Biology subjects

Kageyama, R.

Publications and source records attributed to Kageyama, R..

16 recordsLinked to original sources

bioRxiv2026

Plagl2 unlocks the latent regenerative potential of Müller glia in the adult mouse retina

Muller glia (MG) in the adult mammalian retina have long been recognized as a potential endogenous source for the regeneration of lost retinal neurons. However, existing MG reprogramming strategies yield an incomplete regenerative response by favoring either proliferation or neurogenic differentiation. Here, we show that the zinc-finger transcription factor Plagl2, which has been demonstrated to rejuvenate aged neural stem cells, was sufficient to reprogram adult mouse MG into a progenitor-like state with coupled proliferative and neurogenic competence. Using histology, time-lapse imaging, and single-cell transcriptomics, we found that Plagl2 drove regulated rounds of MG cell cycle re-entry, while N-methyl-D-aspartate-induced retinal injury further promoted the acquisition of neurogenic competence toward inner nuclear layer neuronal identities. These findings identify Plagl2 as a novel rejuvenator of mammalian MG and support the general principle that reprogramming modules can be redeployed across cell types, offering new avenues for unlocking regenerative potential in otherwise non-regenerative tissues.

neuroscience↗

bioRxiv2025

Activation of neurogenesis improves amyloid-β pathology and cognitive function through AMP kinase signaling in Alzheimer's disease model mice

Adult hippocampal neurogenesis declines with aging and in neurological disorders, leading to cognitive impairment. We previously demonstrated that a treatment of inducing Plagl2, a zinc finger transcription factor gene, and antagonizing Dyrk1a, a gene associated with Down syndrome, referred to as iPaD, can functionally rejuvenate aged neural stem cells (NSCs), thereby enhancing neurogenesis and improving cognition in aged mice. Here, we found that NSC-specific iPaD treatment effectively activated neurogenesis, reduced amyloid-{beta} deposition, and improved cognitive function in Alzheimers disease model mice. Transcriptomic analysis revealed widespread significant changes in gene expression in the hippocampus following iPaD treatment. The upregulated genes included those associated with the activation of astrocytes and microglia involved in amyloid-{beta} clearance, while the downregulated genes included several that are upregulated in Alzheimers disease patients, but whose roles in disease progression remain unclear. Among the latter genes, knockdown of Prkag2, a gene encoding protein kinase AMP-activated non-catalytic subunit gamma 2, in the hippocampus most effectively enhanced neurogenesis and reduced amyloid-{beta} accumulation. Notably, both iPaD treatment and Prkag2 knockdown activated AMP-activated protein kinase signaling, thereby upregulating genes involved in autophagy and cellular homeostasis. These results suggest that Prkag2 may represent a promising therapeutic target for neurodegenerative diseases, including Alzheimers disease.

neuroscience↗

bioRxiv2024

Synchronization of the segmentation clock using synthetic cell-cell signaling

Tight coordination of cell-cell signaling in space and time is vital for self-organization in tissue patterning. During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report the reconstitution of synchronized oscillation in PSM organoids by synthetic cell-cell signaling with designed ligand-receptor pairs. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; non-oscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock, and provides direct evidence of oscillatory cell-cell communication in the segmentation clock.

developmental biology↗

bioRxiv2023

Fluctuation of lysosomal protein degradation in neural stem cells of postnatal mouse brain

Lysosomes are intracellular organelles responsible for degrading diverse macromolecules delivered from several pathways, such as the endo-lysosomal and autophagic pathways. Recent reports have suggested that lysosomes are essential in regulating neural stem cells in developing, adult, and aged brains. However, the activity of these lysosomes has not yet been monitored in these brain tissues. Here, we report a new probe to measure lysosomal protein degradation in brain tissue by immunostaining. Our results demonstrate the fluctuation of lysosomal protein degradation in neural stem cells depending on age and brain disorder. Neural stem cells increase lysosomal activity during hippocampal development in the dentate gyrus, but aging and aging-related disease reduces their activity. In addition, physical exercise increases lysosomal activity in neural stem cells and astrocytes. We hypothesize three different stages of lysosomal activity: the increase in development, the stable state for the adult stage, and the reduction by damages with age or disease.

neuroscience↗

bioRxiv2023

An essential role for miR-15/16 in Treg suppression and restriction of proliferation

The miR-15/16 family is a highly expressed group of tumor suppressor miRNAs that target a large network of genes in T cells to restrict their cell cycle, memory formation and survival. Upon T cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector T cells to mediate a sustained immune response. Here, using conditional deletion of miR-15/16 in immunosuppressive regulatory T cells (Tregs) that express FOXP3, we identify new functions of the miR-15/16 family in T cell immunity. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16-deficiency alters Treg expression of critical functional proteins including FOXP3, IL2R/CD25, CTLA4, PD-1 and IL7R/CD127, and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 inhibition of cell cycle programs shifts Treg diversity and produces an effector Treg phenotype characterized by low expression of TCF1, CD25 and CD62L, and high expression of CD44. These Tregs fail to control immune activation of CD4+ effector T cells, leading to spontaneous multi-organ inflammation and increased allergic airway inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance. HighlightsO_LITreg-specific miR-15/16 expression is essential to prevent systemic tissue inflammation C_LIO_LImiR-15/16 restrict Treg proliferation and regulate expression of the key functional Treg molecules FOXP3, IL2R, CTLA4, PD-1 and IL7R C_LIO_LImiR-15/16 limit formation of effector Tregs and is necessary for high suppressive capacity C_LI

molecular biology↗

bioRxiv2023

A spinal synergy of excitatory and inhibitory neurons coordinates ipsilateral body movements

Innate and goal-directed movements require a high-degree of trunk and appendicular muscle coordination to preserve body stability while ensuring the correct execution of the motor action. The spinal neural circuits underlying motor execution and postural stability are finely modulated by propriospinal, sensory and descending feedback, yet how distinct spinal neuron populations cooperate to control body stability and limb coordination remains unclear. Here, we identified a spinal microcircuit composed of V2 lineage-derived excitatory (V2a) and inhibitory (V2b) neurons that together coordinate ipsilateral body movements during locomotion. Inactivation of the entire V2 neuron lineage does not impair intralimb coordination but destabilizes body balance and ipsilateral limb coupling, causing mice to adopt a compensatory festinating gait and be unable to execute skilled locomotor tasks. Taken together our data suggest that during locomotion the excitatory V2a and inhibitory V2b neurons act antagonistically to control intralimb coordination, and synergistically to coordinate forelimb and hindlimb movements. Thus, we suggest a new circuit architecture, by which neurons with distinct neurotransmitter identities employ a dual-mode of operation, exerting either synergistic or opposing functions to control different facets of the same motor behavior.

neuroscience↗

bioRxiv2021

Novel roles of small extracellular vesicles in regulating the quiescence and proliferation of neural stem cells

Neural stem cells (NSCs) quiescence plays pivotal roles in securing sustainable neurogenesis and avoiding stemness exhaustion in the adult brain. The maintenance of quiescence and transition between proliferation and quiescence are complex processes associated with multiple niche signals, and environmental stimuli. Though the mechanisms of the transitions between NSC states have been extensively investigated, they remain to be fully elucidated. Exosomes are small extracellular vesicles (sEVs) containing functional units such as proteins, microRNAs, and mRNAs. It has already been demonstrated that sEVs actively participate in cancer cell proliferation and metastasis. However, the role of sEVs in NSC quiescence has not been investigated. Here, we applied proteomics to analyze the protein cargos of sEVs derived from proliferating, quiescent, and reactivating NSCs. Our findings revealed expression level fluctuations of NSCs sEV protein cargo at different proliferative conditions. We also identified functional clusters of gene ontology annotations from differentially expressed proteins in three sources of exosomes. Moreover, the use of exosome inhibitors revealed the contribution of exosomes to NSC quiescence at the entrance into quiescence, as well as in quiescence maintenance. Exosome inhibition delayed the entrance into quiescence by proliferating NSCs and allowed quiescent NSCs to exit from the G0 phase of the cell cycle. Protein translation was significantly upregulated in both quiescent NSCs and quiescent-induced NSCs via the exosome inhibition. Our results demonstrated that NSC exosomes are involved in regulating the quiescence of NSCs and provide a functional prediction of NSCs exosome protein cargos in terms of cell-cycle regulation and protein synthesis.

developmental biology↗

bioRxiv2020

CLOCK/BMAL1 interferes with segmentation clock oscillation in mouse embryonic organoids

In mammals, circadian clocks are strictly suppressed during early embryonic stages as well as pluripotent stem cells, by the lack of CLOCK/BMAL1 mediated circadian feedback loops. During ontogenesis, the innate circadian clocks emerge gradually at a late developmental stage, then, with which the circadian temporal order is invested in each cell level throughout a body. Meanwhile, in the early developmental stage, a segmented body plan is essential for an intact developmental process and somitogenesis is controlled by another cell-autonomous oscillator, the segmentation clock, in the posterior presomitic mesoderm (PSM). In the present study, focusing upon the interaction between circadian key components and the segmentation clock, we investigated the effect of the CLOCK/BMAL1 on the segmentation clock Hes7 oscillation, revealing that the expression of functional CLOCK/BMAL1 severely interferes with the ultradian rhythm of segmentation clock in induced PSM and gastruloids. RNA sequencing analysis showed that the premature expression of CLOCK/BMAL1 affects the Hes7 transcription and its regulatory pathways. These results suggest that the suppression of CLOCK/BMAL1-mediated transcriptional regulation during the somitogenesis may be inevitable for intact mammalian development.

developmental biology↗

bioRxiv2020

The non-coding RNA miR-17~92 is a central mediator of T cell activation

T cell activation is paramount for productive adaptive immune responses. CD28 is a key clinically targeted immunoregulatory receptor because it provides the prototypical costimulatory signal required for T cell activation. Therefore, a precise understanding of the molecular consequences of CD28 costimulation has direct therapeutic relevance. Here, we uncover that the microRNA cluster miR-17~92 is part of the molecular program triggered by CD28 costimulation and hence T cell activation. Combining genetics, transcriptomics, bioinformatics and biochemical miRNA:mRNA interaction maps we demonstrate that transgenic miR-17~92 can cell-intrinsically largely overcome defects caused by CD28-deficiency. miR-17~92 promotes transcription of a proinflammatory gene signature by enhancing the calcineurin/NFAT pathway. miR-17~92 binds to and represses a network of genes including several negative regulators of T cell activation. Finally, CD28-deficient T cells exhibit derepressed miR-17~92 target genes during activation, demonstrating that this non-coding RNA is required to shape the transcriptome. Thus, we propose that miR-17~92 constitutes a central mediator for T cell activation, integrating signals by the TCR and CD28 costimulation. In this model miR-17~92 facilitates T cell activation by dampening the breaks that prevent T cell activation.

immunology↗

bioRxiv2020

Fgf4 is critical for maintaining Hes7 levels and Notch oscillations in the somite segmentation clock

During vertebrate development, the presomitic mesoderm (PSM) is periodically segmented into somites, which will form the segmented vertebral column and associated muscle, connective tissue, and dermis. The periodicity of somitogenesis is regulated by a segmentation clock of oscillating Notch activity. Here, we examined mouse mutants lacking only Fgf4 or Fgf8, which we previously demonstrated act redundantly to prevent PSM differentiation. Fgf8 is not required for somitogenesis, but Fgf4 mutants display a range of vertebral defects. We analyzed Fgf4 mutants by quantifying mRNAs fluorescently labeled by hybridization chain reaction within Imaris-based volumetric tissue subsets. These data indicate that FGF4 controls Notch pathway oscillations through the transcriptional repressor, HES7. This hypothesis is supported by demonstrating a genetic synergy between Hes7 and Fgf4, but not with Fgf8. Thus, Fgf4 is an essential Notch oscillation regulator and potentially important in a spectrum of human Segmentation Defects of the Vertebrae caused by defective Notch oscillations.

developmental biology↗

bioRxiv2019

The methyl cycle is a conserved regulator of biological clocks.

The methyl cycle is a universally conserved metabolic pathway operating in prokaryotes and eukaryotes. In this pathway, the amino acid methionine is used to synthesize S-adenosylmethionine, the methyl donor co-substrate in the methylation of nucleic acids, histone and non-histone proteins and many other molecules within the cell. The methylation of nucleic acids and proteins is the foundation of epigenetic and epitranscriptomic regulations of gene expression, but whether the methyl cycle centrally regulates gene expression and function by controlling the availability of methyl moieties is poorly understood.\n\nFrom cyanobacteria to humans, a circadian clock that involves an exquisitely regulated transcription-translation-feedback loop driving oscillations in gene expression and orchestrating physiology and behavior has been described. We reported previously that inhibition of the methyl cycle in mammalian cells caused the lengthening of the period of these oscillations, suggesting the methyl cycle may indeed act as a central regulator of gene expression, at least in mammals. Here, we investigated whether the methyl cycle, given its universal presence among living beings, regulates the circadian clock in species across the phylogenetic tree of life.\n\nWe reveal a remarkable evolutionary conservation of the link between the methyl cycle and the circadian clock. Moreover, we show that the methyl cycle also regulates the somite segmentation clock, another transcription-translation negative feedback loop-based timing mechanism that orchestrate embryonic development in vertebrates, highlighting the methyl cycle as a master regulator of biological clocks.\n\nSIGNIFICANCE STATEMENTHere we reveal that the methyl cycle, a universal metabolic pathway leading to the synthesis of S-adenosylmethionine, the methyl donor co-substrate in virtually all transmethylation reactions within the cell, is a conserved regulator of biological clocks. These discoveries highlight the methyl cycle as a metabolic hub that regulates gene expression via the availability of methyl moieties for the methylation of nucleic acids, proteins and many other molecules with the cell.

evolutionary biology↗

bioRxiv2019

Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters

While the mechanisms of embryonic development are similar between mouse and human, the tempo is in general slower in human. The cause of interspecies differences in developmental time remains a mystery partly due to lack of an appropriate model system1. Since murine and human embryos differ in their sizes, geometries, and nutrients, we use in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells between the species in similar culture conditions. As an example of well-defined developmental time, we focus on the segmentation clock, oscillatory gene expression that regulates the timing of sequential formation of body segments2-4. In this way we recapitulate the murine and human segmentation clocks in vitro, showing that the species-specific oscillation periods are derived from cell autonomous differences in the speeds of biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and human PSCs displayed the oscillatory expression of HES7, the core gene of the segmentation clock5,6, with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours (human PSM). Swapping HES7 loci between murine and human genomes did not change the oscillation periods dramatically, denying the possibility that interspecies differences in the sequences of HES7 loci might be the cause of the observed period difference. Instead, we found that the biochemical reactions that determine the oscillation period, such as the degradation of HES7 and delays in its expression, are slower in human PSM compared with those in mouse PSM. With the measured biochemical parameters, our mathematical model successfully accounted for the 2-3-fold period difference between mouse and human. We further demonstrate that the concept of slower biochemical reactions in human cells is generalizable to several other genes, as well as to another cell type. These results collectively indicate that differences in the speeds of biochemical reactions between murine and human cells give rise to the interspecies period difference of the segmentation clock and may contribute to other interspecies differences in developmental time.

developmental biology↗

bioRxiv2019

Clonal replacement of tumor-specific T cells following PD-1 blockade

Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients. However, which tumor-specific T cells are mobilized following checkpoint blockade remains unclear. Here, we performed paired single-cell RNA- and T cell receptor (TCR)-sequencing on 79,046 cells from site-matched tumors from patients with basal cell carcinoma (BCC) or squamous cell carcinoma (SCC) pre- and post-anti-PD-1 therapy. Tracking TCR clones and transcriptional phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction: the T cell response to treatment was accompanied by clonal expansions of CD8+CD39+ T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this expansion did not derive from pre-existing tumor infiltrating T cell clones; rather, it comprised novel clonotypes, which were not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8+ T cells, compared to other distinct T cell phenotypes, and was evident in BCC and SCC patients. These results, enabled by single-cell multi-omic profiling of clinical samples, demonstrate that pre-existing tumor-specific T cells may be limited in their capacity for re-invigoration, and that the T cell response to checkpoint blockade relies on the expansion of a distinct repertoire of T cell clones that may have just recently entered the tumor.

immunology↗

bioRxiv2018

In vitro characterization of the human segmentation clock

The vertebral column is characterized by the periodic arrangement of vertebrae along the anterior-posterior (AP) axis. This segmental or metameric organization is established early in embryogenesis when pairs of embryonic segments called somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock 1,2. While this oscillator has been well characterized in model organisms 1,2, whether a similar oscillator exists in humans remains unknown. We have previously shown that human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells can differentiate in vitro into PSM upon activation of the Wnt signaling pathway combined with BMP inhibition3. Here, we show that these human PSM cells exhibit Notch and YAP-dependent oscillations4 of the cyclic gene HES7 with a 5-hour period. Single cell RNA-sequencing comparison of the differentiating iPS cells with mouse PSM reveals that human PSM cells follow a similar differentiation path and exhibit a remarkably coordinated differentiation sequence. We also demonstrate that FGF signaling controls the phase and period of the oscillator. This contrasts with classical segmentation models such as the \"Clock and Wavefront\" 1,2,5, where FGF merely implements a signaling threshold specifying where oscillations stop. Overall, our work identifying the human segmentation clock represents an important breakthrough for human developmental biology.

developmental biology↗

bioRxiv2018

A global map of RNA binding protein occupancy guides functional dissection of post-transcriptional regulation of the T cell transcriptome

RNA binding proteins (RBPs) mediate constitutive RNA metabolism and gene specific regulatory interactions. To identify RNA cis-regulatory elements, we developed GCLiPP, a biochemical technique for detecting RBP occupancy transcriptome-wide. GCLiPP sequence tags corresponded with known RBP binding sites, specifically correlating to abundant cytosolic RBPs. To demonstrate the utility of our occupancy profiles, we performed functional dissection of 3' UTRs with CRISPR/Cas9 genome editing. Two RBP occupied sites in the CD69 3' UTR destabilized the transcript of this key regulator of lymphocyte tissue egress. Comparing human Jurkat T cells and mouse primary T cells uncovered hundreds of biochemically shared peaks of GCLiPP signal across homologous regions of human and mouse 3' UTRs, including a cis-regulatory element that governs the stability of the mRNA that encodes the proto-oncogene PIM3 in both species. Our GCLiPP datasets provide a rich resource for investigation of post-transcriptional regulation in the immune system.

genomics↗

bioRxiv2018

Dll1 and Jag1 are Differentially Required to Specify Proximal and Distal Pancreatic Duct Compartments

Notch signaling controls proliferation of multipotent pancreatic progenitor cells (MPCs) and their segregation into bipotent progenitors (BPs) and unipotent pro-acinar cells (PACs). Here we uncover fast ultradian oscillations in the ligand Dll1, and the transcriptional effector Hes1, which proved crucial for MPC expansion. Conversely Jag1, a uniformly expressed ligand, curbed MPC growth, but as expression later segregated to PACs it proved critical for specifying all but the most proximal 5% of BPs, while BPs were entirely lost in Jag1, Dll1 double mutants. Moreover, experimentally induced changes in Hes1 oscillation parameters was associated with selective adoption of BP or PAC fates. Anatomically, ductal morphogenesis and organ architecture is minimally perturbed in Jag1 mutants until later stages, when ductal remodeling fails and signs of acinar-to-ductal metaplasia appear. Our study uncovers oscillating Notch activity in the developing pancreas, which along with modulation by Jag1 is required to coordinate MPC growth and fate.

developmental biology↗