The neuroendocrine system of Ciona intestinalis Type A, a deuterostome invertebrate and the closest relative of vertebrates

Deuterostome invertebrates, including echinoderms, hemichordates, cephalochordates, and urochordates, exhibit common and species-specific morphological, developmental, physiological, and behavioral characteristics that are regulated by neuroendocrine and nervous systems. Over the past 15 years, omics, genetic, and/or physiological studies on deuterostome invertebrates have identified low-molecular-weight transmitters, neuro-peptides and their cognate receptors, and have clarified their various biological functions. In particular, there has been increasing interest on the neuroendocrine and nervous systems of Ciona intestinalis Type A, which belongs to the subphylum Urochordata and occupies the critical phylogenetic position as the closest relative of vertebrates. During the developmental stage, gamma-aminobutylic acid, D-serine, and gonadotropin-releasing hormones regulate metamorphosis of Ciona . In adults, the neuropeptidergic mechanisms underlying ovarian follicle growth, oocyte maturation, and ovulation have been elucidated. This review article provides the most recent and fundamental knowledge of the neuroendocrine and nervous systems of Ciona , and their evolutionary aspects.


Introduction
Endocrine systems play crucial roles in the regulation of a variety of biological activities and hemostasis in vertebrates, and their functions are underpinned by the closed circulatory systems and glands that produce and secrete hormones and transport them to their target tissues; for example, the hypothalamus-pituitary-peripheral tissue axes.However, unlike vertebrates, most deuterostome invertebrates are not endowed with closed circulatory systems.Consequently, the neuroendocrine systems of deuterostome invertebrates are responsible for the regulation of biological activities and homeostasis.Moreover, the central nervous systems (CNS) of deuterostome invertebrates possess species-specific morphological characteristics.For example, Ciona intestinalis Type A (this species has recently been suggested to rename Ciona robusta; Brunetti et al., 2015), a cosmopolitan ascidian species (Urochordata), possesses a cerebral ganglion as its CNS and innervates almost all of its tissues.In echinoderms i.e., sea urchins, starfish, and sea cucumbers, the radial nervous system is responsible for major regulation of biological events.These findings indicate that the neuroendocrine and nervous systems, rather than the endocrine system, act as the central regulatory systems in deuterostome invertebrates and that these systems vary among phyla.

Dual transmitter expression in larval neurons of Ciona
An important characteristic of urochordate ascidians is the tadpolelike morphology of the larval stage (Fig. 1).Since the initial discovery of ascidian larva (Kowalevsky, 1866), researchers have been fascinated by the molecular mechanisms responsible for its vertebrate-like morphology.After completing embryogenesis, ascidian larvae swim by beating their tail.This behavior is accomplished through the regulation of muscle contractions by the nervous system.The regulation of this behavior and its comparative analysis with that observed in vertebrates is another important topic of study in ascidian biology.Ciona intestinalis Type A/Ciona robusta has become a model for such research because of its ubiquitous distribution, short generation times, simple body structure, genome decoding, and the introduction of various techniques for manipulating genes (Corbo et al., 1997;Satou et al., 2001;Dehal et al., 2002;Satoh, 2003;Sasakura et al., 2003Sasakura et al., , 2005;;Kawai et al., 2012;Sasaki et al., 2014;Stolfi et al., 2014;Treen et al., 2014).
What benefit does Ciona receive by adopting a dual transmitter system?One plausible hypothesis is maintaining diverse neuronal functions while reducing the number of neurons.The Ciona nervous system comprises a limited number of neurons (Ryan et al., 2016(Ryan et al., , 2018)).The neurons of the nervous system respond to a variety of environmental cues, such as light, gravity, and mechanical stimuli to determine the direction of swimming, and they are also involved in the processes of metamorphosis (Nakagawa et al., 1999;Tsuda et al., 2003;Sakurai et al., 2004;Bostwick et al., 2020).To execute these functions using a small number of cells, neurons likely employ different transmitters depending on the situation.The PC2 mutant larvae are morphologically normal and can swim and adhere to a substrate, thereby initiating the process of metamorphosis (Nakayama-Ishimura et al., 2009).This suggests that neuropeptides are not involved in the regulation of embryogenesis and swimming, at least in culture conditions in the laboratory.Conversely, the small-molecule neurotransmitters like acetylcholine and GABA control swimming behaviors and metamorphosis (Horie et al., 2010;Hirai et al., 2017;Hozumi et al., 2020).The major functions of neuropeptides are observed during metamorphosis.We hypothesize that the dual-transmitter system facilitates the seamless transition from the larval to adult stage using the same neuronal network.If this hypothesis is correct, then classical neurotransmitters and neuropeptides will likely be secreted at different times.Consequently, investigating the regulation of neurotransmitter secretion by Ciona neurons warrants further study (Vaaga et al., 2014).
The molecular mechanisms underlying the acquisition of dualtransmitter properties have been elucidated in dopamine neurons.Ciona larvae have a single cluster of dopamine neurons close to the photoreceptors in the larval brain.The dopamine-positive cells, also known as the coronet cells (Fig. 2D), have a prominent knob that extends into the lumen of the brain.While the function of these dopamine neurons remains to be investigated, experiments using the inhibitors of the monoamine receptors suggest that they play a role in regulating the shadow response (Razy-Krajka et al., 2012).In addition to dopamine pathway genes, larval dopamine neurons express a variety of neuropeptides (Horie et al., 2018).Several lines of evidence, including research on neuropeptide expression, suggest that the cluster dopamine neurons share evolutionary similarities with the vertebrate hypothalamus at the molecular level (Moret et al., 2005;Razy-Krajka et al., 2012).
A recent study showed that Pancreas transcription factor (Ptf) and Meis are necessary and sufficient for the terminal differentiation of dopamine neurons (Horie et al., 2018).Ptf and Meis are considered to directly promote transcription of the genes encoding the enzymes responsible for synthesizing dopamine, as the binding sites of Ptf and Meis are located within their enhancer elements.Ptf and Meis also regulate the expression of the neuropeptide genes, suggesting that the property of dual neurotransmission is closely associated with the core mechanism of dopamine neuron specification.The Ptf-and Meis-binding sites on the neuropeptide genes are adjacent, whereas those located at the enhancer elements of the dopamine pathway genes are separated by a distance of approximately 150 base pairs.Although temporal expression of neuropeptide genes occurs earlier than that of genes in the dopamine pathway, the functional significance of this difference in the timing has yet to be clarified; however, it suggests that the requirements for neuropeptide and dopamine signaling are temporarily distinct.

Functions of neuropeptides in the Ciona larva
A fundamental characteristic of Ciona is its remarkably simple architecture, which is best exemplified by its tadpole larva.A single larva is comprised of less than 3000 cells, including 40 notochord cells, 36 muscle cells, and ~230 neurons (Satoh 1994;Ryan et al., 2016Ryan et al., , 2018)).In this simplified anatomical structure, the circulatory and endocrine systems are underdeveloped, suggesting that the endocrine signaling mechanisms that regulate various physiological functions in vertebrates are not operational in Ciona larvae.
However, numerous genes encoding neuropeptides are expressed in the larval neurons and glial cells (Hamada et al., 2011;Kusakabe et al., 2012).For example, seven GnRHs are expressed and function in Ciona larvae (Adams et al., 2003;Tello et al., 2005;Kamiya et al., 2014;Okawa et al., 2020).GnRHs are highly conserved neuropeptides that play roles in the reproductive development of animals.The seven GnRHs H. Satake and Y. Sasakura in Ciona are encoded by three genes, i.e., Ci-gnrh1, Ci-gnrh2, and Ci-gnrhx (Adams et al., 2003;Kawada et al., 2009).Ci-gnrh1 and Ci-gnrh2 are expressed in the larval nervous system, suggesting that they play a role in larval development (Kusakabe et al., 2012).However, the Ciona larvae are reproductively immature; they do not have a gonad, and only a few primordial germ cells are present on the ventral side of the tail (Okada and Yamamoto, 1999;Takamura et al., 2002b).
This incongruence between GnRH expression and the reproductively immature state of the larva suggests that these neuropeptides have an alternative function beyond reproduction.Although our recent studies showed that GnRH regulates metamorphosis, as discussed in the next section (Kamiya et al., 2014;Hozumi et al., 2020), the roles of neuropeptides including GnRH during the larval period have not been precisely clarified.Recently, the excitation of Ci-gnrh2-positive neurons and glial cells at the larval stage has been characterized using the Ca 2+ indicator G-CaMP8 (Okawa et al., 2020).Excitations in the glial cells of the tail nerve cord were observed to coincide with cessation in the movement of the tail, suggesting that these glial cells negatively regulate swimming.Since Ca 2+ imaging cannot conclusively determine the function of Ci-gnrh2, the role of this hormone at the larval stage requires further investigation using more direct methodologies in future studies.

Ciona metamorphosis
Ascidians undergo a transformation from free-living larvae into sessile adults through a process of metamorphosis (Fig. 3A; Cloney, 1982;Karaiskou et al., 2015).During the metamorphosis of Ciona, the tail regresses toward the anterior trunk and degenerates through apoptosis (Chambon et al., 2002(Chambon et al., , 2007;;Comes et al., 2007).The adult organs, including the digestive and circulatory systems, are underdeveloped at the larval stage.These adult organs become functional through extensive cell proliferation, which is temporally arrested at the larval stage (Nakayama et al., 2005).
Metamorphosis results in the adults becoming sessile, and the initiation of this process is associated with adhesion.The tadpole larva possesses an adhesive organ, known as the adhesive papillae, at its anterior end.During the motile larval stage, the tadpoles seek out sites at which they will settle as sessile adults.The mechanisms of site selection have yet to be fully understood, but environmental conditions have been identified as contributory factors in several ascidian species (Jackson et al., 2002;Sensui and Hirose, 2020).The adhesive organ of Ciona produces mucus substances that interact with the lectin Peanut Agglutinin (Zeng et al., 2019).Using these adhesive substances, the larvae attach themselves to the substrate.The adhesive papillae originate from regions postulated to be homologous to vertebrate neurogenic placodes (Manni et al., 2004).The neurogenic placodes are the primordia of the sensory systems around the facial region (Schlosser, 2006).Likewise, each adhesive papilla of Ciona contains two pairs of sensory neurons, which are required to initiate metamorphosis (Takamura, 1998;Ryan et al., 2018;Zeng et al., 2019;Sakamoto et al., 2022;Johnson et al., 2023).The papillae are considered to be responsive to the mechanical stimuli generated by the adhesion and subsequent tail flicks (Sakamoto et al., 2022).Following adhesion, two round elevations of Ca 2+ are observed in the papilla (Wakai et al., 2021).Although the cells responsible for this Ca 2+ elevation have not yet been fully identified, it is hypothesized that the sensory neurons in the papillae are excited by adhesion, and this excitation is transmitted to the posterior region of the body to initiate metamorphosis.

GnRHs control metamorphic events of Ciona
Our recent studies have shown that neuropeptides are essential for tail regression (Nakayama-Ishimura et al., 2009;Kamiya et al., 2014;Hozumi et al., 2020).The recessive mutants of the gene encoding PC2 exhibit normal embryogenesis and the larvae are indistinguishable from the wild type.However, the PC2 mutant larvae do not initiate tail regression after settlement (Fig. 3B-D).Compared to the wild type, adult organ growth in the PC2 mutants occurs after settlement, albeit to a somewhat weaker extent.As a result, the trunks of the PC2 mutants are characterized by having a post-metamorphosis appearance, but the tails are retained.
The involvement of PC2 in tail regression suggests that neuropeptides play a critical role in this step.Our studies showed that GnRHs play a central role in the regulation of metamorphosis.For example, when administered to larvae, the six GnRH peptides encoded by gnrh1 and gnrh2 induce metamorphosis without adhesion (Kamiya et al., 2014).Moreover, gnrh1-and gnrh2-disrupted larvae do not complete metamorphosis and the larvae have phenotypes that are similar to PC2 mutants (Hozumi et al., 2020).
The primary expression domains of Cignrh1 and -2 are in the larval brain and the tail nerve cord.Therefore, we hypothesize that GnRHs secreted from the CNS exert a direct influence on the tail.Indeed, this is substantiated by the fact that GnRH receptors are mainly expressed in the tail (Kusakabe et al., 2012).Functional analyses of these receptors have yet to be conducted.The remarkable morphological movement of tail cells, which is mediated by actomyosin and apoptosis, results in tail regression in Ciona (Chambon et al., 2002;Yamaji et al., 2020).These cellular events are hypothesized to be activated by GnRH signaling.
The aforementioned observations and hypothesis suggest that GnRHs are not the initial triggers for metamorphosis.Indeed, our research showed that the neurotransmitter GABA is responsible for the initiation of metamorphosis, and that the GnRHs act downstream of GABA (Hozumi et al., 2020).GABA uses its metabotropic receptor (GABABR) for metamorphosis, and GABABR genes are expressed in a subset of the GnRH neurons.This expression pattern suggests that GABA is likely involved in the control of GnRH secretion during metamorphosis.GABA is generally classified as an inhibitory neurotransmitter, and the precise mechanisms by which it activates GnRH secretion remain unclear.In mammals, GABA acts as the primary positive regulator of GnRH secretion from hypothalamic neurons.In such cases, GABA primarily employs its ionotropic receptor; the intracellular concentration of chloride ions is elevated in the GnRH neurons, and binding of GABA to the receptor induces excitations through the efflux of this anion from the neurons (Ben-Ari, 2002; Watanabe et al., 2014).In contrast, Ciona employs a different mechanism involving GABABRs, which are G-protein-coupled receptors as opposed to ionotropic receptors.GABABRs have the capacity to excite neurons by activating the Gq pathway (Karls and Mynlieff, 2015) and it is considered that a similar mechanism is employed in Ciona metamorphosis.
In addition to GABA and GnRH, several other signaling molecules have been suggested to mediate the initiation of Ciona metamorphosis.These include acetylcholine, noradrenaline, L-glutamate, thyroid hormone, fibroblast growth factor, and nitric monoxide (NO) (Coniglio et al., 1998;Patricolo et al., 2001;Kimura et al., 2003;Comes et al., 2007;Treen et al., 2014;Hirai et al., 2017).In other ascidian species, potassium ions and an epidermal growth factor (EGF)-like protein induce metamorphosis (Degnan et al., 1997;Eri et al., 1999;Davidson and Swalla, 2001).Genes encoding proteins having EGF repeats are also expressed in the adhesive papillae of Ciona (Nakayama et al., 2001(Nakayama et al., , 2002)).The characterization of the mechanism of how these molecules interact with GABA-GnRH signaling remains an area for future studies.

D-serine signaling is responsible for tail regression
The mechanisms by which GnRH mediates tail regression are still obscure.However, our recent study identified D-serine as a downstream factor that regulates the important process of tail regression (Krasovec et al., 2022).
Most of the amino acids in organisms are in the L-form.However, a small subset of D-amino acids also plays crucial roles in regulating biological processes (Du et al., 2023).D-serine is a well-known example of such D-amino acids.For example, D-serine is required for some brain functions in mammals and Drosophila where it has been shown to modulate glutamatergic neurotransmission by binding directly to N-methyl-D-aspartic acid (NMDA)-type glutamate receptors (Mothet et al., 2000;Oliet and Mothet, 2009;Park et al., 2022;Inoue et al., 2023).In addition to its role in the CNS, D-serine is present in the peripheral tissues of both vertebrates and invertebrates (Nagata et al., 2006;Horio et al., 2011;Tanigawa et al., 2016).In mammals, D-serine functions in the differentiation and maintenance of the skin (Inoue et al., 2014).The enzyme serine racemase converts L-serine into D-serine (Wolosker et al., 1999).This enzyme is ubiquitously distributed in eukaryotes, including plants, fungi, and animals (Uda et al., 2016), suggesting that the regulatory functions of this amino acid extend beyond the modulation of neurotransmission.
The Ciona genome encodes three homologs of serine racemase (Krasovec et al., 2022).Of these, Ci-SRR3 is expressed at the larval stage.Interestingly, this expression is dependent upon neuropeptide signaling, with expression levels decreasing markedly in PC2 mutants and gnrh2 knockdown larvae (Hozumi et al., 2020;Krasovec et al., 2022).In line with the expression pattern of Ci-SRR3, D-serine is synthesized in Ciona larvae and is implicated in the regulation of the process of tail regression.
Ascidian tail regression involves the compression of long tail tissues into a posterior region of the trunk through actomyosin-mediated morphological alterations (Lash et al., 1973;Yamaji et al., 2020).The epidermis and internal tail tissues, including muscle and notochord, migrate toward the trunk through a different mechanism.While the inner tissues form double coils to shorten their length, the epidermis undergoes cellular shortening along the anterior-posterior axis.Given these differences in the migration mechanisms, the epidermis and inner tail tissues move independently toward the trunk.To facilitate independent migration, the epidermis detaches itself from the internal tissues at the onset of tail regression by secreting a transparent fluid from numerous large vesicles present in the epidermal cells.This fluid also creates a space, or "pocket", in the posterior trunk, which acts as a receptacle for the migrating tail tissues.
Ci-SRR3 and D-serine signaling facilitate formation of the pocket.In Ci-SRR3-disrupted larvae, epidermal cells cannot secrete the transparent fluid and the epidermis does not detach from the internal tissues.Consequently, the subsequent stages of tail regression are not completed.Conversely, D-serine administration to larvae induces exocytosis from the epidermis and pocket formation without initiating the other events associated with metamorphosis.Analogous to it function in other animals, the epidermal cells respond to D-serine via the NMDA-type glutamate receptor.In both Ciona and mammals, D-serine plays a role in mediating secretion from epidermal cells, suggesting that this particular epidermal function of D-serine is a shared trait among chordates.
The NMDA-type receptor is activated by several neurotransmitters, including L-glutamate, D-serine, and glycine (Pace et al., 1992).Structurally, the NMDA receptor is a heterotetramer comprised of two subunits (GluN1 and GluN2).D-serine and glycine bind to the same site on the GluN1 subunit, while the binding site of L-glutamate is on the GluN2 subunit (Hansen et al., 2021).In Ciona, both D-serine and glycine can induce pocket formation, but L-glutamate does not (Hozumi et al., 2020).Interestingly, the Ciona NMDA receptor possesses a substitution of one amino acid residue that is crucial for L-glutamate binding (Laube et al., 1997;Tikhonova et al., 2002).This suggests that the functional attributes of this receptor in Ciona, including its role in pocket formation, are independent of L-glutamate binding.

Neuroendocrine and nervous systems in Ciona adults
Unlike embryos and larvae, Ciona adults perform both reproductive and feeding functions, which are considered to be regulated by the nervous, neuroendocrine, and endocrine systems in an animal.Given the absence of a hypothalamus-pituitary axis, which plays a central role in the endocrine system of vertebrates, the fundamental biological functions in Ciona are expected to be regulated by the nervous and neuroendocrine systems.Indeed, neuroanatomical studies of a transgenic Ciona expressing Kaede reporter under the control of cis-regulatory region of the PC2 gene revealed extensive innervation of cerebral and peripheral peptidergic neurons to a variety of peripheral tissues (Osugi et al., 2017(Osugi et al., , 2020)).These findings demonstrate that a number of biological events are regulated by neuropeptidergic neuroendocrine and nervous systems in adult Ciona.In this section, we provide an overview of Ciona peptides and their biological roles in reproductive processes.

Table 1
Peptide-receptor pairs identified to date in Ciona.
In Ciona, tGnRH-3 to − 8 exhibit a structural similarity to vertebrate GnRHs, since they are composed of 10 amino acids and the complete Nterminal and C-terminal consensus motifs of vertebrate GnRHs are conserved (Sakai et al., 2017(Sakai et al., , 2020)).In contrast, another GnRH-related peptide, CiGnRH-X, is composed of 16 amino acids and lacks the common Pro at the second position from the C-terminus that is conserved in other chordate GnRHs (Kawada et al., 2009;Matsubara et al., 2016;Satake 2023).Taken together, these findings highlight both the evolutionary conservation and Ciona-specific divergence of peptide sequences.
While most Ciona neuropeptide genes are predominantly expressed in the cerebral ganglion, several neuropeptide genes have been shown to be expressed in non-neural tissues.For example, CiTK gene expression was observed not only in the cerebral ganglion, but also in zone 7 of the endostyle, an area that is considered to correspond to the vertebrate thymus harboring secretory vesicles (Satake et al., 2004).Moreover, the expression of the CiCT gene has been detected in a variety of non-neural tissues, including the endostyle and the neural gland (non-neuronal sponge-like tissues) (Sekiguchi et al., 2009).These findings suggest that several Ciona peptides function not only as neurotransmitters, but also as paracrine/autocrine factors.
As shown in Table 1, GPCRs for Ciona-specific peptides, such as CiLFs, CiYFV/Ls, and CiNTLPs, have also been elucidated through a combination of machine learning-based prediction of peptide-GPCR interactions and experimental validation of these predicted outputs (Shiraishi et al., 2019;Kawada et al., 2022;Satake et al., 2023;Satake, 2023).A striking feature of this research is that these GPCRs are not included in clusters of hitherto known GPCRs for peptides; rather, they are included in clusters of GPCRs for non-peptidic ligands or Ciona-specific orphan GPCRs (Shiraishi et al., 2019;Satake et al., 2023;Satake, 2023).These findings indicate that several GPCRs for species-specific peptides have likely evolved from GPCRs for non-peptidic ligands, highlighting the prominent advantages of the machine learning-assisted research strategy in the identification of GPCRs for novel peptides over sequence similarity-and molecular phylogeny-based procedures.

Neuroendocrine regulation of ovarian follicle development
Ciona ovarian follicle development is categorized into four stages (Matsubara et al., 2020;Satake, 2023).Until recently, the molecular mechanisms underlying Ciona ovarian follicle development remained unexplored.Recent studies have revealed that neuropeptides play major roles in the regulation of various follicle development processes in Ciona.
Localization of several GPCRs for peptides in the ovarian follicles has been reported (Aoyama et al., 2008;Matsubara et al., 2019;Osugi et al., 2021).Moreover, neuroanatomical analysis using PC2-transgenic Ciona adults revealed the innervation of neuropeptidergic neurons to the ovary (Osugi et al., 2017(Osugi et al., , 2020)).Collectively, these studies corroborated the hypothesis that neuropeptides participated in the regulation of follicle development in Ciona.Indeed, peptidergic regulation of the follicle development has been verified over the past 15 years (Fig. 4).
Moreover, colocalization of CiTK-R and cathepsin D in test cells, localization of carboxypeptidase B1 and chymotrypsin in follicle cells, and the time course of the gene expression of these proteases all indicate that cathepsin D is directly activated by CiTK in test cells, followed by secondary activation of the two remaining proteases (Aoyama et al., 2012;Kawada et al., 2022;Satake, 2023).This novel regulatory cascade is consistent with the activity of the three proteases.Cathepsin D plays a major role in a proteolytic process of precursors of yolk proteins (e.g., vitellogenin) and follicular component in fish and bird oocytes at the immature stages (Carnevali et al., 2006).Carboxypeptidase B1 participates in the proteolytic processing of various zona pellucida components during early oocyte growth stages in mice (Litscher et al., 1999).Furthermore, the application of chymotrypsin inhibitors inhibited oocyte growth at preGVBD stages in several invertebrates, including an ascidian (Halocynthia roretzi) (Sakairi and Shirai, 1991), starfish (Asterina pectinifera) (Takagi-Sawada et al., 1989;Tanaka et al., 2000), and fruit fly (Drosophila melanogaster) (Jakobsen et al., 2005).These findings are compatible with the view that these proteases, activated by CiTK, are responsible for upregulating the growth of Ciona stage-II follicles via proteolysis of various oocyte and follicle proteins.
Gene expression of CiVP-R has been reported in early stage III follicles (Matsubara et al., 2019;Kawada et al., 2022;Satake, 2023).As shown in Fig. 4, CiVP activates the Ciona-extracellular signal-related kinase (MEK/ERK) homolog in a non-transcriptional manner, leading to the phosphorylation of Ciona maturation promoting factor (MPF, Cdc2).This CiVP-MEK/ERK-cdc2 cascade leads to germinal vesicle breakdown (GVBD), which is a canonical biological signature of oocyte maturation, mediated by CiVP (Matsubara et al., 2019;Kawada et al., 2022;Satake, 2023).The induction of GVBD by CiVP corroborates previous studies which showed that GVBD in ascidian oocytes requires intracellular calcium ions (Lambert, 2011;Deguchi et al., 2015).This is consistent with the observation that CiVP-R mediates intracellular calcium ion levels, but not the production of cAMP, upon binding to CiVP (Kawada et al., 2008).Collectively, these study indicate that the MEK/ERK-MPF signaling pathway to oocyte maturation is conserved across metazoans, whereas the factors that trigger this signaling cascade vary among species (Lambert, 2011;Deguchi et al., 2015;Satake, 2023).Consequently, it is concluded that Ciona species specifically employ CiVP, a VP family peptide, as an oocyte maturation factor, and shares the essential intraoocytic regulation of MPF with other organisms.
CiVP also plays a central role in induction of ovulation (Fig. 4).The activation of the CiVP-ERK/MEK pathway results in upregulation of gene expression and the resulting increase in enzymatic activity of a matrix metalloproteinase (MMP) ortholog, MMP2/9/13, at stage III, leading to ovulation (Matsubara et al., 2019;Kawada et al., 2022;Satake et al., 2023).Notably, ovaries from CiVP gene-edited Ciona exhibit an increased number of immature follicles and a marked decrease in the number of mature follicles (Kawada et al., 2021;Satake, 2023).These findings are in good agreement with the effect of CiVP on the induction of ovulation.Interestingly, cionin upregulates the gene expression and the resultant enzymatic activity of MMP2/9/13 via transcriptional upregulation of receptor tyrosine kinase (RTK) signaling-related genes such as rora, fcol1, and gla3 (Osugi et al., 2020;Kawada et al., 2022;Satake et al., 2023).These findings substantiate the biological role of CiVP and cionin in the induction of ovulation and demonstrate that ovulation in Ciona is regulated by multiple signaling pathways.As shown in Fig. 5, the intraovarian molecular mechanisms that underlie Ciona ovulation are partially conserved in vertebrates.For example, both Ciona and mammals share the MEK/ERK activation pathway, but not upregulation of MMP (Matsubara et al., 2019;Kawada et al., 2022;Satake, 2023).Conversely, the upregulation of MMP, but not an MEK/ERK activation, is conserved in Ciona and teleost fish (Matsubara et al., 2019;Kawada et al., 2022;Satake, 2023).Moreover, induction of ovulation via the CCK (cionin)-RTK-MMP signaling pathway is specific to Ciona (Osugi et al., 2021;Kawada et al., 2022;Satake, 2023).In vertebrates, GnRHs secreted by the hypothalamus stimulate the synthesis and secretion of gonadotropins (luteinizing hormone, LH, and follicle-stimulating hormone, FSH), which subsequently promote oocyte maturation and ovulation within the hypothalamus-pituitary-gonad axis (HPG axis).In contrast, CiVP, not GnRHs, directly induces oocyte maturation and ovulation (Matsubara et al., 2019;Kawada et al., 2021;Satake, 2023).Additionally, the absence of gonadotropins in Ciona is consistent with the absence of tissue corresponding to the pituitary (Lambert, 2011;Deguchi et al., 2015;Satake, 2023;Yang et al., 2023).Collectively, the "hybrid" ovulation molecular mechanisms provide new insights into the evolution of ovulation in chordates; ancestral chordates likely possessed Ciona-like ovulation mechanisms, and some of these mechanisms were conserved while others were lost during the evolution of each species.This diversification likely also entailed the acquisition of species-specific ovulation mechanisms, including the RTK-MMP signaling pathway in Ciona and the HPG axis in vertebrates.

Conclusions and perspectives
Over the past two decades, significant advances have been made in elucidating the fundamental nervous and neuroendocrine regulatory systems in Ciona larva and adults.These discoveries have promoted the exploration of the key molecular mechanisms underlying metamorphosis and follicular development.Ascidian metamorphosis exhibits a variety of unique characteristics that are not observed in vertebrates; the transmitter-directed regulatory systems of metamorphosis highlight the extent of diversification of the nervous system in deuterostomes.The neuropeptidergic follicle development systems in Ciona show both molecular and functional conservation and diversification of signaling pathways for oocyte maturation and ovulation throughout chordates.Further studies are required to verify the net nervous and neuroendocrine systems in Ciona.In particular, although no mature peptides have been detected in Ciona larva, various peptide genes were found to be expressed in the larval brain (Hamada et al., 2011).Consequently, the elucidation of the endogenous role of peptides in Ciona larva awaits further study.Likewise, the biological roles of neurotransmitters have yet to be investigated in Ciona adults.Furthermore, the non-reproductive functions of the nervous and neuroendocrine systems in Ciona adults remain to be clarified.Integration of these biological H. Satake and Y. Sasakura

Fig. 2 .
Fig. 2. Localization of neurons in the larval trunk of Ciona, as revealed by the reporter constructs.(A) GnRH neurons (yellow).This photomicrograph of the stable transgenic line of the gnrh1 reporter construct.MG, motor ganglion (the region including motor neurons); PN, papilla neuron.(B) Glutamate neurons (green).The neurons are labeled with the reporter construct recapitulating the expression of the gene encoding vesicular glutamate transporter (VGLUT).(C) Neurons capable of secreting acetylcholine (blue).The neurons are labeled with the reporter construct of the gene encoding vesicular acetylcholine transporter (VACHT).(D) Dopamine neurons (green).The neurons are labeled with the reporter construct of the gene encoding tyrosine hydroxylase (TH).(E) Schematic illustration of the larval nervous system and the positions of dual-transmitter neurons.

Fig. 3 .
Fig. 3. Metamorphosis of Ciona.(A) Schematic illustration of the life cycle of Ciona.(B) A normal larva.(C) A juvenile after the completion of tail regression.(D) A homozygous PC2 mutant larva.Although this animal started undergoing metamorphosis, the tail was not regressed while the trunk had initiated adult organ growth.

Fig. 4 .
Fig. 4. Neuropeptidergic follicle development.The upper panel shows the development of Ciona ovarian follicles.Black and white arrows indicate a germinal vesicle (GV) and follicular layers, respectively.Permission has been obtained from the publisher to partially reproduce the image of follicles here (Matsubara et al., 2020).The lower panel illustrates the essential molecular mechanisms underlying the follicle growth, oocyte maturation, and ovulation in Ciona.In vitellogenic (stage II) follicles, CiTK induces the expression of Cathepsin D in test cells and Chymotrypsin and Carboxypeptidase B1 in inner follicular cells, and promotes follicle growth from stage II to stage III.CiNTLP6 suppresses CiTK-induced gene expression and subsequent follicle growth.In postvitellogenic (stage III) follicles, CiVP activates MEK/ERK in oocytes and promotes oocyte maturation and ovulation via activation of MPF and MMP2/9/13, respectively.Cionin also induces MMP2/9/13 expression via CioR2 in inner follicular cells and RTK signaling and subsequent ovulation in stage III follicles.

Fig. 5 .
Fig. 5. Comparison of signaling pathways for ovulation.In vertebrates, ovulation is regulated by the HPG axis.GnRH, a hypothalamus peptide hormone, plays a central role in the secretion of gonadotropins from the pituitary.In Ciona, CiVP and cionin, instead of GnRHs, directly induce ovulation.ERK/MEK is responsible for ovulation in the mammalian and Ciona ovary, but not in the teleost ovary.MMPs induce ovulation in the teleost and Ciona ovary.