Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes.
Polyploidization is the most well recognized feature of the liver. Yet, a quantitative and behavioral analysis of centrosomes and DNA content in normal hepatocytes has been limited by the technical challenges of methods available. By using a novel approach employing FISH for chromosomes 18, X and Y we provide, for the first time, a detailed analysis of DNA copies during physiological development in the liver at single cell level. We demonstrate that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features in normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. We show that in murine liver the period between 15 days and 1.5 months marks the transition from a prevalence of mononucleated cells to up to 75% of binucleated cells. Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomes converge with several hepatocytes that contain 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreases concomitantly with the increase of cells with more than 4 centrosomes. Our analysis shows that the extranumerary centrosomes emerge in concomitance with the process of binucleation and polyploidization and maintain α-tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. We speculate that these unique features are relevant to the peculiar biological function of liver cells which are continuously challenged by stress, a condition that could predispose to genomic instability.
Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy.
Francesca
F
Faggioli
Paolo
P
Vezzoni
Cristina
C
Montagna
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002467:Cell Nucleus / Q000378:metabolism; D018385:Centrosome / Q000378:metabolism*; D033481:Chromosomes, Mammalian / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D006657:Histones / Q000276:immunology; D017404:In Situ Hybridization, Fluorescence; D007399:Interphase; D051379:Mice; D011003:Ploidies; D059010:Single-Cell Analysis / Q000379:methods*
D000906:Antibodies; D006657:Histones; D004247:DNA