Biology subjects

Novatchkova, M.

Publications and source records attributed to Novatchkova, M..

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

Cerebral organoids recapitulate interneuron chain migration in the human cortex

The human brain is incomplete at birth, with streams of late-born interneurons migrating into the postnatal cortex. This process is absent in rodents, limiting mechanistic investigation to post-mortem tissue. Here, we show that human cerebral organoids cultured for over six months recapitulate postnatal interneuron migration, enabling its mechanistic and genetic dissection. Live imaging and mathematical modeling revealed that interneurons intrinsically migrate in chains, while astrocyte interactions promote stream organization. Spatial and single-cell transcriptomics identified a distinct molecular signature of human postnatal migratory streams enriched for neurodevelopmental disorder-associated genes. Perturbation of the autism-associated genes CNTN5 and FGF14 differentially disrupted chain organization and migration. Thus, long-term cerebral organoids enable mechanistic analysis of previously inaccessible aspects of postnatal human brain development and their contributions to neurodevelopmental disease.

developmental biology↗

bioRxiv2026

Dissecting human fetal cardiac repair using cardioids

Human cardiac injury responses are governed by dynamic interacting processes that are difficult to resolve. Unlike adults, fetal mammalian hearts regenerate through coordinated remodeling and proliferation supported by a pro-regenerative immune environment, extracellular matrix (ECM), and immature cardiomyocytes, including trabecular subtypes. Here, we establish a modular human cardioid injury platform to dissect these interactions. We show that anti-inflammatory macrophages selectively migrate to the injury, clear debris, and promote ECM remodeling, whereas inflammatory macrophages suppress cardiomyocyte proliferation. Synergistic FGF2-NRG1 signaling induces trabecular identity and morphology in a hyaluronan-dependent manner, conferring enhanced injury repair, characterized by cytoskeletal remodeling and cardiomyocyte proliferation mediated by YAP and WNT signaling. Exogenous YAP, but not WNT, is sufficient to promote repair in non-trabecular cardioids. These findings uncover coordinated immune-ECM-cardiomyocyte interactions governing human fetal regenerative competence and mechanistically resolve remodeling and proliferative components of cardiac repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/735236v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1e647a2org.highwire.dtl.DTLVardef@16911f8org.highwire.dtl.DTLVardef@11ad14eorg.highwire.dtl.DTLVardef@1a35d94_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2026

The SPARK complex forms the molecular basis of vertebrate fertilization

Fertilization requires gamete recognition and membrane fusion, yet the molecular basis of this process in vertebrates remains unknown. Here we identify SPARK (sperm protein assembly and receptor-binding key), a conserved multi-protein complex that integrates all known sperm fertilization factors, including TMEM81-IZUMO1-SPACA6 and DCST1/2, together with two newly identified components, TMDD1 and FAM187A. SPARK subunits are mutually dependent for stability in mature sperm, and disruption of any single component causes male sterility in zebrafish and mice. Incubating zebrafish sperm with soluble egg receptor Bouncer partially rescues fertilization of Bouncer-deficient eggs in a SPARK-dependent manner, consistent with egg receptor binding priming the complex for fusion. Thus, we propose SPARK as a con-served molecular machine that couples gamete recognition to membrane fusion.

developmental biology↗

bioRxiv2025

RNA decay via the nuclear exosome is essential for piwi-mediated transposon silencing

Nuclear Argonaute proteins safeguard genome integrity by directing transcriptional silencing and heterochromatin formation at transposon loci. Yet it remains unclear how Argonautes enforce robust repression while relying on target transcription for their own recruitment. Here we show that transposon silencing by the Drosophila nuclear Piwi-piRNA pathway requires degradation of target RNA by the nuclear exosome. Using proximity proteomics at endogenous Piwi target sites, we identify two previously uncharacterized paralogs, TEsup-1 and TEsup-2, as essential cofactors for Piwi-mediated silencing. TEsup proteins act in part by engaging nuclear exosome adaptor complexes at piRNA-targeted transcripts through a domain that recognizes proline-rich peptides. Disruption of the Piwi-TEsup-exosome axis leads to accumulation and nuclear export of piRNA-targeted transposon RNAs. Notably, the P-element--which evades heterochromatin-based repression--is silenced primarily through this RNA-decay pathway. Thus, the nuclear piRNA pathway couples target recognition to RNA degradation, reconciling small RNA-guided heterochromatin formation with ongoing transcription at target loci.

molecular biology↗

bioRxiv2025

Functional rescue of critical-size bone defect using molecular network analysis of axolotl limb regeneration.

The salamander limb has served as a canonical model for successful regeneration that has yielded numerous molecular insights on its basic mechanism. Harnessing such information to induce regeneration in a non-regenerative setting has been a long-sought goal. The amputated salamander limb efficiently regenerates all of its missing bones, but paradoxically, a large bone gap without amputation - commonly called a Critical Size Defect (CSD) - is not regenerated, similarly to other vertebrates1. This non-regenerating injury provides a human-relevant setting to understand how to rescue lack-of-regeneration. Satoh and colleagues demonstrated in axolotl that transplantation of blastema cells from an amputated limb into a CSD yields cartilage bridging of the CSD2. This work provided a roadmap for rescuing the CSD. Here we asked, what are the crucial molecular differences in cells populating a regenerating blastema versus the CSD and can they be used to elicit CSD bridging? Previous genetic fate mapping and single cell transcriptomics showed that limb regeneration occurs via migration and dedifferentiation of fibroblastic, soft connective tissue (CT) cells that form a multipotent, skeletal stem cell 3, 4. Here using single cell transcriptomics and genetic fate mapping we found that the CSD is populated by CT cells that undergo a divergent molecular transition compared to the blastema. Using gene regulatory network (GRN) modeling, and lipid nanoparticle (LNP) delivery of mRNA we could express a single molecular factor, Wnt3a, to induce cartilage bridging of the CSD. Our results demonstrate the power of using molecular information from successful regeneration to rescue a non-regenerating injury.

cell biology↗

bioRxiv2025

The essential role of connective-tissue cells during axolotl limb regeneration

Axolotls (Ambystoma mexicanum) are known for their remarkable limb-regeneration abilities, which involve the formation of the blastema, a specialized structure consisting of progenitor cells contributed by all major tissues of the limb. Lateral plate mesoderm (LPM)-derived connective tissue (CT) cells dedifferentiate and play a critical role in blastema formation and subsequent limb regeneration. However, the complexity of the blastemas cellular composition and the extent of CT participation and necessity have not been rigorously explored. To address this gap, we conducted spatial transcriptomics using a select array of probes, revealing that CT cells constitute up to 75% of the blastema cells at their peak. Genetic ablation of CT cells significantly delays or truncates limb regeneration, underscoring their necessity during this process. Finally, we analyzed the molecular profile of CT cells throughout the stages of blastema formation and made it accessible through an interactive web platform. Our work reaffirms the central role of CT cells in axolotl limb regeneration and lays the foundation for identifying molecular mechanisms that govern blastema formation during the initial phases of limb regeneration.

developmental biology↗

bioRxiv2025

Direct cell-to-cell transmission of retrotransposons

Transposable elements are abundant in host genomes but are generally considered to be confined to the cell in which they are expressed, with the notable exception of endogenous retroviruses. Here, we identify a group of LTR retrotransposons that infect the germline from somatic cells within the Drosophila ovary, despite lacking the fusogenic Envelope protein typically required for retroviral entry. Instead, these elements encode a short transmembrane protein, sORF2, with structural features reminiscent of viral cell-cell fusogens. Through genetics, imaging, and electron microscopy, we show that sORF2 localizes to invasive somatic protrusions, enabling the direct transfer of retrotransposon capsids into the oocyte. Remarkably, sORF2-like proteins are widespread among insect retrotransposons and also occur in piscine nackednaviruses and avian picornaviruses. These findings reveal a noncanonical, Envelope-independent transmission mechanism shared by retrotransposons and non-enveloped viruses, offering important insights into host-pathogen evolution and soma-germline interactions.

genetics↗

bioRxiv2024

Reconstitution of Human Brain Cell Diversity in Organoids via Four Protocols

Human brain organoids are powerful in vitro models for brain development and disease. However, their variability can complicate use in biomedical research and drug discovery. Both the specific protocol as well as the pluripotent starting cell line influence organoid variability and can result in incomplete representation of brain cell types in an organoid experiment. Here, we systematically analyze the cellular and transcriptional landscape of brain organoids grown from multiple cell lines using four different protocols recapitulating dorsal and ventral forebrain, midbrain, and striatum. We establish the NEST-Score as a quantitative readout for cell line-driven and protocol-driven differentiation propensities by comparing cellular states across multiple cell lines and to in vivo reference data sets. Thereby, we establish a set of organoid protocols that together recreate the vast majority of cell types in the developing human brain and provide a reference for how well cell types are recapitulated across cell lines in each protocol. Additionally, we survey factors contributing to variability during organoid development and identify early gene expression signatures predicting protocol-driven organoid generation at later stages. We provide easy online access to our data through a web-based analysis tool, creating a reference for brain organoid research that allows rapid, straightforward validation of protocol and cell line performance.

developmental biology↗

bioRxiv2024

A dual ribosomal system in the zebrafish soma and germline

Protein synthesis during vertebrate embryogenesis is driven by ribosomes of two distinct origins: maternal ribosomes synthesized during oogenesis and stored in the egg, and somatic ribosomes, produced by the developing embryo after zygotic genome activation (ZGA). In zebrafish, these two ribosome types are expressed from different genomic loci and also differ in their ribosomal RNA (rRNA) sequence. To characterize this dual ribosome system further, we examined the expression patterns of maternal and somatic rRNAs during embryogenesis and in adult tissues. We found that maternal rRNAs are not only expressed during oogenesis but are continuously produced in the zebrafish germline. Proteomic analyses of maternal and somatic ribosomes unveiled differences in core ribosomal protein composition. Most nucleotide differences between maternal and somatic rRNAs are located in the flexible, structurally not resolved expansion segments. Our in vivo data demonstrated that both maternal and somatic ribosomes can be translationally active in the embryo. Using transgenically tagged maternal or somatic ribosome subunits, we experimentally confirm the presence of hybrid 80S ribosomes composed of 40S and 60S subunits from both origins and demonstrate the preferential in vivo association of maternal ribosomes with germline-specific transcripts. Our study identifies a distinct type of ribosomes in the zebrafish germline and thus presents a foundation for future explorations into possible regulatory mechanisms and functional roles of heterogeneous ribosomes.

developmental biology↗

bioRxiv2024

Urinary multi-omics reveal non-invasive diagnostic biomarkers in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the kidney malignancy with the highest incidence and mortality rates. Despite the high patient burden, there are no biomarkers for rapid diagnosis and public health surveillance. Urine would be an ideal source of ccRCC biomarkers due to the low invasiveness, easy accessibility, and the kidneys intrinsic role in filtering urine. In the present work, by combining proteomics, lipidomics and metabolomics, we detected urogenital metabolic dysregulation in ccRCC patients with increased lipid metabolism, altered mitochondrial respiration signatures and increased urinary lipid content. Importantly, we identify three early-stage diagnostic biomarkers for ccRCC in urine samples: Serum amyloid A1 (SAA1), Haptoglobin (HP) and Lipocalin 15 (LCN15). We further implemented a parallel reaction monitoring mass spectrometry protocol for rapid and sensitive detection of SAA1, HP and LCN15 and combined all three proteins into a diagnostic UrineScore. In our discovery cohort, this score had a performance accuracy of 96% in receiver operating characteristic curve (ROC) analysis for classification of ccRCC versus control cases. Our data identifies tractable and highly efficacious urinary biomarkers for ccRCC diagnosis and serve as a first step towards the development of more rapid and accessible urinary diagnostic platforms.

cancer biology↗

bioRxiv2024

CLEC18A interacts with sulfated GAGs and controls clear cell renal cell carcinoma progression

C-type lectins are a large family of proteins with essential functions in both health and disease. In cancer, some C-type lectins have been found to both promote and inhibit tumor growth, but many of the C-type lectins still remain uncharacterised in a tumor context. Therefore, there is growing interst in further elucidating the mechanisms with which C-type lectins control tumor growth. Here, we report a key role of the CLEC18 family of C-type lectins in the progression of clear cell renal cell carcinoma (ccRCC). The CLEC18 family is conserved across the entire Chordata phylum with recent gene duplication events in humans. We found that CLEC18A is exclusively expressed in the proximal tubule of the kidney and the medial habenula of the brain. We further identified sulfated glycosaminoglycans (GAGs) of proteoglycans as the main CLEC18A ligand, making them unique among C-type lectins. In ccRCC patients, high expression of the CLEC18 family lectins in the tumor are associated with improved survival. In mouse models of ccRCC, deletion of the mouse ortholog Clec18a resulted in enhanced tumor growth. Our results establishes CLEC18A as a novel and critical regulators of ccRCC tumor growth and highlights the potential benefit of modulating CLEC18 expression in the renal tumor microenvironment.

cancer biology↗

bioRxiv2024

Increase in ER-mitochondria contacts and mitochondrial fusion are hallmarks of mitochondrial activation during embryogenesis

Mitochondrial ATP production is essential for development, yet the mechanisms underlying the continuous increase in mitochondrial activity during embryogenesis remain elusive. Using zebrafish as a model system for vertebrate development, we comprehensively profile mitochondrial activity, morphology, metabolome, proteome and phospho-proteome as well as respiratory chain enzymatic activity. Our data show that the increase in mitochondrial activity during embryogenesis does not require mitochondrial biogenesis, is not limited by metabolic substrates at early stages, and occurs without an increase in the abundance of respiratory chain complexes or their in vitro activity. Our analyses pinpoint a previously unexplored increase in mitochondrial-ER association during early stages in combination with changes in mitochondrial morphology at later stages as possible contributors to the rise in mitochondrial activity during embryogenesis. Overall, our systematic profiling of the molecular and morphological changes to mitochondria during embryogenesis provides a valuable resource for further studying mitochondrial function during embryogenesis.

developmental biology↗

bioRxiv2023

ARID1B controls transcriptional programs of axon projection in the human corpus callosum

Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC). This condition is characterized by a partial or complete absence of the corpus callosum (CC), an interhemispheric white matter tract that connects distant cortical regions. Using human neural organoids, we identify a vulnerability of callosal projection neurons (CPNs) to ARID1B haploinsufficiency, resulting in abnormal maturation trajectories and dysregulation of transcriptional programs of CC development. Through a novel in vitro model of the CC tract, we demonstrate that ARID1B mutations reduce the proportion of CPNs capable of forming long-range projections, leading to structural underconnectivity phenotypes. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous defects in axonogenesis as a cause of ACC in ARID1B patients. O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/539362v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@dd4e9aorg.highwire.dtl.DTLVardef@153a081org.highwire.dtl.DTLVardef@14e884corg.highwire.dtl.DTLVardef@d64081_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Human callosal projection neurons are vulnerable to ARID1B haploinsufficiency. (Top) During healthy development, callosal projection neurons (CPNs) project long interhemispheric axons, forming the corpus callosum (CC) tract, which can be modeled in vitro. (Bottom) In ARID1B patients, transcriptional dysregulation of genetic programs of CC development reduces the formation of long-range projections from CPNs, causing CC agenesis in vivo and underconnectivity phenotypes in vitro. C_FIG

neuroscience↗

bioRxiv2022

Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous ECM

Establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in cerebral organoids, the effects of different ECM sources in tissue morphogenesis remain unexplored. Here, we show that exposure to exogenous ECM at early neuroepithelial stages causes rapid tissue polarization and complete rearrangement of neuroepithelial architecture within 3 days. In unexposed cultures, endogenous ECM production by NPCs results in gradual polarity acquisition over an extended time. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source. These results advance the knowledge on neuroepithelial biology in vitro, with a focus on mechanisms of exogenously- and endogenously-guided morphogenesis. They demonstrate the self-sustainability of neuroepithelial cultures by endogenous processes, prompting an urgent reassessment of indiscriminate use of exogenous ECM in these model systems.

developmental biology↗

bioRxiv2022

Integrated transcriptome and proteome analysis in human brain organoids reveals translational regulation of ribosomal proteins.

During development of the human cerebral cortex, multipotent neural progenitors generate excitatory neurons and glial cells. Investigations of the transcriptome and epigenome have revealed important gene regulatory networks underlying this crucial developmental event. However, the post-transcriptional control of gene expression and protein abundance during human corticogenesis remains poorly understood. We addressed this issue by using human telencephalic brain organoids grown using a dual reporter cell line to isolate neural progenitors and neurons and performed cell class and developmental stage-specific transcriptome and proteome analysis. Integrating the two datasets revealed modules of gene expression during human corticogenesis. Investigation of one such module uncovered mTOR-mediated regulation of translation of the 5TOP element-enriched translation machinery in early progenitor cells. We show that in early progenitors partial inhibition of the translation of ribosomal genes prevents precocious translation of differentiation markers. Overall, our multiomics approach reveals novel posttranscriptional regulatory mechanisms crucial for the fidelity of cortical development.

developmental biology↗

bioRxiv2022

Cellular stress in brain organoids is limited to a distinct and bioinformatically removable subpopulation.

Organoids enable disease modeling in complex and structured human tissue, in vitro. Like most 3D models, they lack sufficient oxygen supply, leading to cellular stress. These negative effects are particularly prominent in complex models, like brain organoids, where they can prevent proper lineage commitment. Here, we analyze brain organoid and fetal single cell RNA sequencing (scRNAseq) data from published and new datasets totaling over 190,000 cells. We describe a unique stress signature found in all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined organoid cell population, and present Gruffi, an algorithm that uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. Our data show that adverse effects of cell stress can be corrected by bioinformatic analysis, improving developmental trajectories and resemblance to fetal data.

neuroscience↗

bioRxiv2022

PCYT2 controls muscle health and muscle aging

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing, affecting hundreds of millions of people. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases represents an important goal in improving human health. Here, we show that phosphatidylethanolamine cytidyltransferase (PCYT2/ECT), the critical enzyme of the Kennedy branch of phosphatidylethanolamine (PE) synthesis pathway, has an essential role in muscle health. Human genetic deficiency in PCYT2 causes a severe disease with failure to thrive and progressive muscle weakness. Pcyt2 mutant zebrafish recapitulate the patient phenotypes, indicating that the role of PCYT2/PE in muscle is evolutionary conserved. Muscle specific Pcyt2 knockout mice exhibited failure to thrive, impaired muscle development, progressive muscle weakness, muscle loss, accelerated ageing, and reduced lifespan. Mechanistically, Pcyt2 deficiency affects mitochondrial bioenergetics and physicochemical properties of the myofiber membrane lipid bilayer, in particular under exercise strain. We also show that PCYT2 activity declines in the aging muscles of humans and mice. AAV-based delivery of PCYT2 rescued muscle weakness in Pcyt2 knock-out mice and, importantly, improved muscle strength in old mice, offering a novel therapeutic avenue for rare disease patients and muscle aging. Thus, PCYT2 plays a fundamental, specific, and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2 synthesized PE lipids to severe muscle dystrophy, exercise intolerance and aging.

genetics↗