Clarke, Z. A. | Search bioRxiv

bioRxiv2025

A Single-Cell Woodchuck Liver Atlas Identifies Healthy and Disease-Related Cellular Programs Conserved in Human

BackgroundModel organisms allowing for longitudinal examinations of liver disease pathogenesis are pivotal for the development of new therapeutic modalities. The eastern North American woodchuck develops chronic hepatitis and liver cancer after woodchuck hepatitis virus (WHV) infection, mirroring aspects of the natural history of the human hepatitis B virus (HBV). However, the cellular landscape of the woodchuck liver and the cell-level relevance of WHV infection to HBV infection is currently uncharacterized. MethodsWe employed single-cell RNA sequencing (RNA-seq) to generate an atlas of healthy woodchuck liver (63,389 cells, n=8) and peripheral blood mononuclear cells (PBMCs) (26,972 cells, n=7). Cell-specific and hepatic zonation gene signatures were validated using spatial transcriptomics (n=1). We employed our atlas to examine immune activation in stimulated precision cut-liver slices (PCLS) and disease-related pathway activation in chronic WHV infection (11,797 cells, n=3). We further employed our atlas to examine shared disease pathways between WHV infection and human HBV infection. ResultsOur atlas revealed woodchuck hepatic cellular diversity comparable to human and murine livers. Applying single-nucleus RNA-seq to PMA/ionomycin-stimulated precision cut liver slices revealed inflammation-associated activation signatures in T cell, myeloid and endothelial cell compartment. Finally, we describe intrahepatic T cells in chronic WHV hepatitis with both exhaustion and activation-associated signatures that resemble intrahepatic T cell genes signatures described in human chronic HBV. ConclusionsWe present a multi-omic atlas of healthy, diseased and ex vivo stimulated woodchuck liver. By identifying shared pathological processes between WHV and HBV infections, our findings reinforce the value of this preclinical model in translational research. This resource aims to advance studies on HBV pathogenesis and oncogenesis to speed the development of novel therapeutic strategies. Impact and Implications/Lay summaryThe liver plays important roles in metabolism, detoxification, and immune processes; liver transplantation is often the only treatment option for severe chronic liver diseases. Therefore, developing animal models that reflect human liver disease and can be studied throughout the disease course is crucial for the discovery of new treatment options. The woodchuck is an animal that develops chronic hepatitis and liver cancer after infection with woodchuck hepatitis virus, which models the human hepatitis type B virus infection (HBV) and associated hepatic carcinoma. However, our understanding of the cells that compose the woodchuck liver is limited, making it challenging to design and test cell-based therapeutics. In this study, we atlased the healthy and chronically infected woodchuck liver, found that liver cell types in woodchuck resemble those in humans, and employed the atlas to show similarities between WHV and HBV at the cell level, reinforcing the potential of WHV-infected woodchuck as a model for human HBV disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/641192v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@18fdd40org.highwire.dtl.DTLVardef@6ccf2org.highwire.dtl.DTLVardef@7923e1org.highwire.dtl.DTLVardef@122a8d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

An updated reference genome sequence and annotation reveals gene losses and gains underlying naked mole-rat biology

The naked mole-rat (NMR; Heterocephalus glaber) is a eusocial subterranean rodent with a highly unusual set of physiological traits that has attracted great interest amongst the scientific community. However, the genetic basis of most of these traits has not been elucidated. To facilitate our understanding of the molecular mechanisms underlying NMR physiology and behaviour, we generated a long-read chromosomal-level genome assembly of the NMR. This genome was subsequently annotated and incorporated into multiple whole genome alignments in the Ensembl database. Our long-read assembly identified thousands of repeats and genes that were previously unassembled in the NMR and improved the results of routinely used short-read sequencing-based experiments such as RNA-seq, snRNA-seq, and ATAC-seq. We identified several spermatozoa related gene losses that may underlie the unique degenerative sperm phenotype in NMRs (IRGC, FSCB, AKAP3, MROH2B, CATSPER1, DCDC2C, ATP1A4, TEKT5, and ZAN), and an additional gene loss related to the established NK-cell absence in NMRs (PILRB). We resolved several tandem duplications in genes related to pathways underlying unique NMR adaptations including hypoxia tolerance, oxidative stress, and nervous system protection (TINF2, TCP1, KYAT1). Lastly, we describe our ongoing efforts to generate a reference telomere-to-telomere assembly in the NMR which includes the resolution of complex gene families. This new reference genome should accelerate the discovery of the genetic underpinnings of NMR physiology and adaptation.

genomics↗

bioRxiv2024

MALAT1 expression indicates cell quality in single-cell RNA sequencing data

Single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cell types and tissues. However, empty droplets and poor quality cells are often captured in single cell genomics experiments and need to be removed to avoid cell type interpretation errors. Many automated and manual methods exist to identify poor quality cells or empty droplets, such as minimum RNA count thresholds and comparing the gene expression profile of an individual cell to the overall background RNA expression of the experiment. A versatile approach is to use unbalanced overall RNA splice ratios of cells to identify poor quality cells or empty droplets. However, this approach is computationally intensive, requiring a detailed search through all sequence reads in the experiment to quantify spliced and unspliced reads. We found that the expression level of MALAT1, a non-coding RNA retained in the nucleus and ubiquitously expressed across cell types, is strongly correlated with this splice ratio measure and thus can be used to similarly identify low quality cells in scRNA-seq data. Since it is easy to visualize the expression of a single gene in single-cell maps, MALAT1 expression is a simple cell quality measure that can be quickly used during the cell annotation process to improve the interpretation of cells in tissues of human, mouse and other species with a conserved MALAT1 function.

bioinformatics↗