Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch

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Many types of adult stem cells exist in a state of cell-cycle quiescence, yet it has remained unclear whether quiescence plays a role in maintaining the stem cell fate. Here we establish the adult germline of Caenorhabditis elegans as a model for
  *Forcorrespondence: hsseidel@wisc.edu(HSS);jekimble@wisc.edu(JK) Competing interests:  Theauthors declare that nocompeting interests exist. Funding:  See page 23 Received:  13 August 2015 Accepted:  07 November 2015 Published:  09 November 2015 Reviewing editor:  AlejandroSa´nchez Alvarado, StowersInstitute for Medical Research,United StatesCopyright Seideland Kimble.This article is distributed underthe termsof theCreativeCommons Attribution License,whichpermits unrestricted useand redistribution provided thatthe srcinalauthor and source arecredited. Cell-cycle quiescence maintains Caenorhabditis elegans   germline stemcells independent of GLP-1/Notch Hannah S Seidel 1,2 *, Judith Kimble 1,3 * 1 Department of Biochemistry, University of Wisconsin-Madison, Madison, UnitedStates;  2 The Ellison Medical Foundation Fellow of the Life Sciences ResearchFoundation, The Lawrence Ellison Foundation, Mount Airy, United States;  3 HowardHughes Medical Institute, University of Wisconsin-Madison, Madison, United States Abstract  Many types of adult stem cells exist in a state of cell-cycle quiescence, yet it hasremained unclear whether quiescence plays a role in maintaining the stem cell fate. Here weestablish the adult germline of  Caenorhabditis elegans  as a model for facultative stem cellquiescence. We find that mitotically dividing germ cells—including germline stem cells—becomequiescent in the absence of food. This quiescence is characterized by a slowing of S phase, a blockto M-phase entry, and the ability to re-enter M phase rapidly in response to re-feeding. Further, wedemonstrate that cell-cycle quiescence alters the genetic requirements for stem cell maintenance:The signaling pathway required for stem cell maintenance under fed conditions—GLP-1/Notchsignaling—becomes dispensable under conditions of quiescence. Thus, cell-cycle quiescence canitself maintain stem cells, independent of the signaling pathway otherwise essential for suchmaintenance. DOI: 10.7554/eLife.10832.001 Introduction Stem cells in adult tissues were once thought to exist primarily in a state of cell-cycle quiescence.Such quiescence was viewed as an inherent property of the stem cell fate and thus essential for a tis-sue’s long-term self-renewal ( Hall and Watt, 1989  ;  Potten and Loeffler, 1990  ). More recently, how-ever, it has become clear that adult stem cells are not universally quiescent but instead cycle inaccordance with the needs of the tissue: Some types of stem cells proliferate continuously, whereasothers switch from quiescence to rapid proliferation in response to certain stimuli (e.g. wounding orhormones) ( Wabik and Jones, 2015  ). In mammals, for example, hematopoietic and neural stem cellsreversibly switch between quiescence and active proliferation in response to tissue injury( Doetsch et al., 1999  ;  Harrison and Lerner, 1991 ;  Lugert et al., 2010  ), and mammary stem cellsexpand transiently during pregnancy and the estrus cycle ( Asselin-Labat et al., 2010  ;  Joshi et al.,2010  ). Though periods of sustained stem cell proliferation enable rapid tissue growth or turnover,they challenge the view of quiescence as a prerequisite for the stem cell fate. Thus, a long-standingquestion has remained unanswered: Does cell-cycle quiescence play a role in stem cell maintenance?Understanding the relationship between cell-cycle quiescence and stem cell maintenance hasbeen difficult because tractable models of facultative stem cell quiescence have been lacking. Pertur-bations affecting the cell cycle can in some cases impact stem cell maintenance ( Orford and Scad- den, 2008  ;  Pietras et al., 2011 ;  Yilmaz et al., 2012  ), but whether quiescence can maintain stemcells independent of the signals otherwise required for their maintenance has been untested. Such atest requires a system in which cell-cycle quiescence can be readily induced, and in which the signalsotherwise required for stem cell maintenance can be readily removed. In this study, we establish the Seidel and Kimble. eLife 2015;4:e10832. DOI: 10.7554/eLife.10832 1 of 28 RESEARCH ARTICLE  adult germline of   Caenorhabditis elegans   as a model fitting these criteria. We describe a previouslyuncharacterized state of cell-cycle quiescence among adult germline stem cells, emerging underconditions of starvation. We then test whether this quiescence can maintain stem cells, independentof the signal required for their maintenance under conditions of active proliferation.The adult germline of   C. elegans   presents a tractable model for studying stem cell behaviorbecause of its simple, linear organization ( Figure 1A ). Mitotically dividing germ cells—includinggermline stem cells—reside in the distal region of the gonad (the ‘progenitor zone’). Differentiatinggerm cells, in meiotic prophase, are located more proximally. (Here, we use the term ‘progenitorzone’ rather than the earlier term ‘mitotic zone’ or ‘proliferative zone’ to reflect the facultative natureof germ cell divisions.) The progenitor zone has been studied under fed conditions and is composedof a distal pool of germline stem cells and a more proximal pool of cells that have begun to differen-tiate ( Cinquin et al., 2010  ). This proximal pool comprises cells dividing mitotically, as well as cellscompleting their final passage through interphase in preparation for entry into the meiotic cell cycle.We collectively refer to these cells as ‘transient progenitors’, to reflect their continued mitotic divi-sions and transitional state ( Figure 1A ). Under fed conditions, cells throughout the progenitor zonecycle asynchronously and continuously ( Crittenden et al., 2006  ;  Fox et al., 2011 ;  Jaramillo- Lambert et al., 2007  ;  Morgan et al., 2010  ), with transient progenitors undergoing one or tworounds of division as they pass through the proximal progenitor zone ( Fox and Schedl, 2015  ).Prior to this work, germ cell proliferation in  C. elegans   adults had not been examined in detailunder food-limited conditions. However, the effects of such conditions have been examined duringlarval development in  C. elegans  , as well as in adult  Drosophila , and in both contexts, germ cellsrespond robustly to nutritional cues. In  Drosophila , nutrient limitation or changes in nutrient-sensingpathways slow germ cell proliferation, reduce germline stem cell number, or both ( Armstrong et al.,2014  ;  Drummond-Barbosa and Spradling, 2001 ;  Hsu et al., 2008  ;  LaFever et al., 2010  ; McLeod et al., 2010  ;  Roth et al., 2012  ;  Sheng and Matunis, 2011 ). These effects are mediated inpart by changes in the somatic gonad ( Yang and Yamashita, 2015  ), including changes in the size of the somatic niche supporting germline stem cells ( Bonfini et al., 2015  ;  Hsu and Drummond-Bar- bosa, 2011 ). In  C. elegans  , primordial germ cells are born in the early embryo and arrest in the G2phase of the cell cycle until newly hatched larvae begin to feed ( Butuci et al., 2015  ; Fukuyama et al., 2006  ;  Fukuyama et al., 2012  ). This response to feeding has been hypothesized toinvolve food-related signals traveling through soma-to-germline gap junctions, which are requiredearly in larval development for germ cell proliferation and survival ( Starich et al., 2014  ). Later in eLife digest  Adult stem cells can divide to produce cells that can develop into one of manydifferent specialist cell types in a tissue, and so are vitally important for tissue repair andmaintenance. Some types of adult stem cells exist primarily in a non-dividing state known asquiescence, which for a long time was thought to be essential for maintaining the stem cell state.However, researchers have discovered some adult stem cells that are either not quiescent, or onlyenter this state rarely.Until now, biologists have lacked an experimental model in which the role of quiescence inmaintaining stem cells can be easily investigated. Seidel and Kimble have now investigated the roleof quiescence in the germline stem cells – which give rise to egg and sperm cells – of theroundworm  Caenorhabditis elegans  . The results of the study revealed that although the germlinestem cells divide continuously when the worms are well fed, starving the worms causes these stemcells to become quiescent.Maintaining  C. elegans   germline stem cells in a stem cell state normally involves a process calledNotch signaling, which cells use to communicate with each other. However, Seidel and Kimble foundthat the germline quiescence caused by starvation maintains the stem cell state even when Notchsignaling is prevented. This suggests that, in the absence of food, quiescence alone can maintaingermline stem cells, although how it does so remains a question for future work. One possibility isthat quiescence stabilizes other molecules involved in the Notch signaling pathway or prevents theproduction of proteins that enable a stem cell to develop into a specialized cell. DOI: 10.7554/eLife.10832.002 Seidel and Kimble. eLife 2015;4:e10832. DOI: 10.7554/eLife.10832 2 of 28 Research article Cell biology Developmental biology and stem cells  development, germ cells stop dividing if animals enter the non-feeding dauer larval stage( Narbonne and Roy, 2006  ). Even in non-dauer larvae, germ cells proliferate less when food isscarce, an effect mediated in part by communication between food-sensing neurons and the somaticgonad ( Dalfo et al., 2012  ;  Korta et al., 2012  ). In adult  C. elegans  , decreased food intake slowsmitotic and meiotic progression and oogenesis ( Gerhold et al., 2015  ;  Lopez et al., 2013  ; Salinas et al., 2006  ;  Seidel and Kimble, 2011 ), and limited observations suggest that germ cell pro-liferation is also reduced ( Salinas et al., 2006  ). More strikingly, full starvation from the L4 larval stagecauses dramatic germline shrinkage in adult hermaphrodites, and this shrinkage is reversible uponre-feeding ( Angelo and Van Gilst, 2009  ;  Seidel and Kimble, 2011 ). These observations motivatedus to examine in greater detail how mitotically dividing germ cells in adult  C. elegans   respond tofood removal.Here, we report that in the absence of food, mitotically dividing germ cells in adult  C. elegans  stop dividing and become quiescent. This quiescence is characterized by a dramatic slowing of Sphase, cell-cycle arrest in G2, and the ability to re-enter M phase rapidly in response to re-feeding.We investigate these cell-cycle responses in wildtype animals and in germline tumors, and we testwhether this cell-cycle quiescence requires factors controlling larval or behavioral responses to food.We next investigate the control of stem cell maintenance under starved conditions. We uncover amajor difference in the requirement for GLP-1/Notch signaling in the maintenance of actively prolif-erating versus quiescent germline stem cells. This work establishes the  C. elegans   germline as amodel of facultative stem cell quiescence and demonstrates the utility of such a model in clarifyingthe role of quiescence in maintaining the stem cell state. Figure 1.  Fed versus starved adult hermaphrodite gonad of   Caenorhabditis elegans . ( A ) Schematic of an adulthermaphrodite gonadal arm, with the progenitor zone at its distal end and maturing gametes at its proximal end.Germline stem cells and transient progenitors are located in the distal and proximal progenitor zone, respectively.Cells in both pools cycle asynchronously, although they are partially connected via a cytoplasmic core. Filledcircles, germ cell nuclei in the progenitor zone. Open circles, germ cell nuclei in meiotic prophase, includingdeveloping oocytes. Gonads in males and larval hermaphrodites are organized similarly, although their proximalgerm cells differentiate as sperm. This same gonad organization is also seen in starved animals of any stage or sexfor time intervals examined in this work. ( B ) Images of distal gonads dissected from adult hermaphrodites andstained with DAPI to visualize DNA (magenta) and anti-phospho-histone H3 to visualize M-phase chromosomes(green). M-phase cells are outlined and numbered. Left, fed early adult hermaphrodite. Right, hermaphroditestarved from early adult for 8 hr. (See Materials and methods for definition of ‘early adult’.) Asterisks, distal gonadends. Images are maximum-intensity z-projections.DOI: 10.7554/eLife.10832.003 Seidel and Kimble. eLife 2015;4:e10832. DOI: 10.7554/eLife.10832 3 of 28 Research article Cell biology Developmental biology and stem cells  Results M-phase entry in adult germ cells responds rapidly to starvation andre-feeding To investigate how starvation affects germ cell division in adults, we removed food from early adulthermaphrodites and males and monitored the number of germ cells in M phase over the following10.5 hr. Cells in M phase were identified by staining for phospho-histone H3 ( Figure 1B  ), a markerof M phase ( Hans and Dimitrov, 2001 ). Food removal caused a drop in the number of M-phase cells( Figure 2A ), and this response was fast: In hermaphrodites, the number of M-phase cells per pro-genitor zone dropped from an average of 7.6 before food removal to 2.1 after 30 min without food(n = 7 replicates of 220–551 gonadal arms per replicate per time point) ( Figure 2A ). The number of M-phase cells continued to decline thereafter, and after 3.5 hr without food, M-phase cells were vir-tually absent ( Figure 2A ). This drop in M-phase cells did not occur in hermaphrodites fed Figure 2.  Mitotic divisions in adult progenitor zones respond quickly to food removal and re-feeding. Time courses showing the number M-phase cellsper progenitor zone after food removal or re-feeding. Time zero indicates the start of food removal or re-feeding. Animals in  A, B  and  D, E  werestarved from early adult. Animals in  C  were starved from mid L4. Animals in  F  were starved from mid L4 for 24 hr or from early L4 for 72 hr.Independent replicates are overplotted with transparency. For each replicate, lines connect means, and shaded areas show interquartile ranges. Samplesizes indicate numbers of gonadal arms. Source data are available in  Figure 2—source data 1 .DOI: 10.7554/eLife.10832.004The following source data and figure supplement are available for figure 2: Source data 1.  Counts of M-phase cells for starvation and re-feeding time courses of wildtype animals. DOI: 10.7554/eLife.10832.005 Figure supplement 1.  Comparison of numbers of M-phase cells in fed, starved, and re-fed animals.DOI: 10.7554/eLife.10832.006 Seidel and Kimble. eLife 2015;4:e10832. DOI: 10.7554/eLife.10832 4 of 28 Research article Cell biology Developmental biology and stem cells  continuously ( Figure 2—figure supplement 1B, D  ), nor in hermaphrodites exposed to a mock star-vation procedure ( Figure 2—figure supplement 1C  ). In males, M-phase cells also disappeared rap-idly in response to food removal, although the initial drop in M-phase cells was not monotonicallydecreasing ( Figure 2B  ). We conclude that in adults of both sexes, germ cells stop dividing quickly inthe absence of food.We next investigated how germ cell division responds to re-feeding. We removed food from earlyadult hermaphrodites and males, allowed animals to remain in starvation for 12 hr, then re-fed ani-mals, and monitored the number of M-phase cells, as above. In hermaphrodites, this treatment trig-gered a burst of M-phase cells 1.5 hr after the start of re-feeding ( Figure 2D  ). Males showed a similarresponse to re-feeding, but the burst of M-phase cells occurred 1 hr earlier ( Figure 2E  ). In both sexes,these bursts included some individual germlines having approximately twice as many M-phase cells aswere observed among continuously fed animals ( Figure 2—figure supplement 1E, F  ). These resultsdemonstrate that in both sexes, germ cells resume mitotic division rapidly in response to re-feeding.The faster response in males is consistent with germ cells in males having a faster cell-cycle under con-tinuously fed conditions ( Morgan et al., 2010  ). Further, the higher maxima of M-phase cells in re-fedversus continuously fed animals is consistent with germ cells collecting at the G2-to-M transition dur-ing starvation and entering M phase semi-synchronously upon re-feeding. Cessation of M-phase entry in response to starvation coincides withthe molt into adulthood We next extended our results to adult hermaphrodites starved from the L4 larval stage. This exten-sion was motivated by the need to perform certain later experiments in such animals, as starvationfrom L4 prolongs the amount of time that adult hermaphrodites can be maintained without food( Angelo and Van Gilst, 2009  ;  Seidel and Kimble, 2011 ). We removed food from mid-L4 hermaph-rodites and monitored the number of cells in M phase, as above. In starved L4s, M-phase cells per-sisted for  ~ 4–5 hr after food removal, with the average number of M-phase cells only moderatelyreduced relative to fed animals ( Figure 2C  ). Thereafter, the number of M-phase cells declined rap-idly, and after 10.5 hr without food, M-phase cells had virtually disappeared ( Figure 2C  ). The disap-pearance of M-phase cells coincided with the molt into adulthood ( ~ 5–8 hr after food removal), andthe coincidence of these events persisted even under conditions where the timing of this molt waschanged: Hermaphrodites starved from early L4 molted into adulthood  ~ 12–20 hr after foodremoval, and gonads in these animals contained, on average, 1.6 M-phase cells per progenitor zonebefore the molt (n = 57, gonads collected 10.5 hr post food removal) and 0.0 M-phase cells afterthe molt (n = 63, gonads collected 24 hr post food removal). These results demonstrate that germcells in hermaphrodites starved from L4 do not immediately stop dividing in response to foodremoval, but germ cell division eventually ceases, at or near the molt into adulthood. This findingsuggests that mitotically dividing germ cells in L4s are not equivalent to those in adults, a result con-sistent with previous studies ( Crittenden et al., 2002  ;  Dalfo et al., 2012  ;  Gerhold et al., 2015  ; Michaelson et al., 2010  ). Longer starvation does not delay M-phase entry upon re-feeding In other systems, re-entry into the mitotic cell cycle following a period of quiescence occurs moreslowly after longer periods of quiescence ( Lum et al., 2005  ;  Soprano, 1994  ). We therefore testedwhether longer periods of starvation would delay mitotic re-entry upon re-feeding. We repeated there-feeding time course in two types of animals having experienced longer starvation: Adult her-maphrodites starved from mid-L4 for 24 hr and adult hermaphrodites starved from early L4 for72 hr. In both types of animals, re-feeding triggered a burst of M-phase cells 1.5 hr after re-feeding( Figure 2F  ), similar to the re-feeding response in animals starved for only 12 hr (compare  Figure 2D  versus F) . We conclude that the timing of M-phase entry upon re-feeding is largely unaffected bythe duration of preceding starvation, at least during the first 72 hr of starvation. During starvation, germ cells progress slowly through S phase andarrest in G2 We next examined how starvation affects progression of germ cells through G1, S phase, and G2.First, we monitored cell-cycle progression in fed animals. By labeling germlines with the thymidine Seidel and Kimble. eLife 2015;4:e10832. DOI: 10.7554/eLife.10832 5 of 28 Research article Cell biology Developmental biology and stem cells
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