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 unknown1,2,3,4. Polyploidization occurs through failed cytokinesis, begins at weaning in rodents and increases with age2,5,6,7. Previously, we demonstrated that the opposite event, ploidy reversal, also occurs in polyploid hepatocytes generated by artificial cell fusion8,9,10. 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.

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Figure 1: Purified octaploid hepatocytes generate reduced-ploidy daughters in vivo.
Figure 2: Polyploid hepatocytes undergo ploidy reversal and unequal marker segregation in vitro.
Figure 3: Polyploid hepatocyte mitoses with multipolar spindles and chromosome segregation defects.
Figure 4: Live cell imaging of multipolar mitoses in hepatocytes.

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Acknowledgements

We thank P. Canaday (Flow Cytometry Resource at OHSU) for cell sorting; A. Snyder and S. Kaech Petrie (Advanced Light Microscopy Core at OHSU, Core grant S10-RR023432) for microscopy assistance; and the Morphology Core of the Texas Medical Center (DK56338) for histology support. We also thank L. Smith and M. Thayer for discussions. This work was supported by grants from the National Institute of Health to M.G. (R01DK067636) and A.W.D. (F32DK076232).

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Contributions

A.W.D. designed and performed most of the experiments, analysed data and wrote the paper. M.H.T. helped with imaging of dividing hepatocytes. R.D.H. assisted with data analysis. A.E.H.N., M.L.L. and S.B.O. performed all of the cytogenetic analyses. Histological analyses were performed by M.J.F. M.G. supervised all aspects of this work. All authors discussed the results and edited the manuscript.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-14 with legends. (PDF 6377 kb)

Supplementary Movie 1

This movie shows a single binucleated tetraploid hepatocyte undergoing bipolar mitosis. Successful cytokinesis produces 2 mononucleated daughter cells. Time-lapse sequence is annotated in Supplementary Figure 9. (MOV 3051 kb)

Supplementary Movie 2

This movie shows a single mononucleated tetraploid hepatocyte undergoing bipolar mitosis. Failed cytokinesis produces a single binucleated cell. Time-lapse sequence is annotated in Supplementary Figure 10. (MOV 1835 kb)

Supplementary Movie 3

This movie shows a single binucleated tetraploid hepatocyte undergoing tripolar division. Partial failed cytokinesis generates a mononucleated daughter and a binucleated daughter. Time-lapse sequence is annotated in Figure 4b. (MOV 1739 kb)

Supplementary Movie 4

This movie shows a single binucleated tetraploid hepatocyte undergoing tripolar division. Partial failed cytokinesis generates a mononucleated daughter and a binucleated daughter. Both daughter cells proceed to divide again, producing 2 mononucleated daughters each. Time-lapse sequence is annotated in Supplementary Figure 11. (MOV 5689 kb)

Supplementary Movie 5

This movie shows a binucleated tetraploid hepatocyte undergoing double mitosis. Adjacent nuclei migrate apart prior to entering mitosis. Each nucleus undergoes a distinct mitosis, generating 4 daughter nuclei. Successful cytokinesis produces 4 mononucleated daughter cells. Time-lapse sequence is annotated in Supplementary Figure 13. (MOV 2910 kb)

Supplementary Movie 6

This movie shows a mononucleated tetraploid hepatocyte undergoing double mitosis. Following nuclear breakdown, chromosomes align along 2 discrete metaphase plates, which undergo simultaneous bipolar anaphase to produce 4 daughter nuclei. Cytokinesis is partially successful, generating 2 mononucleated daughters and 1 binucleated daughter. Time-lapse sequence is annotated in Supplementary Figure 14. (MOV 2171 kb)

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Duncan, A., Taylor, M., Hickey, R. et al. The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 467, 707–710 (2010). https://doi.org/10.1038/nature09414

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