Conservation of cis-Regulatory Syntax Underlying Deuterostome Gastrulation.

Throughout embryonic development, the shaping of the functional and morphological characteristics of embryos is orchestrated by an intricate interaction between transcription factors and cis-regulatory elements. In this study, we conducted a comprehensive analysis of deuterostome cis-regulatory landscapes during gastrulation, focusing on four paradigmatic species: the echinoderm Strongylocentrotus purpuratus, the cephalochordate Branchiostoma lanceolatum, the urochordate Ciona intestinalis, and the vertebrate Danio rerio. Our approach involved comparative computational analysis of ATAC-seq datasets to explore the genome-wide blueprint of conserved transcription factor binding motifs underlying gastrulation. We identified a core set of conserved DNA binding motifs associated with 62 known transcription factors, indicating the remarkable conservation of the gastrulation regulatory landscape across deuterostomes. Our findings offer valuable insights into the evolutionary molecular dynamics of embryonic development, shedding light on conserved regulatory subprograms and providing a comprehensive perspective on the conservation and divergence of gene regulation underlying the gastrulation process.

Early transcriptional similarities between two distinct neural lineages during ascidian embryogenesis.

In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are ontogenically associated with ectodermal lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we asked to what extent the transcriptional states of the different neural lineages become similar during the course of progressive lineage restriction. We performed single-cell RNA sequencing (scRNA-seq) analyses on hand-dissected neural precursor cells of the two distinct lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from the 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage, including miR-124, Celf3.a, Zic.r-b, and Ets1/2. Altogether, the current study has revealed genome-wide transcriptional dynamics of neural progenitor cells of two distinct developmental origins. Our scRNA-seq dataset is unique and provides a valuable resource for future analyses, enabling a precise temporal resolution of cell types not previously described from dissociated embryos.

Alternative oxidase blunts pseudohypoxia and photoreceptor degeneration due to RPE mitochondrial dysfunction.

Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.

Specification and survival of post-metamorphic branchiomeric neurons in a non-vertebrate chordate.

Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are replaced by adult-specific ones. The regulatory mechanisms underlying this replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the 'neck', a cellular compartment set aside in the larva to give rise to cranial motor neuron-like neurons post-metamorphosis. Using bulk and single-cell RNA-sequencing analyses, we characterize the transcriptome of the neck downstream of Pax2/5/8. We present evidence that neck-derived adult ciliomotor neurons begin to differentiate in the larva and persist through metamorphosis, contrary to the assumption that the adult nervous system is formed after settlement and the death of larval neurons during metamorphosis. Finally, we show that FGF signaling during the larval phase alters the patterning of the neck and its derivatives. Suppression of FGF converts neck cells into larval neurons that fail to survive metamorphosis, whereas prolonged FGF signaling promotes an adult neural stem cell-like fate.

Ascidian embryonic cells with properties of neural-crest cells and neuromesodermal progenitors of vertebrates.

Neural-crest cells and neuromesodermal progenitors (NMPs) are multipotent cells that are important for development of vertebrate embryos. In embryos of ascidians, which are the closest invertebrate relatives of vertebrates, several cells located at the border between the neural plate and the epidermal region have neural-crest-like properties; hence, the last common ancestor of ascidians and vertebrates may have had ancestral cells similar to neural-crest cells. However, these ascidian neural-crest-like cells do not produce cells that are commonly of mesodermal origin. Here we showed that a cell population located in the lateral region of the neural plate has properties resembling those of vertebrate neural-crest cells and NMPs. Among them, cells with Tbx6-related expression contribute to muscle near the tip of the tail region and cells with Sox1/2/3 expression give rise to the nerve cord. These observations and cross-species transcriptome comparisons indicate that these cells have properties similar to those of NMPs. Meanwhile, transcription factor genes Dlx.b, Zic-r.b and Snai, which are reminiscent of a gene circuit in vertebrate neural-crest cells, are involved in activation of Tbx6-related.b. Thus, the last common ancestor of ascidians and vertebrates may have had cells with properties of neural-crest cells and NMPs and such ancestral cells may have produced cells commonly of ectodermal and mesodermal origins.

The alternative enzymes-bearing tunicates lack multiple widely distributed genes coding for peripheral OXPHOS subunits.

The respiratory chain alternative enzymes (AEs) NDX and AOX from the tunicate Ciona intestinalis (Ascidiacea) have been xenotopically expressed and characterized in human cells in culture and in the model organisms Drosophila melanogaster and mouse, with the purpose of developing bypass therapies to combat mitochondrial diseases in human patients with defective complexes I and III/IV, respectively. The fact that the genes coding for NDX and AOX have been lost from genomes of evolutionarily successful animal groups, such as vertebrates and insects, led us to investigate if the composition of the respiratory chain of Ciona and other tunicates differs significantly from that of humans and Drosophila, to accommodate the natural presence of AEs. We have failed to identify in tunicate genomes fifteen orthologous genes that code for subunits of the respiratory chain complexes; all of these putatively missing subunits are peripheral to complexes I, III and IV in mammals, and many are important for complex-complex interaction in supercomplexes (SCs), such as NDUFA11, UQCR11 and COX7A. Modeling of all respiratory chain subunit polypeptides of Ciona indicates significant structural divergence that is consistent with the lack of these fifteen clear orthologous subunits. We also provide evidence using Ciona AOX expressed in Drosophila that this AE cannot access the coenzyme Q pool reduced by complex I, but it is readily available to oxidize coenzyme Q molecules reduced by glycerophosphate oxidase, a mitochondrial inner membrane-bound dehydrogenase that is not involved in SCs. Altogether, our results suggest that Ciona AEs might have evolved in a mitochondrial inner membrane environment much different from that of mammals and insects, possibly without SCs; this correlates with the preferential functional interaction between these AEs and non-SC dehydrogenases in heterologous mammalian and insect systems. We discuss the implications of these findings for the applicability of Ciona AEs in human bypass therapies and for our understanding of the evolution of animal respiratory chain.

Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline.

Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona.

Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the cis-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use Ciona to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed cis-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of Ciona, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.

Characterization of a putative orexin receptor in Ciona intestinalis sheds light on the evolution of the orexin/hypocretin system in chordates.

Tunicates are evolutionary model organisms bridging the gap between vertebrates and invertebrates. A genomic sequence in Ciona intestinalis (CiOX) shows high similarity to vertebrate orexin receptors and protostome allatotropin receptors (ATR). Here, molecular phylogeny suggested that CiOX is divergent from ATRs and human orexin receptors (hOX1/2). However, CiOX appears closer to hOX1/2 than to ATR both in terms of sequence percent identity and in its modelled binding cavity, as suggested by molecular modelling. CiOX was heterologously expressed in a recombinant HEK293 cell system. Human orexins weakly but concentration-dependently activated its Gq signalling (Ca2+ elevation), and the responses were inhibited by the non-selective orexin receptor antagonists TCS 1102 and almorexant, but only weakly by the OX1-selective antagonist SB-334867. Furthermore, the 5-/6-carboxytetramethylrhodamine (TAMRA)-labelled human orexin-A was able to bind to CiOX. Database mining was used to predict a potential endogenous C. intestinalis orexin peptide (Ci-orexin-A). Ci-orexin-A was able to displace TAMRA-orexin-A, but not to induce any calcium response at the CiOX. Consequently, we suggested that the orexin signalling system is conserved in Ciona intestinalis, although the relevant peptide-receptor interaction was not fully elucidated.

Pulsation waves along the Ciona heart tube reverse by bimodal rhythms expressed by a remote pair of pacemakers.

The heart of ascidians (marine invertebrate chordates) has a tubular structure, and heartbeats propagate from one end to the other. The direction of pulsation waves intermittently reverses in the heart of ascidians and their relatives; however, the underlying mechanisms remain unclear. We herein performed a series of experiments to characterize the pacemaker systems in isolated hearts and their fragments, and applied a mathematical model to examine the conditions leading to heart reversals. The isolated heart of Ciona robusta autonomously generated pulsation waves at ∼20 to 25 beats min-1 with reversals at ∼1 to 10 min intervals. Experimental bisections of isolated hearts revealed that independent pacemakers resided on each side and also that their beating frequencies periodically changed as they expressed bimodal rhythms, which comprised an ∼1.25 to 5.5 min acceleration/deceleration cycle of a beating rate of between 0 and 25 beats min-1. Only fragments including 5% or shorter terminal regions of the heart tube maintained autonomous pulsation rhythms, whereas other regions did not. Our mathematical model, based on FitzHugh-Nagumo equations applied to a one-dimensional alignment of cells, demonstrated that the difference between frequencies expressed by the two independent terminal pacemakers determined the direction of propagated waves. Changes in the statuses of terminal pacemakers between the excitatory and oscillatory modes as well as in their endogenous oscillation frequencies were sufficient to lead to heart reversals. These results suggest that the directions of pulsation waves in the Ciona heart reverse according to the changing rhythms independently expressed by remotely coupled terminal pacemakers.

Ovarian Follicle Development in Ascidians.

Ovarian follicle development is an essential process for continuation of sexually reproductive animals, and is controlled by a wide variety of regulatory factors such as neuropeptides and peptide hormones in the endocrine, neuroendocrine, and nervous systems. Moreover, while some molecular mechanisms underlying follicle development are conserved, others vary among species. Consequently, follicle development processes are closely related to the evolution and diversity of species. Ciona intestinalis type A (Ciona rubusta) is a cosmopolitan species of ascidians, which are the closest relative of vertebrates. However, unlike vertebrates, ascidians are not endowed with the hypothalamus-pituitary-gonadal axis involving pituitary gonadotropins and sexual steroids. Combined with the phylogenetic position of ascidians as the closest relative of vertebrates, such morphological and endocrine features suggest that ascidians possess both common and species-specific regulatory mechanisms in follicle development. To date, several neuropeptides have been shown to participate in the growth of vitellogenic follicles, oocyte maturation of postvitellogenic follicles, and ovulation of fully mature follicles in a developmental stage-specific fashion. Furthermore, recent studies have shed light on the evolutionary processes of follicle development throughout chordates. In this review, we provide an overview of the neuropeptidergic molecular mechanism in the premature follicle growth, oocyte maturation, and ovulation in Ciona, and comparative views of the follicle development processes of mammals and teleosts.

A synthetic flavonoid derivate in the plasma membrane transforms the voltage-clamp fluorometry signal of CiHv1.

Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.

Extrinsic apoptosis participates to tail regression during the metamorphosis of the chordate Ciona.

Apoptosis is a regulated cell death ubiquitous in animals defined by morphological features depending on caspases. Two regulation pathways are described, currently named the intrinsic and the extrinsic apoptosis. While intrinsic apoptosis is well studied and considered ancestral among metazoans, extrinsic apoptosis is poorly studied outside mammals. Here, we address extrinsic apoptosis in the urochordates Ciona, belonging to the sister group of vertebrates. During metamorphosis, Ciona larvae undergo a tail regression depending on tissue contraction, migration and apoptosis. Apoptosis begin at the tail tip and propagates towards the trunk as a polarized wave. We identified Ci-caspase 8/10 by phylogenetic analysis as homolog to vertebrate caspases 8 and 10 that are the specific initiator of extrinsic apoptosis. We detected Ci-caspase 8/10 expression in Ciona larvae, especially at the tail tip. We showed that chemical inhibition of Ci-caspase 8/10 leads to a delay of tail regression, and Ci-caspase 8/10 loss of function induced an incomplete tail regression. The specificity between apoptotic pathways and initiator caspase suggests that extrinsic apoptosis regulates cell death during the tail regression. Our study presents rare in vivo work on extrinsic apoptosis outside mammals, and contribute to the discussion on its evolutionary history in animals.

Snail Transcriptionally Represses Brachyury to Promote the Mesenchymal-Epithelial Transition in Ascidian Notochord Cells.

Mesenchymal-epithelial transition (MET) is a widely spread and evolutionarily conserved process across species during development. In Ciona embryogenesis, the notochord cells undergo the transition from the non-polarized mesenchymal state into the polarized endothelial-like state to initiate the lumen formation between adjacent cells. Based on previously screened MET-related transcription factors by ATAC-seq and Smart-Seq of notochord cells, Ciona robusta Snail (Ci-Snail) was selected for its high-level expression during this period. Our current knockout results demonstrated that Ci-Snail was required for notochord cell MET. Importantly, overexpression of the transcription factor Brachyury in notochord cells resulted in a similar phenotype with failure of lumen formation and MET. More interestingly, expression of Ci-Snail in the notochord cells at the late tailbud stage could partially rescue the MET defect caused by Brachyury-overexpression. These results indicated an inverse relationship between Ci-Snail and Brachyury during notochord cell MET, which was verified by RT-qPCR analysis. Moreover, the overexpression of Ci-Snail could significantly inhibit the transcription of Brachyury, and the CUT&Tag-qPCR analysis demonstrated that Ci-Snail is directly bound to the upstream region of Brachyury. In summary, we revealed that Ci-Snail promoted the notochord cell MET and was essential for lumen formation via transcriptionally repressing Brachyury.

Vanadium Accumulation and Reduction by Vanadium-Accumulating Bacteria Isolated from the Intestinal Contents of Ciona robusta.

The sea squirt Ciona robusta (formerly Ciona intestinalis type A) has been the subject of many interdisciplinary studies. Known as a vanadium-rich ascidian, C. robusta is an ideal model for exploring microbes associated with the ascidian and the roles of these microbes in vanadium accumulation and reduction. In this study, we discovered two bacterial strains that accumulate large amounts of vanadium, CD2-88 and CD2-102, which belong to the genera Pseudoalteromonas and Vibrio, respectively. The growth medium composition impacted vanadium uptake. Furthermore, pH was also an important factor in the accumulation and localization of vanadium. Most of the vanadium(V) accumulated by these bacteria was converted to less toxic vanadium(IV). Our results provide insights into vanadium accumulation and reduction by bacteria isolated from the ascidian C. robusta to further study the relations between ascidians and microbes and their possible applications for bioremediation or biomineralization.

Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.

CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.

Distribution of cionin, a cholecystokinin/gastrin family peptide, and its receptor in the central nervous system of Ciona intestinalis type A.

The cholecystokinin (CCK)/gastrin family peptides are involved in regulation of feeding and digestion in vertebrates. In the ascidian Ciona intestinalis type A (Ciona robusta), cionin, a CCK/gastrin family peptide, has been identified. Cionin is expressed exclusively in the central nervous system (CNS). In contrast, cionin receptor expression has been detected in the CNS, digestive tract, and ovary. Although cionin has been reported to be involved in ovulation, its physiological function in the CNS remains to be investigated. To elucidate its neural function, in the present study, we analyzed the expression of cionin and cionin receptors in the CNS. Cionin was expressed mainly in neurons residing in the anterior region of the cerebral ganglion. In contrast, the gene expressin of the cionin receptor gene CioR1, was detected in the middle part of the cerebral ganglion and showed a similar expression pattern to that of VACHT, a cholinergic neuron marker gene. Moreover, CioR1 was found to be expressed in cholinergic neurons. Consequently, these results suggest that cionin interacts with cholinergic neurons as a neurotransmitter or neuromodulator via CioR1. This study provides insights into a biological role of a CCK/gastrin family peptide in the CNS of ascidians.

Dynamics of activation in the voltage-sensing domain of Ciona intestinalis phosphatase Ci-VSP.

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane protein containing a voltage-sensing domain (VSD) that is homologous to VSDs from voltage-gated ion channels responsible for cellular excitability. Previously published crystal structures of Ci-VSD in putative resting and active conformations suggested a helical-screw voltage sensing mechanism in which the S4 helix translocates and rotates to enable exchange of salt-bridge partners, but the microscopic details of the transition between the resting and active conformations remained unknown. Here, by combining extensive molecular dynamics simulations with a recently developed computational framework based on dynamical operators, we elucidate the microscopic mechanism of the resting-active transition at physiological membrane potential. Sparse regression reveals a small set of coordinates that distinguish intermediates that are hidden from electrophysiological measurements. The intermediates arise from a noncanonical helical-screw mechanism in which translocation, rotation, and side-chain movement of the S4 helix are only loosely coupled. These results provide insights into existing experimental and computational findings on voltage sensing and suggest ways of further probing its mechanism.

Polymodal sensory perception drives settlement and metamorphosis of Ciona larvae.

The Earth's oceans brim with an incredible diversity of microscopic lifeforms, including motile planktonic larvae, whose survival critically depends on effective dispersal in the water column and subsequent exploration of the seafloor to identify a suitable settlement site. How their nervous systems mediate sensing of diverse multimodal cues remains enigmatic. Here, we uncover that the tunicate Ciona intestinalis larvae employ ectodermal sensory cells to sense various mechanical and chemical cues. Combining whole-brain imaging and chemogenetics, we demonstrate that stimuli encoded at the periphery are sufficient to drive global brain-state changes to promote or impede both larval attachment and metamorphosis behaviors. The ability of C. intestinalis larvae to leverage polymodal sensory perception to support information coding and chemotactile behaviors may explain how marine larvae make complex decisions despite streamlined nervous systems.

A Novel Hemocyte-Derived Peptide and Its Possible Roles in Immune Response of Ciona intestinalis Type A.

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1-4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates.