Polyploidy in Tissue Repair and Regeneration

Abstract

Polyploidy is defined as a cell with three or more whole genome sets and enables cell growth across the kingdoms of life. Studies in model organisms have revealed that polyploid cell growth can be required for optimal tissue repair and regeneration. In mammals, polyploid cell growth contributes to repair of many tissues, including the liver, heart, kidney, bladder, and eye, and similar strategies have been identified in Drosophila and zebrafish tissues. This review discusses the heterogeneity and versatility of polyploidy in tissue repair and regeneration. Polyploidy has been shown to restore tissue mass and maintain organ size as well as protect against oncogenic insults and genotoxic stress. Polyploid cells can also serve as a reservoir for new diploid cells in regeneration. The numerous mechanisms to generate polyploid cells provide an unlimited resource for tissues to exploit to undergo repair or regeneration.

Throughout the course of an animal's life, its cells need to be replaced due to damage caused by injury, aging, and disease. Tissue repair and regeneration are the two main strategies to compensate for cell loss. Regeneration replicates the missing cells to restore the exact tissue, organ, or limb without scar formation (Goldman and Poss 2020). Whereas tissue repair heals the wound and compensates for cell loss albeit often with scar formation and changes in cellular organization (Gjelsvik et al. 2019; Lazzeri et al. 2019). Cells are then generated by either cell division or cell growth, where cell size increases with a corresponding increase in ploidy (DNA content, C-value). A cell with more than the diploid copy of its genome (>3C) is referred to as a polyploid cell.

Polyploid cell growth is an integral part of development and homeostasis across multiple organisms (Edgar et al. 2014; Orr-Weaver 2015). While polyploidy is commonplace among plants and insects, it is now recognized that vertebrates possess many somatic polyploid cell types in numerous tissues, including within the placenta, muscle, heart, liver, pancreas, bladder, kidney, and eye (Edgar et al. 2014; Gjelsvik et al. 2019). This is in part because of the many advances in cell imaging and genomics in the last decade. Developmentally programmed polyploidy is recognized to be an essential step in many organisms’ cellular differentiation programs and has been extensively reviewed (Edgar et al. 2014; Schoenfelder and Fox 2015; Fox et al. 2020). Here, we focus on the emerging and diverse roles of polyploidy in tissue repair and regeneration. It is now recognized that polyploidization in some cases is desired, or even necessary, to replace lost cells when cell proliferation is limited. This review will focus on the conserved signals that give rise to polyploid cells in tissue repair and regeneration as well as their long-term role in tissue homeostasis and disease progression.

THE STRATEGIES TO GENERATE A POLYPLOID CELL

Polyploid cells can be generated by cell-cycle-dependent and independent mechanisms. Cell fusion occurs independent of cell cycle and involves two or more cells fusing together to form a multinucleated cell, known as a syncytium (Fig. 1A). The most well-studied examples of cell fusion occur in skeletal muscle development and regeneration in mice and fruit fly larvae (Lee and Chen 2019; Lehka and Rędowicz 2020). Muscle cell–cell fusion in Drosophila and mice requires conserved genes that are necessary for remodeling the cytoskeletal network and exerting mechanical forces to facilitate invasion and fusion of the founder cell (fruit fly) or stem cell (mice) with the developing or regenerating myotube. Other mammalian tissues require cell fusion for development including osteoclasts in the bone and syncytiotrophoblasts in the placenta, but the role of cell fusion in tissue repair or regeneration has yet to be determined.

Figure 1.

Polyploid cells are generated by cell fusion and endoreplication. (A) Cell fusion generates a polyploid syncytium when individual cells fuse together. The syncytium's cellular ploidy (C) equals the sum of its nuclear ploidy. This process is mediated by cytoskeletal remodeling proteins, including Rac GTPase in both epithelial and muscle cell types. (B) Mitosis produces two identical daughter cells with 2C ploidy after completing cytokinesis. (C) An endocycle produces a polyploid cell by bypassing M phase and moving between G and S phases only. This generates a daughter cell with double the DNA content as the parent cell. Diploid nuclei (beige) and ≥4C polyploid nuclei (dark blue). (D) Endomitosis results in polyploid cells by failed karyokinesis (nuclear division) generating a mononucleated, polyploid daughter cell with double the ploidy or failed cytokinesis (cell division) creating a binucleated 4C polyploid daughter cell with two diploid nuclei. Cells may use one or more of these processes to form polyploid cells in tissue repair and regeneration. (This figure was created with BioRender.com.)

In Drosophila, cell fusion is dispensable for epithelial development, but required, in part, for wound repair in all postembryonic stages of epithelial development (Galko and Krasnow 2004; Losick et al. 2013; Wang et al. 2015). Needle puncture, pinch, or laser wounds induce epithelial cell–cell fusion. In the adult fruit fly, epithelial cell fusion is dependent on Rac GTPase, which is also required for muscle cell fusion and speeds epithelial wound closure (Fig. 1A; Fernandes et al. 2005; Losick et al. 2013). In development, the fly rectal papilla was found to become multinucleated by a novel Rab5 and Rab11 GTPase-dependent remodeling of the cell apical membranes (Peterson et al. 2020). Overall, it is difficult to detect cell fusion events and there are still many unknowns to how multinucleated cells are generated and contribute to tissue repair.

More often, polyploid cells are generated by endoreplication, an incomplete cell cycle encompassing both the endocycle and endomitosis (Øvrebø and Edgar 2018; Gjelsvik et al. 2019). The endocycle increases nuclear ploidy by allowing cells to bypass M phase of the mitotic cell cycle through elevated expression of an E3 ubiquitin ligase activator, Fizzy-related (Fzr) (Fig. 1B,C; Cohen et al. 2018; Grendler et al. 2019). Fzr, known as Cdh1 in humans, activates the anaphase-promoting complex ubiquitin ligase to mediate the degradation of the M phase CDK components cyclin A and cyclin B (Sigrist and Lehner 1997). In flies, the down-regulation of cyclin A is critical for entry into endocycle, whereas mammals also require the atypical E2F7 and E2F8 repressors to reduce M phase gene expression (Chen et al. 2012; Pandit et al. 2012; Rotelli et al. 2019). The endocycle consists of only G-S phases, which are regulated by oscillations of CycE/Cdk2 (Zielke et al. 2011). Sequential endocycles will then double the cell's nuclear genome content leading to increasing nuclear ploidy (i.e., 4C, 8C, 16C, etc.).

Endomitosis occurs when cells fail to complete either karyokinesis (nuclear division) or cytokinesis (cellular division) (Fig. 1D). Failed cytokinesis occurs when there is a block in chromosome segregation or furrow formation resulting in a binucleated or multinucleated, polyploid cell. Failed cytokinesis has been shown to generate binucleated cells in the liver, heart, and bladder in response to injury and is dependent on the cytokinesis regulators, including Anln, Ect2, and Septin (González-Rosa et al. 2018; Wang et al. 2018; Jiang et al. 2019; Derks and Bergmann 2020; Donne et al. 2020). However, failed karyokinesis will result in a mononucleated polyploid cell, which enters M phase (labeled by P-Histone 3 or CycB), unlike the mononucleated polyploid cells generated by the endocycle, which do not express any mitotic markers (Fig. 1C,D). Lamin B2 was recently shown to be required for cardiomyocyte cell-cycle progression, as its genetic loss led to karyokinesis failure during heart development and regeneration (Han et al. 2020). As will be discussed, any one of these mechanisms can occur simultaneously or in sequence, adding to the complexity in how polyploid cells are generated in tissue repair and regeneration (Fig. 2).

Figure 2.

Polyploid cell growth restores tissue mass and maintains organ size in fruit fly tissues and in mouse liver. Examples of tissues in the fruit fly and mouse where polyploidy has been demonstrated to precisely restore tissue mass. (A) Epithelial cells underlying the abdomen cuticle in the adult fruit fly heal a needle puncture wound by the endocycle and cell fusion. The endocycle boosts epithelial ploidy to >4C and cell fusion speed wound closure (Losick et al. 2013). (B) The hindgut pylorus region of the fruit fly intestine is made of diploid cells that endocycle to repair genetically induced apoptotic cell damage (Cohen et al. 2018). Pyloric cells can be switched to a mitotic cell cycle by knocking down frz to restore organ size. (C) Mammalian liver regeneration relies on a combination of diploid and polyploid division as well as endoreplication of its hepatocytes (Wilkinson et al. 2018; Zhang et al. 2018; Lin et al. 2020). The mouse liver is able to regrow and restore organ size by solely relying on diploid hepatocyte division or polyploid cell growth, as observed in Cdk1 and E2F8 mouse mutants (Diril et al. 2012; Pandit et al. 2012). Total tissue ploidy equals number of cells times each cell's ploidy. (This figure was created with BioRender.com.)

GROWING BIGGER CELLS TO RESTORE TISSUE MASS AND MAINTAIN ORGAN SIZE

The regulation of organ size (mass) is essential to generate a functional and correctly proportioned tissue for both tissue growth and regeneration. An organ's size is determined by the number of its cells and their sizes. Cell proliferation will increase cell number, whereas polyploidization will boost cell size (Fig. 1). Therefore, after injury, tissue mass can be restored by either cell proliferation or polyploidization or more often a combination of both.

In the fruit fly, polyploid cells arise in response to injury or genetically induced damage in many somatic tissues including ovarian follicle cells, hindgut pyloric cells, abdominal epithelial cells, and intestinal enterocytes (Losick et al. 2013; Tamori and Deng 2013; Xiang et al. 2017; Cohen et al. 2018). Likewise, mouse hepatocytes in the liver, tubule epithelial cells in the kidney, urothelial cells in the bladder, and epicardial cells in the zebrafish heart become polyploid or boost ploidy in response to injury (Cao et al. 2017; Lazzeri et al. 2018; Wang et al. 2018; Wilkinson et al. 2018; Zhang et al. 2018). In most of these examples, the polyploid cell growth acts to compensate for cell loss and restore tissue mass as polyploidization enables the cells to grow bigger to maintain total tissue ploidy. This is most evident in the fruit fly and mouse liver, where studies have been able to rigorously measure ploidy, cell number, and/or organ size (Fig. 2).

In the fly adult abdominal epithelium, a puncture wound destroys roughly 100 cells and in turn the surviving cells adjacent to the wound site endocycle and fuse (Fig. 2A; Losick et al. 2013). Remarkably, the epithelial cells were found to boost their ploidy to precisely compensate for the cell loss (Losick et al. 2016). The Hippo-Yorkie signal transduction pathway tunes the extent of endocycling in fly abdominal epithelium in response to injury. The Yorkie transcription factor regulates entry into the endocycle by transcriptional control of G-S phase regulators: Myc, E2F1, and cycE (Grendler et al. 2019). In fly hindgut pylorus, conditional expression of apoptotic genes caused cell loss and a corresponding increase in cell ploidy via the endocycle, which is essential to maintain an epithelial barrier in this organ (Fig. 2B; Fox and Spradling 2009; Sawyer et al. 2017; Cohen et al. 2018). The pyloric cells can be genetically forced to divide by knocking down fzr, which switches the endocycle into a mitotic cell cycle (Fig. 1B,C; Cohen et al. 2018). Remarkably, the pylorus can still regrow after injury to its normal size via cell proliferation instead of polyploid cell growth. However, this sensitizes the pylorus to oncogenic growth and epithelial barrier permeability when a constitutively active Ras GTPase is expressed. Similarly, the mammalian liver relies on the ability of its hepatocytes to polyploidize, as well as divide as either diploid or polyploid cells (Fig. 2C; Duncan et al. 2010; Wilkinson et al. 2018; Zhang et al. 2018). Hepatocyte cell growth was found to precede hepatocyte division following surgical removal of the liver mass (Miyaoka et al. 2012). However, polyploid cells are less efficient at cell division and acute injury or chronic liver injury is mostly repaired by the proliferation of the diploid hepatocytes (Wilkinson et al. 2018; Zhang et al. 2018; Chen et al. 2020).

The hepatocyte ploidy state has been investigated by genetically inhibiting the generation of polyploid or diploid cells in the liver. Genetic loss of cell-cycle regulators, including Cdk1, Skp2, and p53, will cause cells to bypass M phase and default into an endocycle (Figs. 1 and 2C; Minamishima et al. 2002; Diril et al. 2012; Kurinna et al. 2013; Sladky et al. 2020). Likewise, conditional knockout of atypical E2F7 and/or E2F8, which are required for endoreplication in mammals, will prevent generation of polyploid cells (Chen et al. 2012; Pandit et al. 2012). In so doing, it was strikingly discovered that hepatocyte polyploidization or diploid cell proliferation on its own is sufficient to grow and regenerate the liver (Fig. 2C). The decrease in the mouse liver's total cell number and corresponding increase in cell size still allowed the liver to develop or regrow to its normal organ size. Some studies have found that the more polyploid livers were able to function normally without significantly comprising its detoxifying capacity (Fig. 2C; Lin et al. 2020; Sladky et al. 2020). Whereas others observed that enhanced polyploidy in the liver led to a disease-like state characterized by inflammation, fibrosis, and dysregulation of lipid metabolism (Dewhurst et al. 2020; Ow et al. 2020). The difference may lie in the genetic perturbation used to enrich for the polyploid hepatocytes, so further studies are necessary to determine long-term impact of polyploidy on liver function.

Polyploidy is often associated with disease states; however, similar to the Drosophila pylorus, polyploid hepatocytes were found to protect against oncogenic growth. Livers genetically devoid of polyploid cells, by conditionally knocking out E2F7/8, more frequently developed tumors compared with wild-type or Anln mutant enriched for polyploid hepatocytes (Wilkinson et al. 2018; Zhang et al. 2018; Lin et al. 2020). In particular, polyploidy buffered against tumor suppressor mutations, but was equally susceptible to oncogenic activation. The extra gene copies in a polyploid cell acts as a backup to protect against loss of heterozygosity. Overall, polyploid cell growth offers a means to regrow tissue mass and restore organ size, while providing additional protection against tumorigenic insults.

POLYPLOID HETEROGENEITY AND VERSATILITY

Tissue repair and regeneration often rely on a combination of cell division and polyploid cell growth as observed in the mammalian liver. Depending on the animal and its tissue, polyploidy alone or in combination with diploid division can be necessary for optimal tissue repair. This is the case for the Drosophila midgut, which relies on both resident stem cells and polyploid enterocytes for homeostasis and regeneration. The physiological demands of the fly midgut have been found to dictate the cellular mode of repair. InR, PI3K, and Tor signaling stimulate stem cell division and enterocytes to endocycle during homeostasis, whereas tissue damage activates EGFR, Ras GTPase, and MAPK signaling to boost enterocytes ploidy (Xiang et al. 2017). Under conditions of severe stem cell loss caused by starvation, the polyploid enterocytes can then be a source for the generation of new intestinal stem cells. In these rare instances, the immediate stem cell daughter, a 4C polyploid pre-enterocyte, was found to undergo a reductive cell division known as amitosis. Amitosis occurred independently of the mitotic spindle to generate two diploid daughter cells that restored the intestinal stem cell pool (Fig. 3; Lucchetta and Ohlstein 2017). Reductive polyploid divisions, via multipolar spindle formation, have also been observed during mouse liver growth, regeneration, and cancer (Duncan et al. 2010; Wilkinson et al. 2018; Matsumoto et al. 2021). Therefore, amitosis and/or reductive cell division may offer a means to resupply the organ with a diploid cell pool that has more proliferative potential.

Figure 3.

Polyploidy heterogeneity: equal or not? (A) Illustration of many cellular mechanisms in which polyploid cells are generated and in turn generate diploid cells by amitosis, reductive division, or cytokinesis. Polyploid cells can also continue to grow increasing ploidy and cell size or mitotically divide as polyploid. (B) The enigma is whether cells with equivalent ploidy are equal in terms of their structure, gene expression, and physiological function or whether polyploidy and the mechanism used to generate polyploid cells dictates its output. Diploid nuclei (beige) and >4C polyploid nuclei (dark blue). (This figure was created with BioRender.com.)

The complexity of cell-cycle options available provides an unlimited resource for tissues to exploit to undergo repair or regeneration (Fig. 1B–D). But, this also complicates the interpretation of the cellular mechanisms used by a given cell type, as diploid cells can either mitotically divide or endoreplicate, either via the endocycle or endomitosis. Once polyploid, cells can then continue to grow in size by endoreplication, divide to generate new polyploid cells, or even undergo reductive division to be the source for new diploid cells (Fig. 3A). The complexity of possible cell-cycle outcomes has led to the realization that cell-cycle activity does not always equal diploid cell division (Fig. 1). For example, the S phase markers, PCNA and BrdU/EdU, will not distinguish between mitotic cell cycles, endocycles, or endomitosis, nor inform whether the starting cell was diploid or polyploid. To do so, studies have to rely on a combination of cell-cycle markers with ploidy analysis (both number of nuclei per cell and ploidy of each cell's nucleus). In addition, live imaging or lineage analysis is necessary to confirm whether or not a cell can successfully divide and generate daughter cells.

Using these methods, polyploidy is now recognized to be as ubiquitous in mammalian organs as it is in plants and insects. What still remains unanswered is why a cell opts to become polyploid through one mechanism versus another (Figs. 1 and 3A). For example, a tetraploid (4C) cell could be generated by either cell fusion, endomitosis, or the endocycle resulting in mono- or binucleated polyploid cell. This raises the question of whether there are selective pressures that generate a bi- versus mononucleated polyploid cell or whether these polyploid cell types are equivalent (Fig. 3B).

Studies on developmentally programmed polyploidy have begun to provide insights into these questions that have yet to be explored in the context of tissue repair and regeneration. In mice, megakaryocyte maturation in the blood is dependent on endomitosis, but genetic knockdown of Cdk1 (M phase regulator) can reprogram megakaryocytes into an endocycle without compromising platelet production (Trakala et al. 2015). Therefore, a mononucleated polyploid megakaryocyte is functionally equivalent to multinucleated polyploid megakaryocyte in development. Another example, but with the opposite outcome, occurs in development of the blood–brain barrier in Drosophila larvae. The blood–brain barrier is comprised of both mono- and multinucleated polyploid subperineurial glia (SPG) (Unhavaithaya and Orr-Weaver 2012). In development, SPGs first endocycle and then enter either another endocycle or endomitosis generating both mono- and multinucleated polyploid SPGs (Von Stetina et al. 2018). Genetically inhibiting the generation of either mono- or multinucleated SPGs disrupted barrier formation indicating that both polyploid cell states are necessary and the multi- and mononucleated polyploid SPGs cannot compensate for one another in development of the blood–brain barrier.

The recent advances in genomic sequencing could help to elucidate the distinction between mono-, bi-, and multinucleated cells. Indeed, single-cell transcriptomics have been used to compare gene expression in diploid and polyploid hepatocytes, revealing a distinct gene expression program in polyploid rat hepatocytes that was not previously detected in bulk transcriptome analysis (Katsuda et al. 2020). Even single-nuclei RNA sequencing is now being used to identify the gene expression program of individual myonuclei from syncytial skeletal muscle fibers. Several studies have shown both coordinated and differential gene-expression programs in myonuclei depending on muscle fiber type and its physiological state (Dos Santos et al. 2020; Kim et al. 2020; Petrany et al. 2020). Therefore, researchers are on the precipice of identifying the genomic distinction between cellular and nuclear ploidy states.

POLYPLOIDY IN HEART REPAIR: A BARRIER OR POTENTIAL REGENERATIVE RESOURCE?

A major focus in heart regenerative medicine is to elucidate the role and regulation of polyploidy in repair and regeneration. Unlike hepatocytes in the liver, polyploid cardiomyocytes in mammalian heart do not adequately divide or grow to compensate for cell loss. Injury caused by a myocardial infarction leads to scar formation and eventual heart failure. Whereas the zebrafish heart retains diploid cardiomyocytes that rapidly divide to regenerate the heart with minimal scar formation (Poss et al. 2002). Cardiomyocyte ploidy differences were found to be linked to the animal's energy metabolism and thermogenesis, in particular thyroid hormone presence (Hirose et al. 2019). In the mouse, blockade of thyroid hormone receptors reduced the percentage of polyploid cardiomyocytes that form during development, and improved cardiac regenerative capacity. Likewise, incubation of zebrafish with T3 hormone increased the incidence of binucleated cardiomyocytes and disrupted heart regeneration.

Mammalian cardiomyocytes become polyploid during their normal differentiation program and exhibit differences in nuclear number and ploidy depending on the animal species (Hirose et al. 2019; Derks and Bergmann 2020). For example, humans have ∼57% mononucleated and ∼25% multinucleated polyploid cardiomyocytes, whereas mice have predominantly ∼78% multinucleated polyploid cardiomyocytes. However, both species retain a subset (2%–10%) of mononucleated, diploid cardiomyocytes, which are thought to be privileged, like in zebrafish, and retain the capacity, if only latent, to divide (Patterson and Swift 2019). In support of this, mouse strains have natural variation in the number of their mononucleated diploid cardiomyocytes and strains with more diploid cells were found to be more regenerative (Patterson et al. 2017). The percentage of diploid mononuclear cardiomyocytes was found to be dependent on the cardiomyocyte-specific kinase, Tnni3k. Genetic loss of Tnni3k in C57BL/6J mouse strain doubled the diploid cardiomyocyte population, whereas overexpression of Tnni3k in zebrafish cardiomyocytes increased ploidy and impaired heart regeneration similar to expression of dominant-negative Ect2, which blocks cytokinesis in zebrafish (González-Rosa et al. 2018; Gan et al. 2019).

The biological significance of cardiomyocyte ploidy for heart growth and repair still remains to be elucidated. New genetic and cell biological tools have allowed the outcome of cycling cardiomyocytes to be tracked in vivo, and researchers have found that nine out of 10 cardiomyocytes that reenter the cell-cycle endoreplicate, not completing cytokinesis postinjury (Hesse et al. 2012; Bradley et al. 2021). In addition, genetic ablation of these cycling cells compromised myocardial function postinjury demonstrating that endoreplication is beneficial, to some extent, for heart repair (Bradley et al. 2021). Additionally, polyploidy may be a potential resource for new cardiomyocytes, as binucleated cardiomyocytes can be genetically forced to complete cytokinesis via β-catenin-induced expression of Ect2 (Figs. 1 and 3; Jiang et al. 2019). Therefore, like hepatocytes in the liver, stimulating polyploid cardiomyocytes to undergo cytokinesis or endoreplication may offer an alternative means worth exploiting to compensate for lost heart mass.

Still, a major challenge is the ability to stimulate a sufficient number of cardiomyocytes to reenter the cell cycle. Many studies have focused on strategies to boost cardiomyocyte cell-cycle activity (Derks and Bergmann 2020). In the pig heart, a microRNA therapy was shown to stimulate cardiomyocyte proliferation after a myocardial infarction, but this protocol also led to electric uncoupling of the myocardium (Gabisonia et al. 2019). Therefore, functional recovery of the heart tissue mass requires a balance in proliferation and differentiation. Hence, polyploid cell growth may be a necessary means to retain electric coupling and restore heart mass. It is therefore paramount to determine the physiological function of diploid versus polyploid (both mono- versus multinucleated) cardiomyocytes and whether there are benefits to repairing the heart through one cellular mechanism versus another (Fig. 3B).

MECHANICAL FORCES: A DRIVER OF POLYPLOIDY TOO!

Biophysical forces, including shear stress and tension, instruct tissue morphogenesis by controlling cell proliferation, movement, and shape (Coravos et al. 2017). Mechanical forces have been mostly studied under conditions of diploid mitotic cell division, but these forces also regulate polyploid cell growth. Regeneration of the zebrafish epicardium, the outer layer of heart, induces transient endoreplication (both the endocycle and endomitosis) at the wound leading edge, where tension is increased concomitant with accumulation and activation of motor protein myosin (Cao et al. 2017). Endomitosis, via failed cytokinesis, was found to be integrin-dependent, as higher tractional forces delayed closure of cytokinetic ring in zebrafish epicardial cells (Uroz et al. 2019). Similarly, in vitro, a rigid extracellular matrix resulted in binucleation of neonatal cardiomyocytes (Yahalom-Ronen et al. 2015). Other biophysical forces, including shear stress, are also required to induce polyploidization of megakaryocytes during maturation (Jiang et al. 2014).

In Drosophila, integrin signaling was shown to regulate epithelial wound-induced polyploidization (Besen-McNally et al. 2021). Components of focal adhesion complex, including β-integrin, focal adhesion kinase (Fak), and Talin are required for the adult Drosophila abdominal epithelial cells to endoreplicate postinjury. In this model, integrin activates the endocycle via Yorkie and is also necessary for optimal cell fusion and wound closure. Mechanical forces are critical in Drosophila muscle development as well. Myosin increases tension necessary for resistance in the receiving cell and formation of the fusion pore for cell–cell fusion (Kim et al. 2015). Likewise, mechanical forces within the myotube instruct myonuclear position and nuclear ploidy (Windner et al. 2019). There are still many unknowns in how mechanics instruct polyploid cell growth via endoreplication and cell fusion and in how polyploid cells, in turn, affect tissue mechanics as polyploid cells can persist after injury and accumulate with age.

THE INTERPLAY BETWEEN GENOTOXIC STRESS AND POLYPLOIDY

Cells are consistently challenged with genotoxic stress, both exogenously (i.e., radiation, chemical) and endogenously (reactive oxygen species). These stressors can result in DNA damage, including double-stranded breaks (DSBs) that activate the p53-dependent DNA damage response (DDR) resulting in cell-cycle arrest or cell death. However, endoreplication is able to promote cell growth in the presence of DNA damage (Mehrotra et al. 2008). It was discovered that endocycling cells in the Drosophila ovary are resistant to irradiation, whereas mitotically dividing cells undergo apoptosis (Hassel et al. 2014; Zhang et al. 2014). The endocycling follicle cells reduce p53 signaling by epigenetic silencing of its gene targets and via proteolytical degradation p53 itself to prevent apoptosis. This is integral to the endocycle as DNA damage is normally generated due to underreplication of the cell's genome during these cycles (Nordman et al. 2011; Yarosh and Spradling 2014).

In context of tissue repair, endoreplication offers a means to regrow tissues in the presence of genotoxic stress when diploid cell proliferation would otherwise be inhibited. Switching the adult Drosophila abdominal epithelium to the mitotic cycle prevented wound repair, whereas the fly epithelium could still heal via polyploid cell growth even after exposure to UV irradiation (Grendler et al. 2019). DNA damage is known to accumulate with age in many tissues, including the brain, where neurons in Drosophila were found to become polyploid with age via the endocycle (Nandakumar et al. 2020). As in tissue repair, endoreplication offers a means to compensate for cell loss due to normal aging. This also occurs in the human corneal disease Fuchs endothelial dystrophy, where terminally differentiated endothelial cells turn over at a faster rate yet the remaining endothelial cells boost their ploidy to compensate for cell loss, maintaining total tissue ploidy (Losick et al. 2016). Oxidative stress is thought to be one of the drivers of Fuchs dystrophy, causing both mitochondrial and nuclear DNA damage (Jurkunas 2018). Oxidative stress was also found to be a driver of nonalcoholic fatty liver disease, where highly polyploid hepatocytes accumulate (Gentric et al. 2015). Interestingly, oxidative stress-induced DNA damage appears to be a widespread signal to initiate endoreplication. Both the polyploidization of cardiomyocytes in development and generation of giant granuloma macrophages during inflammation are caused by DNA damage (Puente et al. 2014; Herrtwich et al. 2016). However, it is still unknown how specifically polyploidy (via cell fusion, endomitosis, or endocycle) in these different tissue contexts subverts the DDR and whether enhanced polyploidy itself is the cause or consequence of disease progression (Fig. 3).

CONCLUDING REMARKS

From fruit flies to humans, it is now recognized that polyploidy serves as an essential strategy for cells to grow and compensate for cell loss in tissue repair and regeneration. Polyploidy provides versatility as more than one cellular mechanism can be used to restore tissue mass after injury. Polyploid cell growth also enables repair in the presence of genotoxic stress and while protecting against oncogenic stimuli. The remaining enigma lies in why one ploidy state is used over another (mono- vs. multinucleated) and why some polyploid cell types, like liver hepatocytes, are competent to undergo division, reductive division, and growth, whereas others, like mammalian cardiomyocytes, are limited. In the future, it will be critical to understand how the cell's ploidy state is regulated and how it affects cellular and tissue function, as inducing polyploid cell growth may offer a more natural means to restore organ size by maintaining cell contacts and physiological function.