Biology subjects

Irie, N.

Publications and source records attributed to Irie, N..

9 recordsLinked to original sources

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↗

bioRxiv2025

Machine Learning-Assisted Decoding of Temporal Transcriptional Dynamics via Fluorescent Timer

Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. Using the Fluorescent Timer protein (Timer), the Timer-of-cell-kinetics-and-activity (Tocky) system enables analysis of transcriptional dynamics at the single-cell level. However, the complexity of Timer data has limited its broader application. Here, we introduce an integrative approach combining molecular biology and machine learning to elucidate Foxp3 transcriptional dynamics through flow cytometric Timer analysis. We have developed a Convolutional Neural Networks (ConvNet) approach that incorporates image conversion and Gradient-weighted Class Activation Mapping (Grad-CAM) for class-specific feature identification at the single-cell level. Biologically, we developed a novel CRISPR mutant of Foxp3-Tocky lacking the Conserved Non-coding Sequence 2 (CNS2), which has successfully elucidated CNS2-dependent Foxp3 transcription dynamics, revealing novel roles of CNS2 in regulating Foxp3 transcription frequency under specific conditions. Furthermore, generating new data from WT Foxp3 Tocky mice at various ages, the Grad-CAM methods successfully revealed distinct dynamics of Foxp3 expression from neonatal to aged mice, highlighting prominent thymus-like features of neonatal splenic Foxp3+ T cells. In conclusion, our study uncovers previously unrecognised Foxp3 transcriptional dynamics, establishing a proof-of-concept for integrating CRISPR, Tocky, and machine learning methods as advanced techniques to understand transcriptional dynamics in vivo.

immunology↗

bioRxiv2023

Hagfish genome illuminates vertebrate whole genome duplications and their evolutionary consequences

Whole genome duplications (WGDs) are major events that drastically reshape genome architecture and are causally associated with organismal innovations and radiations1. The 2R Hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution2, 3. However, the veracity and timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved4-6 and whether these WGD events underlie vertebrate phenotypic diversification remains elusive7. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and the 2R event occurred in the gnathostome stem-lineage in the late Cambrian after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced two additional, independent genome duplications (herein CR1 and CR2). Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only after the 2R event, questioning the general expectation that WGDs lead to leaps of morphological complexity7.

genomics↗

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↗

bioRxiv2020

Tumors widely express hundreds of embryonic germline genes

We have recently described a class of 756 genes that are widely expressed in cancer, while normally restricted to adult germ cells, referred to as germ cell cancer genes (GC-genes). We hypothesized that carcinogenesis involves reactivation of biomolecular processes and regulatory mechanisms that, under normal circumstances, are restricted to germline development. This would imply that cancer cells share gene expression profiles with primordial germ cells (PGCs). We therefore compared the transcriptomes of human PGCs (hPGCs) and PGC-like cells (PGCLCs) with 17 382 samples from 54 healthy somatic tissues (GTEx) and 11 003 samples from 33 tumor types (TCGA), and identified 672 GC-genes, expanding the known GC-gene pool by 387 genes (51%). Because GC-genes specific to the embryonic germline are not expressed in any adult tissue, targeting these in cancer treatment may result in fewer side effects than targeting conventional cancer/testis (CT) or GC-genes and may preserve fertility. We anticipate that our extended GC-dataset enables improved understanding of tumor development and may provide multiple novel targets for cancer treatment development.

cancer biology↗

bioRxiv2020

Gene expression patterns in chordate embryonic development suggest partial applicability of Haeckel's postulates

The relationship between embryonic development and evolution historically investigated based on embryo morphology, could now be reassessed using mRNA expression endophenotype. Here, we investigated the applicability of von Baers and Haeckels arguments at mRNA expression level by comparing the developmental changes among nine evolutionarily distinct species: from oyster to mouse. In agreement with models based on von Baers postulates, up to 36% of mRNA expression indicated nearly linear conservation of species developmental programs. By contrast, 5-15% of developmental expression profiles, enriched in neural genes, displayed an alignment pattern compatible with the terminal edition paradigm proposed by Haeckel. Thus, the development-evolution relationship based on mRNA expression agrees with early concepts based on embryo morphology and demonstrates that the corresponding patterns coexist in chordate development.

evolutionary biology↗

bioRxiv2019

The Developmental Transcriptome for Lytechinus variegatus Exhibits Temporally Punctuated Gene Expression Changes

Embryonic development is arguably the most complex process an organism undergoes during its lifetime, and understanding this complexity is best approached with a systems-level perspective. The sea urchin has become a highly valuable model organism for understanding developmental specification, morphogenesis, and evolution. As a non-chordate deuterostome, the sea urchin occupies an important evolutionary niche between protostomes and vertebrates. Lytechinus variegatus (Lv) is an Atlantic species that has been well studied, and which has provided important insights into signal transduction, patterning, and morphogenetic changes during embryonic and larval development. The Pacific species, Strongylocentrotus purpuratus (Sp), is another well-studied sea urchin, particularly for gene regulatory networks (GRNs) and cis-regulatory analyses. A well-annotated genome and transcriptome for Sp are available, but similar resources have not been developed for Lv. Here, we provide an analysis of the Lv transcriptome at 11 timepoints during embryonic and larval development. The data indicate that the gene regulatory networks that underlie specification are well-conserved among sea urchin species. We show that the major transitions in variation of embryonic transcription divide the developmental time series into four distinct, temporally sequential phases. Our work shows that sea urchin development occurs via sequential intervals of relatively stable gene expression states that are punctuated by abrupt transitions.

developmental biology↗

bioRxiv2018

Evolutionary transition in accessible chromatin landscapes during vertebrate embryogenesis

The relationship between development and evolution is a central topic in evolutionary biology1,2. Recent transcriptome-based studies support the developmental hourglass model, which predicts that the animal embryogenetic program is most strongly conserved at mid-embryonic stages3-9. This model does not necessarily contradict the classical hypothesis10,11 that animal development recapitulates its evolutionary history after the mid-embryonic stages2,12. However, to date there is no molecular evidence supporting the hypothesis that gene-expression profiles that are more evolutionarily derived appear sequentially in late development. Here, by estimating activated genomic regions and their evolutionary origins, we show that the recapitulative pattern appears during late embryonic stages. We made a genome-wide assessment of accessible chromatin regions throughout embryogenesis in three vertebrate species (mouse, chicken, and medaka) and determined the phylogenetic range at which these regions were shared. In all three species, sequential activation of putative regulatory regions that were more derived occurred later in embryogenesis, whereas ancestral ones tended to be activated early. Our results clarify the chronologic changes in accessible chromatin landscapes and reveal a phylogenetic hierarchy in the evolutionary origins of putative regulatory regions that parallels developmental stages of activation. This relationship may explain, at least in part, the background for morphological observations of recapitulative events during embryogenesis.

evolutionary biology↗