The Cell Cycle and Development: Lessons from C. Elegans David S

Seminars in Cell & Developmental Biology 16 (2005) 397–406

Review The cell cycle and development: Lessons from C. elegans David S. Fay ∗

University of Wyoming, College of Agriculture, Department of Molecular Biology, Dept 3944, 1000 E. University Avenue, Laramie, WY 82071, USA Available online 14 March 2005

Abstract

The invariant developmental cell lineage of Caenorhabditis elegans (and other similar nematodes) provides one of the best examples of how cell division patterns can be precisely coordinated with cell fates. Although the ﬁeld has made substantial progress towards elucidating the many factors that control the acquisition of individual cell or tissue-speciﬁc identities, the interplay between these determinants and core regulators of the cell cycle is just beginning to be understood. This review provides an overview of the known mechanisms that govern somatic cell growth, proliferation, and differentiation in C. elegans. In particular, I will focus on those studies that have uncovered novel genes or mechanisms, and which may enhance our understanding of corresponding processes in other organisms. © 2005 Elsevier Ltd. All rights reserved.

Keywords: Cell cycle; Caenorhabditis elegans; Development; Review

Contents

1. Introduction to C. elegans development...... 397 2. Control of embryonic cell fates and divisions...... 398 3. Core regulators of the cell cycle in C. elegans ...... 399 4. The cell cycle, division, and differentiation ...... 400 4.1. Dependence and independence ...... 400 4.2. Control of vulval cell fate speciﬁcation ...... 400 5. Novel contributions ...... 401 5.1. The cullins ...... 401 5.2. New dogs old tricks and old dogs new tricks ...... 402 5.3. Control of body size...... 403 6. Concluding remarks ...... 403 Acknowledgments ...... 404 References ...... 404

1. Introduction to C. elegans development nearly 40 years. Aiding researchers in this ambitious goal has been the worm itself, both through its amenability to genetic Just how a typical worm goes about generating 558 cells approaches as well as its quasi-fixed or “hard-wired” develop- during embryonic development as well as an additional 401 mental lineage. Briefly, during the first ∼1.5 h of embryogen- somatic cells (plus ∼2000 germ cells) during its four postem- esis, six “founder” cells are generated that will subsequently bryonic larval stages has been a subject of great interest for give rise to all cell types within the embryo [1]. The specific timing of founder cell establishment ranges from ∼30 min (in the case of AB) to ∼90 min (for D and P4; Fig. 1). Three of the ∗ Tel.: +1 307 766 4961; fax: +1 307 766 5098. founder cells will produce differentiated cell types of a single E-mail address: [email protected]. class, e.g. E, from which the intestine is derived. Three others

genes (so named for their striking defects in developmental timing), which include a number of micro RNAs as well as their presumptive targets. Mutations in the heterochronic genes lead to juxtapositions of developmental events, such that divisions typical of the L2 stage may occur during L1, or to patterns that are characteristic of one stage being reiterated throughout multiple stages, producing the equivalent of a developmental stutter [3]. Of particular significance is the implication that heterochronic genes must ultimately interface with cell cycle regulators to control both re-entry into and withdrawal from the cell cycle [4,5]. The exact mechanism by which this occurs, however, remains largely unsolved (also see below). Components of the dauer pathway, the core portion of which includes an insulin-like signaling pathway, provides Fig. 1. Cell lineage of the early C. elegans embryo. Founder cells (red) and their derivatives (blue) are indicated along with the approximate timing of a necessary degree of flexibility to postembryonic develop- divisions after fertilization (at 25 ◦C). Left and right placements of daughter ment, allowing animals to temporarily withdraw from the cells indicate their relative anterior and posterior locations within the embryo, normal course of development in times of food shortage or respectively. Adapted from [1]. other environmental challenges [6]. Similar to the situation for heterochronic genes, entry into the dauer state (following will generate diverse cell types, such as AB, whose descen- L2) must necessarily be coordinated with cell cycle regula- dents include skin, neurons, and muscle cells. After hatch- tors, such that cells withdraw from the cell cycle and remain ing, 53 cells produced during embryogenesis (termed “blast quiescent until re-entry into the L3 stage. cells”) will undergo subsequent rounds of division over the course of four larval stages (designated L1–L4) to generate the cell types and structures associated with the adult animal 2. Control of embryonic cell fates and divisions [2]. Two other embryonically derived cells (Z2 and Z3) are responsible for populating the germ line. Prior to the isolation of informative mutations, several In examining the lineage of Caenorhabditis elegans, early studies suggested a role for asymmetrically distributed three things become apparent: (1) the relative timing of cytoplasmic determinants in governing the duration of indi- all (somatic) cell divisions is invariant; (2) the orientation vidual cell cycles [7,8]. Using several different manipulation planes of the cell divisions (with respect to the major animal techniques, it was found that nuclei within a common cyto- axes) are also highly reproducible; and (3) the ultimate fates plasm divide synchronously, whereas enucleated cells con- assumed by individual cells are invariant and correlate with tinue to cycle (based on surface contractions) with a timing the specific position of a cell within the greater lineage. As that is characteristic of their normal lineage. Furthermore, the described below, by altering any single aspect of the lineage, duration of cell cycles can be greatly altered by the introduc- other characteristics of the lineage may experience conse- tion of cytoplasm from cells with different inherent period- quences. For example, by changing the plane of cell division, icities. Notably, these studies failed to detect alterations in differentiation may be affected because of the abnormal the timing of blastomere divisions based solely on changes segregation of cell fate determinants. In addition, by shifting in nuclear-to-cytoplasmic ratios [7]. cell fates (most commonly through loss of gene function), Initial studies also demonstrated that lineage-specific dif- the subsequent timing of cell divisions will typically (and ferences in cell cycle lengths could be attributed solely to predictably) be altered. It is important to note that whereas disparities in the duration of S phase; the early cycles of C. the fixed lineage of C. elegans is suggestive of a model that elegans, like those of Drosophila, lack detectable gap phases could rely exclusively on the activities of asymmetrically [9]. True gap phases are first observed in the daughters of the segregated differentiation factors, in fact, cell signaling and E cell, and this delay (which corresponds with gastrulation) inductive events play a major role in determining develop- is dependent on embryonic transcription [10]. More recently, mental outcomes during early embryogenesis, as well as it has been reported that the duration of S phase in the two- later during postembryonic development. It is the invariance cell-stage blastomeres AB and P1, depends on the actions of the lineage with respect to both timing and orientation of several conserved checkpoint genes including C. elegans that leads to a reproducible pattern of cell-cell contacts, homologs of Chk1 and ATM/ATR [11]. In the absence of thereby ensuring consistency of the inductive events. checkpoint function, normal differences in the timing of these In addition to the actions of cell fate determinants and divisions were substantially, although not completely, abro- instructive signaling molecules, more global controls exist gated. This study provided further evidence that the longer to guide the relative timing of postembryonic cell divisions. cell cycle associated with P1 may be the indirect effect of Such regulation is provided primarily by the heterochronic the smaller size of P1 relative to AB; a phenomenon that is D.S. Fay / Seminars in Cell & Developmental Biology 16 (2005) 397–406 399 controlled in the first cell cycle by the asymmetric position sequently arrest at the one-cell stage as a result of incomplete of the mitotic spindle apparatus. The authors speculate that a meiotic maturation [20]. Similarly, mutations in the cyclin E cytoplasmic factor required for DNA replication may there- homolog, cye-1, also lead to defects in postembryonic cell fore be limiting in P1, leading to activation of the S-phase divisions (also see below), and like ncc-1, a role during em- checkpoint and a delay in division. bryogenesis is only revealed after RNAi depletion of germline Complementing these studies have been forward and re- cye-1 [21]. In contrast, loss of function (by RNAi or mutation) verse genetic analyses to identify early-acting factors and in either the C. elegans Cdk-4 homolog or cyclin D leads to control mechanisms [12–15]. In general, these fall into three early larval arrest but fails to affect embryogenesis, indicat- broad categories: (1) factors that directly (or indirectly) im- ing that embryonic divisions are not regulated by a classical plement cell fates by controlling the expression of specific G1/S-phase restriction point [22,23]. genes and proteins; (2) factors that regulate the asymmetric Negative regulators of cell cycle progression in C. ele- distribution of maternally-derived cell fate determinants be- gans include Cdk inhibitors, the retinoblastoma protein, a longing to the first category; and (3) factors that control the cdc-25-like phosphatase, mediators of ubiquitin-dependent orientation of the early division planes (through regulation of cyclin degradation, and a number of novel proteins (also see the mitotic spindle position). Typically, mutations in genes below). The two CIP/KIP family members in C. elegans (cki- from the first category lead not only to gross transformations 1 and cki-2) are encoded by adjacent genes, and inactivation in cell fates but also to concomitant changes within the lineage of cki-1 leads to excessive divisions during both embryonic producing these aberrant fates. In other words, the pattern of and larval development [24,25]. Correspondingly, overex- the altered cell divisions often mimics that of the normal lin- pression of cki-1 leads to a block in division at the G1/S-phase eage that would produce this tissue in the wild type. This transition [24]. Reporter analyses indicate that cki-1 levels are phenomenon is also characteristic for transformations of the elevated in G1-arrested blast derivatives and in cells that have postembryonic lineages, and these results indicate that cell undergone terminal differentiation [24]. Interestingly, cki-1 cycle regulators often take their cue from factors controlling expression is itself positively regulated by several known het- cell fate and differentiation. erochronic genes, and cki-1 inactivation results in the prema- It also follows that mutations affecting division planes and ture division of vulval precursor cells (VPCs), a defect also the distribution of maternal determinants would lead to the observed for the heterochronic mutant and positive regulator missexpression of genes that implement cell fate [13,16–18]. of cki-1, lin-14 (for a discussion of VPC divisions, see below) Such defects generally result in gross perturbations of normal [24]. Thus, cki-1 may serve as a link between the cell cycle cell division and differentiation patterns. In some instances, machinery and global regulators of developmental timing. this can result in pseudo-hyperproliferative phenotypes, such Work from my laboratory, together with studies from as mutations in par-1 that give rise to embryos with >800 another group, has in recent years shown a role for the C. cells [14]. In addition, alterations in the activity of genes elegans retinoblastoma protein homolog, LIN-35, in cell that globally control transcription, such as histone acetylase, cycle control. The genome of C. elegans encodes for a can also lead to hyperproliferation along with the absence of single ancestral Rb family member (in contrast to vertebrates many differentiated cell types [19]. Nevertheless, the actions which contain three family members), and animals harboring of such genes on cell cycle control are in most instances quite null alleles of lin-35 are viable and show no systematic indirect. defects in cell division control or differentiation [26,27].A function for lin-35 in G1/S-phase control is indicated by the ability of lin-35 mutations to enhance the hyperproliferative 3. Core regulators of the cell cycle in C. elegans phenotype of cki-1 loss-of-function (LOF) and to partially rescue the postembryonic division defects of cyclin D and In addition to the indirect effectors described above, ap- Cdk4 mutants [22]. In addition, lin-35 also shows a synthetic proaches using forward and reverse genetics have identified hyperproliferation phenotype with mutations in fzr-1, the cellular and developmental roles for a number of conserved C. elegans homolog of the anaphase promoting complex cell cycle components. However, because of the robust ma- (APC) specificity component, Cdh1/fizzy related [27]. lin-35 ternal expression of many cell cycle regulators (as well as also enhances the hyperproliferative phenotype of lin-23 their presumptive S- and M-phase targets), homozygous mu- mutants; lin-23 encodes an F-box protein and constituent of tants may remain unaffected during embryogenesis and do the Skp1–Cullin–Rbx1–F-box (SCF) complex [27]. Both the not show defects until later stages of larval development, at APC and SCF complexes function as (E3) ubiquitin ligases which time maternal stores become depleted. One example and are known to promote the degradation of G1-type cyclins is the presumptive Cdk1 homolog, ncc-1, which encodes one (A and E, respectively). Our results support a model whereby of six cdc-2-related kinases in C. elegans. ncc-1 homozygous the observed synthetic phenotypes result from the inacti- mutants (derived from a heterozygous mother) hatch as vi- vation of two principal mechanisms for regulating cyclin able embryos but generally fail to complete any further cell levels, transcriptional repression (via LIN-35) and targeted divisions. In contrast, RNAi inactivation of ncc-1 results in a destruction (via FZR-1 and LIN-23). In the absence of either depletion of ncc-1 mRNA in the germline, and embryos con- pathway alone, sufficient regulation can be brought about to 400 D.S. Fay / Seminars in Cell & Developmental Biology 16 (2005) 397–406 keep G1 cyclin levels within permissible limits. However, in some period of DNA synthesis, suggesting that entry into S the absence of both pathways, cyclin levels exceed a critical phase (or exit from G1) was necessary for terminal differ- threshold resulting in abnormal cell cycle re-entry. entiation to be initiated (perhaps as a result of changes in The C. elegans genome encodes four members of the chromatin states following replication). For the expression cdc25 phosphatase gene family [28]. Recently, two groups of epidermal and muscle differentiation markers, resistance have identified gain-of-function (GOF) mutations in one of to DNA replication inhibitors occurs at ∼140 and 180 min, the family members (cdc-25.1) that specifically lead to su- respectively, well before terminal divisions and the onset of pernumerary divisions of the E (intestinal) lineage during normal differentiation by these cell types. The precise timing embryogenesis [29,30]. Conversely, loss of cdc-25.1 function of differentiation commitment in these and other embryonic leads to early defects in meiosis and mitosis and in the reduced lineages has, however, not been well explored. mitotic proliferation of the germline [31,32]; see [33] for a Complementary studies have also been carried out using review of proliferation control of the germline. In contrast to mutations that produce variable cell cycle defects in postem- fission yeast, where cdc25 acts at the G2/M-phase transition, bryonic lineages. In several studies, neuronal development cdc-25.1 appears specifically to promote entry into S phase was observed to occur in the absence of complete division cy- [30]. The extra E cell divisions in cdc-25.1 mutants are distin- cles [37,38]. Interestingly, these neurons typically displayed guishable from those caused by LOF in cki-1. For example, the differentiation characteristics of only one of the possible there are significant differences in the timing of the extra di- descendents, indicating that certain fates may be either intrin- visions in the two single mutants and cki-1; cdc-25.1 double sic or dominant. In addition, the dominance of a particular mutants have a partially additive phenotype. Therefore, cki- fate (for a given blocked division) was in part dependent on 1 and cdc-25.1 may be active at different points in the cell the specific mutant used, suggesting that differences in the cycle, or it could be that one or both does more than sim- precise stage of cell cycle arrest may affect developmental ply regulate CDK activity. The functions of the other cdc25 outcomes [38]. Our own studies on the effects of cell cycle family members are currently unknown. perturbations on vulval development are also consistent with As a conclusion to this section, it is worth noting that for proper lineal differentiation occurring in mutants with either the hyperproliferative mutants described above, terminal dif- reduced or excessive divisions [21]. Furthermore, our studies ferentiation into the proper somatic cell types always seems suggest that a differentiation timing mechanism might be set to follow in the wake of the excess division cycles, indicating in motion following the initial induction of vulval cell fates. that although differentiation is delayed, it is not ultimately The timer we observed did not rely on counting cell divisions prevented. This is in apparent contrast to mutations that re- but rather appeared to measure the passage of time. Similar sult in germline hyperproliferation [33–35], and probably re- observations have been made in a number of diverse systems, flects both underlying genetic redundancies and the inherent and it has been suggested that such timers may depend on the difficulty of completely derailing a hardwired developmental progressive expression of Cdk inhibitors [39–42]. lineage. A notable exception to the above findings was a recent study showing that the production of extra distal tip cells (DTCs; a cell type associated with the somatic gonad) in cki- 4. The cell cycle, division, and differentiation 1 mutants does not result from extra divisions of preexisting DTCs [43]. Rather, the origin of the extra DTCs was deduced 4.1. Dependence and independence to be the result of cell fate transformations by several other cells types within the somatic gonad lineage. This finding is Although the differentiation of individual cell types is in contrast to the extra DTCs produced in lin-35; fzr-1 mu- tightly correlated with specific patterns of division in the tants, which arise from preexisting DTCs [27]. The former wild type, a number of studies indicate that terminal differ- study indicates that cki-1 may have roles in tissue-specific entiation for many cell types does not necessarily require a differentiation outside of cell cycle control or that perturba- complete execution of the normal lineage. For example, in tions in the cell cycle can result in some cases in cell fate two-cell-stage embryos treated with the cleavage inhibitor transformations. cytochalasin B, P1 was found to express (after extended in- cubation) at least one marker of gut cell differentiation [36]. 4.2. Control of vulval cell fate specification These studies were complicated, however, by the ability of cytochalasin B-treated embryos to continue nuclear division During the L3 stage of postembryonic development, three cycles, leading to multinucleate cells of indeterminate de- of the six adjacent vulval precursor cells (termed P3.p–P8.p) velopmental age. A more controlled study using aphidicolin are reproducibly induced to acquire vulval cell fates [44]. This (which blocks DNA synthesis) found that intestinal markers event prompts the three most centrally located VPCs (relative were expressed only in those embryos where the replication to the inducing cell) to take on either a 1◦ (P6.p) or 2◦ (P5.p inhibitor was applied after the generation of the intestinal and P7.p) vulval cell fate; the two fates are distinguishable by progenitor cell, E [9]. Furthermore, gut cell differentiation their lineage patterns as well as by several expression mark- occurred only if the E cell had first been allowed to undergo ers (Fig. 2A) This precise pattern of cell fate specification is D.S. Fay / Seminars in Cell & Developmental Biology 16 (2005) 397–406 401

renders VPCs more susceptible to induction by Ras/Map kinase signaling [21]. (An apparent exception to these ﬁndings is that in animals that overexpress LIN-3, acquisition of the 1◦ fate can be extended to later time points [47].) In contrast, the formal acquisition of 2◦ fates by P5.p and P7.p (also via LIN-12) appears to occur only after the com- pletion of S phase, during G2/M of the ﬁrst cell cycle, or early in the subsequent cycle. Thus the timing of 1◦ and 2◦ cell fate acquisition is temporally separated as a result of distinct differences in the competence of VPCs in G1/S versus G2/M to respond to instructive cues [48]. This temporal sequenc- ing of cell fate choices was suggested to provide a means by which to coordinate the selection of different fates by cells with multiple potentials. This would in turn help to ensure the production of a functional organ by preventing more than one cell from acquiring the 1◦ fate.

5. Novel contributions

5.1. The cullins

Perhaps the most notable contribution of C. elegans re- Fig. 2. Summary of vulval induction events. (A) Normal differentiated fates search to the cell cycle field was the discovery in 1996 of the of the six VPCs after multi-step induction has taken place. The inducing anchor cell (AC) is depicted at top. The lineage patterns of P5.p, P6.p, and evolutionarily conserved family of cullins. The first cullin P7.p following induction are indicated below. (B) Model for a two-step VPC mutant to be identified, cul-1, exhibited hyperproliferation induction process that is separated by cell cycle phases (Adapted from [48]). of most or all postembryonic blast lineages, including cells ◦ ◦ Black nuclei (P6.p) indicate the 1 fate; red nuclei (P.7.p), the 2 fate. The of the germline [49]. Since then, cellular and developmen- intermediate pink nuclei (P7.p) indicate an induced vulval cell with a bias ◦ tal functions have been ascribed to an additional three of towards the 2 fate. Because of space considerations, the mirror induction of P5.p by P6.p has been omitted. For additional information, please see the the six family members in C. elegans. Biochemically, cullins text. serve as scaffolds for SCFs and ECSs (Elongin C-Cul2-SOCS box). Both complexes function as multisubunit ubiquitin ligases (E3s), whose targets include a diverse set of proteins. accomplished by two temporally separated inductive events, Based on the large number of predicted Skp1-like and F-box each involving distinct signaling pathways. Initially, an EGF- proteins in C. elegans, the potential for many distinct E3 iso- like ligand (LIN-3; secreted by a gonadal cell just dorsal to forms, each with unique sets of targets, is high [50–52].In P6.p) triggers a graded response of Ras/Map kinase signaling the case of cul-1, one of its targets is likely to include cy- in P5.p–P7.p with highest activation levels occurring in P6.p clin E, since loss of cyclin E function can partially suppress [45]. This leads to P6.p adopting a 1◦ fate, while P5.p and cul-1-associated hyperproliferation [21]. P7.p initially acquire indeterminate vulval fates. Next, P6.p Other cullin family members have been shown to func- activates Notch (LIN-12) signaling in the adjacent cells, P5.p tion in a wide spectrum of developmental processes. For ex- and P7.p, (via several Delta family ligands) leading to these ample, cul-2 is required in germ cells to promote transition cells adopting a 2◦ fate [46]. The outlying VPCs (P3.p, P4.p, through G1/S [53]. In the absence of cul-2 function, CKI- and P8.p) normally fail to receive either signal and instead 1 accumulates at high levels in germ cells, leading to a G1 assume a default epidermal fate (termed the 3◦ fate). Studies arrest. This phenotype can be partially suppressed by a re- from several laboratories indicate that cell cycle position of duction in CKI-1 levels using RNAi. This particular function the VPCs during these induction events is important for the for CUL-2 is somewhat surprising given that the human ho- determination of vulval cell identities [47,48]. Namely, ac- molog of CUL-2 associates with the VHL tumor suppressor quisition of the 1◦ fate (via LIN-3) must normally occur prior protein, implying a negative role for CUL-2 in cell growth or to onset of M phase of the first VPC cell cycle, most likely proliferation in humans [54]. cul-2 mutants are also unable during late G1 and S phases. In addition, lateral signaling (via to complete meiosis (due to a failure to degrade cyclin B1) LIN-12) during the first S-phase cycle by P6.p prevents P5.p and are slow or defective at multiple steps in mitosis, a phe- and P7.p from adopting a 1◦ fate. Thus by late S phase of the notype that probably stems from a requirement for CUL-2 first VPC cell cycle, the 1◦ fate is normally determined and in promoting chromosome condensation [53,55,56].Inad- cannot be reversed. These studies are also consistent with our dition to these functions, cul-2 is necessary for the proper own findings that an extended G1/S phase in cye-1 mutants establishment of embryonic polarity (through the localized 402 D.S. Fay / Seminars in Cell & Developmental Biology 16 (2005) 397–406 targeting of PAR-2 for destruction), and for the degradation pression. At present, the mutual regulatory targets of the Ras of germline-associated proteins in somatic cells during early pathway and the Class B SynMuv genes remain to be eluci- embryogenesis [55,57]. Similarly, cul-3 is also required for dated. multiple cellular processes including the regulation of mi- The role of lin-35 in vulval cell fate specification sug- totic spindle position following the meiosis to mitosis transi- gests a novel and non-cell-cycle-associated function for Rb tion, the control of actomysin contractility during cytokine- family members in C. elegans. In addition to this, our labo- sis, and progression through S phase [58,59]. Recently, it was ratory has uncovered several unique developmental roles for shown that a BTB-domain-containing adapter protein (MEL- lin-35 in genetic screens for novel synthetic phenotypes [27]. 26) serves a bridging function between CUL-3 and its target In one case, LIN-35 was shown to functionally collaborate substrate MEI-1/katanin [60,61]. This finding suggests that with a conserved SWI/SNF family member (XNP-1/ATR-X) BTB proteins may commonly function as substrate adapters to control the generation of several lineally-connected cell for CUL-3-E3 ligases. types in the somatic gonad [68]. In addition to this, we have CUL-4 was recently shown to be required for genome identified an unexpected role for LIN-35 during organogen- stability through the regulation of the DNA-replication li- esis of the C. elegans pharynx [69]. Specifically, LIN-35 ap- censing factor, CDT-1 [62]. cul-4 mutant animals arrest in pears to control an early step in pharyngeal morphogenesis early larval development with undivided but highly polyploid that requires a shift in the apical/basal polarities of several blast cells (up to 100C). The increased ploidy was found pharyngeal epithelial cell precursors. Several other pathways to be due to re-replication, whereby replication origins con- were also identified that act redundantly (and presumably in tinue to fire and cells remain fixed in S phase. Lowering the parallel) with LIN-35 in this process, including a conserved gene dosage of the C. elegans Cdt1 ortholog substantially E2 ubiquitin conjugating enzyme and its E3 ligase partner reduced the re-replication defects of cul-4 mutants. In addi- ([69,70] and X. Qiu and D.S. Fay unpublished observations). tion, antibodies to CDT-1 revealed that CUL-4 is required We are currently working towards the identification of the tar- for the downregulation of CDT-1 following the progression get substrate(s) of these pathways. However, our genetic and of cells through S phase. It is currently unknown if CDT- phenotypic analyses strongly indicate that these new func- 1 is a direct target for ubiquitination by the CUL-4-SCF tions for LIN-35 are unconnected to its established role in complex. cell cycle control. Another “cell cycle” gene for which a non-cell cycle role has been recently ascribed is the SCF 5.2. New dogs old tricks and old dogs new tricks complex component, LIN-23 (also see above). In addition to a hyperproliferation phenotype, strong LOF mutations in One unlikely source for new cell cycle components has lin-23 lead to defects in axon outgrowth in a subset of C. el- come from the identification of genes involved in the spec- egans neurons [71,72]. Interestingly, a partial LOF allele of ification of vulval cell fates (also see above). In the 1980s, lin-23 was identified that displays only neuronal defects, in- the Horvitz laboratory used powerful genetic screens to un- dicating that this mutation effectively uncovered two distinct cover two genetically redundant classes of vulval cell fate functions of LIN-23 [72]. determinants, collectively referred to as the SynMuv genes Several recent studies have also identified new functions [63]. Whereas single mutants in either the A or B class of for CDC-14, a protein phosphatase previously shown to be SynMuv genes failed to show pronounced defects, animals required in budding yeast for mitotic exit [73]. In one analy- containing double mutations in both an A and B class gene sis, CDC-14 was found to be required for the maintenance of displayed a striking multivulval (Muv) phenotype (reviewed G1 arrest in quiescent blast cells during larval development by [64]). This phenotype occurs when >3 VPCs adopt vul- [74]. Several lines of experimental evidence point to a model val fates and is also observed for a number of mutations that whereby CDC-14 functions upstream of CKI-1 to control augment Ras pathway signaling (also see above). Through its stability in quiescent cells. Furthermore, CDC-14 activity the cloning of class B SynMuv genes, several conserved reg- may itself be regulated through its subcellular localization. ulators of cell cycle progression were identified, including CDC-14 was observed to cycle between the cytoplasm and lin-35/Rb and efl-1/E2F [26,65]. More recently, Boxem and nucleus in interphase and mitotic cells and is similarly seg- van den Heuvel [66] tested 18 of the known class B SynMuv regated to the cytoplasm and nucleus in quiescent and post- genes for the ability to suppress division defects in cyclin D mitotic cells, respectively [74]. In a separate line of work, mutants. Interestingly, five class B genes (in addition to lin- CDC-14 was found to act in apparent opposition to Cdk1 to 35) tested positive for cell cycle roles including efl-1/E2F, control binding of the mitotic spindle by ZEN-4, a mitotic its binding partner dpl-1/DP, and three novel genes, one of kinesin-like protein [75]. Earlier studies on CDC-14 also de- which, lin-9, has clear homologs in vertebrates [67]. While tected an association with ZEN-4 at the mitotic spindle, and the precise role of lin-35/Rb and other Class B genes in vul- RNAi of cdc-14 was observed to cause defects in cytokinesis val cell fate determination is currently unknown, a working [76]. These later findings are, however, somewhat compli- model is that LIN-35 (acting in a complex with class B genes cated by issues of strain background, as complete LOF muta- such as E2F, histone deacetylase, and NURD components) tions in cdc-14 (in an otherwise wild-type background) failed acts to antagonize Ras signaling through transcriptional re- to produce the same defects [74]. D.S. Fay / Seminars in Cell & Developmental Biology 16 (2005) 397–406 403

5.3. Control of body size lon-1, a known target for repression by TGF-␤ in C. elegans, leads to an increased size and higher ploidy of hypodermal The isolation of C. elegans mutants with altered body sizes nuclei, whereas overexpression of lon-1 in the hypodermis (both larger and smaller) has provided insights into the path- results in a reduction in body size and a decrease in ploidy ways and mechanisms that regulate cell and organismal mass. [89]. One current model is that TGF-␤, secreted by neuronal Whereas it has been shown in many systems that organis- cells along the major body axis, may directly signal neighbor- mal size more often correlates with variations in cell number ing hypodermal nuclei to undergo endoreduplication [89,90]. than changes in cell size (mice and elephants have equiva- However, other mutations that lead to similar defects in body lently sized cells), this does not appear to be the case for C. size (including both larger and smaller animals) do not show elegans. A number of mutants showing “small” (Sma) and concomitant effects on the ploidy of hypodermal nuclei, or for “long” (Lon) phenotypes have been isolated and these ani- that matter, on total hypodermal DNA content [86,88]. Thus mals appear to contain equivalent numbers of somatic cells hypodermal ploidy (or DNA content) per se is not likely to when compared with wild type [77–80]. Such findings in- be a direct determinant of body size, but may reflect a mech- dicate that for organisms with invariant developmental lin- anism to maintain an optimal nuclear-to-cytoplasmic ratio in eages (and that do not retain somatic division potential into growing syncytial cells. adulthood), changes in organismal size can be attributed to Most recently, a role in the regulation of body size was changes in cell size. Curiously, although Ras activity has been identified for the C. elegans p53 homolog, cep-1 (A.K. Jol- shown to promote cell growth in other organisms including liffe and W.B Derry, personal communication). CEP-1 has Drosophila, Ras appears to have no role in the growth or previously been shown to function in the DNA-damage re- proliferation of somatic cells in C. elegans [81,82]. A recent sponse that leads to apoptosis as well as in chromosome seg- study of Ras pathway mutants in Oscheius tipulae, a related regation during meiosis [91,92]. In addition, cep-1 mutations species of nematode, suggests that Ras may, however, con- are sensitive to a number of environmental stresses including trol cell proliferation in this organism [83]. Another major reduced oxygen concentration (hypoxia; [92]). One outcome regulator of cell growth in mammals and flies, c-Myc, does of hypoxia is that cep-1 mutants (but not wild type) display a not appear to be encoded by the worm genome. significant reduction in body size. Interestingly, inactivation One unique mechanism by which C. elegans may control of the C. elegans homolog of human Krit1, a disease gene body size is through the regulation of cellular ploidy. During associated with vascular abnormalities, results in a synthetic development, a sheet of epithelial cells (termed the hypo- “small” phenotype with cep-1 mutants, even in the presence dermis) forms the outer layer of the animal. By adulthood, of normal oxygen concentrations. Together, these findings the majority of hypodermal nuclei are located within sev- suggest a novel role for CEP-1/p53 in coordinating cell size eral large multinucleate cells that form by cell fusion events and the adaptation to low oxygen. during embryonic and larval development [2,84]. The largest The precise relationship between cep-1 and other size- of these syncytial cell types, hyp-7, contains ∼133 nuclei. control pathways in C. elegans is currently unclear. In ge- Interestingly, most nuclei of hyp-7 undergo multiple rounds netic epistasis tests, the reduced size of cep-1 Krit1 double of endoreduplication (DNA synthesis without karyokinesis) mutants was found to be dominant to the hypertrophic ef- in larvae and adults, leading to an average nuclear ploidy fects normally conferred by mutations in lon-1.Aslon-1 of ∼10C by late adulthood [85,86]. In studies on body size is a known transcriptional target of TGF-␤ signaling [89], regulation in nematodes from the order Rhabditida (which this result suggests that cep-1 may act either downstream or includes C. elegans), Flemming et al. found a striking cor- in parallel to the TGF-␤ pathway. In addition, cep-1 Krit1 relation between body size and hypodermal DNA content (a double mutants show synthetic lethality with certain SMAD measure of both cell number and nuclear ploidy), such that pathway genes (e.g., sma-3), further suggesting that these species containing greater hypodermal DNA contents gen- pathways may function in parallel to carry out overlapping erally achieved larger adult sizes [86]. While this study did functions, including the regulation of body size. not establish a direct causal relationship between hypodermal DNA content and body size, it has been observed that mutations that compromise DNA replication in late-stage larvae, 6. Concluding remarks also display a reduction in body size ([21]; and D.S.F, unpublished observations). Moreover, in C. elegans, an increase in Until recently, the relative contribution of C. elegans stud- ploidy from diploid to triploid results in increased body size ies to the cell cycle field has been somewhat minimal com- [87]. pared with that of yeast or even Drosophila; however, a cur- Several groups have subsequently examined hypodermal rent survey reveals a trend of steadily increasing impact. DNA contents in characterized C. elegans mutants where Moreover, as the focus of cell cycle studies shifts from single adult body size has been altered. In a number of cases, a cells to developing organisms, the worm will undoubtedly direct correlation was observed between hypodermal ploidy occupy a place at the forefront of this emerging discipline. and body size, particularly in mutants that affect TGF-␤ path- Future challenges include connecting the actions of cell fate way signaling [86,88,89]. For example, loss of function in determinants to core components of the cell cycle machin- 404 D.S. Fay / Seminars in Cell & Developmental Biology 16 (2005) 397–406 ery, developing a better understanding of the networks linking [18] Cheeks RJ, Canman JC, Gabriel WN, Meyer N, Strome S, Gold- global regulators of development to cell cycle control, iden- stein B. C. elegans PAR proteins function by mobilizing and tifying additional conserved cell cycle components through stabilizing asymmetrically localized protein complexes. Curr Biol 2004;14:851–62. forward genetics, and continuing to uncover the diverse de- [19] Shi Y, Mello C. 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