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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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

The larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN., (© 2021. The Author(s).)

Many hermaphroditic organisms possess a self-incompatibility system to avoid inbreeding. Although the mechanisms of self-incompatibility in flowering plants are well known, little is known about the mechanisms of self-sterility in hermaphroditic marine invertebrates. Ascidians are hermaphroditic sessile marine invertebrates that release sperm and eggs into the surrounding seawater. Several species, including Ciona intestinalis type A (Ciona robusta), exhibit strict self-sterility. In a previous study, we found that the candidate genes responsible for self-sterility in Ciona reside in chromosome 2q (locus A) and chromosome 7q (locus B). Two pairs of multi-allelic genes, named s(sperm)-Themis-A and v(vitelline-coat)-Themis-A in locus A and s-Themis-B and v-Themis-B in locus B, are responsible for self-sterility. In this study, we identified a third multi-allelic gene pair, s-Themis-B2 and v-Themis-B2, within locus B that is also involved in this system. Genetic analysis revealed that the haplotypes of s/v-Themis-A, s/v-Themis-B and s/v-Themis-B2 play essential roles in self-sterility. When three haplotypes were matched between s-Themis and v-Themis, fertilization never occurred even in nonself crossing. Interestingly, gene targeting of either s/v-Themis-B/B2 or s/v-Themis-A by genome editing enabled self-fertilization. These results indicate that s/v-Themis-A, -B and -B2 are S-determinant genes responsible for self-sterility in the ascidian C. intestinalis type A.

Ascidians are the sister group of vertebrates and occupy a critical position in explorations of the evolution of the endocrine and nervous systems of chordates. Here, we describe the complete ventral peptidergic system in adult transgenic Ciona robusta (Ciona intestinalis Type A) which expresses the Kaede reporter gene driven by the prohormone convertase 2 (PC2) gene promoter. Numerous PC2 promoter-driven fluorescent (Kaede-positive) non-neural cells were distributed in the blood sinus located at the anterior end of the pharynx, suggesting the acquisition of a peptidergic circulatory system in Ciona. Kaede-positive ciliated columnar cells, rounded cells, and tall ciliated cells were observed in the alimentary organs, including the endostyle, pharynx, esophagus, stomach, and intestine, suggesting that digestive functions are regulated by multiple peptidergic systems. In the heart, Kaede-positive neurons were located in the ring-shaped plexus at both ends of the myocardium. Nerve fiber-like tracts ran along the raphe and appeared to be connected with the plexuses. Such unique structures suggest a role for the peptidergic system in cardiac function. Collectively, the present anatomic analysis revealed the major framework of the ventral peptidergic system of adult Ciona, which could facilitate investigations of peptidergic regulation of the pharynx, endostyle, alimentary tissues, and heart.

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

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

In ascidian tunicates, the metamorphic transition from larva to adult is accompanied by dynamic changes in the body plan. For instance, the central nervous system (CNS) is subjected to extensive rearrangement because its regulating larval organs are lost and new adult organs are created. To understand how the adult CNS is reconstructed, we traced the fate of larval CNS cells during ascidian metamorphosis by using transgenic animals and imaging technologies with photoconvertible fluorescent proteins. Here we show that most parts of the ascidian larval CNS, except for the tail nerve cord, are maintained during metamorphosis and recruited to form the adult CNS. We also show that most of the larval neurons disappear and only a subset of cholinergic motor neurons and glutamatergic neurons are retained. Finally, we demonstrate that ependymal cells of the larval CNS contribute to the construction of the adult CNS and that some differentiate into neurons in the adult CNS. An unexpected role of ependymal cells highlighted by this study is that they serve as neural stem-like cells to reconstruct the adult nervous network during chordate metamorphosis. Consequently, the plasticity of non-neuronal ependymal cells and neuronal cells in chordates should be re-examined by future studies.