Biology subjects

Satou, Y.

Publications and source records attributed to Satou, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Multipotent tail bud cells originated with the chordate common ancestor

In vertebrate embryos, the tail bud region generates the neural tube, the notochord, and somites in the posterior part of the body, including the post-anal tail. Here, we traced the fate of tail bud cells during embryogenesis in the cephalochordate, amphioxus, the earliest diverging lineage of extant chordates, and showed that in amphioxus as well, these cells contribute to the neural tube, the notochord, and somites. Our work provides direct evidence to support the hypothesis that ancestral chordates possessed tail bud cells capable of producing both neural and mesodermal derivatives, illuminating the evolutionary origin of posterior body development in chordates.

developmental biology↗

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

Method-dependent biases in cell type detection between single-cell and single-nucleus RNA sequencing in the photosymbiotic acoel Praesagittifera naikaiensis

Background Comparisons of single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) data have been described in some mammalian tissues and, subsequently, in Drosophila, but remain unexplored in most invertebrate lineages. The xenacoelomorphs occupy key phylogenetic positions, yet they differ anatomically from mammals. They have a reduced extracellular matrix, high-salt body fluid, and no circulatory system. Despite these differences, they possess a well-developed nervous system. One of the xenacoelomorphs, the photosymbiotic acoel (Praesagittifera naikaiensis) also harbours symbiotic Tetraselmis algae, whose RNA can be co-captured with host RNA. Results We compared scRNA-seq and snRNA-seq data from whole P. naikaiensis specimens. Both methods yielded high-quality data with comparable gene detection but a larger share of scRNA-seq reads derived from symbiotic algae. Gene-level analyses revealed that neural genes were enriched in snRNA-seq relative to non-neural genes. Cross-method label transfer and integration-based validation identified six snRNA-seq clusters, including some neural populations, that lacked a clear scRNA-seq counterpart. In contrast, three cell populations, including muscle and metabolically active clusters, were reciprocally validated as captured by both methods. Conclusions Our results show that key snRNA-seq advantages, particularly the enhanced recovery of neural transcripts, are recapitulated in our dataset, which is consistent with previous reports in mammals. Furthermore, snRNA-seq reduces symbiont-derived reads and recovers several cell populations underrepresented in scRNA-seq. These findings provide practical guidance for cell atlas construction in non-model, symbiotic invertebrates.

genomics↗

bioRxiv2026

The tripartite neural plate border is a common trait of vertebrates and tunicates

The neural crest and neurogenic placodes, which arise from the neural plate border, give rise to morphological characteristics that distinguish vertebrates from invertebrates (Gans & Northcutt, 1983). Recent studies have suggested that embryos of ascidians, a group of tunicates that are the closest invertebrate relatives of vertebrates, possess cells that share an evolutionary origin with vertebrate neural crest cells (Abitua et al., 2012; Fatieieva et al., 2025; Ishida & Satou, 2024; Stolfi et al., 2015; Todorov et al., 2024; Waki et al., 2015) and neurogenic placode cells (Abitua et al., 2015; Ikeda et al., 2013; Liu et al., 2023; Liu & Satou, 2019; Manni et al., 2004; Mazet et al., 2005; Papadogiannis et al., 2022; Wagner & Levine, 2012). To dissect the neural plate border of ascidian embryos at the molecular level, and to gain deeper insights into evolutionary origins of the neural crest and neurogenic placodes, we comprehensively analyzed expression patterns of transcription factor genes at single-cell resolution. We demonstrated that the ascidian neural plate border consists of three domains with distinct gene expression profiles: the anterior, inner lateral, and outer lateral domains. A cross-species comparison of transcriptomes from ascidians and zebrafish suggests that the anterior domain is homologous to zebrafish neurogenic placodes, the inner lateral domain to the neural crest and tail bud and the outer lateral domain to the median fin fold ectoderm. We propose that a tripartite neural plate border was present in the last common ancestor of vertebrates and tunicates, providing a blueprint for evolutionary emergence of vertebrate morphological novelties.

developmental biology↗

bioRxiv2026

Gata.a, Tbx21, and Klf6/7 function cooperatively for zygotic genome activation in ascidian embryos

In ascidian embryos, gene expression from the zygotic genome begins between the 8- and 16-cell stages. While most zygotic genes are expressed in specific cell lineages at these stages, transcription factors that provide spatial cues for establishing specific expression patterns are insufficient to activate target genes at normal physiological levels. Gata.a is a transcription factor that provides spatial cues for targets expressed specifically in the animal hemisphere. Intriguingly, it is also required for physiological-level expression of many zygotic genes expressed in the vegetal hemisphere. In the present study, we found that Tbx21 and Klf6/7 augment the latter function of Gata.a. To determine the global extent of genes under control of these factors, we identified genes zygotically activated in early embryos using RNA-sequencing of BrU-labelled zygotic mRNAs. Our results revealed that approximately 80% of all zygotically activated genes were under control of these three factors. That is, together, Gata.a, Tbx21, and Klf6/7 are necessary to regulate target gene expression at physiological levels. This requirement for a specific set of broadly distributed factors resembles those of pioneer transcription factors that trigger zygotic genome activation (ZGA) in other animals, including flies and vertebrates. Regulatory factors involved in ZGA vary among animals, and our results indicate that ascidians use a distinct set of transcription factors for ZGA.

developmental biology↗

bioRxiv2026

HIV-1 can undergo Env-independent retrotransposon-like activity

Retrovirus replication requires coordinated interplay between viral proteins and host cellular machinery, including reverse transcription of the viral RNA genome into DNA and its subsequent integration into the host genomes. SOX2-dependent retrotransposon dynamics have been reported for endogenous retrovirus HERV-K; however, whether a similar intracellular pathway exists for exogenous retroviruses remains unclear. To address whether infection-independent intracellular reverse transcription and partial integration can occur, that is, whether retrovirus can exhibit retrotransposon-like activity, we utilized HeLa and 293T cells, which express no HIV-1 entry receptors. We engineered an Env-deficient HIV-1 NL4-3-based reporter encoding a reverse-oriented, intron-disrupted nanoluciferase cassette that becomes expressible only after splicing followed by reverse transcription. We found that reporter activation depends on reverse transcriptase and protease activities. While integrase is dispensable for early expression, it is essential for long-term maintenance of the nanoluciferase signal. Integration site mapping using next-generation sequencing further confirmed that stable reporter activity requires integrase-dependent proviral insertion. Functional analysis of Gag revealed that membrane binding, multimerization, and budding are prerequisite steps for reporter activation. Concentrated virus preparations from culture supernatants failed to activate the reporter in 293T cells, ruling out a role for reinfection. Electron and confocal microscopy suggested that Gag or viral particles traffic through endosomal compartments. Furthermore, inhibition of dynamin- and clathrin-dependent endocytic pathways reduced reporter activity, indicating that these pathways contribute to efficient reporter activity. Collectively, these finding support the conclusion that HIV-1 can undergo intracellular reverse transcription and partial integration in an infection-independent manner, prompting a reconsideration of the boundary between exogenous retroviruses and endogenous retroelements. Author summaryEnv-independent, infection-independent intracellular reverse transcription and integration in HIV-1 may contribute to integration-site diversity within the same cell. More broadly, this phenomenon suggests continuity between the retroviral life cycle and retrotransposition dynamics, therefore informing our understanding of host-virus coevolution, mechanisms of long-term persistence, and the redesign of therapeutic strategies targeting pre-integration steps.

microbiology↗

bioRxiv2026

Canonical Analysis of Fluorescent Timer-Anchored Transcriptomes Resolves Joint Temporal and Developmental Progression

Cell-state transitions during development are shaped by multiple concurrent processes, yet single-cell transcriptomic analyses often rely on static molecular profiles or infer time without an experimental anchor. We developed mCanonicalTockySeq, a systems-level framework that reconstructs temporally resolved developmental state spaces by combining signalling history with single-cell RNA sequencing. Using the Nr4a3-Tocky Fluorescent Timer system in developing thymic T cells, we combined scRNA-seq with a molecular clock of strong T-cell receptor signalling to establish an experimentally anchored temporal reference. Using Timer-defined landmark populations, mCanonicalTockySeq constructs a shared state space in which temporal progression and developmental maturation are jointly represented, identifying a Tocky-defined temporal manifold while resolving progression toward CD4 and CD8 single-positive states. This framework recovered biologically coherent dynamics of immediate TCR-response genes, lineage-associated regulators, and agonist-selection-associated programmes. We then extended the framework across species by translating human thymic single-cell transcriptomes into one-to-one mouse ortholog space and projecting them into the mouse Nr4a3-Tocky reference. The projected human cells occupied an interpretable temporal-developmental geometry, and the inferred Tocky-equivalent temporal coordinate showed a significant donor-level association with chronological age. Together, these results establish mCanonicalTockySeq as a general framework for modelling how signalling history and developmental progression are jointly organised in single-cell state space, and illustrate how experimentally anchored reference systems can support comparative analyses across species.

systems biology↗

bioRxiv2026

Temporal Mechanisms of T-Cell Fate Decisions under Immune Checkpoint Blockade Resolved by CanonicalTockySeq

In cancer immunotherapy, T-cell functional states change dynamically, and no single marker or gene fully defines them. Static molecular profiles may therefore be insufficient to resolve these states. Capturing signalling history together with ongoing activity could provide a temporal framework for their dissection. Here, we developed CanonicalTockySeq, which integrates a molecular clock of T-cell receptor (TCR) signalling based on the Fluorescent Timer reporter Nr4a3-Tocky (Timer-of-cell-kinetics-and-activity) with scRNA-seq to establish an experimentally anchored temporal reference. Using landmark Tocky fractions as biological ground truths, CanonicalTockySeq constructs a transcriptomic manifold in canonical space with conical geometry. In a murine melanoma model treated with combination anti-PD-L1 and anti-CTLA-4, this framework separates temporal progression (geodesic angle) from signalling strength (radial intensity), enabling time-resolved analysis of gene-expression programmes at single-cell resolution. Application to scRNA-seq data from patients with melanoma further identifies distinct temporal programmes associated with clinical response. These analyses indicate that effective combination immunotherapy is associated with decreased persistence of antigen engagement, suppression of exhaustion-associated TCR signalling programmes, and maintenance of progenitor-like features linked to durable antitumour responses. Collectively, our findings identify temporal-state control as a key component of immunotherapy outcome and establish CanonicalTockySeq as a framework for resolving T-cell response states in vivo.

immunology↗

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↗

bioRxiv2024

Involvement of Hepatitis B Core-Related Antigen in Viral Genome Integration in Patients With Prior Hepatitis B Virus Infection

BackgroundThe incidence of non-B non-C hepatocellular carcinoma (NBNC-HCC), which is negative for hepatitis B surface antigen and hepatitis C virus antibodies, is on the rise. Relatively high numbers of NBNC-HCC patients are hepatitis B core antibody (HBcAb) positive, suggesting that previous HBV infection may play a role in NBNC-HCC development, though the exact mechanisms are unclear. This study aimed to investigate whether HBV genomes are integrated into the host genome of HBcAb-positive NBNC-HCC cases and how these integrations may contribute to cancer development and progression. MethodsHBV detection PCR using HBV-specific primers on DNA extracted from HBcAb-positive NBNC-HCC tissue samples was performed. Positive samples were further examined for HBV integration sites using viral DNA-capture sequencing. Additionally, hepatitis B core-related antigen (HBcrAg) serum levels were measured to assess whether they could be predictive for HBV detection PCR results. ResultsAmong 90 HBcAb-positive NBNC-HCC samples, HBV genome amplification was detected in 18 samples, and elevated HBcrAg levels were associated with the HBV detection PCR results. Seventeen of these samples exhibited HBV integration. The HBV genome was integrated near the TERT gene in 7 samples, resulting in significantly increased TERT mRNA levels; in the KMT2B gene (2 samples); and downstream of LOC441666 (2 samples). ConclusionThe integration sites we identified in our samples have been previously reported in HBV-related HCC, suggesting that HBV integration may also contribute to hepatocarcinogenesis in HBcAb-positive NBNC-HCC. Furthermore, HBcrAg could serve as a potential, noninvasive marker for detecting HBV integration in these cases.

microbiology↗

bioRxiv2024

A master regulatory loop that activates genes in a temporally coordinated manner in muscle cells of ascidian embryos

Ascidian larval muscle cells present a classic example of autonomous development. A regulatory mechanism for these cells has been extensively investigated, and the regulatory gene circuit has been documented from maternal factors to a muscle specific gene. In the present study, we comprehensively identified genes expressed specifically in ascidian muscle cells, and found that all of them are under control of a positive regulatory loop of Tbx6-r.b and Mrf, the core circuit identified previously. We also found that several transcription factors under control of the Tbx6-r.b/Mrf regulatory loop resulted in various temporal expression profiles, which are probably important for creating functional muscle cells. These results, together with results of previous studies, provide an exhaustive view of the regulatory system enabling autonomous development of ascidian larval muscle cells. It shows that the Tbx6-r.b/Mrf regulatory loop, but not a single gene, serves a "master" regulatory function. This master regulatory loop not only controls spatial gene expression patterns, but also governs temporal expression patterns in ascidian muscle cells.

developmental biology↗

bioRxiv2024

A heterocyclic compound inhibits viral release by inducing cell surface BST2/Tetherin/CD317/HM1.24

The introduction of combined antiretroviral therapy (cART) has greatly improved the quality of life of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Nonetheless, the ever-present desire to seek out a full remedy for HIV-1 infections makes the discovery of novel antiviral medication compelling. Owing to this, a new late-stage inhibitor, Lenacapavir/Sunlenca, an HIV multi-phase suppressor, was clinically authorized in 2022. Besides unveiling cutting-edge antivirals inhibiting late-stage proteins or processes, newer therapeutics targeting host restriction factors hold promise for the curative care of HIV-1 infections. Notwithstanding, bone marrow stromal antigen 2 (BST2)/Tetherin/CD317/HM1.24, which entraps progeny virions is an appealing HIV-1 therapeutic candidate. In this study, a novel drug screening system was established, using the Jurkat/Vpr-HiBiT T cells, to identify drugs that could obstruct HIV-1 release; the candidate compounds were selected from the Ono Pharmaceutical compound library. Jurkat T cells expressing Vpr-HiBiT were infected with NL4-3, and the amount of virus release was quantified indirectly by the amount of Vpr-HiBiT incorporated into the progeny virions. Subsequently, the candidate compounds that suppressed viral release were used to synthesize the heterocyclic compound, HT-7, which reduces HIV-1 release with less cellular toxicity. Notably, HT-7 increased cell surface BST2 coupled with HIV-1 release reduction in Jurkat cells but not Jurkat/KO-BST2 cells. Seemingly, HT-7 impeded simian immunodeficiency virus (SIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) release. Concisely, these results suggest that the reduction in viral release, following HT-7 treatment, resulted from the modulation of cell surface expression of BST2 by HT-7. ImportanceA collection of scientific strategies has been revealed to find long-term cure for HIV-1 infection. One of these techniques, the therapeutic approach, involves harnessing late events that are not targeted by current medication. The regulator of HIV-1 assembly and release, the HIV-1 Gag protein, has emerged as a prospective inhibitor. We set up a high-efficiency, economically viable, and facile screening system for the identification of late-stage inhibitors. Herein, we discovered a heterocyclic compound that inhibits HIV-1 release. This newly high- performance testing technique can be employed in virological research for investigating HIV- 1 late-stage processes.

microbiology↗

bioRxiv2024

Viral antigen mismatch affects antiviral T-cell response and may impair immunotherapeutic efficacy against ATL

Human T-cell leukemia virus type 1 (HTLV-1) has the potential to transform primary CD4+ T cells in vitro within a short time; however, the majority of infected individuals maintain an asymptomatic and disease-free condition, suggesting the existence of an equilibrium between the proliferation of infected cells and host immunity. The decline in anti-viral immunity contributes to the transformation of the infected cells, leading to the development of adult T-cell leukemia/lymphoma (ATL). This study identified a variation in a major viral antigen, HTLV-1 Tax, in human leukocyte antigen-A24 (HLA-A24) positive individuals. Two variants of Tax301-309 peptides, SFHNLHLLF (Tax301-309 A) and SFHSLHLLF (Tax301-309 B) were found to induce distinct T-cell immune responses in HLA-A24 positive individuals. There was a disparity between two Tax301-309 peptides in the detection of anti-Tax301-309 cytotoxic T-lymphocytes (CTLs) binding to A24/peptide multimers by flow cytometry analysis. More importantly, over half of the anti-Tax TCRs of anti-Tax CTLs from infected individuals did not recognize mismatched Tax301-309 peptides by Enzyme-Linked Immunospot (ELISpot) assay using Jurkat T cells expressing the anti-Tax301-309 specific TCR. These findings underscore the importance of matching the viral antigen epitope type in T-cell-based immunotherapy against ATL by using viral antigen Tax. Key pointsO_LIEpitope heterogeneity in the major viral antigen in HTLV-1 infection causes different T-cell responses in infected individuals. C_LIO_LIRecommended guideline; performing virus typing to obtain optimal efficacy in T-cell-mediated immunotherapy against the viral antigen Tax C_LI

immunology↗

bioRxiv2023

HIV-Tocky system to visualize proviral expression dynamics

The stably integrated pool of HIV-1 proviruses in the host genome stands against curative strategies. This reservoir is extremely heterogeneous with respect to host cell type, anatomical location, integration site, and replication fitness. During the initial phase of infection, only a few infected cells can resist host immune clearance or cytopathic effect and establish this resistant pool. The mechanisms underlying HIV latency initiation are not fully resolved yet. In the current study, we propose and validate a new reporter model for monitoring HIV-1 provirus silencing and reactivation using Timer of cell kinetics and activity (Tocky). HIV-Tocky system uses a fluorescent Timer protein whose emission spectrum spontaneously shifts from blue to red to reveal HIV-1 provirus dynamics. We dissected provirus transcriptional phases into early, persistent, recently silenced, and latent. To our knowledge, this is the first report to distinguish two latent subsets: a directly non-expressing population and a recently silenced after brief expression. In-depth integration site analysis suggested that the distribution of proviruses in directly latent cells was similar to that in actively transcribing cell population, whereas recently silenced cells tended to harbor proviruses integrated into heterochromatin. Furthermore, we established a library of various single integration clones at which we utilized to demonstrate the efficiency of the block-and-lock strategy by capturing the fast dynamics of silencing that were overlooked in previous models. In summary, we propose HIV-Tocky system to serve as a time-sensitive model that can capture the dynamics of provirus expression, making it a useful tool for HIV latency research. Significance StatementDeterminants of HIV-1 latency establishment are yet to be elucidated. This reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allowed; for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies. ClassificationBiological Sciences, Microbiology.

microbiology↗

bioRxiv2023

Ascidian cells with traits of neural crest and tailbud cells of vertebrates

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

developmental biology↗

bioRxiv2022

Single-cell level temporal profiling of tumour-reactive T cells under immune checkpoint blockade

The blockade of the immune checkpoints PD-1 and CTLA-4 enhances T cell response. However, it is largely unknown how antigen-reactive T cells regulate their checkpoint expression in vivo and whether and how the checkpoint blockade can change activation dynamics of tumour-reactive T cells. To address this, here we used Nr4a3-Timer-of-cell-kinetics-and-activity (Tocky), which allows analysis of temporal changes of activated T cells following TCR signalling in vivo. By analysing melanoma-bearing Nr4a3 Tocky mice, we elucidate hidden dynamics of tumour-reactive T cells in the steady-state. Checkpoint blockade depleted highly activated effector Treg, while promoting unique effector T cell populations, and thus differentially modulating activation of tumour-reactive T cell populations. Furthermore, multidimensional analysis and seamless analysis of Tocky and scRNA-seq revealed a full spectrum of T cell dynamics in response to tumour burden and treatment with checkpoint blockade. Lastly, we propose a rational design of combinatorial therapy to further enhance T cell activities.

immunology↗

bioRxiv2022

Lymphoma cells are circulating in blood of Enzootic Bovine Leukosis and clonality value of virus-infected cells is a useful information for the diagnostic test

Bovine leukemia virus (BLV), a retrovirus, causes Enzootic Bovine Leukosis (EBL) in cattle following a latent infection period. The BLV infection results in polyclonal expansion of infected B-lymphocytes and [~]5% of infected cattle develop monoclonal leukosis. Since the clonal expansion of virus-infected cell is a key in the pathogenesis of EBL, assessing the clonality of malignant cells is crucial for both understanding viral pathogenesis, which might be useful for EBL diagnosis. For the investigation of clonality of BLV-infected cells in non-EBL and EBL cattle, two methods were used to evaluate the status of EBL; BLV-DNA-capture-seq method with high sensitivity and specificity and simple and cost-effective Rapid Amplification of Integration Site for BLV (BLV-RAIS) method. We found that the RAIS method efficiently detect expanded clone in EBL tissue sample as BLV-DNA-capture-seq method. Taking advantage of high frequency of BLV-infected cells in blood, we simplified RAIS method and showed that similar to BLV-DNA-capture-seq, this method could reliably provide quantitative value about clonal abundance of BLV-infected cells. Next, we aimed to establish a diagnostic blood test for EBL by using the clonality information. First, we compared clonality of BLV-infected cells in blood with that in tumor tissue in EBL cattle. There was a remarkably similar clonality between blood and tissue in each animal. Furthermore, BLV integration site information clearly showed that the same clone was the most expanded in both blood and tumor tissue, indicating that tumor cells were circulating in blood in the disease cattle. We also analyzed tumor tissue at two independent anatomical regions and found the same clones was most expanded in both regions, supporting the idea that tumor cells are systemically circulating in the diseased cattle. Finally, we compared clonality value between non-EBL and EBL cattle by using BLV-RAIS method and found that there was clear difference between non-EBL and EBL. More importantly, we found that clonality value was low in asymptomatic phase but high in EBL phase in the longitudinal cohort study. These findings have demonstrated that BLV integration site and clonality value are is a useful information to establish diagnostic blood test for EBL. That would contribute to reduction of economic damage caused by EBL and improvement of productivity in cattle industry.

zoology↗

bioRxiv2022

Transcription factors that specify cell fate activate cell cycle regulator genes to determine cell numbers in ascidian larval tissues

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programs. Although cell cycle control in animal development has been studied extensively, such links are not well understood. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. In the present study, we first showed that maternal factors promote approximately 10 rounds of cell division without zygotic gene expression. Notochord and muscle cells stop dividing after fewer than 10 rounds of cell division, and we show that a Cdk inhibitor (Cdkn1.b) is responsible. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells divided more than 10 times, and Myc, which encodes a proto-oncogenic transcription factor, is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by the same developmental programs that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm was under control of the same developmental programs that specify the developmental fate of mesenchyme and endoderm. Because these transcription factors that regulate Cdkn1.b and Myc are essential factors for fate specification of these tissues, cell fate specification and cell cycle control are linked by those transcription factors. In other words, ectodermal, mesodermal, and endodermal tissues in ascidian embryos control the cell cycle through Cdkn1.b and Myc, which are under the control of transcription factors that specify cell fate.

developmental biology↗