BIOLOGICAL OVERVIEW

The anaphase-promoting complex/cyclosome (APC) is a multisubunit ubiquitin ligase that targets several mitotic regulators for degradation and thereby triggers an exit from mitosis. APC activity is restricted to mitotic stages M and G1. This restriction is achieved by the cell cycle-dependent association of proteins encoded by fizzy and fizzy-related genes functioning during M and G1 respectively. fzr bears the accepted FlyBase designation of retina aberrant in pattern, because of a study showing that mutants show a disruption in eye patterning (Karpilow, 1989). Nevertheless, the alternative name fizzy-related is widely used and will be used in this essay. fzr, a conserved eukaryotic gene, negatively regulates the levels of cyclins A, B, and B3. These mitotic cyclins that bind and activate cdk1(cdc2) are rapidly degraded during exit from M and during G1. fzr is required for cyclin removal during G1 when the embryonic epidermal cell proliferation stops and during G2 during the cell cycle preceding salivary gland endoreduplication. Loss of fzr causes progression through an extra division cycle in the epidermis and inhibition of endoreduplication in the salivary gland, in addition to failure of cyclin removal. Conversely, premature fzr overexpression down-regulates mitotic cyclins, inhibits mitosis, and transforms mitotic cycles into endoreduplication cycles (Sigrist, 1997).

Binding of Cdc20 (the homolog of Fizzy in yeast and vertebrates) and Cdh1 (the homolog of Fizzy-related in yeast and vertebrates) to the APC is differentially regulated. APC-Cdc20 activity is present during mitosis and initiates the metaphase-anaphase transition. The association of Cdc20 with the APC requires phosphorylation of at least one subunit of the APC. Several mitotic kinases have been implicated in this phosphorylation. The dependency of APC phosphorylation on Cdc20 binding ensures that APC-Cdc20 is only active during mitosis. During prophase and prometaphase, APC-Cdc20 activity is furthermore restrained by the spindle checkpoint. This system monitors the presence of unattached kinetochores. Until kinetochores are bound by spindles, they serve as an assembly point for active Mad2 protein. Mad2 binds to Cdc20 and inhibits APC activity. Once all kinetochores are attached and chromosomes are aligned on the metaphase plate, Mad2 inhibition of APC-Cdc20 activity is released (Grosskortenhaus, 2002 and references therein).

Cdh1 is found in association with the APC during later stages of mitosis and G1. This interaction depends on the phosphorylation status of Cdh1 (Kramer, 2000; Zachariae, 1998). Only unphosphorylated Cdh1 is able to bind to and activate the APC (Kotani, 1999; Kramer, 2000). Cdk1 and Cdk2 mediate Cdh1 phosphorylation. Thus, only during stages of low Cdk kinase activity will Cdh1 activate the APC. These requirements are fulfilled during later stages of mitosis, when APC-Cdc20 has induced the degradation of mitotic cyclins, and during G1, when Cdk kinase activity is low. However, the G2 stage is also characterized by low Cdk kinase activity. How Cdh1-dependent APC activity is prevented in these situations has not been addressed so far (Grosskortenhaus, 2002 and references therein).

The mitotic cyclins in Drosophila (Cyclin A [CycA], Cyclin B [CycB], and Cyclin B3) are stable in interphase, degraded during mitosis, and continue to be unstable throughout G1. Cdc20^Fzy is required for mitotic cyclin destruction at the metaphase-anaphase transition and is thought to mediate the bulk of cyclin degradation in the first 16 cell cycles in Drosophila. Mutants in fzy arrest in metaphase of cell cycle 16 when the maternal supply of Cdc20^Fzy is exhausted. Overexpression of fzy does not cause abnormal cyclin destruction. Thus, Cdc20^Fzy is not able to activate the APC at other cell cycle stages. This likely reflects the inability of Cdc20 to interact with unphosphorylated APC (Grosskortenhaus, 2002 and references therein).

Analysis in Drosophila demonstrates that fzr is expressed and of crucial importance when cells terminate cell proliferation during embryogenesis. Loss of fzr results in progression through an extra cell cycle in epidermal cells and in inhibition of endoreduplication in salivary glands. These deviations from the normal developmental cell cycle program are accompanied by a failure to down-regulate mitotic cyclins (cyclins A, B, and B3) that bind and activate cdk1(cdc2) kinase. Premature fzr overexpression, when epidermal cells still proliferate, down-regulates mitotic cyclins followed by inhibition of mitosis. All of these findings are consistent with the idea that Fzr activates degradation of mitotic cyclins and thereby prevents ectopic cdk1(cdc2) activity when cells become postmitotic (Sigrist, 1997).

The consequences of loss of fzr were examined with a deficiency deleting other genes in addition to fzr. However, in these deficient embryos, cell cycle defects occurred exclusively at stages and in tissues corresponding to the dynamic developmental program of fzr expression. Moreover, the various cell cycle defects were all corrected by expressing Hs-fzr in the deficient embryos at levels below the endogenous fzr expression as judged by in situ hybridization. Finally, except for correcting the cell cycle defects in tissues that normally express fzr, this moderate Hs-fzr expression has no effects on cell cycle progression in deficient embryos (Sigrist, 1997).

Ectopic cyclin E expression as well as failure to express the cyclin E/cdk2 inhibitor p27^DAP in the embryonic epidermis results in ectopic accumulation of mitotic cyclins and in progression through an ectopic division cycle instead of a G1 arrest. Therefore, ectopic cyclin E/cdk2 activity in the postmitotic epidermal cells has the same phenotypic consequences as loss of fzr, suggesting that Fzr might act primarily as a negative regulator of cyclin E/cdk2 and indirectly on mitotic cyclins. All of the following findings, however, argue strongly against this interpretation. By immunolabeling with anti-cyclin E and anti-DAP antibodies, altered expression in either fzr-deficient embryos or after prd-Gal4-directed UAS-fzr expression was not detected (Sigrist, 1997).

In addition, while ectopic UAS-dap expression in the salivary gland during the larval stages inhibits endoreduplication effectively, UAS-fzr expression has no effect. Finally, when UAS-fzr expression is directed to imaginal disc cells during the larval stages, endoreduplication instead of the normal mitotic proliferation results, as also observed in CycA and Cdc2 mutants, but not in CycE mutants (Sigrist, 1997).

The onset of the developmentally programmed physiological endoreduplication in the salivary gland, therefore, might depend on the inactivation of cdk1 complexes. This inactivation might be achieved by the down-regulation of mitotic cyclins caused by the especially high levels of fzr expression that are observed transiently in the salivary gland before the onset of the first endoreduplication S phase. In the fzr-deficient embryos, mitotic cyclins continue to accumulate and cdk1 activity is expected to be maintained, resulting in the inhibition of endoreduplication (Sigrist, 1997).

While the inactivation of cdk1 is thought to establish the competence to initiate another round of DNA replication, the actual onset of DNA replication is dependent on the activation of cdk1 in yeast and cdk2 in higher eukaryotes. Particular cyclins, Clb5 and Clb6 in yeast and cyclins E and A in higher eukaryotes, are normally involved in this activation under physiological conditions. However, DNA replication can be activated in yeast and higher eukaryotes by cyclin/cdk1 complexes that play little or no role during the G1/S transition under physiological conditions. Moreover, ectopic expression of Drosophila cyclin A, which appears to bind only to cdk1 and not to cdk2, is definitively capable of driving cells from G1 into S. Premature activation of cdk1 must presumably be prevented when cells have to be maintained in the G1 phase. The entry into an additional S phase that is observed in the epidermis of fzr-deficient embryos, therefore, might also result from the failure to down-regulate the mitotic cyclins (Sigrist, 1997).

fzr was identified because of its similarity to fzy, which is required for progression beyond metaphase and mitotic cyclin degradation. fzr and fzy encode highly similar proteins with seven WD repeats in the C-terminal region. WD repeats are found in many proteins with diverse biological function. They are also found in budding yeast Cdc4p, which is required for the ubiquitin-dependent proteolysis of important cell cycle regulators. CDC4 acts in a pathway with CDC53, and interestingly, mutations in cul-1, a C. elegans homolog of CDC53, have recently been shown to result in a similar inability to arrest cell proliferation at the appropriate developmental stage as also apparent in the fzr-deficient embryos (Sigrist, 1997).

The closest yeast relative of fzr, however, is not CDC4 but HCT1, which is required for proteolysis of Clb2p, a budding yeast B-type cyclin with a characteristic destruction box as also present in A- and B-type cyclins of higher eukaryotes. Drosophila fzr appears to be unable to provide HCT1 function in yeast. It remains to be demonstrated that Fzr activates the proteolytic degradation of mitotic cyclins. However, the abnormal cyclin accumulation in both fzr and fzy mutants is not associated with an apparent increase in transcript levels, indicating that Fzr and Fzy down-regulate mitotic cyclins at a posttranscriptional level. If Fzy and Fzr trigger cyclin proteolysis as suspected, it will be important to clarify their functional relationship with proteins known to be required for the ubiquitin-dependent degradation of mitotic cyclins, with Cse1p and the anaphase-promoting complex (APC), a complex composed of several proteins. In addition, it will be interesting to evaluate the relationship of fzr and roughex, an unrelated Drosophila gene with a similar function required at other developmental stages (Sigrist, 1997).

Double mutant analyses demonstrate that Fzy and Fzr are specialized for the down-regulation of mitotic cyclins during either M phase or interphase, respectively. fzy is expressed in proliferating cells and is required for progression beyond metaphase and mitotic cyclin degradation; fzr transcripts accumulate when cells become postmitotic and are required for cyclin down-regulation in G1 during cell cycle exit and in G2 before endoreduplication but not during mitosis. It will be interesting to learn whether fzr is required in proliferating cells progressing through cell cycles with G1 phases (which do not occur during Drosophila embryogenesis). Since Fzy and Fzr promote down-regulation of mitotic cyclins in different cell cycle phases, they might have evolved to respond to different regulatory inputs. A fraction of Fzy is modified precisely during the metaphase/anaphase transition when mitotic cyclin degradation starts. Cyclin degradation during mitosis, and perhaps Fzy activity as well, is controlled by checkpoint mechanisms that monitor spindle integrity and chromosome attachment (Sigrist, 1997)

In contrast, degradation of mitotic cyclins during interphase appears to be important for cell cycle exit and entry into endoreduplication. fzr transcription is regulated by developmental cues that stop embryonic cell proliferation. In addition, Fzr appears to be regulated as well. Observations suggest that Fzr is negatively regulated by cyclin E/cdk2 activity, which in turn is also controlled by the developmental cues that stop embryonic cell proliferation. These developmental cues result in CycE down-regulation and dacapo up-regulation at the stage where cell proliferation is to be arrested. Therefore, the resulting inhibition of cyclin E/cdk2 activity might activate Fzr and thereby prevent mitotic cyclin accumulation and ectopic cdk1 activity when cells become postmitotic (Sigrist, 1997).

It is likely that FZY and Fzr are involved in the degradation of proteins other than the known A- and B-type cyclins. It is thought that the degradation of proteins like the fission yeast cut2 protein is required for sister chromatid separation in mitosis, and fzy is clearly required not only for cyclin degradation but also for sister chromatid separation in mitosis. It is possible, therefore, that Fzy and Fzr trigger the proteolytic degradation of different subsets of proteins (Sigrist, 1997).

In summary, the conserved fzr gene might be generally important in higher eukaryotes for transitions in the developmental cell cycle program (Sigrist, 1997).

APC/C^Fzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. This study reports that APC/C^Fzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. Compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, the data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, these data suggest a model in which the cyclic activity of APC/C^Fzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. It is proposed that, as is observed in mitotic cycles, during endocycles, APC/C^Fzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression (Narbonne-Reveau, 2008).

The endocycle provides a useful model for determining the minimum cell cycle inputs required to achieve a G/S oscillation and the once-per-cell-cycle replication of the genome. This study demonstrates that APC/C activity is required for endocycle progression. During the endocycle, mitotic activities are repressed. This is accomplished, at least in part, by preventing the accumulation of the mitotic activators Cyclin A, Cyclin B and Cdc25, which function to activate the mitotic kinase Cdk1. During the mitotic cycle, the mitotic cyclins are periodically targeted for regulated proteolysis by the E3-Ubiquintin ligase the APC/C. Yet the transcriptional downregulation of several APC/C targets at the mitotic/endocycle boundary, including the mitotic cyclins and String/Cdc25, suggested that the proteolytic activity of the APC/C might not be necessary during endocycles. However, this study found that compromising APC/C activity, after cells have entered the endocycle, results in the accumulation of Geminin and the mitotic cyclins, and in a block of DNA replication. Thus, the transcriptional downregulation of APC/C targets observed at the mitotic/endocycle transition is either downstream of APC/C activity and/or not sufficient to maintain low levels of these proteins. Taken together, these data suggest a model in which APC/C promotes the G/S oscillation of the endocycle by preventing the unscheduled accumulation of Geminin and the mitotic cyclins (Narbonne-Reveau, 2008).

During endocycles, APC/C activity prevents the inappropriate accumulation of Geminin, an inhibitor of the DNA replication-licensing factor Cdt1/Dup. When directly expressed in endocycling cells, Geminin efficiently inhibits DNA replication. These results strongly suggest that an essential function of the APC/C during the endocycle is to prevent the unregulated accumulation of Geminin. A similar role has been proposed for the APC/C during endoreplicative cycles of mouse trophoblasts (Gonzalez, 2006). However, the current data indicate that Geminin is not the only essential target of the APC/C during endocycles. A candidate for a second important target of the APC/C during endocycles is Cyclin A. Previous studies have shown that the overexpression of Cyclin A in the salivary gland, between the first and second endocycle, results in variable inhibitory effects on endoreplication. Although the majority of salivary gland cells that overexpress Cyclin A appear to be unaffected, a small percentage show a marked decrease in ploidy values. The reason for this variability is not clear. However, if the inhibitory influence of Cyclin A is mediated through binding and activation of Cdk1, this effect may be greatly amplified in the presence of high levels of String/Cdc25, which removes an inhibitory phosphate from Cdk1. Recent studies indicate that String/Cdc25, which contains both a consensus Ken box and D-box, is a target of the APC/C (Barbara Thomas, personal communication to Narbonne-Reveau, 2008). Therefore, an essential function of the APC/C during endocycles may involve restricting the activity of the mitotic kinase Cdk1, by preventing the accumulation of both Cyclin A and String/Cdc25. Finally, it is noted that the APC/C may have additional essential targets during the endocycle, which have yet to be identified (Narbonne-Reveau, 2008).

The periodic accumulation of the Orc1 protein during endocycles strongly suggests that the activity of the APC/C^Fzy/Cdh1 may not be continuous but cyclical. Previous work indicates that in Drosophila Cyclin E/Cdk2 inhibits the activity of APC/C^Fzy/Cdh1. These data are consistent with the observation that phosphorylation of Fzr/Cdh1 by Cdks inhibits the ability of Fzr/Cdh1 to bind and activate the APC/C in yeast, Xenopus and mammals. During the endocycle, the levels of Cyclin E oscillate. Taken together, these observations suggest a model in which APC/C^Fzy/Cdh1 is regulated by the periodicity of Cyclin E/Cdk2 activity, with high levels of Cyclin E resulting in the inhibition of APC/C^Fzy/Cdh1 activity and low levels of Cyclin E permitting full APC/C^Fzy/Cdh1 activity. The current data support this hypothesis. First, it was found that the periodicity of Orc1 levels during the endocycle requires a functional O-box, consistent with the cyclic destruction of Orc1 by APC/C^Fzy/Cdh1. Second, the levels of Orc1 are sensitive to Cyclin E. Specifically, overexpressing Cyclin E after cells have entered the endocycle results in the accumulation of APC/C^Fzy/Cdh1 targets, including Orc1, Cyclin A, Cyclin B and Geminin. Thus, the regulatory relationship observed between Cyclin E/Cdk2 and Fzr/Cdh1 that has been reported during mitotic cycles is conserved during endocycles. Finally, in endocycling cells the accumulation of Orc1 occurs during periods of high Cyclin E/Cdk2 activity, when APC/C^Fzy/Cdh1 dependent proteolysis would be predicted to be low. These data support the idea that the oscillations of Cyclin E/Cdk2 activity drive the periodicity of APC/C^Fzy/Cdh1 activity during the endocycle (Narbonne-Reveau, 2008).

Although a requirement for the oscillation of APC/C^Fzy/Cdh1 activity during the Drosophila endocycle has not been formally demonstrated, it is interesting to speculate on how the cyclic, rather than the continuous, activity of the APC/C might serve to facilitate endocycle progression. The data indicate that a period of high APC/C^Fzy/Cdh1 activity is required during the G phase of the endocycle in order to degrade the mitotic cyclins and Geminin, which can function to inhibit the formation of pre-RCs. However, a period of low APC/C activity may also be important. The continuous activation of APC/CCdh1 significantly slows DNA replication in mouse tissue culture cells. This inhibition may reflect the inability of a cell to accumulate adequate levels of proteins required for DNA replication, such as the APC/CCdh1 target and pre-replication complex component CDC6, in the presence of a constitutively active APC/CCdh1. In Drosophila, continuous APC/C^Fzy/Cdh1 activity might prevent the accumulation of two pre-RC components, CDC6 and Orc1. Intriguingly, APC/C activity also appears to oscillate during mammalian endocycles. In endocycling mouse trophoblasts, the levels of Cyclin A oscillate, consistent with the regulated destruction of the Cyclin A protein by the APC/C. Additionally, the inhibition of APC/C activity in endocycling trophoblasts results in the accumulation of the APC/C targets Cyclin A and Geminin. Taken together, these observations support a model in which the oscillation of APC/C^Fzy/Cdh1 activity, which is driven by the regulatory influences of Cdks, promotes efficient cell cycle progression during the endocycle (Narbonne-Reveau, 2008).

The data raises important questions. Why do levels of some APC/C^Fzy/Cdh1 targets, such as Cyclin A, Cyclin B and Geminin, remain below the level of detection while the levels of Orc1 protein oscillate? What might account for these different modes of regulation? Currently, there is no definitive explanation. However, at least three possibilities, which are not mutually exclusive, are envisaged, that may contribute to this differential behavior. First, it was found that relative to the Cyclin A and geminin, the levels of Orc1 transcript are only minimally downregulated upon entry into the endocycle. Transcriptional downregulation, or changes in transcript stability, may help contribute to the low levels of Geminin and Cyclin A proteins observed during the endocycle. Second, the translational efficiency of a subset of transcripts may be reduced upon entry into the endocycle. Finally, it is possible that the Orc1 protein is not as efficiently targeted by the APC/C^Fzy/Cdh1 as the mitotic cyclins or Geminin. Indeed the cis-acting sequences that target these proteins for destruction show considerable variability. Orc1 is targeted for APC/C^Fzy/Cdh1 destruction via a novel motif called the O-box (Araki, 2005). By contrast, Cyclin B and Geminin are targeted by a similar but unique sequence called the destruction-box (D-box), while Drosophila Cyclin A is targeted for destruction by a large complex N-terminal degradation sequence. There is precedence for post-translational regulation of APC/C^Fzy/Cdh1 targets, resulting in differential expression. In mammalian cells the pre-RC component CDC6, which is structurally related to Orc1, is protected from APC/CFzr/Cdh1 degradation by phosphorylation by Cyclin E/Cdk2. One or all of these potential mechanisms may contribute to the differential expression of various APC/CFzr/Cdh1 targets during the endocycle (Narbonne-Reveau, 2008).

Recent evidence from mice indicates that the depletion of the APC/C inhibitor Emi1/Rca1, results in both a strong decrease in E2F target mRNAs, such as geminin and Cyclin A, as well as APC/C activation. This study suggested that the regulation of APC/C activity, by the inhibitor Emi1/Rca1, drives a positive feedback circuit that controls both protein stability and mRNA expression. Thus, the observed decrease in the levels of at least some APC/C targets that occurs upon depletion of Emi1/Rca1, including Geminin and Cyclin A, are controlled at the levels of transcription and protein stability. Developmentally programmed endocycles may provide a natural example where cell cycle progression occurs in the context of increased APC/CFzr/Cdh1 activity. Thus, a similar positive-feedback circuit may be operating during Drosophila endocycles to downregulate the transcription of E2F target genes. Determining the precise regulatory relationships between the upregulation of APC/CFzr/Cdh1 activity and the transcriptional downregulation of genes such as Cyclin A and geminin, during the Drosophila endocycle represents an exciting area for future research (Narbonne-Reveau, 2008).

The requirement for APC/C activity to promote endocycle progression may help answer several longstanding questions concerning the regulation of the Drosophila endocycle. For example, why does the continuous expression of Cyclin E inhibit cell cycle progression during the endocycle but not the mitotic cycle? Several models have been proposed to explain this difference. First, the breakdown of the nuclear envelope that occurs during the mitotic cycle, but not the endocycle, may allow for a transient decrease in local Cyclin E/Cdk2 activity, thus allowing for the relicensing of DNA replication origins. Alternatively, there may be differences in the machinery required to produce a functional pre-RC in mitotic versus endocycling cells. The current results suggest an alternative model for why endocycles are unusually sensitive to continuous Cyclin E expression. This model is based on the demonstration that endocycle progression requires APC/C activity. Both Fzy/Cdc20 and Fzr/Cdh1 function as activators of the APC/C. However, the regulation of these APC/C activators is very distinct. During the mitotic cycle, the binding of Fzy/Cdc20 to the APC/C is dependent on the phosphorylation of several APC/C subunits by the mitotic kinase Cdk1. By contrast, a Cdk-dependent inhibitory phosphorylation on Fzr/Cdh1 relegates APC/CFzr/Cdh1 activity to late M phase and G1. Because of its requirement for Cdk1 activity, APC/C^Fzy/Cdc20 is unlikely to be active during most endocycles. Indeed, Drosophila endocycles proceed normally in fzy mutants. Thus, the only available activator of the APC/C during the endocycle is Fzr/Cdh1. As previously discussed, Fzr/Cdh1 is inhibited by Cyclin E/Cdk2 activity. Therefore, it is proposed that during the endocycle, continuous Cyclin E/Cdk2 activity results in the permanent inhibition of the only available activator of the APC/C, Fzr/Cdh1. This leads to the accumulation of Geminin, Cyclin A and other potential targets, which act to block cell cycle progression. Thus, the ability of continuous Cyclin E to inhibit DNA replication during the endocycle may reflect differences in the available activators of the APC/C present in mitotic versus endocycling cells (Narbonne-Reveau, 2008).

Wound-induced polyploidization is driven by Myc and supports tissue repair in the presence of DNA damage

Tissue repair usually requires either polyploid cell growth or cell division, but the molecular mechanism promoting polyploidy and limiting cell division remains poorly understood. This study finds that injury to the adult Drosophila epithelium causes cells to enter the endocycle through the activation of Yorkie-dependent genes (Myc and E2f1). Myc is even sufficient to induce the endocycle in the uninjured post-mitotic epithelium. As result, epithelial cells enter S phase but mitosis is blocked by inhibition of mitotic gene expression. The mitotic cell cycle program can be activated by simultaneously expressing the Cdc25-like phosphatase String (stg), while genetically depleting APC/C E3 ligase fizzy-related (fzr). However, forcing cells to undergo mitosis is detrimental to wound repair as the adult fly epithelium accumulates DNA damage, and mitotic errors ensue when cells are forced to proliferate. In conclusion, this study finds that wound-induced polyploidization enables tissue repair when cell division is not a viable option (Grendler, 2019).

An unanswered question in tissue repair field is what limits cell proliferation? Why do some tissues retain the capacity to proliferate when injured, yet others fail to do so? Depending on the context (tissue and cell type) signaling pathways, such as the Hippo-Yki pathway, have been found to either promote cell proliferation or polyploidization, but the molecular mechanism regulating this choice of tissue growth has remained poorly understood. This study shows that Yki induces a similar gene set (Myc and E2f1) for polyploid cell growth to that observed for cell proliferation. Myc and E2f1 are known to regulate the cell cycle at the G/S phase transition, but for cells to progress through mitosis, expression of mitotic regulatory genes is required. This study finds that Fzr, an E3 ligase that targets mitotic cyclins for proteolytic degradation, is expressed, while mitotic regulatory genes, including CycA and CycB, are repressed in the adult Drosophila epithelium. As a result, the Yki-dependent expression of Myc and E2f1 induces an endocycle instead of mitosis to repair the adult fly epithelium. Interestingly, the conserved Hippo-Yap pathway has also been found to regulate both liver hepatocyte proliferation and polyploidization through mitotic arrest during tumorigenesis. Therefore, the regulation of the mitotic machinery appears to be a conserved mechanism that may be used to determine whether tissues grow and repair by proliferation or polyploidization (Grendler, 2019).

Some cell types appear to be more permissive than others to switching modes of tissue repair. In the mammalian heart, many studies have been performed to genetically or pharmacologically force cardiomyocytes to proliferate to improve heart regeneration. However, majority of the adult cardiomyocytes are polyploid, which usually inhibits cell division. Only polyploid hepatocytes in the mouse liver and polyploid rectal papillae cells in Drosophila have been demonstrated to retain mitotic competence. A recent study has shown that cardiomyocyte proliferation can be induced to improve heart regeneration by expressing of four cell cycle genes simultaneously. However, it was unclear whether the observed heart regeneration was due to polyploidy- or diploid-induced cardiomyocyte division, and the long-term effects on heart function caused by switching modes of repair (Grendler, 2019).

In the Drosophila hindgut, diploid pyloric cells are induced into the endocycle in response to injury, and fzr knockdown was shown to be sufficient to switch to repair by cell proliferation instead of polyploid cell growth. In adult fly epithelium, this study found that the knockdown of fzr alone was not sufficient to switch to a mitotic cell cycle, but also required the ectopic expression of the mitotic activator stg. In addition, switching to a proliferative response in the fly epithelium significantly impaired wound healing, whereas the hindgut pylorus was not adversely affected by the switch and could still efficiently heal through cell proliferation instead of polyploidization. It was only upon additional oncogenic stress that a defect in tissue integrity in the hindgut was observed. This is also case in the mammalian liver, where polyploid hepatocytes have been shown to protect the liver from tumorigenesis. Therefore, the genetic factors necessary to switch modes of tissue repair are cell/ tissue dependent, with differences in both the short-term and long-term effects on tissue function (Grendler, 2019).

The exposure to either physiological and/or damage-induced cytotoxic stress can result in cellular and genomic damage. Cytotoxic agents, including reactive oxygen species, are known to accumulate with age and injury resulting in DNA damage. This accumulated DNA damage then poses a problem if cells attempt to proliferate by activating the DNA damage checkpoint and causing either apoptosis, cell cycle arrest, or mitotic errors. However, polyploid cells have been found to have a higher resistance to genotoxic stress. Endoreplication was shown in Drosophila to result in chromatin silencing of the p53-responsive genes, allowing polyploid cells to incur DNA damage, but not die. This study has shown that the adult Drosophila epithelium readily accumulates DNA damage, even at 3 days of age, yet the epithelial cells can circumvent this dilemma by inducing polyploid cell growth instead of cell proliferation upon injury (Grendler, 2019).

It remains unclear why the adult epithelium readily accumulates DNA damage and whether WIP works through a similar mechanism to silence p53 targets. This study tested for apoptosis activation in the mitotic-induced epithelial cells (stg, fzr^RNAi), but could not detect any evidence of cell death using either the TUNEL or an active caspase 3 stain. The mitotic errors in the epithelial cells may not activate cell death, as cell fusion was still observed. In the future, it will be interesting to address how polyploid cell generation by fusion contributes to the competence of cells to switch tissue repair modes (Grendler, 2019).

Many tissues lack a resident stem cell population and to undergo efficient repair and regeneration the post-mitotic differentiated cells in the tissue must overcome the controls that restrain the cell cycle entry. A combination of growth factors and cell cycle regulators appears to be required. In case of the Drosophila, Yki-dependent CycE expression was shown to be sufficient to promote cell cycle re-entry, resulting in cell proliferation following tissue damage in the eye imaginal disc. This study shows that Yki-dependent CycE expression is also sufficient to trigger cell cycle re-entry following tissue injury, but results in endocycling instead of mitotic cell cycling. This was unexpected, as overexpression of CycE has been shown to reduce salivary gland cell endoreplication in the Drosophila. Overexpression of CycE blocks the relicensing of S-phase entry required for salivary gland cells to undergo successive endocycles and reach up to 1024C per nuclei. However, it is not a complete block as salivary gland cells still reached 64C with CycE overexpression. Epithelial nuclei increase ploidy up to 32C, suggesting that CycE overexpression is not inhibitory for cells to undergo fewer than five endocycles. The overexpression of CycE without injury, however, was not sufficient to induce endoreplication. Conversely, Myc, another Yki-dependent target, efficiently overcame the cell cycle restraints to drive endoreplication even in the absence of tissue damage (Grendler, 2019).

Myc regulates transcription of a large number of genes, which are required for cell growth, cell cycle and cell death. This study found that Myc is required and sufficient for post-mitotic epithelial cells to enter the endocycle and grow by becoming polyploid; however, ectopic Myc expression does not induce cell death, as has been observed in other systems. Myc has been shown to be activator of endoreplication in other Drosophila cell types, as well as in mammalian epidermal cells and megakaryocytes. Although the Myc targets required to release the adult Drosophila epithelial cells from quiescence remain to be elucidated, Myc appears to be a potent inducer of cell cycle re-activation. Dormant adult muscle precursors in Drosophila larva also require a niche-induced Myc signal to re-enter the cell cycle and proliferate. In summary, activation of Yki by tissue injury induces a potent transcriptional gene set that is sufficient to cause cell cycle entry and is consistent with the previous finding that high levels of CycE and E2f1 are required to overcome cell cycle exit in terminally differentiated cell types (Grendler, 2019).

In the past several years, an increasing number of examples of polyploidy have been observed not only in insects and plants, but also in vertebrate species, including zebrafish, mice and human tissue cell types. Polyploid cells are frequently generated in response to stress and/ or injury and are now recognized to offer an alternative tissue-growth strategy that can prevent acute organ failure. Genotoxic stress is known to accumulate with age and has been observed in the mammalian cornea endothelium, in which multinucleated polyploid cells are generated in response to damage or age-associated diseases. Acute injury to kidney also causes DNA damage and endoreplication in the tubule epithelial cells. Therefore, it remains to be determined whether mitotic arrest allows polyploid cell growth to be the preferred tissue repair strategy to circumvent genotoxic stress in these mammalian tissues as well (Grendler, 2019).

GENE STRUCTURE

cDNA clone length - 2605

Bases in 5' UTR - 467

Exons - 5

Bases in 3' UTR - 701

PROTEIN STRUCTURE

Amino Acids - 478

Structural Domains

Drosophila fizzy is required for cell cycle progression beyond metaphase and for mitotic degradation of A- and B-type cyclins (Dawson, 1993; Dawson, 1995; Sigrist, 1995). The Xenopus fzr gene was identified in an attempt to identify a fzy homolog in Xenopus, an ideal organism for biochemical analyses of cyclin proteolysis. Subsequent analyses have revealed the presence of both, fzy and fzr, in Drosophila and Xenopus. Sequencing projects have identified apparent fzy and fzr homologs in S. pombe, C. elegans, and vertebrates (Sigrist, 1997).

The protein products encoded by the fzy and fzr cDNAs are highly similar in their C-terminal domains, which are composed of seven WD repeats. These repeats, which are found in many functionally diverse proteins, were first identified in ß-transducin. A crystallographic analysis has demonstrated that these WD- or ß-transducin repeats form a seven propeller structure that provides protein-protein interaction faces in trimeric G-protein complexes. In the N-terminal domain, similarities between fzy and fzr are restricted to a few motifs (Sigrist, 1997).

date revised: 25 September 2023

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