Biology subjects

Duan, J.

Publications and source records attributed to Duan, J..

At least 19 recordsLinked to original sources

bioRxiv2026

RnR-ExM: Robust Non-rigid Registration Challenge for Expansion Microscopy Volumes

Expansion Microscopy (ExM) enables nanoscale imaging of biological samples while preserving structural and molecular details. The Robust Non-rigid Registration Challenge for ExM (RnR-ExM) was launched to benchmark 3D registration algorithms on real-world ExM datasets. The challenge released 24 pairs of high-resolution 3D volumes from zebrafish, mouse, and C.~elegans, deliberately selected for their difficulty with standard alignment methods. To evaluate performance, we provided curated segmentation masks of nuclei and blood vessels as landmarks for half of the dataset. To encourage learning-based approaches, we released code for generating synthetic training data reflecting the deformation and appearance of the unlabeled pairs. By submission, 168 participants registered, with six teams submitting results by the deadline; the top three were invited to present at ISBI 2023. Accurate registration of ExM volumes is essential for downstream biological analyses such as multiplexed molecular mapping and cell-type-specific structural profiling. In this paper, we detail the methods and results of these top teams, highlighting current capabilities and future directions for 3D registration in ExM. All datasets and code are available at https://rnr-exm.grand-challenge.org/

neuroscience↗

bioRxiv2026

Co-targeting KRAS and BCL-2 induces pyroptosis and antitumor tertiary lymphoid structures

Mutant KRAS drives lung tumorigenesis through both cell-intrinsic and cell-extrinsic mechanisms, yet mutation subtype-specific differences in metabolic regulation and immune evasion remain incompletely understood. Here we show that the KRAS G12D mutation, particularly in the KRAS/STK11 co-mutant (KL) context, is associated with poor prognosis and a suppressed immune microenvironment characterized by impaired antigen presentation. At the cell-intrinsic level, G12D exhibits weakened interaction with hexokinase 1 (HK1), reduced spare respiratory capacity, and diminished mitochondrial stress resistance compared with G12C. Based on this mitochondrial vulnerability, we screened a panel of clinically available agents that induce mitochondrial stress and identified that co-targeting KRAS and BCL-2 synergistically triggers GSDME-mediated pyroptosis predominantly in G12D tumors. This combination further induces functional tertiary lymphoid structures (TLS) in vivo. Immune cell depletion experiments further demonstrate that the antitumor efficacy is dramatically dependent on B cells. Moreover, this treatment strategy induced membrane attack complex (MAC) deposition in situ and elicited memory antitumor responses. Collectively, these findings establish that combined KRAS and BCL-2 inhibition elicits pyroptosis and TLS formation, leading to B-cell-dependent in situ and memory antitumor immunity, providing a mechanistic rationale for targeting mitochondrial-immune crosstalk in KRAS G12D-mutant lung cancer.

cancer biology↗

bioRxiv2026

A split-control criterion for few-shot adaptation in single-cell perturbation prediction

Few-shot adaptation can help or harm single-cell perturbation prediction, but conventional evaluation shares a control estimate between calibration responses and held-out targets. We show how methods carry control error into their predictions and derive its contribution to score inflation. Nested control-depth experiments support inverse-depth scaling of inner-product inflation; correlation inflation also depends on the control noise-to-signal ratio. Independent-control scoring changed the rank of 40 of 52 configuration-dataset entries. We introduce SafeDelta, a split-control criterion that uses calibration data to decide whether to adapt. Across six datasets, mean gated gain was non-negative under the original two-control evaluation. On a deep plate, fixed predictions and decisions retained positive mean gains under three additional independent control references. The same reproducibility principle identified a fibrosis programme that localises to scar in human liver. These results inform adaptation decisions and control-depth planning.

bioinformatics↗

bioRxiv2026

A foundation model for brain organoid network dynamics uncovers intrinsic computation and convergent disease phenotypes

Brain organoids develop self-organizing neural networks that can be used to model neurodevelopment and disease. Here we introduce PULSE, a foundation model developed using over 900 hours of high-density microelectrode array recordings of in vitro and ex vivo neural tissue. We show that this single model can address two open questions: how disease-associated variants alter network development, and how naive circuits encode information. PULSE revealed accelerated network maturation in held-out human 16p11.2-deletion organoids. It also revealed a change in network coordination shared with SHANK3 deficiency. Because organoids develop without sensory input, they let us ask how circuits encode information before any representation is learned. Closed-loop control of a virtual inverted pendulum engaged existing spontaneous activity patterns rather than creating new ones. PULSE provides a common reference frame for neural circuits across genotypes, protocols and tasks.

neuroscience↗

bioRxiv2026

Impacts of different SETD1A mutations and genetic backgrounds on human induced pluripotent stem cell-derived hippocampal neurons

Abstract Human induced pluripotent stem cells (hiPSCs) provide a translational platform to interrogate the impact of genetic risk factors for various disorders on human biology. It has been a common practice to engineer premature stop codons to model predicted loss-of-function mutations using isogenic hiPSCs, yet effects of mutation position for the same gene and genetic background have been rarely tested systematically and at multiple levels for comparison to those of patient-specific mutations. Ultra-rare, heterozygous, and predicted loss-of-function mutations in the SETD1A gene, which encodes a H3K4 methyltransferase, are highly penetrant genetic risk factors for schizophrenia. Here, we generated a large panel of isogenic heterozygous SETD1A mutant hiPSC lines, including premature stop-codon mutations in one early exon and one late exon in five genetic backgrounds and two patient-specific mutations, to gain insight into molecular, cellular, and functional changes in a disease-relevant and highly pure population of human hippocampal dentate granule cell-like neurons. Our multi-level analyses showed that different SETD1A mutations lead to divergent molecular and cellular phenotypes related to synapses, but convergent neuronal network deficits, while the genetic background influences gene expression with no substantial effects at cellular and network levels. Mechanistic analysis confirmed the early exon patient mutation as loss-of-function, but revealed that the predicted loss-of-function patient mutation in the late exon is likely gain-of-function. Consequently, while all mutations decreased global H3K4me3 levels, deficits were only partially rescued by inhibiting H3K4 demethylase LSD1 for early exon mutations, but not for late exon mutations. Together, these results suggest that highly penetrant mutations can lead to similar cellular and functional phenotypes across different genetic backgrounds, whereas different premature stop-codon mutations could have contrasting impacts due to distinct mechanisms. Our findings have practical implications for applying genome-editing in hiPSCs to model disorders, highlight the value of modeling patient-specific mutations and testing multiple mutations, and suggest the need for tailored mutation-dependent therapeutic strategies.

neuroscience↗

bioRxiv2026

Transposable Elements Profiling Reveals DUXA-associated MLT1D Endogenous Retroviral Elements Activation During Bovine Maternal to Zygotic Transition

Transposable elements (TEs) are a major source of genomic diversity in mammals, yet their regulatory roles in the bovine genome remain poorly understood. Through characterizing bovine TE landscape, despite the substantial proportion (25.6%) of ruminant-specific TEs, we observe age- and class-dependent genomic distribution patterns similar to those observed in other mammals. Next, we profile TE and gene expression dynamics in pre-implantation embryos generated in vivo (IVV), by in vitro fertilization (IVT), and through somatic cell nuclear transfer (SCNT). The zygotic genome activation (ZGA) is shifted from the 4-cell stage to the 8-cell stage in IVT and SCNT embryos compared to IVV embryos. SCNT embryos exhibit impaired initiation of early transcription programs at the 4-cell stage and disrupted developmental trajectories, including abnormal activation of pluripotency-associated genes. A subset of retroviral LTR elements are strongly activated at ZGA in IVV and IVT embryos, whereas their activation is markedly muted in SCNT embryos, suggesting that impaired gene and TE reprogramming may contribute to the developmental defects commonly observed in SCNT embryos. By epigenomic profiling, the MLT1D elements from the ERVL-MaLR LTR family lose repressive marks and gain H3K27ac at ZGA, together with DUXA-binding motif enrichment. Knockdown of DUXA in bovine embryos significantly reduced MLT1D expression and ZGA marker genes. We propose that a subset of DUXA-enriched MLT1D functions as enhancers that promote ZGA. Overall, our study provides new insights into the regulatory roles of TEs during bovine embryogenesis and establishes a framework for comparative studies of TE-mediated gene regulation in early mammalian development.

developmental biology↗

bioRxiv2026

Systemic tumor-cell reprogramming converts solid tumors into in situ biofactories for immunotherapy

A central challenge in oncology is delivering potent drugs to tumors without harming healthy tissues. We therefore asked whether tumor cells themselves could be genetically programmed to produce therapeutic molecules directly within the tumor. We present LENTRA (LENtiviral Tumor-Reprogramming Activator), an antigen-targeted lentiviral platform that enables selective transduction and genetic modification of tumor cells in vivo. Following intravenous administration, LENTRA achieves tumor restricted expression of immune checkpoint inhibitors, cytokines, or therapeutic enzymes in multiple murine solid tumor models. Local production of anti CTLA 4 or IL 12 triggered potent antitumor responses while avoiding systemic toxicities. Tumor restricted secretion of sialidase enhanced dendritic cell and T cell infiltration, and reprogrammed tumor associated macrophages toward an immunostimulatory phenotype, resulting in tumor regression and durable immune memory, including in tumors with heterogeneous antigen expression. In patient derived tumor explants, LENTRA mediated efficient transduction, immune remodeling, and reduced tumor viability. By engineering tumors to produce therapeutic proteins, LENTRA converts resistant tumors into autonomous therapeutic sources and active immune hubs, achieving local TME reprogramming and systemic antitumor immunity with an improved safety profile.

cancer biology↗

bioRxiv2026

Identification of alcohol use disorder-associated and ethanol-responsive proteins and candidate biomarkers using human cortical organoids

Genome-wide association studies of alcohol use disorder (AUD) have identified >100 loci, yet the functional impact and the downstream molecular mechanisms are not well understood. As functional effectors of genes, proteins provide direct insight into disease mechanisms. However, most proteomic alterations in postmortem human brains of AUD likely reflect a combination of inherited susceptibility (AUD-associated), the consequences of alcohol exposure (ethanol-responsive), other environmental and lifestyle factors, as well as sample preparation. Here, we profiled the proteome of human microglia-containing cortical organoids derived from human induced pluripotent stem cells from individuals with AUD (n=11) and without (n=5), paired with or without intermittent ethanol exposure. To maximize statistical power, donors were selected based on high or low genetic liability for AUD as measured by polygenic scores. Among 8,952 proteins that passed quality control, 1,038 were AUD-associated and 718 were ethanol-responsive. AUD-associated proteins were enriched for neuronal signaling, immune, mitochondrial, and extracellular matrix pathways, whereas ethanol-responsive proteins were enriched for RNA processing, ribosome biogenesis, protein translation, vesicle trafficking, and membrane transport pathways. Twenty ethanol-responsive proteins were replicated in a postmortem human prefrontal cortex proteomic dataset. Using UK Biobank plasma proteomics data, we identified 26 candidate AUD-associated biomarkers and 9 candidate ethanol-responsive biomarkers. We also found that major proteomic variation was significantly correlated with AUD polygenic scores. Together, these findings reveal distinct proteomic signatures of inherited susceptibility to AUD and ethanol exposure, providing new insights into mechanisms underlying AUD and identifying candidate circulating biomarkers for further investigation.

cell biology↗

bioRxiv2026

A histidine-mediated, pendulum-like proton transport mechanism is required for the high catalytic activity of -hydrogenases

[FeFe]-hydrogenases are molecular hydrogen (H2) converting enzymes that employ a hexanuclear iron-complex, the H-cluster, as catalytic cofactor, and a proton transfer pathway (PTP) that allows efficient proton coupled electron transfer (PCET). Recent phylogenetic analyses revealed different groups of [FeFe]-hydrogenases. Although only very few members from other groups have been characterized, current knowledge suggests high catalytic activity is predominantly associated with group A members. Here, we show that metal ions inhibit group A [FeFe]-hydrogenases by binding to three specific residues at the entrance of the PTP. Exchanging residue H565 of Clostridium pasteurianum CpI results in a metal-insensitive protein variant with wildtype like activity. In contrast, exchanging S320 and H569 in CpI, and their counterparts in additional group A [FeFe]-hydrogenases results in enzymes with strongly decreased activities and large overpotential requirements. These features are consistent with important roles of both residues in catalytic proton transfer. CpI structures reveal that H569, locally anchored by E278, can be present in two conformations so that we propose a histidine-dependent pendulum mechanism for exchanging protons between bulk solvent and the PTP, which is well supported by theoretical calculations. By that, our study widens information on how the mobility of histidine, governed by residues in the vicinity, contributes to the fundamental concept of PCET.

biochemistry↗

bioRxiv2026

Transthyretin Contributes to Hippocampal Blood-Brain Barrier Recovery Following Intestinal Inflammation, with Reduced Expression in Alzheimer's Disease

Background: The blood-brain barrier (BBB) is increasingly recognized as an active immunoregulatory interface that responds dynamically to systemic inflammation. As intestinal inflammation can influence brain homeostasis through the gut-brain axis, the endogenous mechanisms that preserve BBB integrity during gut-derived inflammatory stress remain poorly understood. Methods and Results: We combined dextran sulfate sodium-induced colitis, fecal microbiota transplantation, aged mice, APP/PS1 mice, human Alzheimer's disease (AD) brain tissue, single-cell RNA sequencing, in vivo BBB permeability assays, and gain- and loss-of-function approaches to investigate adaptive neurovascular responses to gut inflammation and their mechanism in maintaining BBB integrity. Intestinal inflammation induced a region-specific adaptive response characterized by increased hippocampal vascular remodeling and partial restoration of BBB integrity following the initial inflammatory insult. Single-cell transcriptomic analysis identified a transthyretin (TTR)-enriched vascular-associated microglial state accompanying these neurovascular changes. Functional studies demonstrated that TTR contributes to maintaining BBB integrity by promoting endothelial homeostasis and limiting endothelial endocytosis, consistent with modulation of FcRn-associated transport pathways. This adaptive neurovascular response was progressively attenuated in aged mice and APP/PS1 mice and was accompanied by reduced vascular TTR expression in human AD brains. Conclusion: These findings identify TTR as a contributor of adaptive neurovascular homeostasis during gut-derived neuroinflammation. Impairment of this homeostatic response with aging and AD may contribute to persistent BBB dysfunction and chronic neuroinflammation, highlighting neurovascular resilience as a potential therapeutic target.

neuroscience↗

bioRxiv2026

MOLECULAR BASIS FOR PINK1 MATURATION

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), a key regulator of mitophagy, has been linked to the pathogenesis of Parkinson's disease (PD). PINK1 recruits Parkin, an E3 ubiquitin ligase, triggering mitophagy in response to mitochondrial damage. During mitophagy, the quantity, stability, and activity of PINK1 must be strictly regulated; however, the mechanisms governing these parameters under cellular stress are still unclear. Herein, we determined the structural basis for PINK1 maturation mediated by heat shock protein 90/cell division cycle 37/FK506-binding protein 51 (HSP90/CDC37/FKBP51) chaperone complex. We identified PINK1-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and determined the structures of the complexes using Cryo-Electron Microscopy (Cryo-EM). Results showed that FKBP51 potentially interacts with a conserved leucine-proline-phenylalanine (LPF) motif on the activation loop of PINK1 and negatively regulates PINK1 functions in mitophagy. A PINK1 mutation located at the FKBP51 recognition site is linked to mitophagy deficiency, which can be partially rescued by specific inhibition of FKBP51. These findings reveal a general mechanism for PINK1 recognition by the HSP90/CDC37/FKBP51 chaperone complex and suggest a potential approach for upregulating PINK1 activity, which is impaired in PD.

biophysics↗

bioRxiv2026

Direct Binding of Cysteine-367 Thiolate to the Active Site of the -Hydrogenase from Clostridium beijerinckii in the O2-stable State

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

biochemistry↗

bioRxiv2026

CMAPS: Causal Mediation Analysis of Perturbation Screens with Application to Genome-scale Perturb-seq Data

CRISPR-Cas9 perturbation screens coupled with single-cell multi-omic profiling enable dissection of gene regulatory mechanisms, yet existing analyses largely quantify total perturbation effects and offer limited insight into the molecular intermediates that transmit these effects. We introduce CMAPS (Causal Mediation Analysis for Perturbation Screens), a semiparametric framework for robust mediation analysis that accommodates unmeasured mediator-outcome confounding and incorporates an adaptive bootstrap test with false discovery rate control. Simulations and data-driven computational experiments show that CMAPS yields accurate, calibrated mediation estimates and robust mediator identification, as confirmed through negative controls and permutation-based validation. Applied to K562 Perturb-seq, CMAPS recapitulates transcriptional cascades downstream of GATA1. In BT16 MultiPerturb-seq data, CMAPS identifies promoter-centric, enhancer-distributed, and mixed cis-regulatory programs linking chromatin remodeling factors to transcriptional responses. CMAPS provides a rigorous and interpretable framework for mechanistic inference in single-cell perturbation screens. CMAPS is implemented in R and is available at https://github.com/keleslab/CMAPS.

genomics↗

bioRxiv2026

Combinatorial transcription factor interactions drive modular gene regulatory networks

Transcription factors (TFs) cooperatively drive gene regulatory networks (GRNs) to establish transcriptional states. Forced induction of TFs in combination can reprogram cell state by supplanting existing GRNs. Thus, TFs and GRNs are the building blocks to engineering transcriptional state. However, one key challenge is that the relationship between TF combinations and GRNs remains largely uncharacterized and difficult to accurately predict. Here, we apply single-cell overexpression screens to map the combinatorial activities of [~]100 TFs to gene expression states. Our analysis identifies diverse TF combinations driving cell-type specific regulatory programs. Notably, different TF combinations induce shared gene sets with cell-type specific functions, suggesting a modular regulatory architecture of the transcriptome. Furthermore, we define pairwise TF interactions and show that cooperative interactions improve transcriptional reprogramming. Finally, we developed tools to predict combinatorial TF phenotypes. These findings improve our understanding of cell state and how to manipulate it for biomedical applications. HIGHLIGHTSO_LICombinatorial over-expression screens for [~]100 transcription factors (TFs). C_LIO_LIDiverse TF combinations drive cell-type specific regulatory programs. C_LIO_LITF regulatory networks reveal a modular regulatory architecture of the transcriptome. C_LIO_LITF-TF interactions and predictive models enhance reprogramming cocktails. C_LI

genomics↗

bioRxiv2026

Hippocampal Glucuronyl C5-epimerase promotes stress resilience by directly engaging PI3K through a non-enzymatic mechanism

Major depressive disorder (MDD) seriously affects human physical and mental health and causes global socioeconomic burdens. Stress-induced depressive etiology is linked to dendritic atrophy in the hippocampus, however, the underlying mechanism was poorly understood. Here, we identify that glucuronyl C5-epimerase (Glce), a heparan sulfae-modifying enzyme, as a critical regulator of hippocampal dendritic integrity and stress resilience. Genetic ablation of Glce in the hippocampal excitatory neurons is sufficient to induce dendritic atrophy and depressive-like behaviors, whereas its restoration rescues these deficits. Notably, Glce levels are reduced in the plasma of depressed individuals. We further find that Glce functions independently of its enzymatic activity, instead binding directly to and activating PI3K (p85/p110), thereby triggering an Akt/CREB/BDNF signaling cascade. Together, our findings uncover a non-canonical role for Glce and establish that Glce-PI3K-BDNF axis is essential for maintaining hippocampal structure and behavioral resilience, thereby highlighting a critical role of Glce in the pathophysiology of depression.

neuroscience↗

bioRxiv2026

Windthrow-generated tip-up mounds create contrasting regeneration niches for red oak and black cherry in a deer-browsed Carolinian forest

Windstorms are a dominant recurrent natural disturbance type within Carolinian old-growth forests in southwestern Ontario. This study examined how windthrow generated microsites differ from background microsites in soil properties and seedling survival across 84 locations in Hamilton. For each site, soil samples from windthrow generated mound tops and adjacent non-disturbed forest floor were analyzed. Seedlings of red oak (Quercus rubra) and black cherry (Prunus serotina) were planted on mound tops and adjacent ground microsites, and their survival was monitored. Non-disturbed ground microsites had significantly higher soil moisture (p < 0.001) and organic matter (p < 0.001) than disturbed sites, whereas disturbed sites were consistently drier (p < 0.001). Red oak survival was 28% higher on the non-disturbed microsites relative to the disturbed microsites, while black cherry survival was 41% higher on disturbed microsites relative to the non-disturbed ground microsite. Logistic regression analyses reveal that soil moisture was the strongest predictor of seedling survival (p = 0.030), with contrasting responses between the two species (p = 0.019). These results suggest that windthrow generated mounds create distinct environmental conditions that selectively favor unique autecology. As windthrow frequency rises under climate change, mounds could become major mediators of forest canopy composition.

ecology↗

bioRxiv2026

Female iPSC X-chromosome inactivation (XCI) erosion and its transcriptomic effects during CRISPR gene editing and neural differentiation

Human induced pluripotent stem cells (hiPSC) and iPSC-differentiated neural cells, in combination with CRISPR editing, are commonly used for studying neurodevelopmental and other brain disorders. Female iPSCs undergo random X-chromosome inactivation (XCI) via epigenetic silencing by noncoding X inactive specific transcript (XIST). It is known that female iPSCs may lose XIST expression, leading to XCI erosion that affects both X-linked and autosomal gene expression. However, the effects of CRSIPR editing and neural differentiation on XCI erosion in iPSC-derived neurons and how this may confound a real-world transcriptomic analysis of differentially expressed genes (DEGs) are poorly understood. Here, leveraging bulk RNA-seq of hundreds of CRISPR-edited female iPSC lines from four donor lines for 66 genes and single-cell RNA-seq of iPSC-derived neurons of a subset of 42 edited genes, we investigated the effects of XCI erosion during CRISPR editing and in iPSC-derived neurons. We found that XCI erosion was variable in CRISPR-edited female iPSCs and largely preserved in iPSC-derived neurons. Like in iPSCs, XIST in neurons predominately influenced the expression of X-linked genes; however, its effect on autosomal genes was more pronounced in single neurons. Mechanistically, XIST epigenetically causes allelic imbalance of both X-linked and autosomal genes, with the former showing stronger allele-specific expression (ASE) bias. Notably, XIST-induced ASE bias exhibited a conserved positional pattern at loci affecting neurodevelopmental genes across different female lines and cell types. Finally, we demonstrated a confounding effect of XCI erosion on DEG analyses in iPSC-derived neurons. These results have significant implications in hiPSC modeling of neurodevelopmental and other brain disorders.

genomics↗

bioRxiv2026

ASCL1 and OLIG2 Expression Dynamics Control Glial Cell Fate and Regional Diversity in the Dorsal Forebrain

Gliogenesis is a multistep process that begins with the specification of glial progenitors (GPs) into migrating and proliferating precursor cells, which later differentiate into mature astrocytes and oligodendrocytes. How these developmental processes are coordinated to generate the diverse glial lineages in gray matter (GM) and white matter (WM) in the brain remains poorly understood. Here, we show that the basic-helix-loop-helix (bHLH) transcription factor ASCL1 serves as a direct mechanistic link between glial cell fate specification, migration, proliferation, and differentiation in the dorsal forebrain. Notably, ASCL1 is dynamically expressed in GPs, initiating in the ventricular zone (VZ), peaking in the intermediate zone (IZ), but is downregulated once GPs enter the cortical plate. Lineage tracing of ASCL1+ GPs demonstrates that they subsequently co-express OLIG2 to generate both astrocytes and oligodendrocytes in an "outside-in" pattern starting from the upper cortex inward to the corpus callosum, the opposite pattern of neurogenesis. Gain- and loss-of-function experiments further reveal that a sustained ASCL1 expression is essential for inducing sufficient levels of OLIG2 required to specify oligodendrocyte precursor cell (OPC) fate. Interestingly, a persistent ASCL1 expression also maintains OPCs into postnatal stages by promoting their self-renewal while suppressing their differentiation into postmitotic oligodendrocytes. Together, these findings establish ASCL1 as a key regulator of the spatiotemporal order of glial lineage diversity in cortical GM and callosal WM and implicate ASCL1 dysregulation as an underlying mechanism in the pathogenesis of gliomas.

neuroscience↗