Biology subjects

Samakovlis, C.

Publications and source records attributed to Samakovlis, C..

18 recordsLinked to original sources

bioRxiv2026

A repair-associated bronchial epithelial differentiation trajectory through KRT14+ basal and hillock-like cells drives airway inflammation and remodelling in childhood-onset asthma

The bronchial epithelium in asthma is vulnerable to damage and has impaired barrier function, but the mechanisms by which it contributes to airway inflammation and remodelling remain unclear. Here, we dissect these epithelial and immunological disease mechanisms by establishing a comprehensive single cell atlas of the bronchial wall from 21 patients with childhood-onset asthma and 25 matched healthy controls. We identify a novel asthma-associated non-canonical epithelial differentiation trajectory in which a repair-associated KLF4+ basal cell subset differentiates into KRT13+ hillock-like cells through a proliferative KRT14+ intermediate. In vitro cultured matched primary bronchial epithelial cells show that this trajectory is retained in absence of exogenous factors. We find that IL-13 induces hillock-like cell differentiation into CEACAM5hi goblet cells, driving goblet cell metaplasia. Repair-associated basal cells and transitioning CEACAM5hi hillock-like cells strongly contribute to airway inflammation and remodelling. In turn, dendritic cells and mast cells promote a state of highly active epithelial differentiation, which shows increased multiciliated cell fate decisions, in concordance with an increase in multiciliated cell death observed in asthma. Proportions of the epithelial cells of the non-canonical differentiation trajectory are associated with clinical outcomes such as disease severity, FeNO, and small airway function.

immunology↗

bioRxiv2026

Cophenetic Spatial Topology Embedding reveals multiscale tissue architecture in spatial omics

The spatial organization of tissues emerges from cell interactions across multiple scales, yet current spatial omics analysis tools often emphasize local neighborhoods and may not summarize broader tissue architecture. Here we introduce Cophenetic Spatial Topology Embedding (COSTE), a computational framework that embeds directed nearest-neighbor distance profiles into a hierarchical metric space without requiring the user to define a spatial radius or neighborhood cutoff. COSTE can be applied to cell-level and single-transcript inputs without requiring cell segmentation. It constructs directed distance profiles between cell populations and uses hierarchical clustering to quantify tissue topology. This yields a Spatial Separation Score (SSS), a sample-normalized score from 0 to 1 that summarizes relative spatial separation within an analyzed tissue. We apply COSTE to spatial transcriptomics datasets of pulmonary fibrosis and triple-negative breast cancer (TNBC), where it delineates tissue structures, nominates spatially defined cell states, and highlights disease-or treatment-associated architectural patterns that are not readily captured by local neighborhood-based analyses. Our approach provides an interpretable framework for exploring tissue architecture and cell-cell spatial relationships in spatial omics data.

bioinformatics↗

bioRxiv2026

Branch extension and pruning share the same regulatory module in the developing Drosophila airways

Branched tubular organs represent a common solution to the problem of fluid transport in large animals. The growth of new branches has been extensively studied but the regulation of branch pruning remains underexplored. Here, we investigate branch removal in the stereotyped branching patterns of the developing Drosophila airways. After progenitor invagination, the tips of the distal airways generate a stereotyped branching pattern in each central metameric unit. An intriguing exception in the repeating patterns is the lack of branches targeting the visceral mesoderm (visceral primary branch, VB) in the third and nineth metameres. We show that these branches initially form but the localized expression of the pro-apoptotic gene reaper and resultant apoptosis prune them. We reveal that VB3/9 pruning entails four sequential programs. First, a common distal outgrowth program promotes budding and extension of the primary branches, including VB3 and 9. Second, a VB identity program is established representing the ground state of all primary branches. Third, the Bithorax-Complex transcription factors define metameric identities and interfere with the VB program to induce reaper and apoptosis specifically in VB3/9 in a concentration dependent manner. Finally, the default VB cell identity is transformed by extrinsic BMP/Decapentaplegic and WNT/Wingless into more derived primary branch identities and spared from pruning in metamers 3 and 9. Our results demonstrate that molecular and genetic circuits promoting branch emergence and extension can be regionally modified and deployed also for branch pruning. Author summaryMany of our internal organs like the lung, kidney and various glands are composed of epithelial tubes. The function of these organs is sustained by the morphologies of the constituting tubes. The formation of distinct branching patterns in tubular networks including regional variegations can be accomplished by branch ramification and branch pruning. Extensive studies of tube ramification and extension showed that they are typically regulated by extrinsic guidance cues. In contrast to branch extension, branch maintenance and removal is much less understood. Here, we identify a program that intrinsically promotes apoptosis to prune specific branches in the developing Drosophila airways. Intriguingly, the branch extension program is diverted to a branch pruning program. Activation of the branch outgrowth program is prerequisite for both branch extension and pruning. The Hox gene expression code that characterizes regional cell identities along the body axes intrinsically interferes with the branch extension program of visceral primary branches (VB) targeting the visceral muscles. This results in VB pruning only in metameres 3 and 9, where extrinsic WNT and BMP guidance cues direct towards distal branch identities that are spared from apoptotic pruning. Similar interplay of branching and pruning programs may operate in sculpting our lung and vasculature.

developmental biology↗

bioRxiv2026

Mitochondrial COX4I2 drives pericyte-dependent inflammation and emphysema

Chronic obstructive pulmonary disease (COPD) is characterized by neutrophilic inflammation, emphysema, and mild pulmonary hypertension (PH). Oxidative/nitrosative stress are key drivers, but specific mitochondrial mechanisms remain unclear. We show increased expression of the regulatory mitochondrial cytochrome c oxidase subunit 4 isoform 2 (COX4I2) in an early murine model and human COPD. After 8 months of cigarette smoke exposure, Cox4i2-/- mice were completely protected from emphysema but not from PH, associated with reduced nitrosative stress, inflammation, and apoptosis. Using a novel Cox4i2 reporter mouse and in situ hybridization of human lungs, COX4I2 was detected in precapillary ACTA2+ cells and capillary pericytes. COX4I2 promotes mitochondrial reactive oxygen species (mtROS) production in these cells, thereby enhancing neutrophil migration and alveolar type II cell apoptosis, and modulates angiogenesis. In contrast to Cox4i2-/-, mitochondria-targeted antioxidant MitoQ reversed emphysema and PH, suggesting pericyte-specific regulation of COPD pathologies and mtROS inhibition as a therapeutic approach in COPD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/703513v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@83bca4org.highwire.dtl.DTLVardef@d5ebaborg.highwire.dtl.DTLVardef@632d1borg.highwire.dtl.DTLVardef@1267a13_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Single-cell transcriptomics identifies a p21-activated kinase important for survival of the zoonotic parasite Fasciola hepatica

Knowledge on the cell types and cell-specific gene expression of multicellular pathogens facilitates drug discovery and allows gaining a deeper understanding of pathogen biology. By utilizing single-cell RNA sequencing (scRNA-seq), we analyzed 19,581 cells of a globally prevalent parasitic flatworm, the liver fluke Fasciola hepatica, which causes a neglected tropical disease and zoonosis known as fascioliasis. We identified 15 distinct clusters, including cells of the reproductive tract and gastrodermis, and report the identification and transcriptional characterization of potential differentiation lineages of stem cells within this parasite. Furthermore, a previously unrecognized ELF5- and TRPMPZQ-expressing muscle cell type was discovered, characterized by high expression of protein kinases. Among these, the p21-activated kinase PAK4 was essential for parasite survival. These data provide novel insight into the cellular composition of a complex multicellular parasite and demonstrate how gene expression at single-cell resolution can serve as a resource for the identification of new drug targets.

molecular biology↗

bioRxiv2024

Lineage-tracing of Acta2+ cells in aged mice during lung fibrosis formation and resolution supports the lipofibroblasts to myofibroblast reversible switch

Idiopathic pulmonary fibrosis (IPF) develops mostly in old man and is characterized by the irreversible accumulation of excessive extracellular matrix components by activated myofibroblasts (aMYFs) leading to lung failure. Following bleomycin administration in young mice, fibrosis formation associated with efficient resolution takes place, the later limiting the clinical relevance of this model for IPF. In young mice, we previously reported that aMYFs captured during fibrosis formation differentiate towards a lipofibroblast (LIF)-like phenotype during resolution. In this study, we used aged mice in combination with bleomycin administration to trigger enhanced fibrosis formation and delayed resolution, as a more relevant model for IPF and examined the heterogeneity and fate of aMYFs at different time points. Alveolosphere assay were carried out to compare the alveolar resident mesenchymal niche activity for AT2 stem cells in young versus old mice. Lineage tracing of the Acta2+ aMYFs in old mice exposed to bleomycin followed by scRNAseq of the lineage-traced cells isolated during fibrosis formation and resolution was performed to delineate the heterogeneity of aMYFs during fibrosis formation and their fate during resolution. Data mining of human mesenchymal cells from IPF and control datasets were also performed to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis formation. Our results show that alveolar resident mesenchymal cells from old mice display decreased supporting activity for AT2 stem cells. We report that aMYFs consist of four subclusters displaying unique pro-alveologenic versus pro-fibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrosis formation and resolution, respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature. In conclusion, our data indicate that the cellular and molecular bases of aMYFs formation and differentiation towards the LIF phenotype are conserved between young and old mice. Importantly, our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF.

pathology↗

bioRxiv2024

High-parametric protein maps reveal the spatial organization in early-developing human lung

The respiratory system, including the lungs, is essential for terrestrial life. While recent research has advanced our understanding of lung development, much still relies on animal models and transcriptome analyses. In this study conducted within the Human Developmental Cell Atlas (HDCA) initiative, we describe the protein-level spatiotemporal organization of the lung during the first trimester of human gestation. Using high-parametric tissue imaging with a 30-plex antibody panel, we analyzed human lung samples from 6 to 13 post-conception weeks, generating data from over 2 million cells across five developmental timepoints. We present a resource detailing spatially resolved cell type composition of the developing human lung, including proliferative states, immune cell patterns, spatial arrangement traits, and their temporal evolution. This represents an extensive single-cell resolved protein-level examination of the developing human lungs and provides a valuable resource for further research into the developmental roots of human respiratory health and disease.

developmental biology↗

bioRxiv2023

Postnatal FGF-signalling establishes gradients of cell-identities along the proximal-distal axis of the lung airways

Secretory cells are major structural and functional constituents of the lung airways. Their spatial organization and specification mechanisms are partially understood. Here, we labelled major secretory airway cell types and analysed them at single-cell resolution. We found opposing, partially overlapping gene-expression gradients along the proximal-distal airway axis superimposed on a general gene program encoding detoxification. One graded program is elevated proximally and relates to innate immunity, whereas the other is enriched distally, encoding lipid metabolism and antigen presentation. Intermediately positioned cells express low levels of both graded programs and show increased clonogenic potency in vitro, relating cell-plasticity to location in each branch. Single-cell RNA-sequencing following lineage-tracing revealed the sequential and postnatal establishment of the gradients in common epithelial progenitors. Fgfr2b is distally enriched and its postnatal inactivation reduces distal gene expression and expands proximal genes into distally located cells. This suggests a central role of FGFR-signaling in tissue-scale airway patterning.

cell biology↗

bioRxiv2022

Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways

The acquisition of distinct branch sizes and shapes is a central aspect in tubular organ morphogenesis and function. In the Drosophila airway tree, the interplay of apical ECM components with the underlying membrane and cytoskeleton controls tube elongation, but the link between ECM composition with apical membrane morphogenesis and tube size regulation is elusive. Here, we characterized Emp (epithelial membrane protein), a Drosophila CD36-homologue belonging to the scavenger receptor class B protein-family. emp mutant embryos fail to internalize the luminal chitin deacetylases Serp and Verm at the final stages of airway maturation and die at hatching with liquid filled airways. Emp localizes in apical epithelial membranes and shows cargo selectivity for LDLr-domain containing proteins. emp mutants also display over elongated tracheal tubes with increased levels of the apical proteins Crb, DE-cad and phosphorylated Src (p-Src). We show that Emp associates and organizes the {beta}H-Spectrin cytoskeleton and is itself confined by apical F-actin bundles. Overexpression or loss of its cargo protein Serp lead to abnormal apical accumulations of Emp and perturbations in p-Src levels. We propose that during morphogenesis, Emp senses and responds to luminal cargo levels by initiating apical membrane endocytosis along the longitudinal tube axis and thereby restricts airway elongation.

developmental biology↗

bioRxiv2022

Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples

Spatially resolved transcriptomics (SRT) has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of unbiased SRT methods targeting the polyA tail of mRNA, relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available SRT assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), an SRT workflow designed to improve mRNA recovery from fresh frozen (FF) specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we demonstrate the RRST protocol on tissue sections collected from 5 challenging tissue types, including: human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyzed 52 tissue sections and our results demonstrate that RRST is a versatile, powerful, and reproducible protocol for FF specimens of different qualities and origins.

genomics↗

bioRxiv2022

Probe set selection for targeted spatial transcriptomics

Targeted spatial transcriptomics methods capture the topology of cell types and states in tissues at single cell- and subcellular resolution by measuring the expression of a predefined set of genes. The selection of an optimal set of probed genes is crucial for capturing and interpreting the spatial signals present in a tissue. However, current selections often rely on marker genes, precluding them from detecting continuous spatial signals or novel states. We present Spapros, an end-to-end probe set selection pipeline that optimizes both probe set specificity for cell type identification and within-cell-type expression variation to resolve spatially distinct populations while taking into account prior knowledge, as well as probe design and expression constraints. To facilitate data analysis and interpretation, Spapros also provides rules for cell type identification. We evaluated Spapros by selecting probes on 6 different data sets and built an evaluation pipeline with 12 quality metrics to find that Spapros outperforms other selection approaches in both cell type recovery and recovering expression variation beyond cell types. Furthermore, we used Spapros to design a SCRINSHOT experiment of adult lung tissue to demonstrate how probes selected with Spapros identify cell types of interest and detect spatial variation even within cell types. Spapros enables optimal probe set selection, probe set evaluation, and probe design, as a freely available Python package.

bioinformatics↗

bioRxiv2022

Autocrine Sfrp1 inhibits lung fibroblast invasion during transition to injury induced myofibroblasts

Fibroblast to myofibroblast conversion is a major driver of tissue remodeling in organ fibrosis. Several distinct lineages of fibroblasts support homeostatic tissue niche functions, yet, specific activation states and phenotypic trajectories of fibroblasts during injury and repair have remained unclear. Here, we combined spatial transcriptomics, longitudinal single-cell RNA-seq and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. We discovered a transitional fibroblast state characterized by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1+ cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1+ transitional fibroblasts and Cthrc1+ myofibroblasts. Tgf{beta}1 downregulated Sfrp1 in non-invasive transitional cells and induced their switch to an invasive Cthrc1+ myofibroblast identity. Finally, using loss of function studies we showed that autocrine Sfrp1 directly inhibits fibroblast invasion by regulating the RhoA pathway. In summary, our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies Sfrp1 as an autocrine inhibitor of fibroblast invasion during early stages of fibrogenesis.

cell biology↗

bioRxiv2022

FGF10 triggers de novo alveologenesis in a BPD model: impact on the resident mesenchymal niche cells

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease developing in premature babies characterized by arrested alveologenesis and associated with decreased Fibroblast growth factor 10 (FGF10) expression. One-week hyperoxia (HYX) exposure of newborn mice leads to a permanent arrest in alveologenesis. To test the role of Fgf10 signaling to promote de novo alveologenesis following hyperoxia, we used transgenic mice allowing inducible expression of Fgf10 and recombinant FGF10 (rFGF10) protein delivered intraperitoneally. We carried out morphometry analysis, and IF on day 45. Alveolospheres assays were performed co-culturing AT2s from normoxia (NOX) with FACS-isolated Sca1Pos resident mesenchymal cells (rMC) from animals exposed to NOX, HYX+PBS, or HYX+FGF10. scRNAseq between rMC-Sca1Pos isolated from NOX and HYX+PBS were also carried out. Transgenic overexpression of Fgf10 and rFGF10 administration rescued the alveologenesis defects following HYX. Alveolosphere assays indicate that the activity of rMC-Sca1Pos is negatively impacted by HYX and partially rescued by rFGF10 treatment. Analysis by IF demonstrates a significant impact of rFGF10 on the activity of resident mesenchymal cells. scRNAseq results identified clusters expressing Fgf10, Fgf7, Pdgfra, and Axin2, which could represent the rMC niche cells for the AT2 stem cells. In conclusion, we demonstrate that rFGF10 administration is able to induce de-novo alveologenesis in a BPD mouse model and identified subpopulations of rMC-Sca1Pos niche cells potentially representing its cellular target.

cell biology↗

bioRxiv2022

Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice

Fibroblast growth factor receptor 2b (Fgfr2b) signaling is essential throughout lung development to form the alveolar epithelial lineage. However, its role in alveolar epithelial type 2 cells (AT2s) homeostasis was recently considered dispensable. SftpcCreERT2; tdTomatoflox/flox mice were used to delete Fgfr2b expression in cells belonging to the AT2 lineage, which contains mature AT2s and a novel SftpcLow lineage-traced population called "injury activated alveolar progenitors" or IAAPs. Upon continuous tamoxifen exposure for either one or two weeks to delete Fgfr2b, a shrinking of the AT2s is observed. Mature AT2s exit the cell cycle, undergo apoptosis and fail to form alveolospheres in vitro. However, the lung morphometry appears normal, suggesting the involvement of compensatory mechanisms. In mutant lungs, IAAPs which escaped Fgfr2b deletion expand, display enhanced alveolosphere formation in vitro and increase drastically their AT2 signature suggesting differentiation towards mature AT2s. Interestingly, a significant increase in AT2s and decrease in IAPPs occurs after a one-week tamoxifen exposure followed by an eight-week chase period. While mature AT2s partially recover their alveolosphere formation capabilities, the IAAPs no longer display this property. Single-cell RNA seq analysis confirms that AT2s and IAAPs represent stable and distinct cell populations and recapitulate some of their characteristics observed in vivo. Our results underscore the essential role played by Fgfr2b signaling in the maintenance of the AT2 lineage in the adult lung and suggest that the IAAPs could represent a new population of AT2 progenitors.

developmental biology↗

bioRxiv2022

An essential function for autocrine Hedgehog signaling in epithelial proliferation and differentiation in the trachea

The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (Hh) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a new, autocrine function of Hh signaling in airway epithelial cells. Epithelial cell depletion of the ligand Sonic hedgehog (SHH) or its effector Smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, Hh signaling inhibition also hampers cell proliferation and differentiation. Epithelial Hh function is mediated, at least in part, through transcriptional activation as Hh signaling inhibition leads to downregulation of cell-type specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of Hh signaling in epithelial cell proliferation and differentiation during airway development.

developmental biology↗

bioRxiv2022

Developmental origins of cell heterogeneity in the human lung

The lung contains numerous specialized cell-types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a first comprehensive topographic atlas of early human lung development. We report 83 cell states, several spatially-resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated scRNA-Seq and spatial transcriptomics into a web-based, open platform for interactive exploration. To illustrate the utility of our approach we show distinct states of secretory and neuroendocrine cells, largely overlapping with the programs activated either during lung fibrosis or small cell lung cancer progression. We define the origin of uncharacterized airway fibroblasts associated with airway smooth muscle in bronchovascular bundles, and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

developmental biology↗

bioRxiv2020

SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

Changes in cell identities and positions underlie tissue development and disease progression. Although, single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell-states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRISHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity and quantitative qualities of SCRINSHOT facilitate single cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

molecular biology↗

bioRxiv2019

Yorkie controls tube length and apical barrier integrity in the developing Drosophila airways

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication and apical membrane growth. The Drosophila embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional co-activator of the Hippo pathway, Yorkie (YAP in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water-tightness of tracheal tubes by transcriptional regulation of the enzyme{delta} -aminolevulinate synthase (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality. Short SummaryThis work identified an alternative role of the transcriptional co-activator Yorkie (Yki) in controlling water impermeability and tube size of the developing Drosophila airways. Tracheal impermeability is triggered by Yki-mediated transcriptional regulation of{delta} -aminolevulinate synthase, Alas, whereas tube elongation is controlled by binding of Yki to the actin severing factor Twinstar.

developmental biology↗