Fig. 2
From: Circadian clock regulates hepatic polyploidy by modulating Mkp1-Erk1/2 signaling pathway
Nuclear ploidy and binucleation: zone-specific distribution and its developmental change detected by double staining immunohistochemistry with cell membrane marker β-Catenin and nuclear marker Hoechst-33342. By applying the immunohistochemistry of β-Catenin, we analyzed the nuclear ploidy and multinucleation in WT (a, c) and Per-null (b, d) mice (10 weeks). (c) and (d) are high-power field photomicrographs of indicated areas of (a) and (b), respectively. We divide the liver lobule in 15 zones, as indicated by the lines, and counted the mononuclear 2n cells (2n × 1), binuclear 2n cells (2n × 2), mononuclear 4n cells (4n × 1), binuclear 4n cells (4n × 2), mononuclear 8n cells (8n × 1), binuclear 8n cells (8n × 2), mononuclear 16n cells (16n × 1), binuclear 16n cells (16n × 2), mononuclear 32n cells (32n × 1) and binuclear 32n cells (32n × 2) according to the nuclear size of 2n cells in each section. e We show the incidence (%) of each ploidy state, mononucleated and binucleated, in each zone (centrilobular, midlobular and periportal zone) for each hepatic lobe (n = 600 hepatocytes from 5 mice of each genotype), assuming the contribution of each zone to be equal. For developmental analysis f, we analyzed liver from 2, 4, 6, and 8 weeks old WT and Per-null mice (n = 150–360 hepatocytes form 3–5 mice of each genotype at each developmental stage), and analyzed the incidence of each polyploidy state, of mononuclear and binuclear cells, in each developmental stage. Data are analyzed by Two-way ANOVA with Bonferroni’s post-test. All values represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm in (a, b, f) and 20 μm in (c, d). Note hepatocytes increase their polyploidization progressively age-dependently, except for periportal cells, and the prominent polyploidization and binucleation progressively accelerates in pericentral and midlobular zones
