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Biofouling constitutes one of the main obstacles in the sector of shellfish farming. Under this perspective, it is of highly importance to critically combine the updated information regarding the invasive potential of ascidian species, together with the factors contributing toward these events. The biological features of each species in relation to the gathering of the main non-indigenous species in the Mediterranean basin represent the first step toward mitigation of negative effects of the phenomenon. Further, there are limited studies investigating the physiological changes of bivalves caused by biofouling while leading to an increase in stress biomarkers. In the present review, the major ascidian species negatively affecting bivalve culture in the Mediterranean Sea are presented, alongside monitoring of ascidians from four Greek mussel farming locations as typical mussel culture cases. Among the main ascidian species, Styela plicata, Clavelina oblonga, Ciona robusta, Aplidium sp., Didemnum sp., Botryllus schlosseri, and Didemnum drachi are included, with the last three being the most harmful for this aquaculture sector. Based on the existing literature and research conducted so far, future research directions are proposed, in an effort to effectively control or efficiently manage ascidian biofouling organisms. Overall, perspectives toward the way we manage the biofouling phenomenon, such as the use of ascidian’s by-products in feedstuffs, chemical and pharmaceutical industry, or their incorporation in bivalve co-culture and integrated multi-trophic aquaculture systems represent promising alternative approaches.

Lumen formation, as a key process of biological tube construction, is essential in various physiological processes such as nutrient and waste transporting, gas exchanging, and structural supporting. However, the mechanisms underlying tubular lumen development are still not fully understood. In the present study, we identified a matrix metalloproteinase, Nas15, which is enriched in the apical domain of the Ciona embryonic notochord. The expression level of the Nas15 gene significantly increased during notochord lumen formation and expansion. Nas15 loss-of-function resulted in abnormal notochord lumen expansion in Ciona embryos. Besides, yeast two-hybrid screening and CO-IP results indicated a Phosphatase 2 Catalytic Subunit Alpha (PPP2CA) physically interacted with Nas15. PPP2CA also involved in notochord lumen formation via localizing Nas15. Furthermore, we investigated the distribution of laminin in Nas15 disrupted embryos. In conclusion, our results revealed a mechanisms of how notochord cells regulating lumen expansion via metalloproteinase-mediated ECM localization. This findings provide insight into the mechanisms of tubular organ lumen formation and serve as a reference for research on human abnormal lumenogenesis diseases. [ABSTRACT FROM AUTHOR]

The Motor Ganglion (MG) is a small collection of neurons that control the swimming movements of the tunicate tadpole larva. Situated at the base of the tail, molecular and functional comparisons suggest that may be a homolog of the spinal cord and/or hindbrain ("rhombospinal" region) of vertebrates. Here we review the most current knowledge of the development, connectivity, functions, and unique identities of the neurons that comprise the MG, drawn mostly from studies in Ciona spp. The simple cell lineages, minimal cellular composition, and comprehensively mapped "connectome" of the Ciona MG all make this an excellent model for studying the development and physiology of motor control in aquatic larvae. Research Highlights: We review the latest research on the development, circuitry, and function of the Motor Ganglion of tunicate larvae, focused primarily on the most well‐studied species in the Ciona genus. [ABSTRACT FROM AUTHOR]

The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Thalidomide has a dark history as a teratogen, but in recent years, its derivates have been shown to function as potent chemotherapeutic agents. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here, we employ Ciona, a simple invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Mrf and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation. [ABSTRACT FROM AUTHOR]

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.

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.

Cranial neurogenic placodes have been considered vertebrate innovations. However, anterior neural plate border (ANB) cells of ascidian embryos share many properties with vertebrate neurogenic placodes; therefore, it is now believed that the last common ancestor of vertebrates and ascidians had embryonic structures similar to neurogenic placodes of vertebrate embryos. Because BMP signaling is important for specifying the placode region in vertebrate embryos, we examined whether BMP signaling is also involved in gene expression in the ANB region of ascidian embryos. Our data indicated that Admp, a divergent BMP family member, is mainly responsible for BMP signaling in the ANB region, and that two BMP-antagonists, Noggin and Chordin, restrict the domain, in which BMP signaling is activated, to the ANB region, and prevent it from expanding to the neural plate. BMP signaling is required for expression of Foxg and Six1/2 at the late gastrula stage, and also for expression of Zf220, which encodes a zinc finger transcription factor in late neurula embryos. Because Zf220 negatively regulates Foxg, when we downregulated Zf220 by inhibiting BMP signaling, Foxg was upregulated, resulting in one large palp instead of three palps (adhesive organs derived from ANB cells). Functions of BMP signaling in specification of the ANB region give further support to the hypothesis that ascidian ANB cells share an evolutionary origin with vertebrate cranial placodes. [ABSTRACT FROM AUTHOR]

The vertebrate intestinal system consists of separate segments that remarkably differ in morphology and function. However, the origin of intestinal segmentation remains unclear. In this study, we investigated the segmentation of the intestine in a tunicate ascidian species, Ciona savignyi, by performing RNA sequencing. The gene expression profiles showed that the whole intestine was separated into three segments. Digestion, ion transport and signal transduction, and immune-related pathway genes were enriched in the proximal, middle, and distal parts of the intestine, respectively, implying that digestion, absorption, and immune function appear to be regional specializations in the ascidian intestine. We further performed a multi−species comparison analysis and found that the Ciona intestine showed a similar gene expression pattern to vertebrates, indicating tunicates and vertebrates might share the conserved intestinal functions. Intriguingly, vertebrate pancreatic homologous genes were expressed in the digestive segment of the Ciona intestine, suggesting that the proximal intestine might play the part of pancreatic functions in C. savignyi. Our results demonstrate that the tunicate intestine can be functionally separated into three distinct segments, which are comparable to the corresponding regions of the vertebrate intestinal system, offering insights into the functional evolution of the digestive system in chordates. [ABSTRACT FROM AUTHOR]

Studying the generation of biomechanical force and how this force drives cell and tissue morphogenesis is challenging for understanding the mechanical mechanisms underlying embryogenesis. Actomyosin has been demonstrated to be the main source of intracellular force generation that drives membrane and cell contractility, thus playing a vital role in multi-organ formation in ascidian Ciona embryogenesis. However, manipulation of actomyosin at the subcellular level is impossible in Ciona because of the lack of technical tools and approaches. In this study, we designed and developed a myosin light chain phosphatase fused with a light-oxygen-voltage flavoprotein from Botrytis cinerea (MLCP-BcLOV4) as an optogenetics tool to control actomyosin contractility activity in the Ciona larva epidermis. We first validated the light-dependent membrane localization and regulatory efficiency on mechanical forces of the MLCP-BcLOV4 system as well as the optimum light intensity that activated the system in HeLa cells. Then, we applied the optimized MLCP-BcLOV4 system in Ciona larval epidermal cells to realize the regulation of membrane elongation at the subcellular level. Moreover, we successfully applied this system on the process of apical contraction during atrial siphon invagination in Ciona larvae. Our results showed that the activity of phosphorylated myosin on the apical surface of atrial siphon primordium cells was suppressed and apical contractility was disrupted, resulting in the failure of the invagination process. Thus, we established an effective technique and system that provide a powerful approach in the study of the biomechanical mechanisms driving morphogenesis in marine organisms. [ABSTRACT FROM AUTHOR]

Constitutive expression of the chemokine Mcp1 in mouse cardiomyocytes creates a model of inflammatory cardiomyopathy, with death from heart failure at age 7-8 months. A critical pathogenic role has previously been proposed for induced oxidative stress, involving NADPH oxidase activation. To test this idea, we exposed the mice to elevated oxygen levels. Against expectation, this prevented, rather than accelerated, the ultrastructural and functional signs of heart failure. This result suggests that the immune signaling initiated by Mcp1 leads instead to the inhibition of cellular oxygen usage, for which mitochondrial respiration is an obvious target. To address this hypothesis, we combined the Mcp1 model with xenotopic expression of the alternative oxidase (AOX), which provides a sink for electrons blocked from passage to oxygen via respiratory complexes III and IV. Ubiquitous AOX expression provided only a minor delay to cardiac functional deterioration and did not prevent the induction of markers of cardiac and metabolic remodeling considered a hallmark of the model. Moreover, cardiomyocyte-specific AOX expression resulted in exacerbation of Mcp1-induced heart failure, and failed to rescue a second cardiomyopathy model directly involving loss of cIV. Our findings imply that mitochondrial involvement in the pathology of inflammatory cardiomyopathy is multifaceted and complex.

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programmes. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. Notochord and muscle cells stop dividing after eight or nine rounds of cell division depending on their lineages. In the present study, we showed that a Cdk inhibitor, Cdkn1.b, is responsible for stopping cell cycle progression in these lineages. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells continue to divide even after hatching, and Myc is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by transcription factors that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm is under control of transcription factors that specify the developmental fate of mesenchyme and endoderm. Thus, cell fate specification and cell cycle control are linked by these transcription factors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Abstract Background Inductive signaling interactions between different cell types are a major mechanism for the further diversification of embryonic cell fates. Most blastomeres in the model chordate Ciona robusta become restricted to a single predominant fate between the 64-cell and mid-gastrula stages. The deeply stereotyped and well-characterized Ciona embryonic cell lineages allow the transcriptomic analysis of newly established cell types very early in their divergence from sibling cell states without the pseudotime inference needed in the analysis of less synchronized cell populations. This is the first ascidian study to use droplet scRNAseq with large numbers of analyzed cells as early as the 64-cell stage when major lineages such as primary notochord first become fate restricted. Results and conclusions We identify 59 distinct cell states, including new subregions of the b-line neural lineage and the early induction of the tail tip epidermis. We find that 34 of these cell states are directly or indirectly dependent on MAPK-mediated signaling critical to early Ciona patterning. Most of the MAPK-dependent bifurcations are canalized with the signal-induced cell fate lost upon MAPK inhibition, but the posterior endoderm is unique in being transformed into a novel state expressing some but not all markers of both endoderm and muscle. Divergent gene expression between newly bifurcated sibling cell types is dominated by upregulation in the induced cell type. The Ets family transcription factor Elk1/3/4 is uniquely upregulated in nearly all the putatively direct inductions. Elk1/3/4 upregulation together with Ets transcription factor binding site enrichment analysis enables inferences about which bifurcations are directly versus indirectly controlled by MAPK signaling. We examine notochord induction in detail and find that the transition between a Zic/Ets-mediated regulatory state and a Brachyury/FoxA-mediated regulatory state is unexpectedly late. This supports a “broad-hourglass” model of cell fate specification in which many early tissue-specific genes are induced in parallel to key tissue-specific transcriptional regulators via the same set of transcriptional inputs.

Abstract Background Left-right asymmetries are a common feature of metazoans and can be found in a number of organs including the nervous system. These asymmetries are particularly pronounced in the simple central nervous system (CNS) of the swimming tadpole larva of the tunicate Ciona, which displays a chordate ground plan. While common pathway elements for specifying the left/right axis are found among chordates, particularly a requirement for Nodal signaling, Ciona differs temporally from its vertebrate cousins by specifying its axis at the neurula stage, rather than at gastrula. Additionally, Ciona and other ascidians require an intact chorionic membrane for proper left-right specification. Whether such differences underlie distinct specification mechanisms between tunicates and vertebrates will require broad understanding of their influence on CNS formation. Here, we explore the consequences of disrupting left-right axis specification on Ciona larval CNS cellular anatomy, gene expression, synaptic connectivity, and behavior. Results We show that left-right asymmetry disruptions caused by removal of the chorion (dechorionation) are highly variable and present throughout the Ciona larval nervous system. While previous studies have documented disruptions to the conspicuously asymmetric sensory systems in the anterior brain vesicle, we document asymmetries in seemingly symmetric structures such as the posterior brain vesicle and motor ganglion. Moreover, defects caused by dechorionation include misplaced or absent neuron classes, loss of asymmetric gene expression, aberrant synaptic projections, and abnormal behaviors. In the motor ganglion, a brain structure that has been equated with the vertebrate hindbrain, we find that despite the apparent left-right symmetric distribution of interneurons and motor neurons, AMPA receptors are expressed exclusively on the left side, which equates with asymmetric swimming behaviors. We also find that within a population of dechorionated larvae, there is a small percentage with apparently normal left-right specification and approximately equal population with inverted (mirror-image) asymmetry. We present a method based on a behavioral assay for isolating these larvae. When these two classes of larvae (normal and inverted) are assessed in a light dimming assay, they display mirror-image behaviors, with normal larvae responding with counterclockwise swims, while inverted larvae respond with clockwise swims. Conclusions Our findings highlight the importance of left-right specification pathways not only for proper CNS anatomy, but also for correct synaptic connectivity and behavior.

Pax3/7 factors play numerous roles in the development of the dorsal nervous system of vertebrates. From specifying neural crest at the neural plate borders, to regulating neural tube closure and patterning of the resulting neural tube. However, it is unclear which of these roles are conserved in non-vertebrate chordates. Here we investigate the expression and function of Pax3/7 in the model tunicate Ciona. Pax3/7 is expressed in neural plate border cells during neurulation, and in central nervous system progenitors shortly after neural tube closure. We find that separate cis-regulatory elements control the expression in these two distinct lineages. Using CRISPR/Cas9-mediated mutagenesis, we knocked out Pax3/7 in F0 embryos specifically in these two separate territories. Pax3/7 knockout in the neural plate borders resulted in neural tube closure defects, suggesting an ancient role for Pax3/7 in this chordate-specific process. Furthermore, knocking out Pax3/7 in the neural impaired Motor Ganglion neuron specification, confirming a conserved role for this gene in patterning the neural tube as well. Taken together, these results suggests that key functions of Pax3/7 in neural tube development are evolutionarily ancient, dating back at least to the last common ancestor of vertebrates and tunicates.

Wnt signaling is essential during animal development and regeneration, but also plays an important role in diseases such as cancer and diabetes. The canonical Wnt signaling pathway is one of the most conserved signaling cascades in the animal kingdom, with the T‐cell factor/lymphoid enhancer factor (TCF/LEF) proteins being the major mediators of Wnt/β‐catenin‐regulated gene expression. In comparison with invertebrates, vertebrates possess a high diversity of TCF/LEF family genes, implicating this as a possible key change to Wnt signaling at the evolutionary origin of vertebrates. However, the precise nature of this diversification is only poorly understood. The aim of this study is to clarify orthology, paralogy, and isoform relationships within the TCF/LEF gene family within chordates via in silico comparative study of TCF/LEF gene structure, molecular phylogeny, and gene synteny. Our results support the notion that the four TCF/LEF paralog subfamilies in jawed vertebrates (gnathostomes) evolved via the two rounds of whole‐genome duplications that occurred during early vertebrate evolution. Importantly, gene structure comparisons and synteny analysis of jawless vertebrate (cyclostome) TCFs suggest that a TCF7L2‐like form of gene structure is a close proxy for the ancestral vertebrate structure. In conclusion, we propose a detailed evolutionary path based on a new pre‐whole‐genome duplication vertebrate TCF gene model. This ancestor gene model highlights the chordate and vertebrate innovations of TCF/LEF gene structure, providing the foundation for understanding the role of Wnt/β‐catenin signaling in vertebrate evolution. [ABSTRACT FROM AUTHOR]

Here we present a parallel study of mRNA and microRNA expression during oral siphon (OS) regeneration in Ciona robusta, and the derived network of their interactions. In the process of identifying 248 mRNAs and 15 microRNAs as differentially expressed, we also identified 57 novel microRNAs, several of which are among the most highly differentially expressed. Analysis of functional categories identified enriched transcripts related to stress responses and apoptosis at the wound healing stage, signaling pathways including Wnt and TGFβ during early regrowth, and negative regulation of extracellular proteases in late stage regeneration. Consistent with the expression results, we found that inhibition of TGFβ signaling blocked OS regeneration. A correlation network was subsequently inferred for all predicted microRNA-mRNA target pairs expressed during regeneration. Network-based clustering associated transcripts into 22 non-overlapping groups, the functional analysis of which showed enrichment of stress response, signaling pathway and extracellular protease categories that could be related to specific microRNAs. Predicted targets of the miR-9 cluster suggest a role in regulating differentiation and the proliferative state of neural progenitors through regulation of the cytoskeleton and cell cycle.

The vertebrate intestinal system consists of separate segments that remarkably differ in morphology and function. However, the origin of intestinal segmentation remains unclear. In this study, we investigated the segmentation of the intestine in a tunicate ascidian species, Ciona savignyi, by performing RNA sequencing. The gene expression profiles showed that the whole intestine was separated into three segments. Digestion, ion transport and signal transduction, and immune-related pathway genes were enriched in the proximal, middle, and distal parts of the intestine, respectively, implying that digestion, absorption, and immune function appear to be regional specializations in the ascidian intestine. We further performed a multi−species comparison analysis and found that the Ciona intestine showed a similar gene expression pattern to vertebrates, indicating tunicates and vertebrates might share the conserved intestinal functions. Intriguingly, vertebrate pancreatic homologous genes were expressed in the digestive segment of the Ciona intestine, suggesting that the proximal intestine might play the part of pancreatic functions in C. savignyi. Our results demonstrate that the tunicate intestine can be functionally separated into three distinct segments, which are comparable to the corresponding regions of the vertebrate intestinal system, offering insights into the functional evolution of the digestive system in chordates.

Studying the generation of biomechanical force and how this force drives cell and tissue morphogenesis is challenging for understanding the mechanical mechanisms underlying embryogenesis. Actomyosin has been demonstrated to be the main source of intracellular force generation that drives membrane and cell contractility, thus playing a vital role in multi-organ formation in ascidian Ciona embryogenesis. However, manipulation of actomyosin at the subcellular level is impossible in Ciona because of the lack of technical tools and approaches. In this study, we designed and developed a myosin light chain phosphatase fused with a light-oxygen-voltage flavoprotein from Botrytis cinerea (MLCP-BcLOV4) as an optogenetics tool to control actomyosin contractility activity in the Ciona larva epidermis. We first validated the light-dependent membrane localization and regulatory efficiency on mechanical forces of the MLCP-BcLOV4 system as well as the optimum light intensity that activated the system in HeLa cells. Then, we applied the optimized MLCP-BcLOV4 system in Ciona larval epidermal cells to realize the regulation of membrane elongation at the subcellular level. Moreover, we successfully applied this system on the process of apical contraction during atrial siphon invagination in Ciona larvae. Our results showed that the activity of phosphorylated myosin on the apical surface of atrial siphon primordium cells was suppressed and apical contractility was disrupted, resulting in the failure of the invagination process. Thus, we established an effective technique and system that provide a powerful approach in the study of the biomechanical mechanisms driving morphogenesis in marine organisms.

This paper presents a deep-learning-based workflow to detect synapses and predict their neurotransmitter type in the primitive chordate Ciona intestinalis (Ciona) electronmicroscopic (EM) images. Identifying synapses fromEMimages to build a fullmap of connections between neurons is a labor-intensive process and requires significant domain expertise. Automation of synapse classificationwould hasten the generation and analysis of connectomes. Furthermore, inferences concerning neuron type and function fromsynapse features are inmany cases difficult tomake. Finding the connection between synapse structure and function is an important step in fully understanding a connectome. Class ActivationMaps derived fromthe convolutional neural network provide insights on important features of synapses based on cell type and function. The main contribution of this work is in the differentiation of synapses by neurotransmitter type through the structural information in their EM images. This enables the prediction of neurotransmitter types for neurons in Ciona, which were previously unknown. The prediction model with code is available on GitHub. [ABSTRACT FROM AUTHOR]

Synthesis of cellulose and formation of tunic structure are unique traits in the tunicate animal group. However, the regulatory mechanism of tunic formation remains obscure. Here, we identified a novel microRNA cluster of three microRNAs, including miR4018a, miR4000f, and miR4018b in Ciona savignyi. In situ hybridization and promoter assays showed that miR4018a/4000f/4018b cluster was expressed in the mesenchymal cells in the larval trunk, and the expression levels were downregulated during the later tailbud stage and larval metamorphosis. Importantly, overexpression of miR4018a/4000f/4018b cluster in mesenchymal cells abolished the cellulose synthesis in Ciona larvae and caused the loss of tunic cells in metamorphic larvae, indicating the regulatory roles of miR4018a/4000f/4018b cluster in cellulose synthesis and mesenchymal cell differentiation into tunic cells. To elucidate the molecular mechanism, we further identified the target genes of miR4018a/4000f/4018b cluster using the combination approaches of TargetScan prediction and RNA-seq data. Left–right determination factor (Lefty) was confirmed as one of the target genes after narrow-down screening and an experimental luciferase assay. Furthermore, we showed that Lefty was expressed in the mesenchymal and tunic cells, indicating its potentially regulatory roles in mesenchymal cell differentiation and tunic formation. Notably, the defects in tunic formation and loss of tunic cells caused by overexpression of miR4018a/4000f/4018b cluster could be restored when Lefty was overexpressed in Ciona larvae, suggesting that miR4018a/4000f/4018b regulated the differentiation of mesenchymal cells into tunic cells through the Lefty signaling pathway during ascidian metamorphosis. Our findings, thus, reveal a novel microRNA-Lefty molecular pathway that regulates mesenchymal cells differentiating into tunic cells required for the tunic formation in tunicate species.

A major goal in biology is to understand the rules by which cis-regulatory sequences control spatially and temporally precise expression patterns. Here we present a systematic dissection of the proximal enhancer for the notochord-specific transcription factor brachyury in the ascidian chordate Ciona. The study uses a quantitative image-based reporter assay that incorporates a dual-reporter strategy to control for variable electroporation efficiency. We identified and mutated multiple predicted transcription factor binding sites of interest based on statistical matches to the JASPAR binding motif database. Most sites (Zic, Ets, FoxA, RBPJ) were selected based on prior knowledge of cell fate specification in both the primary and secondary notochord. We also mutated predicted Brachyury sites to investigate potential autoregulation as well as Fos/Jun (AP1) sites that had very strong matches to JASPAR. Our goal was to quantitatively define the relative importance of these different sites, to explore the importance of predicted high-affinity versus low-affinity motifs, and to attempt to design mutant enhancers that were specifically expressed in only the primary or secondary notochord lineages. We found that the mutation of all predicted high-affinity sites for Zic, FoxA or Ets led to quantifiably distinct effects. The FoxA construct caused a severe loss of reporter expression whereas the Ets construct had little effect. A strong Ets phenotype was only seen when much lower-scoring binding sites were also mutated. This supports the enhancer suboptimization hypothesis proposed by Farley and Levine but suggests that it may only apply to some but not all transcription factor families. We quantified reporter expression separately in the two notochord lineages with the expectation that Ets mutations and RBPJ mutations would have distinct effects given that primary notochord is induced by Ets-mediated FGF signaling whereas secondary notochord is induced by RBPJ/Su(H)-mediated Notch/Delta signaling. We found, however, that ETS mutations affected primary and secondary notochord expression relatively equally and that RBPJ mutations were only moderately more severe in their effect on secondary versus primary notochord. Our results point to the promise of quantitative reporter assays for understanding cis-regulatory logic but also highlight the challenge of arbitrary statistical thresholds for predicting potentially important sites.

Conserved transcription factors termed "terminal selectors" regulate neuronal sub-type specification and differentiation through combinatorial transcriptional regulation of terminal differentiation genes. The unique combinations of terminal differentiation gene products in turn contribute to the functional identities of each neuron. One well-characterized terminal selector is COE (Collier/Olf/Ebf), which has been shown to activate cholinergic gene batteries in C. elegans motor neurons. However, its functions in other metazoans, particularly chordates, is less clear. Here we show that the sole COE ortholog in the non-vertebrate chordate Ciona robusta , Ebf, controls the expression of the cholinergic locus VAChT/ChAT in a single dorsal interneuron of the larval Motor Ganglion, which is presumed to be homologous to the vertebrate spinal cord. We propose that, while the function of Ebf as a regulator of cholinergic neuron identity conserved across bilaterians, its exact role may have diverged in different cholinergic neuron subtypes (e.g., interneurons vs. motor neurons) in chordate-specific motor circuits. [ABSTRACT FROM AUTHOR]

Neural tube closure (NTC) is a complex multi-step morphogenetic process that transforms the flat neural plate found on the surface of the post-gastrulation embryo into the hollow and subsurface central nervous system (CNS). Errors in this process underlie some of the most prevalent human birth defects, and occur in about 1 out of every 1000 births. Previously, we discovered a mutant in the basal chordate Ciona savignyi (named bugeye) that revealed a novel role for a T-Type Calcium Channel (Cav3) in this process. Moreover, the requirement for CAV3s in Xenopus NTC suggests a conserved function among the chordates. Loss of CAV3 leads to defects restricted to anterior NTC, with the brain apparently fully developed, but protruding from the head. Here we report first on a new Cav3 mutant in the related species C. robusta. RNAseq analysis of both C. robusta and C. savignyi bugeye mutants reveals misregulation of a number of transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane, which are aberrantly upregulated in the mutant, are expressed in the closing neural tube, and when disrupted by CRISPR gene editing lead to the open brain phenotype displayed in bugeye mutants. We speculate that these molecules play a transient role in tissue separation and adhesion during NTC and failure to downregulate them leads to an open neural tube. [Display omitted] • In the basal chordate Ciona, loss of Cav3 results in an exencephaly-like phenotype. • Loss of Cav3 results in misregulation of many transcripts including two cell adhesion molecules, Selectin and Flrt. • Components of neurite outgrowth machinery are essential for NTC in Ciona. [ABSTRACT FROM AUTHOR]

Conserved transcription factors termed “terminal selectors” regulate neuronal sub-type specification and differentiation through combinatorial transcriptional regulation of terminal differentiation genes. The unique combinations of terminal differentiation gene products in turn contribute to the functional identities of each neuron. One well-characterized terminal selector is COE (Collier/Olf/Ebf), which has been shown to activate cholinergic gene batteries in C. elegans motor neurons. However, its functions in other metazoans, particularly chordates, is less clear. Here we show that the sole COE ortholog in the non-vertebrate chordate Ciona robusta, Ebf, controls the expression of the cholinergic locus VAChT/ChAT in a single dorsal interneuron of the larval Motor Ganglion, which is presumed to be homologous to the vertebrate spinal cord. We propose that, while the function of Ebf as a regulator of cholinergic neuron identity conserved across bilaterians, its exact role may have diverged in different cholinergic neuron subtypes (e.g., interneurons vs. motor neurons) in chordate-specific motor circuits.

Sea squirts (Tunicata) are chordates and develop a swimming larva with a small and defined number of individually identifiable cells. This offers the prospect of connecting specific stimuli to behavioral output and characterizing the neural activity that links these together. Here, we describe the development of a microfluidic chip that allows live larvae of the sea squirt Ciona intestinalis to be immobilized and recorded. By generating transgenic larvae expressing GCaAMP6m in defined cells, we show that calcium ion levels can be recorded from immobilized larvae, while microfluidic control allows larvae to be exposed to specific waterborne stimuli. We trial this on sea water carrying increased levels of carbon dioxide, providing evidence that larvae can sense this gas., (© 2024 The Author(s). Journal of Experimental Zoology Part B: Molecular and Developmental Evolution published by Wiley Periodicals LLC.)

Invertebrate chordates, such as the tunicate Ciona , can offer insight into the evolution of the chordate phylum. Anatomical features that are shared between invertebrate chordates and vertebrates may be taken as evidence of their presence in a common chordate ancestor. The central nervous systems of Ciona larvae and vertebrates share a similar anatomy despite the Ciona CNS having ~180 neurons. However, the depth of conservation between the Ciona CNS and those in vertebrates is not resolved. The Ciona caudal CNS, while appearing spinal cord-like, has hitherto been thought to lack motor neurons, bringing into question its homology with the vertebrate spinal cord. We show here that the Ciona larval caudal CNS does, in fact, have functional motor neurons along its length, pointing to the presence of a spinal cord-like structure at the base of the chordates. We extend our analysis of shared CNS anatomy further to explore the Ciona "motor ganglion", which has been proposed to be a homolog of the vertebrate hindbrain, spinal cord, or both. We find that a cluster of neurons in the dorsal motor ganglion shares anatomical location, developmental pathway, neural circuit architecture, and gene expression with the vertebrate cerebellum. However, functionally, the Ciona cluster appears to have more in common with vertebrate cerebellum-like structures, insofar as it receives and processes direct sensory input. These findings are consistent with earlier speculation that the cerebellum evolved from a cerebellum-like structure, and suggest that the latter structure was present in the dorsal hindbrain of a common chordate ancestor.

Background: Inductive signaling interactions between different cell types are a major mechanism for the further diversification of embryonic cell fates. Most blastomeres in the model chordate Ciona robusta become restricted to a single predominant fate between the 64-cell and mid-gastrula stages. The deeply stereotyped and well-characterized Ciona embryonic cell lineages allow the transcriptomic analysis of newly established cell types very early in their divergence from sibling cell states without the pseudotime inference needed in the analysis of less synchronized cell populations. This is the first ascidian study to use droplet scRNAseq with large numbers of analyzed cells as early as the 64-cell stage when major lineages such as primary notochord first become fate restricted. Results and conclusions: We identify 59 distinct cell states, including new subregions of the b-line neural lineage and the early induction of the tail tip epidermis. We find that 34 of these cell states are directly or indirectly dependent on MAPK-mediated signaling critical to early Ciona patterning. Most of the MAPK-dependent bifurcations are canalized with the signal-induced cell fate lost upon MAPK inhibition, but the posterior endoderm is unique in being transformed into a novel state expressing some but not all markers of both endoderm and muscle. Divergent gene expression between newly bifurcated sibling cell types is dominated by upregulation in the induced cell type. The Ets family transcription factor Elk1/3/4 is uniquely upregulated in nearly all the putatively direct inductions. Elk1/3/4 upregulation together with Ets transcription factor binding site enrichment analysis enables inferences about which bifurcations are directly versus indirectly controlled by MAPK signaling. We examine notochord induction in detail and find that the transition between a Zic/Ets-mediated regulatory state and a Brachyury/FoxA-mediated regulatory state is unexpectedly late. This supports a "broad-hourglass" model of cell fate specification in which many early tissue-specific genes are induced in parallel to key tissue-specific transcriptional regulators via the same set of transcriptional inputs. [ABSTRACT FROM AUTHOR]

Visual processing transforms the complexities of the visual world into useful information. Ciona , an invertebrate chordate and close relative of the vertebrates, has one of the simplest nervous systems known, yet has a range of visuomotor behaviors. This simplicity has facilitated studies linking behavior and neural circuitry. Ciona larvae have two distinct visuomotor behaviors – a looming shadow response and negative phototaxis. These are mediated by separate neural circuits that initiate from different clusters of photoreceptors, with both projecting to a CNS structure called the posterior brain vesicle (pBV). We report here that inputs from both circuits are processed to generate fold change detection (FCD) outputs. In FCD, the behavioral response scales with the relative fold change in input, but is invariant to the overall magnitude of the stimulus. Moreover, the two visuomotor behaviors have fundamentally different stimulus/response relationships – indicative of differing circuit strategies, with the looming shadow response showing a power relationship to fold change, while the navigation behavior responds linearly. Pharmacological modulation of the FCD response points to the FCD circuits lying outside of the visual organ (the ocellus), with the pBV being the most likely location. Consistent with these observations, the connectivity and properties of pBV interneurons conform to known FCD circuit motifs, but with different circuit architectures for the two circuits. The negative phototaxis circuit forms a putative incoherent feedforward loop that involves interconnecting cholinergic and GABAergic interneurons. The looming shadow circuit uses the same cholinergic and GABAergic interneurons, but with different synaptic inputs to create a putative non-linear integral feedback loop. These differing circuit architectures are consistent with the behavioral outputs of the two circuits. Finally, while some reports have highlighted parallels between the pBV and the vertebrate midbrain, suggesting a common origin for the two, others reports have disputed this, suggesting that invertebrate chordates lack a midbrain homolog. The convergence of visual inputs at the pBV, and its putative role in visual processing reported here and in previous publications, lends further support to the proposed common origin of the pBV and the vertebrate midbrain. [ABSTRACT FROM AUTHOR]

In early embryos of Ciona , an invertebrate chordate, the animal-vegetal axis is established by the combinatorial actions of maternal factors. One target of these maternal factors, Foxd , is specifically expressed in the vegetal hemisphere and stabilizes the animal-vegetal axis by activating vegetal hemisphere-specific genes and repressing animal hemisphere-specific genes. This dual functionality is essential for the embryogenesis of early ascidian embryos; however, the mechanism by which Foxd can act as both a repressor and an activator is unknown. Here, we identify a Foxd binding site upstream of Lhx3/4 , which is activated by Foxd, and compare it with a repressive Foxd binding site upstream of Dmrt.a. We found that activating sites bind Foxd with low affinity while repressive sites bind Foxd with high affinity. Reporter assays confirm that this qualitative difference between activating and repressive Foxd binding sites is sufficient to change Foxd functionality. We therefore conclude that the outcome of Foxd transcriptional regulation is encoded in cis -regulatory elements. [Display omitted] • Foxd activates endomesodermal genes and represses ectodermal genes in Ciona embryos. • Activating and repressing sites bound Foxd with different affinity in vitro. • This qualitative difference was sufficient to change Foxd functionality in vivo. • The outcome of Foxd transcriptional regulation is encoded in cis -regulatory elements. [ABSTRACT FROM AUTHOR]

Background: Left-right asymmetries are a common feature of metazoans and can be found in a number of organs including the nervous system. These asymmetries are particularly pronounced in the simple central nervous system (CNS) of the swimming tadpole larva of the tunicate Ciona, which displays a chordate ground plan. While common pathway elements for specifying the left/right axis are found among chordates, particularly a requirement for Nodal signaling, Ciona differs temporally from its vertebrate cousins by specifying its axis at the neurula stage, rather than at gastrula. Additionally, Ciona and other ascidians require an intact chorionic membrane for proper left-right specification. Whether such differences underlie distinct specification mechanisms between tunicates and vertebrates will require broad understanding of their influence on CNS formation. Here, we explore the consequences of disrupting left-right axis specification on Ciona larval CNS cellular anatomy, gene expression, synaptic connectivity, and behavior. Results: We show that left-right asymmetry disruptions caused by removal of the chorion (dechorionation) are highly variable and present throughout the Ciona larval nervous system. While previous studies have documented disruptions to the conspicuously asymmetric sensory systems in the anterior brain vesicle, we document asymmetries in seemingly symmetric structures such as the posterior brain vesicle and motor ganglion. Moreover, defects caused by dechorionation include misplaced or absent neuron classes, loss of asymmetric gene expression, aberrant synaptic projections, and abnormal behaviors. In the motor ganglion, a brain structure that has been equated with the vertebrate hindbrain, we find that despite the apparent left-right symmetric distribution of interneurons and motor neurons, AMPA receptors are expressed exclusively on the left side, which equates with asymmetric swimming behaviors. We also find that within a population of dechorionated larvae, there is a small percentage with apparently normal left-right specification and approximately equal population with inverted (mirror-image) asymmetry. We present a method based on a behavioral assay for isolating these larvae. When these two classes of larvae (normal and inverted) are assessed in a light dimming assay, they display mirror-image behaviors, with normal larvae responding with counterclockwise swims, while inverted larvae respond with clockwise swims. Conclusions: Our findings highlight the importance of left-right specification pathways not only for proper CNS anatomy, but also for correct synaptic connectivity and behavior. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) regulate multicellular processes and diverse signaling pathways in organisms. The detection of the spatiotemporal expression of miRNA in vivo is crucial for uncovering the function of miRNA. However, most of the current detecting techniques cannot reflect the dynamics of miRNA sensitively in vivo. Here, we constructed a miRNA-induced CRISPR-Cas9 platform (MICR) used in marine chordate Ciona. The key component of MICR is a pre-single guide RNA (sgRNA) flanked by miRNA-binding sites that can be released by RNA-induced silencing complex (RISC) cleavage to form functional sgRNA in the presence of complementary miRNA. By using the miRNA-inducible CRISPR-on system (MICR-ON), we successfully detected the dynamic expression of a miRNA csa-miR-4018a during development of Ciona embryo. The detected patterns were validated to be consistent with the results by in situ hybridization. It is worth noting that the expression of csa-miR-4018a was examined by MICR-ON to be present in additional tissues, where no obvious signaling was detected by in situ hybridization, suggesting that the MICR-ON might be a more sensitive approach to detect miRNA signal in living animal. Thus, MICR-ON was demonstrated to be a sensitive and highly efficient approach for monitoring the dynamics of expression of miRNA in vivo and will facilitate the exploration of miRNA functions in biological systems. [ABSTRACT FROM AUTHOR]

Visual processing transforms the complexities of the visual world into useful information. Ciona, an invertebrate chordate and close relative of the vertebrates, has one of the simplest nervous systems known, yet has a range of visuomotor behaviors. This simplicity has facilitated studies linking behavior and neural circuitry. Ciona larvae have two distinct visuomotor behaviors – a looming shadow response and negative phototaxis. These are mediated by separate neural circuits that initiate from different clusters of photoreceptors, with both projecting to a CNS structure called the posterior brain vesicle (pBV). We report here that inputs from both circuits are processed to generate fold change detection (FCD) outputs. In FCD, the behavioral response scales with the relative fold change in input, but is invariant to the overall magnitude of the stimulus. Moreover, the two visuomotor behaviors have fundamentally different stimulus/response relationships – indicative of differing circuit strategies, with the looming shadow response showing a power relationship to fold change, while the navigation behavior responds linearly. Pharmacological modulation of the FCD response points to the FCD circuits lying outside of the visual organ (the ocellus), with the pBV being the most likely location. Consistent with these observations, the connectivity and properties of pBV interneurons conform to known FCD circuit motifs, but with different circuit architectures for the two circuits. The negative phototaxis circuit forms a putative incoherent feedforward loop that involves interconnecting cholinergic and GABAergic interneurons. The looming shadow circuit uses the same cholinergic and GABAergic interneurons, but with different synaptic inputs to create a putative non-linear integral feedback loop. These differing circuit architectures are consistent with the behavioral outputs of the two circuits. Finally, while some reports have highlighted parallels between the pBV and the vertebrate midbrain, suggesting a common origin for the two, others reports have disputed this, suggesting that invertebrate chordates lack a midbrain homolog. The convergence of visual inputs at the pBV, and its putative role in visual processing reported here and in previous publications, lends further support to the proposed common origin of the pBV and the vertebrate midbrain.

In the ascidian Ciona intestinalis, basal body parts regenerate distal structures but distal body parts do not replace basal structures. Regeneration involves the activity of adult stem cells in the branchial sac, which proliferate and produce migratory progenitor cells for tissue and organ replacement. Branchial sac-derived stem cells also replenish recycling cells lining the pharyngeal fissures during homeostatic growth. Apoptosis at injury sites occurs early during regeneration and continuously in the pharyngeal fissures during homeostatic growth. Caspase 1 inhibitor, caspase 3 inhibitor, or pan-caspase inhibitor Z-VAD-FMK treatment blocked apoptosis, prevented regeneration, and suppressed branchial sac growth and function. A pharmacological screen and siRNA-mediated gene knockdown indicated that regeneration requires canonical Wnt signaling. Wnt3a protein rescued both caspase-blocked regeneration and branchial sac growth. Inhibition of apoptosis did not affect branchial sac stem cell proliferation but prevented the survival of progenitor cells. After bisection across the mid-body, apoptosis occurred only in the regenerating basal fragments, although both fragments contained a part of the branchial sac, suggesting that apoptosis is unilateral at the wound site and the presence of branchial sac stem cells is insufficient for regeneration. The results suggest that apoptosis-dependent Wnt signaling mediates regeneration and homeostatic growth in Ciona.

The Ciona larva has served as a unique model for understanding the development of dopaminergic cells at single-cell resolution owing to the exceptionally small number of neurons in its brain and its fixed cell lineage during embryogenesis. A recent study suggested that the transcription factors Fer2 and Meis directly regulate the dopamine synthesis genes in Ciona, but the dopaminergic cell lineage and the gene regulatory networks that control the development of dopaminergic cells have not been fully elucidated. Here, we reveal that the dopaminergic cells in Ciona are derived from a bilateral pair of cells called a9.37 cells at the center of the neural plate. The a9.37 cells divide along the anterior-posterior axis, and all of the descendants of the posterior daughter cells differentiate into the dopaminergic cells. We show that the MAPK pathway and the transcription factor Otx are required for the expression of Fer2 in the dopaminergic cell lineage. Our findings establish the cellular and molecular framework for fully understanding the commitment to dopaminergic cells in the simple chordate brain. [ABSTRACT FROM AUTHOR]

Chromosomal rearrangements can reduce fitness of heterozygotes and can thereby prevent gene flow. Therefore, such rearrangements can play a role in local adaptation and speciation. In particular, inversions are considered to be a major potential cause for chromosomal speciation. There are two closely related, partially sympatric lineages of ascidians in the genus Ciona , which we call type-A and type-B animals in the present study. Although these invertebrate chordates are largely isolated reproductively, hybrids can be found in wild populations, suggesting incomplete prezygotic barriers. Although the genome of type-A animals has been decoded and widely used, the genome for type-B animals has not been decoded at the chromosomal level. In the present study, we sequenced the genomes of two type-B individuals from different sides of the English Channel (in the zone of sympatry with type-A individuals) and compared them at the chromosomal level with the type-A genome. Although the overall structures were well conserved between type A and type B, chromosomal alignments revealed many inversions differentiating these two types of Ciona ; it is probable that the frequent inversions have contributed to separation between these two lineages. In addition, comparisons of the genomes between the two type-B individuals revealed that type B had high rates of inversion polymorphisms and nucleotide polymorphisms, and thus type B might be in the process of differentiation into multiple new types or species. Our results suggest an important role of inversions in chromosomal speciation of these broadcasting spawners. [ABSTRACT FROM AUTHOR]

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. • Long- and short-range chemical cues influence larval attachment and metamorphosis • Ciona larvae use polymodal sensory cells to sense mechanical and chemical cues • Individual sensory neurons can encode stimulus-specific information • Brain dynamics act as a framework for stimulus-driven behavioral action choice Ciona is a marine chordate with a biphasic life cycle. Hoyer et al. identify a set of short- and long-range chemical cues that influence attachment and metamorphosis of Ciona larvae. These cues are detected by polymodal sensory cells, which encode the valence of the stimulus and drive brain-wide dynamics to elicit robust behavioral actions. [ABSTRACT FROM AUTHOR]

The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona. Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes is unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators, and find that combinatorial cocktails aremore effective at reprogramming other cell types than Bra alone.We reassess the network relationships between Bra, Foxa.a and other components of the notochord gene regulatory network, and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically defined master regulator. [ABSTRACT FROM AUTHOR]