Keywords:Drosophila, midgut, intestinal stem cell, regeneration, dedifferentiation, depolyploidization, amitosis, starvation, injury, aging

ISC number remains at a steady-state in the PMG of young flies

Multipotent ISCs have been identified in both vertebrates (Barker et al., 2007) and invertebrates (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006) and give riseto all cell types in the epithelium. ISCs divide asymmetrically to give rise toan ISC and either a secretory enteroendocrine (ee) cell expressing thetranscription factor Prospero (Pros) (Guo andOhlstein, 2015;Zeng and Hou,2015), or an enteroblast (EB) daughter cell that has high activeNotch (N)-signaling (Micchelli and Perrimon,2006;Ohlstein and Spradling,2007) (FigureS1A). The EB subsequently enters the endocycle, and the resulting4n polyploid pre-EC (Jianget al., 2011;Zhou et al.,2015) further differentiates into an absorptive enterocyte (EC),ploidies of which typically range from 4n to16n (Figure S1A).

To probe alternate mechanisms of ISC replacement, we first defined a timewindow during which ISC number is stable and perturbations can therefore bemeasured reliably. We used flies of the genotypeNRE-lacZ; esg-GAL4,UAS-GFP (esg>GFP);, previously reported asa precise method of quantifying ISC and EB number (McLeod et al., 2010;O’Brien et al., 2011). In these flies, ISCs and EBs aremarked byescargot (esg)-driven GFP expressionand EBs are marked by strong lacZ expression under the control of aNotch response element (NRE) (Micchelli and Perrimon, 2006) (Figure S1A, B).Age-matched females were collected and maintained on a rich diet of standardcornmeal-molasses supplemented with yeast. Midguts were dissected at differenttime points, and the total number of ISCs per PMG was quantified by subtractingthe number of diploid EBs (lacZ+ cells) from the total number of diploidISCs and EBs (GFP+ cells) (Figure S1C). ISC number remained relatively constant inadults from 3 to 17 days PE (Figure S1D, steady-state phase), enabling us to precisely quantifyand interpret any induced ISC loss and subsequent recovery during this time.

Starvation induces a rapid and severe regional loss of ISCs in thePMG

In a previous study, McLeod et al. observed a loss of ISCs in fliesmaintained on a restricted diet of 10% sucrose from 3 to 18 days PE.Recovery of ISC number was subsequently observed when animals were re-fed aprotein-rich diet (McLeod et al., 2010),suggesting that ISC number changes in response to food availability. However,the interpretation of these results was confounded by the length of therestricted-diet assay, which extended into the period of the normal age-inducedincrease in ISC number reported in the PMG of well-fed animals (Biteau et al., 2008;Choi et al., 2008a;Choi et al.,2008b). In addition, the mechanism of ISC replacement was notexplored.

Based on the study of McLeod et al., we supposed that completestarvation with only water may induce a more rapid loss of ISCs within thesteady-state phase (FigureS1D). To this end, we developed an assay in which flies weremaintained on a rich diet from 0–3 days PE and then transferred to vialscontaining only water for two days (Figure1A).

Maintaining flies on water resulted in a dramatic shrinkage of the PMG(defined as regions R4a-R5) (Buchon et al.,2013) (Figure 1B) toapproximately one-half the length (Figure1C,FigureS2A–B) of age-matched controls (Figure 1D,FigureS2B). Tissue shrinkage resulted from regional muscle contraction(Figure 1C (bracket)), visualized bythe condensed fibers of the visceral muscle in R4b-c, a subset of the PMG, ofstarved flies (Figure 1E, left panel, 25muscle bands per region) relative to controls (Figure 1F, left panel, 15 muscle bands per region). Within the areaof muscle contraction, cells in the epithelium were severely overcrowded (Figure 1E, right panel) compared to controls(Figure 1F, right panel). In addition,ISCs within R4b-c of starved flies delaminated from the basement membrane (Figure 1G, yellow arrow), likely bymechanical extrusion, while ISCs in control flies maintained direct contact withthe basement membrane (Figure 1H).

To determine the extent of ISC loss in starved flies, the total numberof ISCs was quantified as described above (Figure S1C). After 2 daysof starvation, the total number of ISCs per PMG decreased to an average of 420± 95, compared to an average of 740 ± 85 in controls (Figure 1I). EBs were also significantlydepleted (Figure 1I). However, the numberof differentiated ees (Pros+) and ECs (polyploid) was not affected(Figure 1I), giving rise to the severecellular overcrowding in areas of muscle contraction.

As the number of ISCs and EBs appeared to be most severely depleted inR4b-c, we further quantified the percent of ISCs or EBs of total cells in a 100μm × 100 μm region within R4b-c (Figure 1J,Figure S2C). ISCs and EBs constituted an average 16± 2 and 20 ± 3 percent of total cells per region in controlflies, respectively (Figure 1J). Incontrast, ISCs and EBs only constituted an average 3 ± 2 and 10± 4 percent of total cells per region in starved flies (Figure 1J), a severe decrease in the density ofprogenitor cells and particularly of ISCs.

To confirm ISC loss, we induced ISC clones using the Mosaic Analysiswith a Repressible Cell Marker (MARCM) system (Lee and Luo, 2001) one day prior to starvation and quantified thenumber of ISC clones remaining in starved flies relative to controls. Inaccordance with our ISC number quantification, ISC clones were lost in starvedflies, with the most dramatic loss in R4b-c (Figure 1K), corroborating that ISCs are lost in this region.

ISC number rapidly increases in re-fed animals

We next determined if ISCs are replaced by re-feeding flies a rich diet.Within 24 hours, the length of the PMG expanded to that of well-fed flies (Figure S2B). Followingtissue expansion, a significant increase in ISC number was observed by 16 hoursof re-feeding. ISC number expanded from an average of 420 ± 95 att = 0 to 490 ± 62, 550 ± 70, 680± 90, and 760 ± 65 att = 16, 24, 48,and 72 hours of re-feeding, respectively (Figure2A), and was indistinguishable from that of age-matched controls by72 hours of re-feeding.

Symmetric ISC divisions and mitoses are negligible at 16 hours of re-feeding,when a significant increase in ISC number is observed

The ISC number increase in re-fed flies is reminiscent of that observed0–3 days PE in the distal hairpin, which is linked to developmentaltissue growth and governed by symmetric divisions of existing ISCs (O’Brien et al., 2011). However,R4b-c of starved flies was severely depleted of ISCs, as well as their immediateEB daughter cells (Figure 1I–K). Asapproximately 70 ISCs are replaced within the first 16 hours of re-feeding, wepredicted that 70 symmetric ISC divisions would have to occur within this shorttime with few remaining ISCs in R4b-c.

To determine the contribution of symmetric ISC divisions to ISCreplacement, we conducted twin-spot clonal analysis (Figure 2B) using flies of the genotypehsFLP;; FRT2A His-GFP/His2AV-mRFP, FRT2A. In these flies, heat-shock(hs)-induced FLP/FRT-mediated recombination results in one GFP-marked and oneRFP-marked daughter cell (Figure 2B). Anasymmetric outcome is visualized by one multi-cellular ISC clone adjacent to onedifferentiated daughter cell of differing colors (Figure 2C). In contrast, a symmetric outcome is visualized by twoadjacent multi-cellular ISC clones of differing colors (Figure 2D).

In control flies at 6 d PE, symmetric division of ISCs was negligible(Figure 2C, E). In re-fed flies, nosignificant increase in symmetric divisions was detectable at 8 or 16 hours ofre-feeding (Figure 2E), despite an increasein ISC number from an average of 420 to 490 by 16 hours of re-feeding (Figure 2A). Symmetric divisions were onlyobserved beginning at 24 hours of re-feeding (Figure 2D, E). Furthermore, by using phospho-histone-H3 (PH3) as amarker of mitosis, no increase in the number of mitoses were observed prior to24 hours of re-feeding in the entire PMG (Figure2F), suggesting that new ISCs did not arise from mitosis of remainingISCs outside of R4b-c. Finally, while quantifying the number of PH3+ISCs, we observed no evidence of ISCs migrating into this region. Together,these data demonstrate that symmetric ISC divisions cannot account for theinitial increase in ISC number from 0–16 hours and point to analternative initial source of new ISCs.

Polyploid cells undergoing amitosis are observed during the first 16 hours ofre-feeding

The Snail-family of zinc-finger transcription factors are highlyconserved across species, and are expressed in many types of stem cells in bothDrosophila and mammals. Indeed, the Snail homologescargot (esg) is expressed in ISCs andEBs of the fly midgut (Micchelli and Perrimon,2006), and has been shown to maintain progenitor cells in anundifferentiated state (Korzelius et al.,2014;Loza-Coll et al., 2014).During the first 24 hours of re-feeding, we observedesg-GAL4-driven expression of GFP (Figure S3A) or themembrane-reporter CD2 (Figure 3A–E)in an average of 36 ± 25 4n polyploid cells in R4a-5(Figure S3B),suggesting that ECs may adopt a progenitor-like state during this time.

To characterize the ontogeny of these cells, midguts were dissectedevery 4 hours during the first 24 hours of re-feeding. Beginning at 8 hours ofre-feeding, this subset of 4n polyploid CD2+ cellsappeared to undergo a ploidy reduction (depolyploidization) (Figure 3A–E) exclusively in R4b-c where ISCdepletion was most severe. Notably, these cells were negative for the mitoticmarker PH3 (Figure 3A–D). The fateof these cells progressed over time, as follows:

At 8 hours of re-feeding, a subset of 4n polyploidCD2+ cells appeared to be undergoing karyokinesis and partitioningchromosomes within an intact nuclear lamina (Figure 3A–B), markedly differing from prophase and metaphaseof a mitotic ISC division (Figure3F–G), during which chromosomes condense and the nuclearlamina dissociates.

At 16 hours of re-feeding, a subset of 4n polyploidCD2+ cells, approximately 1 ± 2 per R4b-c (Figure S3C), progressedthrough a binucleate intermediate containing two nuclei, each with a fullyintact nuclear lamina, which were often bridged (Figure 3C–D, yellow arrow head), in contrast to anaphase ortelophase of a mitotic division, during which the nuclear lamina remainscompletely or partially dissociated (Figure3H–I). Binucleate cells lacked an organizedα-tubulin-rich spindle (Figure 3E),in contrast to mitotic cells (Figure 3J),which rely on the spindle apparatus for proper separation of chromosomes.Finally, binucleate cells appeared to resolve by invagination of the cellmembrane (Figure 3D, yellow arrows) in theabsence of an anillin-rich contractile ring (Figure S3D), in contrastto a mitotic cell, in which anillin localizes to the cleavage furrow in anaphase(Figure S3E).Binucleate cells were fully laminated to the basement membrane (Figure S3F), and not anartifact of the membrane marker CD2, used as a reporter ofesg-GAL4 expression, as these cells were also observed inNRE-lacZ; esg>GFP; flies (Figure S3G), in contrastto controls (FigureS3H).

The ontogeny of CD2+ polyploid cells suggested a process ofdepolyploidization unbeknownst to us. However, in searching the literature, wewere able to identify each step of this process, documented in several differentorganisms. First, the nuclear invagination that we observed at 8 hours ofre-feeding, more prominent on one side of the nucleus (Figure 3A–B), was reminiscent of that reportedin theTetrahymena macronucleus (Endo and Sugai, 2011), cricket egg follicle cells(Conklin, 1903), scorpion serosa(Johnson, 1892), rainbow trouterythrocytes (Wang et al., 2010), rattrophoblast cells (Zybina and Zybina,2008), and human adrenal cells (Magalhaes et al., 1991) undergoing ploidy reduction. Second, thebinucleate cell and characteristic bridge between the two nuclei that weobserved at 16 hours of re-feeding (Figure3C–D, yellow arrow heads) was like that reported in theTetrahymena macronucleus (Endo and Sugai, 2011), scorpion serosa (Johnson, 1892), rainbow trout erythrocytes (Wang et al., 2010), mouse (Kuhn et al., 1991) and spotted skunk trophoblastcells (Isakova and Mead, 2004), and humanfibroblasts (Walen, 2005) undergoingploidy reduction. Finally, the lack of an α-tubulin-rich mitotic spindlewas consistent with reports of cells in all above mentioned species and celltypes. Together, the progression of depolyploidizing cells in re-fed fliesmatched the process of amitosis described in the literature across phyla anddefined as a cell division in which nuclear invagination separates geneticmaterial in the absence of a mitotic spindle, resulting in a binucleate cellthat resolves into two daughter cells (Figure3K). The localization of amitotic cells to R4b-c, and theirappearance from 8 to 16 hours of re-feeding, the time at which we observed asignificant increase in ISC number in the absence of symmetric ISC divisions ormitoses, pinpoints amitosis of 4n polyploid cells as the mostlikely source of new ISCs.

Cells in the EC-lineage serve as an alternate source of new ISCs

To confirm the contribution of cells in the EC-lineage to new ISCs, weconducted lineage analysis based on a flipout system (Theodosiou and Xu, 1998), in which a stop cassetteflanked byFRT sites is excised upon GAL4-driven expression ofUAS-flippase (UAS-FLP), resulting inAct5C-lacZ expression (Figure4A). Two drivers,Myo1A-GAL4 (Morgan et al., 1995) andMex-GAL4(Phillips and Thomas, 2006), arecommonly used to mark and genetically manipulate gene expression in ECs.However, we foundMyo1A-GAL4 drove reporter expression in asub-set of EBs after 5 d PE (Figure S4A) and in a sub-set of ISCs and EBs after 14 d PE (Figure S4B). In addition,Mex-GAL4-driven reporter expression was observed only in asubset of ECs in R4b-c and absent in Esg+ polyploid cells within thisregion in re-fed flies (FigureS4C). Therefore, to maintain stringency in our clonal analysis, weused28E03-GAL4, identified in a screen of Janelia Farm lines(Jenett et al., 2012) as a driverexpressed at high levels (Figure S4D–E) in a subset of EBs. Notably,28E03-GAL4 is absent in all ees and ISCs within ouranalyses from 0–21 days PE. When coupled to the flipout system, the EB,pre-EC and fully differentiated ECs of increasing ploidy (EC-lineage) are marked(Figure 4B).

Flies of the genotype; Act5C FRT draf⁺ FRTlacZ/pBID-UASC-FLP; 28E03-GAL4/NRE-GFP (EC-lineage flipout) (Figure 4A) were subjected to our starvationassay to determine the contribution of cells in the EC-lineage to new ISCs. Nolineage-labeled ISCs were observed in control flies (Figure 4B, D). Likewise, no marked ISCs were observedimmediately after re-feeding, ruling out the possibility that starvation inducedthe expression of28E03-GAL4 in ISCs (FigureS4F–G″). In contrast, an average of 5 ± 3, 6± 3, and 5 ± 2 lineage-labeled ISCs were observed in flies thatwere starved and re-fed for 5, 7, and 11 days, respectively (Figure 4C–D), demonstrating that cells in theEC-lineage contribute to ISC replacement and that these newly formed ISCs arestable over time. In addition, lineage-labeled ISCs gave rise to multi-cellclones of increasing size over time, averaging 2 ± 1, 2 ± 1 and6 ± 4 cells per clone at day 5, 7 and 11 of re-feeding, respectively(Figure 4E). Together, these datademonstrate that new, functional ISCs originate from cells in the EC-lineage,either by direct dedifferentiation of the EB or by amitosis of a 4n polyploidEC.

The frequency of flipout events is low in our EC-lineage analysis.Therefore, we sought to determine the percent of potential amitotic eventsrepresented by the number of ISC clones generated in our lineage analysisexperiment. To this end, flies of the genotype; esg-GFP/Act5C FRTdraf⁺ FRT lacZ; 28E03-GAL4/pBID-UASC-FLP,incorporating a reporter ofesg expression, were subjected toour starvation assay. We then quantified the total number of Esg+4n cells (42 ± 19) and the number oflineage-labeled Esg+ 4n cells (4 ± 2) in fliesthat were re-fed for 16 hours (Figure4F–G). As 10% of the total Esg+4n cells were also lineage-labeled (Figure 4G), we estimate that the number of ISC clonesoriginating from the EC-lineage represent 10% of the total potentialamitotic events. Therefore, the 5 ± 3 ISC clones observed in fliesre-fed for 5 days (Figure 4D) represent20–80 ISCs originating from cells in the EC-lineage, consistent with anaverage of 70 ISCs gained within the first 16 hours of re-feeding (Figure 2A).

Amitosis gives rise to functional ISCs, but is also a source for loss ofheterozygosity

During mitosis, spindle assembly ensures fidelity of chromosomesegregation into each daughter cell. As such, heterozygous cells remainheterozygous. However, it has been shown that chromosomes can be randomlysegregated into daughter cells during amitosis in ciliates (Prescott, 1994). In this case, a heterozygouspolyploid cell could give rise to two daughter cells homozygous for one or morechromosomes. We took advantage of this property of amitosis in two ways. First,we hypothesized that random segregation of homologous chromosomes into eachdaughter cell could be used as lineage-analysis to directly determine ifamitosis gives rise to ISCs. Second, we sought to probe the possible deleteriousconsequence amitosis may have in a fly carrying a heterozygous mutation.

We genetically probed for amitosis-induced random homologous chromosomesegregation as follows. Flies of the genotypetub-GAL4, UAS-dsRed(tub>dsRed); ; +/TM6B, tubP-GAL80 were subjected toour starvation assay. In these flies,tub-GAL80 repressestub>dsRed expression. In the event of mitoticdivision or direct dedifferentiation of a diploid cell to an ISC, the resultingISC should remain heterozygous on the third chromosome (+/TM6B,tubP-GAL80) (Figure 5A), andtub>dsRed expression will remain repressed bytub-GAL80. However, upon amitosis, a fraction of theresulting ISCs may inherit two copies of thewild-type thirdchromosome (+/+), restoringtub>dsRedexpression and giving rise to a positively-labeled ISC clone (Figure 5A). Indeed, flies that were starved and re-fedcontained an average of 6 ± 4 ISC clones with an average of 2 ±2 cells per clone, per PMG (Figure5C–D) (n = 10), supporting the ideathat amitotic events give rise to new ISCs upon re-feeding. ISC clones containedall differentiated cell types (Figure 5C),demonstrating that ploidy reduction can result in fully functional, multipotentISCs. In contrast, control flies contained none to rare positively-labeledclones (1 ± 1 ISC clones per PMG, with an average of 4 ± 2 cellsper clone) (Figure 5B, D)(n = 10), presumably resulting from rare amitoticevents occurring during other phases of ISC number expansion (Figure 6B).

As amitosis resulted in loss of heterozygosity inwild-type flies, we presumed that homozygous ISC mutantclones could be generated in starved and re-fed flies harboring a heterozygousmutation. To test this, we subjected flies heterozygous for a mutation inkuzbanian (kuz^e29-4/CyO,WeeP-GFP), an ADAM family metalloprotease involved in the cleavageof Notch (Lieber et al., 2002), to ourstarvation assay. Again, upon amitosis, a fraction of the resulting ISCs wouldinherit two copies of thekuz^e29-4 mutation on thesecond chromosome (kuz^e29-4/kuz^e29-4),resulting in a negatively-labeled ISC clone with a Notch-mutant phenotype (Figure 5E). Indeed, starved and re-fed fliescontained an average of 1 ± 1 negatively-labeled mutant clones per PMGcontaining a single ISC and cluster of Pros+ ee cells (FigureG–H) (n = 10), a Notch-mutant phenotypepreviously reported in theDrosophila midgut (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006), and consistent withthe loss ofkuz-mediated S2 cleavage (Pan and Rubin, 1997). In control animals,negatively-labeled mutant clones were not observed (Figure 5F, H) (n = 10).

Amitosis occurs in response to ISC dysfunction during periods ofproliferative demand

We hypothesized that, akin to physical loss, functional saturation orfunctional loss of the ISC during periods of increased demand may serve as aninitiating factor of amitosis. Three additional periods of ISC number increaseshave been established. First, a developmental increase in ISC number, governedby symmetric ISC divisions, occurs in response to feeding and organ growth innewly eclosed flies (O’Brien et al.,2011). Second, a transient increase in ISC number is invoked byinjury of the midgut and the ensuing proliferative demand necessary to repairthe epithelium (Chatterjee and Ip, 2009;Choi et al., 2008a). Finally, anincrease in ISC number has been reported in aging flies, due to thedysregulation of tissue homeostasis (Biteau etal., 2008;Choi et al., 2008a;Choi et al., 2008b). We sought toassess the generality by which amitosis is initiated throughout the lifetime ofthe fly in response to the conditional state of the ISC during these periods ofISC number increase.

We first quantified the number of PH3+ cells per PMG in newlyeclosed (1 day PE), injured (7 days PE), and aging (26 days PE) flies relativeto control adult flies (7 days PE). Consistent with previous reports, the rateof proliferation significantly increased during all three phases of ISC numberexpansion and proliferative demand (Figure6A). We next quantified the number of binucleate cells, an indicatorof amitosis, per PMG of flies in each condition. While not observed in controladult or newly eclosed flies, amitosis was observed at a low frequency ininjured flies and significantly in aging flies (Figure 6B), suggesting that amitosis can be initiated by functionalsaturation or functional loss of the ISC, respectively.

To determine if amitosis initiates in response to ISC dyfunction, wesought to chemically inhibit mitosis by oral administration of demecolcine, amicrotubule depolymerizing agent, in the following two assays. In the firstassay, newly eclosed flies were fed a rich diet from 0–24 hours PE toenable organ growth and subsequent demand for an expansion of the ISC pool.Flies were then transferred to a rich diet containing 32 μg/mLdemecolcine (Rebollo et al., 2004) (Figure 6C) for 16 hours. In the second assay,flies were fed a rich diet from 0–6 days PE and then transferred to arich diet containing 25 μg/mL bleomycin, a damaging agent. After 24hours of feeding, flies were removed from the damaging agent and allowed torecover for 16 hours on a rich diet containing demecolcine (Figure 6C).

Demecolcine effectively inhibited mitosis, as the number of PH3+cells per PMG significantly increased in demecolcine-treated flies relative tonon-treated cohorts (Figure 6D, treated,relative toFigure 6A, non-treated).Amitosis was not significantly observed in demecolcine-treated uninjured7-day-old adults, in which an expansion of ISC number is not normally required(Figure 6I). Consistent with ourhypothesis, blocking ISC mitoses in newly eclosed or injured flies led to theinitiation of amitosis (Figure6E–H). The number of binucleate cells increased from an averageof zero (Figure 6B) to 2 ± 1 (Figure 6I) in newly eclosed flies and from anaverage of 0.2 ± 0.3 (Figure 6B) to3 ± 2 (Figure 6I) in injured flies.Notably, amitosis only occurred in areas where all ISCs were blocked in mitosis(FigureS5A–B, asterisks), supporting that amitosis is initiated inresponse to ISC dysfunction during proliferative demand. By contrast, amitosisoccurred in the presence of functional, PH3+, ISCs (Figure S5C, asterisk) inaging flies, pointing to dysregulation in the initiation of this process withage. Finally, rare tri-nucleate amitotic cells were observed when blocking ISCmitoses in newly eclosed or injured flies, suggesting that the process ofamitosis can be error-prone (Figure S5D–E). Together, these data establish that amitosisis initiated in response to either the physical or functional loss of the ISCduring periods of tissue demand and ISC number increases throughout the lifetimeof the fly.

Drosophila intestinal stem cell number is regulated bymultiple mechanisms

Replenishment of differentiated cell types by stem cells has been anintensive focus of tissue homeostasis (Fuchs andChen, 2013). However, homeostatic regulation of stem cell number, thevery foundation of tissue homeostasis, remains poorly understood (Korinek et al., 1998). Increases in thenumber of stem cells can either be permanent, following stem cell loss, ortransient, in response to increased tissue demand. Symmetric division ofexisting stem cells is viewed as the classical mode of stem cell expansionfollowing loss, injury, or disease (Morrison andKimble, 2006). However in the event of widespread stem cell loss orexcessive demand for new cells, alternate mechanisms for generating new stemcells likely exist to satisfy short and long term tissue needs. Indeed,experiments involving genetic ablation of resident stem cells in a wide host oftissues and species, reveal that in the absence of stem cells, early stem celldaughter cells directly convert, by a process known as dedifferentiation, to newfunctional stem cells (Brawley and Matunis,2004;Kai and Spradling, 2004;Nakagawa et al., 2007;Tata et al., 2013;Tetteh et al., 2016;van Es et al., 2012), underscoring the ability of tissues to employalternative strategies to maintain stem cell number and tissue homeostasis.

Our data uncover an alternate mode of new stem cell production by aspecialized process of depolyploidization known as amitosis. Following severeISC loss, an increase in ISC number can be achieved in the absence of ISCmitoses. During this time, polyploid cells in the enterocyte lineage undergoamitosis to fully functional ISCs. Amitosis is also initiated in aging flies andin response to blocking the ISC’s ability to symmetrically divide innewly eclosed or injured adult flies. Together, these data underscore amitosisas a general alternative mode of stem cell replacement in the intestinalepithelium.

Means of achieving polyploidy and ploidy reduction

Two modes of ploidy reduction have been reported. Reductive mitoticdivision has been described in the mammalian liver (Duncan et al., 2009). Amitosis has been described ina host of cell types from primitive ciliates (Prescott, 1994) to plants (Miller,1980), fish (Wang et al.,2010), birds (Patterson, 1908),skunk (Isakova and Mead, 2004), rodents(Kuhn et al., 1991;Zybina and Zybina, 2008), and humans (Magalhaes et al., 1991). In the absence of diploidprogenitor cells, why might theDrosophila midgut replace ISCsthrough an amitotic division rather than a mitotic one? The answer may lie inhow polyploidy arises in this tissue. Cells can achieve polyploidy by a varietyof means including stalled mitoses, in which the mitotic program is active, orvariations of endomitosis or endocycling, in which the mitotic program ispartially or completely inactive. In the mammalian liver, hepatocytes arethought to achieve polyploidy through a stalled mitosis due to failedcytokinesis (Margall-Ducos et al., 2007),and subsequently undergo ploidy reduction through a reductive mitotic division(Duncan et al., 2010). In contrast,rodent trophoblast cells achieve polyploidy by exiting the mitotic program andentering the endocycle (MacAuley et al.,1998), subsequently undergoing ploidy reduction through amitosis.Along these lines, while polyploid cells in theDrosophilarectum can exit the endocycle and reenter the mitotic program, polyploid mitoticdivisions are not reductive in this system (Foxet al., 2010). From work in these cell types, we would speculate thatcertain means of achieving polyploidy may direct the means of achievingdepolyploidization. As is the case with mammalian trophoblasts, cells in theDrosophila enterocyte lineage become polyploid by enteringthe endocycle. Because the genetics underlying amitosis remain an enigma,nuances into how enterocytes achieve polyploidy may provide insight intosignaling that also initiates the “amitotic cell cycle”.

If the initiation of amitosis is linked to the endocycle, can cells ofany ploidy greater than 4n undergo amitosis in the midgut? Wehypothesize that amitotic cells are at a specific stage within the lineage, justexiting the first endocycle, at which point they are still capable of expressingthe stemness factorescargot. At this point, the4n cell, presumably at a programmed checkpoint, can eitherprogress to terminal differentiation or can immediately revert to diploidprogenitors, depending on the needs of the tissue (Figure 6J). As such, we do not expect that terminallydifferentiated, highly polyploid (8n to 16n)cells would be capable of this process. Consistent with this hypothesis, it hasbeen reported that ISC replacement is not observed upon genetic ablation of allcells expressingesg-GAL4 (Luand Li, 2015), which includes progenitor cells, a sub-population of4n ECs and the 4n amitotic cells observedherein. Finally, polyploid genomes are often not 2n replicatesof the diploid genome, as certain genomic regions are underreplicated duringendocycling (Gall et al., 1971). Asunderreplication often occurs in high ploidy cells with polytene chromosomes,such as theDrosophila salivary gland, or mammalian gianttrophoblast cells (Schoenfelder and Fox,2015), the first endocycle may give a cellular product with the mostfidelity.

Amitosis as a conserved biological and pathological mechanism of cellpropagation

Since Remak’s initial account of amitosis in 1841 (Remak, 1841), its occurrence has been foundacross phyla. Yet, the functional significance of this distinctive form of celldivision remains largely unexplored. Our data demonstrate that amitosis is anintegral component of stem cell homeostasis under physiological stress in thehigh-turnover tissue of theDrosophila midgut. Likewise,amitosis may also bear functional significance in maintaining homeostasis underphysiological and pathological conditions in other tissues containingpopulations of polyploid cells, in which amitosis has not been previouslyexplored. While polyploidy was once thought to be a rarity in vertebrates,polyploid cells have been identified in the uterus, placenta, myocardium, bonemarrow, and perhaps most notably, the liver (Zielke et al., 2013). Although hepatocytes have been shown toundergo reductive mitotic divisions, it is worth noting that amitosis has beendescribed in both the damaged rodent liver and human liver cancer tissue (Yiquan and Binkung, 1986), suggesting thatamitosis may play a role in liver homeostasis.

In addition to developmentally-programmed polyploidy, polyploidy isoften associated with tumorigenesis (Fox andDuronio, 2013). Cancer cells utilize endoreplication as a means toescape mitotic catastrophe, and subsequently undergo ploidy reduction to producehighly proliferative, aggressive cells. Following γ-irradiation,tetraploidization (Davoli and de Lange,2011) has been reported in Barrett’s esophagus (Galipeau et al., 1996), prostate cancer(Montgomery et al., 1990) and colonadenomas (Park et al., 2011). Ploidyreversion from the tetraploid state results in two outcomes: aneuploidy (Davoli and de Lange, 2011) ordepolyploidization, with all of the features of amitosis, to give rare buthighly proliferative diploid cells that are morphologically indistinguishablefrom other diploid cells within a tumor (Erenpreisa et al., 2005a;Erenpreisaet al., 2005b;Erenpreisa et al.,2000).

As suggested in cancerous tissue, the process of amitosis bearsramifications. Our results demonstrate a beneficial outcome of amitosis– the generation of new, functional stem cells. However, our resultsalso show a potential danger in the use of this mechanism, loss ofheterozygosity and generation of cells homozygous for deleterious mutations.Mutation-driven endocycling leading to polyploid enterocytes has been identifiedin the human gastrointestinal tract (Clarke etal., 2007). However, whether these mutant enterocytes are capable ofgiving rise to new stem cells by amitosis under an array of physiologicalcontexts has not been explored. Our findings raise the possibility thatendocycling followed by amitosis may function as a cancer-initiating factor.Such errors, even at a low frequency, could have profound implications on tissuehomeostasis and initiation of cancer.

Contact for Reagent and Resource Sharing

Further information and requests for resources and reagents should bedirected to and will be fulfilled by the Lead Contact, Benjamin Ohlstein(bo2160@cumc.columbia.edu).

Experimental Model and Subject Details

Method Details

Quantification and Statistical Analysis

For all quantifications,n represents the number ofmidguts analyzed, error bars represent standard deviation, and statisticalsignificance was determined using the Welch’s T-test with equal samplesize and unequal variance, unless otherwise noted, and expressed as P-values.(^*) denotes p < 0.05,(^**) denotes p < 0.01,(^***) denotes p < 0.001 and(ns) denotes values whose difference was notsignificant.

With the exception of total cell number counts per PMG (Figure 1I andFigure2A), all graphs are scatter plots of raw data to present the fulldistribution of values observed, with averages reported as a black line.

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Supplementary Materials