Biology subjects

Yamazaki, S.

Publications and source records attributed to Yamazaki, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Developmental Gfi1 Dynamics Define Hematopoietic Emergenceand Adult Hematopoietic Stem Cell Potency

Gfi1 regulates endothelial-to-hematopoietic transition (EHT) and hematopoietic stem cell (HSC) maintenance; however, its expression dynamics remain unclear. We generated a non-disruptive Gfi1-T2A-mScarlet reporter to track Gfi1 expression. At E10.5, reporter selectively marked EHT in the dorsal aorta, umbilical artery, and vitelline artery. At E9.5, mScarlet-positive endothelial cells were present in the umbilical and vitelline arteries but were scarce in the dorsal aorta, indicating vascular-bed-specific differences in hemogenic activation. Gfi1 was broadly expressed in fetal liver HSCs, while higher expression levels identified HSCs with enhanced multilineage reconstitution and preferential T-cell output. Although Gfi1 expression declined during fetal-to-adult maturation, a subset of adult bone marrow HSCs retained expression and exhibited superior repopulating and self-renewal capacity. Bulk and single-cell transcriptomic analyses linked this subset to dormant and fetal-associated programs, including imprinted genes. Reduced chromatin accessibility at a conserved Gfi1 +26.5kb putative enhancer correlated with developmental Gfi1 downregulation. Thus, Gfi1 dynamics define EHT onset and functionally distinct HSC stemness.

cell biology↗

bioRxiv2026

DNA barcoding-based paired daughter cell analysis reveals division preferences of hematopoietic stem cells

Hematopoietic stem cells(HSCs) maintain their pools by stem-stem division and produce mature blood cells through stem-progenitor or progenitor-progenitor division. A paired daughter cell(PDC) assay combined with single cell transplantation is a powerful method to compare the lineage outputs of two HSC daughter cells. However, single-cell transplantation precludes large-scale analysis of daughter-cell pairs, as only one cell can be transplanted per recipient. Here, we developed a DNA barcoding-based PDC assay to overcome this limitation, enabling simultaneous analysis of 476 daughter pairs from individual HSC divisions and revealing that daughter-cell fates are coordinated and that HSC division patterns are biased toward stem-stem and progenitor-progenitor divisions rather than stem-progenitor divisions. These findings indicate that HSC fate outcomes are directed toward symmetric division outcomes. Integration of single-cell RNA sequencing with DNA barcoding revealed a continuum of HSC states--from balanced HSCs to myeloid-biased HSCs and ultimately to a low-output HSC subset--in which progressively reduced production of mature blood cells relative to stem cell expansion, a proxy for stem-stem division bias, exhibits distinct activities of transcription factors and signaling pathways. Overall, our analysis uncovers characteristic patterns of HSC division and links stem maintenance with distinct molecular features.

cell biology↗

bioRxiv2026

HSF1-mediated Proteostasis Decline Links Aging and Sleep Disruption

Sleep disruption increases with age and is associated with adverse age-related outcomes, yet the molecular mechanisms linking these phenomena remain unclear. Here, through integrative analysis of human and mouse transcriptomic and proteomic datasets, we identify proteostasis-related pathways whose aging trajectories align with transcriptional responses to chronic sleep disruption across tissues and cell types. In the human prefrontal cortex, gene expression exhibits coherent age-associated directional shifts. Across human peripheral blood following sleep restriction and multiple aging mouse tissues and cell types, proteostasis pathways exhibit concordant downregulation. Among these, heat shock response pathways emerge as the most persistent and cross-modal signatures, with components of the heat shock factor 1 (HSF1)-mediated proteostasis network displaying diminished inducibility with age and chronic sleep insufficiency, in contrast to transient activation following short-term sleep deprivation. This attenuation is particularly pronounced in neurons, where age-associated suppression of HSF1 target programs indicates selective vulnerability of neuronal proteostasis. Spatial and single-cell analyses map this vulnerability to hippocampal circuits during aging and to superficial cortical layers and glutamatergic neurons in Alzheimers disease. These findings support a model in which repeated sleep disruption progressively reduces the inducible capacity of proteostatic stress responses, shifting from adaptive activation to progressive attenuation and accelerating age-related decline in proteome maintenance. Consistent with emerging functional evidence, this identifies HSF1-mediated proteostasis as an integrative axis linking sleep stability and molecular aging, suggesting a self-reinforcing relationship in which sleep disruption and proteostasis decline reciprocally exacerbate one another. These results connect transient molecular responses to sleep perturbations with long-term aging trajectories, revealing a systems-level mechanism through which cumulative sleep disruption may increase vulnerability during aging.

neuroscience↗

bioRxiv2026

Genetic Identification of Dopamine Neurons Required for Circadian Food Anticipatory Activity in Mice

Anticipating daily food availability is a conserved circadian behavior that persists even in animals lacking the suprachiasmatic nucleus, yet its neural substrates remain poorly defined. Previous studies implicated dopamine signaling in food anticipatory activity (FAA) but lacked the resolution to identify the responsible neuronal population. Here, we conditionally deleted tyrosine hydroxylase (Th) from molecularly defined dopamine neuron populations in mice. Broad deletion of Th in dopamine transporter-expressing neurons nearly abolished FAA, whereas restoration of Th in substantia nigra dopamine neurons rescued anticipatory locomotion. Surprisingly, deletion of Th from several large dopamine neuron populations had little effect on FAA. In contrast, deletion using Calb1Cre, targeting only [~]25% of substantia nigra dopamine neurons, produced a profound FAA deficit. Notably, these mice exhibited a profound loss of anticipatory locomotion while retaining substantial anticipatory food-seeking behavior. These findings identify a small Calbindin1+ dopamine population required for anticipatory locomotion and demonstrate that distinct behavioral components of food anticipation can be genetically dissociated.

neuroscience↗

bioRxiv2026

Optogenetic Modeling of Wake-Like Transcriptional Progression in Human SH-SY5Y Neuronal Cells

Sleep deprivation induces extensive transcriptional remodeling in the brain, yet it remains unclear how much of this response arises directly from sustained neuronal excitation versus systemic and behavioral confounds. Here we establish a reductionist human neuronal cell model to isolate the cell-autonomous consequences of prolonged depolarization. Controlled optogenetic stimulation of human SH-SY5Y neuronal cells elicited scalable calcium influx and robust induction of canonical activity-dependent genes, enabling precise manipulation of excitation history independent of receptor-specific pleiotropy. Time-resolved RNA sequencing revealed that sustained excitation does not produce a uniform transcriptional shift. Instead, responses partitioned into a small set of reproducible temporal modules including early-, mid-, and late-peaking induction, sustained induction, biphasic responses, and suppression, each associated with distinct functional and regulatory signatures. At the systems level, pathway activities evolved through three sequential transcriptional states, each defined by a distinct mixture of these temporal gene modules within individual programs. State transitions were directional and asymmetric across stimulation and recovery, consistent with cumulative excitation-history-dependent reorganization rather than simple reversal after stimulus offset. Cross-dataset mapping with in vivo mouse sleep datasets showed selective concordance between defined temporal modules and independent sleep-deprivation signatures. Together, these findings show that sustained neuronal excitation alone is sufficient to generate staged, wake-like transcriptional progression, linking gene-level temporal structure to higher-order regulatory states shaped by excitation history.

neuroscience↗

bioRxiv2026

Chromid-like secondary replicons as key sites of biosynthetic gene clusters in Ktedonobacteria

Soils harbour immense biosynthetic gene cluster (BGC) diversity that can mediate microbial interactions, yet this potential is still mapped unevenly across the tree of life. Ktedonobacteria--a class of actinomycete-like bacteria within phylum Chloroflexota--are widespread in terrestrial environments and repeatedly dominate pioneer communities in extremely oligotrophic volcanic bare-ground soils; however, their secondary metabolism and genome architecture remain poorly characterised. Here, we integrate targeted cultivation using volcanic soils from Mount Zao with genome-resolved metagenomics and public genomes to analyse 183 ktedonobacterial genomes. Using antiSMASH and BiG-SLiCE, we identified 1,546 BGCs comprising 1,162 non-redundant gene-cluster families (GCFs). In our dataset, nearly one quarter of genomes encode [≥]10 distinct GCFs, and several family-level clades show mean GCF counts comparable to those in genus Streptomyces. Most ktedonobacterial BGCs are highly divergent from reference collections and exhibit unusually low intra-genomic redundancy, suggesting broad, underexplored chemotypes. Long-read assemblies from ten strains reveal recurrent 1.6-3.5 Mb chromid-like secondary replicons with chromosome-like composition but distinct maintenance signatures. These replicons are consistently enriched in BGCs and mobility-associated genes, with mobility loci concentrated near BGC boundaries. Collectively, our results expand the current knowledge of the phylogenetic landscape of soil biosynthetic diversity and highlight chromid-like secondary replicons as major genomic reservoirs for specialised metabolism in Ktedonobacteria.

microbiology↗

bioRxiv2026

Intrinsic Circadian Organization of the Spinal Cord and Dorsal Root Ganglia

Circadian rhythms coordinate daily fluctuations in physiology and behavior, yet their organization within primary sensory pathways remains poorly defined. Although somatosensory responsiveness varies across the day-night cycle, it is unclear whether peripheral sensory circuits possess molecular mechanisms for temporal regulation. Here, we demonstrate that the spinal-peripheral sensory axis harbors robust, tissue-autonomous circadian clocks. Using real-time bioluminescence imaging, we observed sustained oscillations of the core clock protein PER2 in the spinal dorsal horn and dorsal root ganglia (DRGs), indicating autonomous circadian timing within these tissues. To define the molecular scope of this regulation, we performed RNA sequencing across a 52-hour circadian time course in DRGs. Circadian analysis identified 626 rhythmic transcripts, representing 3.6% of expressed genes. These genes exhibited non-uniform phase distributions and segregated into discrete temporal clusters. Functional annotation revealed phase-specific enrichment of biological processes related to transport, neuronal structure, and proteostasis, suggesting coordinated temporal deployment of distinct molecular programs rather than uniform oscillations across the circadian cycle. Cross-referencing circadian genes with neuropathic pain-associated gene sets revealed limited overlap; however, overlapping genes aligned to specific baseline phase windows enriched for regenerative annotations. Potassium channel-related signaling components implicated in neuropathic pain also showed baseline circadian modulation. Together, these findings establish the spinal dorsal horn and DRGs as intrinsically circadian tissues and reveal a temporally structured molecular landscape in primary sensory neurons, providing a framework for understanding how peripheral sensory processing, plasticity, and homeostatic regulation are coordinated across the day-night cycle.

neuroscience↗

bioRxiv2025

Asymmetric Cross-Reactivity of Nuclear Receptors Reveals an Evolutionary Buffer Between Estrogen and Androgen Signaling

A comprehensive all-by-all receptor ligand affinity screen using Boltz-2, a deep learning framework for protein-ligand interaction prediction, reveals a previously unrecognized asymmetry in steroid hormone receptor binding. Using systematic in silico affinity prediction, we show that estradiol binds the androgen receptor with higher predicted affinity than testosterone and also displays strong affinity for multiple related steroid hormone receptors, whereas the reciprocal interaction - binding of non-aromatized steroids to estrogen receptors - is not observed. Structural modeling demonstrates that estradiol and testosterone occupy the same canonical ligand-binding pocket within the androgen receptor, indicating a conserved steroid-recognition architecture rather than a specialized binding mode. Analysis of a clinically relevant androgen receptor mutation shows modest and broadly distributed stabilization of ligand engagement, consistent with tuning of a pre-existing estradiol-compatible interaction rather than generation of a novel binding mechanism. Reconstruction of ancestral steroid receptors indicates that estradiol maintains high predicted affinity across both ancestral and modern receptors, while other steroids progressively diversify in their receptor preferences following lineage expansion. Together, these results support an evolutionary model in which estradiol represents an early steroid ligand, with younger receptors retaining ancestral estrogen compatibility while evolving specificity for upstream steroid hormones. Functionally, this asymmetric architecture provides a mechanism by which estradiol may modulate androgen receptor signaling under physiological conditions and may contribute to altered receptor activation in pathological contexts such as advanced prostate cancer. These findings define a coherent biochemical and evolutionary framework for estradiol cross-reactivity and highlight the estradiol-androgen receptor interface as a potential therapeutic target.

cell biology↗

bioRxiv2025

A Novel Cell-Type Specific Circadian Reporter Mouse Reveals Self-Sustained Food Entrainable Nature in Enteric Neurons

Global luciferase reporter gene technology is an important real-time readout for the analysis of circadian oscillations. However, because nearly all cells in the body possess cell-autonomous circadian oscillators, developing cell-type-specific reporter systems is essential to dissect how these oscillators interact within complex multicellular tissues and are modulated by brain-body circadian signals. The intestine is a complex organ composed of diverse cell types with distinct origins and functions, and it exhibits robust daily rhythms in physiological activities driven by intrinsic circadian clocks. While the circadian regulation of the mucosal compartment, including the intestinal epithelium, has been relatively well characterized, the mechanisms governing the circadian rhythms in the cells of the muscularis externa remain largely unexplored. Here, we report a novel Cre-dependent Per2-luciferase reporter mouse that enables precise measurement of circadian oscillations in specific peripheral cell populations ex vivo and demonstrate its utility in revealing the hierarchical circadian chrono-architecture within the intestine. Ex vivo gut explants from mice expressing the reporter in one of five major cell types of the muscularis externa--enteric neurons (ENs), enteric glial cells (EGCs), interstitial cells of Cajal (ICCs), smooth muscle cells (SMCs), and muscularis macrophages (MMs)--exhibited robust, self-sustained circadian bioluminescence rhythms, indicating that all of these cell types possess cell-autonomous circadian oscillators. Notably, ENs in the small intestine entrained more rapidly to feeding schedules than did those in the colon, revealing regional differences in food entrainment among the gut clocks. Moreover, the clocks of ENs, SMCs, and MMs shifted their phase in response to daytime-restricted feeding, whereas ICC clocks remained unaffected even after three weeks of restricted feeding. These findings demonstrate that distinct intestinal cell types possess unique entrainment properties and that feeding rhythms can induce heterogeneous phase shifts within the gut clocks. Given that circadian disruption, such as that caused by shift work, contributes to intestinal disorders, this reporter system could provide a powerful platform to dissect the mechanisms linking intercellular circadian desynchrony to intestinal homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/688705v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@11250fforg.highwire.dtl.DTLVardef@18397f2org.highwire.dtl.DTLVardef@6ccd24org.highwire.dtl.DTLVardef@10fffef_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA cell-type-specific circadian reporter mouse enables dissection of the chronoarchitecture of complex multicellular organs (e.g., the intestine) C_LIO_LIAll five major intestinal cell types contain autonomous circadian oscillators peaking at night C_LIO_LIEnteric neurons in small intestine entrain to feeding cycles faster than that in colon C_LIO_LIInterstitial cells of Cajal in both the small intestine and colon do not entrain to feeding cycles C_LIO_LIChemogenetic activation of enteric neurons mimics food entrainment, shifting their circadian phase. C_LI

neuroscience↗

bioRxiv2025

Integration of Hematopoietic and Thymus-like Niches in a Human iPSC-derived Bone Marrow Organoid

Human bone marrow generates blood cells but critically lacks the thymic environment required for T cell development. Developing an integrated in vitro platform that reconstitutes both functions simultaneously remains a major challenge in regenerative and immune medicine. We asked whether a synthetic marrow could be engineered to provide both capacities. Using human induced pluripotent stem cells, we created self-organizing bone marrow organoids (iBMOs) that faithfully reproduced native stromal and vascular structures and, remarkably, supported robust thymus-like T cell differentiation. iBMOs directed hematopoietic progenitors toward functional T and dendritic cells. When engrafted into immunodeficient mice, they autonomously sustained human erythropoiesis and de novo bone formation in vivo. Single-cell transcriptomics revealed a complex niche architecture, including a hybrid cluster co-expressing stem, endothelial, and stromal markers, key to understanding this dual functionality. This bioengineered organoid-stromal system unifies bone marrow and thymic functions in a single human-derived platform, offering a scalable source of immune cells for adoptive cell therapies and regenerative applications, as well as a versatile model for studying human hematopoiesis and immunity.

systems biology↗

bioRxiv2025

From model to field: predictive design of microbial communities for resilient crop performance

Synthetic microbial communities (SynComs) represent a promising approach to enhance crop growth and stress resilience through microbiome engineering. However, the systematic design and field validation of SynComs remain limited. Here, we present a predictive framework for SynCom optimization, integrating plant phenotyping, microbial genomics, and machine learning. Using tomato as a model, we tested over 800 SynCom-temperature combinations consisting of root endophytic bacteria and rhizosphere metabolites. An Elastic Net regression model trained on plant biomass data accurately predicted the performance of unseen SynComs, with prediction accuracy plateauing at [~]5% (301/6144) of all possible SynCom-temperature combinations. Incorporating genomic features significantly improved model performance, whereas microbiome compositional data alone were not informative. We applied the model to design novel SynComs, which were tested in both laboratory and field conditions using a commercial tomato cultivar. The model-informed SynCom enhanced plant growth in field trials and improved heat stress tolerance under controlled laboratory conditions. Multi-omics analyses and feature importance metrics identified specific microbial taxa, including Sphingobium sp., whose enrichment was linked to host plant metabolite (e.g., tomatine) and stress-responsive gene expression. Our results demonstrate a scalable strategy for the predictive design of beneficial microbiomes to improve resilient crop performance under real-world conditions.

systems biology↗

bioRxiv2025

A Conserved Stress-Inducible Hub Enhancer Governs Natriuretic Peptide Expression and Undergoes Dynamic and Reversible Activation in Human Heart Failure

BackgroundNatriuretic peptides (NPs), encoded by the NPPA and NPPB genes, serve as both diagnostic biomarkers and cardioprotective hormones in heart failure. Their expression is tightly regulated by mechanical load, yet the upstream enhancer mechanisms translating hemodynamic stress into transcriptional activation remain incompletely understood. MethodsWe investigated the Nppa/Nppb super-enhancer (SE), which resides in a well-insulated topologically associating domain (TAD) and regulates stress-inducible expression of Nppa and Nppb. We applied CRISPR-based enhancer perturbation, chromatin accessibility and histone profiling, chromatin conformation assays, genetic mouse models, human iPSC-derived cardiomyocytes, and paired failing human hearts before and after left ventricular assist device (LVAD) unloading. ResultsThrough integrative epigenomic and genetic analyses, we identified a conserved element, CR9, as a dominant hub enhancer within this SE. In neonatal rat cardiomyocytes, CRISPR-based activation and inhibition established that CR9 was both necessary and sufficient for NP induction, whereas neighboring elements (CR7/CR8) displayed modest activity but synergized with CR9, establishing a hierarchical and cooperative SE architecture. In vivo, CR9 deletion in mice markedly suppressed Nppa/Nppb expression and diminished chromatin accessibility and histone acetylation across adjacent enhancers and promoters, underscoring its structural as well as transcriptional role. Reinsertion of CR9, even in reverse orientation, restored transcription, confirming its orientation-independent activity. CR9 function remained confined within TAD boundaries, supporting a locus-specific regulation. RNAscope revealed spatial proximity between CR9 enhancer RNA and Nppa/Nppb nascent transcripts. Developmental profiling revealed a switch in enhancer usage from CR6/CR7 in the fetal heart to CR9 in adulthood, indicating a transition from developmental to stress-inducible regulation. In human induced pluripotent stem cell-derived cardiomyocytes, deletion of CR9 nearly abolished NPPA/NPPB expression, establishing its essential role in a human cellular context. Most notably, in paired human failing hearts before and after LVAD unloading, chromatin accessibility at CR9 was dynamically reversed, providing the first direct evidence that enhancer states are plastic and therapeutically modifiable in the human myocardium. ConclusionsCR9 functions as a stress-inducible hub enhancer that coordinates NP transcription under pathological load. This enhancer exhibits reversible activation in human hearts, underscoring enhancer plasticity as a modifiable regulatory layer mediating stress-responsive transcriptional adaptation in the diseased myocardium. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIWe identified CR9 as a stress-inducible hub enhancer within the Nppa/Nppb super-enhancer that is both necessary and sufficient for natriuretic peptide induction, defining a hierarchical and cooperative regulatory architecture controlling Nppa and Nppb expression. C_LIO_LIWe uncovered a developmental switch in enhancer usage from CR6 in the fetal heart to CR9 in adulthood, revealing how stress-inducible enhancer activity emerges as the heart transitions from developmental to adaptive regulation. C_LIO_LIUsing paired failing human hearts before and after LVAD unloading, we provide the first direct evidence that enhancer states are dynamically reversible in the human myocardium. C_LI What are the clinical implications?O_LIThe reversibility of CR9 activity in human heart failure provides a molecular basis for how natriuretic peptide levels decrease when cardiac function improves with effective therapy, reflecting the underlying enhancer plasticity of the diseasesd myocardium. C_LIO_LIEnhancer plasticity is an emerging and targetable mechanism in heart failure, raising the possibility that stress-inducible enhancers like CR9 could be engineered to switch on cardioprotective genes when the heart is under stress, offering a new strategy for precision gene therapy. C_LI

molecular biology↗

bioRxiv2025

Plcl1 Regulates Hematopoietic Stem Cell Function During Aging and Stress by Modulating Calcium Dynamics

Long-term hematopoietic stem cells (HSCs) can generate all blood lineages but typically remain quiescent, becoming activated only in response to acute stress. We previously demonstrated that quiescent HSCs exhibit heterogeneity in intracellular calcium levels. However, the mechanisms underlying this heterogeneity and its physiological relevance remain unclear. Herein, we identify phospholipase C-like 1 (Plcl1), a noncatalytic protein that binds inositol 1,4,5-trisphosphate (IP3), as being selectively enriched in the most quiescent HSC subset. Loss-of-function studies revealed that Plcl1 deficiency at steady state reduced basal intracellular calcium levels and skewed the HSC compartment toward CD41 subsets while preserving overall HSC numbers and long-term reconstitution capacity. Under acute hematopoietic stress, Plcl1 loss accelerated and amplified platelet rebound and the expansion of non-canonical megakaryocyte progenitors (ncMkPs), indicating activation of the thrombopoietic bypass pathway. In aged HSCs, Plcl1 deficiency exacerbated aging-related features, including expansion of the HSC pool, accumulation of CD41 HSCs and ncMkPs, and myeloid-skewed differentiation with impaired competitive reconstitution. These changes were accompanied by diminished induction of calcium-responsive immediate-early genes. Collectively, we identified Plcl1 as an intrinsic regulator that stabilizes calcium dynamics in HSCs, thereby restraining stress- and aging-associated megakaryocytic priming and preserving stem cell function.

cell biology↗

bioRxiv2025

Hematopoietic stem cells undergo bidirectional fate transitions in vivo.

Differentiation of haematopoietic stem and progenitor cells (HSPCs) is widely considered unidirectional in vivo. Here, we developed clonal phylogenetic tracing (CP-tracer) by sequential genetic barcoding, which enabled high-resolution analysis of 181,695 subclones derived from 847 individually labelled HSPCs. This approach uncovered bidirectional fate transitions between myeloid-biased (My-) and lineage-balanced haematopoietic stem cells (HSCs). Individual HSC clones, distinguished by temporally unique serial barcodes, exhibited durable lineage bidirectionality, with dynamics favouring progressive accumulation of My-HSCs over time. CRISPR-Cas9 screening identified the homeobox gene Hhex as a suppressor of myeloid differentiation that enables pivoting toward balanced output. Hhex function is age-dependent, with its expression declining in aged HSCs. Together, these findings demonstrate unexpected plasticity in HSC differentiation, modulated in part by Hhex-mediated repression, that shifts over time to drive the myeloid bias characteristic of ageing.

cell biology↗

bioRxiv2025

Selection of pre-leukemic hematopoietic stem cells driven by distinct extracellular matrix molecules

Despite rapid advances in mapping genetic drivers and gene expression changes in hematopoietic stem cells (HSCs), there is a relative paucity of studies at the protein level. Here, we perform a deep, multi-omic characterization (epigenome, transcriptome and proteome) of HSCs carrying a loss-of-function mutation in Tet2, a key driver of increased self-renewal in blood cancers. Using state-of-the-art, multiplexed, low-input mass spectrometry (MS)-based proteomics, we profile wildtype (WT) and TET2-deficient (Tet2-/-) HSCs and show that the proteome captures previously unrecognized molecular processes which define the pre-leukemic HSC molecular landscape. Specifically, we obtain more accurate stratification of WT and Tet2-/- HSCs than transcriptomic approaches and identify extracellular matrix (ECM) molecules as novel points of dysregulation upon TET2 loss. HSC expansion assays using ECM-functionalized hydrogels confirm a selective effect on the expansion of Tet2-mutant HSCs. Taken together, our study represents a comprehensive molecular characterization of Tet2-mutant HSCs and identifies a previously unanticipated role of ECM molecules in regulating self-renewal of disease-driving HSCs.

cell biology↗

bioRxiv2024

Heat Shock Factor 1 Governs Sleep-Wake Cycles Across Species

Heat Shock Factor 1 (HSF1) is a critical transcription factor for cellular proteostasis, but its role in sleep regulation remains unexplored. We demonstrate that nuclear HSF1 levels in the mouse brain fluctuate with sleep-wake cycles, increasing during extended wakefulness and decreasing during sleep. Using CUT&RUN and RNA-seq, we identified HSF1-regulated transcriptional changes involved in synaptic organization, expanding its known functions beyond traditional heat shock responses. Both systemic and brain-specific Hsf1 knockout mice exhibit altered sleep homeostasis, including increased delta power after sleep deprivation and upregulation of sleep-related genes. However, these knockouts struggle to maintain sleep due to disrupted synaptic organization. In Drosophila, knockout of HSF1s ortholog results in fragmented sleep patterns, suggesting a conserved role for HSF1 in sleep regulation across species. Our findings reveal a novel molecular mechanism underlying sleep regulation and offer potential therapeutic targets for sleep disturbances.

neuroscience↗

bioRxiv2024

Quantitative phase imaging with temporal kinetics predicts hematopoietic stem cell diversity

Innovative identification technologies for hematopoietic stem cells (HSCs) have advanced the frontiers of stem cell biology. However, most analytical techniques capture only a single snapshot, disregarding the temporal context. A comprehensive understanding of the temporal heterogeneity of HSCs necessitates live-cell, real-time and non-invasive analysis. Here, we developed a prediction system for HSC diversity by integrating single-HSC ex vivo expansion technology with quantitative phase imaging (QPI)-driven machine learning. By analyzing single-cell kinetics with QPI, we discovered previously undetectable diversity among HSCs that snapshot analysis fails to capture. Our QPI-driven algorithm quantitatively evaluates the stemness of individual HSCs and incorporates temporal information to significantly improve prediction accuracy. This platform marks a paradigm shift from "identification" to "prediction", enabling us to forecast HSC status by analyzing their past temporal kinetics.

cell biology↗

bioRxiv2024

Reducing oxidative stress improves ex vivo polymer-based human haematopoietic stem and progenitor cell culture and gene editing

Self-renewing multipotent haematopoietic stem cells (HSCs) have the unique capacity to stably regenerate the entire blood and immune systems following transplantation. HSCs are used clinically to reconstitute a healthy blood system in patients suffering from a range of haematological diseases. However, HSCs are very rare and have been challenging to grow ex vivo, which has hampered efforts to collect large numbers of HSCs for both basic research and clinical therapies. Polymer-based culture conditions have recently been developed to support expansion of mouse and human haematopoietic stem and progenitor cells (HSPCs). While mouse HSPCs expanded rapidly in polymer-based cultures, growth speeds for human HSPCs in polymer-based cultures was limited to [~]70-fold over 4-weeks. Here we have found that reducing oxidative stress improves human HSPC growth in these conditions. We describe an optimised culture condition that improves growth to 250-1400-fold over 4-weeks through reducing oxygen tension and the addition of antioxidants. These conditions also enable efficient gene editing in these polymer-based cultures. We envision these improved culture conditions will support a range of research into human HSPC biology and provide a platform for clinical-scale HSPC expansion and gene editing.

cell biology↗