Development 126, 4849-4860 (1999) 4849 Printed in Great Britain © The Company of Biologists Limited 1999 DEV5301

Regulation of postembryonic G1 cell cycle progression in Caenorhabditis elegans by a cyclin D/CDK-like complex

Morgan Park and Michael W. Krause* Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room B1-04, Bethesda, Maryland 20892-0510, USA *Author for correspondence (e-mail: [email protected])

Accepted 13 August; published on WWW 6 October 1999

SUMMARY

In many organisms, initiation and progression through the transgenic animals is sufficient to activate a S-phase G1 phase of the cell cycle requires the activity of G1-specific reporter gene. We observe no embryonic defects associated cyclins (cyclin D and cyclin E) and their associated cyclin- with depletion of either of these two gene products, dependent kinases (CDK2, CDK4, CDK6). We show here suggesting that their essential functions are restricted to that the Caenorhabditis elegans genes cyd-1 and cdk-4, postembryonic development. We propose that the cyd-1 encoding proteins similar to cyclin D and its cognate cyclin- and cdk-4 gene products are an integral part of the dependent kinases, respectively, are necessary for proper developmental control of larval cell proliferation through division of postembryonic blast cells. Animals deficient the regulation of G1 progression. for cyd-1 and/or cdk-4 activity have behavioral and developmental defects that result from the inability of the postembryonic blast cells to escape G1 cell cycle arrest. Key words: Caenorhabditis elegans, Cell cycle, Cyclin D, CDK, cyd- Moreover, ectopic expression of cyd-1 and cdk-4 in 1, cdk-4

INTRODUCTION CDK2, respectively, are responsible for initiating progression from G1 to S phase by phosphorylating cellular substrates. D- The formation of a complex multicellular organism requires type cyclins act as growth factor sensors and regulators of G1 the control of cell proliferation within the context of initiation and progression (Matsushime et al., 1992; Baldin et environmental and developmental signaling. The G1 phase of al., 1993; Sherr, 1993, 1994). Mitogens induce the synthesis of the cell cycle is a key regulatory point that controls cell cycle cyclin D and the assembly of cyclin D with its catalytic CDK decisions and previous studies in several different systems have partners to form an active complex that will promote passage defined the basic machinery involved in the initiation of cell through the restriction point (Matsushime et al., 1991, 1992, cycle entry and progression through the cell cycle (Sherr, 1993, 1994). The formation of the G1 cyclin/CDK complex is the 1994; Hunter and Pines, 1994). During G1, cells are responsive rate-limiting step in early G1 progression (Ohtsubo and to extracellular cues that control proliferation, differentiation, Roberts, 1993; Quelle et al., 1993; Resnitzky et al., 1994). quiescence, senescence and apoptosis (Sherr, 1994). The Once formed, the activity of this complex is also regulated by commitment to cell division is linked to the restriction point CDK inhibitory proteins (p21/27, INK4; Sherr and Roberts, (START) in late G1 (Pardee, 1989). In mammalian cells, 1995), by phosphorylation state (through the action of passage through this regulatory point requires the presence of CAK, CDC25, WEE1; Lew and Kornbluth, 1996), and by mitogenic signaling, but once past this point, the cells no longer degradation via ubiquitin-mediated pathways (King et al., require mitogenic signaling and they are irreversibly 1996; Hoyt, 1997). committed to enter S phase and to complete the cell cycle Although the molecular mechanisms of cell cycle control are (Pardee, 1989; Sherr, 1994, 1996). well understood and evolutionarily conserved, there currently Progression through the cell cycle is controlled by the is a poor understanding of how regulators of the cell cycle are activity of cyclin-dependent kinases (CDKs) in association integrated into the development of a multicellular organism to with a regulatory cyclin subunit (Hunt, 1991; Nigg, 1995). ensure the correct number of cells are generated at the correct Previous studies have shown that a distinct set of cyclin/ CDK place and time. Recently, organisms such as mice, Xenopus and complexes act during the G1 phase to drive cells through Drosophila have been utilized to study cell cycle regulation in START (Dulic et al., 1992; Koff et al., 1992; Xiong et al., the context of animal developmental programs. Caenorhabditis 1992). D-type and E-type cyclins, in association with their elegans is also emerging as an attractive system to address cognate cyclin-dependent kinases, CDK4 (or CDK6) and questions of developmental cell cycle control. The entire 4850 M. Park and M. W. Krause lineage and timing of cell divisions in C. elegans is known, however, we have been unable to confirm SL1 or SL2 trans-splicing both embryonically and postembryonically, providing single- to this site. The coding sequences corresponding to the cyd-1 and cdk- cell resolution to studies of cell division (Sulston and Horvitz, 4 genes have been deposited in Genbank (AF053067 and AF083878, 1977; Sulston et al., 1983). For example, there are 55 respectively). embryonically born blast cell nuclei that can divide during Gene expression constructs larval development in response to developmental cues. All of Genomic sequence corresponding to the putative promoter regions of these divisions can be monitored by direct observation and with cyd-1 and cdk-4 were cloned separately into a GFP expression vector reporter genes or antibodies specifically marking a subset of (pPD 95.67; A. Fire, G. Seydoux, J. Ahnn, and S. Q. Xu, personal these blast cell lineages. This comparatively simple and defined communication). Regions containing non-coding and coding pattern of cell divisions, coupled with the molecular, genetic sequences were amplified from genomic wild-type N2 DNA by and genomic resources, makes C. elegans an ideal system to PCR. cyd-1 was amplified using primers MP135 (5′-GAAGGCTC- study cell cycle regulation. GCATGTCGAGTTG-3′) and MP136 (5′-CGCGGATCCATAGTA- Several genes affecting the initiation and progression of the GGAACGATGCGCAC-3′). cdk-4 was amplified using primers cell cycle during postembryonic development have been MP147 (5′-CGCGGATCCCGAAGGATCTCCATTTTCTAC-3′) and ′ ′ identified in C. elegans. The heterochronic genes define a MP148 (5 -CGCGGATCCCAATTCATACATCCATTCTGTGG-3 ). developmental pathway regulating the timing of many Each reporter gene construct (cyd-1::GFP and cdk-4::GFP) contains 3.3 kb of non-coding sequence upstream of the ATG start and the first postembryonic cell cycles (Ambros and Horvitz, 1984; Euling (cdk-4) or first two (cyd-1) coding exon sequences fused in frame to and Ambros, 1996; Ambros, 1997). Precocious and retarded the green fluorescent protein (GFP). To rescue the cdk-4(gv3) heterochronic mutants accelerate or delay, respectively, the mutation, the cdk-4::GFP construct was modified by inserting the timing of postembryonic divisions. In addition, two negative cdk-4 cDNA, containing a FLAG tag, between the putative cdk-4 regulators of G1 cell cycle progression have been identified. promoter and GFP-encoding sequences. Transgenic animals The cul-1 gene, encoding one of several cullins in C. elegans, containing these reporter or rescuing constructs were generated by is required for cell cycle exit (Kipreos et al., 1996). The cullins germline transformation using a dominant roller marker (pRF4) as a are part of the ubiquitin-mediated degradation pathway and co-transformation marker (Mello and Fire, 1995). For cdk-4(gv3) rescue, the construct was introduced into the szTI balanced strain presumably act to degrade G1 cyclins like the related yeast (S. cerevisiae) gene product CDC53 (Mathias et al., 1996, KM48 (see below) and viable Rollers cloned to select for rescued homozygous cdk-4(gv3) lines. Jackson, 1996). The cki-1 gene product that is related to the CIP/KIP family of cyclin-dependent kinase inhibitors (Hong et Immunoprecipitations al., 1998) encodes a second negative regulator of G1 A HA-tagged CYD-1 expression construct was generated by PCR using progression in C. elegans. Reduction or loss of function for the cyd-1 EST yk118d3 as template and primers MP155 (5′-CGC- either cul-1 or cki-1 activity results in hyperproliferation of the TCTAGAATGCACTTTGAGTCGTCGTCGGC-3′) and MP156 (5′- postembryonic blast cells. GCGTCTAGAAGCGTAATCTGGAACATCGTATGGGTATAAAGT- In this study, we show that two C. elegans proteins similar CTTGAAGATCTTC-3′). The HA-tag is encoded in primer MP156 and to the G1-specific cyclin, cyclin D, and its cognate cyclin- is added to the carboxy-terminus of CYD-1. A FLAG-tagged CDK-4/6 construct was generated by PCR using cdk-4 EST yk492e2 as template dependent kinases, CDK4 and CDK6, are responsible for ′ initiating postembryonic cell division. By analogy to other and primers MP161 (5 -CGCGGATCCACCATGTGCGAGAATCT- TTATGGAGAGGAGTAC-3′) and MP158 (5′-GCGTCTAGACTTGT- systems, these two factors may function in a complex and we CATCGTCGTCCTTGTAGTCCTTGTTGAAGTTGATTTGC-3′). The show by co-immunoprecipitation that they can interact when FLAG tag is encoded in primer MP158 and is added to the carboxy- co-expressed in insect cells. Depletion of these factors in a cdk- terminus of CDK-4/6. These tagged constructs were cloned into the 4 deletion mutant or by RNA-mediated interference (RNAi) for Fastbac expression vector in the BAC-to-BAC system (Gibco-BRL). cyd-1 or cdk-4, leads to phenotypic defects that are restricted Sf9 cells were infected with Baculovirus extracts and cells harvested to postembryonic development. Our results suggest that cyd-1 48 hours postinfection. Immunoprecipitation used anti-HA antibody and cdk-4 are critical in C. elegans for the developmental (Santa Cruz Biotechnology) and anti-FLAG (Sigma) antibodies. control of G1 cell cycle progression in postembryonic blast Immunoprecipitates were separated by SDS-PAGE using a 4-20% cells. gradient gel (Novex) and analyzed by Western blot using the ABC detection system (Vector labs). Strains MATERIALS AND METHODS The following strains were used in these studies. N2, cyd-1::GFP reporters KM25 and KM26, ckd-4::GFP reporters KM33 and KM34, Cloning of cyd-1 and cdk-4 heat shock cyd-1-expressing strains KM38 and KM39, heat shock A cyd-1 cDNA clone was isolated and mapped to the C. elegans cdk-4-expressing strains KM40 and KM41, rnr::GFP S-phase physical map by YAC grid analysis; the cDNA maps to two reporter VT765, heat shock cyd-1 and cdk-4 expression in the overlapping YACs Y38F1 and Y43F9 on the right arm of chromosome rnr::GFP reporter background KM 42 and KM 43, elt-2::GFP II. We identified two additional cyd-1 clones in the C. elegans EST intestinal reporter JM63, hlh-8::GFP M reporter PD4667 dpy- database (yk118d3 and CEESH01). Our cyd-1 cDNA and the two cyd- 20(e1282); ayIs7, lin-29::GFP reporter RG174, vit::GFP reporter 1 ESTs did not contain the initiator methionine. RT-PCR was used to BL3219, and NJ582 cul-1(e1756). determine the 5′ end of the cyd-1 transcript and to determine that the The methodology for the PCR-based deletion screen is described transcript is trans-spliced to SL1 (Krause and Hirsh, 1987). The cdk- in Dernburg et al. (1998). A mutant library (kindly provided by Hilary 4 clones were identified as EST clones in the Kohara C. elegans EST Ellis and Andy Golden) was screened with nested cdk-4 primers in database (yk76f3, yk456a2, yk492e2) and were kindly provided by two sequential rounds of PCR. First round external primers were MP Yuji Kohara. The genomic sequence for cdk-4 reveals a potential 162 (5′-CTGCGAGTTCTGGCAAATTGCCAAC-3′) and MP163 (5′- trans-splice acceptor sequence upstream of the putative ATG, GAACACGAGATTTTGTTGCCACCGC-3′); second round internal C. elegans G1 cyclin/CDK 4851 primers MP 164 (5′-CCAAAACAATGCCGCATATTCCGAC-3′) and A. MP165 (5′-GGCTTCAAGAACGGATGGCTCATTGC-3′). A single CYD-1 FTGVQENITPFHREQAIDWIYDVAKEENCDGDVFLLAVSLIDRFMSVQ.N cyclin D1 F--VQ-++-P--R-----W+-+V--E+-C+++VF-LA+--+DRF+S++.- mutant allele, cdk-4(gv-3), was identified and cloned by sib-selection. cyclin D2 F--VQ-+IQP+-R-----W+-+V--E+-C+++VF-LA+--+DRF+---.- The cdk-4(gv-3) mutation was subsequently backcrossed to N2 three cyclin D3 F--VQ-+I-P--R-----W+-+V--E+-C++DVF-LA+--+DR++S--.- CYA-1 --++Q--+----R---IDW+-DV-KE-N-+-+-F-LAVSL+DR-+S+-.N times and balanced using szT1(I;X) (Edgley et al., 1995) to generate CYB-1 -----++-TP--R---+DW+V-V----+---+-+-L-V-++DR++---.- strain KM48. cyclin E F---+--+-P--R---+DW+-+V------+-F-LA--++DRFM--Q-N ---V------C---V------Staining and visualization of wild-type and (RNAi) progeny CYD-1 ILKHDIQMIAGVALFIASKLKAPHPMTASKIAYYSDNSCPIDMILQWELL For DAPI and antibody staining, animals were placed in a drop of cyclin D1 +-K--+Q++++--+F+ASK+K---P+TA-K+--Y-D-S---+-+LQ-ELL cyclin D2 --K--+Q++++V-+F+ASKLK---P+TA-K+--Y-DNS---+-+L+WEL+ water on poly-lysine-treated slides, covered with a coverslip and then cyclin D3 --K-++Q+++-V-L++ASKL+---P+T--K+--Y-D---+---+-+WE+L frozen on dry ice. After removing the coverslips, the animals were CYA-1 I-K--+Q+++---++IA-K-----P-----+A---DN-+-+--IL--E-+ CYB-1 --K-D+Q+++--A-F-ASK+------+--+N-+----IL--E-- fixed in methanol and then acetone for 5 minutes each at −20¡C, and cyclin E ++K--+Q+I+---LFIA-KL----P-----+-+--D--C--D-I---EL+ then allowed to air dry at room temperature. DNA was stained using ------T--K---Y------µ DAPI (Sigma) at 1 g/ml in PBS. Antibody staining was done in TBS CYD-1 IVTTLQWETESPTAFSFFNFL.....ASRIPQIHN....TRGDFQTV with 0.1% Triton X-100 (Krause et al., 1997) using rabbit polyclonal cyclin D1 +V--L-W+----T+--F++-+.....-S++P+--+....-+--+--- cyclin D2 ++--L-W+----T+--F++-+.....--++PQ-++-----R---QT- antisera against LIN-26 (1:500) and rhodamine-conjugated donkey cyclin D3 ++--L-W+-----A--F+-++.....--R+-...... --+D-Q-+ anti-rabbit antibody (1:500, Jackson Laboratories). For DIC CYA-1 ++--+++----PT---F---+.....A-R+-----....-R---+-+ CYB-1 I+--L-++--+P--+-F+--L.....---+-+---....---D-Q-- microscopy and visualization of GFP in transgenic reporter lines, cyclin E I+--L-W-----T--S+FNV+------+-Q+--....-----+-- animals were anesthetized using 0.1-1 mM levamisol and mounted on 2% agarose pads. Images were captured using Ektachrome 1600 film or a SenSys CCD camera (Scanalytics, Inc) and IP Labs software and B. % compiled using Adobe Photshop software. (identity/similarity) human cyclin D1 31/59 Assay for S phase and DNA content human cyclin D2 43/59 rat cyclin D3 30/54 To assay inhibition of G1 progression to S phase, animals containing C.elegans CYA-1 28/50 a S-phase reporter rnr::GFP construct (VT765) were treated by cyd- C.elegans CYB-1 21/43 1 (RNAi) or cdk-4(RNAi) as described above. To assay induction of S X.laevis cyclin E 28/44 phase, animals harboring the rnr::GFP reporter alone, or in Fig. 1. Sequence comparisons of cyclins. (A) Comparison of CYD-1 combination with heat-inducible cyd-1 and cdk-4 expression to other cyclins. The CYD-1 cyclin box is compared to the cyclin constructs, were starved by hatching out embryos in sterile water or boxes from human cyclin D1 (Xiong et al., 1991), human cyclin D2 on 2% agarose NGM plates with no OP50 bacteria as a food source. (Palmero et al., 1993), rat cyclin D3 (Hosokawa et al., 1994), C. Starved L1 animals that did, or did not, contain a heat-inducible cyd- elegans cyclin A (CYA-1; Kreutzer et al., 1995), C. elegans cyclin B 1 (hsp16-41::cyd-1) and/or cdk-4(hsp16-41::cdk-4) construct(s) were (CYB-1; Kreutzer et al., 1995) and Xenopus cyclin E (Rempel et al., then heat shocked for 3 hours at 33¡C and allowed to recover for 3 1995). In the alignment, identical residues are shown and similar hours at room temperature and assayed for the expression of the GFP residues are noted by +. Gaps introduced into the sequence alignment S-phase reporter. are shown as (.). Residues that are unique to CYD-1 and cyclin D1, Nuclei from cyd-1 (RNAi) and wild-type N2 animals were analyzed D2 and D3 are noted below the alignment. CYD-1 is more similar to for DNA content using DAPI to stain DNA. Images from DAPI- the cyclin Ds and appears to be most similar to cyclin D2. In stained animals were captured with a CCD camera and then analyzed addition, CYD-1 and cyclin D1, D2 and D3 share common residues using NIH image 1.6.2 software. The fluorescence of DAPI was used within the cyclin box that are not found in other cyclins. (B) The per as a measure of the amount of DNA in each nucleus and was measured cent of identical and similar residues in the cyclin box of CYD-1 in × as optical density units (area of nucleus mean optical density). The the comparison with other cyclins optical density measurements were converted to DNA content by comparison with measurements of body wall muscle nuclei with a known DNA content of 2C (Hedgecock and White, 1985). In our studies of cyd-1, we were also interested in identifying any potential partner proteins. In analyzing available genomic RESULTS information from the C. elegans Genome Consortium, a potential open reading frame on chromosome X encoding a cyd-1 and cdk-4 encode C. elegans homologs of CDK homolog was identified (H06A10.1). In addition, three cyclin D and CDK4/6 ESTs were identified that correspond to this potential CDK We have identified a C. elegans cDNA that encodes a product coding region in the Kohara EST database. We sequenced the that is similar to cyclin D from other organisms (Fig. 1); we available ESTs and compared the predicted coding region with have designated the cyclin D-like gene cyd-1 and the protein other CDKs. The product encoded by the ESTs was most product CYD-1. A comparison of the cyclin boxes, a conserved similar to CDK4 and CDK6 (as originally reported by Boxem 150 amino acid stretch found in all cyclins, showed that CYD- et al., 1999) when we compared a region of the predicted 1 is more similar to cyclin D than any other cyclin (Fig. 1). protein corresponding to the putative serine/threonine kinase Southern blot analysis of wild-type N2 DNA from mixed domain. We have termed this C. elegans protein CDK4/6 and staged animals using the cyd-1 cDNA as a probe suggests that named the gene encoding it cdk-4. this is a single copy gene (data not shown) and no other coding regions more similar to cyclin D have been identified in the C. cyd-1 and cdk-4 have similar expression patterns elegans genome sequence database (>95% complete). We To compare the spatial and temporal transcription pattern of determined the physical map position of this clone in the C. the genes encoding cyd-1 and cdk-4, we generated reporter elegans genome by YAC grid analysis on the right arm of gene constructs (see Methods) containing genomic sequence chromosome II (data not shown). corresponding to each of the genes fused in frame to GFP. The 4852 M. Park and M. W. Krause

Fig. 2. Embryonic expression of cyd-1::GFP and cdk-4::GFP Fig. 3. Postembryonic expression of cyd-1::GFP and cdk-4::GFP constructs. Two images are shown for each embryo. One image of constructs. Paired images are again shown as described in Fig. 2. the pair is false-colored GFP expression merged with Nomarski (top Similar expression patterns are seen for both cyd-1::GFP (A-C) and of each pair) and the second image is the corresponding GFP cdk-4::GFP (E-G) reporter genes in most of the postembryonic blast fluorescence alone (bottom of each pair). Expression of cyd-1::GFP cell lineages. Shown is expression in a row of P cell descendents (A-C) and cdk-4::GFP (E-G) reporter genes is very similar, each constituting part of the ventral nerve cord in L1 animals (A,E), the L4 beginning at about the 300-cell stage and continuing throughout the lateral hypodermal (seam) blast cells (arrows; B,F) and dividing cells remainder of embryogenesis. Expression of both reporters in comma of the forming vulva in L4 animals (arrows; C,G). (B,E) and 1.5-fold (C,F) embryos is concentrated primarily in postproliferative neurons in the head, ventral cord and tail as well as in hypodermal cells. Most of the embryonic expression ceases in late stage embryos and becomes restricted to seam cells in 3-fold embryos (not shown).

expression patterns of the reporter constructs for the two genes were very similar and high level expression of each was occasionally associated with embryonic or larval lethality. Both reporter genes were initially expressed during mid- embryogenesis, primarily in postproliferative hypodermal cells and neurons in the head, ventral cord and tail (Fig. 2). This Fig. 4. Co-immunoprecipitation of CYD-1 and CDK-4. Extracts expression fades during morphogenesis so that when the larvae from Sf9 cells infected with Baculovirus expressing CDK-4-FLAG alone (lanes 1, 2), or in combination with CYD-1-HA (lanes 3-6), hatch expression is seen primarily in lateral hypodermal cells were immunoprecipitated with either an anti-FLAG or anti-HA known as seam cells. antibody. The immunoprecipitated proteins were analyzed by The postembryonic expression patterns of the cyd-1::GFP western blot using anti-FLAG or anti-HA antibodies as probes. The and ckd-4::GFP reporter genes were spatially and temporally CDK-4-FLAG protein was co-precipitated with the anti-HA antibody co-incident in many of the blast cell lineages (Fig. 3). Common only when co-expressed with CYD-1-HA (lane 3) indicating that sites of expression included the P cells and their descendents, CYD-1 and CDK-4 proteins can specifically interact. The converse lateral seam cells, and somatic gonad and vulva. Expression in immunoprecipitation is shown in lanes 4 and 5 demonstrating that the P lineage, whose descendents give rise to numerous ventral immunoprecipitation of CDK-4 with the anti-FLAG antibody pulls cord neurons, often disappeared once proliferation of the down CYD-1-HA. lineage was complete whereas expression in the seam cells and somatic gonad often persisted. Seam cell expression of cdk- 4::GFP was more intense and consistent than that observed spermathecal cells and their precursors whereas cdk-4::GFP with cyd-1::GFP and for both reporters this expression was was biased towards the uterine cells with minor spemathecal seen throughout larval development. cell expression. The cdk-4::GFP reporter gene was also Although there was substantial overlap in expression of the expressed at high levels throughout the intestine. In contrast, two reporters, some differences were also observed. The the cyd-1::GFP reporter only showed sporadic expression in somatic gonad expression of cyd-1::GFP was restricted to the intestinal cells reminiscent of ectopic expression sites C. elegans G1 cyclin/CDK 4853

Fig. 5. Defects in cyd-1(RNAi), cdk-4 (RNAi) and cdk-4(gv3) deletion mutant animals. (A) Nomarski and DAPI images of wild-type and mutant animals. A wild-type L2 larvae (a) is compared to an arrested cyd-1 (RNAi) animal (b) and an arrested cdk-4 (RNAi) animal (c). The cyd-1 (RNAi) animal (b) is arrested at L2 size and is uncoordinated and slightly dumpy. The cdk-4 (RNAi) animal (c) progressed to a later larval stage (L4) but was uncoordinated and had a protruding vulva defect that is indicative of problems in vulva precursor cell proliferation. (d) A schematic of the cdk-4 gene structure is shown. A black line above the gene indicates the 745 bp region that is deleted in the cdk-4(gv3) allele; this region encodes the putative ATP- binding domain and catalytic residues of serine/threonine kinase. (e-h) Nuclei stained by DAPI in a wild- type L2 animal (e) and similarly staged mutant animals (g-h). In the wild-type animal (e), the ventral nerve cord is visible as a row of small nuclei along the ventral side of the animal (bracketed by arrowheads). The ordered row of tightly spaced ventral nuclei is not seen in arrested cyd-1 (RNAi) animals (f) due to a failure in P blast cell divisions. An intermediate phenotype is seen in an arrested cdk-4 (RNAi) animal (g) that displays variable defects in the proliferation of the P blast cells; few DAPI-staining nuclei are seen on the ventral side of the animal posterior to the gonad whereas some P cell descendents are seen anterior to the gonad (bracketed by arrowheads). A cdk-4(gv3) animal showing a strong L2 arrest phenotype is shown in h; note the lack of P cell descendents as seen with cyd-1(RNAi) in f. The gonad region overlies the asterisk in all DAPI-stained images. In all panels, the anterior side of the animal is to the left and dorsal to the top. (B) Analysis of cyd-1 (RNAi) and cdk-4(RNAi) mutant animals using reporter gene constructs. Two images are shown for each animal as described in Fig. 2. (a-c) Divisions of the blast cell M are visualized using hlh-8::GFP (Harfe et al., 1998). Wild-type (a) and cdk-4(RNAi) mutant animals (c) have multiple M cell descendents whereas the M cell fails to divide in cyd-1(RNAi) mutant animals (b). (d-f) Divisions of the intestinal cell nuclei (without cytokenesis), which normally occur during the L1 stage, are visualized using an elt-2::GFP reporter (Fukushige et al., 1998). Sister nuclei from the division of a single progenitor intestinal nucleus are indicated by paired arrowheads as seen in the wild-type animal (d). The intestinal nuclei fail to divide in cyd-1(RNAi) mutant animals (e; single arrowheads) and only rarely divide in cdk-4(RNAi) mutant animals (f; double arrowhead and single arrowheads). commonly seen among many different reporter genes (Krause immunoprecipitation with an anti-HA antibody. Western blot et al., 1994). analysis of immunoprecipitation products using anti-FLAG antibodies to detect CDK-4/6 show specific co-precipitation of The cyd-1 and cdk-4 gene products interact CDK-4/6 with cyclin D demonstrating that CYD-1 and CDK- In other systems, cyclin D and CDK4 (or CDK6) interact and 4/6 can interact when co-expressed in insect cells. Conversely, form an active complex that phosphorylates cellular substrates immunoprecipitation with anti-FLAG antibody followed by to initiate G1 progression (Matsushime et al., 1994; Meyerson probing a Western blot with an anti-HA antibody showed and Harlow, 1994). The co-incident expression of cyd-1::GFP CDK-4/6 can pull down CYD-1 when co-expressed in insect and cdk-4::GFP raised the possibility that these two proteins cells (Fig. 4). may interact during development in C. elegans. To determine if CYD-1 interacts with CDK-4/6, we used a Baculovirus Depletion of cyd-1 activity affects the divisions of system incorporating tagged versions of each of the proteins postembryonic blast cells with co-precipitation assays (Fig. 4). Co-expression of HA- To determine the function of cyd-1 during development, we tagged CYD-1 and FLAG-tagged CDK-4/6 was followed by utilized the reverse genetic technique of RNA-mediated 4854 M. Park and M. W. Krause interference (RNAi) to generate a gene-specific loss of function proliferating mitotically during L1 development. Continued (Fire et al., 1998). Double-stranded RNA (dsRNA) that was proliferation of the germ cells is dependent on signals derived transcribed in vitro from the cyd-1 cDNA was injected into the from the distal tip cells (DTCs) of the somatic gonad, which syncitial gonad of N2 hermaphrodites and the progeny from are derived from divisions of the Z1 and Z4 blast cells. Laser these injected worms were analyzed for defects in ablation of the DTCs destroys this requisite signaling and the development. germ cell nuclei enter meiosis resulting in the formation of an We did not observe any obvious embryonic defects in the abnormally small number of germ cells (Kimble and Hirsh, cyd-1 (RNAi) progeny; the animals hatched and gave rise to 1979, Kimble and White, 1981). The arrested cyd-1 (RNAi) normal looking L1 larvae. After the L1 stage, the cyd-1(RNAi) progeny initiate germ cell nuclei proliferation but cannot progeny became increasingly uncoordinated in their maintain mitotic divisions and the gonad of arrested animals movements and ultimately stopped moving, arrested growth fails to elongate, arresting as a ball of cells. Using an antibody and died. Importantly, we observed the same defects using against LIN-26 (Labouesse et al., 1996), which marks the dsRNA corresponding to a portion of the cyd-1 cDNA that did somatic gonad blast cells (Z1 and Z4) and some of their not contain the cyclin box, suggesting that the defects are not descendants, we observed that Z1 and Z4 did not divide in the associated with other cyclin-box-containing proteins and are cyd-1 (RNAi)-arrested animals and consequently the DTCs that specific to this cyclin D-like gene. We believe that the cyd- are normally derived from Z1 and Z4 were not formed (data 1(RNAi) progeny are arrested at the L2 stage based on the not shown). The gonad defect in cyd-1(RNAi) animals thus following observations: (1) the progeny underwent the first appears to be due largely, if not entirely, to a somatic gonad larval molt and arrested at a L2 size, (2) the gonad initiated the proliferation defect. The initiation of germline proliferation in germline proliferation normally observed in L2 animals, and cyd-1(RNAi) progeny appeared normal suggesting that the (3) a reporter construct that initiates expression at L2 early divisions of the germ cells might be controlled by (lin29B::lacZ; Bettinger et al., 1996) was still expressed in the mechanisms distinct from those regulating the soma. cyd-1 (RNAi)-arrested progeny, whereas a reporter expressed at late L4 (vit::GFP; MacMorris and Blumenthal, personal Depletion or loss of cdk-4 activity results in communication) was not expressed (data not shown). phenotypes similar to cyd-1 (RNAi) We determined that the observed defects in cyd-1 (RNAi) To determine the function of cdk-4, we performed RNAi and animals were due to problems in cell proliferation. Several analyzed the progeny in a similar manner as in our studies with somatic nuclear divisions normally occur during L1 cyd-1 (RNAi). The defects that we observed in cdk-4 (RNAi) development and could be assayed in the L2-arrested cyd- were not as penetrant as those seen in the cyd-1 (RNAi) 1(RNAi) animals. These include the P blast cells that give rise experiments but they were qualitatively the same and restricted to ventral cord neurons, the postembryonic mesoblast M, and to the cells that proliferate postembryonically (Fig. 5). the somatic gonad precursors Z1 and Z4. In addition, the Although some cdk-4 (RNAi) progeny were phenotypically intestinal cell nuclei undergo a single nuclear division without indistinguishable from cyd-1 (RNAi) progeny, the majority of cytokenesis during L1. We directly analyzed each of these sets the cdk-4 (RNAi) progeny grew up to later larval stages and had of postembryonic divisions by several approaches including variable defects related to problems in cell proliferation. One direct observation, staining with the DNA-binding dye DAPI, common phenotype that we observed was a protruding vulva, antibody staining and reporter gene expression. All of these L1 indicative of defects in the proliferation/differentiation of the nuclear divisions failed to occur in cyd-1(RNAi) progeny. Pn.p blast cells, which give rise to the vulva (Fig. 5A). DAPI The most easily observed division defects in cyd-1(RNAi) staining and reporter gene assays revealed that the proliferation progeny are seen in the 12 P cells that migrate to the ventral of the postembryonic blast cells varied, but animals that looked side of the animal and proliferate during L1. These cells and like cyd-1 (RNAi) phenotypically had the same defects in cell their descendents are easily identified by Nomarski optics or division of the P cells and somatic gonad precursors (Fig. 5A). DAPI staining as a row of small nuclei along the ventral side There was some tissue preference for division defects of the animal. This ventral region contained many fewer nuclei following cdk-4(RNAi) that could reflect tissue-specific in the arrested cyd-1 (RNAi) progeny as compared to wild-type patterns of cdk-4 expression, the effectiveness of RNAi or both. (Fig. 5A) and the number and position of these nuclei was For example, proliferation of the mesoblast cell M was seldom consistent with P cell migration but no P cell divisions. The affected in cdk-4(RNAi) progeny whereas the intestinal nuclear lack of P cell divisions in cyd-1(RNAi) progeny was confirmed divisions and cell divisions of P cells and Z1 and Z4 were by direct observation, staining with anti-LIN-26 antibody consistently and effectively inhibited (Fig. 5B). (Labouesse et al., 1996) and the absence of expression of a To address the possibility that our results with cdk-4 reporter that is expressed in the motorneurons that are derived (RNAi) were due to inefficient reduction of CDK4/6 function from the division of the P blast cells (unc-4::GFP, Miller and by the RNAi technique, we screened for a deletion in cdk-4 Niemeyer, 1995; data not shown). We have used reporter gene using a PCR-based technique developed by Barstead and expression to assay directly the divisions of the mesoblast cell Moulder (see Materials and Methods). We isolated a cdk-4 M (hlh-8:: GFP, Harfe et al., 1998) and the intestinal nuclei mutant allele (gv3) that contains a deletion that removes the (elt-2::GFP, Fukushige et al., 1998). These two sets of putative ATP-binding domain and serine/threonine kinase divisions are completely blocked following cyd-1(RNAi) (Fig. catalytic residues from the protein (Fig. 5A). It is likely that 5B). the cdk-4(gv3) mutant is a null for CDK4/6 activity because The arrested cyd-1(RNAi) progeny had an additional defect the deleted regions are essential for kinase function in in the germline. The hermaphrodite germline is derived from mammalian CDKs (Hanks et al., 1988; Meyerson et al., two embryonically born precursor cells, Z2 and Z3, that begin 1992). We have been able to rescue and maintain a C. elegans G1 cyclin/CDK 4855 homozygous cdk-4(gv3) strain by transformation rescue with to express the S-phase marker by overexpression of either gene the wild-type cDNA expressed under the control of cdk-4 alone or in combination. The S-phase marker that we used was upstream sequences used in our reporter gene studies. This a GFP reporter gene under the control of the ribonucleotide demonstrates that the phenotypes that we observe are due to reductase promoter (rnr::GFP) that is expressed in most loss of cdk-4 activity and that our cdk-4::GFP reporter gene proliferating blast cells during postembryonic development expression pattern includes at least the minimum set of (Hong et al., 1998). Previous work has shown that the tissues requiring cdk-4 activity for viability and normal postembryonic blast cells require the animal to eat food in development. order to initiate postembryonic cell divisions (Wood, 1988; Homozygous cdk-4(gv3) deletion mutants animals were Hong et al., 1998). L1 animals that have hatched out in the phenotypically identical to the cdk-4(RNAi) animals although absence of food are developmentally arrested and do not the penetrance of cell division defects was much higher in the express rnr::GFP in the postembryonic blast cells, presumably mutant. Most homozygous animals arrested as L2 larvae with because these cells in hatching embryos are arrested at a stage a phenotype indistinguishable from cyd-1 (RNAi) animals earlier than S phase, such as G0 or G1 (Hong et al., 1998). (Fig. 5A) whereas others animals displayed a variable There is sporadic ‘background’ expression of rnr::GFP in a phenotype reflecting the extent to which postembryonic blast few non-proliferative cells in L1 animals even in the absence cell divisions had occurred in the particular animal. The of food. We introduced heat-shock-inducible constructs of cyd- fraction of progeny from heterozygous cdk-4(gv3) animals 1 (hsp16-41::cyd-1) and cdk-4 (hsp16-41::cdk-4) either alone that arrest at L2 due to a lack of most, if not all, blast cell or together into worms carrying the rnr::GFP reporter. divisions was 21% (n=682). This is less than the expected Embryos carrying the expression construct(s) were allowed to frequency (25%) if all homozygous progeny were equally hatch in the absence of food and then the starved L1 animals affected, consistent with the observation that some mutant were heat shocked to stimulate the expression of cyd-1 and/or animals progress to later larval stages. We did observe a small cdk-4. The rnr::GFP reporter expression was detected in fraction of sickly L3 animals that presumably escaped L2 starved, heat-shocked L1 only when both cyd-1 and cdk-4 arrest due to limited proliferation of some postembryonic blast expression constructs were present (Fig. 6A); heat-shocked cells. We never observed animals with a protruding vulva that animals without both hsp16-41::cyd-1 and hsp16-41::cdk-4 would indicate problems associated with vulval cell did not express the reporter above background (Fig. 6A). This proliferation suggesting that few, if any, of the animals that result indicates that the postembryonic blast cells can be driven escape L2 arrest reach the L4 stage. The ability of some to express a S-phase reporter gene by the combined activity of homozygous cdk-4(gv3) animals to escape L2 arrest may be cyd-1 and cdk-4. due to the function of one or more factors that can partially To determine if cyd-1 or cdk-4 activity was necessary for compensate for the loss of CDK4/6. Nevertheless, cdk-4(gv3) postembryonic blast cells to express the S-phase reporter gene, were qualitatively similar to cyd-1(RNAi) and suggested that we assayed rnr::GFP gene expression following cyd-1 (RNAi) in vivo cdk-4 is acting in concert with cyd-1 to regulate or cdk-4(RNAi). The cyd-1(RNAi)-arrested progeny did not postembryonic cell cycles. express the rnr::GFP reporter, suggesting that the blast cells did not enter S phase in the absence of cyd-1 activity (Fig. 6B). cyd-1 (RNAi) can suppress the hyperproliferative As expected, cdk-4(RNAi) blocked expression of the reporter defects associated with a mutation in the CDC53 gene in only a fraction of the blast cells, consistent with the homolog cul-1 variable penetrance of cell division defects seen in cdk-4(RNAi) The C. elegans gene cul-1 encodes a cullin that is a mutant animals (Fig. 6B). component of the ubiquitin-mediated degradation pathway To confirm that the postembryonic blast cells are arrested in (Kipreos et al., 1996). Mutants in the yeast homolog of cul- the G1 phase of the cell cycle in cyd-1 (RNAi) mutant animals, 1, CDC53, fail to degrade G1 cyclins (Mathias et al., 1996; we measured the DNA content of the P blast cell nuclei Jackson, 1996). A mutation in the C. elegans gene cul-1 compared with embryonically born bodywall muscle nuclei causes the postembryonic blast cells to hyperproliferate, (2C) and seam cell nuclei (4C; Hedgecock and White, 1985). presumably due to the inability of cells to exit the cell cycle The undivided P blast cells in cyd-1 (RNAi) animals had a 2C (Kipreos et al., 1996). We performed cyd-1 (RNAi) in a C. DNA content (Fig. 7A), which is the expected result if these elegans cul-1(e1756) mutant and observed that the progeny cells did not enter S phase. This result, together with the arrested with the cyd-1 (RNAi) phenotype, suggesting that the inhibition of rnr::GFP expression, strongly suggests that the hyperproliferation of blast cells in cul-1 mutants requires undivided P cells in cyd-1(RNAi) animals are arrested at G1. cyclin D activity (data not shown). This result supports a We have also assayed the effects of cyd-1(RNAi) on model in which cul-1(+) activity is necessary to regulate the endoreduplication. In wild-type animals, the intestinal nuclei postembryonic blast cell divisions once proliferation has been undergo one round of nuclear division followed by one round initiated. In the absence of cyclin D, the blast cells never enter of endoreduplication during L1 development. As a result, at the the proliferation phase and the cul-1(e1756) phenotype is start of L2 each intestinal nucleus has a 4C DNA content. ‘suppressed’. Interestingly, the undivided intestinal nuclei in cyd-1 (RNAi)- arrested animals had a 4C DNA content (Fig. 7B), cyd-1 and cdk-4 are necessary for cell cycle demonstrating that, although the nuclei are unable to divide, progression they can still undergo one round of endoreduplication just To determine if cyd-1 and/or cdk-4 activity was sufficient to as in wild-type animals. This result suggests that initiate cell cycle progression, we asked whether the non- endoreduplication can occur by a cyd-1-independent proliferating blast cells in starved L1 animals could be induced mechanism (see Discussion). 4856 M. Park and M. W. Krause

DISCUSSION C. elegans where only a single cyclin D-like gene has been identified in the nearly complete genome sequence. The In C. elegans, the postembryonic blast cells undergo temporary developmentally restricted defects that result from the loss of and prolonged G1 arrest during development and these cells cyd-1 activity in C. elegans suggest that cyclin D only become competent to re-enter the cell cycle upon hatching and functions after embryonic cell proliferation is complete or its after each larval molt. We have shown that C. elegans proteins function is redundant to that of a distinct cyclin(s) or other gene related to the G1 cell cycle regulators cyclin D and product(s). CDK4/CDK6 are necessary for G1 cell cycle progression and Loss of CDK-4/6 activity also results in cell divisions the proliferation of many of these postembryonic blast cells. defects that are restricted to postembryonic development in C. We believe that cyd-1 and cdk-4 gene products act together to elegans. The incomplete penetrance of both cdk-4(RNAi) and regulate the G1 phase of postembryonic cell cycles by analogy the putative null cdk-4(gv3) allele suggests that one or more to other systems and based on the co-incident expression of additional factors can partially compensate for the loss of this cyd-1::GFP and cdk-4::GFP reporter genes and the ability of cyclin-dependent kinase. Five additional CDKs have been the two gene products to specifically interact. identified in C. elegans (Boxem et al., 1999) and it is possible Developmentally restricted cell proliferation defects have that one or more of these proteins can partially compensate for also been reported for cyclin D knockouts in the mouse. There the loss of cdk-4. Such compensation, if present, is not very are multiple mouse cyclin D genes (D1, D2, D3) and loss of effective, however, as postembryonic blast cell divisions fail to at least two of these can also result in proliferation defects. occur in the vast majority of homozygous cdk-4(gv3) animals. Nullizygous cyclin D1 mice have abnormalities in neurogenesis, mammary epithelium development and retinal The postembryonic developmental program cell proliferation (Fantl et al., 1995; Sicinski et al., 1995) while continues in the absence of cyd-1 or cdk-4 activity loss of cyclin D2 results in proliferation defects restricted to The regulation of G1 progression in C. elegans by cyd-1 and gonadal cells (Sicinski et al., 1996). Developmentally cdk-4 is not surprising, given that similar roles have been restricted defects due to the loss of mammalian cyclin D were described previously for related factors in other systems. not unexpected due to anticipated redundancy of function However, the developmentally restricted mutant phenotypes among cyclin D gene family members. This is not the case in arising following the reduction or loss of cyd-1 and/or cdk-4

Fig. 6. cyd-1and cdk-4 are necessary and sufficient for expression of a S-phase reporter gene. (A) Ectopic expression of both cyd-1 and cdk-4 is sufficient to induce expression of rnr::GFP, a S-phase reporter gene (Hong et al., 1998). Animals harboring an integrated rnr::GFP transgene alone (strain VT765), or in combination with extrachromosomal arrays of heat-shock-inducible cyd-1 and cdk-4 expression constructs, were hatched out into sterile water and maintained at room temperature or heat shocked as described in the methods section. All populations were then assayed for rnr::GFP expression. Two images are shown for each animal as described in Fig. 2. A rnr::GFP only (VT765) L1 animal that was not (a) or was (b) heat shocked showing the normal background expression of this reporter gene in starved L1 animals (Hong et al., 1998). (c) A heat-shocked animal harboring both cyd-1 and cdk-4 expression constructs as extrachromosomal arrays in addition to the integrated rnr::GFP reporter. Note induced expression of rnr::GFP in a bilateral row of hypodermal cells (seam cells) indicated with arrowheads. (B) Inhibition of S-phase reporter gene expression following cyd-1(RNAi) and cdk-4(RNAi). Two images are shown for each as described in Fig. 2. The S-phase reporter gene rnr::GFP was assayed in a wild-type L1, cyd-1 (RNAi)-arrested and cdk-4(RNAi)-arrested animals. Expression is normally seen in proliferating blast cells such as the descendents of P (arrowheads) and M (arrow) as shown for a wild-type animal (a). The rnr::GFP reporter is not expressed in cyd-1(RNAi) animals and only background gut fluorescence is seen (b). The rnr::GFP reporter is seen only sporadically in cdk-4(RNAi) animals in a few cells that undergo limited proliferation (c; arrowheads) C. elegans G1 cyclin/CDK 4857 elegans CDK, encoded by the ncc-1 gene, that is required for M phase (Boxem et al., 1999). However, inhibition of cyd-1 and/or cdk-4 results in L2 growth arrest and death whereas animals lacking ncc-1 activity continue to be healthy and grow to 80% the normal adult size (Boxem et al., 1999). Continued growth of homozygous ncc-1 mutant animals is seen with multiple loss-of-function or null alleles, and no postembryonic divisions occur in homozygous mutant animals. The differences between the consequences of loss of cyd-1 and cdk- 4 versus loss of ncc-1 may be due to the phase of the cell cycle that is blocked. Our results suggest that depletion of cyd-1 and/or cdk-4 activity blocks postembryonic G1 progression whereas loss of ncc-1 has been shown to result in a block at M phase (Boxem et al., 1999). Perhaps transition through G1 and S in homozygous ncc-1 mutant animals is sufficient to allow expression of one or more genes that may be essential for postembryonic viability. These genes might not be expressed in cyd-1(RNAi) and cdk-4(gv3) mutant animals resulting in growth arrest and death. It is also interesting to note that the first round of endoreduplication in the intestinal cells (ints) is unaffected by cyd-1(RNAi) even though nuclear division is completely blocked. During the L1 stage in wild-type animals, the ints undergo one round of nuclear division followed by one round of endoreduplication to generate nuclei with a 4C DNA content (Hedgecock and White, 1985; Wood, 1988). The ints in the cyd- Fig. 7. DNA content of nuclei in wild-type and cyd-1(RNAi) mutant 1 (RNAi) animals do not divide but have a DNA content (4C) animals. The DNA content was determined by measuring the optical equal to the endoreduplicated ints in wild-type early L2 larvae. density of DAPI-stained nuclei. The number of nuclei assayed is Further cycles of endoreduplication presumably do not occur in indicated parenthetically below each cell type. (A) The undivided P these animals because they arrest and die. Because both nuclear cells of cyd-1(RNAi) animals have a G1 DNA content. Bodywall division and endoreduplication occur in the same tissue, the muscle, P cell descendent and seam cell nuclei in wild-type L2 differential sensitivity of these two processes to inhibition by animals (black vertical bars) were assayed. Bodywall and P cell cyd-1(RNAi) is unlikely to be an artifact of the RNAi technique. descendent nuclei have a 2C DNA content whereas the hypodermal Instead, endoreduplication may be regulated by developmental seam cells have a 4C content as previously reported (Hedgecock and controls that are independent of cyd-1 and cell division. In White, 1985). The undivided P cells of arrested cyd-1(RNAi) animals Drosophila, endoreduplication is regulated by another G1 cyclin, (grey vertical bar) have a G1 DNA content of 2C. (B) Endoreduplication of the intestinal cells. The intestinal cells cyclin E (Knoblich et al., 1994; Duronio and O’Farrell, 1995; undergo developmentally controlled endoreduplication at the end of Sauer et al., 1995; Lilly and Spradling, 1996; Britton and Edgar, each larval stage resulting in DNA contents of 4C, 8C, 16C and 32C 1998) and a cyclin E-like gene (M. P. and M. W. K., unpublished; at L1, L2, L3 and L4 stages, respectively (Hedgecock and White, GenBank accession #AF058331) has been identified in the C. 1985). We determined the DNA content of intestinal cells in wild- elegans genome sequence. It is possible that endoreduplication type animals at several larval stages (vertical black bars). Our in C. elegans may also be regulated by cyclin E. measurements are lower than expected for L3 and L4 animals due to Finally, initiation of nuclear division in the germline appears inclusion of data from animals that had not initiated and/or to be independent of cyd-1 and cdk-4 activity. The factors that completed the endoreduplication cycle for the given larval stage. The control the initiation of germ cell proliferation are unknown, intestinal nuclei of arrested cyd-1(RNAi) animals had a 4C DNA content indicating that one round of endoreduplication has occurred but the continued proliferation of the germline is known to be in the absence of intestinal cell division. The optical density is given dependent on signaling from the somatic gonad. Perturbation in arbitrary units. of normal somatic gonad development, either by laser ablation of the distal tip cells (DTCs) (Kimble and White, 1981) or by genetic mutation (Austin and Kimble, 1987; Sun and Lambie, activity was unexpected. In addition, the resulting absence of 1997), results in a germline phenotype similar to that seen in postembryonic divisions revealed several interesting aspects of cyd-1 (RNAi) and cdk-4(gv3) mutant animals. It may be that C. elegans development. For example, the arrest point that we cyd-1 and cdk-4 have no direct role in regulating germ cell observe in these mutant animals that fail to initiate proliferation and that the mutant germline phenotype that we postembryonic divisions is at the L2 stage, not the L1 stage, observe is a secondary consequence of the failure of the even though there are defects in nuclear divisions that normally somatic gonad precursors to divide to generate DTCs that will occur during the L1 stage. Thus, progression through the L1 signal the germ cells. developmental program, including the first molt, is not dependent on any of the numerous somatic cell divisions that Inhibition of cyd- 1and/or cdk-4 activity has no effect normally occur during this stage of development. Similar embryonically results have been reported recently in a study of another C. Although cyd-1 and cdk-4 reporter genes are expressed 4858 M. Park and M. W. Krause beginning in embryogenesis we have been unable to determine 1988). The earliest reported Gap phase in C. elegans is a G2 any function for these gene during this period of development. phase that is seen the E blastomeres just prior to gastrulation Neither cyd-1(RNAi) nor homozygous cdk-4(gv3) animals have and no G1 phase has been reported for any embryonic cell cycle any obvious embryonic defects and animals hatch out as (Hedgecock and White, 1985; Edgar and McGhee, 1988). The apparently normal L1s. We have raised antibodies against first identified G1 phases in C. elegans are in the cell cycle of CYD-1 but they have not been successful in detecting the postembryonic blast cells (Euling and Ambros, 1996). G1- endogenous levels of the protein in situ or by Western blot so independent cell cycling during embryogenesis in C. elegans we have been unable to validate the embryonic expression could be driven by a constitutively active cyclin E/CDK patterns of the reporter genes. Our ability to rescue the cdk- complex similar to that in early nuclear divisions of the 4(gv3) mutation with a cdk-4 cDNA clone expressed under the Drosophila embryo (Edgar and Lehner, 1996). Such a control of the same sequences used for GFP expression mechanism in C. elegans could eliminate the need for cyclin suggests that the observed cdk-4::GFP expression pattern D and associated CDKs during embryonic cell proliferation. includes at least the minimal tissues required for viability and normal development. Developmental control of postembryonic G1 cell The lack of embryonic cell division defects in cyd-1(RNAi) cycle progression or cdk-4(gv3) mutant animals may be due to sufficient maternal Several C. elegans factors and pathways have been identified contributions of both factors that sustain early development. that are likely to be closely linked to the regulation of cyd-1 Although RNAi is typically very efficient at eliminating and cdk-4 activity. These links are based on coincident patterns maternal RNA (Fire et al., 1998), there are some genes that are of expression, mutant phenotypes and information on not inhibited by this technique and RNAi would not be homologous factors in other systems. For example, cyclin predicted to be effective at depleting stable maternal D/CDK-4-specific inhibitors (INK4 and p21/27) act to regulate protein contributions. Similarly, heterozygous cdk-4(gv3) the activity of the kinase (Serrano et al., 1993; Hirai et al., hermaphrodites could contribute sufficient maternal cdk-4 gene 1995; Xiong et al., 1993; LaBaer et al., 1997). Although no products to function throughout embryogenesis in homozygous INK4 homologs have been identified in C. elegans, the cki-1 mutant progeny. Our ability to rescue the cdk-4(gv3) allele with gene product is related to the cyclin-dependent kinase the wild-type cDNA minimizes the likelihood that there is an inhibitors p21/27 and has been shown to function in essential maternal contribution because transgenes are usually postembryonic development (Hong et al., 1998). Ectopic expressed poorly, or not at all, in the germline (Mello and Fire, expression of cki-1 is sufficient to arrest cells in G1 and 1995; Kelly and Fire, 1998). Regardless, for both cyd-1 and depletion by RNAi leads to extra larval cell divisions. This cdk-4 gene products, any maternal contributions must be finding is consistent with the inhibitory role that these CKIs insufficient for early larval development as inhibition of either play in the regulation of the cyclin/CDK activity in mammals cyd-1 or cdk-4 activity results in L1 division defects. and specifically for the regulation of G1/S progression (Sherr Another possible explanation for the lack of embryonic and Roberts, 1995). It is reasonable to think that the cki-1 gene defects when these two activities are depleted is that embryonic product would negatively regulate the proposed CYD-1/CDK- divisions are cyclin D- and CDK4/6-independent. CYD-1 and 4/6 activities in the control of cell cycle progression from G1 CDK4/6 may be present during embryogenesis but not active to S phase. due to the action of negative regulators such as the CIP/KIP- The gene cul-1 has also been shown to affect the G1 phase related cki-1 gene product (Hong et al., 1998). Alternatively, of postembryonic blast cells in C. elegans and is likely to be early divisions could be initiated by the action of one or more involved in regulating CYD-1 activity (Kipreos et al., 1996). cyclin- or CDK-related factors that are sufficient for embryonic The cul-1 gene product, a cullin, is related to S. cerevisiae G1 progression, making CYD-1 and CDK4/6, if present, CDC53. In yeast, CDC53 is part of the ubiquitin-mediated dispensable. As previously noted, five additional CDKs have degradation pathway required for elimination of G1 cyclins been identified in C. elegans (Boxem et al., 1999) providing (Mathias et al., 1996; Jackson, 1996). In C. elegans, cul-1 several potential candidate gene products to compensate for the mutant animals have a hyperproliferation of the postembryonic loss of cdk-4 activity. Although no other cyclin D-like genes blast cells, presumably because G1 cyclins are not degraded at have been identified by the C. elegans genome sequence the appropriate time resulting in the failure of cells to exit the project, there is a cyclin E-like gene. In other systems, both cell cycle (Kipreos et al., 1996). Our epistasis experiment with cyclins D and E act with their associated CDKs to promote G1 cyd-1 (RNAi) and cul-1 demonstrates that cyd-1 is necessary progression. Perhaps the C. elegans cyclin E-like gene product for the cul-1 (e1756) mutant phenotype. Therefore, CUL-1 and is sufficient for embryonic cell divisions. Preliminary results components of the ubiquitin-mediated degradation pathway suggest this may be the case since inhibition of the C. elegans might be necessary to regulate the activity of CYD-1 to cyclin E-like gene by (RNAi) results in embryonic cell division promote withdrawal from the cell cycle. defects (M. P. and M. W. K., unpublished). Previous results in other systems have suggested that D-type In Drosophila, a constitutively active maternal cyclin E- cyclins are controlled by extracellular signals. Knockin dependent kinase activity allows rapid nuclear divisions experiments into the cyclin D1 locus in the mouse suggest that without an intervening G1 phase (Edgar and Lehner, 1996). It cyclin E is the rate-limiting target of cyclin D function. This is possible that C. elegans has adopted a similar strategy to led the authors to speculate that cyclin D control of cell cycle accommodate the rapid proliferation of embryonic progression may have evolved as a mechanism to link blastomeres. The cell divisions in the early C. elegans embryo extracellular signals to the control of cyclin E (Geng et al., (up to the 28-cell stage) are known to cycle between S and M 1999). It seems reasonable to think that in C. elegans the cyclin phases with no intervening Gap phases (Edgar and McGhee, D-dependent pathway of cell cycle progression also serves to C. elegans G1 cyclin/CDK 4859 link extracellular signals to cell proliferation. This could Edgar, B. A. and Lehner, C. F. (1996). Developmental control of cell cycle include signals accessing food supply as well as temporal regulators; A fly’s perspective. Science 274, 1646-1652. developmental cues. For example, the proper timing of Edgar, L. G. and McGhee, J. D. (1988). DNA synthesis and the control of embryonic gene expression in C. elegans. Cell 53, 589-599. postembryonic developmental events is controlled by the Edgley, M., Baillie, D. L., Riddle, D. L. and Rose, A. M. (1995). Genetic heterochronic genes (Ambros and Horvitz, 1984; Ambros, balancers. In Caenorhabditis elegans; Modern Biological Analysis of an 1997). Mutations in these genes cause defects in the correct Organism. (ed. H. F. Epstein and D. C. 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Potent and specific genetic interference by double-stranded with the regulation of cyd-1 and cdk-4. The challenge now RNA in Caenorhabditis elegans. Nature 391, 806-811. becomes to identify direct links between the genes of these Fukushige, T., Hawkins, M. G. and McGhee, J. D. (1998). The GATA-factor regulatory hierarchies and the activity of the highly conserved elt-2 is essential for formation of the Caenorhabditis elegans intestine. Dev. Biol. 198, 286-302. cell cycle factors so that we can better understand how division Geng, Y., Whoriskey, W., Park, M. Y., Bronson, R. T., Medema, R. H., is regulated during animal development. Tiansen, L., Weinberg, R. A. and Sicinski, P. (1999). Rescue of cyclin D1 deficiency by knockin cyclin E. Cell 97, 767-777. We would like to thank Alan Couslon for a YAC grid for mapping; Hanks, S., Quinn, A. and Hunter, T. (1988). 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