Down-regulation of tricarboxylic acid (TCA) cycle genesblocks progression through the first mitotic division inCaenorhabditis elegans embryosMohammad M. Rahman, Simona Rosu, Daphna Joseph-Strauss, and Orna Cohen-Fix1

Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

Edited* by Angelika Amon, Massachusetts Institute of Technology, Cambridge, MA, and approved January 9, 2014 (received for review June 19, 2013)

The cell cycle is a highly regulated process that enables the accuratetransmission of chromosomes to daughter cells. Here we uncover apreviously unknown link between the tricarboxylic acid (TCA) cycleand cell cycle progression in the Caenorhabditis elegans early em-bryo. We found that down-regulation of TCA cycle components, in-cluding citrate synthase, malate dehydrogenase, and aconitase,resulted in a one-cell stage arrest before entry into mitosis: pronu-clear meeting occurred normally, but nuclear envelope breakdown,centrosome separation, and chromosome condensation did not takeplace. Mitotic entry is controlled by the cyclin B–cyclin-dependentkinase 1 (Cdk1) complex, and the inhibitory phosphorylation ofCdk1 must be removed in order for the complex to be active. Wefound that following down-regulation of the TCA cycle, cyclin Blevels were normal but CDK-1 remained inhibitory-phosphorylatedin one-cell stage-arrested embryos, indicative of a G2-like arrest.Moreover, this was not due to an indirect effect caused by check-point activation by DNA damage or replication defects. These obser-vations suggest that CDK-1 activation in the C. elegans one-cellembryo is sensitive to the metabolic state of the cell, and thatdown-regulation of the TCA cycle prevents the removal of CDK-1inhibitory phosphorylation. The TCA cycle was previously shown tobe necessary for the development of the early embryo in mammals,but the molecular processes affected were not known. Our studydemonstrates a link between the TCA cycle and a specific cell cycletransition in the one-cell stage embryo.

The developmental program of any organism must be preciselyexecuted. In Caenorhabditis elegans embryos, immediately after

fertilization, two pronuclei form at opposite poles of the embryo:one containing the maternal chromosomes and the other con-taining the paternal ones (1, 2). These pronuclei then move towardeach other, and at the same time centrosomes separate and beginto assemble a spindle. After pronuclear meeting, the cell enters itsfirst mitosis, resulting in nuclear envelope breakdown, chromatincondensation, and the subsequent alignment of chromosomes onthe metaphase plate, followed by chromosome segregation (2).Entry into mitosis depends on the mitotic cyclin B–cyclin-dependentkinase 1 (Cdk1) complex. The activity of this complex is regu-lated by both cyclin B levels and regulatory phosphorylation ofCdk1. In particular, Cdk1 activity is inhibited by Wee1 phos-phorylation, which is removed at the onset of mitosis by theCdc25 phosphatase (3, 4). Cdk1 activation is also subjected tovarious checkpoints that inhibit mitotic progression in the pres-ence of intracellular damage (5). However, in organisms thatundergo rapid embryonic divisions, including C. elegans, check-points are inoperative during the first few cell cycles (6).Although it is clear that cell cycle progression requires energy,

the link, if any, between metabolic pathways and progressionthrough mitosis is poorly understood. Genes and proteins in-volved in various aspects of metabolism (e.g., nucleotide bio-synthesis and lipid metabolism) are regulated by the cell cyclemachinery, and cells will not commit to a new cell cycle ifnutrients are scarce (7). However, to what extent the metabolicstate of the cell is sensed by the cell cycle machinery once cellshave passed into S phase is not clear (8).

We have previously conducted a visual screen in C. elegansembryos for genes that when down-regulated by RNAi lead to anabnormal nuclear morphology (9). Most genes whose inactiva-tion affected early embryonic development did so without ar-resting cell cycle progression. It was therefore striking when wecame across a set of genes, coding for enzymes of the tricarboxylicacid (TCA) cycle, that when down-regulated, led to a one-cellstage arrest with paired nuclei. The TCA cycle, also known as theKrebs cycle, uses the oxidation of acetate (in the form of acetylCoA) derived from carbohydrates, proteins, or lipids, to generateintermediates (i.e., NADH and FADH2) that are used by theelectron transport chain for ATP production. Intermediates ofthe TCA cycle are also important for various anabolic pathways,such as fatty acid synthesis, and the synthesis of nucleotides. Inthis study, we examine the relationship between TCA cycle down-regulation and cell cycle progression in the one-cell C. elegansembryo. Our data suggest that down-regulation of the TCA cycleleads to a G2-like arrest at the one-cell stage embryo by pre-venting the activation of cyclin B–Cdk1.

ResultsDown-Regulation of the C. elegans Citrate Synthase Ortholog Leadsto a One-Cell Stage Embryonic Arrest Before Nuclear EnvelopeBreakdown. Recently we conducted an RNAi screen in C. elegansfor genes that affect nuclear morphology by targeting genes thatwere reported to cause embryonic lethality when mutated ordown-regulated by RNAi (9). In the course of these studies weuncovered an unusual phenotype caused by the down-regulationof ORF T20G5.2 that codes for CTS-1, an ortholog of the eu-karyotic citrate synthase (Fig. S1). When cts-1 was down-regulated

Significance

Cell division is driven by the cell cycle machinery, whichresponds in an unknown fashion to the metabolic and nutrientstate of the cell. We uncovered a previously unknown linkbetween the cell cycle machinery and the tricarboxylic acid(TCA) cycle (also known as the Krebs cycle), which formsintermediates required for ATP production and other anabolicpathways. We show that in Caenorhabditis elegans embryos,down-regulation of the TCA cycle inhibits entry into the firstmitotic division by preventing the removal of inhibitoryphosphorylation on cyclin-dependent kinase 1. Our data sug-gest that in the one-cell stage embryo, the cell cycle machineryis sensitive to the metabolic state of the cell, a phenomenonthat may also exist in mammalian embryos.

Author contributions: M.M.R., S.R., D.J.-S., and O.C.-F. designed research; M.M.R., S.R., andD.J.-S. performed research; M.M.R., S.R., and D.J.-S. contributed new reagents/analytictools; M.M.R., S.R., D.J.-S., and O.C.-F. analyzed data; and M.M.R., S.R., D.J.-S., and O.C.-F.wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: ornac@helix.nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1311635111/-/DCSupplemental.

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by RNAi, embryos accumulated at the one-cell stage with pairednuclei (Fig. 1A).During normal development, embryos at the one-cell stage

with paired nuclei are relatively rare: they are present transientlyfollowing maternal and paternal pronuclear meeting and beforenuclear envelope breakdown (1). Adult C. elegans animals haveone or no such embryos on each side of the uterus (that is, theuterine half that is between a spermatheca and the vulva); mostembryos in the uterus contain four or more cells, as was alsoobserved in our control RNAi-treated animals (Fig. 1 A and Band Fig. S2). In contrast, a 40 h treatment with RNAi against cts-1resulted in a dramatic increase in one-cell stage embryos withpaired nuclei (Fig. 1 A and B and Fig. S2). The percentage ofanimals exhibiting this phenotype (i.e., at least two or more one-cell stage embryos with paired nuclei per uterine half; hence-forth, “animals with arrested embryos”) varied from experimentto experiment, ranging from 50% to 90%. The reason for thispartial penetrance is not known. The RNAi treatment resulted ina ∼60% reduction in CTS-1 levels (Fig. S3). Although CTS-1 levelsin embryos from cts-1 RNAi-treated animals that did not accu-mulate one-cell embryos was slightly higher than that of arrestedembryos (Fig. S3), the difference was very small. This differencemay be sufficient to bypass the one-cell stage arrest, but it is alsopossible that embryos that can progress past the one-cell stagedespite low CTS-1 levels have somehow adapted to low CTS-1levels. In animals with arrested embryos, the average fraction ofembryos at the one-cell stage with paired nuclei was over 50% ofthe total number of embryos per each uterine half (Fig. 1B and Fig.S2). This was not accompanied by an accumulation of two- andfour-cell stage embryos (Fig. 1B and Fig. S2), arguing that thedown-regulation of cts-1 did not cause a general slowing down ofembryonic development. Embryos from all cts-1 RNAi-treatedanimals that did not arrest at the one-cell stage continued to de-velop but arrested before hatching, with the exception of a smallfraction of embryos that hatched but arrested as larvae. Thus,down-regulation of cts-1 resulted in one of two fates: an arrest atthe one-cell stage with paired nuclei, or a developmental arrest ata multicellular embryo or early larva stage.Cell cycle events that preceded fertilization were also exam-

ined. The germ line of cts-1 RNAi animals containing one-cell

stage-arrested embryos were indistinguishable from the germline of control animals (Fig. S4A). Meiosis appeared to havetaken place normally, as all six chromosome pairs connected bychiasmata were visible at diakinesis in oocytes from animalstreated with either control RNAi or cts-1 RNAi (Fig. S4B).Moreover, 100% of the one-cell stage-arrested embryos (n = 60)contained two polar bodies, indicating that both meiosis I and IIdivisions took place. Finally, polarity cues, as judged by thedistribution of the asymmetrically localized P granules (10) andthe polo-like kinase PLK-1 (11), appeared normal in one-cellstage-arrested embryos (Fig. S5). The one difference we noticedbetween control and cts-1 RNAi-treated animals at the pointwhen the one-cell stage-arrested embryos were assayed (i.e., 40 hof RNAi treatment) was a reduced rate of egg laying in the cts-1RNAi-treated worms: 39% fewer eggs laid per animal per 40 h inthe cts-1 RNAi-treated worms (n = 38) compared with controlworms (n = 16). The reduced rate of egg laying could be in-dicative of starvation or muscle/neuronal dysfunction (12), bothplausible consequences of TCA cycle down-regulation. None-theless, the cell division events leading to the formation of one-cell stage embryos appeared to be overall normal.To examine mitotic cell cycle events after fertilization, we

followed chromosomes using histone H2B fused to mCherry(H2B::mCR) and nuclear envelope dynamics using nuclear poreprotein NPP-1::GFP. In embryos from animals treated withcontrol RNAi, chromosome condensation happened as the ma-ternal and paternal pronuclei met, followed immediately by nu-clear envelope breakdown and metaphase plate formation (lessthan 7 min after pronuclear meeting; Fig. 1C). In contrast, inembryos from cts-1(RNAi) animals, maternal and paternalpronuclear meeting occurred normally, but nuclear envelopebreakdown did not happen, even after a prolonged time (greaterthan 30 min, n = 8; for example, Fig. 1D). In these animals,chromatin condensation did not take place and a metaphaseplate never formed (compare Fig. 1 C with D).We also examined nuclear envelope dynamics using additional

markers, including LMN-1 (the single C. elegans B-type laminhomolog) (13), NPP-9 (another nuclear pore complex component)(14), and LEM-2 (a LEM-domain protein that resides in thenuclear envelope and is a component of the nuclear lamina)

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Fig. 1. Down-regulation of cts-1 results in an accumulation of one-cell stage embryos with paired nuclei. (A) Embryos from animals after 40 h of RNAitreatment with either control RNAi (Left) or cts-1 RNAi (Right). The animals (strain OCF3) expressed NPP-1 fused to GFP (NPP-1::GFP) and histone H2B fused tomCherry (H2B::mCR). (B) The percentages of one-, two-, four-, or multicell stage embryos per uterine half of OCF3 animals treated with control or cts-1 RNAi(n = 16 and n = 19 animals, respectively) are shown. For cts-1 RNAi-treated animals, only those that accumulated one-cell stage embryos (i.e., two or more pergonad arm) were included. Shown are the results from a typical experiment, where 50% of animals accumulated one-cell stage-arrested embryos followingcts-1 RNAi (see Fig. S2 for additional experiments). Error bars indicate SD. The difference in the percentage of embryos in the one-cell stage between controland cts-1(RNAi) animals was statistically significant (P < 0.05, Student t test). (C) Time-lapse images of an embryo from a control RNAi-treated OCF3 animal.Time 0 was defined as the time point when the maternal and paternal pronuclei met. (D) Time-lapse images of an embryo from a cts-1 RNAi-treated OCF3animal. Outlines indicate the edges of the embryos. (E) The uterus of a cts-1 RNAi-treated animal expressing NPP-9::GFP. (F) Embryos from cts-1 RNAi-treatedanimals expressing the indicated fusion proteins, imaged 30 min following pronuclear meeting. (Scale bar: 10 μm.)

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(15). Following cts-1 RNAi, all three proteins remained at thenuclear envelopes of the paired nuclei in one-cell stage-arrestedembryos for at least 30 min following pronuclear meeting (Fig. 1E and F). Thus, down-regulation of cts-1 causes a general defectin nuclear envelope breakdown.

Down-Regulation of Additional TCA Cycle Components Leads to anEmbryonic One-Cell Stage Premitotic Arrest. To determine whetherthe one-cell stage embryonic arrest observed following cts-1RNAi was due to CTS-1’s function in the TCA cycle, we exam-ined whether down-regulation of other TCA cycle componentsresulted in a similar phenotype. Acetyl CoA is made from py-ruvate by the pyruvate dehydrogenase (PDH) complex. A re-duction in PDH complex activity would likely limit the amount ofacetyl CoA available for conversion to citrate by citrate synthase,thus reducing overall TCA cycle activity (16). The PDH complexconsists of three enzymes: PDH, dihydrolipoyl transacetylase,and dihydrolipoyl dehydrogenase (17). The sequence of C. elegansORF F23B12.5, coding for DLAT-1, is highly similar to verte-brate dihydrolipoyl dehydrogenase, which is also part of theoxoglutarate dyhedrogenase complex (also in the TCA cycle)and the alpha-keto acid dehydrogenase complex. Down-regula-tion of dlat-1 by RNAi resulted in a paired-nuclei phenotype inone-cell stage-arrested embryos, similar to the phenotype ob-served following cts-1 down-regulation (Fig. 2 A and B). More-over, the fraction of animals with arrested embryos and thepercentage of one-cell stage-arrested embryos in these animalswas similar to that of cts-1(RNAi) animals (Fig. 2B). A doubleRNAi treatment against dlat-1 and cts-1 resulted in a similarphenotype as cts-1 RNAi or dlat-1 RNAi alone (Fig. 2B and Fig.S6), suggesting that DLAT-1 and CTS-1 affect mitotic progressionin the one-cell stage embryo through the same metabolic pathway.Consistent with this, we found that down-regulation of additionalTCA cycle components, malate dehydrogenase, aconitase, andsuccinate dehydrogenase, also resulted in an accumulation ofone-cell stage embryos with paired nuclei (Fig. S7). Thus, down-regulating the TCA cycle blocks the first embryonic cell cyclebefore mitotic entry.

Down-Regulation of the TCA Cycle Affects Centrosome Separation inthe C. elegans One-Cell Stage Embryos. Centrosomes are necessaryfor the formation of a bipolar spindle in the C. elegans embryo(2, 18). After fertilization, the paternally contributed centriolesduplicate, generating two centrosomes that separate and migrateto the center of the embryo, where they nucleate microtubulesthat ultimately form a bipolar spindle (2). To follow spindledynamics in vivo we used transgenic animals expressing β-tubulinfused to GFP (TBB-2::GFP) and NPP-1 fused to mCherry (NPP-1::mCR). In control RNAi-treated animals, before nuclear en-velope breakdown, the two centrosomes align on either side ofthe plane where the two pronuclei meet (Fig. 3A). In contrast, inembryos from cts-1 RNAi-treated animals, the position of thecentrosomes was abnormal: over half of the embryos had eitherone or two closely associated centrosomes (53.8%, n = 78),whereas in the remaining embryos the centrosomes were sepa-rated but abnormally positioned (Fig. 3B). Live imaging oftransgenic animals expressing a centrosomal component, SPD-2,fused to GFP (SPD-2::GFP) (19) confirmed that cts-1 down-regulation prevented the timely separation of centrosomes in theone-cell stage embryo (n = 4; Fig. 3C, Insets). A similar trend wasobserved following dlat-1 down-regulation by RNAi: of the 57embryos arrested in the one-cell stage with paired nuclei, 50.9%had centrosomes that failed to separate, 7% had centrosomesthat separated but were abnormally positioned, and in 42.1% thecentrosomes were separated and positioned correctly. Thus,down-regulation of the TCA cycle not only blocked nuclear en-velope breakdown and chromosome condensation, but also inhibi-ted the timely separation of centrosomes, another event associatedwith mitotic entry.

Down-Regulation of the TCA Cycle Prevents CDK-1 Activation in theOne-Cell Stage Embryo. Because centrosome separation, chro-mosome condensation and nuclear envelope breakdown aremechanistically independent (20, 21), it was likely that down-regulation of the TCA cycle prevented mitotic entry by blockinga common upstream regulatory event. In metazoans, entry intomitosis is largely dependent on increased activity of Cdk1,which involves the accumulation of nuclear cyclin B and theremoval of inhibitory phosphorylation from threonine (Thr14)and tyrosine (Tyr15) residues of Cdk1 (residue numbers ac-cording to the human Cdk1) by the Cdc25 phosphatase (3, 4).Cyclin B builds up in the nucleus in G2 and begins to be de-graded upon entry into mitosis (22). Thus, in G2 cells, Cdk1activity is held in check primarily by inhibitory phosphorylationon Thr14/Tyr15 (4). To examine the status of Cdk1 in the one-cell stage arrest induced by down-regulation of the TCA cycle,we examined the levels of C. elegans cyclin B (CYB-1) and thephosphorylation state of CDK-1 in embryos following controlor cts-1 RNAi using antibodies against CYB-1 (23) and anti-bodies against human inhibitory phosphorylated Cdk1(Thr14/Tyr15) that also recognize the C. elegans phospho-Cdk1(Thr32/Tyr33) (Fig. S8).In control RNAi-treated animals, cyclin B was visible at the

one-cell stage before nuclear envelope breakdown (Fig. 4A), andit exhibited the expected pattern in later cell cycle stages (see, forexample, its absence from a metaphase cell in the four-cell stageembryo). One-cell stage-arrested embryos from cts-1(RNAi)animals also had significant levels of CYB-1 (in 59 embryos of 65

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Fig. 2. Down-regulation of TCA cycle components results in a paired-nucleiphenotype in a one-cell stage-arrested embryo. (A) Embryos from an OCF3animal treatedwithRNAiagainstdlat-1 (Left) ordlat+ cts-1 (Right). Controlandcts-1 RNAi alone are the same as shown in Fig. 1A. (Scale bar: 10 μm.) (B) OCF3animals were treated with control RNAi or RNAi against cts-1, dlat-1, or both,and the percentages of embryos in the indicated stages in each uterine halfwere scored. Shown is a typical RNAi experiment (additional experiments are inFig. S6). Foreach treatment, uterinehalves fromat least 10animalswere scored.The percentages of animals with arrested embryos in this experiment were50%, 61.9%, and 40% for RNAi against cts-1, dlat-1, and cts-1 + dlat-1, re-spectively. Error bars indicate SD. cts-1, dlat-1, and cts-1 + dlat-1 RNAi treat-ments led to the accumulation of one-cell stage embryos that was statisticallysignificantly higher than in the control (P < 0.05, Student t test). The differencebetweencontroland thethreeRNAi treatments in thepercentageofmultistageembryos was also statistically significant (P < 0.05, Student t test).

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analyzed; Fig. 4B), comparable to interphase nuclei in embryosfrom control RNAi-treated animals. Thus, the inability to entermitosis following cts-1 down-regulation was not due to a defect inCYB-1 accumulation. However, although in one-cell stage em-bryos from the control RNAi-treated animals the levels of in-hibitory phosphorylated CDK-1 diminished as the two pronucleirotated (Fig. 4A, Center) and were absent once mitosis had begun(Fig. S9), the levels of inhibitory phosphorylated CDK-1 in one-cell stage-arrested embryos from cts-1(RNAi) animals were un-usually high (Fig. 4B). This suggested that the one-cell stagearrest is due to persistent inhibitory phosphorylation of CDK-1.The high level of inhibitory phosphorylated CDK-1, along withthe simultaneous presence of nuclear localized CYB-1, suggeststhat the one-cell stage-arrested embryos from cts-1 RNAi-treated animals are in a G2-like state.

The One-Cell Stage Embryonic Arrest Following cts-1 Down-RegulationIs Not Due to the Activation of the DNA Damage Checkpoint or theSpindle Assembly Checkpoint. The G2-like arrest could have beenthe result of a cell cycle defect that led to checkpoint activation

and consequently inhibition of CDK-1 activation. The G2/Mtransition is subjected to regulation by DNA damage responsepathways, and specifically by the Chk1 protein kinase (24, 25). Inthe presence of DNA damage, activated Chk1, which is phos-phorylated on Ser345 (residue number according to the humanprotein), inhibits the Cdc25 protein phosphatase (26). This, inturn, prevents the removal of inhibitory phosphorylation ofCdk1 that is critical for Cdk1 activation, as discussed in the In-troduction. In C. elegans, a similar CHK-1 activation pathway,through the phosphorylation of the corresponding Ser344, hasbeen reported (27, 28). Although the DNA damage and repli-cation checkpoints are thought to be inactive in the one-cellstage embryo (28, 29), it was formally possible that down-regu-lation of the TCA cycle led to unscheduled CHK-1 activation atthis stage. To test this, we examined one-cell stage-arrestedembryos from cts-1 RNAi-treated animals for the presence ofCHK-1 phosphorylated at Ser344 (p-CHK-1S344) by immuno-fluorescence using antibodies against human phospho-Chk1 atSer345 (27). As a positive control for the presence of p-CHK-1S344, we down-regulated the large subunit of ribonucleotide

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Fig. 3. Down-regulation of cts-1 results in failure in centrosome separation.Embryos from a control (A) or cts-1 (B) RNAi-treated animals (strain OCF28)expressing β-tubulin fused to GFP and NPP-1 fused to mCherry. (C, Upper) Anembryo from a control RNAi-treated animal expressing SPD-2::GFP (strainOC534) showing two centrosomes shortly following duplication (0 min), and asthey move to the center of the embryo. Images were taken at the indicatedtime points. (C, Lower) An embryo from the same strain treated with cts-1(RNAi), showing duplicated centrosomes (see Insets, which are an enlargementof the boxed areas in the first and last time points). (Scale bar: 10 μm.)

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Fig. 4. Down-regulation of cts-1 results in a G2-arrest in the one-cell stageembryo. (A) One-cell stage embryos before (Left) or after (Center) pro-nuclear rotation, and a four-cell stage embryo, all from control RNAi-treatedanimals, were coimmunostained with antibodies against cyclin B (Middle)and phosphospecific antibodies against Cdk1 inhibitory phosphorylation atThr14 and Tyr15 (Bottom). Note that neither cyclin B nor phospho-Cdk1 isdetected in the metaphase cell in the four-cell stage embryo, as shown byDAPI staining (Top). (B) Two typical examples of embryos arrested in theone-cell stage from cts-1 RNAi-treated animal. Embryos were coimmunos-tained as in A. (Scale bar: 10 μm.)

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reductase (rnr-1) by RNAi, thereby reducing de novo synthesis ofdeoxyribonucleotides, leading to stalling of DNA replication forkprogression and delaying mitotic entry via CHK-1 activation (30,31). As expected, down-regulation of rnr-1 resulted in activationof CHK-1 (Fig. S10 A and B). In contrast, p-CHK-1S344 waslargely absent in embryos from control RNAi-treated animalsand never detected in the one-cell–arrested embryos from cts-1(RNAi) animals (Fig. S10 A and B). Thus, the persistence ofCdk1 inhibitory phosphorylation following cts-1 down-regulationis not due to the activation of the DNA damage or replicationcheckpoint through CHK-1 activation.We also examined whether the one-cell stage arrest following

TCA cycle down-regulation depended on the spindle assemblycheckpoint, which prevents progression through mitosis in theabsence of chromosome-microtubule attachments (32). If thatwere the case, then the inactivation of this checkpoint wouldbypass the one-cell arrest. To test this possibility we inactivatedthe C. elegans MAD3 homolog by using a san-1 mutant (33, 34)and treated the worms with RNAi against cts-1. The absence ofa functional spindle assembly checkpoint did not bypass the one-cell stage arrest following cts-1 RNAi (Fig. S10C), consistentwith a cell cycle arrest before mitotic entry.

DiscussionMetabolism affects cell cycle progression, but the extent to whichthis happens during embryogenesis, and the pathways that linkmetabolism and the cell cycle circuitry, are largely unknown. Herewe show that in C. elegans, down-regulation of TCA cycle com-ponents results in a one-cell stage embryonic arrest with pairednuclei. The arrested embryos fail to break down the nuclear en-velope, chromosomes remain decondensed, and centrosomes areunseparated or abnormally separated. Taken together, these phe-notypes are consistent with a defect in mitotic entry. Indeed, thearrested embryos had high levels of both cyclin B and inhibitory-phosphorylated CDK-1, indicative of a G2-like stage arrest. Thus,our data suggest that the cell cycle machinery of the one-cell stageembryo is sensitive to the metabolic state of the cell, and that whenthe TCA cycle is down-regulated, the one-cell stage embryo fails toenter the first mitotic division following fertilization.One of the earliest defects caused by TCA cycle down-regu-

lation was a defect in centrosome separation. Previously, Hachetet al. proposed that centrosomes affect timely entry into mitosisin the C. elegans early embryo (35). However, it is unlikely thatcentrosomes are the main target affected by down-regulation ofthe TCA cycle because defects in centrosome maturation, causedby down-regulation of SPD-2 or SPD-5 and which blocks cen-trosome separation and spindle assembly, do not prevent nuclearenvelope breakdown or entry into mitosis (19, 35, 36).At present we do not know how the TCA cycle affects the cell

cycle machinery; it may cause a metabolic imbalance in theembryo that blocks Cdk1 activation, but it may also cause animbalance earlier, in the germ line, which is maintained post-fertilization. Even if the TCA cycle perturbation occurs in thegerm line, our data suggest that its effect on the cell cycle ma-chinery occurs in the embryo rather than earlier. This is based onthe observations that the germ line and oocytes of cts-1 RNAianimals appear morphologically normal and that all of thearrested embryos had two polar bodies, indicating that thecompletion of meiosis was not perturbed. Had Cdk1 been inac-tivated in the germ line, meiosis would not have been completed(37). Thus, the most straightforward explanation for the one-cellembryonic arrest is that down-regulation of TCA cycle activityled, either directly or indirectly, to a defect in mitotic Cdk1 ac-tivation in the one-cell stage embryo, and this, in turn, blockedfurther progression through the cell cycle.An interesting observation is that embryos that did not arrest

in the one-cell stage following TCA cycle down-regulation con-tinued to develop to multicellular embryos. This indicates thatalthough the first mitotic division is sensitive to perturbations inthe TCA cycle, subsequent divisions are not. One possibility isthat the first mitotic division is inherently different from later

divisions, either in its regulation of Cdk-1 activation or in itsdependence on TCA cycle activity. For example, in mammalianembryos the TCA cycle is active during early embryonic divisionswhereas glycolysis is activated only postimplantation (see below).Down-regulation of enzymes in other metabolic pathways, in-cluding glycolysis [the C. elegans homologs of hexokinase (codedby F14B4.2), glyceraldehyde 3-phosphoate dehydrogenase and6-phosphofructokinase], the electron transport chain (includingthe CYC-1 and UCR-1 subunits of complex III and the CCO-1and CCO-2 subunits of complex IV) and the PHI-37, ATP-4, andATP-2 subunits of ATP synthase, all led to embryonic lethalitybut did not lead to an increase above control of one-cell stageembryos. This suggests that either the TCA cycle is predominantduring this phase of development, or that it has a unique in-termediate that affects cell cycle progression. It was previouslyshown that depolarizing mitochondria in human colorectal car-cinoma HTC116 tissue culture cells (38) or reducing ATP levelsin Drosophila cells (39) blocks the G1/S transition. We, however,found that down-regulating the TCA cycle arrests cells in G2-likestate. It is possible that the first cell division cycle in the C. ele-gans embryo has unique features that allow it to progress pastG1/S and on to G2 in the presence of low ATP levels. Alterna-tively, the arrest in the one-cell stage embryo following TCAcycle down-regulation could be due to changes in the levels ofa metabolite other than ATP; either the accumulation of a toxicintermediate or the absence of a critical one. Alterations in theTCA cycle may also affect intracellular pH or the redox state ofthe cytoplasm, both of which can have a profound effect on manydifferent processes.The Cdc25 phosphatase, which removes the Wee1-dependent

inhibitory phosphates from Cdk1, is an obvious candidate forbeing affected by the TCA cycle (40, 41). In C. elegans, CDC-25.1, which is homologous to the mammalian Cdc25A, is ma-ternally contributed and is proposed to promote mitotic pro-gression in the early embryo (42, 43). If our model is correct,then down-regulation of CDC-25 should phenocopy cts-1 RNAiwhereas inactivation of WEE-1 should bypass the one-cell stagearrest. Unfortunately, in C. elegans, the activities of both CDC-25and WEE-1 are necessary for gametogenesis (44, 45), precludingus from testing these predictions at this time.To what extent can our findings in C. elegans embryos extend

to mammalian systems? Studies conducted mostly in the 1960sand 1970s have shown that two-cell stage mammalian embryosuse pyruvate and lactate through the TCA cycle, whereas gly-colysis is activated at a later embryonic, postimplantation, stage(46–48). Moreover, inhibitors of the TCA cycle block mamma-lian embryonic development at the one-cell stage, whereas in-hibition of glycolysis affects the morula to blastocyst transition(49). When these studies were conducted, very little was knownabout the molecular details of the cell cycle machinery, and thusthe molecular link between the TCA cycle and the ability toexecute the early embryonic divisions was not known. The presentstudy gains insight into the link between metabolism and cell cycleprogression in the early embryo; at least in C. elegans, down-reg-ulation of the TCA cycle blocks Cdk1 activation by preventingthe removal of its inhibitory phosphorylation.

Materials and MethodsStrains. All C. elegans strains were maintained at 20 °C using standardmethods except where noted otherwise (50). A list of strains and theirgenotypes is in Table S1.

RNAi Experiments. RNAi constructs were isolated from the RNAi feeding library(Open Biosystems) and performedusing standard feedingmethods. The identityof each RNAi clonewas verified by sequencing. L4-stage larvae were transferredto RNAi plates, and embryos were examined after 40 h of RNAi treatment at20 °C. For each treatment, at least 10 animals were scored, and each experi-ment was repeated multiple times. Because the two gonad arms act in-dependently of each other, we scored the phenotypes of embryos per uterinehalf, that is, between each spermatheca and the vulva. To determine embry-onic lethality, 8–10 animals were transferred to new RNAi plates following 40 htreatment and removed 3–6 h thereafter. Hatching was scored 24 h later.

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Immunofluorescence. Animals from RNAi plates were dissected on poly-L-lysine–coated slides and embryos were opened by the freeze-crackingmethod as described previously (9). Samples were fixed in −20 °C coldmethanol for 2 min and blocked in 5% (vol/vol) BSA in PBS for 30 min at 4 °Cbefore overnight incubation at 4 °C with primary antibody diluted in 1%BSA. The following antibodies were used: anti-cyclin B at 1:50 dilution(Development Studies Hybridoma Bank, University of Iowa, Iowa City, IA),anti–phospho-Cdk1 Thr14/Tyr15 1:200 dilution (Santa Cruz Biotechnology;sc-28435-R). Samples were washed three times in PBS containing 0.1%Tween20 followed by 45 min incubation in secondary antibodies, AlexaFluor 488 and 568 (Invitrogen), each at a 1:2,500 dilution. Samples werethen washed three times in PBS containing 0.1% Tween20 and mountedin Vectashield with DAPI (Vector Laboratories).

Microscopy. Confocal images of live embryos were taken on a Nikon EclipseTE2000U (spinning-disk confocal) microscope using IPLab 4.0.8 software(BioVision Technologies). The microscope is equipped with a 60 × 1.4 N.A.

Apo objective, four LMM5 laser merge module with diode lasers (excitationat 405, 491, 561, and 655 nm) from Applied Research, a CSU10 spinning-diskunit by Yokogawa, and a C9100-13 EM-CCD camera by Hamamatsu. Immu-nostained embryo images were taken on a Nikon E800 microscope usingIPLab 3.9.5 software (BioVision Technologies). The microscope is equippedwith a 60 × 1.4 N.A. Apo objective and a C4742-95 CCD camera by Hamamatsu.Images were processed with IPLab 3.9.5 and 4.0.8, ImageJ 1.44o (http://imagej.nih.gov/ij), Adobe Photoshop CS Version 8.0, and Adobe Illustrator CS5Version 15.1.0.

ACKNOWLEDGMENTS. We thank Andy Golden, Amy Fabritius, and EdwardKipreos for comments on the manuscript. We also thank Andy Golden andKevin O’Connell for advice, strains, and antibodies; and Geraldine Seydouxfor the PGL-1::GFP strain. Some strains were provided by the CaenorhabditisGenetics Center, which is funded by the National Institutes of Health Officeof Research Infrastructure Programs (P40 OD010440). M.M.R., S.R., D.J.-S.and O.C.-F. were funded by an intramural grant from National Institute ofDiabetes and Digestive and Kidney Diseases.

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