技术资料

Deleterious germline DDX41 variants constitute the most common inherited predisposition disorder linked to myeloid neoplasms (MNs),yet their role in MNs remains unclear. Here we show that DDX41 is essential for erythropoiesis but dispensable for other hematopoietic lineages. Ddx41 knockout in early erythropoiesis is embryonically lethal,while knockout in late-stage terminal erythropoiesis allows mice to survive with normal blood counts. DDX41 deficiency induces a significant upregulation of G-quadruplexes (G4),which co-distribute with DDX41 on the erythroid genome. DDX41 directly binds to and resolves G4,which is significantly compromised in MN-associated DDX41 mutants. G4 accumulation induces erythroid genome instability,ribosomal defects,and p53 upregulation. However,p53 deficiency does not rescue the embryonic death of Ddx41 hematopoietic-specific knockout mice. In parallel,genome instability also activates the cGas-Sting pathway,impairing survival,as cGas deficiency rescues the lethality of hematopoietic-specific Ddx41 knockout mice. This is supported by data from a DDX41-mutated MN patient and human iPSC-derived bone marrow organoids. Our study establishes DDX41 as a G4 resolvase,essential for erythroid genome stability and suppressing the cGAS-STING pathway. Germline DDX41 mutations are linked to myeloid neoplasms,but their roles in the disease is unclear. Here,the authors show that DDX41 resolves G-quadruplex structures to maintain erythroid genome stability and prevent cGAS-mediated cell death. These functions are lost in disease-associated variants. View Publication

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across developmental lineages. Here,we engineer barcoded induced pluripotent stem cells to generate an atlas of multilineage differentiation from pluripotency,encompassing an eight-day time course with modulation of WNT,BMP,and VEGF signalling pathways. Annotation of in vitro cell types with reference to in vivo development reveals diverse mesendoderm lineage cell types including lateral plate and paraxial mesoderm,neural crest,and primitive gut. Interrogation of temporal and signalling-specific gene expression in this atlas,evaluated against cell type-specific gene expression in human complex trait data highlights the WNT-inhibitor gene TMEM88 as a regulator of mesendodermal lineages influencing cardiovascular and anthropometric traits. Genetic TMEM88 loss of function models show impaired differentiation of endodermal and mesodermal derivatives in vitro and dysregulated arterial blood pressure in vivo. Together,this study provides an atlas of multilineage stem cell differentiation and analysis pipelines to dissect genetic determinants of mammalian developmental physiology. Shen et al. report a method for multiplexing isogenic iPSCs for single-cell RNA-seq. With it,they created an atlas of in vitro differentiation and identified TMEM88 as a regulator of cardiovascular development,impacting blood pressure in adult mice. View Publication

Disrupted glucose metabolism and protein misfolding are key characteristics of age-related neurodegenerative disorders including Parkinson’s disease,however their mechanistic linkage is largely unexplored. The hexosamine biosynthetic pathway utilizes glucose and uridine-5’-triphosphate to generate N-linked glycans required for protein folding in the endoplasmic reticulum. Here we find that Parkinson’s patient midbrain cultures accumulate glucose and uridine-5’-triphosphate,while N-glycan synthesis rates are reduced. Impaired glucose flux occurred by selective reduction of the rate-limiting enzyme,GFPT2,through disrupted signaling between the unfolded protein response and the hexosamine pathway. Failure of the unfolded protein response and reduced N-glycosylation caused immature lysosomal hydrolases to misfold and accumulate,while accelerating glucose flux through the hexosamine pathway rescued hydrolase function and reduced pathological ?-synuclein. Our data indicate that the hexosamine pathway integrates glucose metabolism with lysosomal activity,and its failure in Parkinson’s disease occurs by uncoupling of the unfolded protein response-hexosamine pathway axis. These findings offer new methods to restore proteostasis by hexosamine pathway enhancement. Reduced glucose flux via the hexosamine pathway contributes to lysosomal dysfunction and protein accumulation in Parkinson patient iPSC-neurons. Enhancing the hexosamine pathway rescues lysosome activity and restores proteostasis. View Publication

AbstractThe fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here,we implemented a 3D model of cardiac vasculogenesis,incorporating endothelial cells (EC),stromal cells,and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues,resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis,including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs,the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast,supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly,enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization,illustrating the use of this approach in the engineering of enhanced,perfusable,microfluidic models of the myocardium. View Publication

AbstractNeurodegeneration in the autoimmune disease multiple sclerosis still poses a major therapeutic challenge. Effective drugs that target the inflammation can only partially reduce accumulation of neurological deficits and conversion to progressive disease forms. Diet and the associated gut microbiome are currently being discussed as crucial environmental risk factors that determine disease onset and subsequent progression. In people with multiple sclerosis,supplementation of the short-chain fatty acid propionic acid,as a microbial metabolite derived from the fermentation of a high-fiber diet,has previously been shown to regulate inflammation accompanied by neuroprotective properties. We set out to determine whether the neuroprotective impact of propionic acid is a direct mode of action of short-chain fatty acids on CNS neurons. We analysed neurite recovery in the presence of the short-chain fatty acid propionic acid and butyric acid in a reverse-translational disease-in-a-dish model of human-induced primary neurons differentiated from people with multiple sclerosis-derived induced pluripotent stem cells. We found that recovery of damaged neurites is induced by propionic acid and butyric acid. We could also show that administration of butyric acid is able to enhance propionic acid-associated neurite recovery. Whole-cell proteome analysis of induced primary neurons following recovery in the presence of propionic acid revealed abundant changes of protein groups that are associated with the chromatin assembly,translational,and metabolic processes. We further present evidence that these alterations in the chromatin assembly were associated with inhibition of histone deacetylase class I/II following both propionic acid and butyric acid treatment,mediated by free fatty acid receptor signalling. While neurite recovery in the presence of propionic acid is promoted by activation of the anti-oxidative response,administration of butyric acid increases neuronal ATP synthesis in people with multiple sclerosis-specific induced primary neurons. In human multiple sclerosis-specific neurons,differentiated via induced pluripotent stem cells,Gisevius et al. display neuroregeneration mediated by the short-chain fatty acids propionic and butyric acid. Intracellularly,free fatty acid receptor signalling leads to inhibition of histone deacetylase activity,thereby altering the oxidative stress response and cellular protein biosynthesis. Graphical Abstract Graphical Abstract View Publication

SUMMARY Increasing evidence suggests that the mechanics of chromatin and nucleoplasm regulate gene transcription and nuclear function. However,how the chromatin and nucleoplasm sense and respond to forces remains elusive. Here,we employed a strategy of applying forces directly to the chromatin of a cell via a microinjected 200-nm anti-H2B-antibody-coated ferromagnetic nanoparticle (FMNP) and an anti-immunoglobulin G (IgG)-antibody-coated or an uncoated FMNP. The chromatin behaved as a viscoelastic gel-like structure and the nucleoplasm was a softer viscoelastic structure at loading frequencies of 0.1–5 Hz. Protein diffusivity of the chromatin,nucleoplasm,and RNA polymerase II (RNA Pol II) and RNA Pol II activity were upregulated in a chromatin-stretching-dependent manner and stayed upregulated for tens of minutes after force cessation. Chromatin stiffness increased,but the mechanomemory duration of chromatin diffusivity decreased,with substrate stiffness. These findings may provide a mechanomemory mechanism of transcription upregulation and have implications on cell and nuclear functions. Graphical abstract In brief Rashid et al. show that chromatin and nucleoplasm in cells behave as viscoelastic materials. Chromatin stretching mediates the mechanomemory of chromatin and nucleoplasm diffusivity as well as of RNA polymerase II activity. The mechanomemory of RNA polymerase II activity provides a mechanism for sustained transcription upregulation tens of minutes after force cessation. View Publication

ABSTRACTLoss of Cdx2 in vivo leads to stunted development of the allantois,an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here,we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT),heterozygous (CDX2-Het),and homozygous null CDX2 (CDX2-KO) genotypes,differentiating these cells in a 2D gastruloid model,and subjecting these cells to single-nucleus RNA and ATAC sequencing,we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile,suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo,we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together,these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo. Summary: Using 2D human gastruloids,CDX2 is shown to dose-dependently influence genes related to tissue permeability,cell-cell adhesions,and cytoskeletal architecture during extraembryonic mesoderm development. View Publication

Astroglia are integral to brain development and the emergence of neurodevelopmental disorders. However,studying the pathophysiology of human astroglia using brain organoid models has been hindered by inefficient astrogliogenesis. In this study,we introduce a robust method for generating astroglia-enriched organoids through BMP4 treatment during the neural differentiation phase of organoid development. Our RNA sequencing analysis reveals that astroglia developed within these organoids exhibit advanced developmental characteristics and enhanced synaptic functions compared to those grown under traditional two-dimensional conditions,particularly highlighted by increased neurexin (NRXN)-neuroligin (NLGN) signaling. Cell adhesion molecules,such as NRXN and NLGN,are essential in regulating interactions between astroglia and neurons. We further discovered that brain organoids derived from human embryonic stem cells (hESCs) harboring the autism-associated NLGN3 R451C mutation exhibit increased astrogliogenesis. Notably,the NLGN3 R451C astroglia demonstrate enhanced branching,indicating a more intricate morphology. Interestingly,our RNA sequencing data suggest that these mutant astroglia significantly upregulate pathways that support neural functions when compared to isogenic wild-type astroglia. Our findings establish a novel astroglia-enriched organoid model,offering a valuable platform for probing the roles of human astroglia in brain development and related disorders.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-024-00219-5. View Publication

Understanding how embryonic progenitors decode extrinsic signals and transform into lineage-specific regulatory networks to drive cell fate specification is a fundamental,yet challenging question. Here,we develop a new model of surface epithelium (SE) differentiation induced by human embryonic stem cells (hESCs) using retinoic acid (RA),and identify BMP4 as an essential downstream signal in this process. We show that the retinoid X receptors,RXRA and RXRB,orchestrate SE commitment by shaping lineage-specific epigenetic and transcriptomic landscapes. Moreover,we find that TCF7,as a RA effector,regulates the transition from pluripotency to SE initiation by directly silencing pluripotency genes and activating SE genes. MSX2,a downstream activator of TCF7,primes the SE chromatin accessibility landscape and activates SE genes. Our work reveals the regulatory hierarchy between key morphogens RA and BMP4 in SE development,and demonstrates how the TCF7-MSX2 axis governs SE fate,providing novel insights into RA-mediated regulatory principles.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05525-4. View Publication

Neural stem cells have intact innate immune responses that protect them from virus infection and cell death. Yet,viruses can antagonize such responses to establish neuropathogenesis. Using a forebrain organoid model system at two developmental time points,we identified that neural stem cells,in particular radial glia,are basally primed to respond to virus infection by upregulating several antiviral interferon-stimulated genes. Infection of these organoids with a neuropathogenic Enterovirus-D68 strain,demonstrated the ability of this virus to impede immune activation by blocking interferon responses. Together,our data highlight immune gene signatures present in different types of neural stem cells and differential viral capacity to block neural-specific immune induction.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03275-5. View Publication

CSMD1 (Cub and Sushi Multiple Domains 1) is a well-recognized regulator of the complement cascade,an important component of the innate immune response. CSMD1 is highly expressed in the central nervous system (CNS) where emergent functions of the complement pathway modulate neural development and synaptic activity. While a genetic risk factor for neuropsychiatric disorders,the role of CSMD1 in neurodevelopmental disorders is unclear. Through international variant sharing,we identified inherited biallelic CSMD1 variants in eight individuals from six families of diverse ancestry who present with global developmental delay,intellectual disability,microcephaly,and polymicrogyria. We modeled CSMD1 loss-of-function (LOF) pathogenesis in early-stage forebrain organoids differentiated from CSMD1 knockout human embryonic stem cells (hESCs). We show that CSMD1 is necessary for neuroepithelial cytoarchitecture and synchronous differentiation. In summary,we identified a critical role for CSMD1 in brain development and biallelic CSMD1 variants as the molecular basis of a previously undefined neurodevelopmental disorder. View Publication

SummaryIn Drosophila,long noncoding RNA Hsr? rapidly assembles membraneless organelle omega speckles under heat shock with unknown biological function. Here,we identified the distribution of omega speckles in multiple tissues of adult Drosophila melanogaster and found that they were selectively distributed in differentiated enterocytes but not in the intestinal stem cells of the midgut. We mimicked the high expression level of Hsr? via overexpression or intense heat shock and demonstrated that the assembly of omega speckles nucleates TBPH for the induction of ISC differentiation. Additionally,we found that heat shock stress promoted cell differentiation,which is conserved in mammalian cells through paraspeckles,resulting in large puncta of TDP-43 (a homolog of TBPH) with less mobility and the differentiation of human induced pluripotent stem cells. Overall,our findings confirm the role of Hsr? and omega speckles in the development of intestinal cells and provide new prospects for the establishment of stem cell differentiation strategies. Graphical abstract Highlights•LncRNA Hsr? is differentially expressed in different cell types of fly midguts•Omega speckles nucleate TPBH and promote the differentiation of ISCs to ECs•Heat shock treatment induces the assembly of omega speckles and paraspeckles•Heat shock treatment accelerates the differentiation of fly midguts and human iPSCs Molecular biology; Cell biology; Developmental biology View Publication