Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes

Francesca Faggioli,Cristina Montagna,Paolo Vezzoni,Ferdinando Di Cunto

doi:10.1371/journal.pone.0026080

Abstract

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.

Highlights

Despite the body of work investigating the mechanisms leading to liver polyploidization [1,2,3,4], a detailed analysis of hepatocytes at the single cell level during their physiological development has not yet been described
Nelsen and colleagues reported similar data in normal hepatocytes; they detected a small fraction of normal hepatocytes with three centrosomes, while .4 centrosomes were detected only in cells transfected with cyclin D
To test the hypothesis that adult polyploid hepatocytes are able to generate progeny with halved chromosome content, Duncan et al [13] demonstrated that after 5 days in culture tetraploid hepatocytes produced daughters cells with 8n and 2n DNA content. They were the first to show that tetraploid liver cells are able to divide and that, during cell division, centrosomes can polarize multipolar spindles, this is a temporary step followed by its reorganization into a bipolar spindle

Summary

Introduction

Despite the body of work investigating the mechanisms leading to liver polyploidization [1,2,3,4], a detailed analysis of hepatocytes at the single cell level during their physiological development has not yet been described. To test the hypothesis that adult polyploid hepatocytes are able to generate progeny with halved chromosome content (a phenomenon known as ‘‘ploidy conveyor’’), Duncan et al [13] demonstrated that after 5 days in culture tetraploid hepatocytes produced daughters cells with 8n and 2n DNA content. They were the first to show that tetraploid liver cells are able to divide and that, during cell division, centrosomes can polarize multipolar spindles, this is a temporary step followed by its reorganization into a bipolar spindle. Even in Duncan’s paper a deep analysis of the number of centrosomes for mono and binucleated hepatocytes during different developmental stages has not been shown

Methods

Results

Conclusion