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Bintu, B.

Publications and source records attributed to Bintu, B..

14 recordsLinked to original sources

3D MERFISH reveals cell-type diversity generated by mRNA partitioning in spiralian embryogenesis

Cell type diversification during development is often attributed to differential gene transcription. Here, we show that, during Crepidula atrasolea embryogenesis, transcriptomically distinct cell types arise through differential distribution of pre-existing mRNAs. Combining immunofluorescence, live imaging, and whole-mount 3D spatial transcriptomics (MERFISH) of over 40 embryos, we show that many mRNAs form subcellular aggregates during mitosis, which segregate asymmetrically between daughter cells through microtubule associations. Cell divisions thus simultaneously increase cell number and diversity, producing six transcriptomically distinct cell quartets in the 24-cell stage embryo. We modeled aggregate compositions across cell divisions using transcript abundance and quantified transcript-specific affinity. These results establish mRNA sorting and inheritance as a scalable cell type patterning mechanism and provide a framework for investigating how mRNA segregation shapes animal development.

molecular biology↗

MERFISHEYES: Web-based Visualization and Sharing Platform for Single-cell and Single-molecule Spatial Transcriptomics

Interactive exploration and sharing of hybridization-based spatial transcriptomics datasets remain limited by large file sizes, specialized software, and computationally intensive preprocessing, restricting rapid access to publicly available resources such as the Brain Image Library. Here we present MERFISHEYES (https://merfisheyes.com), a browser-based platform for interactive visualization of single-cell and single-molecule spatial transcriptomics data across commercial and emerging high-throughput platforms. MERFISHEYES enables marker gene exploration, cross-dataset and cross-species comparison, visualization of subcellular RNA localization, and collaborative sharing through browser-accessible links generated directly from drag-and-drop datasets. We use MERFISHEYES to enable browser-based visualization of all 158 spatial transcriptomics datasets in the Brain Image Library and demonstrate its capabilities in handling high-throughput 3D data with genome-scale imputation of gene expression and DNA accessibility. MERFISHEYES provides a scalable framework for rapid exploration, sharing, and collaborative analysis of spatial transcriptomics data.

bioinformatics↗

Single-molecule spatial genomics reveals the multi-scale organization and plasticity of extrachromosomal DNA in glioblastoma

Extrachromosomal DNA (ecDNA) is a major driver of intratumoral heterogeneity and is associated with poor clinical outcomes across cancers, yet how individual ecDNA molecules are organized and regulated within intact tumors remains unknown. Here, we leveraged single-molecule, multi-modal spatial genomics to resolve the three-dimensional chromatin organization and transcriptional activity of individual EGFR-containing ecDNA molecules in glioblastoma (GBM) cells in vitro, in orthotopic xenografts, and in patient-derived GBM tissue. At the larger scale, we find that distinct GBM molecular and functional states emerge depending on the local cellular environment. EGFR expression was markedly different between GBM subpopulations, and perturbations of EGFR dosage shifted GBM cellular states. ecDNA expression was modulated by multiple mechanisms, including variation in copy number, chromatin organization, DNA sequence, and chromosomal reintegration, which were simultaneously measured within the same cells. At the single-molecule scale, ecDNA adopts a physically expanded chromatin configuration with larger ecDNA molecules having higher transcriptional activity and interaction with active transcriptional machinery. ecDNA regulation was coordinated within cells and across GBM states, and ecDNA copy number, structure, and transcription were spatially organized across the tumor architecture. Co-culturing GBM cells with neurons recapitulated key features of infiltrative regions, including lower EGFR expression, reduced ecDNA copy number, and increased chromosomal reintegration, suggesting a causal role for the microenvironment in shaping ecDNA regulation. Collectively, these findings support a model in which GBM states and ecDNA are linked, plastic, and influenced by microenvironmental contexts, revealing a previously inaccessible layer of genome organization underlying tumor heterogeneity and malignant cell behavior.

genomics↗

PantheonOS: An Evolvable Multi-Agent Framework for Automatic Genomics Discovery

The convergence of large language model-powered autonomous agent systems and single-cell biology promises a paradigm shift in biomedical discovery. However, existing biological agent systems, building upon single-agent architectures, are narrowly specialized or overly general, limiting applications to routine analyses. We introduce PantheonOS (https://PantheonOS.stanford.edu), an evolvable, privacy-preserving multi-agent framework designed to reconcile generality with domain specificity. Critically, PantheonOS enables agentic code evolution, allowing evolving state-of-the-art batch correction and our reinforcement-learning augmented gene panel selection algorithms to achieve super-human performance. PantheonOS drives biological discoveries across systems: uncovering asymmetric paracrine Cer1-Nodal inhibition in proximal-distal axis formation of novel early mouse embryo 3D data; integrating human fetal heart multi-omics with whole-heart data to reveal molecular programs underpin heart diseases; and adaptively selecting virtual cell models to predict cardiac regulatory and perturbation effects. Together, PantheonOS points towards a future where scientific discoveries are increasingly driven by self-evolving AI systems across biology and beyond. Websitehttps://pantheonos.stanford.edu Ecosystemhttps://github.com/aristoteleo SummaryLarge language model-powered agent systems are driving a paradigm shift in scientific discovery by automating, scaling, and accelerating data analysis. This transformation is particularly profound in biology, where the rapid expansion of single-cell and spatial genomics has effectively reshaped the field into a data-intensive science. However, existing biological agent systems are typically constrained to single-agent designs, are narrowly specialized, or are overly general without sufficient domain expertise, limiting their applicability to routine or shallow analyses. Here, we introduce PantheonOS (https://pantheonos.stanford.edu), an evolvable, privacy-preserving, and general-purpose multi-agent framework designed to reconcile generality with deep domain specificity. PantheonOS provides an abstract, extensible architecture that enables customized agent composition and supports end-to-end single-cell and multi-omics analysis, spanning reinforcement-learning-augmented gene panel design, raw FASTQ processing, multimodal data integration, and three-dimensional spatial genomics reconstruction. Central to this framework, Pantheon-Evolve enables agentic code evolution, allowing the system to autonomously improve state-of-the-art batch-correction algorithms and new reinforcement-learning based gene panel design algorithms, achieving performance beyond manually designed baselines. We demonstrate the power of PantheonOS across multiple biological domains. In early mouse embryogenesis, PantheonOS automatically reconstructs three-dimensional spatial gene expression landscapes and resolves asymmetric Cer1 expression and paracrine Cer1-Nodal inhibition, revealing a robust proximal-distal axis at embryonic day six (E6.0). In human development, PantheonOS integrates fetal heart single-cell multi-omics with whole-heart 3D MERFISH+ data at post-conception week 12, uncovering spatially resolved molecular programs underlying heart disease ontogeny. Finally, an intelligent model-routing mechanism enables PantheonOS to adaptively select optimal virtual cell models across heterogeneous tasks, revealing minimal regulatory networks of cardiogenesis and predicting spatially resolved perturbation effects in the developing heart. Together, PantheonOS establishes a foundation for fully automated, evolvable, and domain-aware agentic analysis in genomics, and points toward a future in which scientific discovery is increasingly driven by self-improving AI systems across biology and beyond.

bioinformatics↗

A Single-Cell and Spatial 3D Multi-omic Atlas of Developing Human Basal Ganglia and Inhibitory Neurons

The human basal ganglia (BG), subcortical nuclei fundamental to motor regulation and cognitive modulation, is constructed from neurons produced during gestation in the adjacent ganglionic eminences (GEs). GEs are transient structures in the ventral prenatal brain that also generate GABAergic inhibitory neurons which migrate to destinations in the BG, cortex and other destinations. This study aims to elucidate the epigenomic and 3D-genomic dynamics involved in the specification and maturation of GEs and GE-derived neurons, using single-nucleus methyl-3C sequencing (snm3C-seq), highly-multiplexed spatial transcriptomics, and chromatin+RNA single-molecule imaging. Our multi-modal data support a heterogeneous temporal progression across GE subregions, with the lateral GE (LGE) showing declining neurogenic activity in mid-gestation and caudal GE (CGE) exhibiting ongoing developmental progression through infancy. We identified regulatory programs that specify subtypes of BG principal cells, medium spiny neurons (MSN), via synchronized maturation of the 3D-epigenome. In infant brains, we found a transient short-range enriched (SE) chromatin conformation during the transition between oligodendrocyte progenitors (OPCs) and oligodendrocytes (ODCs), and a temporary shift toward Long-range Enriched (LE) chromatin conformation in projection neurons, extending previous works showing the differentiation of neurons and glial cells is associated with permanent SE and LE conformation, respectively. Lastly, we found that gene regulatory regions active in MSNs were enriched in loci associated with genetic risk for neuropsychiatric disease. Our study delineates the highly complex, lineage-specific 3D genomic dynamics in ventral progenitors and basal ganglia populations of the perinatal human brain. HighlightsO_LIJoint 3D genome and DNA methylome analysis of ventral brain progenitor zones C_LIO_LIHeterogeneous developmental progressions of the ganglionic eminences C_LIO_LIDistinct development dynamics and regulatory landscape of MSNs and interneurons C_LIO_LITransient remodeling of the 3D-genome in neurons and oligodendrocyte progenitors C_LI

genomics↗

Multimodal spatial transcriptomics determines repeat expansion, huntingtin aggregation, and selective cortical neuron loss in Huntington Disease

Huntingtons disease (HD) is caused by CAG expansion in HTT, yet how somatic repeat instability and huntingtin aggregation relate to selective cell loss in the human brain remains unclear. We have developed a multimodal spatial transcriptomics approach that enables defining transcriptional programs with subcellular resolution, somatic CAG repeat lengths, and six other pathology marks including huntingtin aggregates in every cell of intact brain sections. Imaging 428,173 cells in HD cortex revealed selective vulnerability: L5-6 NP and L6b deep-layer excitatory neurons undergo >50% loss, closely linked to very large (>380{+/-}55) somatic expansions. Intranuclear aggregation was most prevalent at intermediate somatic repeat expansion (220-300 CAGs) and was accompanied by broader transcriptional changes. In contrast, chandelier and somatostatin+ inhibitory interneurons are lost despite only modest repeat expansion or aggregation. These data provide a comprehensive resource and establish a broadly applicable framework for connecting repeat expansion and protein pathology across diverse cell types. Short Bullet pointsO_LIDevelopment of a novel, multimodal spatial transcriptomics platform enables definition of RNA transcriptomes with subcellular resolution, somatic repeat expansions, and protein accumulation in cells within tissue sections C_LIO_LIGeneration of two complementary datasets for HD and control cortex: 428,173 cells to quantify comprehensively cell-type vulnerability, and a deeper multimodal dataset in which CAG repeat expansion, huntingtin aggregation, six additional pathological readouts, and expression of 1,128 genes were measured in 185,721 cells, providing a comprehensive resource for the neurodegeneration community. C_LIO_LIDeep layer excitatory neuron loss (L5-6 NP and L6b) was associated with very large somatic CAG expansions (>380{+/-}55), while selective inhibitory neuron loss (chandelier and somatostatin interneurons) occurred with modest CAG repeat expansion or huntingtin intranuclear aggregation C_LIO_LIIntranuclear aggregation, not somatic repeat expansion, was more predictive of transcriptional changes, including chromatin remodeling and RNA export factors C_LI

neuroscience↗

Multiscale Spatial Transcriptomic Atlas of Human Basal Ganglia Cell-Type and Cellular Community Organization

We generated a multi-region, subcellular-resolution spatial transcriptomic atlas of the human basal ganglia by integrating MERFISH+ and Stereo-seq across four neurotypical donors. These datasets profiled [~]7 million cells spanning the caudate, putamen, nucleus accumbens, and globus pallidus, resolving 60 transcriptionally distinct cell types. We show region-selective, molecular and spatial diversification of medium-spiny-neuron cell types and multiple non-neuronal populations with distinct molecular identities and spatial localizations. Subcellular RNA localization captures somatic size and projection-inferred signatures that reflect direct and indirect pathway topology. Cellular community analyses reveal the enrichment of sub-clusters of astrocytes and oligodendrocytes at striosome-matrix borders, while primate-expanded interneurons are confined to matrix territories. Cross-species mapping uncovers orthologous striosome-matrix organization and conserved dorsolateral-ventromedial gene expression gradients. This atlas provides a foundational molecular and spatial framework for studying human basal ganglia architecture, offering a multi-centimeter scale resource that links cell types, spatial architecture, and subcellular transcript topography across multiple nuclei. HighlightsO_LIOur multi-centimeter scale spatial taxonomy identifies the precise locations of 60 neuronal and glial cell types of human basal ganglia. C_LIO_LIMERFISH+ and Stereo-seq platforms map consistent spatial modules that align with classical neuroanatomical nuclei. C_LIO_LID1D2 hybrid MSNs and primate-expanded interneurons show regional and domain specific organization C_LIO_LISubcellular RNA localization reports soma morphology and projection-inferred signatures. C_LI

neuroscience↗

MERFISH+, a large-scale, multi-omics spatial technology resolves the molecular holograms of the 3D human developing heart

Hybridization-based spatial transcriptomics technologies have advanced our ability to map cellular and subcellular organization in complex tissues. However, existing methods remain constrained in gene coverage, multimodal compatibility, and scalability. Here, we present MERFISH+, an enhanced version of Multiplexed Error-Robust Fluorescence in Situ Hybridization (MERFISH), which integrates chemical probe anchoring in protective hydrogels with high-throughput microfluidics and microscopy. This optimized design supports robust and repeated hybridization cycles across an entire centimeter-scale tissue sample. MERFISH+ allowed to simultaneously quantify over 1,800 genes and resolve the 3D organization of chromatin loci and their associated epigenomic marks in developing human hearts. Using a generative integration framework for spatial multimodal data (Spateo-VI), we harmonized these MERFISH+ transcriptomic and chromatin data to reconstruct a 3D spatially-resolved multi-omic atlas of the developing human heart at subcellular resolution capturing 3.1 million cells across 34 distinct populations. This 3D atlas provides a holistic view of an entire organ enabling the characterization of 3D cellular neighborhoods and transcriptional gradients of substructures such as the descending arteries. Thus, MERFISH+ offers a robust, large-format platform for spatial multi-omics that enables high resolution mapping of gene expression at subcellular resolution and the characterization of cellular organization within 3D organs. One Sentence SummaryMERFISH+ is an spatial multi-omics platform that integrates hydrogel-based probe anchoring, automated high-throughput microfluidics, and large-format multimodal data production to enable comprehensive, subcellular resolution mapping of gene expression and chromatin organization across millions of cells within complex developing human organs. HighlightsO_LIMERFISH+ expands MERFISH capabilities to measure >1,800 genes and at whole-organ 3D imaging scale C_LIO_LICombines chemical probe anchoring with high-throughput volumetric microscopy and microfluidics C_LIO_LIGenerates a 3D molecular atlas of a developing human heart with > 3.1 million cells at subcellular resolution C_LIO_LIIntroduces Spateo-VI, a novel generative framework integrating 3D multimodal datasets C_LI

genomics↗

A spatial code governs olfactory receptor choice and aligns sensory maps in the nose and brain

Although topographical maps organize many peripheral sensory systems, it remains unclear whether olfactory sensory neurons (OSNs) choose which of the [~]1100 odor receptors (ORs) to express based upon their spatial location in the olfactory epithelium (OE) or instead ORs are scattered randomly. Here we reveal that each OR is expressed at a precise mean position along the OE dorsoventral axis, thereby instantiating a receptor map. This patterning reflects the differential use, by precursors and mature OSNs, of a coherent gene expression program controlled by a spatially-varying retinoic acid gradient; this program -- which includes key transcription factors and axon guidance genes -- translates position into a spatially appropriate distribution of OR choices and aligns the epithelial map of OR identity with the glomerular map present in the olfactory bulb. These results identify a transcriptional code that distinguishes and spatially organizes the vast array of sensory channels that comprise the olfactory system.

neuroscience↗

Molecular and Spatial Organization of the Primary Olfactory System and its Responses to Social Odors

The detection of olfactory cues is essential to signal food, predators, and social encounters. To determine how the sensory detection of physiologically relevant odors is systematically mapped into the mouse primary olfactory system, we used Multiplexed Error Robust Fluorescent In Situ Hybridization (MERFISH) to construct a molecular atlas of odorant receptor (OR) expression in the main olfactory epithelium (MOE) and olfactory bulb (OB). We comprehensively quantified the expression of the mouse OR repertoire and uncovered stereotypical gradients of sensory neuron distribution in the MOE along two, central-to-peripheral and basal-to-apical, axes. Projections of sensory neurons mirror MOE gradients along the dorsal-ventral and anterior-posterior axes of the OB, respectively. Integration with sequencing data revealed candidate signaling molecules underlying this spatial organization. Co-imaging OR and activity marker expression identified distinct spatial domains of sensory responses in the MOE and OB, providing a topographical basis for olfactory responses to ethologically relevant odors.

neuroscience↗

Combined transcriptomic, connectivity, and activity profiling of the medial amygdala using highly amplified multiplexed in situ hybridization (hamFISH)

In situ transcriptomic technologies provide a promising avenue to link gene expression, connectivity, and physiological properties of neural cell types. Commercialized methods that allow the detection of hundreds of genes in situ, however, are expensive and therefore typically used for generating unimodal reference data rather than for resource-intensive multimodal analyses. A major bottleneck is the lack of a routine means to efficiently generate cell type data. Here, we have developed hamFISH (highly amplified multiplexed fluorescence in situ hybridization), which enables the sequential detection of 32 genes using multiplexed branched DNA amplification. We used hamFISH to profile the projection, activity, and transcriptomic diversity of the medial amygdala (MeA), a critical node for innate social and defensive behaviors in mice. In total, we profiled 643,834 cells and classified neurons into 16 inhibitory and 10 excitatory types, many of which were found to be spatially clustered. We then examined the organization of outputs of these cells and activation profiles during different social contexts. Therefore, by facilitating multiplexed detection of single molecule RNAs, hamFISH provides a streamlined and versatile platform for multimodal profiling of specific brain nuclei.

neuroscience↗

Whole-embryo Spatial Transcriptomics at Subcellular Resolution from Gastrulation to Organogenesis

Spatiotemporal patterns of gene expression underlie embryogenesis. Despite progress in single-cell genomics, mapping these patterns across whole embryos with comprehensive gene coverage and at high resolution has remained elusive. Here, we introduce a whole-embryo imaging platform using multiplexed error-robust fluorescent in-situ hybridization (weMERFISH). We quantified the expression of 495 genes in whole-mount zebrafish embryos at subcellular resolution. Integration with single-cell multiomics data generated an atlas detailing the expression of 25,872 genes and the accessibility of 294,954 chromatin regions, explorable with an online interface MERFISHEYES (beta version). We found that temporal gene expression aligns with cellular maturation and morphogenetic movements, diverse expression patterns correspond to composites of tissue-specific accessible elements, and changes in gene expression generate sharp boundaries during gastrulation. These results establish a novel approach for whole-organism spatial transcriptomics, provide a comprehensive spatially resolved atlas of gene expression and chromatin accessibility, and reveal the diversity, precision and emergence of embryonic patterns.

developmental biology↗

Re-activation of neurogenic niches in aging brain

Recent studies proposing induced glia-to-neuron conversion raised the potential for generating new neurons to replace those lost due to injury, aging or neurodegenerative diseases. Here, single-cell spatial transcriptomics [Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH)] is used to construct a spatial cell atlas of the subventricular and dentate gyrus neurogenic niches of young and aged adult murine brain. RNAs that encode the RNA binding protein Polypyrimidine Tract-Binding Protein (PTBP1) in the aged murine brain are determined to be highest in glia that line previously active neurogenic niches. A glial cell population with ependymal character within an initially quiescent subventricular neurogenic niche in the aged murine brain is identified that upon transient suppression of PTBP1 reenters the cell cycle, replicates DNA, and converts into neurons through a canonical adult neurogenesis pathway. Glia-derived neurons migrate from this niche, with some neurons transiting to the striatum and acquiring a transcriptome characteristic of GABAergic inhibitory neurons. Similar PTBP1 expressing quiescent glia are identified in the corresponding neurogenic niche of aged human brain. Thus, transient reduction of PTBP1 holds potential for inducing the generation of new neurons in quiescent neurogenic niches of the aged nervous system, thereby offering promising therapeutic applications. Bullet point summary1) Single-cell spatial transcriptomics is used to validate active neurogenesis in the two neurogenic niches of the young adult murine brain, determine that those niches are quiescent in the aging adult brain of mice, and demonstrate the absence of neurogenesis in the aging human brain. 2) The RNA binding protein PTBP1 is determined to be most highly expressed within glia that line the aged murine and human neurogenic niches, with its transient reduction sufficient in mice to activate/re-activate expression of genes characteristic of immature neurons. 3) Suppression of PTBP1 using a single intra-cerebral-ventricular injection of PTBP1-targeting antisense oligonucleotide (ASO) induces generation of new immature neurons in the neurogenic niches of the aged mouse brain via a canonical adult neurogenesis pathway. 4) Single-cell RNA signature tracing is used to identify a) a subclass of ependymal cells in a previously quiescent neurogenic niche of the aged mouse brain that convert into GABAergic inhibitory neurons following transient suppression of PTBP1, and b) the molecular steps in the conversion process including cell cycle re-entry, DNA replication, and transcriptome changes that mimic canonical neurogenesis. 5) A similar class of PTBP1-expressing ependymal cells lining the ventricle of the aging non-human primate and human brains is identified, suggesting the promise of re-activation of neurogenesis as a therapeutic approach in humans.

neuroscience↗

Super-resolution stimulated Raman Scattering microscopy with A-PoD

Unlike traditionally-mapped Raman imaging, stimulated Raman scattering (SRS) imaging achieved the capability of imaging metabolic dynamics and a greatly improved signal-noise-ratio. However, its spatial resolution is still limited by the numerical aperture or scattering cross-section. To achieve super-resolved SRS imaging, we developed a new deconvolution algorithm - Adam optimization-based Pointillism Deconvolution (A-PoD) - for SRS imaging, and demonstrated a spatial resolution of 52 nm on polystyrene beads. By changing the genetic algorithm to A-PoD, the image deconvolution process was shortened by more than 3 orders of magnitude, from a few hours to a few seconds. By applying A-PoD to spatially correlated multi-photon fluorescence (MPF) imaging and deuterium oxide (D2O)-probed SRS (DO-SRS) imaging data from diverse samples, we compared nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in lipid droplets using A-PoD coupled with DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal the metabolic change in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic co-localization of biomolecules and metabolic dynamics in organelles. We expect that the A-PoD algorithm will have a wide range of applications, from nano-scale measurements of biomolecules to processing astronomical images.

bioengineering↗