bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.02.12.705355

Multi-gene biomarkers reveal spatial organization and subpopulation-specific damage response in intrahepatic biliary epithelial cells

Kanakanui, K. G.·Hantelys, F.·Hrncir, H. R.·Bombin, S.·Gracz, A. D.
View original source ↗Export metadata ↓

Abstract

Background & AimsIntrahepatic biliary epithelial cell (BEC) heterogeneity remains challenging to define. Here, we sought to identify BEC subpopulations and biomarkers in mouse liver. MethodsWe performed scRNA-seq on Sox9EGFP+ liver epithelium from mice subjected to bile duct ligation (BDL) and sham controls. A machine learning algorithm, NSForest, identified minimal, multi-gene signatures for BEC subpopulations. These "metagenes" were validated using hybridization chain reaction (HCR) FISH in tissue sections from wild-type mice and on primary BECs expanded in vitro. Metagenes were used to match BDL subpopulations to their corresponding sham subpopulations for differential gene expression (DGE) analysis. ResultsWe identified 4 BEC subpopulations in sham controls, each associated with 1-2 gene metagenes. Spatial localization of metagene-defined BEC subpopulations by HCR FISH revealed heterogeneous cellular composition of intrahepatic bile ducts. BECs belonging to a given subpopulation were most likely to have neighbors of the same identity, forming homogenous cellular compartments within ducts. BDL downregulated subpopulation-specific genes and upregulated a damage-associated gene set. BDL samples also included a proliferative subpopulation not found in sham controls, which contained populations enriched for three of the four metagenes. All BEC subpopulations were also found in monolayers in vitro, where they clustered spatially with BECs of the same subtype. ConclusionsNovel metagene biomarkers of BEC subpopulations facilitated spatial localization of BECs in situ, identified subpopulation specific injury responses, and confirmed that BEC heterogeneity is preserved in vitro. The presence of locally homogenous BEC "neighborhoods" in vitro suggests some degree of BEC organization may be epithelial-autonomous.

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Kanakanui, K. G., Hantelys, F., Hrncir, H. R., Bombin, S., Gracz, A. D.. 2026-02-15. Multi-gene biomarkers reveal spatial organization and subpopulation-specific damage response in intrahepatic biliary epithelial cells. https://doi.org/10.64898/2026.02.12.705355

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Deep generative embeddings of gene expression and splicing reposition the interpretation of single-cell transcriptomic signatures

Single-cell transcriptomic analysis predominantly derives cell identity from gene expression analysis, while alternative splicing is processed separately despite its fundamental role for cell homeostasis. To overcome the limits of separate investigations, we developed a probabilistic deep learning framework, Crecerelle, enabling resolution of the contributions of gene expression and alternative splicing in each cell. Crecerelle learns cell embeddings from gene expressions and alternative splicing isoforms, to decipher their mutually dependent impact on the functional characterisation of cells in a data-driven manner, exemplified for the Tabula Muris dataset. This is enabled through a zero-and-N-inflated Dirichlet-Multinomial for a variational autoencoder that learns cell embeddings solely from splicing profiles, as well as a bi-modal variational autoencoder with a relevance-weighted mixture-of-experts variational posterior to consolidate the modality-specific contribution at single-cell level. Crecerelle reveals cell-type-specific isoform markers as well as subpopulations with unique isoforms and uncovers regulatory and disease-associated pathways not detected by gene expression analyses alone. This scalable and interpretable framework thus allows a more holistic study of transcriptomic regulation and will open a route to modality-relevance-weighted investigations across single-cell multiomics datasets and their influence on cellular homeostasis, tissue development and disease phenotypes.

cell biology↗
bioRxiv2026

MHC Molecules on B Cell Microvilli Are Spatially Associated with IL-15Rα

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor (IL-15R) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B cell-T-cell contact, the distribution of IL-15R and MHC molecules within these structures may be important during the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15R with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET). Both MHC class I and class II molecules showed significant spatial association with IL-15R; however, the extent of colocalisation decreased as spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15R and MHC class I, whereas STORM did not detect this association. In contrast, IL-15R and MHC class II remained significantly colocalised at both resolutions. FLIM-FRET further demonstrated molecular proximity between IL-15R and both MHC class I and class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15R is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B-cell-T-cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B-cell-T-cell interactions.

cell biology↗
bioRxiv2026

Pulsed-SILAC in single mouse embryos reveals early embryonic protein synthesis dynamics and phosphosite regulation

Early embryogenesis relies extensively on maternally deposited products until zygotic genome activation, yet the dynamics for the synthesis of new proteins in mammalian embryos remains poorly characterized. To address this, we applied pulsed stable isotope labelling by amino acids in cell culture (pSILAC) combined with narrow-window data-independent acquisition mass spectrometry to single mouse oocytes and embryos to resolve de novo protein synthesis during early embryogenesis. This revealed that the maternal proteome is not a static reservoir, with components of the subcortical maternal complex and amino acid transporters SLC7A1/2 being actively synthesized during the earliest developmental stages. Furthermore, phosphoproteomic analysis identified hundreds of previously unreported phosphosites and extensive regulation during the oocyte-to-embryo transition. Notably, phosphorylation of the PRC2-interacting KLP motif of EZHIP emerged as a potential regulatory mechanism, with modification of this region reducing EZHIP-PRC2 interaction and coinciding with H3K27me3 remodelling. Together, single embryo pSILAC revealed a maternal proteome that is continuously synthesized, recycled, and post-translationally regulated during early embryogenesis.

cell biology↗