Functional Dissection of Caenorhabditis Elegans Clk-2/ Tel2 Cell Cycle Defects during Embryogenesis and Germline Development

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Introduction
CLK-2/TEL2 is a DNA damage checkpoint gene which is essential for viability in budding yeast, C. elegans and vertebrates. DNA damage checkpoints are essential for maintaining genome stability in response to DNA damage and act by coordinating DNA repair and by triggering a transient cell cycle arrest, or apoptosis of affected cells. The loading of a pair of highly conserved PI3 kinase-related kinases (PIKKs), ATM and ATR, to sites of DNA damage acts at the apex of DNA damage response pathways [1]. These kinases have overlapping substrate specificity and phosphorylate multiple targets including the kinases Chk1 and Chk2 [2,3]. The first C. elegans clk-2 allele initially referred as rad-5 (mn159), was isolated in a screen for C. elegans mutants hypersensitive for ionizing irradiation [4]. C. elegans clk-2 temperature sensitive mutants are embryonic lethal at the restrictive temperature of 25uC [5][6][7]. However, the cause of this embryonic lethality is not known. At the ''permissive temperature'' of 20uC both known clk-2 temperature sensitive alleles lead to a slow growth phenotype that is particularly evident in the clk-2 (qm37) allele, which also shows a reduction in cyclic behaviours such as pharyngeal pumping [5,6]. Furthermore, both alleles are defective in various DNA damage responses including DNA damage-induced germ cell apoptosis and cell cycle arrest when propagated at 20uC [5,6]. CLK-2/TEL2 has been implicated in S-phase regulation and DNA damage checkpoint responses in fission yeast [8,9], and human CLK-2/TEL2 is required for the DNA replication checkpoint and for DNA crosslink repair [10]. Human and yeast CLK-2/TEL2 directly bind to all PI3K-related protein kinases (PIKKs) and are considered to be required for maintaining their stability [8,9].
Here we use the C. elegans experimental system to assess the essential functions of CLK-2 during development and cell cycle control. In worms cell cycle progression in early embryos occurs very rapidly, with alternating M and S phases and an apparent lack of gap phases [11]. The timing and pattern of cell division and differentiation is invariant and has been fully characterized [12]. Aberrant embryonic development can therefore be traced by cell lineage analysis and resolved at a cellular level [13]. A relatively high level of DNA damage is tolerated during rapid embryonic cell divisions, possibly as a result of natural selection that favours a rapid pace of replication at the expense of genome integrity [14]. Only high levels of DNA damage or replication failure lead to a DNA damage checkpoint-dependent slowing of cell cycle progression [14]. Interestingly, the DNA damage checkpoint is used during early embryogenesis to contribute to the asymmetry of the first zygotic cell division [15]. In contrast to this, cell proliferation is much slower in the C. elegans germline and DNA damage checkpoint signalling is much more sensitive [11]. The germline is the only proliferative tissue in adult worms. The gonad contains various germ cell types that are arranged in an ordered distal to proximal gradient of differentiation [16,17]. The distal end of the gonad is comprised of a mitotic stem cell compartment, which is followed by the transition zone where entry into meiotic prophase occurs. Proximal to the transition zone most germ cells are in meiotic pachytene and subsequently complete meiosis and concomitantly undergoing oogenesis in the proximal gonad. DNA replication failure and DNA double strand breaks lead to a prolonged cell cycle arrest of mitotic germ cells and to apoptosis of meiotic pachytene germ cells [18]. In this DNA damage response pathway CLK-2 and ATL-1 act as upstream DNA damage signalling molecules, while the worm p53 like gene cep-1 is only required for apoptosis [19,20]. Thus, CLK-2 and ATL-1 are part of sensitive germ cell DNA damage checkpoint pathways that ensure the faithful transmission of genetic information from one worm generation to the next.
C. elegans clk-2 ts mutants show that CLK-2 is required for embryonic development [6,7]. As these mutants show an increased level of DNA damage in germ cells at the restrictive temperature, the embryonic lethality might be caused by the accumulation of DNA damage that ultimately may result in the death of the embryo [21]. Given that ATR stability depends on CLK-2 [8,9], the depletion of CLK-2 might phenocopy the atl-1 (worm ATR) mutant phenotype, which is germline sterility associated with massive levels of DNA double strand breaks [22]. Furthermore, given that CLK-2 is required for the stability of all PIKKs clk-2 mutations might mimic the phenotype of depleting other PIKKs such as TOR-1, implicated in nutrient sensing [23] and SMG1, a kinase involved in nonsense-mediated mRNA decay [24]. Finally, loss of CLK-2 function might result in distinct developmental defects not directly predicted from failing to maintain normal levels of PIKKs or from potential DNA replication and/or DNA damage signalling defects. In this study, we assess the essential defects associated with clk-2 by analysing embryonic cell divisions by cell lineage analysis and by exploiting the C. elegans germline system. We show that clk-2 mutants exhibit defects in early embryonic development and in germline cell cycle progression. These phenotypes do not overlap with reported C. elegans PIKK deletion phenotypes.

Results
Embryonic Cell Cycle Delay in clk-2 Mutants Depends on atl-1/chk-1 We wished to determine why clk-2 mutants fail to complete embryogenesis. We therefore started our analysis by following the embryonic development of the two available recessive clk-2 thermosensitive (ts) mutants, mn159 and qm37, ( Figure S1) by cell lineage analysis using 4D microscopy. Analysis of clk-2 mutant lineages at the restrictive temperature of 25uC revealed that asymmetric cell divisions occurred normally during the first three embryonic cell cycles as previously reported [5,6] but that cell division timing of all cells was delayed compared to wild type ( Figure 1A, B, Table S1). This delay was more pronounced in clk-2 (qm37) than in clk-2 (mn159) ( Figure 1B, Table S1). In the depicted recordings, the wild type embryo is at the 4-cell stage 11 min after cytokinesis of the P0 cell while the clk-2 (mn159) embryo is about to reach the three cell stage with the AB cell approaching cytokinesis ( Figure 1C). The depicted clk-2 (qm37) embryo is at the two cell stage with the AB blastomere just having undergone nuclear envelope breakdown ( Figure 1C). Thirty-one minutes after P0 cytokinesis wild type embryos are at the 8-cell stage while both clk-2 mutants are in the 6-cell stage. We next aimed to determine the cause of the cell cycle delay associated with clk-2 mutants. Given that clk-2 (mn159) worms show increased DNA double strand breaks in the mitotic zone of the adult C. elegans germline at the restrictive temperature [22], we reasoned that the cell cycle delay in clk-2 (mn159) and (qm37) embryos might be due to excessive DNA damage, potentially resulting from compromised DNA replication. We therefore tested whether RAD-51 foci, which are indicative of processed DNA double strand breaks or stalled replication forks [25], accumulate in clk-2 embryos at the restrictive temperature. We indeed observed increased levels of RAD-51 foci in embryos examined between the 100 and 200 cell stage in both clk-2 (mn159) (2.1460.62 foci/nucleus n = 7 embryos) and clk-2 (qm37) (0.9760.19 foci/nucleus n = 8) mutants compared to wild type (0.260.02 foci/nucleus n = 6) ( Figure 2A). These results indicate that clk-2 mutants display a delay in embryonic cell cycle timing and increased genomic instability.
Given the delay in cell division timing and the accumulation of RAD-51 foci in clk-2 mutants, we asked if the delay is due to the activation of the DNA replication checkpoint. Previous studies showed that the ATL-1/CHK-1 checkpoint is needed for sensing replication failure in C. elegans embryos [15]. Furthermore, the ATL-1/CHK-1 checkpoint contributes to developmental asymmetry by being in part responsible for the DNA replication delay in the P1 cell. Co-depletion of atl-1 and chk-1 is needed to fully inactivate the DNA replication checkpoint [15]. We observed that Author Summary PI3K-related protein kinases (PIKKs) ATM and ATR are essential upstream components of DNA damage signalling pathways, while TOR-1 acts as a nutrient sensor. CLK-2/ TEL2 is a conserved gene initially implicated in budding yeast telomere length regulation and uncovered in the same genetic screen as the yeast TEL1 ATM like kinase. CLK-2/TEL2 was first implicated in DNA damage response signalling by C. elegans genetics, a function confirmed in yeast and human cells. In addition, CLK-2/TEL2 is essential for cellular and organismal survival from yeasts to vertebrates, but the essential phenotypes were not defined. A direct interaction between CLK-2/TEL2 and all PI3K-related protein kinases and the reduction of PIKK protein levels upon CLK-2/TEL2 depletion lead to the widely discussed notion that CLK-2/TEL2 mutants might phenocopy PIKK depletion phenotypes. We take advantage of embryonic lineage analysis and germline cytology to dissect developmental and cell cycle related functions of CLK-2. CLK-2 depletion does not phenocopy PIKK kinase depletion. We rather link CLK-2 to multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development. Furthermore, we implicate CLK-2 in a distinct cell lineage decision and show that its depletion leads to a novel germline cell cycle arrest phenotype.
upon atl-1/chk-1 depletion cell cycle timing is faster beyond the first embryonic cell division ( Figure 2B, Table S1). We therefore conclude that the ATL-1/CHK-1 pathway acts in normal C. elegans early embryonic development to slow down cell cycle progression. As expected, atl-1/chk-1 (RNAi) rescued the prolonged cell cycle delay associated with depleting the DIV-1 DNA polymerase primase alpha-subunit [15] ( Figure 2B, Table  S1). Importantly, RNAi-mediated atl-1/chk-1 depletion largely rescued the delay in cell division timing associated with both clk-2 mutants ( Figure 2B, Table S1). Our results thus indicate that clk-2  Table S1. C) Nomarski images of early wild type and clk-2 embryos undergoing the second and third round of mitotic divisions. All embryos are shown with anterior to the left and dorsal up. Scale bar: 10 mm. doi:10.1371/journal.pgen.1000451.g001 (mn159) and clk-2 (qm37) mutations result in increased DNA damage, which triggers the ATL-1/CHK-1 checkpoint.
It has previously been shown that embryonic lethality associated with dut-1 (RNAi), which leads to the misincorporation of uracil during DNA replication is partially rescued by clk-2 (RNAi) and chk-1 (RNAi) as well as by the clk-2 (mn159) mutation [26]. These results hint towards a checkpoint function of CLK-2 in embryonic cell divisions. We therefore assessed if CLK-2 functions in DNA damage checkpoint signalling in embryos and asked if the cell cycle delay caused by div-1 (RNAi) depends on clk-2. We found that the delay in S-phase progression of the P1 cell caused by div-1 (RNAi) is partially rescued by both clk-2 ts alleles (Table S2). These results suggest that CLK-2 has a checkpoint function in early embryos. However, the AB cell cycle delay is not rescued likely due to the above described cell cycle delay associated with clk-2 ts mutations.
The Cell Cycle Delay of clk-2 Mutants Depends on cep-1 It was reported that CLK-2 and CEP-1, the single C. elegans p53 homolog, cooperate in pathways leading to germ cell apoptosis upon treatment with ionizing irradiation (IR) [19,20]. cep-1 mutants are defective in IR induced apoptosis but are wild type for IR induced cell cycle arrest and DNA repair suggesting that CEP-1 acts downstream of CLK-2 in the DNA damage response pathway. Derry et al. also observed that a cep-1 deletion partially rescues the slow growth phenotype associated with clk-2 (mn159) and clk-2 (qm37) [27]. We first confirmed the reported partial rescue of the slow growth phenotype of clk-2 (mn159) and (qm37) by the cep-1 (lg12501) deletion ( Figure S2A/B) [27].
Given the rescue of the clk-2 slow growth phenotype by cep-1 we wondered if cep-1 (lg12501) would suppress the embryonic cell cycle delay of clk-2 mutants. cep-1 (lg12501), which results in a slightly slower developmental rate compared to wild type, partially rescued the embryonic cell cycle delay associated with both clk-2 alleles ( Figure 2C, Table S1). In contrast, the cell cycle delay in div-1 embryos was not rescued by cep-1 (lg12501) ( Figure 2C, Table  S1). This may indicate that distinct DNA lesions occurring in clk-2 mutant embryos but not a general failure of DNA replication as it occurs in div-1 mutations leads to the activation of a cep-1 dependent checkpoint during early C. elegans embryogenesis. In addition, we found that clk-2 (mn159) or (qm37); cep-1 (lg12501) double mutants develop to a later embryonic stage and often arrest in morphogenesis stage, with clear signs of tissue differentiation such as the formation of the pharynx or the appearance of gut granules. This late arrest never occurs in either clk-2 single mutant or atl-1/chk-1 (RNAi) depleted clk-2 embryos ( Figure 3A, B).
Given the rescue of the clk-2 mutant cell cycle delay by a cep-1 deletion, we asked if CEP-1 might be modified in clk-2 mutant worms and assayed for changes in its abundance by western blotting. We found that the levels of CEP-1 protein were markedly increased in extracts prepared from synchronised adult clk-2 (mn159) and clk-2 (qm37) worms compared to wild type, indicating that the checkpoint triggered by clk-2 mutations leads to the accumulation of CEP-1 ( Figure 3C). This accumulation of CEP-1 likely results from increased CEP-1 in embryos. CEP-1 germline levels are not increased in clk-2 mutants (data not shown) and besides embryonic and germline expression CEP-1 is only expressed in very few cells in the pharynx [19] (data not shown).
In summary, we show that deleting cep-1 partially rescues the slow growth phenotype associated with clk-2 mutants and that CEP-1 accumulates in clk-2 mutants. It will be interesting to determine the mechanism of CEP-1 accumulation and if other embryonic defects also lead to CEP-1 accumulation. Figure 2. DNA double strand break accumulation in clk-2 worms and attenuation of clk-2 cell cycle delay by atl-1/chk-1 (RNAi) and cep-1. A) Wild type and clk-2 embryos were stained with anti RAD-51 antibodies (red) and DAPI (blue). Scale bar: 10 mm. B) Cell cycle timing is advanced in atl-1/chk-1 (RNAi) embryos (blue) and atl-1/chk-1 (RNAi) suppresses the cell cycle delay associated with clk-2 and div-1 mutants. C) The cep-1 (lg12501) deletion partially rescues the clk-2 cell cycle delay but does not rescue the div-1 cell cycle delay. doi:10.1371/journal.pgen.1000451.g002 We speculated that there might also be phenotypes occurring in early clk-2 (mn159) and clk-2 (qm37) embryos that are not linked to the cell cycle delay of clk-2 mutants. Indeed, our lineage analysis revealed that 2 out of 7 clk-2 (mn159) and 6 out of 12 clk-2 (qm37) mutant embryos recorded at 25uC exhibit a distinct lineage defect ( Figure 4). We found an abnormal spindle rotation of the ABar cell (the anterior right granddaughter of the AB founder cell) at the 8cell stage in clk-2 mutants. In the six clk-2 (qm37) embryos showing the abnormal spindle rotation ABar divided on average 4866u off the a-p axis placing ABarp towards the ventral side of the embryo. In five wild-type embryos ABar divided on average by 54614u off the a-p axis placing ABarp towards the dorsal side of the embryo. The ABar spindle in the six affected clk-2 (qm37) embryos thus derived 102u from wild type. This abnormal rotation gives rise to mispositioned ABarp and ABara daughters at the 12 cell stage, bringing ABarp instead of ABara in contact to the MS blastomere ( Figure 4, Videos S1, S2, and S3). The MS blastomere emits an inductive signal which in wild type is part of the left versus right cell fate decision ( Figure 4, arrows, Videos S1, S2, and S3) [28][29][30]. As a consequence cell fates of the early founder cells are changed in the clk-2 mutants, the ABara and ABarp blastomeres adopt the fates of their left counterparts, ABala and ABalp, respectively (data not shown). This change in cell fate identity leads to embryonic death. A failure of the ABar blastomere to rotate the spindle properly can be taken as an indication that spindles are generally not polarised properly [31], which is a hallmark of mutants in mom-2 (wnt) and mom-5 (frizzled) [32]. Future work will reveal, if clk-2 influences the Wnt pathway directly or if the observed clk-2 phenotype is independent of this pathway.

clk-2 Is Required for Germ Cell Proliferation
To further assess potential developmental and cell proliferationassociated defects of clk-2 mutants, we analysed the germline of clk-2 mutants. clk-2 ts mutants are deficient in responding to DNA damaging agents [5] at the ''permissive temperature'' of 20uC and shifting clk-2 (mn159) mutants to 25uC at the L4 stage leads to the accumulation of DNA damage in affected germ cells [22]. However, these studies were done with the clk-2 ts alleles. As it is not clear whether they act as null alleles at 25uC we analysed a clk-2 deletion allele.
The clk-2 (tm1528) deletion allele provided by the Japanese C. elegans knockout consortium lacks part of the 59 region, the first three exons, and a part of the fourth exon ( Figure S1A). Western blotting with a CLK-2 specific antibody provided by Simon Boulton failed to detect any CLK-2 protein in clk-2 (tm1528) worm extracts ( Figure S1B). We found that the major phenotype associated with the clk-2 (tm1528) deletion mutant kept at 20uC is not embryonic lethality but germline sterility ( Figure 5A, see below) and that the same phenotype occurs when the clk-2 (tm1528) deletion mutant is kept at 25uC (data not shown). The clk-2 (tm1528) phenotype is recessive (data not shown). Given that clk-2 (tm1528) worms go through embryogenesis whereas clk-2 (mn159) and (qm37) worms arrest during embryogenesis at the restrictive temperature, we assume that clk-2 (tm1528) worms are rescued by maternal contribution. To ascertain that the missing embryonic lethality of the clk-2 (tm1528) mutant is indeed caused by the maternal supply we reviewed the phenotype of clk-2 (mn159) and clk-2 (qm37) worms by shifting those mutants to 25uC at the L1 stage. Under these conditions we found that clk-2 (qm37) worms are 100% sterile similar to clk-2 (tm1528) worms, while the weaker allele mn159 does not lead to sterility ( Figure 5A). Both ts alleles, as well as the deletion, lead to a protruding vulva phenotype (pvl) ( Figure 5A). This phenotype is often associated with sterile germlines and general problems in postembryonic cell cycle progression [33]. clk-2 (qm37) and clk-2 (tm1528) gonades are significantly smaller in size than those of wild type and clk-2 (mn159) mutants and clk-2 (qm37) and (tm1528) gonads showed a dramatic reduction of germ cell numbers ( Figure 5B, Figure S3). This reduction in germ cell numbers and germline sterility was also obtained upon clk-2 RNAi in the weaker clk-2 (mn159) mutant, further indicating that the clk-2 (qm37) and clk-2 (tm1528) germline phenotypes represent the clk-2 null phenotype ( Figure 5B). These results are in contrast to a previous report which stated that no sterility of clk-2 (qm37) germlines was observed [6].
The reduced germ cell number raised the question whether CLK-2 is required for germ cell proliferation or germ cell differentiation. To address this question we performed a time course analysis of germline development in wild type and clk-2 (tm1528) worms. We found that both strains have similar numbers of germ cells up to the L4 stage at which point germlines of clk-2 (tm1528) worms stop proliferating ( Figure 5C). To further assess if this phenotype is caused by a proliferation defect we took advantage of gld-2 (q497) gld-1 (q485) double mutants which have germlines that do not enter meiosis and are thus entirely proliferative. Comparing gld-2 (q497) gld-1 (q485) germlines to gld-2 (q497) gld-1 (q485); clk-2 (tm1528) triple mutant germlines we found that germ cell numbers are dramatically reduced in the triple mutant indicating that clk-2 has a role in germ cell proliferation rather than in germ cell differentiation ( Figure 5D). In addition, clk-2 (tm1528) and clk-2 (qm37) germ cells are larger than wild type. This phenotype, which is reminiscent of arrested mitotic germ cells after ionizing irradiation, indicates that cells might stop cell division but continue with cellular growth [18] ( Figure 5B, arrowheads). In summary, our data suggest that CLK-2 is required for cell cycle progression in germ cells.

clk-2 Germ Cells Arrest in an Early Prophase-Like Stage Independent of Checkpoint Activation
Given that clk-2 mutations lead to a DNA damage checkpoint dependent delay of embryonic cell cycle progression ( Figure 2B) and given that clk-2 (mn159) germ cells showed elevated levels of RAD-51 foci indicative of faulty replication when shifted to the restrictive temperature at the L4 stage [22], we suspected that the germ cell cycle arrest of the clk-2 (tm1528) mutant might be due to the activation of the DNA damage checkpoint. We therefore examined if RAD-51 foci occur in the mitotic compartment of clk-2 (tm1528) germ cells. To our surprise we found that like in wild type germ cells, RAD-51 was mainly localized in the cytoplasm of clk-2 (tm1528) germ cells and did not form nuclear foci ( Figure 6A, Video S4, Table 1). In contrast, clk-2 (mn159) shifted to the restrictive temperature of 25uC at the L1 or the L4 stage accumulated RAD-51 foci ( Figure 6A, Video S6, Table 1) while clk-2 (qm37) formed fewer foci (Video S5, Table 1). Thus RAD-51 foci accumulate mostly Figure 5. clk-2 is required for germ cell proliferation. A) Fertility and Pvl phenotype of various clk-2 mutants. clk-2 (mn159) and (qm37) were grown at 25uC, clk-2 (tm1528) was grown at 20uC. B) Nomarski DIC images of adult mitotic germlines. The distal tip cell is to the right of each germline. Arrowheads depict individual germ cell nuclei which are enlarged in clk-2 (tm1528), clk-2 (qm37) and in irradiated wild type animals. C) Number of germ cell nuclei per gonad arm in clk-2 (tm1528) (n = 10) and wild type (n = 10) worms. Worms were synchronised by L1 starvation, transferred to seeded NGM plates and grown at 20uC, fixed at indicated time points and stained with DAPI. Error bars represent SD. D). Images of gld-2 (q497) gld-1 (q485) (left) and gld-2 (q497) gld-1 (q485); clk-2 (tm1528) (right) gonads stained with DAPI. Loss of CLK-2 results in a reduction of mitotic germ cells. Worms were grown at 20uC. Scale bar: 10 mm. doi:10.1371/journal.pgen.1000451.g005 in the weak clk-2 (mn159) allele as reported previously [22], while less foci formation is observed in clk-2 (qm37) and only very few RAD-51 foci can be found in clk-2 (tm1528) ( Table 1).
The defect in RAD-51 foci formation in clk-2 (tm1528) might be due to a cell cycle arrest outside of S-phase or due to a failure to process DNA double strand breaks, which is needed for RAD-51 focus formation. To test whether DNA double stand break processing is defective in clk-2 (tm1528) mutants we tested whether focus formation occurred after inducing DNA double strand breaks by exposing worms to ionizing irradiation. Irradiationinduced RAD-51 focus formation indicated that double strand break processing occurs normally in clk-2 (tm1528) worms ( Figure 6B). Summing up, these results indicate that the clk-2 (tm1528) deletion does not lead to excessive DNA damage and that CLK-2 is not needed for DNA double strand break processing.
To further analyse the cell cycle arrest associated with CLK-2 depletion, we asked if clk-2 (tm1528) germ cells arrest in a distinct cell cycle stage. To facilitate this analysis we first established G2 Figure 6. RAD-51 foci do not accumulate in clk-2 (tm1528) germlines. A) Representative images of fixed germlines from adult worms stained with anti-RAD-51 antibody (red) and DAPI (blue). Wild type and clk-2 (tm1528) were analysed after growth at 20uC, clk-2 (mn159) and (qm37) were analysed 24 hours after reaching the L4 stage upon shifting to 25uC at the L1 stage. Scale bar: 10 mm. Representative inserts depict mostly cytoplasmic RAD-51 staining in wild type and clk-2 (tm1528) germlines. B) RAD-51 nuclear foci formation in wild type and clk-2 (tm1528) mitotic germlines upon ionising irradiation. doi:10.1371/journal.pgen.1000451.g006 and M phase cell cycle markers. Prior to mitotic entry Cdk1 is kept inactive by Tyr-15 phosphorylation [34,35]. An antibody recognizing Tyr-15 phosphorylation of mammalian Cdk1 cross reacts with the corresponding phospho-epitope of C. elegans NCC-1/CDK-1. Phospho-NCC-1/CDK-1 can be detected until late prophase in worm embryonic divisions [36]. To confirm that phospho-NCC-1 is indeed indicative of G2/M arrested germ cells, we irradiated wild type germlines and found that all germ cells arrested in G2 with high levels of NCC-1 P-Tyr15 ( Figure 7A). We observed NCC-1 Tyr-15 phosphorylation in only few wild type and clk-2 (mn159) mitotic germ cells but found that all clk-2 (tm1528) cells and clk-2 (qm37) cells showed high levels of NCC-1 Tyr-15 phosphorylation even in the absence of ionizing irradiation ( Figure 7A).
The clk-2 prophase arrest phenotype might be caused by a direct prophase defect or alternatively by replication defects which could trigger a checkpoint-dependent late G2/M cell cycle arrest. To assess these possibilities, we depleted atl-1/chk-1 in clk-2 (tm1528) worms. The efficiency of atl-1/chk-1 (RNAi) depletion was confirmed by observing germ cell micronuclei [22] and by the embryonic lethality of the progeny of RNAi depleted wild type worms (data not shown). We found that all cells of clk-2 (tm1528) atl-1/chk-1 (RNAi) germlines were NCC-1 Tyr-15 phosphorylation-positive ( Figure 7B). We therefore conclude that cell cycle arrest is unlikely to be mediated by activation of the ATL-1/CHK-1 DNA damage checkpoint ( Figure 7B).
To further analyze the cell cycle stage of clk-2 (tm1528) germ cells we also used antibodies against phosphorylated histone H3 (P-H3). In C. elegans P-H3 staining can be observed in cells from prophase/early prometaphase to late telophase [37]. When wild type gonads were stained with anti-P-H3 antibody only 2-5 nuclei per gonad arm were stained and all stained cells displayed a metaphase-like morphology. While we observed the same phenotype for clk-2 (mn159) worms grown at 25uC, all germ cells were P-H3 positive in clk-2 (qm37) worms propagated at 25uC and in clk-2 (tm1528) worms ( Figure 7C). However, P-H3 positive cells did not show a metaphase-like morphology. Rather, in most nuclei chromosomes appear to be partially condensed but not aligned at the metaphase plate suggesting a prophase or very early prometaphase arrest. This arrest neither depends on atl-1/chk-1 ( Figure 7D), nor on cep-1 ( Figure S4). Thus while cep-1 and atl-1/ chk-1 are required for delaying cell cycle progression in clk-2 embryos, the germ cell cycle arrest observed in clk-2 (tm1528) mutants does not depend on either of these genes.
Given the early prophase arrest we also assessed centrosome behaviour in clk-2 (tm1528) germ cells. Centrosome duplication occurs during S-phase and centrosomes split during late G2 phase. In prophase, centrosome maturation is an essential prerequisite for the assembly of the mitotic spindle, and centrosomes can be visualized through the accumulation of a and c-tubulin (for review see, [38]). Increased a-tubulin nucleation is followed by the formation of mitotic spindles [38]. When gonads were immunostained for c-tubulin [39] to label centrosomes we found that centrosome duplication occurs normally in clk-2 (tm1528) worms ( Figure 8A). Furthermore, double immunostaining for c-tubulin and a-tubulin ( Figure 8B) showed that several wild type germ cells exhibited accumulated a-tubulin, indicative of centrosome maturation and imminent spindle formation. In contrast, no a-tubulin accumulation and no spindle formation could be observed in clk-2 (tm1528) germ cells, although germ cells with duplicated and separated centrosomes were present ( Figure 8B). These results raise the possibility that the prophase-like cell cycle arrest phenotype of clk-2 (tm1528) germ cells might be due to the activation of the spindle assembly checkpoint, which responds to defects in spindle formation and kinetochore-microtubule attachment and blocks anaphase progression until correct bi-orientation has occurred [40]. We therefore tested if the RNAi depletion of the C. elegans MAD1 spindle checkpoint gene ortholog mdf-1 [41] rescues the cell cycle arrest phenotype observed in clk-2 (tm1528) worms. Even though both wild type and clk-2 (tm1528) strains displayed the typical previously described pre-meiotic like morphology of mdf-1 (RNAi) germ cells [41] (Figure 8C), mdf-1 (RNAi) clk-2 (tm1528) germ cells still uniformly stained P-H3 positive ( Figure 8C). In summary, our analysis of clk-2 germlines suggests that clk-2 is essential for cell proliferation and that cells deficient in CLK-2 arrest in prophase without forming a mitotic spindle. The CLK-2 cell cycle arrest phenotype is independent of DNA damage and spindle checkpoint activation.

Discussion
In aiming to define the developmental and cell cycle-related functions of clk-2, we found multiple roles of this conserved gene (summarized in Table 2). Aided by C. elegans cell lineage analysis, we found spindle orientation defects that can lead to cell fate transformations. Furthermore, dsDNA breaks accumulate in clk-2 point mutations and embryonic cell cycle progression is retarded starting from the very first cell division. During early embryonic cell divisions the CLK-2 cell cycle delay can be rescued by depleting the ATL-1/CHK-1 pathway. CEP-1 accumulates in clk-2 mutants and deletion of cep-1 partially rescues the cell cycle delay associated with clk-2 point mutations. Analysis of the clk-2 (tm1528) deletion reveals that these worms progress through embryogenesis (due to maternal rescue), but then halt cell cycle progression in the germline. This arrest phenotype, which occurs at an early prophase-like stage, appears to be independent of DNA damage and spindle checkpoint activation.

clk-2 Mutant Worms Do Not Phenocopy PIKK Depletion
It has recently been shown that CLK-2/TEL2 interacts with all PIKKs in budding and fission yeast as well as in mammals [8,9,42,43]. CLK-2/TEL2 depletion leads to reduced levels of PIKKs, and using CLK-2/TEL2 mouse knockout lines it was shown that the half life of PIKKs is reduced in those cell lines [9]. This finding together with the notion that PIKK dependent checkpoint signalling is reduced in cells lacking CLK-2/TEL2 led to the hypothesis that CLK-2/TEL2 might function in checkpoint signalling by regulating PIKK kinase levels. Given the conserva-  Figure 7. clk-2 (tm1528) and clk-2 (qm37) mitotic germ cells arrest in a distinct cell cycle stage. A) CDK-1/NCC-1 P-Tyr15 staining. Germlines of wild type, clk-2 (tm1528), as well as clk-2 (qm37) and clk-2 (mn159) (shifted to 25uC at the L1 stage) were stained with NCC-1 P-Tyr15 antibody (red) and DAPI (blue). B) CDK-1/NCC-1 P-Tyr15 staining after atl-1/chk-1 RNAi depletion in wild type and clk-2 (tm1528) worms C) PhosphoH3 staining. Germlines of wild type and clk-2 (mn159, qm37 and tm1528), propagated as described in A) were stained with phospho-H3 antibody (red) and DAPI (blue). D) Phospho-H3 staining of wild type and clk-2 (tm1528) depleted of atl-1/chk-1 by RNAi. Efficiency of atl-1/chk-1 RNAi depletion was confirmed by observing micronuclei and by scoring dead embryos in the next generation [22] (data not shown). For each representative picture shown in Figure 7 at least 15 germ lines were analysed. doi:10.1371/journal.pgen.1000451.g007 tion of the CLK-2 PIKK interaction it is likely that this interaction also occurs in C. elegans, albeit we could not confirm this since we were unable to generate specific CLK-2 and ATR antibodies suitable for immunoprecipitation from worm extracts (data not shown). Nevertheless, our genetic results suggest that, at least in C. elegans, CLK-2 depletion does not phenocopy PIKK depletion phenotypes (summarized in Table 2). atl-1/ATR and clk-2 mutations have opposite phenotypes during embryonic develop-ment and a clk-2 deletion, unlike atl-1 depletion [22], does not lead to mitotic germ cell catastrophe. Concerning ATM, this worm PIKK is primarily involved in responding to UV-induced DNA damage where, like CLK-2 it is required for UV-induced apoptosis [44]. Furthermore, an atm-1 deletion only shows weak defects in responding to ionizing irradiation [44], unlike clk-2 (qm37) and clk-2 (mn159) point mutations. Similarly, clk-2 deleted worms do not resemble worms depleted for tor-1, which arrest in Figure 8. Centrosome duplication occurs but spindle formation is abolished in clk-2 (tm1528). A) Images of fixed mitotic germlines of adult worms stained with c-tubulin (red) and DAPI (blue). Arrowheads indicate centrosomes. Germ cells with one as well as two centrosomes can be found as indicated in the representative magnified panels. B) Germlines were stained for a-tubulin (green), and c-tubulin (red) and DAPI (blue). In contrast to wild type, no mitotic spindles can be observed in clk-2 (tm1528) as indicated in the representative magnified panels. C) mdf-1 (RNAi) does not abolish phospho-H3 staining (red) of clk-2 germlines. mdf-1 RNAi depletion was confirmed by the premeiotic like appearance of germ cells (data not shown) [41]. For each representative picture shown in Figure 8 at least 15 germ lines were analysed. doi:10.1371/journal.pgen.1000451.g008 the L3 larval stage and show concomitant gonadal and intestinal degradation [45]. It is possible that partial tor-1 depletion which results in a slow growth phenotype and enhanced longevity [46], overlaps with the clk-2 (qm37) phenotypes that include a slow growth and a relatively weak longevity phenotype [6,7,21]. However, the enhanced life span of clk-2 (qm37) worms is rather weak and clk-2 (tm1528) life span is dramatically reduced compared to wild type (data not shown). Our evidence suggesting that CLK-2/TEL2 might not predominately act by regulating PIKK stability is also supported by recent evidence from the budding yeast system. While steady state levels of the budding yeast ATR homologue TEL1 are somewhat reduced in tel2-1 mutants, it was shown that TEL2 is required for the loading of TEL1 to sites of DNA damage [47]. In addition, the finding that TEL2 binding to the budding yeast MEC1 ATM like kinase is lost in tel2-1 mutants while MEC-1 remains functionally intact [48], points towards the possibility that CLK-2 be able to regulate ATM and ATR PIKKs by mechanisms not directly related to TEL2 PIKK interaction.

Is CLK-2 Required for DNA Replication in the Embryo?
We observed that cell cycle progression in early clk-2 mutant embryos is generally delayed and is associated with DNA damage accumulation (for summary see Table 2). The clk-2 cell cycle delay is partially suppressed by atl-1/chk-1 and cep-1 deletion. These results are surprising in the light of previous reports suggesting that CLK-2 and ATL-1 might act together in C. elegans DNA damage response signalling in germ cells [22]. These two proteins might thus act in different pathways during C. elegans embryogenesis. Our results suggest that ATL-1 is active in clk-2 ts mutants. Thus even if there is a reduced level of ATL-1 protein in clk-2 mutant worms, enough ATL-1 is left to cause a cell cycle delay.
In embryos depleted for DNA replication factors cell cycle progression is delayed starting from the very first cell cycle and upon division of the zygote the posterior daughter, referred to as the P1 cell, is particularly strongly affected [49]. This delay depends on the ATL-1/CHK-1 dependent DNA damage checkpoint. The relatively weak replication defect of CLK-2 could be due to partial loss of function in the clk-2 (mn159) or (qm37) point mutants or due to CLK-2 being required for faithful DNA replication rather than replication per se.
Our genetic analysis implicates the C. elegans p53-like gene cep-1 in the cell cycle delay associated with clk-2 mutants during embryonic cell divisions. Interestingly, deleting cep-1 alleviates the cell cycle delay of clk-2 mutants but does not have an effect on the delay caused by div-1 mutants. Thus distinct DNA replication defects caused by div-1 and clk-2 depletion might lead to differential checkpoint activation. Our results implicate cep-1 in an embryonic DNA integrity checkpoint. Future studies will be required to address how cep-1 can slow embryonic cell cycle progression and which exact replication defects trigger CEP-1 accumulation.

CLK-2 Is Required for C. elegans Germ Cell Cycle Progression
Despite a possible role of clk-2 in embryonic DNA replication, clk-2 (tm1528) germ cells still undergo replication and do not display overt signs of genome instability. Analysis of clk-2 (tm1528) deletion mutants reveals that these worms progress through embryogenesis due to maternal rescue but then halt cell cycle progression in the germline. This arrest is distinct from the cell cycle arrest induced by DNA damage and does not require the ATL-1/CHK-1 DNA damage checkpoint and CEP-1. Similarly, this arrest does not require the spindle checkpoint. It will be interesting to assess if the cell cycle arrest is due to the requirement of clk-2 in G2 cell cycle progression or due to the activation of a further checkpoint such as the p38 stress activated checkpoint [50]. clk-2 (tm1528) worms arrest in a phosphohistone H3 positive pro-metaphase like stage with partially condensed chromosomes, while DNA damage leads to a G2 arrest characterized by high levels of phosphorylated CDK-1 Tyr 15 and the absence of phosphorylated histone H3 in wild type worms. Interestingly, CDK-1 Tyr 15 is still phosphorylated in these arrested germ cells, indicating that these cells arrest with low CDK-1 activity. Thus our data suggest that there might be an uncoupling of mitotic events in clk-2 (tm1528) germ cells. Clk2/Tel2 has also been shown to be required for cellular proliferation in mouse embryonic fibroblasts. The arrest after CLK-2/TEL2 depletion is not uniform in TEL2 deficient MEFs. These cells arrest with an increased proportion of cells with a 2N or 4N DNA content, and a reduced S and M phase index, and were reported to show a 'senescence-like flattened morphology' [8]. Thus CLK-2/TEL2 might have additional functions in mammalian cells that are not directly related to cell cycle regulation. Alternatively, a cell cycle regulatory function of CLK-2/TEL2 might not be uniformly needed in all cell types.
Our analysis of clk-2 mutant phenotypes reveals distinct CLK-2 functions in embryonic cell cycle progression and in germ cell cycle progression. The clk-2 (tm1528) null allele results in the most severe germline phenotype. At present we do not know if clk-2 ts alleles are completely inactive when shifted to the restrictive temperature during early embryonic cell cycle progression. Indeed, as is the case for the germ cell cycle arrest phenotype, a complete inhibition of clk-2 might result into earlier or more severe defects during embryonic cell divisions potentially resembling the clk-2 (tm1528) germ cell cycle arrest phenotype. We extensively tried RNAi to completely inhibit clk-2 during embryogenesis using both RNAi injection and feeding procedures but never found a phenotype stronger than the phenotype of either clk-2 ts allele propagated at 25uC (data not shown). clk-2 RNAi injections did not result in any phenotype [5], and only the RNAi feeding construct introduced by the Nilsen laboratory worked for RNAi feeding. Only, when we analyzed clk-2 mutants kept at 25.5uC combined with clk-2 (RNAi) we observed more severe defects as seen in clk-2 (qm37) and clk-2 (mn159) mutants at the restrictive temperature. Under these conditions we observe a further delay of cell cycle progression (particularly in the P lineage) as compared to clk-2 ts mutants kept at the restrictive temperature ( Figure S5). This delay appears as atl-1 independent. ATL-1 dependence was, however, difficult if not almost impossible to study due to severely abnormal cell divisions (data not shown), that often resulted in cell divisions where only one daughter cell received an intact nucleus. Even without atl-1 (RNAi) treatment, nuclei often appeared as disorganized and at times fragmented under DIC optics ( Figure  S5), but we never observed uniform defects starting from the very first cell cycle, further complicating a detailed analysis (data not shown). Obviously, these findings will raise the question as to how CLK-2 might affect early embryonic cell divisions, which will be the subject of further studies. These studies will however, require new clk-2 alleles as we currently can not rule out the possibility of off target effects associated with clk-2 RNAi that might unspecifically enhance clk-2 mutant defects. At present, we can only speculate if the developmental, cell cycle related and DNA damage response pathway defects associated with clk-2 mutations are due to a single molecular defect. We, indeed, favour an alternative model according to which CLK-2 affects multiple molecular processes. Our analysis which is based on an allelic series of clk-2 mutants with increasing strength clearly indentifies distinct functions associated with CLK-2 during embryonic and germ cell cycle progression as well as during embryonic development. It was recently shown that TEL2/CLK-2 belongs to the ARM repeat superfamily of structurally related proteins [47] (Alexander Schleiffer, personal communication). Tandem ARM repeats fold together into a superhelical fold to form a surface for protein-protein interactions (for review see, [51,52]). ARM repeat proteins are structurally related to proteins containing tandem HEAT motifs [51]. The demonstrated interactions between Tel2/CLK-2 and the HEAT repeat containing PIKKs suggests that TEL2/CLK-2 might act as an adaptor protein that impinges on multiple signalling pathways besides PIKKs through ARM/HEAT domain mediated protein-protein interactions. Our dissection of CLK-2 phenotypes in C. elegans is likely to stimulate future studies in mammalian cells addressing developmental and cell cycle-related functions of CLK-2/TEL-2.

RNAi Analysis
RNAi was performed by using the feeding procedure [56]. RNAi-expressing bacteria were seeded on NGM agar plates containing 3 mM IPTG and 50 mg/ml ampicillin, and worms were added as L4 larvae the following day. Animals were fed with bacteria carrying an empty L4440 feeding vector [57] or atl-1, chk-1 [15] and mdf-1 feeding (MRC geneservice) constructs. Phenotypes were observed in F1 animals. F1 animals in the L4 stage were placed onto RNAi plates. F2 embryos were analysed after 24 h of incubation, and F1 animals were analysed after 48 h to observe germline phenotypes.

Germ Cell Counts
Worms at the indicated time post-L1 were stained by DAPI using the following procedure. Animals were transferred to 100 ml M9 buffer and washed 36with M9 buffer and resuspended in 1 ml 96% ethanol containing DAPI (200 ng/ml) for 1 h and rehydrated with 1 ml M9 buffer for 1 h. Worms were transferred into 3 ml of mounting solution (90% glycerol, 20 mM Tris pH 8.0, 1 mg/ml pphenylenediamine) and mounted on slides. Germ cells were identified by nuclear morphology according to DAPI staining.

4-D Microscopy, Lineage Analysis, and Cell Cycle Timing
Methods for 4D-microscopy were described in [13]. Modifications of the 4D-microscope system are described in [31]. Embryos were recorded at 25uC and stacks of 25 DIC-images, viewing different focal planes of the developing embryo, were taken every 35 sec. The 4D-recordings were analysed using the SIMI Biocell program (SIMI Reality Motion Systems, Unterschleissheim, Germany; http://www.simi.com) [31,13]. Cell cycle timing was determined by measuring the time between the two mitotic divisions (completion of cytokinesis).

Fluorescence Microscopy
Deltavision microscopy was used to examine germlines using either a 606 or a 1006, UPlanSApo objective (Olympus; NA 1.40), Soft-WoRx software (Applied Precision), and a CoolSnap HQ (Photometrics) OCD camera. Three-dimensional datasets were computationally deconvolved, and regions of interest were projected onto one dimension.

Supporting Information
Video S1 Video depicting early embryonic development of wild type embryo shown in Figure 4.