The ploidy conveyor of mature hepatocytes as a source of genetic variation

Abstract

Mononucleated and binucleated polyploid hepatocytes (4n, 8n, 16n and higher) are found in all mammalian species, but the functional significance of this conserved phenomenon remains unknown. Polyploidization occurs through failed cytokinesis, begins at weaning in rodents and increases with age. Previously, we demonstrated that the opposite event, ploidy reversal, also occurs in polyploid hepatocytes generated by artificial cell fusion. This raised the possibility that somatic 'reductive mitoses' can also happen in normal hepatocytes. Here we show that multipolar mitotic spindles form frequently in mouse polyploid hepatocytes and can result in one-step ploidy reversal to generate offspring with halved chromosome content. Proliferating hepatocytes produce a highly diverse population of daughter cells with multiple numerical chromosome imbalances as well as uniparental origins. Our findings support a dynamic model of hepatocyte polyploidization, ploidy reversal and aneuploidy, a phenomenon that we term the 'ploidy conveyor'. We propose that this mechanism evolved to generate genetic diversity and permits adaptation of hepatocytes to xenobiotic or nutritional injury.

Fig. 1. Purified octaploid hepatocytes generate reduced-ploidy daughters in vivo

a, Hepatocytes were separated into ploidy populations by FACS with 2c, 4c and 8c DNA content, corresponding to diploid, tetraploid and octaploid hepatocytes, respectively. Highly pure viable octaploid hepatocytes were collected. b, Hepatocytes isolated from mice repopulated by 8c donors were loaded with Hoechst + FDG. All ploidy classes expressed ß-gal (n=6 recipients). c, d, Chromosome number (c) and representative aneuploid karyotypes (d) of donor-derived mChrY+ hepatocytes in repopulated livers (n=3). Chromosomal gains/losses are described relative to the nearest ploidy. e, Percentage of numerical aneuploidy in donor-derived hepatocytes from repopulated mice or wild-type hepatocytes from non-transplanted mice (average ± s.e.m.; see Supplementary Fig. 5 for details). * P<0.006; ** P=0.01.

Fig. 2. Polyploid hepatocytes undergo ploidy-reversal and unequal marker segregation in vitro

a, b, DNA content of cultured hepatocyte populations was determined by FACS. Representative plot is shown for freshly-sorted 4c hepatocytes and cells expanded for 5d (a). Results are summarized over multiple experiments (b). Data points represent average values ± s.e.m. c, ∼ 99% of freshly-isolated 2c or 4c hepatocytes had the expected number of hCD46 and mChr9 signals. Cultured hepatocytes displayed chromosome missegregation. *, P < 0.0001 compared to freshly-isolated cells. d, Schematic of the observed FISH signals in 2c daughters derived from cultured 4c hepatocytes.

Fig. 3. Polyploid hepatocyte mitoses with multipolar spindles and chromosome segregation defects

a-h, Mitotic figures were detected in cultured hepatocytes by visualizing DNA (blue) and microtubules (green). Centrioles (red) were detected in a-d; centromeres (red) were detected in h. (n=5 experiments). 4c hepatocytes contained bipolar (a) or multipolar spindles (b, c). Multipolar spindles were also seen in 8c hepatocytes (d). Three daughter nuclei emerged from a tripolar telophase (e). Double mitosis was detected in metaphase (f) and anaphase (g). Lagging chromosomes (arrow and inset) were also seen (h). i-l, Hepatocytes dividing in vivo formed similar mitotic figures: multipolar spindles (i), tripolar division (j), double mitosis (k) and lagging chromosomes (l) (n=4 mice). Scale bars are 10 μm.

Fig. 4. Live cell imaging of multipolar mitoses in hepatocytes

a, Summary of mitotic events captured by time-lapse microscopy. The percentage (average ± s.e.m.) of dividing mononucleated (red) and binucleated (blue) hepatocytes is shown. * P<0.001; ** P<0.0001. b, Images (related to Supplementary Movie 3) of a binucleated tetraploid hepatocyte undergoing tripolar division. Time (hours : minutes) is indicated. Mitosis was tracked with DIC and Hoechst (pseudocolored green). Cell boundaries are marked. Scale bar is 10 μm. c-e, Schematic depiction of divisions completed by polyploid (mono- or binucleated) hepatocytes. The outcome of each division depends on spindle reorganization and completion/failure of cytokinesis. Metaphase chromosomes (blue), mitotic spindles (green) and centrosomes (red) are indicated.