Mutations in cye-1, a Caenorhabditis elegans cyclin E homolog, reveal coordination between cell-cycle control and vulval development

Transcription

1 Development 127, (2000) Printed in Great Britain The Company of Biologists Limited 2000 DEV Mutations in cye-1, a Caenorhabditis elegans cyclin E homolog, reveal coordination between cell-cycle control and vulval development David S. Fay and Min Han Howard Hughes Medical Institute and Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder CO , USA Authors for correspondence ( fayd@alpha.colorado.edu; mhan@colorado.edu) Accepted 28 June; published on WWW 22 August 2000 SUMMARY We have identified strong loss-of-function mutations in the C. elegans cyclin E gene, cye-1. Mutations in cye-1 lead to the underproliferation of many postembryonic blast lineages as well as defects in fertility and gut-cell endoreduplication. In addition, cye-1 is required maternally, but not zygotically for embryonic development. Our analysis of vulval development in cye-1 mutants suggests that a timing mechanism may control the onset of vulval cell terminal differentiation: once induced, these cells appear to differentiate after a set amount of time, rather than a specific number of division cycles. cye-1 mutants also show an increase in the percentage of vulval precursor cells (VPCs) that adopt vulval cell fates, indicating that cell-cycle length can play a role in the proper patterning of vulval cells. By analyzing cul-1 mutants, we further demonstrate that vulval cell terminal differentiation can be uncoupled from associated changes in vulval cell division planes. Key words: Cyclin E, Caenorhabditis elegans, Vulval development INTRODUCTION Development of the C. elegans vulva has been described as a microcosm for events that occur during metazoan organogenesis (Greenwald, 1997). This portrayal reflects the utilization of several ubiquitous processes including cell signaling, migration and fusion, to generate the 22-cell organ that serves as a passage for incoming sperm and outgoing eggs. Many studies have enhanced our understanding of the process by which a subset of hypodermal cells termed vulval precursor cells (VPCs) is specified to acquire vulval cell fates. However, considerably less is known about how these cells, once specified, carry out a set of characteristic division cycles and terminally differentiate. In wild-type animals, an induction event during the third larval stage induces three of the six VPCs to acquire vulval cell fates (reviewed by Greenwald, 1997). The induced VPCs then undergo three stereotypical division cycles to produce 22 cells. At the time of induction, VPCs (designated P3.p-P8.p) are arranged linearly along the ventral midline. The centermost VPC to acquire a vulval cell fate (P6.p) adopts a primary fate (1 ), generating eight cells, while the two flanking VPCs (P5.p and P7.p) take on secondary (2 ) fates, producing seven cells each (Sulston and Horvitz, 1977). Determination of 1 versus 2 fates is controlled by both a graded induction signal of LIN-3 (Sternberg and Horvitz, 1986; Katz et al., 1995), and by a lateral signaling mechanism which employs LIN-12 (Greenwald et al., 1983; Sternberg, 1988), a member of the Notch family of receptors (Yochem et al., 1988). Once generated, vulval cells carry out characteristic migrations and fusions, form attachments to non-vulval cell types and undergo eversion to form the mature organ (Sulston and Horvitz, 1977; Sharma-Kishore et al., 1999). A large number of mutations affecting vulval development have been isolated, and can be divided into four general categories based on their stage-specific defects (Ferguson et al., 1987; Greenwald, 1997). Mutations in the first class result in a reduction in the number of cells capable of responding to the vulval inductive signal (VPC generation mutants). The second class affects the efficiency by which VPCs are induced, leading to either an increase or decrease in the number of VPCs that acquire vulval fates (reviewed by Sternberg and Han, 1998). Mutants of the third class undergo proper VPC induction but display a range of defects in the execution of vulval cell fates, as evidenced by abnormal vulval cell lineage patterns (e.g., Fergusen et al., 1987; reviewed by Greenwald, 1997). A fourth class displays normal induction and lineage patterns, but is defective in later stages of vulval morphogenesis (e.g., Hanna- Rose and Han, 1999; Herman and Horvitz, 1999). To identify new genes from these latter two classes, we conducted a screen for mutations that confer both a protruding vulva (Pvl) and sterile phenotype. Such mutants were previously reported to display a range of vulval morphogenetic and lineage defects (Seydoux et al., 1993). We report here the identification of mutations in the conserved cell-cycle regulator, cyclin E, which results in an abnormal pattern of vulval cell divisions. Cyclin family proteins regulate cell-cycle progression in all eukaryotes through their association with cyclin-dependent kinases (CDKs) (Reviewed by Nigg, 1995). Cyclin E functions in

2 4050 D. S. Fay and M. Han multiple systems to regulate the G 1 - to S-phase transition (Ohtsubo and Roberts, 1993; Knoblich et al., 1994; Strausfeld et al., 1996). However, the role of cyclin E, as well as other cell-cycle regulators, in controlling developmental processes is less well understood. Our analysis of the cyclin E mutation has revealed an apparent timing mechanism that controls the onset of vulval cell terminal differentiation. Interestingly, this means of control may be similar to regulatory mechanisms described for differentiating mammalian oligodendrocytes (reviewed by Raff et al., 1998). Further studies with another cell-cycle regulator, cul-1, demonstrate that the execution of specific terminal division planes can be separated from other aspects of vulval cell differentiation. MATERIALS AND METHODS Strains and genetic methods Maintenance, culturing and genetic manipulations of C. elegans strains were carried out according to standard procedures (Sulston and Hodgkin, 1988), and conducted at 20 C unless otherwise noted. Strains used to map cye-1(ku256) included: MH1249 [cye- 1(ku256)/dpy-5(e61), let-381(h107), unc-13(e450)] (16/25 Unc non Let recombinants picked up the ku256 mutation); [cye-1(ku256), unc- 13(e450)/ sem-4(ku200)] (11/17 Unc non Pvl recombinants picked up the sem-4 mutation); [cye-1(ku256)/ dpy-14(e188), unc-13(e51)] (10/10 Unc non Dpy recombinants picked up the ku256 mutation). After mapping cye-1(ku256) to approximate map position 1.35 on LGI, cosmid rescue (Mello and Fire, 1995) was carried out using strain MH1328 [cye-1(ku256), unc-13(e450)/dpy-14(e188)]. GFP strains used were: GR1314 (mgis21)[lin-11::gfp+prf4] (Hobert et al., 1998), NH2466 (ayis4) [egl-17::gfp+pmh86]; dpy-20(e1282) (Burdine et al., 1998), VT765 [rnr::gfp] (Hong et al., 1998), JR672 (seam-cell GFP), SU93 [jam-1::gfp(jcis1)]. Other strains and mutations include: MH1220 [cye-1(ku256)/unc-11(e47), dpy-5(e61)], GS307 [cye-1(ar95), dpy-5(e61)/ unc-13(e51)] (Seydoux et al., 1993), let-60(n1046), lin-15(n765ts) (Ferguson and Horvitz, 1985), NJ582 [cul-1(e1756)/unc-69(e587)] (Kipreos et al., 1996), [dpy-17(e164), cul-1(e1756)/+; cye-1(ku256), unc-13(e450)/+]. To facilitate identification, a marked strain [cye-1(ku256), unc-13(e450)] was used in the construction of double mutant strains with let-60(n1046), lin- 15(n765ts), cul-1(e1756); dpy-17(e164) and GFP marker strains (NH2466, GR1314, SU93). unc-13(e450) single mutants are wildtype for both vulval development and fertility. Generation and identification of mutant alleles Ethylmethanesulfonate (EMS) mutagenesis was conducted on N2 animals (Sulston and Hodgkin, 1988) and recessive Pvl-sterile mutations were clonally isolated. Single worm PCR was carried out on strains MH1328 and GS307 using standard methods (Barstead et al., 1991). Sequencing of both strands spanning the cye-1-coding region was carried out for each allele. RNAi Double-stranded RNA (dsrna) was transcribed from PCR products spanning cye-1 exons 1-2 and exon 5 using T7 RNA polymerase. Following injection of dsrnas ( µg/µl) into gravid wild-type adults, F 1 progeny were scored for effects. RNAs from both regions produced identical phenotypes and contained no extensive homology to sequences in the database other than cye-1. Vulval lineage analysis Lineage analysis of cye-1 vulval division planes was conducted using the marked strain MH1328. The following orientation plane percentages were derived for the second division cycle (n=14): L- lineages, P5.pa and P7.pp (L=93%, N=7%); T-lineages P6.pa and P6.pp (L=17%, T=75%, N=8%); N/T-lineage, P5.pp and P7.pa (L=41%, T=40%, N=19%). Similar results were obtained using the unlinked strain MH1220. The vulval lineage of cul-1 mutants was obtained using strain NJ582 (n=10), and by analyzing cul-1 strains containing GFP markers (NH2246 and GR1314, n>50). Lineage analysis of vulval cell division timing was carried out using strains MH1220, NJ582 and N2. To maintain the health of the animals, individual worms were transferred to standard NGM-bacterial plates between viewing times ( minutes). Determination of cell-cycle rates in populations Wild-type and MH1220 [cye-1(ku256)/unc-11(e47), dpy-5(e61)] gravid adults were treated with a NaOCl solution to facilitate the release of early stage embryos (Sulston and Hodgkin, 1988). Vulval cell divisions were assayed in synchronized populations beginning approx. 30 hours after NaOCl treatment, taking time points every 2-4 hours and scoring >25 animals per time point. cye-1(ku256) homozygous animals were positively identified within populations by their characteristic germline phenotype. Approximate vulval cell division times were derived from three independent experiments. Cellcycle times were determined in individual animals by moving animals between slides and bacteria plates and examining cell-cycle progress every minutes. DAPI staining and DNA quantitation DAPI staining was carried out using standard procedures (Sultston and Hodgkin, 1988). Quantitation of DNA content was carried out using Scion Image 1.62, subtracting for background. Gut cell DNA content in wild-type (n>30) and mutant animals (n=21) was derived by comparison to body wall muscle cells. Hydroxyurea treatment Late L2 animals were placed on 40 mm HU plates containing a bacterial food source as previously described (Ambros, 1999). Animals were then transferred back to normal plates after 8-24 hours and scored for the presence of extra vulvae 24 hours later. For lineage analysis of HU-treated animals, L3-stage animals that had undergone the first round of vulval cell divisions were transferred to HU plates and monitored periodically for progress. RESULTS ku256 uncovers a mutation in the C. elegans cyclin E homolog, cye-1 EMS mutagenesis was used to generate a collection of mutations conferring a Pvl-sterile phenotype. One allele, ku256, was mapped to a small region on the right arm of LGI, and full rescue of the mutant phenotype was obtained by injection of a single cosmid in the region (C37A2) (for details see Materials and Methods). A 6,500 bp subfragment from cosmid C37A2 conferred complete rescue and was predicted to encode a single complete gene product, the C. elegans cyclin E homolog, previously named cye-1. Rescue of sterility was most robust in the F 1 and F 2 progeny of injected animals and diminished over subsequent generations. Because a progressive silencing of transgene expression occurs within the germline (Kelly et al., 1997), our observation suggests that rescue of cye-1 sterility requires germline expression. cye-1 encodes a predicted polypeptide of 524 amino acids with a cyclin-box domain spanning amino acids (Fig. 1). To confirm the molecular identity of the mutant locus, DNA encompassing the cye-1 genomic region was amplified from

3 C. elegans cyclin E 4051 * ar95(w234 Amb) C. elegans 232 KVWSLMVKRDE--IPRATRFLLGNHPDMDDEKRRILIDWMMEVCESEKLHRETFHLAVDY 100 EVWKIMLNKEK--TYLRDQHFLEQHPLLQPKMRAILLDWLMEVCEVYKLHRETFYLAQDF Drosophila 223 DVWRLMCHRDEQDSRLRSISMLEQHPGLQPRMRAILLDWLIEVCEVYKLHRETFYLAVDY Fig. 1. Alignment of the cyclin-box regions of human cyclin E from C. elegans, human and Drosophila melanogaster. Black-shaded boxes denote identity; gray, similarity. cye-1 is 46% identical to both human and Drosophila cyclin E genes in the region human of the cye-1 cyclin-box (aa ), while human and Drosophila share a 62% identity over this * ku256(w357 Amb) same region. Significant but lower levels of C. elegans 350 LIVKYIGWSLGPITSIQWLSTYLQL homology with the human and Drosophila genes human 217 MIMKALKWRLSPLTIVSWLNVYMQV can also be found in the regions C-terminal to the Drosophila 342 ILLQALDWDISPITITGWLGVYMQL cyclin-box (data not shown). Locations of the identified molecular lesions for mutants cye-1(ar95) and cye-1(ku256) are indicated by asterisks. The cye-1 genbank accession number is AF (submitted by M. Park and M. Krause). C. elegans 290 VDRYLESSNVECSTDNFQLVGTAALFIAAKYEEIYPPKCIDFAHLTDSAFTCDNIRTMEV 158 FDRYMAT-QENVVKTLLQLIGISSLFIAAKLEEIYPPKLHQFAYVTDGACSGDEILTMEL Drosophila 383 LDRYLHV-AHKVQKTHLQLIGITCLFVAAKVEEIYPPKIGEFAYVTDGACTERDILNHEK ku256 animals and sequenced. A single nucleotide substitution was found within the coding region of cye-1, leading to a premature termination signal following amino acid 356 (Fig. 1). Based on the location of the termination signal and the likely instability of the resultant mrna (Pulak and Anderson, 1993), ku256 would be predicted to encode a strong loss-offunction (lf) or null allele. This is supported by the finding that ku256 over a regional chromosomal deficiency (hdf8) results in animals that are Pvl-sterile, indicating that a more severe phenotype is not obtained if the gene dose of ku256 is halved. Genetic analysis with ku256 identified a noncomplementing mutation, evl-10(ar95), an allele previously isolated that also confers a Pvl-sterile phenotype (Seydoux et al., 1993). Sequence analysis of evl-10(ar95) identified a single nucleotide substitution in the cye-1 gene, resulting in a stop codon within the N terminus of the cyclin-box domain (Fig. 1). This allele, also predicted to encode a strong lf or null allele, produces a phenotype identical to ku256 (see text below for details). Based on the molecular identity of the mutant loci, we designate these alleles cye-1(ku256) and cye-1(ar95) (J. Hodgkin, personal communication). resulting in gonads of abnormal shapes (data not show). A striking phenotype of cye-1 mutants is the unusually large cells present in the germline gonad (compare Fig. 2C,D). Since the total number of cells in the germline of cye-1 animals is relatively small compared to wild type (Fig. 2C,D, and data not shown), this indicates that germ cells in cye-1 mutants are defective in cell division or progression through the cell cycle. cye-1 mutants display cell division defects in the vulva and somatic and germline gonads Unlike wild-type animals, which generate 22 cells during the course of vulval development, cye-1 mutants produce approx. 12 vulval cells, resulting in a small and often asymmetric L4 invagination (Fig. 2B,E; Table 1). Following eversion, vulvae from cye- 1 mutants protrude abnormally (data not shown). This phenotype may result from structural defects within the vulva itself, or could be due to improper attachments of vulval cells to the somatic gonad or neighboring muscle and hypodermal cells. cye-1 mutants also show abnormalities in both somatic and germline gonad tissues, and often lack a detectable uterus or contain a uterus that is unusually small (compare Fig. 2A,B,E; Seydoux et al., 1993). In addition, cye-1 mutants do not manufacture sperm or oocytes and consequently never produce embryos (data not shown and Seydoux et al., 1993). Migration of the gonad arms is often aberrant in cye-1 mutants, Fig. 2. Vulval and gonad phenotypes of cye-1 mutants. (A) Mid-L4-stage wildtype vulva containing 22 cells. The location of the uterine space is indicated (ut). (B) cye-1(ku256) and (E) cye-1(ar95) L4-stage vulva containing approx. 12 cells each. (C,D) Turn region of the germline gonad in a (C) wild-type and (D) cye- 1(ku256) mutant. Black arrowheads indicate the position of a single germline nucleus containing a large nucleolus. Note the presence of large nuclei in the cye- 1(ku256) germline compared to wild type. (F) cye-1(rnai) terminally arrested embryo at approx. 100-cell stage. Size bar, 10 µm.

4 4052 D. S. Fay and M. Han Table 1. cye-1(ku256) a double mutants with let-60/ras(n1046) and lin-15(n765ts) Average induction Average no. of Strain (%) b vulval cells c N2 (wild type) d cye-1(ku256) 103 (n=25) e 11.9(±1.5) (n=50) cye-1(ar95) 104 (n=25) 11.7(±1.3) (n=15) let-60(n1046) 148 (n=25) 34.0(±5.7) (n=5) f cye-1(ku256); let-60(n1046) 184 (n=25) g 21.3(±1.7) (n=15) 25 C lin-15(n765ts) 200 (n=20) 43.4(±1.5) (n=5) cye-1(ku256); lin-15(n765ts) 200 (n=15) 23.7(±2.1) (n=10) 15 C lin-15ts(n765) 101 (n=25) ND cye-1(ku256); lin-15(n765ts) 135 (n=25) ND cye-1(ku256); lin-15a(n767) 103 (n=33) ND cye-1(ku256); lin-36(n766) 102 (n=30) ND a For ease of analysis a marked strain, cye-1(ku256), unc-13(e450), was used. b Induction was calculated as (#VPC cells induced/3) 100. c Includes vuval cells present in both normal and ectopic locations. Number expected if all VPCs (6/6) are fully induced is d According to Sulston and Horvitz (1983). e Two animals were observed to have additional partial inductions, one in P4.p and the other in P8.p. f Variation in vulval cell numbers correlated precisely with the extent of VPC induction observed in individual animals, e.g., two animals with a 167% induction (5/6 VPCs) contained 37 and 38 cells while one animal with a 133% induction (4/6 VPCs) contained 30 cells. g Two animals were observed to contain an induction of P9.p. cye-1(rnai) results in embryonic lethality While both mutant alleles of cye-1 likely possess no zygotic cye-1 activity, mutant animals would contain maternal stores of cye-1 mrna. Since RNAi effectively abolishes the activities of transcripts from both maternal and zygotic sources, we tested the effect of RNAi on cye-1 (Fire et al., 1998). Injection of double-stranded RNAs from several regions of cye-1 yielded F 1 progeny that were arrested as early as the approx. 100 cellstage (Fig. 2F). The majority of embryos arrested prior to visible signs of morphogenesis, although some differentiation of tissues was observed (data not shown and E. Kipreos, personal communication). Interestingly, we observed no difference in the length of embryogenesis between cye-1 mutants and wild-type animals (data not shown). We conclude that cye-1 is required for embryogenesis but that, in the absence of zygotic cye-1, maternal stores are sufficient for normal embryonic development to occur. cye-1 mutants show a slight increase in VPC induction and act synergistically with mutations that confer a Multivulval phenotype While cye-1 mutants showed a marked reduction in the total number of vulval cells generated versus wild type, we observed a modest increase in the number of VPCs induced to adopt vulval cell fates. Whereas, in wild-type animals, only three of the six VPCs (P5.p, P6.p and P7.p) are induced to form vulval tissue, a low percentage of cye-1(ku256) and cye-1(ar95) animals (<10%) displayed additional inductions in P4.p and P8.p (Table 1). Some cye-1(ku256) animals contained up to 16 vulval cells, the result of partial inductions in both P4.p and P8.p. These ectopic vulval cells displayed similar lineage defects to those derived from P5.p-P7.p, resulting in small invaginations containing about half the number of expected cells. To further analyze the induction defect of cye-1 mutants, we generated double mutants with each of two mutations that confer a pronounced Multivulval (Muv) phenotype. VPCs in animals containing a let-60ras gain-of-function (gf) allele, adopt vulval cell fates at an increased frequency (Fig. 3A; Table 1) (Ferguson and Horvitz, 1985; reviewed by Sternberg and Han, 1998). Interestingly, double mutants with let-60ras and cye-1 showed a degree of VPC induction that was significantly above what is normally observed in let-60ras single mutants (Table 1). Both the central and ectopic invaginations produced in these double mutants showed a similar lineage defect to cye-1 single mutants. Thus the vulval lineage defect of cye-1 is epistatic in a mutant background that contains high levels of the inductive signaling activity (Fig. 3B; Table 1). Mutations in a separate class of genes known as Syn Muvs (for Synthetic Multivulva) alleviate an activity that inhibits vulval induction, causing a highly penetrant Muv phenotype (Lu and Horvitz, 1998; reviewed by Fay and Han, 2000). At 25 C, animals homozygous for a temperature-sensitive (ts) allele of lin-15(n765), adopt vulval cell fates in all six VPCs (Fig. 3C; Table 1) (Ferguson and Horvitz, 1985). Double mutants with cye-1 and lin-15(ts) grown at 25 C also adopt vulval cell fates at the same high frequency, but exhibit lineage defects characteristic of cye-1 mutants (Fig. 3D; Table 1). At the permissive temperature for lin-15(ts) (15 C), the percentage of VPCs that adopt vulval cell fates is essentially equal to wild type (Table 1). In contrast, double mutants of cye- 1 and lin-15(ts) showed a high degree of ectopic VPC induction at this temperature (Table 1), with 73% of the double mutant animals displaying a Muv phenotype. Expression of the SynMuv phenotype has been shown to require the inactivation of two distinct classes of gene products, referred to as class A and class B (Ferguson and Horvitz, 1989; reviewed by Fay and Han, 2000). In the case of the lin-15(ts) allele, the activities of two adjacent genes, lin-15a (a class A gene) and lin-15b (a class B gene), are compromised at the non-permissive temperature (25 C), while at the permissive temperature (15 C), only the activity of lin-15b is reduced (Ferguson and Horvitz, 1989). It was therefore possible that cye-1 was itself acting as a class A SynMuv gene, leading to the expression of the Muv phenotype. To test this, we constructed a double mutant strain with cye-1 and a mutation in the class B SynMuv gene, lin-36. cye-1, lin-36 double mutants were not Muv, nor were double mutants with cye-1 and lin-15a (Table 1) indicating that cye-1 functions neither as a class A or class B SynMuv gene. The ability of cye-1 to increase the percentage of induced VPCs in both let-60ras and lin-15 mutant backgrounds suggested that an alteration in cell-cycle length could affect the efficiency of VPC induction. Based on other systems, mutations in cye-1 would be predicted to lead to an increase in the length of G 1 phase, the cell-cycle period during which VPCs normally become induced (Ambros, 1999). To see if G 1 is extended in cye-1 mutants, we examined expression of a ribonucleotide reductase (RNR) GFP reporter (Hong et al., 1998) in cye-1 mutant and wild-type backgrounds. RNR expression is initiated following the cyclin D execution point

5 C. elegans cyclin E 4053 and continues through late G 1 and into S phase (reviewed by Dyson, 1998). Compatible with a G 1 delay, high levels of rnr::gfp expression were detected in 87% of dividing vulval cells in cye-1 mutants (n=46) compared with 45% in wild-type animals (n=40). Consistent with these findings, we also observed uniformly high levels of rnr::gfp expression in many other cell types of larval-stage cye-1 mutant animals, and in cye-1 RNAi-arrested embryos (data not shown). This apparent cell-cycle delay is also consistent with our observations regarding vulval cell-cycle lengths in cye-1 mutants (see below). We also sought to determine whether or not extensions in other phases of the cell cycle would lead to an increase in the Muv phenotype. Treatment of wild-type L2- to L3-stage animals with hyroxyurea (HU), a compound that inhibits DNA replication resulting in an extended S phase, led to a highly penetrent Pvl-sterile phenotype with many animals displaying vulval and germline lineage defects. However HU did not affect the induction pattern of wild-type animals (n=50) and led only to a modest increase in the percentage of Muv animals in lin-15(ts) mutants at 15 C (7%, n=45). Similarly, a double mutant with lin-15(ts) and a mutation affecting M phase (dynein light intermediate chain, J. Yoder and M. H., unpublished results) resulted in <5% (n=44) increase in Muv animals at the permissive temperature. We conclude that extensions in S and M phases are less effective in leading to increased vulval cell-fate adoption then extensions in G 1 phase. Following induction by the anchor cell, the progeny of VPCs in wild-type animals execute division cycles approximately every 2 hours, until divisions are complete (Sulston and Horvitz, 1977). However, while lineaging cye-1 mutants for division plane defects, we observed what appeared to be abnormally long cell-cycle times (data not shown). To accurately determine vulval cell-cycle rates in cye-1 mutants, we followed cell divisions in growing cye-1 mutant animals (see Materials and Methods). Out of 25 cye-1 mutant animals, 18 completed division cycles in hours, 2/25 executed divisions in hours, while 5/25 had cell-cycle lengths 5.5 hours. Thus, while somewhat variable, we find that the average vulval cell-cycle length in cye-1 mutants to be hours (4.3±0.9). In contrast wild-type animals showed consistent division times of hours (n=8). We also observed that terminal divisions in both mutant and wild-type animals coincided with the L3-L4 molt, indicating that differentiation occurs at approximately the same life-cycle stage in wild-type and cye-1 mutant animals. To further analyze this timing defect, we followed vulval cell divisions in synchronized populations of animals (see Materials and Methods). VPCs in cye-1 mutants were observed to commence divisions during the L3 stage with approximately cye-1 mutants uncouple terminal differentiation from the number of cell cycles To determine more precisely the nature of the vulval lineage defect of cye-1 mutants, we followed vulval cell division patterns in L3-stage larval mutants. The induced VPCs of wildtype animals undergo two rounds of proliferative divisions, followed by a single round of terminal divisions. While the proliferative divisions occur longitudinally along the anteroposterior axis, a subset of the terminal divisions occurs transversely along the left-right body axis (Fig. 4). In contrast, cye-1 mutants undergo only one round of proliferative divisions before executing a terminal division pattern that is analogous to wild type (Fig. 4). This pattern produces the observed 12-cell vulva, which contains radially arranged cells reminiscent of the wild-type 22-cell vulva. Our finding indicates that mechanisms that normally couple the orientation of division planes to the number of vuval cell cycles can be separated. We also examined the timing of vulval cell divisions in cye-1 mutants. Fig. 3. cye-1(ku256) double mutants with let-60(n1046) and lin-15(n765ts). Double mutants with cye-1(ku256) were analyzed to determine the induction percentage of VPCs and vulval cell numbers. For details, see Table 1. (A-D) White arrowheads identify positions of the central vulval invagination (derived from the progeny of P5.p, P6.p and P7.p); white arrows, ectopic invaginations. Anterior is to the left. (A) let-60(n1046): note the presence of a normal appearing L4-stage vulva containing 22 cells, and an ectopic anterior invagination (derived from P3.p and P4.p) containing approx. 15 cells. (B) cye-1(ku256); let-60 (n1046): note the central vulva containing 12 cells while the ectopic anterior (P3.p and P4.p.-derived) and posterior (P8.pderived) invaginations contain approx. 8 and 4 cells respectively. (C) lin-15(n765ts) at 25 C: the central invagination contains 22 cells while the ectopic posterior invagination (P8.p derived) contains approx. 8 cells. (D) cye-1(ku256); lin-15(n765ts) at 25 C: the central invagination contains approx. 12 cells while the ectopic posterior invagination (P8.p derived) contains approx. 4 cells. Size bar, 10 µm.

6 4054 D. S. Fay and M. Han A. Wild type L B. cye-1 P5.p P6.p P7.p L T N T T T T N T L L P5.p P6.p P7.p L V T/O T/O V L C. cul-1 P5.p P6.p P7.p L L V V T T T T V V L L Fig. 4. Consensus wild-type and mutant vulval cell lineages. (A) Vulval lineage pattern in wild-type animals depicting the three division cycles that generate 22 cells (Sulston and Horvitz, 1977). By convention, anterior daughters appear below and left of progenitors, posterior daughters below and right. Terminal division planes are indicated: L, longitudinal; T, transverse; N, no division. (B) Consensus vulval lineage pattern of cye-1 mutants, which generally produce approx. 12 cells over two division cycles. T/O, transverse or oblique; V, variable. For exact division plane percentages, see Materials and Methods. (C) Derived consensus vulval lineage of cul-1 mutants. Division plane orientations refer only to the third cell cycle. Dotted lines indicate one or more additional divisions. Vulvae in cul-1 mutants reportedly contain on average approx. 80 cells (Kipreos et al., 1996), however, for simplicity, only 44 cells are depicted. (A-C) The expression pattern of the egl-17::gfp reporter in wild-type and mutant backgrounds is indicated by green-filled ovals. the same timing as VPCs in wild-type animals, indicating that cye-1 mutants are not significantly delayed in the overall timing of their developmental stages. However, while >90% of wild-type animals executed two division cycles during the 4 hour time period following VPC induction, the majority of cye-1 mutants (>85%) carried out only a single division cycle during this same period. Thus, consistent with the above results, completion of vulval cell cycles requires approx. 4 hours in cye-1 mutants versus approx. 2 hours in wildtype animals. As a result, terminal vulval cell divisions are initiated in cye-1 mutants with about the same timing as those in wild-type animals, but with mutant vulval cells having completed only one round of proliferative divisions. The terminal division planes for vulval cells in cye-1 mutants suggest that these cells adopt their proper fates and are capable of terminal differentiation. To test this further, we analyzed cye-1 mutants in background strains containing integrated GFP reporters with well-characterized patterns of vulval-cell expression. lin-11::gfp (gift from G. Ruvkun and O. Hobert) serves as a stable marker for the N and T lineages of P5.p Fig. 5. Expression of vulval differentiation markers in cye-1 mutants. cye-1(ku256) was crossed into strains containing integrated GFP reporter arrays to determine the expression pattern of characterized vulval markers in the mutant background. Panel sets show Nomarski and corresponding GFP fluorescence in L3- (A,B) and L4- stage (C-E) vulvae (anterior to the left). (A,B) cye-1(ku256); lin-11::gfp. Arrows indicating positions of GFP-positive vulval cells (from left to right) P5.ppa, P5.ppp and P7.pa. In addition, several fluorescing cells of the developing uterus can be detected. (C,D) cye-1(ku256); egl-17::gfp. Arrows indicate positions of GFP-positive cells derived from P5.pp and P7.pa that have undergone transverse divisions. (E,F) cye- 1(ku256); jam-1::gfp showing the location of adherens junctions, which serve to delineate vulval cell boundaries. Size bar, 10 µm.

7 C. elegans cyclin E 4055 and P7.p during vulval development (Freyd et al., 1990; Hobert et al., 1998). Expression of lin-11::gfp in cye-1 mutants was observed only in proper cell types (Fig. 5A,B), suggesting that vulval cells in cye-1 mutants acquire and maintain their correct identities. Another vulval marker, egl-17::gfp (gift from M. Stern) shows a dynamic pattern of expression during the course of vulval development (Burdine et al., 1998). Initially expressed during the early L3 stage in P6.p, egl-17::gfp expression continues in the progeny of P6.p until shortly after the execution of terminal divisions in the third cycle, at which point expression shifts to the N and T lineages of P5.p and P7.p (Fig. 4). In cye-1 mutants, the egl-17::gfp reporter is also initially expressed in P6.p and its daughters and, like wild type, expression shifts to the inner progeny of P5.p and P7.p following the terminal division round for cye-1 (Fig. 5C,D; Fig. 4). This precise correlation in the shift of egl-17::gfp expression suggests, that despite fewer division cycles, vulval cells in cye-1 mutants can execute differentiation programs and acquire their correct terminal fates. Cells of the wild-type vulva form a series of seven concentric toroid rings that can be viewed during the L4 stage using antibodies or markers that stain epithelial cell borders (Francis and Waterston, 1991; Sharma-Kishore et al., 1999). We examined the arrangement and shape of vulval cells in cye-1 mutants using the jam-1::gfp reporter transgene (gift of J. Hardin and J. Simske), which expresses a fusion protein that localizes to adherens junctions (Mohler et al., 1998). Ring-like structures are easily detected in cye-1 mutant vulvae, although they appear somewhat more disorganized than those of wildtype animals (Fig. 5E,F; data not shown). Additionally, cye-1 mutants generally appear to be missing one or more rings, Fig. 6. cul-1(e1756) double mutants with egl-17::gfp and cye-1(ku256). The expression pattern of the egl-17::gfp reporter was assayed in cul-1 mutants. (A,B) Nomarski and corresponding GFP images in a late L3-stage cul-1 mutant vulvae. Unlabeled arrows indicate the positions of two GFP-positive cell nuclei (derived from P6.p) that were visible within this focal plane. Also indicated is the non-fluorescing gonad anchor cell (ac). (C,D) Nomarski and corresponding GFP images in an L4-stage cul-1 mutant vulva. Arrows indicate the positions of three GFP-positive nuclei derived from the N and T lineages of P5.p and P7.p. (E-H) Double mutants with cul-1 and cye- 1(ku256) were examined and found to fall into three classes: class I (E), which closely resemble cye-1 single mutants for both vulval and gonad defects; class II (F), which show an intermediate vulval phenotype but contain gonads characteristic of cye-1 single mutants; and class III (G,H), which contain vulvae that resemble cul-1 single mutants and gonads that appear intermediate. Depicted double mutants contained: (E) 12; (F) 23; and (G) >35 vulval cells. (H) Note the presence of large distal germ cells (arrowheads) and smaller proximal germ cells (arrows) in the double mutant gonad. Size bar, 10 µm.

8 4056 D. S. Fay and M. Han consistent with the observed lineage defect. Like wild type, we observed fusion of half rings to form multinucleated toroid cells during the late L4 stage. Taken together, these findings indicate that vulval cells in cye-1 mutants undergo proper lineage-specific differentiation and proceed to form structures characteristic of the mature wild-type vulva. cul-1 mutants uncouple cell division planes and terminal differentiation In contrast to cye-1, cul-1(lf) alleles show a pronounced hyperproliferation of many postembryonic blast lineages including those that generate the vulva (Kipreos et al., 1996). cul-1 encodes a member of the cullin family of genes, which function in a conserved protein degradative pathway utilizing ubiquitination to target proteins (including cyclins) to the proteosome (Kipreos et al., 1996; reviewed by Krek, 1998). The hyperproliferation phenotype presumably results from the inability of cul-1 mutants to degrade factors that promote continued cell cycling. Cell division rates were observed to be normal in cul-1 mutants, although cul-1 mutants display an overall elongation in the length of each postembryonic developmental stage (during which time extra cell divisions occur). Vulvae in cul-1 mutants were reported to contain approx. 80 cells, the result of several additional division cycles (Kipreos et al., 1996). To extend our analysis of the relationship between terminal differentiation and division cycles, we further analyzed vulval development in cul-1 mutants. Interestingly, we find that vulval cell division planes in cul-1 mutants are similar to those of wild type during the first three cell cycles (Fig. 4). Following two rounds of longitudinal divisions, grandaughters of P6.p generally divided transversely, while the outer progeny of P5.p and P7.p divided longitudinally. The inner progeny of P5.p and P7.p (N and T lineages) showed variable division patterns including transverse, longitudinal and no divisions. Following the third cycle, vulval cell division planes appeared randomized in cul-1 mutants, perhaps due in part to the steric effects of neighboring cells. Consistent with previous findings (Kipreos et al., 1996), we observed vulval cell-cycle lengths to be similar to that of wildtype animals ( hours, n=14). We also note several differences between vulval cell divisions in cul-1 mutants and wild type: (1) Whereas wild-type vulval cells begin to invaginate following the second division cycle, vulval cells in cul-1 mutants do not initiate ingression until after the third cycle, (2) vulval cell divisions in cul-1 mutants occur in a somewhat less synchronous manner than those generally observed in wild type, and (3) the morphology of vulval cell nuclei in cul-1 mutants appeared somewhat indistinct relative to wild-type animals. We also examined lin-11::gfp and egl-17::gfp expression in cul-1 mutant vulvae. Similar to wild type, lin-11::gfp expression was observed predominantly in the N and T lineages of P5.p and P7.p, although some expression was detected in the progeny of P6.p (data not shown). Expression of egl-17::gfp was observed in P6.p progeny throughout the extended proliferative phase of vulval development in cul-1 mutants (Fig. 6A,B), switching to the N and T lineages of P5.p and P7.p only after divisions had ceased (Fig. 6C,D; Fig. 4). This latter finding indicates that, while the expression of a differentiation marker is delayed in cul-1 mutants, proper vulval cell identities are maintained. Collectively, these findings demonstrate that the regulatory mechanisms connecting division plane orientations to other components of terminal differentiation are separable in cul-1 mutants. cul-1 can partially suppress the vulval and germline defects of cye-1 Because cul-1 and cye-1 display essentially opposite phenotypes, we tested whether or not cul-1 could suppress either the vulval or germline defects of cye-1 mutants. While cye-1; cul-1 double mutants were uniformly sterile and generally Pvl, a careful examination revealed three distinct classes of double mutants. Class I double mutants, the majority (approx. 70%), appeared phenotypically similar to cye-1 single mutants displaying characteristic cye-1 vulval and gonad defects (Fig. 6E). Class II double mutants (approx. 15%) showed a germline defect that was largely indistinguishable from cye-1 single mutants (data not shown) but contained an intermediate number of vulval cells (14-23) that were not attributable to ectopic VPC inductions (Fig. 6F). The final, Class III double mutants (approx. 15%) displayed vulvae that appeared similar to those of cul-1 single mutants (Fig. 6G) and contained germline gonads that were intermediate between cul- 1 and cye-1 (Fig. 6H). Specifically, large cells typical of cye-1 animals were observed in the distal portion of Class III gonads, while smaller cells were detected in the region proximal to the vulva. The existence of Class II and III double mutants indicates that cul-1 can partially suppress the cell proliferation defect of cye-1 mutants (see Discussion). cye-1 mutants show abnormalities in multiple postembryonic lineages Seam cells are specialized hypodermal cells arranged bisymmetrically along the anteroposterior axis. During postembryonic development, a set of 20 blast cells is expanded to produce 32 seam cells (as well as other cell types), which fuse during the late L4 stage to produce two large syncitial cells, each containing 16 nuclei (Sulston and Horvitz, 1977). We examined seam cell generation in cye-1 mutants using a reporter that expresses GFP in seam cell nuclei (Terns et al., 1997). Whereas wild-type adults contained an average of 15.9±0.5 (range=14-17) GFP-positive seam cell nuclei per side, cye-1 mutants averaged only 14.0±1.7 (range=10-16), indicating a partially penetrant lineage defect. (An approximate t-test indicated these values to be significantly different, P<<0.001.) Using the jam-1::gfp marker, we observed that seam cells in cye-1 mutants fuse at the correct time during the late L4 stage. In addition, cye-1 mutants generate relatively normal appearing cuticular structures, termed alae, which are produced by seam cells in adult animals. These latter findings suggest that, despite lineage defects, seam cells in cye-1 mutants differentiate with normal timing and adopt proper cell fates. Like the hermaphrodite vulva, the mating structures of the C. elegans male tail arise from hypodermal precursor cells during the L3 and L4 larval stages (Sulston and Horvitz, 1977). cye-1 mutants display a number of abnormalities in the male tail including crumpled spicules, missing rays and ray fusions (data not shown). This phenotype suggests a proliferation defect in the progenitor cells that form these structures. Many cye-1 mutants also display a mild to moderate uncoordinated

9 A. wild type C. elegans cyclin E 4057 Fig. 7. Proposed developmental clock controlling vulval cell differentiation. (A) Schematic representations of the P6.p (1 ) lineage during vulval development in wild-type, cye-1 and cul-1 mutants. Transverse and longitudinal divisions as depicted. Red cells indicate terminal differentiation; yellow cells, non-terminally differentiated. As depicted, cye-1 cellcycle rates are approximately twice as long as those for wild type (approx. 4 hours versus approx. 2 hours, respectively). (B) A timing mechanism, possibly operating within the vulval cells, is initially set in motion by VPC induction. Following a set period of time (approx. 4-5 hours at 20 C), terminal differentiation is then triggered by an unknown mechanism. cul-1 and B. cye-1(lf) cul-1(lf) wild type cye-1(lf) Dauer entry Terminal division planes Cessation of division? cul-1 CKIs? possibly CDK inhibitors (CKIs) are required to prevent further divisions. Under certain circumstances, entry into the dauer-stage can reset the timing mechanism back to zero. See text for further details. phenotype (Unc) that is manifested by abnormal kinking and coiling. Though we have not analyzed neuronal lineages in cye- 1 mutants, this phenotype suggests defects in the generation or function of neurons. Intestinal cells in wild-type animals normally undergo four cycles of endoreduplication (once during each larval stage), to produce polyploid gut cells with a 32N DNA content (Hedgecock and White, 1985). We measured the DNA content of wild-type and mutant gut cells using DAPI staining (see Materials and Methods). In wild-type animals, gut cells were readily visible with calculated DNA contents of approx. 30N, close to the expected value of 32N. In contrast, gut cells in cye-1 mutants were often difficult to detect, precluding a straightforward and unbiased comparison. However, quantitation of visible DAPI-staining gut cells in cye-1 mutants revealed a range of DNA contents from approx. 2N to 8N (avg=4.2±2.4) indicating that endoreduplication is severely compromised in cye-1 mutants. DISCUSSION Zygotic cyclin E expression is dispensable for embryonic but not postembryonic cell divisions We have isolated an allele of the Pvl-sterile mutant cye-1, which results from a strong lf mutation in the C. elegans cyclin E homolog, cye-1. cye-1 mutants exhibit defects in multiple postembryonic blast lineages, but carry out what appears to be a completely normal embryonic developmental program. Given that cye-1(rnai), a method that would deplete both maternal and zygotic cye-1 mrna results in embryonic lethality, we conclude that cye-1 is required for cell divisions during embryogenesis but that, in the absence of zygotic cye- 1 activity, maternal cye-1 mrna is sufficient. Consistent with this, zygotic expression of the C. elegans Cdk1/Cdc2 homolog, ncc-1, is required for postembryonic but not embryonic cell cycles (Boxem et al., 1999). In contrast, RNAi experiments indicate that cyclin D is not required during C. elegans embryonic divisions, perhaps reflecting the absence of a true G 1 phase during these cycles (Park and Krause, 1999). Our failure to detect embryonic arrest at very early stages (<100 cells) using RNAi methods could reflect either the lack of a requirement for cye-1 during early divisions, or more likely that such divisions can occur in the presence of maternal CYE-1 protein, which would be unaffected by RNAi treatments. Our observation that a cye-1::gfp reporter is expressed at high levels during embryogenesis, as well in all dividing cells during larval stages (data not shown), suggests that zygotic cye-1 may, however, play a role in ensuring maximal developmental success under certain conditions. The ability of maternal cye-1 to consistently drive embryonic but not postembryonic cell divisions may reflect a fundamental difference in the regulation of cyclin E during these stages. Interestingly, such differences have been observed in Drosophila where cyclin E levels show characteristic fluctuations only after completion of the first 16 embryonic cell cycles (Richardson et al., 1993). Correspondingly, loss of zygotic cyclin E activity in Drosophila results in a late embryonic arrest (Knoblich et al., 1994). We hypothesize that cye-1 may become limiting during C. elegans postembryonic development following reductions in levels of the maternal cyclin E transcript and/or protein. An extended G 1 phase may increase the likelihood of VPC induction cye-1 single mutants show a slight increase in the number of VPCs that adopt vulval cell fates compared to wild type (Table 1). cye-1 double mutants with either a let-60ras(gf) allele or a

10 4058 D. S. Fay and M. Han lin-15(lf) mutation, significantly enhanced the Muv phenotype of let-60ras and lin-15 mutants (Table 1). Because let-60ras and lin-15 function in different cell types and signaling pathways (Herman and Hedgecock, 1990), it is probable that cye-1 promotes the increased adoption of vulval cell fates through a mechanism that is not directly connected to either pathway. We suggest that this general effect is the result of an extended window of time during which VPCs can receive sufficient levels of the induction signal to trigger the adoption of vulval cell fates. VPCs are normally receptive to the signals that promote vulval cell fates during late G 1 phase of the first cell cycle (Ambros, 1999). Based on the well established role of cyclin E in G 1 - to S-phase cell-cycle progression, and our finding that an rnr-1::gfp reporter is expressed at elevated levels in cye- 1 mutants, we propose that an elongated G 1 phase results in an increased probability that VPCs will acquire vulval cell fates. This hypothesis is also supported by our findings that VPC induction is enhanced only slightly by treatments that extend S or M phases, although it remains possible that an extension in S phase could lead to the observed increase in induction. The finding that an elongation in S phase or M phase does lead to a modest increase in the percentage of Muv animals is consistent with previous findings showing that, under certain conditions, VPCs can be induced to adopt vulval fates through the end of the first cell cycle (Wang and Sternberg, 1999). The signal for vulval cell terminal differentiation depends on time and not the number of cell divisions Wild-type vulvae undergo terminal differentiation during the third round of VPC divisions. This differentiation process can be described as having three sequential components: (1) the execution of characteristic cell division planes, (2) the cessation of further cell divisions, and (3) the acquisition of mature vulval-cell traits including cell shape changes, cell movements and the expression of terminal differentiation markers. Interestingly, terminal differentiation of vulval cells in cye-1 mutants initiates during the second division cycle (Figs 4, 5, 7A). While this event is premature with respect to the normal number of cell divisions, the slower cell-cycle rate observed for vulval cells in cye-1 mutants means that terminal differentiation is initiated with roughly the same timing as in wild type, approximately 4 hours after the induction of VPCs. We also observed vulval lineage defects reminiscent of cye- 1 mutants in animals treated with HU (data not shown) indicating that disruption of the cell cycle by an alternate means can lead to a similar phenotype. Though phenotypically quite variable (we observed from 3-20 vulval cells in HUtreated animals), vulval differentiation in HU-treated animals occurred in a similar manner to that of wild type, despite defects in cell division (data not shown). Because HU also caused a variable slowing of growth and life cycle progress, it was difficult to compare the timing of differentiation in HUtreated and wild-type animals directly. However, our findings on HU-treated animals do support our previous observations that vulval cell divisions and differentiation are separable. We propose that a timing mechanism, independent of the cell cycle, controls the onset of vulval cell terminal differentiation (Fig. 7B). Following the initial induction event, vulval cells are instructed to divide with characteristic terminal division planes after a specified amount of elapsed time. This differentiation stopwatch could function intrinsically within vulval cells, possibly through the steady accumulation of a differentiationpromoting-factor. Alternatively, differentiation could be triggered by the controlled degradation (or dilution) of a differentiation-inhibitor. A variation on this model places the actual timing mechanism in surrounding tissues, which would then emit a differentiation signal. In support of an intrinsic mechanism, cell ablation experiments indicate that following induction, vulval cell divisions are unaffected by the absence of the somatic gonad including the anchor cell (Kimble, 1981). It was previously shown by Euling and Ambros (1996) that entry into an alternative larval stage known as the dauer can under certain circumstances reset the identities of dividing vulval cells back to a pluripotent state. Using a specialized genetic background these researchers found that dividing vulval cells could revert to a multipotential state ( reversal of fate ) following a forced entry into the dauer stage, provided that vulval cells had not already undergone terminal divisions. These previously committed cells could then be re-induced to form vulval tissue at a later stage through the normal inductive pathway. This finding leads to an interesting implication for our timing model. Namely, the clock controlling vulval cell differentiation can apparently be set back to zero, provided it is still ticking. However, once terminal differentiation has been initiated, there is no possibility of going back. Importantly, this result also implies that the proposed timer would necessarily be mechanistically linked somehow to factors controlling life cycle stages such as genes identified through screens for heterochronic mutants (Ambros and Horvitz, 1984). Aspects of terminal differentiation can be uncoupled An analysis of vulval development in cul-1 mutants revealed that the execution of terminal division planes can be uncoupled from both cell-cycle withdrawal and the expression of terminal differentiation markers (Figs 4, 6A-D, 7A). We therefore propose that further divisions are normally prevented by the action of CUL-1 protein, presumably by degrading cyclins that promote transit through G 1 (Fig. 7B). A failure to withdraw from the cell cycle in turn prevents normal differentiation processes. The finding that cul-1 can variably suppress cye-1 vulval and (to a lesser extent) germline defects, can be explained in several ways. Suppression could result in part from an increase in the stability of cyclin E protein (transcribed from maternal cye-1 mrna), or possibly other G 1 -phase cyclins, leading to faster cell-cycle transit times and an increased number of divisions. Consistent with this hypothesis, cullins are required for the degradation of G 1 cyclin family members (Yu et al., 1998; Singer et al., 1999; reviewed by Krek, 1998). It is also possible that cell-cycle rates are largely unaltered in the double mutants but, by preventing vulval cells from exiting the cell cycle, a greater number of divisions can occur. The poor health of the marked cye-1; cul-1 double mutant strain precluded direct measurements of cell-cycle times in these animals. However, given that cell-cycle division rates are roughly normal in cul- 1 single mutants and that life-cycle stages are extended (Kipreos et al., 1996), it is likely that this second mechanism plays a significant role in the observed suppression. The finding