ABSTRACT
Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. Here, using Drosophila, we uncover two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindlePindependent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2’s control of overall mitotic timing ensures efficient SIRS. Our results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention.
INTRODUCTION
Regulating mitotic chromosome structure is critical to preventing genomic instability (Gordon et al., 2012; Pfau & Amon, 2012). During mitosis, chromatids associate in sister pairs, which facilitates their biorientation and subsequent segregation to opposite spindle poles. A frequently occurring and long-recognized departure from this paired chromosome structure occurs when the genome reduplicates without chromatid separation (hereafter: genome reduplication). Following a single extra S-phase, cells frequently form diplochromosomes: four sister chromatids conjoined at centromeres (White, 1935). A more general term for chromosomes formed by any degree of genome reduplication without chromatid separation is “polytene” (Painter, 1934; Zhimulev et al., 2004). Mitosis in polytene cells is considered “ill-advised for mechanical reasons” (Edgar & Orr-Weaver, 2001). Indeed, separation of polytene diplochromosomes at anaphase causes chromosome mis-segregation (Vidwans et al., 2002).
Given this association of polytene chromosomes with mitotic errors, it is not surprising that these structures are often associated with aberrant development and disease. Polytene chromosomes have been observed in cells from spontaneous human abortions (Therman et al., 1978), in muscular dystrophy patients (Schmidt et al., 2011), in a variety of tumors (Biesele & Poyner, 1943; Erenpreisa et al., 2009; Levan & Hauschka, 1953; Therman et al., 1983) and can also precede tumor formation in mice (Davoli & de Lange, 2012). Polytene chromosomes also occur after treatment with currently used anti-mitotic chemotherapeutics such as those that inhibit Topoisomerase II (Cantero et al., 2006; Sumner, 1998). Disruption of numerous other processes crucial for mitosis, including spindle formation (Goyanes & Schvartzman, 1981; Takanari et al., 1985) sister chromatid cohesion (Wirth et al., 2006) or genome integrity control (Davoli et al., 2010) also cause genome reduplication and polyteny. Thus, polytene chromosomes, a source of mitotic instability, are a conserved and common outcome of ectopic genome reduplication.
To understand how cells adapt the cell cycle machinery to the challenge of segregating polytene chromosomes, naturally occurring models of this problem can prove useful. Programmed genome reduplication cycles of successive S-phase without M-phase (endocycles, Edgar et al., 2014; Fox & Duronio, 2013 see nomenclature) produce polytene chromosomes in many plant, insect, and mammalian species, including humans (Zhimulev et al., 2004; Zybina & Zybina, 1996). However, many cells with programmed genome reduplication do not subsequently divide, preventing study of how nature has circumvented the issue of segregating polytene chromosomes. In contrast, we previously demonstrated that rectal papilla (hereafter: papillar cells), ion-absorbing structures in the Drosophila hindgut, are built entirely by mitosis of endocycled cells (Fox et al., 2010; Schoenfelder et al., 2014). Surprisingly, we never observed polytene chromosomes in hundreds of papillar metaphases (Fox et al., 2010; Schoenfelder et al., 2014), suggesting papillar cells are programmed to either avoid or eliminate polyteny and its associated mitotic defects. Interestingly, previous studies suggest that polyteny can be at least partially undone without anaphase in both normal and tumorous tissue (Dej & Spradling, 1999; Grell, 1946; Levan & Hauschka, 1953). Thus, in some cases of genome reduplication, polyteny may be actively regulated or eliminated.
Taken together, the potential negative impact of genome reduplication on mitotic chromosome structure is clear. However, the responses that enable either developing or tumorous cells to continue dividing after reduplication, despite profound chromosome structure changes, remain unclear. Here, using Drosophila tissue models of both ectopic and naturally occurring genome reduplication, we uncover two distinct cellular responses to reduplicated chromosomes. Both reduplication responses require the conserved spindle assembly checkpoint (SAC) protein Mad2, which inhibits the Anaphase Promoting Complex to both delay anaphase in response to unattached or tensionless kinetochores and to also regulate overall mitotic timing from nuclear envelope breakdown (NEBD) to anaphase onset (London & Biggins, 2014; Musacchio, 2015). In reduplicated cells that retain polytene chromosomes at metaphase, we show Mad2 is involved in a SAC wait-anaphase response. This anaphase delay does not fully prevent the mitotic errors and the resulting aneuploidy associated with mitosis of polytene chromosomes, but it substantially prevents apoptosis, tissue malformation, and organismal death. In contrast to this SAC response, we also define a second response in reduplicated cells that actively eliminates polyteny before anaphase. In this response, polytene chromosomes undergo a dynamic, spindle-independent process we term Separation into Recent Sister chromatid pairs (SIRS), which eliminates any trace of polyteny before anaphase. Unlike mitosis with metaphase polytene chromosomes, mitosis with SIRS does not trigger a Mad2-dependent anaphase delay. Yet, we find Mad2 promotes efficient SIRS by allowing sufficient time between nuclear envelope breakdown and anaphase, which allows polytene chromosomes to separate into conventional mitotic sister chromatid pairs. Our results therefore define two distinct responses to reduplicated chromosomes, each of which depends on a distinct Mad2 response.
RESULTS
To understand the mechanisms employed by cells with reduplicated chromosomes, we took advantage of accessible developmental models and in vivo genetic tools in Drosophila. While ectopic genome reduplication was previously established to generate polytene diplochromosomes and subsequent mitotic errors in Drosophila, the effects were examined within the time-frame of the terminal embryonic mitotic cell cycle (Vidwans et al., 2002). Thus, the long-term effects of ectopic genome reduplication on cell viability and tissue development, and key molecular regulation of reduplicated chromosomes has remained unexplored. In parallel, our development of rectal papillae as a non-ectopic, naturally occurring model of mitosis after genome reduplication enabled us to also study how cells programmed to undergo genome reduplication can regulate polytene chromosome structure during mitosis.
Ectopic genome reduplication yields polyteny and continued aneuploid cell division
We first ectopically induced genome reduplication in proliferating tissues of developing larvae by transiently re-programming mitotic cycles to endocycles. fizzy-related (fzr, mammalian Cdhl) plays a conserved role in endocycles by targeting the anaphase promoting complex to destroy the mitotic Cyclins A, B, and B3 (Larson-Rabin et al., 2008; Sigrist & Lehner, 1997). fzr overexpression was previously shown to transform mitotic cycles into endocycles (Sigrist & Lehner, 1997). To transiently induce endocycles, we used a brief heat shock (HS) pulse to express ectopic fzr(HS>fzr, Fig1A). Using the cell cycle marker system Fly-FUCCI (Zielke et al., 2014 Fig1B) we find that pulsed fzr overexpression temporarily eliminates expression of the S/G2/M mRFP-CyclinB reporter in wing imaginal disc cells (Fig1C vs. C’,D). This same population of mRFP-CyclinB-negative cells continues to express the G2/M/G1 GFP-E2F1 reporter, but in greater proportion (Fig1C vs. C’, D). Together, these data suggest HS>fzr promotes G1 accumulation (91% of cells compared to 36% in controls, Fig1B-D). To test whether this G1 accumulation is due to direct conversion of G2 cells to G1, as opposed to an acceleration of the cell cycle through G2/M, we stained for the mitotic marker Phospho-Histone H3 at several time points after pulsed fzr expression. For up to 7 hours after fzr overexpression, there is essentially no mitosis in the wing imaginal disc, whereas wing cells in heat shocked wild type flies continue to divide after heat shock (FigS1A,B). Based on previous studies of fzr function and our FUCCI and Phospho-Histone H3 data, we conclude that pulsed fzr expression converts G2 cells to a G1 state by eliminating mitotic cyclins (Fig1A).
To determine if the G2 cells re-programmed to G1 proceed through a second genome duplication, we examined mitotic chromosome number when mitosis of HS>fzr tissue first resumes (ten hours after heat shock). At this time-point, we observe frequent tetraploidy (41% of mitotic cells, equivalent to 93% of all G2 cells prior to heat shock, Fig1F,F’,H). We obtained similar results when examining the results of fzr overexpression in diploid brain progenitors (FigS1C). These results confirm our ability to induce genome reduplication in normally diploid tissues. We also examined chromosome structure in our induced tetraploid cells. When HS>fzr-induced tetraploid DNA first condenses post-heat shock, all chromatids of each chromosome type are closely aligned in a polytene configuration, as evidenced by having the haploid number of distinguishable chromosomes (four in females, Fig1F). Frequently, we observe un-pairing of the homologous groups of centromeres within each polytene chromosome (Fig1F, asterisk, also see discussion). Later, at the first metaphase, homologous chromosomes of each polytene are now completely separated, but the four centromeres of each group of sister chromatids remain conjoined within diplochromosomes (observed for 96% of tetraploid cells, Fig1F’ inset, H). Thus, HS>fzr induces ectopic genome reduplication, resulting in tetraploid cells with metaphase polytene diplochromosomes.
We next examined the mitotic fidelity of cells with diplochromosomes by two independent means: chromosome karyotype analysis and live imaging. By examining the metaphase chromosomes of the division immediately following diplochromosome division, we could detect whether aneuploidy results from diplochromosome segregation. Following the division of cells with diplochromosomes, we observe tetraploid-aneuploid cells with one or two extra or missing chromosomes (8.6% of mitotic cells). In these cells, diplochromosomes are no longer present and instead chromatids are found in distinct sister pairs (Fig1G). This suggests that during or after anaphase of the first post-reduplication division, diplochromosomes can separate into individual chromatids. Further, these diplochromosome divisions can produce aneuploid daughter cells, many of which continue to divide (Fig1G,H,K, FigS1D).
We also live imaged mitosis of wing imaginal disc and brain progenitor (neuroblasts and ganglion mother) cells, both with and without ectopic genome reduplication. In addition to using a histone marker to observe chromosomes, we used the Cenp-C-Tomato marker to observe kinetochores. Control diploid cells divide without errors (Fig1I,J No HS, MovieS1A). In contrast, most (85%) tetraploid divisions with diplochromosomes exhibit lagging chromosomes (Fig1I,J, HS+10hr, Movie S1B). In some of these divisions we clearly observe four chromatids of a diplochromosome segregating 3:1 (in agreement with prior work in embryos by Vidwans et al., 2002), suggesting incomplete or imprecise sister chromatid disjunction is the cause of chromosome gains and losses (Fig1I, FigS1D, Movie S1B). These errors appear to result primarily from diplochromosomes and not tetraploidy itself, as tetraploid cells in the subsequent divisions (which lack diplochromosomes) have a substantially reduced error rate (Fig1J, S1E, MovieS1C). In spite of the high initial error rate caused by separation of diplochromosomes, tetraploid-aneuploid cell divisions continue to occur for at least 5 days after genome reduplication, as determined by cytology (Fig1H HS+120hr). We conclude that division of diplochromosomes in the mitotically expanding diploid progenitor tissues that we surveyed can lead to the generation of aneuploid cells, which can continue to divide (Fig1K).
To determine the long-term effect of tetraploid-aneuploid divisions on tissue development, we took advantage of the fact that expression of HS>fzr occurs in adult progenitor tissues. We thus examined the survival of these animals to adulthood. Survival is only subtly affected in animals with mild (23.0% tetraploid [S.E.M. 4.9%]) levels of induced error-prone tetraploid progenitor division (Fig2A), and resulting adult tissues appear normal (Fig2B). In contrast, when tetraploidy is further increased by increasing the duration of heat shock, organism survival decreases in a tetraploid-dependent fashion (Fig2A, FigS2A). Together, these results show that ectopic genome reduplication yields tetraploid cells with polytene metaphase diplochromosomes, which are aneuploid-prone (Fig1K). These conclusions are in agreement with a previous study (Vidwans et al., 2002). We further show that such aneuploid-prone cells can continue to propagate, and that only at high frequencies are these error-prone tetraploid mitotic events lethal to the organism.
Polyteny response 1: Spindle assembly checkpoint (SAC)-mediated anaphase delay
Our data (Fig1H, FigS1C) and those of others (Vidwans et al., 2002) suggest that many polytene diplochromosome divisions in a variety of tissues do not lead to aneuploidy. Little is known about aneuploidy prevention mechanisms in cells with polytene chromosomes, despite the numerous mechanisms that can generate these aberrations. Through live imaging, we uncovered one such aneuploidy-prevention mechanism in cells with metaphase diplochromosomes. Because our heat shock protocol only affects cells in G2, a single HS>fzr pulse creates a mixed population of unaltered diploid cells and diplochromosome-containing tetraploid cells. Allowing us to simultaneously live image both cell types in the same tissue. Metaphase in cells with diplochromosomes (Fig2C, yellow dotted outline) is significantly longer than in diploid cells (Fig2C blue dashed outline, Fig2D), consistent with previous work (Pandey et al., 2005). We thus hypothesized that diplochromosomes trigger the SAC, which activates a wait-anaphase signal until all kinetochores attach to microtubules and are under tension (London & Biggins, 2014; Musacchio, 2015). To test this model, we examined SAC-defective mad2 null animals (Buffin et al., 2007; Emre et al., 2011 FigS2B). Using live imaging of wing imaginal discs before and after heat shock, we find that loss of mad2 eliminates the lengthened period of metaphase caused by diplochromosomes (Fig2D). From these data, we conclude that diplochromosomes trigger a SAC wait-anaphase response.
Although important for mitosis in cultured Drosophila S2 cells (Orr et al., 2007), the Mad2-directed SAC is reported to be dispensable in Drosophila tissue mitosis (Buffin et al., 2007; Emre et al., 2011). mad2 null animals are viable with no obvious tissue defects (Fig2B Buffin et al., 2007) due in part to an apoptotic response (Morais da Silva et al., 2013). In contrast, the Mad2 SAC response is essential during development of HS>fzr animals. Even at low levels of tetraploidy, which affect the survival of HS>fzr animals only slightly, few HS>fzr, mad2 animals survive to adulthood (15.4%, Fig2A, S2A). To understand why HS>fzr, mad2 animals have defects, we analyzed the surviving animals. In these animals, we find a variety of developmental defects in normally diploid tissues, including smaller eyes, ectopic wing veins, and melanotic abdominal masses (Fig2B). Increased apoptosis is associated with these tissue malformation phenotypes, as progenitor tissue from HS>fzr, mad2 animals have much higher rates of apoptotic cell death as shown by both TUNEL labeling (Fig2E,F), and cleaved caspase staining (FigS2C,D). Taken together, our data identify an important role for the Mad2-dependent SAC in delaying anaphase in the presence of metaphase polytene diplochromosomes.
Polyteny response 2: Separation Into Recent Sisters (SIRS)
Having defined a response to mitosis after ectopic genome reduplication, we next asked if this same mechanism operates in a tissue that we previously found to divide after programmed genome reduplication. In earlier work we found Drosophila rectal papillar cells (hereafter: papillar cells) naturally undergo two fzr-dependent endocycles during the 2nd larval instar to generate octoploid cells and then divide, on average, two times during pupal development. An intervening S-phase accompanies these polyploid divisions, and cells at the papillar base undergo one additional S-phase after the final polyploid mitosis (Fig3A Fox et al., 2010; Schoenfelder et al., 2014). Thus, as with HS>fzr induction in diploid tissues, papillar development naturally involves genome reduplication followed by mitosis. Our previous work established that papillar mitoses can be error prone, so the same problems with dissociating polytene chromosomes in HS>fzr tissues could be responsible for a portion of these errors during papillar divisions. However, we previously did not see, in hundreds of observed cells, any instances of metaphases with persistent polyteny in papillar cells, suggesting that papillar cells somehow avoid mitosis of polytene chromosomes. If papillar cells were to behave like cells that undergo ectopic genome reduplication, one would expect first division papillar chromosomes to be arranged in quadruplochromosomes, reflecting one additional genome doubling relative to cells with diplochromosomes. Instead, through careful re-examination of the first octoploid metaphase, we confirmed that papillar chromosomes do not have quadruplochromosomes, but instead chromatids in these octoploid cells are arranged in individual sister chromatid pairs (Fig3B’’ inset, C). This suggested two possibilities: 1) papillar cells never form polytenes, or 2) papillar cells form polytenes, but somehow separate into recent sister pairs prior to the first metaphase.
To distinguish these two possibilities, we examined papillar karyotypes from the moment chromosome condensation could be detected. Papillar cells center mitosis from a G2-like state, as evidenced by expression of the G2/M regulator Cdc25/string just before the onset of pupal cell cycles (Fox et al., 2010) (Fig S3A). At time points early in the first mitosis, we indeed find that papillar chromosomes are polytene (Fig3B, Polytene). In these polytenes, we again see examples of cells where the centromeric regions are no longer tightly associated, as we did in our studies of HS>fzr-induced polyteny. However, unlike in cells with induced polyteny, the centromeres in papillar polytene cells are able to not only separate into groups of homologs, but to further separate into individual sister chromatid pairs (asterisks in Fig 3B and FigS3C vs. Fig 1F, also see discussion). At this early mitotic time point, we also observe cells where polytenes are absent. Instead, in these cells, sister chromatid pairs and homologs of each chromosome type are separated but remain clumped closely together, as if the polytene chromosome recently separated into pairs containing only the most recent sister chromatids (Fig3B’, D, Clumped). Neither the polytene nor the clumped configurations remain during the second division (Fig3C, D), suggesting a specific chromosome structure is present early in the first division of papillar cells. Similarly, by Fluorescent In Situ Hybridization (FISH) we find examples of both closely associated (polytene) and dispersed (separated/non-polytene) signals during the period of the first papillar mitosis (FigS3B). Thus, a key difference between the response to genome reduplication between papillar and HS>fzr cells is the elimination of polyteny before anaphase in papillar cells.
To examine if a majority of (if not all) papillar cells transition from polytene to separate/non-polytene chromosomes during the first mitosis, we used drug treatment to isolate specific chromosome structures during the transition into the first papillar division. To enrich for early mitotic and pre-mitotic chromosomes, we induced Premature Chromosome Compaction (PCC) in papillar cells at a time point just prior to the first mitosis (Methods). PCC causes interphase chromosomes to condense and makes it possible to visualize interphase chromosome structure by standard cytological methods (FigS3C). Using this technique we find that in pre-mitotic papillar tissue, clear polytene chromosomes are present in nearly every cell (Fig 3D Pre 1st Div, FigS3C). If we instead enrich for cells in metaphase of the first mitosis by treating with the spindle poison colcemid, we find zero examples where chromosomes are still polytene. In these metaphase-enriched samples, all chromosomes are separated into recent-sister pairs, and even cells with clumped chromosomes are rare (Fig3D, 1st Div +colc). Thus, our pharmacological studies further suggest that essentially all genome-reduplicated papillar cells are programmed to completely eliminate polytene chromosomes as cells progress into the first metaphase.
To observe the temporal dynamics of the pre-anaphase elimination of papillar polyteny, we used live imaging, using the same markers used to image diplochromosome division. Prior to the first papillar mitosis, the kinetochores from each homolog are closely associated into an average of 4.1 large foci, close to the haploid number of distinct chromosomes (4 for females and 5 for males due to X/Y un-pairing, Fig 3E). As time progresses in the first division, it is possible to watch these large kinetochore foci disperse into many smaller foci prior to metaphase (Fig3F, inset). In contrast, prior to the second division kinetochores are already separated into many more foci (an average of 15.1 observably distinct foci per cell) before entry into mitosis (Fig3E). During the second division, the number of resolvable foci remains essentially constant (Fig3G). Additionally, the histone marker reveals that polytene chromosomes are visible when chromosomes first condense and can then be seen to disperse during the first but not the second division (Fig3F v. Fig3G, -l8:00 min). This result confirms the model that genome-reduplicated papillar cells eliminate polyteny during the first mitosis, then undergo an intervening S-phase before the next division (Fig3A). We also confirmed that each clump of 4 or 5 pre-first division centromeres only contains a single chromosome type. To do so, we took advantage of the fact that dosage compensation in flies relies on upregulation of transcription on the male X chromosome via the Dosage Compensation Complex (DCC, Conrad & Akhtar, 2012). By live imaging papillar cells expressing the DCC complex protein MSL3 tagged with GFP, which localizes only to the male X-chromosome (Strukov et al., 2011), we find that indeed only a single Cenp-C-Tomato focus is MSL3-GFP positive prior to polytene dissociation (FigS3D,Movie S3C). Taken together, we find papillar cells avoid mitosis of polytene chromosomes in part by undergoing a pre-anaphase chromosome separation process we term Separation Into Recent Sisters (SIRS, Fig3H).
Previously, we confirmed that a similar polyploid mitotic program occurs in the developing hindgut of the mosquito Culex pipiens (Fox et al., 2010). In this tissue, we observe mitotic polytene chromosomes only during the early period of the first polyploid mitosis (FigS3E, MovieS3D). This result is consistent with previous classical descriptions of this process that suggested a polytene organization is present only early in the first polyploid mitosis (Grell, 1946). From our Drosophila and Culex studies, we conclude that unlike cells that enter metaphase with polytene chromosomes, a separate mechanism, SIRS, can eliminate polyteny as cells enter metaphase.
Spindle-independent mitotic timing by Mad2 promotes efficient SIRS
Our dual genome-reduplication systems identified two distinct cellular responses to polytene chromosomes: a Mad2 SAC response that delays anaphase when polytenes remain at metaphase, and a SIRS response that eliminates polyteny as cells enter metaphase. Despite the lack of metaphase polytene chromosomes in papillar cells, we also identified an important role for Mad2 during SIRS. Because mad2 loss has no reported mitotic defects in Drosophila animals (Buffin et al., 2007), we were surprised to find that first division mad2 papillar cells exhibit a substantial increase in DNA bridges (Fig4A,B). We did not detect similar defects during papillar mitosis of animals null for mad1, another SAC component (Fig4A,B). Thus, a Madl independent function of Mad2 is important in cells during SIRS.
In Drosophila, Mad2 plays a conserved, cell type-dependent role in regulating NEBD-to-anaphase onset timing (Buffin et al., 2007; Meraldi et al., 2004; Rodriguez-Bravo et al., 2014; Yuan & O’Farrell, 2015). As for the wait-anaphase response, this mitotic timing role involves Mad2 inhibition of the Anaphase Promoting Complex. However, Mad2’s control of overall mitotic timing is irrespective of SAC kinetochore attachment surveillance (Meraldi et al., 2004; Rodriguez-Bravo et al., 2014). Interestingly, Drosophila Mad1 is reported to be dispensable for regulation of NEBD-to-anaphase timing (Emre et al., 2011). Given the lack of mad1 phenotypes with respect to the first papillar division, we thus hypothesized that SIRS enables papillar cells to bypass the SAC-mediated anaphase delay, and that Mad2 control of overall mitotic timing is important during SIRS. If so, one would predict cells undergoing SIRS to not trigger an anaphase delay, but to still depend on mitotic timing.
To first test if papillar cells employ the SAC wait-anaphase in response to polytene chromosomes, we treated animals with colcemid, a known SAC wait-anaphase trigger. This treatment increases the mitotic index of wild type papillar cells, whereas the mitotic index of mad2 null animals is unaffected (Fig4C,D) Thus, spindle defects trigger the SAC wait-anaphase response in papillar cells. We next asked if the SAC wait-anaphase responds to polytene chromosomes during SIRS. If so, the first divisions (polytenes present) should have a longer metaphase than the second division (polytenes absent). However we find that metaphase is not any longer in the first papillar division than in the second papillar division, while in contrast metaphase is almost twice as long in wing cells with diplochromosomes (+10hr) than in those that are polyploid but lack diplochromosomes (+24hr, Fig4E). We then tested if triggering the SAC wait-anaphase response can prevent or delay SIRS completion. We find SIRS occurs on schedule even in the presence of colcemid concentrations that are sufficient to eliminate a detectable spindle and inhibit anaphase (Fig4F,G). We conclude that: a) SIRS is not regulated by the SAC wait-anaphase response, and b) chromosome separation during SIRS does not require a mitotic spindle.
We next tested whether Mad2-dependent control of overall mitotic timing is crucial for efficient SIRS. Using an NEBD marker, we first confirmed that Mad2 regulates NEBD-to-anaphase timing (Fig5A,B). We also co-imaged NEBD and kinetochores to monitor SIRS completion in wild type and mad2 papillar cells. Interestingly, in both mad2 and wild type animals, SIRS begins synchronously with NEBD (Fig5C,D). However, SIRS fails to complete before anaphase in mad2 animals, leading to a high variance of centromere intensity signal across the metaphase plate (reflecting failure of centromere dissociation/SIRS completion). This high variance disrupts the bilateral symmetry of the metaphase plate in mad2 cells that form DNA bridges (Fig5E,F). Taken together, our data show mitotic fidelity after genome reduplication is improved by one of two Mad2-dependent functions: 1) in the presence of metaphase polytene chromosomes a Mad2 SAC dependent wait-anaphase signal is generated, 2) the efficient elimination of polytenes as cells enter metaphase by SIRS requires a Mad2 (but SAC wait-anaphase-independent) NEBDp anaphase timer (Fig5G).
DISCUSSION
Two Mad2 responses to genome reduplication
Despite a large body of literature describing reduplicated chromosomes in development and disease, the cellular and molecular responses enabling cells to progress through mitosis after genome reduplication have remained essentially unknown. Here, we define two such responses‐ one that prevents malformation of tissues with polytene chromosomes that persist until anaphase onset, and another (SIRS) that eliminates polyteny before anaphase onset. Both polyteny responses require the conserved mitotic fidelity regulator Mad2, yet Mad2’s role in each response is distinct (anaphase delay vs. control of overall mitotic timing). These findings identify new roles for Drosophila Mad2, for which few roles have been identified. Further, our findings illuminate a likely recurring role for Mad2 in response to genome reduplication.
In cells with metaphase polytenes (e.g. diplochromosomes), our data suggest polytene chromosomes present a challenge for the mitotic spindle, leading to a prolonged period of unattached/tensionless kinetochores. What particular feature of diplochromosomes triggers the SAC wait-anaphase response is unclear. It seems likely that diplochromosome structure is at least partially incompatible with attachment to the spindle. For example, it may be that the outer kinetochores block spindle attachment to the inner kinetochores within a diplochromosome, or it could be that the spindle has trouble generating tension on four kinetochores simultaneously, both of which would trigger the SAC wait-anaphase response. Eventually, the spindle appears able to attach and bi-orient all kinetochores, but the resulting anaphase is frequently error prone. These data are consistent with the known ability of cells with erroneous merotelic kinetochore attachments to satisfy the SAC and proceed to anaphase with lagging chromosomes (Gregan et al., 2011), the latter of which we observe (Fig1i). Potentially, then, the AuroraB-mediated mechanism that can correct merotely is overwhelmed/inoperable in cells with reduplicated chromosomes (Cimini et al., 2006; Knowlton et al., 2006). Despite the inability of the SAC to prevent all instances of mitotic errors in cells with polyteny, our data suggest that development of normally diploid tissues with an operable SAC is not noticeably altered by up to 23% ± 4.9% of error prone tetraploid divisions. Given the conserved nature of SAC signaling, and the widespread occurrence of diplochromosomes in disease, it will be interesting to explore whether the SAC wait-anaphase response is a general mechanism used to enable the expansion of aneuploid cells formed by aberrant genome reduplication.
In contrast to cells with polyteny at anaphase, in cells such as papillar cells, SIRS vastly improves mitotic fidelity. This process does not require the Mad2 SAC wait-anaphase response, but the efficient completion of SIRS before anaphase requires the Mad2-dependent mitotic timer. Little is known about distinct, checkpoint-independent regulation of the Mad2 timer. In the future, papillar cells may prove useful in further study of the timer, given the dependence of SIRS completion on this Mad2 function.
We previously described the error-prone nature of papillar divisions, as well as the high tolerance of this tissue for chromosome mis-segregation. This raises the question of why papillar cells employ SIRS, if papillar aneuploidy is well tolerated (Schoenfelder et al., 2014). Based on our study of HS>fzr cells, which lack SIRS, we propose that SIRS is required to prevent extreme polytene chromosome mis-segregation events during papillar development, which could result in inviable nullisomic cells. Additionally, we have recently found that papillar cells actively prevent accumulation of micronuclei resulting from broken DNA (Bretscher and Fox, unpublished). Thus, while mitotic genome-reduplicated cells such as papillar cells do tolerate some degree of aneuploidy, processes such as micronucleus prevention and SIRS may act to ensure a viable degree of mitotic fidelity.
A model for SIRS
Our results identify SIRS as a spindle-independent chromosome separation process that remarkably eliminates polytene chromosomes before anaphase. This process is distinct from another spindle-independent chromosome separation process known as C-mitosis, which involves complete sister chromatid separation before anaphase (Levan, 1938; Östergren, 1944). While future work will determine what differentiates cells capable of SIRS from cells with polytenes that persist until anaphase, our data thus far has examined three layers of polytene chromosome organization that either are or are not eliminated during papillar and HS>fzr mitosis, and has pinpointed one of these layers of polytene organization as distinct to cells undergoing SIRS.
The first layer of polytene organization is homologphomolog pairing. Given that we observe the haploid number of chromosomes after both papillar (Fig3B) and HS>fzr (Fig1F) endocycles, it is clear that homologous chromosomes associate within both types of polytene chromosomes by somatic homolog pairing (Metz, 1916; Painter, 1933). Both papillar and HS>fzr polytenes separate homologs before dividing (Fig3B’’, FiglF’), and this process appears to initiate at centromeres (Fig1F, Fig3B, asterisks, most obvious for the acrocentric X chromosome). Thus, homologphomolog dissociation is not unique to SIRS. The second layer of polytene organization is interaction between sister chromatid pairs. Importantly, the arrangement of chromatid pairs at metaphase differs between cells that do or do not undergo SIRS. In SIRSpcapable (e.g. papillar) cells, only the product of the single most recent round of replication (recent sisters, see nomenclature) remain attached at metaphase whereas the products of previous rounds of replication are no longer attached. In contrast, in SIRSpincapable (e.g. HS>fzr) cells, all sister chromatids remain attached. Thus, the separation into chromatid pairs appears to be the critical function of SIRS. Future work can test if this separation requires the Condensin II complex activity during SIRS, which was shown previously to enable partial polytene chromosome dissociation (Hartl et al., 2008). The third layer of organization within polytenes are contacts between recent sister chromatid arms. These are equally undone by metaphase in both papillar and HS>fzr cells (Fig3B’’, FiglF’) so that, at metaphase, chromatids are only attached at the centromere. This process likely involves the prophase cohesin removal pathway (Losada et al., 2002;Sumara et al., 2002). Taken together, we conclude the key difference between SIRS-capable (e.g. papillar) and SIRS-incapable (e.g, HS>fzr) cells is the ability to separate into sister chromatid pairs before metaphase (Fig5G).
We further hypothesize that a key prerequisite to SIRS is the careful regulation of chromosome structure during genome reduplication/endocycles. During the endocycle, papillar cells show no evidence of karyokinesis, which suggests these cells lack a mechanical method of separating chromosomes during the endocycle (Fox et al., 2010). However, we speculate that during endocycles, periodic cohesin removal occurs at centromeres after each S-phase. Such cohesin removal would then allow each chromatid to both eliminate its cohesins between its sister from a previous S-phase and then establish cohesins with a new sister during the subsequent S-phase. While alterations in cohesins do not noticeably perturb interphase polytene structure (Pauli et al, 2008), future work can determine if such endocycle-mediated cohesin regulation confers cells with polytene chromosomes with the ability to undergo SIRS during a later mitosis. Future work can also determine if cohesin regulation differs during endocycles of cells that are destined to later divide. We previously defined features of a distinct pre-mitotic variant of the endocycle (Schoenfelder et al., 2014), and cohesin regulation may be distinct during this endocycle variant.
SIRS is likely frequent and conserved. Consistent with classical reports (Berger, 1938; Grell, 1946; Holt, 1917), we find polytene chromosomes are present before polyploid mitosis in the ileum of the mosquito Culex pipiens, and are later apparent as individual chromosomes during mitosis (FigS3E). Polyploid Drosophila ovarian nurse cells undergo an incomplete SIRS-like process (Dej & Spradling, 1999), and can further separate if mitotic cyclins are experimentally elevated (Reed & Orr-Weaver, 1997). In polyploid trophoblasts of some mammalian species, polytene chromosomes separate into numerous bundles of paired chromosomes at the polykaryocyte stage (Zybina & Zybina, 1996; Zybina et al., 2011). SIRS may also act to eliminate polyteny in some polyploid tumors. One of the first descriptions of polyteny in tumors noted diplochromosomes “fall apart” before anaphase (Levan & Hauschka, 1953). Whole genome duplication is common (~37%) in human tumors (Zack et al., 2013). Given the transient nature of polytene chromosomes in mitotic tissues demonstrated here, we suggest future studies of whole genome duplication in cancer models should closely examine the initial mitosis after multiple S-phases to identify potential polytene chromosome origins of tumor aneuploidy. Finally, while our studies agree with the notion that multiple S-phases and polyploidy precede aneuploidy (Davoli & de Lange, 2012; Gordon et al., 2012), they also underscore the need for aneuploidy-prevention responses including SIRS and the SAC for continued propagation of viable polyploid/aneuploid cells. Future studies can reveal additional SIRS regulation, and other critical genome instability controls in normal or tumorous cells following genome reduplication.
MATERIALS AND METHODS
Drosophila genetics
All flies were raised at 25° on standard media (Archon Scientific, Durham, NC). For experiments to measure the length of mitosis larvae or pupae were shifted to 29° for at least 18 hrs before dissection. Heat shocks were performed on third instar larvae. Vials were heat shocked in a 37° water bath for 15min, 30min, or 60min. Flybase (flybase.org) describes full genotypes for the following stocks used in this study: engrailed Gal4 (Bloomington stock 1973); w1118 (Bloomington stock 3605); His-2av-GFP (Bloomington stock 24163); UAS>GFP.E2f1.1-230, UAS>mRFP1.CycB.1-266 (Bloomington stock 55117). Kyoto DGRC (kyotofly.kit.jp) describes the genotype for the following stock: S/G2/M-Azami (Kyoto stock 109678). The Other stocks were generous gifts: tomato-Cenp-C (Althoff et al., 2012); HS>fzr (Sigrist & Lehner, 1997); byn>gal4 (Singer et al., 1996); mad11, Df (2R) W45-30n (Emre et al., 2011); mad2p (Buffin et al., 2007), msl3-GFP (Strukov et al., 2011), stg>LacZ49 (Bruce Edgar), jupiter-GFP (Karpova et al., 2006).
Mosquito culture
Culex pipiens larvae were obtained from Carolina Biological (Burlington, NC). Culturing conditions were as in Fox et. al, 2010. Larvae were monitored hourly for pupation, and the hindgut was dissected beginning 7 hours post-puparium formation. Antibody staining was as for Drosophila tissue.
Survival analysis
20 wandering 3rd instar larvae per replicate of the indicated genotype were placed into a fresh vial with food and then heat shocked for 15 or 30 minutes. The number of adults that eclosed was counted.
Chromosome Cytology and FISH
Chromosome preparations were based on previous protocols with modifications for the pupal hindgut (Fox et al., 2010; Gatti et al., 1994). For colcemid treatment to enrich for metaphase cells tissue was first incubated in colcemid (Sigma, St. Louis, MO) at 50μg/ml for 30min in PBS. For pre-mitotic chromosome spreads with Premature Chromosome Compaction, tissue was incubated in Calyculin A (Cell Signaling Technology, Danvers, MA) at 200nM in PBS for 30min,(Gotoh et al., 1995; Miura & Blakely, 2011). FISH was performed as in Dej & Spradling, 1999. BAC clone #BACN04H23 (Chromosome 3L, region 69C3-C8) from the PacMan collection (Venken et al., 2006) was labeled using the BioNick labeling system (Invitrogen, Carlsbad, CA). BAC probe signal was amplified through sequential labeling with Peroxidase-labeled Streptavidin followed by the TSA Peroxidase detection kit (Perkin Elmer, Waltham, MA). Imaging was performed on a Zeiss Axio Imager 2 with a 63x oil immersion lens.
Live Imaging
Tissue was dissected and cultured based on previous protocols with modifications for the pupal hindgut (Fox et al., 2010; Prasad et al., 2007). For colcemid live imaging experiments, pupae were dissected and imaged in media containing 50μg/ml of colcemid (Sigma), from the initiation of dissection to the first frame was at least 15 minutes and up to 1 hour. Imaging was performed on a spinning disc confocal (Yokogawa CSU10 scanhead) on an Olympus IX-70 inverted microscope using a 60×/1.3 NA UPlanSApo Silicon oil, 100×/1.4 NA U PlanSApo oil, or a 40×/1.3 NA UPlanFl N Oil objective, a 488 nm and 568 nm Kr-Ar laser lines for excitation and an Andor Ixon3 897 512 EMCCD camera. The system was controlled by MetaMorph 7.7.
Fixed Imaging
Tissue was dissected in PBS and immediately fixed in 3.7% formaldehyde + 0.3% Triton-X for 15 minutes. Immunostaining was performed in 0.3% Triton-X with 1% normal goat serum as in Fox et al., 2010. The Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes (Zielke et al., 2014) and mouse anti-Phospho-Histone H3 (ser 10) (Cell Signaling Technology, 1:1000) were used to determine cell cycle stages. Rabbit anti-RFP (MBL, Woburn, MA, 1:500) was used to detect Cenp-C-Tomato Foci. Rabbit anti-DCP1 (Cell Signaling Technology), 1:500 was used to measure cleaved caspases. TUNEL staining was performed with the in situ cell death detection kit (Roche, Basel, Switzerland) according to the protocol in Schoenfelder et al., 2014. Tissue was stained with DAPI at 5μg/ml. Images were obtained using a Leica SP5 inverted confocal with a 40× oil objective. Emission was done using a 405nm diode laser, an argon laser tuned to 488nm emission, a 561nm Diode laser, and a 633 HeNe laser.
Image Analysis
All image analysis was performed using ImageJ (Schneider et al., 2012). nuclear envelope brightness was calculated by measuring Geminin-Azami intensity or a single cell. The brightness for each cell was normalized from 1 to 0, with 1 being the highest pixel intensity value and 0 being the dimmest value for a single cell across all time-points. Anaphase was determined as the first frame with poleward movement of the kinetochores as evident by Cenp-C-Tomato. Time from NEBD to anaphase was determined as the point from half-maximal Azami signal to anaphase. To calculate kinetochore clustering we closely cropped around the cell of interest for all frames. We then used a thresholding approach to outline each centromere or group of centromeres and stored those as ROIs. We then used those ROIs to measure the average intensity of each centromere pixel and the total area of all the pixels. We reasoned that as centromeres disperse the total area they cover increases and there is a corresponding decrease in fluorescence intensity from each individual point, therefore we divided the average pixel intensity by the area and normalized that on a scale from 1 to 0. To measure symmetry of metaphase centromere alignment (Fig5E-F), we generated a line plot of each metaphase plate at the frame immediately prior to metaphase. We then generated aggregate plots of each genotype.
Nomenclature
For chromosomes we use “homolog” to distinguish maternally and paternally contributed chromosomes of the same chromosome type. All chromatids of the same homolog are considered “sisters.” We use “recent sister” to refer to two chromatids that are the product of the most recent S-phase. “Polytene” refers to the chromosome state of any cell formed by genome reduplication that has not fully separated its chromosomes into recent sisters. 4 chromatids conjoined at centromeres are “diplochromosomes”. 8 chromatids conjoined at centromeres are “quadruplochromosomes". We use the term “endocycle” to refer to any cell cycle involving successive genome reduplication without any sign of M-phase. We note the use in the literature of terms such as “endoreduplication", “rereplication”, and “endoreplication” to often refer to the same phenomenon.
SUPPLEMENTAL MATERIAL
Supplemental Figure Legends
Supplemental Movies
Supplemental Movie for Figure 1)
A) Movie from Figure 1I showing His2av-GFP in magenta, and Cenp-C-Tomato in cyan from a diploid HS>fzr wing imaginal disc cell dividing prior to HS>fzr expression.
B) Movie from Figure 1I showing His2av-GFP in magenta, and Cenp-C-Tomato in cyan from a teteraploid HS>fzr wing imaginal disc cell with diplochromosomes 10hr after HS>fzr expression.
C) Movie from Figure S1E showing His2av-GFP in magenta, and Cenp-C-Tomato in cyan from a tetraploid HS>fzr wing imaginal disc cell without diplochromosomes 24hr after fzr overexpression.
Time Indicates minutes to the last frame of metaphase, scale bars represent 5μm.
Supplemental Movie for Figure 2)
A) Movie from Figure 2C showing His2av-GFP from a HS>fzr animal 10hr after heat shock with mitosis by a polyploid diplochromosome-containing wing imaginal disc cell (yellow dotted line) and a diploid cell (blue dotted and dashed line) in the same field.
Time Indicates minutes from the start of filming, scale bars represent 5μm.
Supplemental Movie for Figure 3)
A) Movie from Figure 3F showing His2av-GFP in magenta, and Cenp-C-Tomato in cyan from a papillar cell undergoing a first division, including the SIRS process.
B) Movie from Figure 3G showing His2av-GFP in magenta, and Cenp-C-Tomato in cyan from a papillar cell undergoing a second division.
C) Movie from Figure S3D showing MSL3-GFP in magenta and Cenp-C-Tomato in cyan from a male papillar cell undergoing a first division.
D) Movie from Figure S3E’ and S3E’’ showing sequential z-planes from a fixed ileum of Culex pipiens with mitotic cells that are pre-SIRS and a post-SIRS.
Time Indicates minutes to the last frame of metaphase, scale bars represent 5μm.
Supplemental Movie for Figure 4)
A) Movie From Figure 4A showing His2av-GFP in magenta, and Cenp-C-Tomato in cyan from a wild type papillar cell during anaphase of the first mitosis.
B) Movie From Figure 4A showing His2av-GFP in magenta, and Cenp-C-Tomato in cyan from a mad2 papillar cell from DNA condensation through anaphase of the first mitosis, including formation of a DNA bridge.
C) Movie From Figure 4A showing His2av-GFP in magenta, and Cenp-C-Tomato in cyan from a mad1 papillar cell during from DNA condensation through anaphase of the first mitosis.
D) Movie from Figure 4F showing Jupiter-GFP in magenta and Cenp-C-Tomato in cyan from a first division papillar cell undergoing SIRS in the presence of a vehicle control.
E) Movie from Figure 4F showing Jupiter-GFP in magenta and Cenp-C-Tomato in cyan from a first division papillar cell undergoing SIRS in the presence of a colcemid.
Supplemental Movies for Figure 5)
A) Movie From Figure 5A showing Geminin-Azami in magenta, and Cenp-C-Tomato in cyan from a wild type papillar cell from prior to Nuclear Envelope Breakdown through anaphase.
B) Movie From Figure 5A showing Cenp-C-Tomato in cyan and Geminin-Azami in magenta from a mad2 papillar cell from prior to NEBD through anaphase.
Time indicates minutes from the first frame after NEBD. Scale bar represents 5μm
ACKNOWLEDGMENTS
The following kindly provided reagents used in this study: The Bloomington Stock Center, the Vienna /Drosophila Resource Center, the Kyoto Drosophila Genetic Resource Center, Roger Karess (U. Paris), Christian Lehner (U. Zurich), Mitzi Kuroda (Harvard University), and Bill Sullivan (U.C. Santa Cruz). We thank David MacAlpine, Eda Yildirim, Beth Sullivan, Joshua Bembenek, and Fox lab members for valuable comments on the manuscript. Work on polyploidy in the Fox lab is supported by a Pew Scholar Award, (Pew Charitable Trusts), a Basil O’ Connor Scholar Award (The March of Dimes) a Whitehead Scholar Award (Whitehead Foundation), and Scientist Development Award (American Heart Association), and a Creative and Novel Ideas in HIV Research Award (NIH Centers For AIDS Research/International AIDS Society).