively expressed in the chamber-like bulging stomach. In the development of the gastrointestinal tract, genes for the pancreas-related transcription factors, namely Ptf1a, Nr5a2, and Pdx, are expressed near the stomach. Recent organ/tissue RNA-seq studies on two Ciona species reported that transcripts of the XDE homologs exist in the intestinal regions, as well as in the stomach. In the present study, we investigated the spatial gene expression of XDE homologs in the gastrointestinal region of the C. intestinalis type A. Whole-mount in situ hybridization using adult and juvenile specimens revealed apparent expression signals of XDE homologs in a small number of gastrointestinal epithelial cells. Furthermore, two pancreas-related transcription factor genes, Nr5a2 and Pdx, exhibited multi-regional expression along the Ciona juvenile intestines. These results imply that ascidians may form multiple digestive regions corresponding to the vertebrate pancreas.

Nkx2-1 and FoxE regionalize glandular (mucus-producing) and thyroid-equivalent traits in the endostyle of the chordate Oikopleura dioica.

The endostyle is a ventral pharyngeal organ used for internal filter feeding of basal chordates and is considered homologous to the follicular thyroid of vertebrates. It contains mucus-producing (glandular) and thyroid-equivalent regions organized along the dorsoventral (DV) axis. Although thyroid-related genes (Nkx2-1, FoxE, and thyroid peroxidase (TPO)) are known to be expressed in the endostyle, their roles in establishing regionalization within the organ have not been demonstrated. We report that Nkx2-1 and FoxE are essential for establishing DV axial identity in the endostyle of Oikopleura dioica. Genome and expression analyses showed von Willebrand factor-like (vWFL) and TPO/dual oxidase (Duox)/Nkx2-1/FoxE as orthologs of glandular and thyroid-related genes, respectively. Knockdown experiments showed that Nkx2-1 is necessary for the expression of glandular and thyroid-related genes, whereas FoxE is necessary only for thyroid-related genes. Moreover, Nkx2-1 expression is necessary for FoxE expression in larvae during organogenesis. The results demonstrate the essential roles of Nkx2-1 and FoxE in establishing regionalization in the endostyle, including (1) the Nkx2-1-dependent glandular region, and (2) the Nkx2-1/FoxE-dependent thyroid-equivalent region. DV axial regionalization may be responsible for organizing glandular and thyroid-equivalent traits of the pharynx along the DV axis.

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.

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.

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.

Hox-mediated endodermal identity patterns pharyngeal muscle formation in the chordate pharynx.

The chordate pharynx, possessing gill slits and the endostyle, is a complex of multiple tissues that are highly organized along the anterior-posterior (AP) axis. Although Hox genes show AP coordinated expression in the pharyngeal endoderm, tissue-specific roles of these factors for establishing the regional identities within this tissue have not been demonstrated. Here, we show that Hox1 is essential for the establishment of AP axial identity of the endostyle, a major structure of the pharyngeal endoderm, in the ascidian Ciona intestinalis We found that knockout of Hox1 causes posterior-to-anterior transformation of the endostyle identity, and that Hox1 represses Otx expression and anterior identity, and vice versa. Furthermore, alteration of the regional identity of the endostyle disrupts the formation of body wall muscles, suggesting that the endodermal axial identity is essential for coordinated pharyngeal development. Our results demonstrate an essential role of Hox genes in establishment of the AP regional identity in the pharyngeal endoderm and reveal crosstalk between endoderm and mesoderm during development of chordate pharynx.

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.

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians.

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

Introduction.

The chordate ascidians, the major group of tunicate s, is the best animal group for studying molecular and cellular processes underlying formation of a chordate body plan. For these studies, transgenic technologies are powerful. Transgenesis of ascidians has a long history of more than 20 years, and many practical tips have been accumulated. This book is aimed at summarizing the accumulated techniques in ascidians in addition to concrete research in which transgenic techniques have played pivotal roles. This book is useful for fast assimilation of the techniques and for learning the unique devices developed by the enthusiasm of ascidian researchers.

Germline Transgenesis in Ciona.

Transgenesis is an indispensable method for elucidating the cellular and molecular mechanisms underlying biological phenomena. In Ciona, transgenic lines that have a transgene insertion in their genomes have been created. The transgenic lines are valuable because they express reporter genes in a nonmosaic manner. This nonmosaic manner allows us to accurately observe tissues and organs. The insertions of transgenes can destroy genes to create mutants. The insertional mutagenesis is a splendid method for investigating functions of genes. In Ciona intestinalis, expression of the gfp reporter gene is subjected to epigenetic silencing in the female germline. This epigenetic silencing has been used to establish a novel method for knocking down maternal expression of genes. The genetic procedures based on germline transgenesis facilitate studies for addressing gene functions in Ciona.

The Enhancer Trap in Ciona.

Enhancer trap is a famous application of transposons. This method is useful for the creation of marker transgenic lines that express a reporter gene in tissue- or organ-specific manner, characterization of enhancers in the genome, finding novel patterns of gene expression, and mutagenesis. In Ciona intestinalis, efficient enhancer traps with Minos and Sleeping beauty transposons have been reported. With the enhancer trap lines, the intronic enhancers regulating the expression of the Musashi gene, the compartment in the digestive tube, the presence of enhancers sensitive to the orientation of the gene that they regulate, and the functions of the Hox1 gene have been revealed. The enhancer trap lines generated with the transposon vectors are valuable resources for use as visual markers.

Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona.

Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.