Biology subjects

Wong, A.

Publications and source records attributed to Wong, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Phosphoregulation of hierarchical protein condensate formation during clathrin-mediated endocytosis

The sequential assembly of over 50 proteins during clathrin-mediated endocytosis (CME) is well documented in yeast. However, the mechanisms governing this ordered recruitment remain poorly understood. Biomolecular condensation is proposed to orchestrate CME, but defining biophysical parameters in vivo remains challenging. Here, we developed a quantitative in vivo titration strategy to systematically determine CME protein assembly profiles. Beyond the previously reported Ede1 (yeast Eps15), seven additional proteins formed enlarged, concentration-dependent assemblies with distinct biophysical properties. Single-cell profiling revealed two biophysical classes among these eight proteins: homeostatic buffers with defined saturation boundaries and continuous assemblers that scale linearly. Acting as scaffolds and clients, these conserved proteins form an interconnected network of kinases, phosphatase recruiters, and substrates. Within this network, reversible phosphorylation modulates assembly stability to drive recruitment transitions. Collectively, this work establishes a two-stage model of protein condensation that drives CME progression from initiation to maturation and offers a general framework for how protein phosphorylation dynamically modulates biomolecular condensation in vivo.

cell biology↗

bioRxiv2026

Salinity is associated with habitat use and mitochondrial lineage diversity in halophilic Ephydra flies (Diptera: Ephydridae)

Brine flies of the genus Ephydra (Diptera: Ephydridae) are ubiquitous extremophile animals whose larval forms are able to thrive in high-stress environments, including hypersaline and high carbonate waters. Despite their historical and ecological importance, little is known about their evolutionary history. Here, we focus on an understudied population of Ephydra living in former industrial salt evaporation ponds of the San Francisco Bay Area (SFBA), a region which has undergone significant environmental changes in the past century. We observed and collected Ephydra specimens and took salinity measurements of waters they inhabited during a 6-month period in 2024. We found that E. millbrae and E. gracilis demonstrate strong salinity preferences. We also analyzed DNA from Ephydra spp. specimens from distant sites and a timespan of ~40 years, generating the most comprehensive gene genealogy of the group to date based on a single mitochondrial locus. Our analysis of phylogenetic signals suggests that salinity envelopes are influenced by evolutionary relationships in Ephydra. This study provides new information about the natural history of extremophile flies in the SFBA and fills in current gaps in our knowledge about the evolutionary history of the genus more broadly.

evolutionary biology↗

bioRxiv2026

Identification and characterization of inflammatory LILR and fibrotic SPP1 macrophages in chronic lung allograft dysfunction

Chronic lung allograft dysfunction (CLAD) is a major cause of death after lung transplantation and manifests principally as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Although pulmonary macrophages are key regulators of lung injury and repair, their contributions to CLAD pathogenesis remain under-examined. We performed single-cell RNA sequencing-based transcriptomic analysis of CLAD (7 BOS, 7 RAS) and control lung tissue (n = 6), complemented by comparative and functional analyses. Two distinct macrophage populations were identified in CLAD: inflammatory macrophages expressing leukocyte immunoglobulin-like receptors (LILR), and fibrotic macrophages characterized by expression of osteopontin (SPP1). LILR macrophages were present in both BOS and RAS, whereas SPP1 macrophages were selectively enriched in RAS. Both populations were identified in idiopathic pulmonary fibrosis and cross-tissue comparisons. Surface marker-based sorting strategies were developed to isolate both populations. Functional studies demonstrated that LILR macrophages exhibited enhanced phagocytic activity, promoted T cell chemotaxis and secreted inflammatory mediators, whereas SPP1 macrophages produced soluble factors that drove fibroblast activation and contraction. These findings identify distinct inflammatory and fibrotic macrophage programs associated with CLAD, link RAS to conserved fibrotic macrophage states observed in pulmonary fibrosis and highlight macrophage populations as potential therapeutic targets for inflammatory and fibrotic allograft injury.

immunology↗

bioRxiv2026

Scalable spatial DNA sequencing from archival tissue maps copy number subclones

Spatially resolved DNA sequencing holds promise due to its potential utility in understanding cancer intra-tumour heterogeneity and tumour evolution in relation to tissue architecture. However, it has so far been used to a limited extent due to technical challenges and high cost of existing methods. Hence we aimed to develop a high throughput spatial genomic assay to obtain copy number alteration (CNA) information at user-defined spatial resolution. We derived CNA profiles from ultra-low coverage whole genome sequencing at sub-millimetre resolution from archival samples using a novel method called Adaptive Resolution Multiscale Spatial DNA sequencing (ARMS DNAseq). We used it to profile CNAs from more than 766 regions (tiles) from 3 patients, covering a total area of over 300 mm2, with 1.2-2.6 million mapped reads per tile and tile sizes of 0.1-0.99mm2. Using ARMS DNAseq, we delineate tumour evolution in a spatial context, and identify more tumour subclones that were obscured or incompletely represented in bulk multi-region whole genome sequencing. Next, we show associations between tumour subclones and morphology, and prediction of subclone identity from deep learning-derived image representations. Finally, we demonstrate multi-omic integration by alignment with spatial transcriptomic data, showing subclone-specific immune cell co-occurrence as well as transcriptional programmes cutting across subclone boundaries. ARMS DNAseq converts low-throughput, region-by-region profiling into a scalable and adaptable workflow for direct spatial copy number profiling from archival tissue sections.

genomics↗

bioRxiv2026

NC4touch: An open-source networked touchscreen apparatus for rodent behavioral testing

In recent years, rodent touchscreen-based paradigms have gained popularity for their flexible task design, automated data collection and improved standardization. These devices minimize experimenter intervention and contribute to more replicable and reliable research. However, the high costs and proprietary hardware / software associated with commercially available systems present a financial barrier for smaller labs or researchers with limited resources. To address this issue, we developed "NC4Touch", an open-source scalable, modular rodent testing apparatus. It features three independent touchscreens and a feeding port for easy use in a wide array of tests comparable to those available with standard operant chambers, with the added benefit that visual stimuli can be highly customized. The system includes a user-friendly graphical interface that offers real-time control, task customization, video recording, and data management. We demonstrate its effectiveness in a visual discrimination task using both rats and mice. Several devices can be operated in parallel from the same computer and user interface, allowing high- throughput data collection. We provide detailed assembly instructions for the hardware and well- documented and easily-configurable software. We hope that the affordable open-source nature of NC4Touch will expand the scope of behavioral testing, allowing researchers to overcome traditional financial barriers and create a more collaborative community. Significance StatementTouchscreen-based testing is a powerful tool for assessing cognition in rodent models, however commercial systems remain cost-prohibitive for many researchers. NC4Touch is a rodent touchscreen apparatus that provides an open-source, customizable, and affordable alternative that enables high-throughput, cognitive testing in both mice and rats. By lowering financial barriers and promoting hackability, this system enables greater accessibility and collaboration in research.

animal behavior and cognition↗

bioRxiv2026

Mitochondrial-targeted therapy with elamipretide preserves cardiac function and prevents late mortality in murine sepsis-induced cardiac dysfunction.

Sepsis-induced cardiac dysfunction (SICD) occurs in nearly half of septic patients, is associated with increased mortality, and lacks targeted therapy. Emerging evidence implicates impaired mitochondrial function and metabolic inflexibility as central contributors to myocardial depression. Here, we characterized SICD in a murine model of polymicrobial sepsis and evaluated the therapeutic potential of the cardiolipin-stabilizing peptide elamipretide (Ela). Sepsis induced marked impairments in cardiac performance, accompanied by reductions in cardiac cardiolipin content, impaired mitochondrial respiratory capacity localized to complex I, and altered substrate utilization. Integration of stable isotope metabolic flux tracing with lipidomic, metabolomic, and proteomic analyses identified a convergent metabolic bottleneck at the level of the electron transport system. This defect was associated with upstream accumulation of acetyl-CoA, Co-A esters, and ketone bodies, consistent with impaired oxidative flux and energetic failure. Administration of a single early dose of Ela restored cardiolipin content, complex I function, normalized metabolic flux, improved cardiac function during both acute sepsis and recovery, and completely prevented late sepsis-related mortality. These findings identify cardiolipin-dependent mitochondrial dysfunction as a central pathogenic mechanism underlying SICD and position mitochondrial-targeted therapy as a promising therapeutic strategy in sepsis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/736409v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1ca5b47org.highwire.dtl.DTLVardef@2ecfc2org.highwire.dtl.DTLVardef@149ccb9org.highwire.dtl.DTLVardef@1fbcb6_HPS_FORMAT_FIGEXP M_FIG C_FIG Ela improves SICD by stabilizing cardiolipin species and improving mitochondrial complex I function. SICD depicted in red denotes conditions altered compared to healthy control cardiomyocyte, SICD+Ela depicted in green denotes changes relative to SICD. SICD, sepsis-induced cardiac dysfunction; ELA, elamipretide; ADP, adenosine diphosphate; ATP, adenosine triphosphate; ROS, reactive oxygen species.

physiology↗

bioRxiv2026

A Dual-Locus-Targeting Strategy to Enhance CRISPR/Cas9-mediated CFTR Replacement via Helper-Dependent Adenoviral vector in porcine genome

Gene therapy has been the subject of extensive research following the advent of gene-editing technologies. Genetic disorders with difficult-to-target tissues, such as cystic fibrosis (CF), still face many challenges in developing efficacious gene therapy. The potential universal approach of gene replacement involves inserting a functional CFTR gene after generating DNA double strand breaks using gene editors such as CRISPR/Cas9. However, this strategy has not achieved clinical significance, as CRISPR/Cas9-mediated integration of CFTR is limited primarily by the infrequent activity of the homology-directed repair (HDR) pathway. To circumvent this limitation and improve CFTR transgene integration and expression, we explored a method of adding a second integration site, which we termed the dual-locus-targeting method. Using a helper-dependent adenoviral vector (HDAd)-delivered CRISPR/Cas9 system in porcine epithelial cells, we found that sequential delivery of two vectors, one targeting the CFTR locus and the other the genomic safe harbour site GGTA1, enhanced the integration efficiency of lacZ and CFTR donor genes to 16.5% and 3.4%, respectively. These results demonstrated a potential strategy to improve the efficacy of CFTR replacement for the development of a universal and permanent gene therapy treatment for CF lung disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/731381v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@14c8583org.highwire.dtl.DTLVardef@d1862eorg.highwire.dtl.DTLVardef@1c0594eorg.highwire.dtl.DTLVardef@1271c10_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2026

Comparative profiling of carnitine palmitoyltransferase 1 isoforms reveals vincamine as a selective carnitine palmitoyltransferase 1b inhibitor

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the rate-limiting step of fatty acid oxidation and has emerged as a therapeutic target for metabolic diseases and cancer. CPT1 exists in three isoforms, CPT1a, CPT1b, and CPT1c, with distinct tissue distributions and enzymatic properties; however, limitations of previous platforms enabling parallel isoform comparison have undermined efforts to identify selective inhibitors that could minimize off-target effects. Here, we describe a DTNB-based enzyme activity assay adapted for high-throughput screening of CPT1b, the predominant isoform in cardiac and skeletal muscle. Mitochondrial extracts from Expi293F cells transfected with CPT1a or CPT1b expression plasmids served as sources of catalytically active enzymes. The assay was validated using three previously confirmed CPT1b inhibitors: (R)-(+)-etomoxir, perhexiline, and malonyl-CoA. We then generated side-by-side inhibitory profiles for both isoforms, identifying vincamine as a lead selective inhibitor of CPT1b. Furthermore, chlorpromazine, previously characterized only as a broad CPT1 inhibitor and subsequently shown to inhibit CPT1a, is demonstrated here to also inhibit CPT1b, expanding its known isoform profile. Together, these results establish a robust platform for comparative isoform profiling and demonstrate that selective modulation of CPT1b is achievable, with implications for targeted therapeutics in metabolic and oncological disease.

cell biology↗

bioRxiv2026

Novel tool use does not depend on mechanical reasoning: evidence from apraxia

A hallmark of human intelligence is the ability to use tools. Yet the cognitive processes supporting this ability remain debated. One contemporary view holds that mechanical reasoning is central for tool use, especially in the case of tools with which we have no prior experience. However, previous support for the role of mechanical reasoning often relies on circular logic, wherein poor performance on novel tool-use tasks is taken as evidence that impaired mechanical reasoning causes tool-use deficits in limb apraxia. To address this limitation, we independently assessed mechanical reasoning and novel tool use in separate tasks in individuals with limb apraxia, and compared their performance to individuals without apraxia. We also examined whether these two abilities are similarly associated with other cognitive abilities including motor imagery, mental rotation of non-body objects, general reasoning, and spatial working memory. Finally, we explored brain-behavior relationships using support vector regression lesion-symptom mapping. Our behavioral and imaging data together showed that mechanical reasoning does not underlie novel tool-use deficits in apraxia. Graphical analysis further revealed that novel tool use and mechanical reasoning loaded onto distinct latent clusters: novel tool use was strongly associated with other praxis abilities yet separable from cognitive abilities that require reasoning and mental simulation, whereas mechanical reasoning was primarily linked to other high-level reasoning abilities but not tool use. These findings challenge the notion that mechanical reasoning is central to tool-use ability, and instead suggest that tool use is more likely to be an intuitive or automatic process.

neuroscience↗

bioRxiv2026

Maternal iron deficiency remodels cardiac mitochondria and alters stress responses in hypertensive pregnancy

Maternal iron deficiency (ID) during pregnancy is associated with cardiovascular adaptations, including reduced blood pressure and improved cardiac efficiency in hypertensive pregnancy. However, whether these apparent functional gains are accompanied by preserved cardiac mitochondrial function remains unclear. Given the high metabolic demands of the maternal heart and irons central role in oxidative metabolism, we examined how maternal ID affects cardiac mitochondrial ultrastructure, respiration, dynamics, and redox status in pregnant spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Female SHR and WKY rats were fed iron-replete or iron-restricted diets before and throughout gestation. On gestational day 21, cardiac mitochondrial ultrastructure was assessed by transmission electron microscopy, respiration by high-resolution respirometry, mitochondrial dynamics and quality control proteins by immunoblotting, and antioxidant gene expression by RT-qPCR. Iron restriction reduced maternal hemoglobin levels in both strains. ID dams exhibited enlarged, morphologically heterogeneous mitochondria with reduced cristae density and lower succinate-supported respiration. SHR dams exhibited reduced fusion signalling, reflected by a lower L-OPA1:S-OPA1 ratio, lower MFN2 abundance, and further ID-associated reductions in MFN1 and MFN2. In contrast, DRP1 phosphorylation increased in ID-WKY dams. Iron restriction increased LC3-II:I ratio and BNIP3 in SHR, increased PINK1 in both strains, and increased antioxidant gene expression in ID-SHR but decreased in ID-WKY dams. Despite these alterations, downstream apoptosis activation was not observed. Maternal ID was associated with remodelling of myocardial mitochondrial ultrastructure and selectively constrains iron-dependent respiration in hypertensive pregnancy, suggesting favourable hemodynamic adaptations may coexist with underlying bioenergetic constraints in the maternal heart.

physiology↗

bioRxiv2026

Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

A preeminent goal of virology is to discover cellular genes that mediate virus entry. Genome-wide loss-of-function screens can illuminate single genes necessary for virus entry, but are stymied by genetic redundancy. Here we report a genome-wide CRISPR activation screening strategy to discover single genes that are sufficient for viral entry into normally-uninfectable cells. Sequential rounds of viral infection vastly enhanced screening sensitivity. This sequential screening strategy was generalizable to two unrelated viruses--Ebola and rabies viruses--and could broadly accelerate the discovery of viral entry factors.

microbiology↗

bioRxiv2026

Modeling patient variants of Cnot1 and Cdc42bpb results in distinct forms of congenital diaphragmatic hernia in mice

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly characterized by impairment of both diaphragm and lung development in utero. CDH presents as a spectrum of forms and severities, with diaphragm defects arising in the dorsal/posterior region typically correlating with more severe pulmonary disease and higher risk of mortality than those appearing in ventral/anterior regions. The genetic etiology underlying CDH is complex, with many genes implicated showing variable expressivity and incomplete penetrance in both human patients and mouse models. Here we present in vivo validation of two genes previously unassociated with CDH: the CDC42-interacting kinase CDC42BPB; and CNOT1, a scaffolding protein of the CCR4-NOT protein complex, critical for mRNA regulation through modifications such as deadenylation. Each gene was found to have a damaging, de novo missense variant in a recent large-scale CDH patient sequencing screen. Loss of Cdc42bpb leads to ventral diaphragmatic hernias, heart septal defects and minor lung epithelial differentiation defects in mouse embryos. Installation of the orthologous patient-specific missense variant through CRISPR/Cas9 editing leads to less severe ventral diaphragm defects. Mouse embryos with either one or two copies of the orthologous Cnot1 variant, c.1867C>T (p.R623W), develop dorsal diaphragmatic hernias with low (<50%) penetrance, and mutants showed alterations in mRNA isoform expression consistent with the molecular role of Cnot1 in RNA splicing. These results underscore the power of in vivo functional modeling to validate genes and patient-specific variants uncovered by patient sequencing, reveal two previously unrecognized genetic causes of CDH, and highlight the heterogeneity of different patient anatomic presentations.

genetics↗

bioRxiv2026

Abscisic acid binds to an Arabidopsis thaliana phosphodiesterase and tunes its activity

Growing evidence suggests that plant proteomes contain numerous proteins that specifically bind abscisic acid (ABA). Many of them are complex multidomain proteins where specific ABA-binding can cause biochemical and physiological changes. Here we show that the Arabidopsis thaliana K+ transporter AtKUP5 contains both a functional cytoplasmic N-terminal adenylate cyclase (AC) enabling the synthesis of 3,5-cAMP from ATP and a C-terminal phosphodiesterase (PDE) that hydrolyses 3,5-cAMP to 5-AMP. We found that ABA binds in a ligand-specific manner to the catalytic center of the PDE thereby causing a reduction of 3,5-cAMP hydrolysis in vitro. The hydrolytic activity of the PDE is ABA concentration-dependent, biphasic and requires the presence of an intact ABA-binding site similar to the one in the canonical Pyrabactin resistance 1/PYR-like/Abscisic acid receptors, with Vmax of 1.19 pmole min{square}1 g{square}1 in the absence of ABA, increasing to 1.58 pmole min{square}1 g{square}1 at 2 nM ABA, and decreasing to 0.75 pmole min{square}1 g{square}1 at 50 nM ABA. These findings are therefore consistent with a direct role of ABA in PDE activity modulations and form a functional link between 3,5-cAMP signaling and K+ flux. Furthermore, we predict that a growing number of such receptor-like proteins that specifically and directly interact with ABA will be discovered thereby uncovering complex and ancient layers of signaling and metabolic regulation.

plant biology↗

bioRxiv2026

Using a GPT-5-driven autonomous lab to optimize the cost and titer of cell-free protein synthesis

We used an autonomous lab, comprising a large language model (LLM) and a fully automated cloud laboratory, to optimize the cost efficiency of cell-free protein synthesis (CFPS). By conducting iterative optimization, the LLM-driven autonomous lab was able to achieve a 40% reduction in the specific cost ($/g protein) of CFPS relative to the state of the art (SOTA). This cost reduction was accompanied by a 27% increase in protein production titer (g/L). Iterative experimental design, experiment execution, data capture and analysis, data interpretation, and new hypothesis generation were all handled by the LLM-driven autonomous lab. The interface between OpenAIs GPT-5 LLM and Ginkgo Bioworks cloud laboratory incorporated built-in validation checks via a Pydantic schema to ensure that AI-designed experiments were properly specified. Experimental designs were translated into programmatic specification of multi-instrument biological workflows by Ginkgos Catalyst software and executed on Ginkgos Reconfigurable Automation Cart (RAC) laboratory automation platform, with human intervention largely limited to reagent and consumables preparation, loading and unloading. By integrating LLMs with programmatic control of a cloud lab, we demonstrate that an LLM-driven autonomous lab can successfully perform a real-world scientific task, highlighting the potential of AI-driven autonomous labs for scientific advancement.

synthetic biology↗

bioRxiv2025

Combined antagonism of Oncostatin M (OSM) and Interleukin 6 (IL-6) provides both anti-fibrotic and anti-inflammatory benefit in pulmonary fibrosis

Interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated ILD (SSc-ILD), are irreversible fibrosing diseases with a mean survival time of less than 5 years for IPF. Poorly understood etiology and complex pathogenesis of these diseases have hampered the identification and development of effective therapeutics. Existing treatments can slow progressive fibrosis and lung function decline but do not stop it entirely, resulting in a minimal impact on patient survival. Thus, novel therapeutic interventions are needed. Tocilizumab, an anti-IL-6 receptor antibody, was recently approved by FDA for the treatment of SSc-ILD based on evidence demonstrating a reduction in the rate of lung function decline. In this study, we have characterized an IL-6-driven feed-forward myeloid axis contributing to lung inflammation providing a mechanistic hypothesis for tocilizumab. Concomitantly we have identified an oncostatin M (OSM) orchestrated lung injury response contributing to epithelial and endothelial cell disruption, myofibroblast activation and fibrosis. Despite dysregulated expression of IL11 in IPF, SSc-ILD, and murine models of fibrosis, we found no evidence for a pro-fibrotic role for IL-11 in vitro or in vivo. Instead, in pre-clinical models of IPF we demonstrate that antagonism of OSM alone, or to a greater degree in combination with IL-6, reduced lung fibrosis and inflammation. Translating these observations, we validated gp130:OSMR, rather than gp130:LIFR, as the dominant human receptor complex used by OSM, identifying OSMR as a potential therapeutic target to stall fibrosis.

immunology↗

bioRxiv2025

Symbiotic State Affects Microbiome Recovery in a Facultatively Symbiotic Cnidarian

The cnidarian holobiont consists of host cells, algal symbionts, and a complex microbiome that resides in and on the host tissue and the algal symbionts. To investigate the interactions between a host cnidarian, its algal symbionts, and its bacterial microbiome, we used antibiotics to deplete the microbiome of the facultatively symbiotic sea anemone Exaiptasia pallida (Aiptasia) in both symbiotic and aposymbiotic states. To understand the dynamics of microbiome reestablishment following disruption, we used 16S gene sequencing of the bacterial microbiome to characterize communities during recovery following antibiotic treatment. We assessed the host molecular response to microbiome depletion and recovery using RNA-seq and Western blotting of the immune transcription factor NF-{kappa}B. 16S results demonstrate that, following depletion, symbiotic Aiptasia can reestablish bacterial communities (based on diversity metrics and community composition) alike to those of controls before microbiome depletion. However, the microbiomes of aposymbiotic Aiptasia fail to reestablish to control conditions even after seven days of recovery. Bacteria in the Family Endozoicomonadaceae reestablish to control levels in symbiotic, but not aposymbiotic, Aiptasia, suggesting an association between Endozoicomonadaceae and algal symbionts. Our molecular analyses show that host immune system gene expression is downregulated during recovery from antibiotic treatment, but that NF-{kappa}B protein levels increase during recovery, suggesting mechanisms for microbiome reestablishment following disruption. This study shows the dynamics of microbiome recovery following depletion and the influence of microbiome community members on host gene expression in a cnidarian model, providing a foundation for future research involving pairwise interactions between microorganisms and cnidarian hosts.

microbiology↗

bioRxiv2025

Uncovering the Heterogeneity and Ontogeny of Mouse Thymic Macrophages Reveals an Unexpected Early Checkpoint Role

Thymic macrophages (TMs) maintain tissue homeostasis by clearing the large numbers of apoptotic cells generated during T cell development, but how TM heterogeneity relates to their developmental origin and role in thymocyte maturation remains incompletely understood. Using complementary flow-cytometric, single-cell transcriptomic, and genetic approaches, we resolved two major TM populations corresponding to TIMD4+ cortical and CX3CR1+ medullary/cortico-medullary macrophages. TIMD4+ VCAM1+ TMs displayed a prominent efferocytosis and apoptotic-cell-clearance program, whereas TIMD4- VCAM1+ TMs were enriched for antigen-presentation and interferon-response pathways. Fate mapping revealed unequal progenitor contributions to these populations, and CCR2 deficiency selectively reduced TIMD4- VCAM1+ TMs and thymic monocytes, supporting ongoing input from circulating precursors. Exploratory pseudotime analysis further identified a transcriptional continuum from Ly6c2+ Ccr2+ monocytes toward macrophage states. Using MaFIA fetal thymic organ cultures, AP20187-mediated depletion of Csf1r-expressing myeloid cells reduced CD4+CD8+ thymocyte differentiation and produced a coordinated accumulation of DN3 cells, loss of DN4 cells, and reduction in CD27 expression. These convergent changes identify the DN3-to-DN4 transition as a developmental stage that requires an intact Csf1r-expressing myeloid compartment, and establish a functional connection between the thymic myeloid niche and early {beta} T cell development. Together, our study refines the phenotypic and developmental organization of mouse TMs and reveals a previously under-appreciated requirement for myeloid-cell support during progression through the {beta}-selection checkpoint.

immunology↗

bioRxiv2025

Two parallel lineage-committed progenitors contribute to the developing brain

The hindbrain is a life-sustaining brain region. In one model, a common neural progenitor generates all brain regions. Here our studies of mouse embryos and human pluripotent stem cells (hPSCs) support a different model: two parallel brain progenitors emerge simultaneously during gastrulation, anterior neural ectoderm (forebrain/midbrain progenitor) and posterior neural ectoderm (hindbrain progenitor). Not only are they lineage-committed to respectively form forebrain/midbrain vs. hindbrain in vitro, but they also have diverging chromatin landscapes foreshadowing future forebrain/midbrain vs. hindbrain identities. Leveraging these differences, we differentiated hPSCs into hindbrain rhombomere 5/6-specific motor neurons, hitherto difficult to generate in vitro. We postulate the brain is a composite organ emanating from two lineage-restricted progenitors; these dual progenitors may be evolutionarily conserved across 550 million years from hemichordates to mammals.

developmental biology↗