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

Plcl1 Regulates Hematopoietic Stem Cell Function During Aging and Stress by Modulating Calcium Dynamics | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Plcl1 Regulates Hematopoietic Stem Cell Function During Aging and Stress by Modulating Calcium Dynamics Tomohiro Yabushita , View ORCID Profile Yosuke Tanaka , Tsuyoshi Fukushima , Kanako Wakahashi , Terumasa Umemoto , View ORCID Profile Hitoshi Takizawa , Susumu Goyama , Toshio Kitamura , Akira Nishiyama , Tomohiko Tamura , Satoshi Yamazaki , Toshio Suda doi: https://doi.org/10.1101/2025.09.15.674672 Tomohiro Yabushita 1 International Research Center for Medical Sciences (IRCMS), Kumamoto University , Kumamoto, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yosuke Tanaka 1 International Research Center for Medical Sciences (IRCMS), Kumamoto University , Kumamoto, Japan 2 Division of Cell Regulation, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yosuke Tanaka For correspondence: yosuketagm{at}gmail.com sudato{at}keio.jp Tsuyoshi Fukushima 2 Division of Cell Regulation, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kanako Wakahashi 1 International Research Center for Medical Sciences (IRCMS), Kumamoto University , Kumamoto, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Terumasa Umemoto 1 International Research Center for Medical Sciences (IRCMS), Kumamoto University , Kumamoto, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hitoshi Takizawa 1 International Research Center for Medical Sciences (IRCMS), Kumamoto University , Kumamoto, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hitoshi Takizawa Susumu Goyama 3 Division of Molecular Oncology, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Toshio Kitamura 4 Department of Physiological Chemistry, Graduate School of Pharmaceutical Science, The University of Tokyo , Tokyo, Japan 5 Institute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation at Kobe , Hyogo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Akira Nishiyama 6 Department of Immunology, Yokohama City University Graduate School of Medicine , Yokohama, Kanagawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tomohiko Tamura 6 Department of Immunology, Yokohama City University Graduate School of Medicine , Yokohama, Kanagawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Satoshi Yamazaki 2 Division of Cell Regulation, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo , Tokyo, Japan 7 Division of Cell Engineering, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo , Tokyo, Japan 8 Laboratory for Stem Cell Therapy, Faculty of Medicine, Tsukuba University , Ibaraki, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Toshio Suda 1 International Research Center for Medical Sciences (IRCMS), Kumamoto University , Kumamoto, Japan 9 State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: yosuketagm{at}gmail.com sudato{at}keio.jp Abstract Full Text Info/History Metrics Preview PDF Abstract 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 (IP 3 ), 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. Introduction Hematopoietic stem cells (HSCs) sustain lifelong hematopoiesis by leveraging their ability to self-renew and differentiate into multiple lineages. A hallmark of long-term HSCs (LT-HSCs) is their quiescent state, defined by arrest in the G 0 phase of the cell cycle, which preserves genomic integrity, prevents replicative exhaustion, and maintains their long-term regenerative capacity 1 , 2 . This dormant state, in turn, is actively sustained by complex transcriptional, epigenetic, and metabolic programs and extrinsic niche-derived factors 3 – 7 . However, the complete set of molecular pathways governing the G 0 state in HSCs remains unclear. Previous studies have shown that LT-HSCs exhibit substantial functional heterogeneity 8 , 9 . Recently, calcium (Ca 2+ ) signaling has emerged as a key determinant of HSC fate 10 , 11 . Cytokines such as stem cell factor (SCF) and thrombopoietin (TPO) increase intracellular Ca 2+ levels, promoting HSC activation and differentiation 10 . Consistent with their quiescent and undifferentiated state, LT-HSCs maintain substantially lower basal Ca 2+ levels than lineage-committed progenitors 10 , 12 . However, in our previous study, when focusing on the quiescent LT-HSC compartment defined by G 0 markers, HSCs with relatively elevated intracellular Ca 2+ levels exhibited enhanced dormancy, bone marrow reconstitution, and self-renewal capacity 13 . Taken together, these findings indicate that intracellular Ca 2+ dynamics play multifaceted roles in HSC biology and highlight the importance of elucidating how intracellular Ca 2+ levels are tightly regulated in HSCs and how their disruption affects stem cell function. Recent evidence implicates dysregulated intracellular Ca 2+ dynamics as a major contributor to HSC aging 14 . During the physiological HSC aging, abnormal intracellular Ca 2+ enrichment promotes mitochondrial dysfunction, impairs regenerative capacity, and intensifies myeloid-biased differentiation in HSCs 14 . Another hallmark of aged HSCs is the expansion of megakaryocyte-biased subsets, which preferentially support thrombopoiesis via a non-canonical bypass pathway 15 – 18 . Beyond the classical route through multipotent progenitors (MPPs) and canonical CD48⁺ megakaryocyte progenitors (MkPs), lineage-tracing and single-cell analyses have revealed that HSCs can directly generate CD48 − /low non-canonical MkPs, accelerating platelet production 19 – 21 . This bypass pathway is likewise engaged during acute stress hematopoiesis, such as 5-fluorouracil (5-FU) or poly I:C, enabling rapid platelet replenishment 21 , 22 . Although such acute response is essential for short-term recovery, repeated or chronic stress accelerates HSC functional decline and worsens age-associated lineage bias 22 , 23 . Phospholipase C-like 1 ( Plcl1 ) is a non-catalytic member of the phospholipase C family that retains an inositol 1,4,5-trisphosphate (IP 3 )-binding domain but lacks enzymatic activity 24 , 25 . In non-hematopoietic tissues, Plcl1 has been demonstrated to fine-tune intracellular Ca 2+ dynamics by modulating IP 3 receptor signaling. When IP 3 concentrations are elevated, Plcl1 can attenuate receptor-mediated Ca 2+ release by competing for IP 3 binding; under physiological conditions, however, it stabilizes IP 3 and facilitates efficient receptor signaling 26 – 28 . Despite these insights, the functional relevance of Plcl1 in HSC regulation remains uncharacterized. In this study, we examine Plcl1 as an intrinsic regulator of Ca 2+ homeostasis in HSCs by integrating transcriptomic analyses, genetic loss-of-function models, and transplantation assays to delineate its role in maintaining quiescence, balanced lineage output, and stress responses during aging and acute hematopoietic stress. Materials and Methods Mice The Plcl1 knockout ( Plcl1 -KO) mouse line, also known as the Prip1 -deficient model, was first established and characterized by Dr. Kanematsu and Dr. Hirata 29 . To assess HSC quiescence in vivo, we used Vav1 -Cre; Rosa R26R-mVenus-p27K-/R26R-wt mice, wherein quiescent hematopoietic cells are visualized by the expression of mVenus–p27K − , a fluorescent reporter that does not bind Cdks and thereby specifically marks G 0 -phase cells without altering cell cycle dynamics 13 . All experiments were conducted using male mice aged 12–16 weeks. For aging studies, male mice aged 18–21 months were used. Acute stress hematopoiesis was induced by intraperitoneal injection of 150 mg/kg 5-FU (Kyowa Kirin) or by intravenous injection of 2 μg anti-mouse CD42b antibody (R300, Emfret Analytics). All animals were maintained under specific pathogen-free conditions at the Center for Animal Resources and Development (CARD), Kumamoto University. All procedures were approved by the Animal Care and Use Committee of Kumamoto University. Cell preparation Bone marrow (BM) cells were isolated by gently crushing the pelvis, femurs, and tibias in Dulbecco’s Modified Eagle Medium (Sigma) supplemented with 10% fetal bovine serum (FBS; Biowest). Following red blood cell lysis with ACK buffer (Thermo Fisher Scientific), the suspensions were filtered through 100 µm strainers and washed once with Dulbecco’s phosphate-buffered saline (PBS; Sigma) containing 2% FBS. Nucleated cells were subsequently counted using Turk’s solution. For HSC enrichment, the BM cells were magnetically labeled with microbead-conjugated anti–c-Kit antibodies (Miltenyi Biotec), fractionated using the autoMACS Pro Separator (Miltenyi Biotec), and stained with fluorescence-conjugated antibodies for analysis and sorting. BM and peripheral blood analysis Fluorescence-activated cell sorting (FACS) staining was performed using the following antibodies: anti-c-kit (2B8), anti-CD150 (TC15-12F12.2), anti-CD48 (HM48-1), anti-EPCR (eBio1560), anti-CD41 (MWReg30), anti-Sca-1 (D7), anti-Ter-119 (TER-119), anti-CD4 (GK1.5), anti-CD8a (53-6.7), anti-B220 (RA3-6B2), anti-Gr-1 (RB6-8C5), and anti-Mac-1 (M1/70). All antibodies were purchased from BioLegend unless stated otherwise. Flow cytometric analysis and cell sorting were conducted using the FACSAria III instrument (BD Biosciences). Data were analyzed using FlowJo software, version 10.9.0 (Beckman Coulter). Cell populations were defined as follows: KSL (Lineage − c-Kit + Sca-1 + ), MPP2 (CD150 + CD48 + KSL), MPP3/4 (CD150 − CD48 + KSL), ST-HSC (CD150 − CD48 − KSL), LT-HSC (HSC; CD150 + CD48 − KSL), LK (Lineage − c-Kit + Sca-1 − ), MkP (CD150 + CD41 + LK), cMkP (canonical MkP; CD48 + MkP) and ncMkP (non-canonical MkP; CD48 −/low MkP). Following 5-FU treatment, HSCs were defined as Lin⁻EPCR⁺CD150⁺CD48 − cells (L − ESLAM), based on previously validated criteria that reliably identify functionally pure HSCs under both steady-state and stress conditions 30 , 31 . Cell cycle analysis The cell cycle status of HSCs and haematopoietic stem and progenitor cells (HSPCs) was assessed using the PerFix EXPOSE Kit (Phospho-Epitopes Exposure Kit; Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. Freshly isolated BM cells were surface-stained with lineage and HSC markers, following which they were fixed and permeabilized using the kit’s reagents. Intracellular Ki-67 was detected using Alexa-Fluor-647-conjugated anti–Ki-67 antibody (Ki-67, BioLegend), and DNA content was assessed by staining with propidium iodide (BioLegend). Samples were analyzed by flow cytometry as described above. Transplantation For competitive transplantation, FACS-purified LT-HSCs (CD150⁺CD48⁻ KSL) from wild-type (WT) or Plcl1 -KO (CD45.2) mice were intravenously co-injected with 2 × 10 6 unfractionated CD45.1 + competitor BM cells into lethally irradiated CD45.1 + recipients (9.5 Gy), using 100 LT-HSCs for young mice and 250 for aged mice. Donor chimerism and lineage contribution were assessed in peripheral blood every 4 weeks up to 16 weeks using antibodies against CD45.1 (A20), CD45.2 (104), Gr-1(RB6-8C5), CD11b (M1/70), B220 (RA3-6B2), CD4(GK1.5), and CD8a(53-6.7). These antibodies were purchased from BioLegend. Intracellular calcium analysis Freshly isolated HSCs were incubated with 0.5 μM Fluo-4 AM and 0.04% Pluronic F-127 (both from Thermo Fisher Scientific) for 1 h at 37 °C in 5% CO 2 . Following staining, the cells were washed with PBS containing 2% FBS and resuspended in the same buffer for analysis. Fluorescence was measured using the FITC channel on a BD FACSAria III cell sorter, and flow cytometric data were analyzed using FlowJo software (BD Biosciences). RNA extraction and quantitative polymerase chain reaction (qPCR) Total RNA was extracted from LT-HSCs using the RNeasy Micro Kit (QIAGEN). First-strand cDNA was synthesized using the ReverTra Ace™ qPCR RT Kit (Toyobo) according to the manufacturer’s instructions. qPCR was performed on a Roche LightCycler96 system using the Luna Universal qPCR Master Mix (New England Biolabs). Gene expression levels were normalized to Gapdh . The primer sequences were as follows: Plcl1 forward 5′-AGAAGAAGATTAGCAGTGCCCA-3′ and reverse 5′-TCGGTTGTAGATGCGAGAGTT-3′; Gapdh forward 5′-CATCACTGCCACCCAGAAGACTG-3′ and reverse 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′. RNA-seq As previously described 10 , 100 FACS-purified LT-HSCs were used for RNA-seq library construction. First-strand cDNA synthesis was performed using the PrimeScript RT reagent kit (Takara) and not-so-random primers. Second-strand synthesis was carried out using Klenow Fragment (3′–5′ exo⁻; New England Biolabs) and complementary primers. Following purification, the resulting double-stranded cDNA was used for library preparation and amplification with the Nextera XT DNA Sample Preparation Kit (Illumina), according to the manufacturer’s protocol. Sequencing was conducted on an Illumina NextSeq 500 platform to generate 75-bp single-end reads. Subsequently, FASTQ files were uploaded to the Galaxy platform ( https://usegalaxy.org ) for downstream analysis. Sequencing reads were aligned to the mouse reference genome (mm39) using HISAT2, and gene-level counts were generated with FeatureCounts. These count data were then analyzed using the RNAseqChef web-based transcriptome platform 32 . Enrichment analysis was conducted in RNAseqChef using the Pathway Interaction Database and Gene Ontology (GO) Biological Process categories to identify pathways associated with differentially expressed genes. Moreover, previously generated RNA-seq datasets (GEO: GSE139013 and GSE138884) were reanalyzed to compare gene expression profiles across four HSC fractions: G 0 M-negative HSCs, G 0 M-low HSCs, G 0 M-high & Ca 2+ -low HSCs, and G 0 M-high & Ca 2+ -high HSCs. Complementary gene expression data were also obtained from the Blood Stem Cell Atlas 33 and Gene Expression Commons 34 for broader validation. Statistical analysis All statistical analyses were performed using GraphPad Prism software. Data are presented as mean ± standard deviation (SD) or standard error of the mean (SEM), as indicated. Comparisons between two groups were performed using unpaired two-tailed Student’s t-tests. For comparisons involving multiple groups, one-way or two-way analysis of variance followed by Bonferroni post hoc testing was conducted. A p -value < 0.05 was considered statistically significant. Results Plcl1 Expression Characterizes the Most Functionally Competent HSC Subset To identify novel regulators associated with HSC quiescence and functional capacity, we reanalyzed our previously published transcriptomic dataset obtained from G 0 marker (G 0 M) reporter mice ( Vav1-Cre;Rosa R26R-G0M/R26R-wt mice). In this model, G 0 -phase HSCs were further subdivided according to intracellular Ca 2+ levels using CaSiR, a cell-permeant fluorescent Ca 2+ indicator 13 . This analysis identified Plcl1 as one of the top differentially expressed genes in the G 0 M-high/Ca 2+ -high HSC subset, which corresponds to the most quiescent and functionally potent population ( Figure 1A ) . To validate this observation, we analyzed a publicly available single-cell RNA-seq dataset of adult murine hematopoiesis 33 . Plcl1 expression was significantly higher in LT-HSCs (Lin⁻Sca-1⁺c-Kit⁺CD34⁻Flk2⁻) than in multipotent and lineage-committed progenitors ( Figure S1A–B ) . Single-cell expression analysis also demonstrated a strong positive correlation with canonical HSC markers (CD150, EPCR, and Sca-1) and an inverse correlation with Flk2, a marker of progenitor commitment ( Figure S1C ) . We next queried Gene Expression Commons 34 , a platform that assigns binary activity states (“active” or “inactive”) across diverse hematopoietic populations. This approach identified an HSC-specific gene set (active in HSCs but inactive in other HSPCs and differentiated lineages), comprising 117 candidates, including Hoxb5 and Tcf15 ( Figure S1D–E ) . To further enrich for the most quiescent population, we compared vWF + and vWF⁻ HSCs and identified 14 vWF⁺-HSC-specific genes. Notably, Plcl1 was among these vWF⁺-HSC-specific genes ( Figure 1B , Figure S1E ). Finally, consistent with its association with stem cell dormancy, Plcl1 expression was rapidly downregulated upon in vitro culture of HSCs, suggesting that its expression is closely linked to the maintenance of stem cell quiescence ( Figure S1F ) . Download figure Open in new tab Supplementary Figure 1. Plcl1 marks quiescent HSCs in the G₀ phase and associates with elevated intracellular calcium levels. ( A–C ) Single-cell RNA-seq analysis from the Single-Cell Gene Expression Atlas database. (A) PCA plot depicting enrichment of Plcl1 expression (red) in HSCs. (B) Comparison of Plcl1 expression across gated populations: Prog (Lin⁻ Sca-1⁻ c-Kit⁺), HSPCs (Lin⁻ Sca-1⁺ c-Kit⁺), and HSCs (Lin⁻ Sca-1⁺ c-Kit⁺ CD34⁻ Flk2⁻). ****p < 0.0001. (C) Correlative analysis of Plcl1 expression with cell surface antigens (CD150, EPCR, Flk2, Sca-1, c-Kit, and CD34) at the single-cell level. (D) Venn diagram from the Gene Expression Commons database showing that Plcl1 is among the Vwf⁺-HSC-specific active genes shared between HSC-specific genes (n = 117) and Vwf⁺-restricted genes (n = 854). (E) Summary from Gene Expression Commons illustrating an example of an HSC-specific gene, with Hoxb5 displayed as a heatmap showing peak expression in HSCs. Cell population abbreviations: MPP, multipotent progenitor; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/erythrocyte progenitor; CMP, common myeloid progenitor; MkP, megakaryocyte progenitor; CLP, common lymphoid progenitor; BasoE, basophilic erythroblast; PolyE, polyorthochromatic erythroblast; Mono, monocyte; Gra, granulocyte; BLP, B-lymphoid progenitor; preproB, pre-pro B cell; MzB, marginal zone B cell; FoB, follicular B cell; iNK, intermediate natural killer cell; mNK, mature natural killer cell. For further details, see Chen et al. (Nature 530, 223–227, 2016). (F) Bar graph depicting rapid downregulation of Plcl1 expression in HSCs after ex vivo culture for 3, 6, 12, and 24 h. Download figure Open in new tab Figure 1. Plcl1 is enriched in quiescent HSCs and regulates their Ca 2+ dynamics and subset composition. (A) Quantitative comparison of Plcl1 expression across HSC subsets gated by mVenus–p27K⁻ intensity (a G 0 marker consisting of mVenus fused to a CDK-binding–defective p27) 54 and CaSiR fluorescence (intracellular Ca 2+ indicator). ** p < 0.01; *** p < 0.001. The gating strategy shown on the right was adapted from our previous study. (B) Heatmap from the Gene Expression Commons database showing that Plcl1 expression is highest in vWF⁺ HSCs (see Supplementary Fig. 1E for population abbreviations). (C) Quantification of Plcl1 expression in SLAM-KSL HSCs from WT and Plcl1 -deficient (KO) mice, assessed by qPCR (n = 5). **p < 0.01. (D) Frequency of LT-HSCs as a percentage of total BM cells in WT and Plcl1 -KO mice (n = 6). ns: not significant. (E) CD41 mean fluorescence intensity (MFI) (left), frequency (% of total BM cells, middle), and absolute number (right) of CD41⁺ HSCs (n = 6). *p < 0.05, **p < 0.01. (F) Intracellular Ca 2+ levels measured by Fluo-8 in HSCs (left), CD41⁺ HSCs (middle), and CD41⁺ HSCs displayed in a representative histogram (right) (n = 6). *p < 0.05; ns, not significant. (G) Cell cycle states of HSCs (left) and CD41⁺ HSCs (right) in WT and Plcl1 -KO mice (n = 6). ns, not significant. (H) Schematic overview of the competitive BM transplantation experiment. CD45.2⁺ donor cells from WT or Plcl1 -KO mice were mixed with CD45.1⁺ competitor cells and transplanted into lethally irradiated Ly5.1 recipient mice. (I) Donor-derived peripheral blood chimerism at 4, 8, 12, and 16 weeks after competitive transplantation of WT or Plcl1 -KO HSCs (n = 5). ns, not significant. (J) Donor-derived peripheral blood lineage distribution at 16 weeks post-transplantation in recipients of WT or Plcl1 -KO HSCs (n = 5). ns, not significant. Plcl1 Deficiency Shifts the HSC Compartment Toward a CD41⁺ Bias and Reduces Intracellular Ca 2+ Levels under Steady-State Conditions To explore the physiological role of Plcl1 in maintaining HSC homeostasis, we analyzed Plcl1 -deficient ( Plcl1 -KO) mice under steady-state conditions. We confirmed that Plcl1 was expressed in LT-HSCs from WT mice but absent in Plcl1 -KO mice ( Figure 1C ) . Peripheral blood counts, including white blood cells, hemoglobin, and platelets, were comparable between genotypes ( Figure S2A ) . Likewise, both the frequency and absolute number of HSCs remained unchanged ( Figure 1D ). In contrast, Plcl1 -KO mice exhibited a significant increase in CD41⁺ HSCs, a subset associated with megakaryocytic lineage bias, 35 – 37 while EPCR⁺ HSCs, which are linked to deeper quiescence, remained unchanged ( Figure 1E , Figure S2B ). To determine whether these phenotypic changes reflected altered intracellular signaling, we measured intracellular Ca 2+ levels in HSCs using Fluo-8 fluorescence. Intracellular Ca 2+ levels were significantly reduced in Plcl1 -KO HSCs, including within the CD41⁺ subset ( Figure 1F ). This finding is consistent with the proposed biphasic role of Plcl1 in IP 3 -dependent Ca 2+ regulation, where its absence impairs IP 3 stabilization and reduces Ca 2+ levels under steady-state conditions 26 . Subsequently, we assessed downstream megakaryocytic commitment. Although the total number of MkPs was unchanged, CD41 expression was significantly elevated in ncMkPs (CD48 −/low ) 19 – 21 from Plcl1 -KO mice, while cMkPs (CD48⁺) 19 – 21 remained unaffected ( Figure S2C–D ) . Other HSPC populations, including short-term HSCs (ST-HSCs) and MPPs, displayed no differences in abundance or intracellular Ca 2+ levels ( Figures S2D–E ) . Cell cycle analysis revealed no differences in the proportion of G 0 -phase cells within LT-HSCs or CD41⁺ HSCs ( Figure 1G ), although a modest decrease in quiescence was observed in MPP2 cells ( Figure S2F ). To assess long-term functional capacity, we performed competitive BM transplantation by co-injecting CD45.2⁺ WT or Plcl1 -KO donor cells with CD45.1⁺ competitor cells into lethally irradiated recipients ( Figure 1H ). Over a 16-week period, donor chimerism and multilineage reconstitution, including B cells, T cells, and myeloid cells, were comparable between the WT and Plcl1 -KO groups ( Figure 1I–J and Figure S2G ), indicating preserved self-renewal and multilineage output in the absence of Plcl1 . Collectively, these results demonstrate that Plcl1 is dispensable for overall HSC maintenance under homeostatic conditions but plays a selective role in stabilizing intracellular Ca 2+ levels and limiting the expansion of megakaryocyte-biased HSC subsets. Download figure Open in new tab Supplementary Figure 2. Plcl1 Attenuates Emergency Thrombopoiesis During Acute Hematopoietic Stress Given its role in regulating intracellular Ca 2+ and limiting megakaryocyte-biased HSC expansion under steady-state conditions, we next examined how Plcl1 influences hematopoietic output under regenerative stress. Acute hematopoietic stress promotes platelet-biased hematopoiesis by activating a non-canonical thrombopoietic bypass pathway that originates directly from megakaryocyte-biased HSCs 21 , 22 . To model this response, we administered 5-FU, which induces acute hematopoietic stress followed by a pronounced platelet rebound 38 . In WT mice, Plcl1 expression in HSCs was markedly downregulated after 5-FU administration ( Figure 2A ), suggesting a suppressive effect of Plcl1 in stress-induced thrombopoiesis. Plcl1 -KO mice displayed an exaggerated platelet rebound, marked by accelerated recovery, higher peak platelet counts, and extended rebound duration ( Figure 2B–C ). Recovery of other hematopoietic lineages, including erythroid and myeloid cells, was comparable between genotypes ( Figure S3A ), indicating a thrombopoiesis-specific effect. On day 6 after 5-FU administration, when HSCs typically exhibit a transient calcium surge, intracellular Ca 2+ levels were significantly elevated in Plcl1 -KO HSCs, particularly in the CD41⁺ subset, despite similar total HSC counts ( Figure 2D , Figure S3B–C ). By day 12, intracellular Ca 2+ levels had normalized; nevertheless, CD41⁺ HSCs and ncMkPs remained expanded in Plcl1 -KO mice ( Figure 2E , Figure S3D–E ), consistent with the sustained activation of the non-canonical thrombopoietic bypass pathway. To investigate the underlying molecular mechanisms, we performed RNA-seq on Lineage⁻ EPCR⁺ CD150⁺ CD48⁻ (L - ESLAM) HSCs isolated on days 0, 5, and 10 following 5-FU treatment. This population was selected because conventional markers such as Sca-1 and c-Kit are dynamically regulated during stress, while L - ESLAM HSCs provide a stable definition of LT-HSCs 30 , 31 . Although principal component analysis (PCA) and clustering analyses revealed substantial temporal variation in gene expression ( Figure S3F–G ), transcriptional differences between WT and Plcl1 -KO mice remained minimal ( Figure S3H ). These findings suggest that the enhanced platelet output observed in Plcl1 -KO mice may arise from non-transcriptional mechanisms, such as altered Ca 2+ dynamics or post-transcriptional regulation, rather than from global transcriptional rewiring. To further validate these observations, we employed an anti-CD42b-antibody-induced immune thrombocytopenia model, which similarly engages the non-canonical bypass pathway 39 . Consistent with the 5-FU model, Plcl1 -KO mice exhibited accelerated and sustained platelet rebound accompanied by the expansion of CD41⁺ HSCs and ncMkPs ( Figure 2F–G and Figure S3I ). Collectively, these results identify Plcl1 as a critical negative regulator of stress-induced thrombopoiesis, functioning in part through intracellular calcium buffering and restriction of platelet-biased HSC expansion. Download figure Open in new tab Supplementary Figure 3. Loss of Plcl1 selectively enhances platelet-biased responses without affecting global hematopoiesis under acute hematopoietic stress. ( A ) Peripheral blood white blood cell (WBC) counts (left) and hemoglobin (HGB) levels (right) during hematopoietic recovery following 5-FU (150 mg/kg) administration in WT and Plcl1 -KO mice (n = 6). ( B and C ) Frequencies ( B ) and absolute numbers ( C ) of HSCs (L-ESLAM; Lineage⁻ EPCR⁺ CD150⁺ CD48⁻) and CD41⁺ HSCs at day 6 after 5-FU administration in WT and Plcl1 -KO mice (n = 6). ns: not significant. ( D and E ) Frequencies ( D ) and absolute numbers ( E ) of MkPs at day 12 after 5-FU administration in WT and Plcl1 -KO mice (n = 6). * p < 0.05; *** p < 0.001. ( F and G ) UMAP projection ( F ) and unsupervised clustering ( G ) of transcriptomic profiles obtained by RNA-seq from HSCs isolated at days 0, 5, and 10 following 5-FU administration in WT and Plcl1 -KO mice (n = 3). ( H ) MA plot displaying DEGs between WT and Plcl1 -KO L-ESLAM cells at days 0, 5, and 10 after 5-FU treatment based on RNA-seq data. ( I ) Hematopoietic response at day 2 after administration of anti-CD42b antibody (R300) in WT and Plcl1 -KO mice. The absolute number of CD41⁺ HSCs, intracellular calcium levels (ΔFluo-8 MFI) in CD41⁺ HSCs, MkP and ncMkP frequencies (as a percentage of MkPs), and absolute number of ncMkPs (n = 6) are indicated. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Download figure Open in new tab Figure 2. Plcl1 deficiency accelerates platelet recovery under acute stress hematopoiesis. (A) Temporal dynamics of Plcl1 expression in HSCs at 0, 4, 6, and 10 days following 5-FU administration, profiled using publicly available RNA-seq datasets (n = 4 for day 0, day 4, and day 6; n = 6 for day 10). (B) Schematic representation of the 5-FU-induced acute hematopoietic stress model used to assess peripheral blood recovery in WT and Plcl1 -KO mice. (C) Peripheral blood platelet counts during recovery after 5-FU administration (150 mg/kg) in WT and Plcl1 -KO mice (n = 6). (D) Intracellular Ca 2+ concentrations in HSCs and CD41⁺ HSCs from WT and Plcl1 -KO mice on day 6 post-5-FU, measured by Fluo-8 fluorescence intensity (n = 5). **p < 0.01. (E) CD41 mean fluorescence intensity (MFI) in HSCs and frequencies of CD41⁺ HSCs and non-canonical megakaryocyte progenitors (ncMkPs) in WT and Plcl1 -KO mice on day 12 post-5-FU (n = 6). (F) Temporal analysis of peripheral platelet reconstitution following anti-CD42b-antibody-induced thrombocytopenia in WT and Plcl1 -KO mice (n = 6). (G) Frequencies of CD41⁺ HSCs and ncMkPs in WT and Plcl1 -KO mice on day 2 after anti-CD42b antibody administration (n = 5). Plcl1 Deficiency Amplifies Age-Associated Platelet-Biased Hematopoiesis Because aging and acute hematopoietic stress share common features, we examined the effects of Plcl1 deficiency on age-associated HSC behavior. Analysis of public transcriptomic datasets revealed that Plcl1 expression increases with age, alongside platelet-associated markers such as Itga2b (CD41), Gp1ba (CD42b), Slamf1 (CD150), Vwf , Itgb3 (CD61), and Cd9 ( Figure S4A ) 40 , suggesting its potential role in age-associated hematopoietic remodeling. In aged Plcl1 -KO mice, both the frequency and absolute number of HSCs were significantly elevated relative to those in aged WT controls, while ST-HSC, MPP2, and MPP3/4 compartments remained unchanged ( Figure 3A , Figure S4B ). This expansion of the HSC compartment was primarily driven by CD41⁺ HSCs, which exhibited a marked increase in both frequency and absolute number ( Figure 3A , Figure S4C ). Thus, aging and Plcl1 deficiency cooperatively exacerbate megakaryocytic lineage bias. In line with these findings, aged Plcl1 -KO mice exhibited a significant expansion of ncMkPs, while cMkPs remained unchanged ( Figure 3B , Figure S4D–E ), further supporting enhanced engagement of the bypass pathway with age in the absence of Plcl1 . Functionally, competitive transplantation assays demonstrated that CD45.2⁺ donor cells from Plcl1 -KO mice yielded significantly reduced peripheral blood chimerism across all lineages ( Figure 3C–G ). However, this defect was transient, as myeloid lineage reconstitution recovered to WT levels by four months post-transplantation ( Figure 3E ). These findings suggest that the observed impairment results from the dilution of functional HSCs within the expanded phenotypic pool, rather than from exhaustion or irreversible dysfunction. Analysis of lineage distribution further revealed a pronounced myeloid bias in donor-derived CD45.2⁺ cells, particularly at two months post-transplantation ( Figure 3H ), consistent with the aging-associated myeloid skewing of HSCs. Collectively, these results indicate that Plcl1 deficiency accelerates key features of HSC aging, including HSC expansion, compromised reconstitution capacity, and myeloid-biased differentiation. Download figure Open in new tab Supplementary Figure 4. Plcl1 deficiency exacerbates age-associated megakaryocyte bias and alters HSC composition. (A) Gene expression analysis using the Gene Expression Commons platform (Seita et al., PLoS One, 2012) indicates that Plcl1 expression increases with age, in parallel with other platelet-biased HSC markers including Itga2b (CD41), Gp1ba (CD42b), Cd150 , Vwf , Cd9 , Itgb3 (CD61), and Gabrr1 . (B) Frequencies of KSL subfractions, namely ST-HSCs, MPP2, and MPP3/4, expressed as percentages of total bone marrow cells (n = 6). ns, not significant. (C) Absolute numbers of HSCs and CD41⁺ HSCs in aged WT and Plcl1 -KO mice (n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. (D) Frequencies of MkPs (% of LK) and ncMkPs (% of MkPs) in aged WT and Plcl1 -KO mice. * p < 0.05. (E) Absolute numbers of MkPs, ncMkPs, and cMkPs in aged WT and Plcl1 -KO mice (n = 6). * p < 0.05; ** p < 0.01; ns, not significant. (F) Intracellular calcium levels (ΔFluo-8 mean fluorescence intensity) in HSCs, CD41⁺ HSCs, MkPs, ncMkPs, and cMkPs in aged WT and Plcl1 -KO mice (n = 6). ns, not significant. * p < 0.05. Download figure Open in new tab Figure 3. Plcl1 mitigates HSC aging by suppressing the direct megakaryopoiesis bypass pathway. (A) Frequency of HSCs (% of total BM cells, left), CD41 mean fluorescence intensity (MFI) in HSCs (middle), and frequency of CD41⁺ HSCs (right) in aged WT and Plcl1 -KO mice (n = 6). *p < 0.05; ***p < 0.001. (B) Frequencies of megakaryocyte progenitors (MkPs), canonical MkPs (cMkPs), and non-canonical MkPs (ncMkPs) as percentages of total BM cells in aged WT and Plcl1 -KO mice (n = 6). *p < 0.05; **p < 0.01. (C) Schematic overview of competitive transplantation of aged WT or Plcl1 -KO HSCs into young Ly5.1 recipient mice. (D–G) Donor-derived peripheral blood chimerism at 4, 8, 12, and 16 weeks post-transplantation in recipients of aged WT or Plcl1 -KO HSCs (n = 5), showing chimerism in total peripheral blood cells (D), myeloid cells (E), B cells (F), and T cells (G). *p < 0.05; **p < 0.01; n.s., not significant. (H) Lineage distribution of donor-derived cells in peripheral blood at 4, 8, 12, and 16 weeks post-transplantation. *p < 0.05; **p < 0.01; not significant where not indicated. Plcl1 Supports Calcium-Responsive Transcriptional Programs in Aged HSCs To investigate the molecular basis of impaired calcium buffering in aged Plcl1 -KO HSCs, we conducted RNA-seq-based transcriptomic analysis of HSCs from aged WT and Plcl1 -KO mice ( Figure 4A–B ). Gene ontology analysis identified substantial downregulation of calcium-associated transcriptional programs, most notably the term “cellular response to calcium ion,” in Plcl1 -KO HSCs ( Figure 4C–D ). The majority of these downregulated genes, including Fos , Fosb , Jun , Junb , Nr4a1 , and Egr1 , are members of the immediate-early gene (IEG) class 41 . These IEGs are well-established calcium-responsive transcriptional regulators 42 , indicating that Plcl1 deficiency disrupts calcium-driven gene expression programs in aged HSCs ( Figure 4E ). Moreover, HSC-specific loss of Junb , Nr4a1 , or Egr1 has been reported to disrupt quiescence, promote aberrant cycling, expand the phenotypic HSC pool, and skew output toward myeloid lineages 43 – 45 . The reduced expression of these genes in Plcl1 -KO HSCs may therefore contribute to aging-associated expansion and the preferential accumulation of CD41⁺ HSCs. To further dissect subset-specific transcriptional changes, we stratified aged HSCs into CD41⁻ and CD41⁺ subsets and conducted transcriptome profiling. Upon examination, CD41⁺ HSCs exhibited elevated expression of platelet-and proliferation-associated genes ( Itga2b, Itgb3, Vwf, Rrm1, Dnmt1 ), consistent with their megakaryocytic priming and heightened cell cycle activity ( Figure S5A–E ) 37 , 46 . Importantly, calcium-signaling-related pathways remained suppressed in Plcl1 -KO CD41⁺ HSCs, consistent with findings in bulk HSCs ( Figure 4E , Figure S5F ). Collectively, these results demonstrate that Plcl1 is essential for proper transcriptional adaptation to calcium signaling in aged HSCs. Its absence diminishes HSC responsiveness to calcium-dependent cues, which exacerbates aging-associated expansion and megakaryocytic bias. Altogether, our findings establish Plcl1 as a molecular brake that preserves HSC quiescence and lineage balance under age-related stress ( Figure 4F ) . Download figure Open in new tab Supplementary Figure 5. Transcriptomic profiling of aged CD41⁺ HSCs and Plcl1 -deficient CD41⁺ HSCs. (A) Transcriptomic profiles of aged CD41⁻ and CD41⁺ WT HSCs analyzed via RNA-seq and visualized using multi-dimensional scaling (MDS) and hierarchical clustering (n = 3). (B) MA plot and hierarchical clustering heatmap depicting results of DEG analysis comparing aged CD41⁻ and CD41⁺ WT HSCs (n = 3). ( C, D ) GO biological process enrichment analysis of DEGs between aged CD41⁻ and CD41⁺ HSCs, visualized as an over-representation bar plot ( C ) and a gene–concept network (cnet) plot (D) (n = 3). (E) Normalized expression levels of surface markers associated with platelet-biased HSCs ( Itga2b , Itgb3 , Vwf ) and cell cycle–related genes ( Rrm1 , Dnmt1 , Cdc45 , Procr ) in aged CD41⁺ and CD41⁻ HSCs (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001. (F) GO biological process enrichment analysis of DEGs between aged CD41⁺ WT and Plcl1 -KO HSCs, visualized as an over-representation bar plot (n = 3). Download figure Open in new tab Figure 4. Plcl1 safeguards calcium-dependent immediate early gene responses in aged HSCs. (A) Multidimensional scaling (MDS) and hierarchical clustering of transcriptomic profiles in HSCs from aged WT and Plcl1 -KO mice (n = 3). (B) MA plot and hierarchical clustering heatmap illustrating differential gene expression (DEG) of aged HSCs from WT and Plcl1 -KO mice (n = 3). (C) Enrichment analysis of GO biological processes based on DEGs between aged WT and Plcl1 -KO HSCs, visualized using an over-representation bar plot and a gene–concept network (cnet) plot (n = 3). (D) Pathway interaction database enrichment analysis of DEGs from aged WT and Plcl1 -KO HSCs (n = 3). (E) Normalized expression of calcium-responsive IEGs, namely Junb , Nr4a1 , and Egr1 , in aged HSCs from WT and Plcl1 -KO mice (n = 3). n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001. (F) Schematic model depicting how Plcl1 deficiency in aged HSCs promotes platelet-biased HSC expansion and bypass megakaryopoiesis, resulting in altered hematopoiesis. Discussion Extracellular calcium influx has been reported to regulate HSC behavior via L-type voltage-gated calcium channels, the calcium-sensing receptor, and purinergic receptors 10 , 47 , 48 . In contrast, the intracellular regulation of IP 3 -dependent Ca 2+ release in HSCs remains unclear. Here, we demonstrate that Plcl1 , a catalytically inactive IP 3 -binding protein, is enriched in the most quiescent HSC subset and modulates intracellular Ca 2+ dynamics. Functionally, loss of Plcl1 increased CD41⁺-megakaryocyte-primed HSCs at steady state, exaggerated platelet rebound during acute stress, and intensified aging-associated HSC expansion, and myeloid skewing, followed by diminished activation of Ca 2+ -responsive transcriptional programs. Together, these findings suggest that Plcl1 acts as an intracellular modulator of Ca 2+ signaling that helps restrain premature megakaryocytic priming during stress and aging. Under steady-state conditions, Plcl1 deficiency resulted in diminished basal Ca 2+ levels in LT-HSCs. Consistent with this, previous biochemical studies indicate that Plcl1 binds IP 3 with high affinity, prolonging its half-life by competing with IP 3 5-phosphatase 26 , 49 , 50 . This binding is thought to sustain localized IP 3 –IP 3 R interactions and support basal Ca 2+ release from the endoplasmic reticulum (ER), thereby enabling Ca 2+ -dependent transcriptional programs in quiescent HSCs. In contrast, following 5-FU treatment, intracellular Ca 2+ levels in HSCs typically surge around day 6 as part of the regenerative rebound 10 , and in our study this Ca 2+ surge was further amplified in Plcl1 -KO HSCs. These findings suggest that Plcl1 attenuates stress-induced Ca 2+ release by sequestering IP 3 and limiting excessive propagation of IP 3 R-mediated signaling 26 , 51 . Furthermore, analogous modulatory roles for Plcl1 in IP 3 -mediated Ca 2+ signaling have been documented in other non-hematopoietic contexts 26 , 28 . These support the view that Plcl1 functions not as a canonical signaling effector but rather as a context-dependent modulator that restrains excessive Ca 2+ transients and prevents premature or maladaptive lineage commitment. Thus, Plcl1 acts primarily to modulate Ca 2+ signaling dynamics rather than fundamentally altering core transcriptional networks and may influence the platelet-biased output of HSCs. During physiological aging, cytosolic and mitochondrial Ca 2+ levels progressively increase in HSCs, promoting a gradual loss of quiescence and accelerating HSC aging 14 , 52 . A key mechanism driving this increase involves elevated CD38 activity in aged HSCs, which generates metabolites such as cADPR that stimulate ER-based Ca 2+ release, primarily through ryanodine receptors 52 . Our findings suggest that the pronounced aging-associated phenotypes in aged and Plcl1 -KO HSCs likely result from this synergistic disruption of Ca²⁺ homeostasis. Notably, although Plcl1 deletion reduces basal cytosolic Ca 2+ levels in young HSCs, this effect was not observed in aged HSCs. This phenomenon is presumably due to the dominant influence of aging-associated intracellular Ca 2+ influx 52 . Furthermore, aged Plcl1 -KO HSCs display reduced expression of Ca 2+ -responsive IEGs, including JunB , Egr1 , and Nr4a1 , which are critical for maintaining quiescence and enabling appropriate stress responses 43 , 45 , 53 . Because IEG activation depends not on global Ca 2+ elevation but on the finely tuned dynamics of Ca 2+ spikes, including their amplitude, frequency, and duration, 53 disruption of these parameters in aged Plcl1 -KO HSCs likely decouples Ca 2+ signaling from transcriptional output, thereby impairing adaptive transcriptional programs. Together, these findings identify Plcl1 as a critical gatekeeper of Ca 2+ dynamics that preserves Ca 2+ -dependent transcriptional responses in aged HSCs, thereby maintaining stem cell stability under aging-associated stress. This study has several limitations. We were unable to directly assess IP 3 buffering or Ca ²⁺ oscillatory dynamics in primary HSCs. Moreover, potential redundancy with other regulators such as Plcl2 or IP 3 Rs remains unresolved. Analysis of a publicly available human hematopoiesis dataset (GSE42519) indicated that PLCL1 transcript levels in human HSCs (Lin⁻ CD34⁺ CD38⁻ CD90⁺ CD45RA⁻) were approximately 1.5-fold higher than in MPPs and granulocyte/macrophage progenitors and 1.7-fold higher than in common myeloid progenitors and megakaryocyte/erythrocyte progenitors. These data suggest that Plcl1 may also be enriched in human HSCs at the transcript level. However, Plcl1 expression at the protein level, as well as its functional contribution to human HSC regulation, remains to be validated in future studies. In summary, our findings identify Plcl1 as a critical modulator of Ca 2+ signaling that preserves HSC quiescence and mitigates age- and stress-associated megakaryocyte priming. These insights establish a mechanistic link between Ca 2+ homeostasis and stem cell fate and highlight Plcl1 as a potential target for sustaining balanced hematopoiesis during aging and following therapeutic intervention. Authorship T.Y. and Y.T. conceived the project, designed and performed most of the experiments, analyzed and interpreted the data, and wrote the manuscript. T.F., T.I., R.I., M.K., S.S., K.W., K.K., D.K., Y.A., T.U., assisted with the experiments. T.F., H.T., S.G., T.K., A.N., advised on the data interpretation, discussed and made suggestions for this study. T.S. supervised the project, interpreted the data, and participated in writing the manuscript. Disclosure of conflicts of interest The authors declare no competing interests. Acknowledgments We thank Dr. Masato Hirata (Fukuoka Dental College) for generously providing the Plcl1 knockout mice. We are also grateful to Takako Ideue, Rina Iwata, Miho Kataoka, and Shiori Shikata for their excellent technical assistance. We appreciate the support of the core facilities of the International Research Center for Medical Sciences (IRCMS), Kumamoto University, for technical support, and the Center for Animal Resources and Development (CARD), Kumamoto University, for excellent animal care. We also thank the FACS Core and the Mouse Core at the Institute of Medical Science, the University of Tokyo. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Young Scientists (TY), a JSPS Grant-in-Aid for Young Scientists (TY, 24K19227), a JSPS Grant-in-Aid for Challenging Explanatory Research (YT, 24K11520), a JSPS Grant-in-Aid for (21K19500), a grant from SENSHIN Medical Research Foundation (TY), a Grant-in-Aid for Scientific research from MOCHIDA Memorial Foundation (TY and YT), the Japanese Society of Hematology Research grant (TY), and Kanehara Ichiro Award (TS). This research was also supported by MEXT Promotion of Distinctive Joint Usage/Research Center Support Program Grant Numbers JPMXP0724020288 at the Advanced Medical Research Center, Yokohama City University. Funder Information Declared Japan Society for the Promotion of Science, https://ror.org/00hhkn466 , 24K19227 , 24K11520 , 21K19500 SENSHIN Medical Research Foundation, https://ror.org/021tj4g88 Mochida Memorial Foundation For Medical And Pharmaceutical Research, https://ror.org/050rsje66 The Japanese Society of Hematology, https://ror.org/00qembr49 The Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care, https://ror.org/043s0fn12 References 1. ↵ Wilson A , Laurenti E , Oser G , et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair . Cell . 2008 ; 135 ( 6 ): 1118 – 1129 . OpenUrl CrossRef PubMed Web of Science 2. ↵ Foudi A , Hochedlinger K , Van Buren D , et al. 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Share Plcl1 Regulates Hematopoietic Stem Cell Function During Aging and Stress by Modulating Calcium Dynamics Tomohiro Yabushita , Yosuke Tanaka , Tsuyoshi Fukushima , Kanako Wakahashi , Terumasa Umemoto , Hitoshi Takizawa , Susumu Goyama , Toshio Kitamura , Akira Nishiyama , Tomohiko Tamura , Satoshi Yamazaki , Toshio Suda bioRxiv 2025.09.15.674672; doi: https://doi.org/10.1101/2025.09.15.674672 Share This Article: Copy Citation Tools Plcl1 Regulates Hematopoietic Stem Cell Function During Aging and Stress by Modulating Calcium Dynamics Tomohiro Yabushita , Yosuke Tanaka , Tsuyoshi Fukushima , Kanako Wakahashi , Terumasa Umemoto , Hitoshi Takizawa , Susumu Goyama , Toshio Kitamura , Akira Nishiyama , Tomohiko Tamura , Satoshi Yamazaki , Toshio Suda bioRxiv 2025.09.15.674672; doi: https://doi.org/10.1101/2025.09.15.674672 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17690) Bioengineering (13892) Bioinformatics (41936) Biophysics (21451) Cancer Biology (18588) Cell Biology (25499) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88603) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15152) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)