Fig. 1: Hepatocyte renewal and ploidy during homeostasis in the healthy mouse liver.

A Schematic showing the 3 mouse models of our study; (Rosa26-Rainbow (Rosa26^rbw) Cre-mediated recombination mice, TMX-inducible albumin Cre mice (Alb-CreERT2) and Alb-CreERT2 Rosa26^rbw mice) (created with BioRender.com) and representative images of liver sections from 10-week-old male Rosa26^rbw and Alb-CreERT2 Rosa26^rbw mice (n = 4). The bottom panel shows a detail view of the liver lobule from a Alb-CreERT2 Rosa26^rbw male mouse, showing the 3 zones along the portal-central axis; zone 1 or periportal area (PP) that encircles the portal tracts identified by bile ducts (arrowhead); zone 3 or pericentral area (PC) that is located around central veins; and zone 2 or midlobular area (Mid) that is located in between. The Cre-activated fluorophores (CAG-EGFP, mCerulean, mOrange and mCherry) plus DAPI staining are shown in the images. CV central vein, PV portal vein. TMX tamoxifen. Scale bars, 100 μm. B Representative Glutamine Synthetase (GS) and Cytokeratin 19 (CK19) IHC analysis in liver sections from a 10-week-old Alb-CreERT2 Rosa26^rbw mouse (n = 4). Scale bar, 100 μm. C Schematic of the heterozygous multicolor Alb-CreERT2 Rosa26^rbw mice, that allow us to trace the fate of polyploid or diploid hepatocytes in vivo. Created with BioRender.com. D Total area in μm² per fluorophore and per photo or field of view (10×) of liver sections from Alb-CreERT2 Rosa26^rbw mice. 6–7 photos per mouse; n = 3 mice. Data are presented as mean values +/− SEM. One-way ANOVA, Tukey’s multiple comparisons post-test. E Colored hepatocytes in % per Rainbow fluorophore and per photo or field of view (10×). Data are presented as mean values +/− SEM. 6–7 photos per mouse; n = 3 mice. F Colored hepatocytes in % per Rainbow fluorophore and per area of the liver lobule (PP, Mid and PC). 27 liver lobules analyzed from 3 mice. Data are presented as mean values +/− SEM. One-way ANOVA, Tukey’s multiple comparisons post-test. G Quantification of number of cells per clone (y-axis) and number of clones (x-axis) per area of the liver lobule and per Rainbow fluorophore. Data are presented as mean values. 32 liver lobule areas analyzed from 3 mice (7–8 photos per mouse). One-way ANOVA, Tukey’s multiple comparisons post-test. H Relative frequency in % of each clone size per area of the liver lobule and per Rainbow fluorophore. Data are presented as mean values +/− SEM. 32 liver lobule areas analyzed from 3 mice (7–8 photos per mouse). I Number of total Ki67 positive hepatocytes per area within the liver lobule and per photo or field of view (10×). 48 photos analyzed from 3 mice. Data are presented as mean values +/− SEM. One-way ANOVA, Tukey’s multiple comparisons post-test. Ki67 IHC showing a liver lobule. Gray, red and blue arrows show PP, Mid and PC hepatocytes positive for Ki67. Scale bar, 100 μm. J Hepatocyte ploidy in the liver lobule of 10-week Alb-CreERT2 Rosa26^rbw mice. % of total polyploid and diploid hepatocytes within the liver lobule (top left graph) and per area of the liver lobule (top right graph). % of diploid hepatocytes (hepatocytes that express only 1 fluorophore) being mononucleated or multinucleated (bottom left graph). % of polyploid hepatocytes (hepatocytes that express more than 1 fluorophore) being mononucleated or multinucleated (bottom right graph). Data are presented as mean values +/− SEM. Right: Immunofluorescence showing 4 of 5 individual PC hepatocytes expressing more than 1 color. 5 photos from 3 mice were analyzed. Scale bar, 100 μm. Source data are provided as a Source Data file.