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Liu, Z.

Publications and source records attributed to Liu, Z..

At least 109 records · Page 6Linked to original sources

Vision-Based Genomic Model for Copy Number Variant Pathogenicity Prediction

Copy number variants (CNVs) are a major class of structural genomic alterations underlying rare disease, including neurodevelopmental delay and intellectual disability, yet predicting their pathogenicity remains challenging. Existing methods reduce CNVs to region-level numerical features, discarding the positional structure and cross-track patterns that expert clinical reviewers use to interpret genomic evidence. To address this, we introduce TO_SCPLOWESSERACTC_SCPLOW for CNV, a track-based spatial representation for CNV pathogenicity prediction, which represents each variant as a base-pair-resolution multi-track image and models spatial genomic patterns across annotation tracks while preserving positional structure and cross-track dependencies. Trained on a chromosome-level hold-out split of the ClinVar dataset, TO_SCPLOWESSERACTC_SCPLOW outperforms prior methods on held-out and curated noncoding benchmarks, improving AUROC by up to 0.10 over the state-of-the-art baseline. On the independent DECIPHER cohort, the model demonstrates generalizability by maintaining the highest AUROC and the highest F1 score across baselines. Furthermore, our model localizes pathogenic signals to clinically meaningful genomic subregions, providing track-annotated evidence that supports practical clinical interpretation.

bioinformatics↗

Interpretable morphology mapping of peripheral blood leukocytes using annotation-efficient artificial intelligence

BackgroundPeripheral blood smears (PBS) review is labor-intensive, subjective, and challenging for rare or morphologically heterogeneous cell types in hematologic malignancies. Artificial intelligence (AI) offers a scalable alternative, but broader clinical translation is constrained by annotation burden and limited interpretability. MethodsWe developed an interpretable, annotation-efficient AI framework that learns leukocyte morphology through a two-stage process: label-free representation learning to construct a morphological embedding space, followed by supervised fine-tuning for cell type and morphological attribute classification. The model was trained and evaluated on 5,952 PBS images from cancer patients at MD Anderson Cancer Center, including blast cells, and 17,092 images from public sources. Active learning strategies were assessed to improve label efficiency, and interpretability was examined using saliency and embedding visualization. An interactive web application, HemoSight, was developed to support clinical review. FindingsThe framework achieved a macro-F1 score of 0{middle dot}96 for 9-way leukocyte classification on the internal test split and 0{middle dot}83 on the held-out patient cohort. Active learning substantially reduced annotation requirements, reaching peak performance with only 13{middle dot}3% of available labels and significantly improving learning efficiency across 8 of 9 cell types. The model generalized to classifying 11 leukocyte morphological attributes with a mean F1 score of 85{middle dot}8% and revealed structured morphological landscapes. Saliency maps, embedding visualizations, and the HemoSight application enabled transparent morphological inspection of model predictions, supporting confidence in model behavior and feasibility for clinical integration. InterpretationOur framework enables scalable, annotation-efficient, and interpretable modeling of leukocyte morphology, supporting the integration of AI-assisted PBS review for hematopathology workflows. FundingSeed funding from The University of Texas MD Anderson Cancer Center. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSPeripheral blood smear review is essential for diagnosing and monitoring hematologic malignancies, but manual case review is time-consuming and variable, particularly for rare or abnormal leukocyte types. Automated hematology analyzers are widely used to flag abnormal cells; however, they provide limited morphological insight and often require frequent manual correction, especially in cancer settings where disease and treatment alter cell appearance. Previous artificial intelligence approaches for leukocyte classification have shown promise, but most rely on fully supervised learning, require extensive expert annotation, focus on a limited set of cell types, and frequently exclude diagnostically important rare cells such as blasts. Interpretability is inconsistently addressed, and few studies provide tools that allow clinicians to inspect and interpret model outputs within routine workflows. Added value of this studyThis study introduces an annotation-efficient framework trained on a large collection of peripheral blood smear images, including cancer patient samples with hematopathologist-verified rare cell types such as blasts. The framework learns leukocyte morphology from unlabeled images and adapts to multiple classification tasks with minimal expert labeling. Performance is evaluated on both internal test splits and a held-out patient cohort to provide a realistic estimate of generalization. Iterative, uncertainty-guided annotation substantially reduces labeling requirements while improving learning efficiency across most leukocyte classes. Beyond cell-type classification, the framework is extended to 11 clinically relevant morphological attributes and reveals a structured morphological landscape. These capabilities are integrated into a web application, HemoSight, enabling real-time inference and transparent morphological inspection of predictions within hematopathology workflows. Implications of all the available evidenceAdvancing artificial intelligence for hematology requires methods that reduce expert labeling demands, provide interpretable outputs, and perform reliably across clinically diverse patient samples. This study shows that learning from largely unlabeled data combined with iterative expert annotation can support scalable and flexible modeling of leukocyte morphology for classification tasks. Integrating quantitative predictions and interactive visualization supports the use of artificial intelligence as an assistive tool for diagnostic peripheral blood smear review, with potential to improve efficiency, consistency, and reviewer confidence.

pathology↗

Oncofetal RNA-binding proteins of IGF2BP family suppress IRF-3 and NF-kB dependent transcription downstream of cytosolic RNA sensors

Activation of innate inflammatory signaling and tumor-specific antigen presentation in cancer cells provides a foundation for anti-cancer immunotherapies. Here, we show that the insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3), which are upregulated across diverse human malignancies, including acute myeloid leukemia (AML), suppress RNA-sensing pattern recognition receptor signaling and downstream ISRE-and NF-{kappa}B-dependent transcription. Among these pathways, RIG-I signaling is particularly sensitive to IGF2BP-mediated inhibition. This suppressive effect is strongest when all three IGF2BP paralogs are co-expressed, especially in embryonic-like hematoendothelial cells and leukemia stem cells. Mechanistically, IGF2BPs suppress innate immune signaling by directly binding to TNFAIP3 mRNA and promoting its expression. Consequently, inhibition of IGF2BPs activates innate immune signaling and induces MHC class I gene expression in AML cells, highlighting IGF2BPs as promising therapeutic targets to enhance RIG-I- and TLR-based cancer immunotherapies. HighlightsO_LIIGF2BP family of proteins regulate PRR responses C_LIO_LIIGF2BP levels affect chromatin accessibility and pan-transcriptome C_LIO_LISV40 large T antigen induces de novo expression of IGF2BPs C_LIO_LITNFAIP3 mRNA levels and stability are regulated by IGF2BPs C_LI

cancer biology↗

Nuclear SUN2 coordinates endothelial cell-matrix interactions to regulate blood vessel homeostasis and barrier function

Vascular endothelial cells respond to environmental forces to remodel vessels during development and to achieve homeostasis, and mis-regulated responses lead to vascular dysfunction and disease. The nucleus participates in force transduction to cell-matrix junctions via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that resides in the nuclear envelope, but how these forces are regulated and relayed is incompletely understood. We found that the LINC complex protein SUN2 is required for proper endothelial cell-matrix interactions that occur far from the nucleus and affect angiogenic expansion, vascular responses to flow, and barrier integrity. Endothelial cells lacking SUN2 had inappropriate flow responses and reduced expression of flow-mediated transcription factors in vitro and in vivo. Expression of several matrix and adhesion genes was reduced in SUN2-depleted cells, leading to defective extracellular matrix, dysmorphic focal adhesions resistant to dynamic turnover, and disturbed cell-matrix force distribution. Mechanistically, nuclear SUN2 affected dynamic regulation of the microtubule cytoskeleton that correlated with matrix metalloprotease-dependent barrier dysfunction. These findings indicate that nuclear SUN2 establishes and maintains blood vessel homeostasis by controlling microtubule-mediated effects on focal adhesion turnover and extracellular matrix properties, with implications for cardiovascular aging and diseases such as Marfan syndrome that affect vessel wall integrity.

cell biology↗

Microbial network rewiring mediates the function-biosafety trade-off in transboundary Pamir lakes

The Pamir Plateau is a transboundary water tower whose source lakes serve as critical biogeochemical hubs with implications for downstream freshwater security. However, it remains unclear how environmental shifts in these high-altitude lakes reshape the microbial communities that drive ecosystem functioning and water safety. Here, we conducted a multi-omics survey across 20 lakes spanning Chinese and Tajikistani Pamir. Our results revealed that prokaryotes exhibited lower diversity but higher among-lake connectivity in China, while eukaryotes showed higher diversity but stronger dispersal limitation. These contrasting biogeographic responses triggered profound rewiring of microbial associations. Under intensified anthropogenic pressures, Chinese cross-kingdom networks decoupled from environmental constraints and became more centralized and complex. Conversely, Tajikistani lakes maintained more modular networks governed by hydrochemical filtering. Critically, this rewiring mediated a trade-off between multifunctionality and potential biosafety risk, with higher element cycling abundances in Chinese lakes, whereas Tajikistani lakes harbored larger biosafety burden dominated by virulence, pathogen, and toxic-algae potential. Incorporating network topology also substantially improved the prediction of these ecological consequences. These findings highlight the importance of network-informed monitoring and management strategies to safeguard ecosystem sustainability in transboundary Pamir lakes under global change.

microbiology↗

cfMIND: A read-level methylation framework for accurate non- invasive disease detection using cell-free DNA

Plasma cell-free DNA (cfDNA) emerged as a promising non-invasive biomarker for cancers. However, reliable detection remains challenging due to the low abundance of tumor-derived cfDNA fragments and the dilution of informative methylation signals when aggregated into region-level features. Here, we propose a novel approach cfMIND, an efficient and robust machine-learning framework that leverages stratified read-level methylation signals to preserve rare cell-type-specific information and enhance detection sensitivity. By avoiding information loss inherent to conventional aggregation strategies, cfMIND enables more accurate and stable inference across diverse conditions. cfMIND is compatible with various cfDNA methylation sequencing technologies and cancer types. Across multiple cancer datasets (n = 868), cfMIND achieves high performance (AUROC up to 0.966) and maintains strong accuracy even at ultra-low sequencing depth (0.2x) and in early-stage cancers. Notably, cfMIND demonstrates exceptional robustness, generalizing effectively across cohorts and platforms without the need for model retraining. These results highlight its potential utility in heterogeneous experimental and clinically relevant settings. Beyond cancer detection, cfMIND is readily extendable to non-malignant diseases, as demonstrated by its ability to capture disease-associated methylation alterations in amyotrophic lateral sclerosis (ALS). Functional investigations on cfMIND-identified features further reveal enrichment in key regulatory regions implicated in disease pathogenesis and recapitulate tissue- and single-cell-level methylation and transcriptional programs underlying tumor biology. Collectively, cfMIND represents a significant advancement in the field, offering a broadly applicable, functionally interpretable, and high-resolution framework for non-invasive disease detection.

bioinformatics↗

Large-scale activity analysis of gut prophages reveals significantly active lineages in the human gut

Prophages dominate the human gut virome and their transition from lysogeny to lytic replication profoundly affects gut microbial community structure. However, the global activity landscape of gut prophages remains largely uncharted. Here, we developed a workflow to assess prophage activity across more than 30,000 gut bacterial isolates cultured in vitro under standard conditions. Using this approach, 4,033 prophages, corresponding to a small fraction (7.5%) of the overall prophage reservoir, were detected as active, indicating a high-lysogeny, low-activity landscape. Prophage activity varied markedly across host taxa, with Bacillota exhibiting notably elevated active rates. Active prophages derived from 183 viral families (vFAMs), 55 of which were assigned as significantly active (SA-vFAMs), including: SA-vFAM7, a novel, hyper-prevalent lineage with expanded host range into Tannerellaceae and substantial intra-lineage diversity; and Candidatus Mubacviridae (SA-vFAM1), a newly defined, hankyphage-inclusive, highly active Mu-like transposable phage family infecting Bacteroidales, characterized by conserved MuA-MuB operons and cross-family host range. Together, this work provides a large-scale, activity-integrated atlas of human gut prophages, demonstrating that prophage activity is driven by both host phylogeny and viral lineage identity, and establishing a foundational resource for understanding prophage ecology.

microbiology↗

Metabolic Coherence of the Mouse Brain

The brains metabolic demands are well established, but how metabolism is coordinated across anatomically distinct regions remains poorly understood. Here, using matrix-assisted laser desorption/ionization (MALDI) imaging integrated with the Allen Brain Atlas and optimal transport-based computational analysis, we map the spatial metabolome across twelve major mouse brain divisions. We define an optimal-transport-derived inter-regional metabolite similarity metric and refer to it as metabolic coherence. This structure is largely preserved in an amyloid mouse model of Alzheimers disease despite widespread changes in individual metabolite and lipid levels. Individual metabolites and lipids shift in a coordinated manner across regions, sustaining inter-regional relationships even as absolute levels change in patterns indicative of mitochondrial dysfunction. To test whether the coherence metric is responsive to local intervention, we targeted the left hippocampus of mice from this model via lentiviral shHIF1 knockdown or neuronal AAV-mediated AOX expression. Both interventions were associated with metabolite normalization at the injection site. More importantly, normalization extended across distal regions sharing high metabolic similarity with the hippocampus and was accompanied by improved social memory in a single behavioral assay. Gene modulation and amyloid plaque reduction localized to the injection site.

neuroscience↗

A massively parallel reporter assay of MECP2 cis-regulatory elements reveals genetic candidates for male-biased autism

Autism affects males four times more often than females, yet the basis of this sex bias remains unclear. One hypothesis is that hypomorphic variants in X-linked genes--genes where loss-of-function alleles cause syndromic neurodevelopmental disorders (NDDs) predominantly in females--produce milder, non-syndromic phenotypes in hemizygous males. We tested this by investigating cis-regulatory elements (CREs) of MECP2, a dosage-sensitive X-linked gene. Using a massively parallel reporter assay in human neurons, we mapped transcription factor binding sites within MECP2 CREs and tested autism-associated variants for functional impact. We identified two noncoding variants that change CRE activity, each with a male-biased phenotype. One of these, a promoter variant, disrupts NFY binding and reduces MECP2 expression by [~]30%, a magnitude that produces autism-like phenotypes in mice. These findings suggest noncoding MECP2 variants can cause non-syndromic, male-biased autism, and provide a framework for uncovering regulatory variants in other X-linked NDD genes that may contribute to autisms missing heritability.

genetics↗

spatiAlytica: Viewer-Grounded Multimodal Agentic System for Interactive Spatial Omics Analysis

Spatial transcriptomics and proteomics map tissue architecture and cellular interactions, but analysis remains limited by programming demands and text-centered AI agents that lack viewer grounding and cross-turn context. We present spatiAlytica, a viewer-centric multimodal interactive agentic system embedded in the Napari viewer that enables non-programmer biologists to perform iterative, hypothesis-driven spatial omics analysis via natural language. spatiAlytica couples viewer-state serialization, agentic memory, biological concept-to-data-field mapping, code generation and debugging, Spatial VQA, and grounded interpretation to support an exploratory analysis and interpretive reasoning workflow. We introduce spatiAlyticaBench, a comprehensive benchmark spanning 222 single-turn spatial analytical coding questions, 178 multi-turn sequential workflow questions, and 7,350 image-grounded reasoning questions. spatiAlytica outperformed strong agentic baselines, while using less time and tokens. Case studies across Kaposis sarcoma, colorectal cancer, and ovarian cancer recapitulated known spatial patterns and uncovered progressive CD8 T-cell dysfunction during KS progression.

bioinformatics↗

Molecular mechanism of M1-mediated influenza A virus assembly

Influenza A virus assembly is orchestrated by matrix protein 1 (M1), which engages ribonucleoproteins, mediates glycoprotein incorporation, and scaffolds lipid envelopment, but how these activities are performed and coordinated is unclear. We determined high-resolution structures of M1 within virions, virus-like particles, and in vitro. We found that M1 binds phosphatidylinositol 4,5-bisphosphate at the membrane, oligomerizes through an electrostatic interface, and switches between three alternative conformational states in different regions of the virion. The M1 conformer at the front associates with ribonucleoproteins and initiates budding. In the body of the virion, two conformers alternate to create an extended filament with seam-like discontinuities. Enrichment of the seam-prone conformation at the virion rear induces envelope closure and exposes a binding site for the cytoplasmic tail of neuraminidase (NA) to promote virus release. Conformational switching within a polarized M1 lattice thereby couples organization of the viral components with shaping of the virion architecture.

microbiology↗

Lesion-Level-Dependent Neuroendocrine Surge Precedes Neuroinflammation and Endoplasmic Reticulum Stress in the Hypothalamus After Spinal Cord Injury: Dual-Cohort Transcriptomic Evidence for a Temporally Ordered AVP Cascade

BackgroundSpinal cord injury (SCI) triggers remote pathological changes in supraspinal regions, including neuroendocrine dysfunction that manifests clinically as hyponatremia and central diabetes insipidus. Clinical observations of lesion-level dependency and sequential transformation between these disorders suggest a temporally ordered hypothalamic cascade in which a compensatory arginine vasopressin (AVP)-driven neuroendocrine surge may precede a later neuroinflammation and endoplasmic reticulum (ER) stress-mediated neuronal exhaustion. Direct transcriptomic evidence for the temporal ordering of these events, however, has been lacking. MethodsWe performed a dual-cohort transcriptomic analysis. A discovery cohort (NCBI Sequence Read Archive PRJNA953752) comprised hypothalamic tissue from adult male Sprague-Dawley rats subjected to high-thoracic (T3) SCI, low-thoracic (T10) SCI, or sham surgery, sampled at post-injury day 7 and analyzed with edgeR/DESeq2 (|log2FC| > 1, Padj < 0.05). An independent chronic-phase validation cohort (Gene Expression Omnibus GSE297887) of hippocampal tissue from SCI and sham mice was interrogated as a sensitive supraspinal proxy for remote neuroinflammatory and ER-stress signatures. Pre-defined gene panels covered neuroendocrine, neuroinflammation, and ER-stress/unfolded-protein-response categories. ResultsIn the discovery cohort, high-thoracic SCI produced a lesion-level-dependent neuroendocrine surge in the hypothalamus: Avp (fold change 7.23; Padj = 0.002), Oxt (fold change 14.25; Padj = 2.3 x 10-7), and Ucn3 (fold change 9.22; Padj = 0.002) were among the most significantly upregulated genes genome-wide, whereas low-thoracic SCI failed to reach significance for any of these targets. Classical neuroinflammation markers and canonical ER-stress effectors remained transcriptionally silent (all Padj > 0.69). The PERK-pathway sentinel genes Trib3 and Ppp1r15a/GADD34 exhibited coordinated sub-threshold trends indicative of early activation, and Avp expression was tightly correlated with Mmp9 (r = 0.833; P = 0.0004). In the chronic-phase validation cohort, microglial P2ry12 and ferroptosis signatures were significantly upregulated (P2ry12 fold change 1.33; P = 0.008) suggesting a primed microglial state, while ER-stress effectors remained silent. ConclusionsThese data support a temporally ordered hypothalamic cascade after SCI in which an early compensatory neuroendocrine surge precedes -- and may precipitate, through biosynthetic overload and blood-brain-barrier disruption -- a subsequent neuroinflammation and ER-stress crisis. The defined molecular window between neuroendocrine activation and inflammatory/ER-stress engagement identifies a candidate therapeutic window for early neuroprotective intervention in acute SCI.

neuroscience↗

CD164 is an endolysosomal host factor for entry of Clade A New World Arenaviruses

Arenaviruses are divided into Old World (OW) and New World (NW) groups. OW arenaviruses enter cells through a pH-dependent receptor switch from a plasma-membrane factor to an endolysosomal receptor for subsequent membrane fusion, whereas clade B NW arenaviruses use transferrin receptor 1 without a secondary receptor. Using a vesicular stomatitis virus (VSV) chimera expressing the glycoprotein complex (GPC) of the clade A NW arenavirus Pichinde virus, we performed a genome-wide CRISPR loss-of-function screen and identified the endolysosomal sialomucin CD164 as an essential host factor. CD164 knockout cells were resistant to VSV chimeras bearing the GPCs of Pichinde, Parana, and Flexal viruses, and to authentic Pichinde and Parana virus, with susceptibility restored by complementation. The requirement mapped to the cysteine-rich domain of CD164, which bound GP1 in a pH-dependent manner through main-chain interactions. These findings define CD164 as an endolysosomal receptor for clade A NW arenaviruses expanding the receptor switching paradigm.

microbiology↗

Bacterial internalins exploit E-cadherin to promote head-neck tumor metastasis and drug resistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy characterized by local invasion, lymph node metastasis, and therapeutic resistance. Chronic periodontal disease has been linked to HNSCC progression, yet the responsible pathogens and underlying molecular mechanisms remain unclear. Here, we show that the keystone periodontal pathogen Porphyromonas gingivalis promotes HNSCC metastasis and chemoresistance through two internalin proteins that are secreted via the type IX secretion system (T9SS). These internalin proteins specifically bind the EC1 domain of E-cadherin through their curved solenoid-like leucine-rich repeats (LRRs), facilitating bacterial invasion and inducing epithelial-to-mesenchymal transition (EMT). Mechanistically, internalin-E-cadherin engagement drives {beta}-catenin nuclear translocation and activates p38 and JNK1/2 MAP kinase signaling pathways, enhancing tumor cell migration, metastatic dissemination, and resistance to cisplatin-induced apoptosis. Tissue microarrays detect internalin antigens in HNSCC specimens, supporting their in vivo relevance. Together, these findings establish a direct mechanistic link between an oral pathogen and HNSCC progression and extend the paradigm of internalin-E-cadherin interactions from microbial pathogenesis to cancer biology.

microbiology↗

The BraALA3 Homologs Mediate Propiconazole-Modulated Plant Growth in flowering Chinese cabbage

In agriculture, propiconazole (PCZ) controls excessive growth in flowering Chinese cabbage but poses dietary safety risks due to residue accumulation. Therefore, identifying novel PCZ targets and breeding PCZ-free cultivars is critical for the safe production of flowering Chinese cabbage. Here, we identified three P4-ATPase flippase homologs aminophospholipid ATPase 3 (BraALA3a/b/c) in flowering Chinese cabbage that function as sensitive targets for PCZ. These proteins exhibit high binding affinity for PCZ, which directly inhibits their ATPase activity. Overexpression of the BraALA3 homologs enhanced plant growth and increased sensitivity to PCZ, whereas knockdown led to dwarfism and reduced sensitivity. Based on these findings, we identified editable active sites via protoplast-based screening. Genetic transformation of one such site yielded BraALA3a/braala3aK200T mutant lines, which displayed a dwarf and compact architecture. These findings provide a precise molecular target for developing PCZ-free germplasm in flowering Chinese cabbage through gene editing.

plant biology↗

Expanding the genetic code with diverse backbone structures across diverse sequence contexts

Expanding the genetic code to enable the selective and specific incorporation of non-canonical monomers (ncMs), beyond -L amino acids with variant sidechains, is a key outstanding challenge. Here we discover orthogonal aminoacyl-tRNA synthetases that selectively and specifically acylate their cognate orthogonal tRNA in vivo with eleven new ncMs spanning five different chemical classes: ,-disubstituted-amino acids, malonic acids, carboxylic acids, {beta}2-amino acids and N-cyclic amino acids. We demonstrate that co-translational incorporation of ,-disubstituted-amino acids, {beta}2-amino acids, {beta}3-amino acids and N-cyclic amino acids is strongly dependent on the codons either side of the codon used to direct ncM incorporation, with several ncMs incorporated at less than 1% of sequence contexts. We evolve orthogonal tRNAs that enable the incorporation of previously unincorporated ncMs, enable the incorporation of ncMs at >95% of sequence contexts and, increase the incorporation efficiency at challenging sequence contexts up to 40-fold. We demonstrate the encoded cellular synthesis of proteins and macrocycles containing ncMs and, explicitly demonstrate that our evolved tRNAs provide direct access to a wider range of genetically encoded macrocyclic sequences containing ncMs. Our results provide a foundation for composing, discovering and manufacturing proteins and peptides with functions augmented by ncMs.

synthetic biology↗

In utero transduction resolves gut cell lineages and enables conditional gene perturbation in the developing enteric nervous system

How diverse cell lineages emerge and are genetically regulated during organogenesis are central questions in understanding the developmental origins of disease. However, the mouse gut, including its intrinsic enteric nervous system (ENS) derived from migratory neural crest, has remained difficult to experimentally target. Here, we introduce an in utero lentiviral nano-injection strategy that enables early and efficient access to progenitor cells of all major cell types within the developing gut as well as gut-innervating ganglia. Leveraging this approach in combination with DNA barcoding and single cell transcriptomics, we resolve clonal relationships in all gut lineages, including epithelial, neural, immune, and mesenchymal cell types. Clonal coupling between distinct subsets of fibroblasts and either pericytes, mesothelial cells, or interstitial cells of Cajal, suggested a developmental logic whereby the mesenchymal compartment arises from a set of fate-biased progenitors. Yet, mesenchymal regionalization along the anterior-posterior axis establishes early, whereas the ENS displays broad clonal dispersion across gut regions and acquires subsequent regional identities. We further adapted the platform for temporally controlled cell-type specific gene manipulation and, as a proof-of-principle, show that induced expression of the proneural factor Ascl1 biases ENS progenitor cells toward neuronal differentiation. Together, this work provides insights into refined spatiotemporal lineage relationships within a multigerm-layer organ and establishes a broadly applicable in vivo framework for probing gene function during gastrointestinal and neural crest development. SIGNIFICANCEThe gastrointestinal tract comprises diverse cell types originating from all three germ layers and includes the neural crest-derived enteric nervous system (ENS). Progress in defining these lineages and their gene regulation is challenged by the limited experimental access to the developing gut. Here, we establish in utero lentiviral transduction as an efficient approach to resolve clonal lineages and address gene functions in defined gut cell types. We show that mesenchyme assumes positional allocation early and differentiates through fate-restricted progenitors, linking specialized mesenchymal cell types to different fibroblasts. In contrast, the ENS differentiates stochastically and acquires late regional identities. Our study reveals fundamental principles of multi-lineage organogenesis and provides a framework to dissect the contribution of developmental programs to visceral dysfunction.

developmental biology↗

Tartrazine clears live cells while preserving viability at high refractive indices and osmolality

Tissue-clearing techniques have transformed optical imaging of fixed specimens, yet their application to living systems remains limited by toxicity and removal of key tissue components. We recently demonstrated that absorbing molecules such as tartrazine can reversibly render live mouse skin transparent. Subsequently, it was reported that isotonic protein solutions can achieve ex vivo and in vivo cellular clearing. However, discrepancies remain regarding the optimal refractive index (RI) for live-cell clearing and the impact of elevated osmolality on cell viability. Here, using cultured mammalian cells, we systematically examine the dependence of optical contrast on medium RI and the effects of hyperosmolality. We find that, contrary to the recent report of an optimal RI of 1.36[~]1.37 for suspended cells, densely-packed adherent cells exhibit a monotonic decrease in phase contrast up to an RI of 1.41 with tartrazine. Moreover, even under highly hyperosmotic conditions ([~]1200 mOsm/kg), cultured cells exhibit minimal deformation and negligible loss of viability for up to 30 min in the clearing solution. These results demonstrate that tartrazine enables effective live-cell clearing at RI up to 1.41 while preserving viability under elevated osmolality, and motivate future studies to define optimal conditions for in vivo optical clearing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/717314v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ec9fbforg.highwire.dtl.DTLVardef@1ebe9e6org.highwire.dtl.DTLVardef@1492c3corg.highwire.dtl.DTLVardef@f75559_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗