Biology subjects

Adameyko, I.

Publications and source records attributed to Adameyko, I..

At least 19 recordsLinked to original sources

bioRxiv2026

Connectomics by Sequencing Reveals Self-Organizing Principles of Neuronal Networks in Cerebral Organoids

Functional neural networks emerge as developing neurons form synaptic connections. Revealing how these connections are organized and perturbed in disease requires linking single-neuron connectivity to molecular state across thousands of networks, which is challenging with current methods. We developed COSCO (Connectomics by Sequencing in Cerebral Organoids), combining barcoded monosynaptic rabies tracing with single-cell RNA sequencing to reconstruct over 10,000 networks across organoids. We demonstrate that organoid circuitry self-organizes into stereotyped network motifs governed by cell-intrinsic wiring rules aligning with features of mouse cortical connectivity. Projecting network topology onto transcriptomic space reveals that wiring correlates with cell-adhesion and synaptic gene expression programs. In a Tuberous Sclerosis Complex model, pathological hyperexcitability coincides with topological rewiring: mutant networks become more heterogeneous, driven by the transcriptional distribution of upper layer excitatory cell states. This work establishes cerebral organoids as a model to study self-organization of human connectivity, and its disruption in neurodevelopmental disease. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/755792v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@50ade8org.highwire.dtl.DTLVardef@c97320org.highwire.dtl.DTLVardef@1a53a47org.highwire.dtl.DTLVardef@9520ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

In utero barcoding of mouse mesoderm reveals the clonal architecture of organ mesenchyme

The mesoderm coordinates organogenesis and generates the cardiovascular, visceral and musculoskeletal systems, yet in mammals this germ layer is difficult to access, impeding its genetic manipulation and study. Here, we establish embryonic day (E) 7.5 exocoelomic cavity nano-injection as a scalable route for selective genetic targeting of mouse mesoderm. Combining in utero lentiviral barcoding with single-cell and spatial transcriptomics, we reconstructed mesodermal and neural crest clonal relationships across >590,000 cells and 31,000 multicellular clones from E9.5-E10.5 embryos and E16.5 livers and hearts. In liver, capsular mesothelium was closely related to hepatic stellate cells, whereas fibroblast and vascular smooth muscle lineages diverged earlier. In heart, epicardium was clonally coupled to three spatially restricted mesenchymal sub-lineages but not to cardiomyocytes, and lineage tracing separated epicardial- and neural crest-derived populations within transcriptionally convergent valve mesenchyme. This approach enables Cre-independent manipulation of mammalian mesoderm and resolves organ mesenchyme development at clonal resolution.

developmental biology↗

bioRxiv2026

A uniform tissue-clearing framework and mesoSPIM-ultra enable cm-scale single-neuron tracing

Tissue-clearing and light-sheet microscopy have transformed volumetric imaging of intact organs, yet limited mechanistic understanding of dehydration-based clearing continues to constrain rational protocol design and broader applicability. Here, we define the cardinal chemical and physical principles underlying dehydration-based tissue-clearing and establish a new pipeline for large-volume imaging. To maximize imaging performance, we developed the "mesoSPIM-ultra", an upgraded mesoSPIM platform with a temperature-controlled sample chamber, a large field-of-view (FoV) camera and specialized optics to achieve long-working-distance, high-resolution imaging of cleared samples. We applied this approach to investigate the projectome of Chx10+ neurons, a cell population with complex axonal morphologies along the entire mouse spinal-cord and brain, and implicated in ipsilateral orienting behaviors. By combining behavioral analysis with post-hoc single-neuron reconstructions, we revealed previously inaccessible branching architectures and long-range projections extending from the brainstem to the spinal cord. Together, our work establishes a mechanistic foundation for tissue-clearing and scalable imaging.

neuroscience↗

bioRxiv2026

Fibroblast-derived thrombospondin-1 shapes macrophage polarization in advanced human co-culture models

BackgroundTumor-associated macrophages (TAMs) are key drivers of the immunosuppressive tumor microenvironment (TME), supporting tumor progression through diverse functions. However, mechanistic studies of TAM polarization remain limited by the lack of physiologically relevant human model systems that capture stromal-immune interactions and macrophage heterogeneity. MethodsWe established advanced human co-culture systems that integrate healthy donor-derived macrophages with patient-derived organoids and tumoroids (PDOs and PDTs), as well as matched normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). These multicellular models enabled the investigation of interactions among stromal, epithelial, and immune cells within tumor and adjacent normal tissue environments. ResultsThe co-culture systems recapitulated distinct macrophage states associated with tumor and adjacent normal environments and identified fibroblasts as major regulators of macrophage phenotypes. CAFs promoted macrophage metabolic remodeling characterized by altered lipid handling and enrichment of TAM-like signatures. Mechanistically, we identified thrombospondin 1 (TSP1) as a CAF-secreted factor linked to metabolic priming. Recombinant TSP1 induced transient lipid accumulation followed by mitochondrial remodeling. In tumor co-culture conditions, CD36 inhibition reduced lipid accumulation in macrophages, supporting a role for TSP1-linked lipid crosstalk in stromal-immune interactions. ConclusionOur study establishes advanced patient-derived co-culture models as a platform to investigate human TAM biology and stromal-immune interactions in CRC. Using these systems, we identify a fibroblast-associated TSP1-lipid axis linked to macrophage metabolic remodeling and TAM-like polarization, highlighting stromal metabolic communication as a potential targetable feature of the CRC microenvironment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=176 HEIGHT=200 SRC="FIGDIR/small/728363v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@11b8c9borg.highwire.dtl.DTLVardef@1ac3aa2org.highwire.dtl.DTLVardef@3da223org.highwire.dtl.DTLVardef@5fc3a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

Combinatorial transcription factor (TF) codes specify neuronal fates, yet how quantitative differences in interacting TFs shape these decisions remains unresolved. We address this in the developing basal ganglia, where a pool of undifferentiated progenitors gives rise to both D1 and D2 medium spiny neurons (MSNs). Combining sparse in vivo CRISPR perturbation, lineage barcoding, and single-cell transcriptomics in mice, we find that loss of Sp9 shifts clonal fate bias from D2 toward D1 MSNs and intercalated cells. Mechanistically, chromatin profiling and biochemical assays show that at GC-rich promoters, SP9 binds DNA directly and activates transcription. At distal enhancers, SP9 binds indirectly, tethered by DLX factors, represses activity, and associates with the NuRD corepressor complex. The relative abundance of SP9 and DLX selects between these modes. These findings extend the combinatorial TF code beyond factor identity to relative proportions, with relevance to neurodevelopmental disorders.

developmental biology↗

bioRxiv2026

Clonal embeddings allow exploratory analysis of lineage-resolved single-cell data

Assays coupling high-throughput lineage tracing with single-cell transcriptomics are transforming studies of development and disease biology, revealing not only major differentiation routes but also continuous fate biases and their putative regulators. Yet, analysis of such data at scale presents challenges due to the sparse nature of clonal data and annotation dependencies. Towards that aim we developed a machine learning approach - clone2vec - which learns informative clone embeddings directly from the cellular expression manifold, bypassing discrete cell-type labels and remaining stable when clones are represented by few cells. This representation summarizes clonal variation as an interpretable geometry that supports exploration, statistics for clone-gene associations, and cross-dataset alignment. In prospective barcoding datasets spanning embryogenesis, tumorigenesis, and hematopoiesis, clone2vec recapitulates established clonal patterns and uncovers new axes of continuous variation that implicate regulatory programs and developmental pathways. In tumor microenvironments profiled with TCR sequencing, clone2vec robustly recovers distinct Treg lineages as well as conserved CD8+ T cell sublineages across cancer types, including several bystander-like clonal subsets. Overall, clone2vec provides a robust, general solution for the exploratory analysis of lineage-coupled scRNA-seq data.

bioinformatics↗

bioRxiv2026

Single-cell, clonal and spatial atlases of cranial placodes illuminate their specification and evolution

The vertebrate head is defined by complex sensory structures derived from cranial placodes. Placodes arise alongside the neural crest at the neural plate border, yet the mechanisms governing their identity, diversification, and evolutionary origins are unclear. We present an integrated single-cell, spatial, and clonal atlas of placode development to resolve the dynamics of their lineage segregation. Combining single-cell RNA-sequencing, spatial transcriptomics, and high-resolution clonal tracing, we show that placodal and neighboring progenitors form a continuous transcriptional landscape with gradual transitions between domains. Domain boundary cells co-express markers of adjacent territories, suggesting transient bipotent states. Consistent with this, clonal analysis reveals sharing of progenitors between neighboring placodes, supporting a model of competitive segregation. Comparisons with amphioxus suggests that vertebrate olfactory placodes emerged from an ancestral neuroectoderm that later partitioned into distinct neural and olfactory domains. Our findings provide a unified framework for understanding the developmental and evolutionary origins of vertebrate sensory organs.

developmental biology↗

bioRxiv2026

High-resolution retrospective single cell lineage tracing with mutable homopolymers

Retrospective reconstruction of cell lineages from somatic mutations holds substantial promise for understanding development, tissue homeostasis, and disease. Yet, current approaches are often constrained by low marker density, technical noise, and incomplete molecular readouts that limit the resolution and accuracy of inferred lineages. Here, we present RETrace2, a single-cell dual-omic method for simultaneous lineage tracing and cell-type identification. By identifying and targeting highly mutable homopolymers, which we show are approximately twice as informative as conventional microsatellite repeats, combined with technical improvements to increase marker coverage and reduce experimental errors of reading and amplifying homopolymers, RETrace2 can trace lineage continuously at an estimated resolution of fewer than five cell divisions in microsatellite-unstable organisms. Simultaneously, sparse methylation profiles were obtained from the same cells for cell-type identification. We validated the method using in vitro ground-truth models and demonstrated its utility in vivo by reconstructing multi-organ lineage trees from the brain, kidney and liver of Msh2-deficient mice.

genetics↗

bioRxiv2025

Cell Cycle Arrest of a "Zippering" Epithelial Cell Cluster Shapes the Face and is Disrupted in Craniofacial Disorders

Facial features identify individuals, but the mechanisms shaping the human face remain elusive. Orofacial clefting (OFC), the most common craniofacial abnormality, results from failed fusion of the facial prominences that is in part caused by persistence of the cephalic epithelium. Here we uncover the identity, behaviors, and molecular blueprints of a novel craniofacial epithelial population, the Zippering Lambda (ZL), which mediates prominence fusion and is characterized by cell cycle arrest in mouse and human embryos. Remarkably, cell cycle is unleashed in the ZL of Pbx1/2 and p63 mutant mice with OFC. Intersection of ZL-enriched genes with human OFC whole-genome sequencing datasets identifies ZFHX3 variants in affected individuals and cephalic epithelial Zfhx3 deletion causes murine OFC. ZFHX3 and PBX1 genetically interact and synergistically regulate cell cycle inhibitor genes within a complex in embryonic faces. Collectively, we deconstruct new mechanisms that pattern the face, connecting cell cycle arrest to developmental tissue fusion.

developmental biology↗

bioRxiv2025

A transcriptional atlas of the pubertal human growth plate reveals direct stimulation of cartilage stem cells by growth hormone.

The cartilaginous growth plate is a critical organ responsible for longitudinal bone growth. It remains open throughout life in mice but closes in humans after puberty. Growth hormone (GH) is a widely used therapy for children with growth retardation and open growth plates. However, it remains unclear whether GH directly targets human growth plates. Furthermore, while cartilage stem cells have recently been identified in mouse growth plates, their presence and GH responsiveness in human growth plates are unknown. To address these gaps, we characterized the cellular and molecular organization of early pubertal human growth plates using unique tissue samples obtained during growth-restricting surgeries. Our analysis identified two distinct populations of stem cells differing in cycling activity, molecular profiles, and regulatory factors. Quiescent stem cells were localized within a niche characterized by low Wnt and TGF{beta} signaling. To investigate the direct effects of GH, we developed a human growth plate explant culture system. GH directly stimulated explant growth and promoted stem cell proliferation by activating the JAK/STAT, TGF{beta}, and ERK pathways while inhibiting the AKT pathway. Notably, activation of the TGF{beta} pathway occurred in an autocrine manner. These findings provide critical new insights into human longitudinal growth and the mechanisms of GH action, with potential implications for optimizing treatments for growth disorders. One Sentence SummaryThis study reveals that growth hormone (GH) directly promotes proliferation within the human growth plate and activates TGF{beta} and ERK signaling pathways in cartilage stem cells, providing critical insights into human longitudinal growth and potential improvements in treatments for growth disorders.

cell biology↗

bioRxiv2024

The main duct of von Ebner's glands is a source of Sox10+ taste bud progenitors and susceptible to pathogen infections

We have recently demonstrated that Sox10-expressing (Sox10+) cells give rise to mainly type-III neuronal taste bud cells that are responsible for sour and salt taste. The two tissue compartments containing Sox10+ cells in the surrounding of taste buds include the connective tissue core of taste papillae and von Ebners glands (vEGs) that are connected to the trench of circumvallate and foliate papillae. In this study, we used inducible Cre mouse models to map the cell lineages of connective tissue (including stromal and Schwann cells) and vEGs and performed single cell RNA-sequencing of the epithelium of Sox10-Cre/tdT mouse circumvallate/vEG complex. In vivo lineage mapping showed that the distribution of traced cells in circumvallate taste buds was closely linked with that in the vEGs, but not in the connective tissue. Sox10, but not the known stem cells marker Lgr5, expression was enriched in the cell clusters of main ducts of vEGs that contained abundant proliferating cells, while Sox10-Cre/tdT expression was enriched in type-III taste bud cells and excretory ductal cells. Moreover, multiple genes encoding pathogen receptors are enriched in the vEG main ducts. Our data indicate that the main duct of vEGs is a source of Sox10+ taste bud progenitors and susceptible to pathogen infections.

cell biology↗

bioRxiv2024

Spatial Dynamics of the Developing Human Heart

Heart development relies on a topologically defined interplay between a diverse array of cardiac cells. We finely curated spatial and single-cell measurements with subcellular imaging-based transcriptomics validation to explore spatial dynamics during early human cardiogenesis. Analyzing almost 80,000 individual cells and 70,000 spatially barcoded tissue regions between the 5.5th and 14th postconceptional weeks, we identified 31 coarse- and 72 fine-grained cell states and mapped them to highly resolved cardiac cellular niches. We provide novel insight into the development of the cardiac pacemaker-conduction system, heart valves, and atrial septum, and decipher heterogeneity of the hitherto elusive cardiac fibroblast population. Furthermore, we describe the formation of cardiac autonomic innervation and present the first spatial account of chromaffin cells in the fetal human heart. We support independent exploration of our datasets by an open-access, spatially centric interactive viewer. In summary, our study delineates the cellular and molecular landscape of the developing hearts architecture, offering links to genetic causes of heart disease.

developmental biology↗

bioRxiv2022

β-catenin-dependent endomesoderm specification appears to be a Bilateria-specific co-option

Endomesoderm specification based on a maternal {beta}-catenin signal and axial patterning by interpreting a gradient of zygotic Wnt/{beta}-catenin signalling was suggested to predate the split between Bilateria and their evolutionary sister Cnidaria. However, in Cnidaria, the roles of {beta}-catenin signalling in both these processes have not been proven directly. Here, by tagging the endogenous {beta}-catenin protein in the sea anemone Nematostella vectensis, we show that the oral-aboral axis in a cnidarian is indeed patterned by a gradient of {beta}-catenin signalling. Unexpectedly, in a striking contrast to Bilateria, Nematostella endoderm specification takes place opposite to the part of the embryo, where {beta}-catenin is translocated into the nuclei. This suggests that {beta}-catenin-dependent endomesoderm specification is a Bilateria-specific co-option, which may have linked endomesoderm specification with the subsequent posterior-anterior patterning.

developmental biology↗

bioRxiv2022

Decoding spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin

The human spinal cord contains diverse cell types, governed by a series of spatiotemporal events for tissue assembly and functions. However, the spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. Single-cell RNA sequencing and spatial transcriptomics techniques have advanced the understanding of human organ development considerably. By performing integrated analysis of single-cell and spatial multi-omics methods, we created a comprehensive developmental cell atlas of the first trimester human spinal cord. Our data revealed that the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. Beyond this resource, we unexpectedly discovered unique events in human spinal cord development compared to rodents, including earlier quiescence of active neural stem cells, different regulation of stem cell differentiation, and distinct spatiotemporal genetic regulations of cell fate choices. In addition, using our atlas we identified specific gene expression in cancer stem cells in ependymomas. Thus, we demonstrate spatiotemporal genetic regulation of human spinal cord development as well as its potential to understand novel disease mechanisms and to inspire new therapies.

developmental biology↗

bioRxiv2022

Early pre-neural serotonin modulates balance of late monoamines and behavioral patterns in fish model system

The presence of serotonergic system during early pre-neural development is enigmatic and conserved amongst all studied invertebrate and vertebrate animals. We took advantage of zebrafish model system to address what is the role of early serotonin before first neurons form. Unexpectedly, we experimentally revealed the existence of delayed developmental neurogenic and behavioral effects resulting from the manipulations of pre-neural (zygote, blastula and gastrula) serotonergic system. In particular, the delayed effects included differences in the synthesis of serotonin in early serotonergic neurons in the central nervous system as well as in behavioral alterations after habituation in zebrafish larvae. These effects appeared as highly specific and did not coincide with any major abnormalities. The same manipulations of the serotonergic system at neural developmental stages did not show such effects, which confirms that early effects of serotonergic system manipulation are not based on retained serotonin in embryonic cells. Accordingly, gene expression analysis demonstrated specific changes only in response to the elevation of early pre-neural serotonin, which included the delayed and pre-mature onsets of different gene expression programs. Taken together, our results introduce a novel function of early pre-neural serotonergic system in a vertebrate embryo - tuning and fine control of specific mechanisms at later neural developmental stages that result in a mild variation of a behavioral adaptive spectrum.

developmental biology↗

bioRxiv2022

A branching model of cell fate decisions in the enteric nervous system

How neurogenesis and gliogenesis are coordinated during development and why mature glial cells often share properties with neuroectodermal progenitors remains unclear. Here, we have used single cell RNA sequencing to map the regulatory landscape of neuronal and glial differentiation in the mammalian enteric nervous system (ENS). Our analysis indicates that neurogenic trajectories branch directly from a linear gliogenic axis defined by autonomic neural crest cells adopting sequential states as they progressively lose their strong neurogenic bias and acquire properties of adult enteric glia. We identify gene modules associated with transcriptional programs driving enteric neurogenesis and cell state transitions along the gliogenic axis. By comparing the chromatin accessibility profile of autonomic neural crest and adult enteric glia we provide evidence that the latter maintain an epigenetic memory of their neurogenic past. Finally, we demonstrate that adult enteric glia maintain neurogenic potential and are capable of generating enteric neurons in certain contexts by activating transcriptional programs employed by early ENS progenitors. Our studies uncover a novel configuration of enteric neurogenesis and gliogenesis that enables the coordinate development of ENS lineages and provides a mechanistic explanation for the ability of enteric glia to be functionally integrated into the adult intestine and simultaneously maintain attributes of early ENS progenitors.

neuroscience↗

bioRxiv2022

scFates: a scalable python package for advanced pseudotime and bifurcation analysis from single cell data

scFates provides an extensive toolset for analysis of dynamic trajectories comprising tree learning, feature association testing, branch differential expression and with a focus on cell biasing and fate splits at the level of bifurcations. It is meant to be fully integrated into scanpy ecosystem for seamless analysis of trajectories from single cell data of various modalities (e.g. RNA, ATAC). Availability and implementationscFates is released as open-source software under the BSD 3-Clause "New" License and is available from the Python Package Index at https://pypi.org/project/scFates/. The source code is available on Github at https://github.com/LouisFaure/scFates/ Supplementary informationA supplementary document is provided with a complete explanation of the underlying statistics, and two figures showing examples of analysis.

bioinformatics↗

bioRxiv2022

Contributions of cell behavior to geometric order in embryonic cartilage

During early development, cartilage provides shape and stability to the embryo while serving as a precursor for the skeleton. Correct formation of embryonic cartilage is hence essential for healthy development. In vertebrate cranial cartilage, it has been observed that a flat and laterally extended macroscopic geometry is linked to regular microscopic structure consisting of tightly packed, short, transversal clonar columns. However, it remains an ongoing challenge to identify how individual cells coordinate to successfully shape the tissue, and more precisely which mechanical interactions and cell behaviors contribute to the generation and maintenance of this columnar cartilage geometry during embryogenesis. Here, we apply a three-dimensional cell-based computational model to investigate mechanical principles contributing to column formation. The model accounts for clonal expansion, anisotropic proliferation and the geometrical arrangement of progenitor cells in space. We confirm that oriented cell divisions and repulsive mechanical interactions between cells are key drivers of column formation. In addition, the model suggests that column formation benefits from the spatial gaps created by the extracellular matrix in the initial configuration, and that column maintenance is facilitated by sequential proliferative phases. Our model thus correctly predicts the dependence of local order on division orientation and tissue thickness. The present study presents the first cell-based simulations of cell mechanics during cranial cartilage formation and we anticipate that it will be useful in future studies on the formation and growth of other cartilage geometries. Author SummaryIn embryos, the initial skeleton is made out of cartilage. As the embryo grows, this cartilage needs to increase in size while correctly maintaining shape. A recent study revealed that for cartilage found in growing skulls, a flat sheet-like geometry is reflected in a distinct arrangement of cells at the microscopic level. Cells sharing a common ancestor are arranged into short columns such that the sheet grows in thickness by lengthening columns, and expands length-wise by adding new columns from single precursor cells. In this work we investigate the mechanical principles underlying column formation and insertion using a computational model that individually represents cells and their behavior. We confirm that arrangement of clonal columns perpendicular to the main expansion direction of the sheet requires oriented cell division. Moreover, we find that column order benefits from an increased amount of extracellular matrix between cells. Similarly, our model suggests that new clonal columns are able to insert themselves into pre-existing cartilage if sufficient matrix is available. Our model constitutes an important step to study cartilage formation and growth in different geometries which will be useful for understanding skeletal developmental disorders as well as for applications in tissue engineering.

developmental biology↗