Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.

Department of Cell and Molecular Biology Karolinska Institutet Stockholm SE 171 77 Solna Sweden

Department of Cell Biology Faculty of Science Charles University Viničná 7 128 00 Prague 2 Czech Republic

Department of Clinical Science Intervention and Technology Karolinska Institutet 141 86 Stockholm Sweden

Department of Molecular Biosciences the Wenner Gren Institute Stockholm University Svante Arrhenius Väg 20C SE 106 91 Stockholm Sweden

Microbial Single Cell Genomics facility SciLifeLab Biomedical Center Uppsala University SE 751 23 Uppsala Sweden

Science for Life Laboratory Department of Gene Technology KTH Royal Institute of Technology Tomtebodavägen 23a SE 171 65 Solna Sweden

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Glycolysis in hepatic stellate cells coordinates fibrogenic extracellular vesicle release spatially to amplify liver fibrosis

Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver