Biology subjects

Sasakura, Y.

Publications and source records attributed to Sasakura, Y..

11 recordsLinked to original sources

bioRxiv2026

Antenna cells of ascidian larvae are gravity-sensing neurons

Ascidians are the closest living relatives of vertebrates, and they share a basic body plan including the dorsal central nervous system. The central nervous system of ascidian tadpole larvae controls their swimming behavior. In particular, for the first few hours after hatching, larvae swim upward. Gravity is sensed by an organ called the otolith, which resides in a brain vesicle. Although this sensory information is thought to be transmitted to two antenna cells abutting the otolith, direct evidence that the antenna cells function as gravity-sensory neurons has been lacking. In the present study, we first identified a regulatory element of the Vglut gene that drives expression in antenna cells and otolith-associated ciliated cells. Using this upstream regulatory element, we specifically overexpressed the apoptosis-inducing gene, Bax, in antenna cells and otolith-associated ciliated cells, and succeeded in their targeted ablation. These Bax-overexpressing larvae lost their preference for upward swimming. Because antenna cells have axons and otolith-associated ciliated cells lack neurites, our observation indicates that antenna cells are the sensory neurons for gravity. In addition, our experimental method for targeted disruption of cells using an enhancer element and Bax provides a valuable tool to examine neural circuit functions at cellular resolution.

zoology↗

bioRxiv2026

Possible function of Hox2 in atrial siphon fusion of the ascidian Ciona

The hallmark of sessile adult ascidians is a vase-like shape with a single oral and atrial siphon. Ciona, however, develops two atrial siphons after metamorphosis, which subsequently fuse into one. The mechanisms underlying this fusion are unknown. This study suggests that Hox2 controls this process. Hox2-knockout animals using Transcription-Activator-Like Effector Nuclease (TALEN) retain two atrial siphons throughout their lives. During normal fusion, epidermal cells between the siphons flatten along the anterior-posterior axis. This cellular flattening does not occur in Hox2-knockout animals, suggesting that the shape change in the epidermal cells produces tension, allowing the atrial siphon openings to converge at the midline for fusion. Hox2-knockout animals lack cupular organs, which are suspected hydrodynamic sensors in the internal epithelium of the fused atrial siphon and on the sperm duct. Among several knockout attempts, atrial siphon fusion was reproduced by only one TALEN pair, suggesting that this phenotype is driven by a mutation having a broader effect than those abolishing protein function. Many ascidians, unlike Ciona, develop a single atrial siphon shortly after metamorphosis. Our findings suggest that a phylogenetically conserved gene, Hox2, establishes this group-specific atrial siphon formation mechanism in Ciona.

developmental biology↗

bioRxiv2026

Establishing genetically controlled, closed 1 colonies of an ascidian

Recent technological advances have made many "non-model" organisms accessible for experimental studies. However, reference inbred strains were not necessarily available, especially in marine invertebrates, and genetic background of organisms used for experiments are often non-uniform. This situation potentially affects experimental reproducibility. Although ascidians, Ciona intestinalis (type A, or C. robusta), are a widely used marine animal for many areas of experimental biology including developmental studies, no reference strains have been obtained despite extensive efforts. As an alternative way to improve reproducibility, we have established and maintained ascidian colonies through intra-population breeding every year from 2016 to 2020, and monitored genomic variants of these colonies. This method does not reduce genetic variations but instead manages and monitors genetic variations in the colonies, providing an easy and cost-effective way of increasing experimental reproducibility. Furthermore, we recently upgraded these genetically isolated, closed colonies that were re-established every year, and have maintained them for more than three years only through intra-population breeding and occasional back-cross using cryopreserved sperm. Genetic variants that we revealed using 3.7 tera-bases of sequence data will help to design future experiments in this species. Our data also show that two wild-populations, which were used to establish the colonies, have maintained distinct genetic backgrounds, although their habitats are directly linked to the Pacific Ocean and only 170 km apart. More importantly, genetic information regarding these colonies will undoubtedly improve experimental reproducibility and traceability, and our method will provide a realistic solution for performing reproducible experiments using non-model organisms.

developmental biology↗

bioRxiv2025

Gamete release in Ciona robusta: roles of gonadotropin-releasing hormone and the photoreception system

Gamete release, an essential event for animals, is regulated by various factors including environmental stimuli, neural circuit, and endocrine molecules. In this study, we investigated the mechanisms regulating gamete release in the ascidian Ciona robusta, part of the sister group of vertebrates. Ciona is a hermaphrodite, releasing sperm first from orange-pigmented organ (OPO) at the end of the spermiduct, followed by eggs from the oviduct beneath the spermiduct knob-like structure. In this study, behavioral and morphological analyses revealed that this sequential release occurs because the knob-like structure compresses the oviduct opening at the onset of gamete release. Observations of transgenic Ciona expressing Kaede under the gonadotropin-releasing hormone 2 (Gnrh2) promoter showed that Gnrh2-expressing neurons and fibers accumulate in the epithelium around the spermiduct openings. In contrast, Gnrh1-expressing neurons are localized in the cerebral ganglion and project toward the ovary, suggesting distinct roles of GnRH1 and GnRH2 in reproductive regulation. RNA-seq and real-time PCR analyses revealed that Opsin2, Opsin3, beta-carotene-15,15-monooxygenase (Bco), and several ion channel genes are specifically expressed in the OPO, along with Gnrh2. In situ hybridization showed that these genes are localized in the innermost OPO layer, suggesting that the OPO functions as a photoreceptive organ. Although, Gnrh2 expression has been considered low in adult Ciona, our study revealed strong OPO-specific expression with photoreceptive genes. Collectively, these findings suggest that GnRH2 plays a central role in gamete release regulation, potentially in coordination with the photoreceptive system, while GnRH1 may regulate ovarian functions. These results indicate that C. robusta employs distinct GnRH systems in a tissue-specific manner to regulate reproduction.

evolutionary biology↗

bioRxiv2025

Neural signaling contributes to heart formation and growth in the invertebrate chordate, Ciona robusta.

Neurons contribute to the complex interplay of signals that mediate heart development and homeostasis. Although a limited set of studies suggest that neuronal peptides impact vertebrate heart growth, the specific contributions of these peptides to cardiomyocyte progenitor differentiation or proliferation have not been elucidated. Here we show that the neuropeptide tachykinin along with canonical Wnt signaling regulate cardiomyocyte progenitor proliferation in the chordate model Ciona robusta. In C. robusta, the heart continues to grow throughout adulthood and classic histological studies indicate that a line of undifferentiated cells may serve as a reserve progenitor lineage. We found that this line of cardiomyocyte progenitors consists of distinct distal and midline populations. Distal progenitors divide asymmetrically to produce distal and midline daughters. Midline progenitors divide asymmetrically to produce myocardial precursors. Through single cell RNA sequencing (scRNA-seq) of adult C. robusta hearts, we delineated the cardiomyocyte progenitor expression profile. Based on this data we investigated the role of Wnt signaling in cardiomyocyte progenitor proliferation and found that canonical Wnt signaling is required to suppress excessive progenitor proliferation. The scRNA-seq data also identified a number of presumptive cardiac neural-like cells. Strikingly, we found that a subset of these neuronal cells appears to innervate the distal cardiomyocyte progenitors. Based on the expression of the tachykinin receptor in these neuronal cells, we blocked tachykinin signaling using pharmacological inhibitors and found that this drove reduced proliferation in the distal progenitor pool. Through targeted CRISPR-Cas9 knockdown we then demonstrated that both extrinsic tachykinin and intrinsic, cardiac tachykinin receptors are required for formation of the myocardial heart tube. This work provides valuable insights into how organisms may deploy neural signals to regulate organ growth in response to environmental or homeostatic inputs.

developmental biology↗

bioRxiv2024

cryoMDM; Molecular simulation Driven structural Matching Approach to Estimate Variety of Atomic Three-dimensional Model Based on Noisy cryoEM Single Particle Images.

Understanding the structure of biomolecules is essential for comprehending life phenomena. In recent years, cryo-electron microscopy (cryoEM) single-particle structure analysis has made remarkable advancements. The single-particle images obtained from cryoEM measurements have a poor signal-to-noise ratio, and existing methods rely on multi-image analysis, which averages multiple measurement data to achieve high-resolution structures. However, this multi-image approach statistically processes several measurement images, causing the unique structural features of individual data to be lost. To overcome this inherent challenge, we developed a molecular simulation-driven structural matching method, cryoMDM, which identifies the plausible structure for cryoEM two-dimensional images from a vast number of three-dimensional (3D) structural model candidates generated through molecular simulations. The innovative aspect of this method is that it enables the estimation of 3D atomic models from a single-particle image, going beyond merely generating a 3D electron density map. By linking each cryoEM particle image to a 3D structure in the simulation space, we can directly connect cryoEM measurement data to continuous structural changes in biomolecules. Applying this method to the spike protein derived from SARS-CoV-2, we successfully captured various intermediate structures of the spike protein, revealing critical information about the early stages of viral entry.

biophysics↗

bioRxiv2024

Tunicate-specific protein Epi-1 is essential for conferring hydrophilicity to the larval tunic in the ascidian Ciona

Animals must avoid adhesion to objects in the environment to maintain their mobility and independence. The marine invertebrate chordate ascidians are characterized by an acellular matrix tunic enveloping their entire body for protection and swimming. The tunic of ascidian larvae consists of a surface cuticle layer and inner matrix layer. Hydrophilic substances coat the cuticle; this modification is thought to be for preventing adhesion. However, the molecule responsible for regulating this modification has not been clarified. We here found that the tunicate-specific protein Epi-1 is responsible for preventing adhesiveness of the tunic in the ascidian Ciona intestinalis Type A. Ciona mutants with homozygous knockouts of Epi-1 exhibited adhesion to plastic plates and to other individuals. The cuticle of the Epi-1 mutants was fragile, and it lost the glycosaminoglycans supplied by test cells, the accessory cells that normally attach to the tunic surface. Although it has an apparent signal peptide for membrane trafficking, we showed that the Epi-1 protein is localized to the cytosol of the epidermal cells. Our study demonstrated that the emergence of the tunicate-specific protein Epi-1 accelerated the immediate ancestor of tunicates to evolve a tunic by providing a way to avoid the adhesiveness of this structure. HighlightsO_LIThe ascidian Ciona tunic has a glycosaminoglycan (GAG)-coated hydrophilic cuticle. C_LIO_LIEpi-1 is a protein expressed in the epidermis of tunicates during embryogenesis. C_LIO_LIEpi-1 Ciona mutants have GAG-free, hydrophobic and therefore sticky cuticles. C_LIO_LIEpi-1 acquisition may have prevented adhesion of tunicates to environmental objects. C_LI

developmental biology↗

bioRxiv2024

Stimulatory and inhibitory G-protein signaling relays drive cAMP accumulation for timely metamorphosis in the chordate Ciona

Larvae of the ascidian Ciona initiate metamorphosis tens of minutes after adhesion to a substratum via their adhesive organ. The gap between adhesion and metamorphosis initiation is suggested to ensure the rigidity of adhesion, allowing Ciona to maintain settlement after losing locomotive activity through metamorphosis. The mechanism producing the gap is unknown. Here, by combining gene functional analyses, pharmacological analyses, and live imaging, we propose that the gap represents the time required for sufficient cAMP accumulation to trigger metamorphosis. Not only the Gs pathway but also the Gi and Gq pathways are involved in the initiation of metamorphosis in the downstream signaling cascade of the neurotransmitter GABA, the known initiator of Ciona metamorphosis. The mutual crosstalk of stimulatory and inhibitory G-proteins functions as the accelerator and brake for cAMP production, ensuring the faithful initiation of metamorphosis at an appropriate time and in the right situation.

developmental biology↗

bioRxiv2023

Uncovering novel endolysins against methicillin-resistant Staphylococcus aureus using microbial single-cell genome sequencing

Endolysins, peptidoglycan hydrolases derived from bacteriophages (phages), are being developed as a promising alternative to conventional antibiotics. To obtain highly active endolysins, a diverse library of endolysins is vital. We here propose microbial single-cell genome sequencing as an efficient tool to discover dozens of previously unknown endolysins, owing to its culture-independent sequencing method. As a proof-of-concept, we analyzed and recovered endolysin genes within prophage regions of Staphylococcus single-amplified genomes (SAGs) in human skin microbiome samples. We constructed a library of chimeric endolysins by shuffling domains of the natural endolysins and performed high-throughput screening against Staphylococcus aureus. One of the lead endolysins, bbst1027, exhibited desirable antimicrobial properties such as rapid bactericidal activity, no detectable resistance development, and in vivo efficacy. We foresee that this endolysin discovery pipeline is in principle applicable to any bacterial target, and boost the development of novel antimicrobial agents.

microbiology↗

bioRxiv2022

Error-acceptable vertical transmission fostered uncoupled phylogenetic relationships between longicorn beetles and their symbionts

The Coleoptera Cerambycidae (longicorn beetles) use wood under different states (living healthy, freshly snapped, completely rot, etc.) in a species-specific manner for their larval diet. Larvae of some Cerambycidae groups have mycetomes, accessory organs associated with the midgut that harbor fungal symbiont cells. The symbionts are thought to improve nutrient conditions; however, this has yet to be shown experimentally. To deduce the evolutionary history of this symbiosis, we investigated the characteristics of the mycetomes in the larvae of longicorn beetles collected in Japan. Lepturinae, Necydalinae, and Spondylidinae are the only groups that possess mycetomes, and these three groups mycetomes and corresponding fungal cells exhibit different characteristics between the groups. However, the phylogenetic relationship of symbiont yeasts does not coincide with that of the corresponding longicorn beetle species, suggesting they have not co-speciated. The imperfect vertical transmission of symbiont yeasts from female to offspring is a mechanism that could accommodate the host-symbiont phylogenetic incongruence. Some Lepturinae species secondarily lost mycetomes. The loss is associated with their diet choice, suggesting that different conditions between feeding habits could have allowed species to discard this organ. We found that symbiont fungi encapsulated in the mycetomes are dispensable for larval growth if sufficient nutrients are given, suggesting that the role of symbiotic fungi could be compensated by the food larvae take. Aegosoma sinicum is a longicorn beetle classified to the subfamily Prioninae, which does not possess mycetomes. However, this species contains a restricted selection of yeast species in the larval gut, suggesting that the symbiosis between longicorn beetles and yeasts emerged before acquiring the mycetomes.

ecology↗

bioRxiv2021

CatSper mediates the chemotactic behavior and motility of the ascidian sperm

Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca2+ concentration. The cation channel of sperm (CatSper), plays an important role in the regulation of intracellular Ca2+ concentration. In mammals, CatSper is the only Ca2+ channel that functions in the sperm, and the mice that lack the genes for the subunits of CatSper, which make up the pore region of the Ca2+ channel, are infertile due to the inhibition of hyperactivation of the sperm. CatSper is also thought to be involved in chemotaxis in sea urchins. In contrast, in the ascidian, Ciona intestinalis, the sperm-activating and -attracting factor (SAAF) interacts with Ca2+/ATPase, which is a Ca2+-pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is the specific Ca2+ channel that functions in the ascidian sperm. Therefore, in this study, we generated Catsper3 knockout (KO) animals that found that they were significantly less motile, with few motile sperms not exhibiting any chemotactic behavior. These results suggest that CatSper plays important roles in the spermatogenesis and basic motility mechanisms of sperms in both ascidians and mammals.

developmental biology↗