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Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
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Introduction |
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In C. elegans embryogenesis, the separation of somatic and germline cells occurs through a series of asymmetrical divisions in which embryonic blastomeres divide unequally to produce one somatic daughter and one germline daughter (Sulston et al. 1983). Three general properties distinguish germline blastomeres from somatic daughters: first, the presence of germ cell-specific granules, called P granules, second, the suppression of embryonically transcribed mRNAs and third, activation of protein expression of germline-specific maternal RNAs (Tenenhaus et al. 2001). P granules are maternally contributed to the embryo and migrate through the cytoplasm to the posterior region of the one-cell embryo, where they associate with the cortex prior to the first cleavage; P granules that remain in the anterior region are disassembled or degraded by an unknown mechanism (Hird et al. 1996). P granules are progressively partitioned to the germ lineage during each of the unequal divisions that generate a somatic founder cell and a germline blastomere (Strome & Wood 1983). This asymmetric localization ensures that P granules are segregated only to the germline blastomere P1. A P granules-positive blastomere at the 28-cell stage is called P4 and it produces only germ cell descendants. The continuous association of P granules with the germline suggests that they function in important aspects of germline development (Kawasaki et al. 1998). Recently, several proteins encoded by maternal-effect lethal genes have been shown to transiently associate with P granules during early stages of embryogenesis. These include several CCCH-type zinc-finger proteins, PIE-1, MEX-1, MEX-5/6 and POS-1, that possess the characteristic cysteine-X8–10-cysteine-X5-cysteine-X3-histidine sequence of the CCCH-type zinc-finger domain (Bai & Tolias 1996; Clarke & Berg 1998). Genetic studies have shown that they are important for differentiation of germ cells in C. elegans (Mello et al. 1992, 1996; Schnabel et al. 1996; Guedes & Priess 1997; Tenenhaus et al. 1998; Tabara et al. 1999; Reese et al. 2000; Schubert et al. 2000). PIE-1 is a predominantly nuclear protein but also associates with P granules (Mello et al. 1996), and that appears to repress transcription in the germline blastomere (Seydoux et al. 1996; Batchelder et al. 1999; Seydoux & Strome 1999; Tenenhaus et al. 2001). MEX-1, a related CCCH-type zinc-finger protein required for correct localization of PIE-1, is both dispersed in the cytoplasm and associated with P granules in the germline blastomeres (Guedes & Priess 1997). Although the biochemical function of MEX-1 is not clear, mutant analysis has suggested that MEX-1 protein regulates the translation of maternal mRNAs in the germline blastomere. Besides these genetically identified proteins, there are many as-yet-uncharacterized CCCH-type zinc-finger proteins in the database of the C. elegans genome. The functions and significance of these CCCH-type zinc-finger proteins in embryonic development have not been adequately elucidated.
In our previous study, we identified three novel redundant CCCH-type zinc-finger genes, named by us moe-1, -2 and -3, as a group related by functions and nucleotide sequences (Shimada et al. 2002). Similar results were reported by Detwiler et al. (2001), and they gave the names OMA-1 and OMA-2 to MOE-1 and MOE-2, respectively. OMA-1 and OMA-2 have about 63% amino acid similarity, and OMA-2 and MOE-3 (OMA-3) have about 45% amino acid similarity (Shimada et al. 2002). Each of them contains two copies of the conserved CCCH-type zinc-finger domain. We and Detwiler et al. (2001) have found that they have overlapping functions that are crucial for oocyte maturation. The results of our in situ hybridization have revealed that each of the oma transcripts is expressed from the distal to proximal region of the gonad, while their corresponding proteins accumulate specifically in the cytoplasm of growing oocytes as well as on P granules. These observations indicate that oma gene products function at the maturing oocyte stage. Thus, our previous results suggest that oma family gene products are unique CCCH-type zinc-finger proteins in terms of their characteristic behavior during the course of oogenesis.
In the present study, we focused on the embryonic localization and function of OMA proteins. We found that maternally accumulated OMA-1 protein is rapidly removed from the cytosol after fertilization, while it continued to associate with P granules until the formation of P4 blastomere. OMA-1 and OMA-2 play pivotal roles in the determination of the distribution of P granules and related proteins in the early stages of embryonic development. Thus, our approach has revealed a new class of regulatory roles of OMA family proteins that are collectively required for the correct specification of germline cells. We conclude that OMA family proteins are unique CCCH-type zinc-finger proteins that are essential for two vital events, oocyte maturation and proper germline determination during the course of C. elegans development.
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Results |
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To investigate the possible functions of OMA-family proteins, we tried to prepare several polyclonal antibodies to synthetic peptides derived from specific sequences of either OMA-1 or OMA-2 (Fig. 1A). The peptides used corresponded to OMA-1 N-terminal amino acids 1–14 (designated anti-OMA-1a) and amino acids 28–45 (designated anti-OMA-1b), both of which are regions specific to OMA-1 among the predicted C. elegans protein sequences from the genome database. Similarly, the region of OMA-2 N-terminal amino acids 12–37 (designated anti-OMA-2a) in the predicted OMA-2 amino acid sequence was used for production of the anti-OMA-2 antibody.
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To further evaluate the specificity of the antibodies for immuno-cytochemical analysis, we immunostained gonads of adult hermaphrodites. It has been reported that both OMA-1 and OMA-2 are specifically expressed in proximal oocytes (Detwiler et al. 2001; Shimada et al. 2002). As anticipated, all of the anti-OMA antibodies strongly and exclusively stained wild-type developing oocytes (Fig. 1C-c,g). The OMA-1a, OMA-1b and OMA-2a signals in oocytes were completely abolished by absorption using their corresponding antigenic peptides (data not shown). Furthermore, RNAi treatment of oma-1 and oma-2 abolished the fluorescence signals from anti-OMA-1b (Fig. 1C-a), anti-OMA-1a (data not shown) and anti-OMA-2a antibodies (Fig. 1C-e). It should be noted that RNAi of oma-1 did not influence OMA-2a immunosignals and that oma-2 RNAi did not influence OMA-1a signals. Furthermore, we also confirmed that double RNAi of oma-1 and oma-2 did not influence the staining of related CCCH-type zinc-finger proteins, PIE-1 and MEX-1 (Fig. 1C-i,m), in oocytes under the same experimental conditions as those used in OMA staining. All of these results indicate that there is no cross-reaction within our RNAi treatments and that a series of anti-OMA antibodies specifically and exclusively recognized the corresponding target proteins in our immunocytochemical analysis.
Cytoplasmic OMA protein decreases rapidly after fertilization
As mentioned above, large amounts of OMA proteins accumulate in the cytoplasm of maturing oocytes (Fig. 1 C-c,g). Although it has been shown that cytoplasmic OMA proteins are required for meiotic maturation (Detwiler et al. 2001; Shimada et al. 2002), OMA-1 proteins that have accumulated in oocytes may be also required for preparation for postfertilization events, as is the case with many other maternally accumulated proteins, such as PIE-1 and MEX-1. To determine whether the cytoplasmic accumulation of OMA protein is also important for postmaturation stages, we examined the expression levels and localization of OMA proteins after fertilization.
We found that the amount of accumulated cytoplasmic OMA-1 proteins in pronuclear-stage embryos (Fig. 2A-a, indicated by an arrow) had decreased markedly in dividing embryos (Fig. 2A-a, indicated by an arrowhead), as assessed by immunostaining with anti-OMA-1a antibody. A similar reduction in staining was also observed with the other anti-OMA antibodies, anti-OMA-1b (Fig. 2B) and anti-OMA-2a (Fig. 2D, Shimada et al. 2002) and was also reported using OMA-1::GFP fusion protein (Lin 2003; Nishi & Lin 2005; Shirayama et al. 2005; Stitzel et al. 2005). With both anti-OMA-1b and anti-OMA-2a antibodies, strong immunostaining was observed in pronuclear-stage fertilized eggs (Fig. 2B-a–f, in which two pronuclei are visible), whereas only a trace amount of OMA-1 signal was observed in the cytoplasm of fertilized embryos at the mitotic stage (Fig. 2B-g–n, chromosomes having already condensed). These results indicate that OMA proteins disappear from the cytoplasm of one-cell stage fertilized embryos after the initiation of first mitosis. Notably, this phenomenon was observed in all of the fertilized embryos. It is unlikely that the reduction in intensity of anti-OMA-1 and OMA-2 immunostaining after fertilization was due to a change in antibody permeability because the dividing embryos were simultaneously stained clearly by both anti-P granules (Fig. 2A-c) and anti-tubulin antibodies (Fig. 2D-b).
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We previously reported that OMA-1 protein is co-localized with P granules in growing oocytes (Shimada et al. 2002). In fertilized embryos, dotted structures in the posterior regions of embryos became visible as the intensity of cytoplasmic signals decreased with both anti-OMA-1a and anti-OMA-1b immunostaining (right half of each embryo in Fig. 2B-g,i,k,m). These granules seemed to correspond to P granules in size, number and distribution. This speculation is supported by the results of our double immunostaining experiments. As shown in Fig. 2C, anti-OMA-1b immunostaining associated on P granules in first mitotic stage fertilized eggs (Fig. 2C), although the most of cytoplasmic OMA-1 signal has been removed. These observations indicate that OMA-1 proteins remain in P granules even when free OMA-1 in the cytoplasm has disappeared after fertilization and that OMA-1 protein on P granules is able to escape from the cytoplasmic clearance and is possibly inherited to subsequent developmental processes.
To determine the possible functions of oma gene products during embryogenesis periods, we examined the distribution of OMA-1 proteins in developing embryos of various stages. We found that OMA-1 proteins continued to co-localize with P granules in the germ cell lineage blastomeres in 2 cell (P1 blastomere), 4 cell (P2 blastomere) (Fig. 3) and in the P3 germ cell lineage blastomere in blastula-stage embryos (Fig. 4A-b,B-b, indicated by arrows). RNAi treatment of oma-1 resulted in almost complete elimination of the anti-OMA-1 signals on P granules (Fig. 4A-e) compared to that in the wild-type embryos (Fig. 4A-b). Furthermore, the anti-OMA-1b signals were completely absorbed by the corresponding OMA-1b antigenic peptide and were not detected by preimmune serum (our unpublished observations). There was no cross-reaction between secondary antibodies used in these experiments, and the staining is not merely derived from false recognition of antigens. Although P granules are present at all stages of the life cycle of C. elegans, OMA-1 is not detectable in daughters of P4. Similarly, OMA-1 is not detectable on P granules of mitotic germ cells in adult gonads of hermaphrodites. These findings indicate that OMA-1 is actually a component of P granules during the early embryogenesis stages. Since P granules play a pivotal role in germline development (Kawasaki et al. 1998), OMA-1 protein might be an candidate for regulator of germline determination during the embryo stage.
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Since OMA-1 shows a characteristic distribution in developing embryos, we examined in detail the abnormalities in OMA-1- and OMA-2-compromised embryos. As reported previously, inactivation of oma-1; oma-2 genes results in arrest of oocyte maturation (Detwiler et al. 2001; Shimada et al. 2002). To determine the effect on early embryos with avoiding the defect in oocyte maturation, we employed a partially compromised RNAi approach starting from the L4 larval stage by a feeding RNAi method using unique sequences of oma-1 and oma-2 genes as targets for RNAi. Using this method, at 18 h after RNAi treatment, there were no detectable morphological abnormalities in oocyte growth, meiotic maturation and first asymmetric mitotic cleavages. Nevertheless, we found the high penetrant lethality (93%; n = 170) in these embryos thereafter and the embryos eventually did not proceed to develop beyond the embryonic stages of 80- to 100-cells.
In wild-type embryos, P granules are inherited exclusively by germ lineage blastomeres. There is a single germline cell (called the P1–P4 series of blastomere) during the early cleavage stage (Fig. 6A), and the P-blastomere then divides into two cells (Z2 and Z3) around the 100-cell stage (Fig. 6I, indicated by arrows). Z2 and Z3 blastomeres are maintained through to the L1 larval stage, until germ cells begin to proliferate to form a newly established gonad (Sulston et al. 1983). Thus, P granules are confined to no more than two cells until the larval stage (Fig. 6I). Interestingly, we found that P granules (detected by OIC1D4 antibodies) in oma-1; oma-2-compromised embryos are much more widely distributed (Fig. 6C,K,M). Most of embryos contained more than two P granule-positive blastomeres at the 10-cell stage (85.7%, n = 42) (Fig. 6C). Embryos at later stages contained four or more germline cells (Fig. 6M) and P granules were sometimes dispersed throughout the posterior half of the embryo (87.5%, n = 48) (Fig. 6K). This is not derived from a pleiotropic impairment of early embryonic polarity. It should be noted that the segregation of P granules in 1 cell-stage oma-1; oma-2 (RNAi) embryos is normal (see next section, Fig. 7A-a), ruling out the possibility that the abnormality in distribution of P granules in a later embryonic stage is due to a defect in one-cell embryos or a defect in an earlier maturation stage. Such an abnormal distribution of P granules was never observed in control embryos (Fig. 6A, I) or in embryos from single RNAi treatment of either oma-2 (n = 48) (Fig. 6G,O) or oma-1 (Fig. 6E). These results indicate that OMA-1 and OMA-2 collectively regulate the distribution of P granules among embryonic blastomeres during C. elegans development.
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Since germ cell-specific granules were found to be improperly distributed in oma-1; oma-2-suppressed embryos, we analyzed three proteins that are required for germline specification, PGL-1 (Kawasaki et al. 1998), MEX-1 (Guedes & Priess 1997) and PIE-1 (Mello et al. 1996; Seydoux et al. 1996), in oma-1; oma-2-suppressed embryos. In wild-type 2-cell and 4-cell embryos, PGL-1 is present at high levels in the posterior blastomere. We found that the germline protein PGL-1 was localized inappropriately in blastomeres in oma-1; oma-2-suppressed embryos (Fig. 7A). In all of the oma-1; oma-2-suppressed embryos, PGL-1 successfully migrated to the posterior region in one-cell embryos and was specifically inherited by the P1 blastomere as was the case in wild-type embryos (100%, n = 48) (Fig. 7A-a). However, PGL-1 frequently failed to be partitioned in the P1 blastomere, and this resulted in incorrect distribution in descendants of P2 and EMS blastomeres (Fig. 7A-b,c). Eventually, PGL-1 became distributed abnormally in dividing embryos (76.8%, n = 148) (Fig. 7A-b,c).
It is known that the behavior of P granules is influenced by MEX-1 protein (Mello et al. 1992; Schnabel et al. 1996). We therefore tried to determine the distribution of MEX-1 protein. Although MEX-1 normally migrated to the posterior blastomere of 2 cell-stage oma-1; oma-2-suppressed embryos, MEX-1 failed to be distributed exclusively to a P2 blastomere (66.7%, n = 72) (Fig. 7B-d), as was found in the case of PGL-1 (Fig. 7A). It has been reported that MEX-1 is required for restriction of PIE-1 expression and that an abnormality in MEX-1 affects the nuclear localization of PIE-1 proteins (Guedes & Priess 1997). This is also the case in oma-1; oma-2-suppressed embryos. PIE-1 was diffusely distributed in both P2 and EMS blastomeres in such embryos (50.0%, n = 32) (Fig. 7C-d), and the nuclear distribution of PIE-1 in P granules-positive blastomeres is obscure compared to that in wild-type embryos. These observations imply that the transcriptional suppression by PIE-1 (Tenenhaus et al. 1998) in a germline blastomere might be abolished in oma-1; oma-2-suppressed embryos (see Discussion). All of these results suggest that oma-gene products are required for proper distribution of PGL-1, MEX-1 and PIE-1 proteins, all of which are crucial regulators of germline specification in C. elegans embryos. The two main characteristics of germline cells, exclusive distribution of P granules and proper nuclear localization of PIE-1 protein, seem to be lost in oma-1; oma-2-suppressed embryos.
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Discussion |
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OMA proteins and germ cell development
In this study, we found that OMA-1 continued to associate with P granules during embryogenesis even though the cytoplasmic pool had been largely removed at the time of first mitosis. Thus, OMA-1 might be stabilized by association with P granules in the developing C. elegans embryo. An intriguing finding in this study is that oma-gene products are important for specification of an aspect of germline development. Since OMA-1 is a component of P granules (Figs 2C, 3 and 4), it is possible that this protein directs P granules to the germline blastomere during cell division. Alternatively, OMA-1 and OMA-2 may play important regulatory roles in germ-lineage cell proliferation/cell division. The third and most likely possibility is that dysfunction of OMA-1 and OMA-2 influences the regulation of other germline determinants, such as MEX-1 and PIE-1 proteins (Mello et al. 1992, 1996; Guedes & Priess 1997), and therefore induces mislocation of P granules. Our analysis indicates that oma-1; oma-2 (RNAi) did not influence the asymmetric distribution of PAR proteins at 1 cell stage, resulting in proper establishment of anterior-posterior orientation in the fertilized embryos. We compared the relative size of the anterior blastomere in 2 cell stage wild-type embryos with that in oma-1; oma-2 (RNAi) embryos. The size of the anterior blastomere/total was 57.6% for wild-type embryos and 57.9% for oma-1; oma-2 (RNAi) embryos. These results indicate that oma-1; oma-2 (RNAi) embryos have normal first cleavage and cell cycles, distinguishing them from par embryos. In accordance with this, we found that several posterior proteins are normally segregated in P1 blastomere at first mitosis in oma-1; oma-2 (RNAi) embryos even in a condition OMA-proteins are depleted (Supplementary Fig. S1). In contrast, we found the high penetrant missegregation of PGL-1, PIE-1 and MEX-1 to EMS blastomere with moderate abnormality in the second cleavage planes in oma-1; oma-2 (RNAi) embryos (Fig. 7 and Supplementary Fig. S2). In mex-5(-); mex-6(-) mutant embryos, it was reported that germline proteins are mislocalized in anterior somatic blastomeres (Schubert et al. 2000). We never observed anterior localization of PIE-1 and MEX-1 in oma-1; oma-2 (RNAi) embryos, and thus we speculate that OMA-1/OMA-2 works in a different manner to the MEX-5/MEX-6 proteins.
An abnormal distribution of P granules similar to the case in oma-1; oma-2 (RNAi) was observed in a mutant of mex-1 (Mello et al. 1992; Schnabel et al. 1996; Guedes & Priess 1997). In newly fertilized mex-1 mutant eggs, P granules accumulated posteriorly but they did not associate properly with the cortex and thus spread throughout the posterior half of the egg. In the next and subsequent cleavages, this incomplete localization led to mispartitioning of P granules into somatic blastomeres. Since the defect in distribution of P granules in early stage oma-1; oma-2 (RNAi) embryos is quite similar to the defect in mex-1 family mutants, we speculate that the roles of OMA-1 and OMA-2 in the embryo are closely related to MEX-1 functions. There may be concern about simple cross-reaction between mex-1 and oma-1; oma-2 (RNAi). However, this is not the case from the following reasons. First, the amounts of MEX-1 protein and a similar CCCH-type zinc-finger protein, PIE-1, were not reduced in the oocytes by our RNAi treatment of oma-1; oma-2 genes (Fig. 1C-i,k,m,o). Second, single RNAi treatment of either the oma-1 or oma-2 gene did not cause obvious defects in germline specification. If any of the oma RNAi cross-reacted with mex-1, pie-1, or any other related genes, treatment with a single RNAi would cause germline defects. However, such defects were not observed in our single RNAi experiments.
Although we have confirmed that the expression levels of MEX-1 proteins in oma-1; oma-2 (RNAi)-treated embryos were not reduced, we found that the distribution of MEX-1 protein in early stage embryos was not correct (Fig. 7B-d), partly explaining the defects in germline formation. It was reported that MEX-1 controls its downstream target PIE-1. In the case of PIE-1 protein, we also found that it was mislocalized in P2 and EMS blastomeres with reduced amounts in these blastomeres of oma-1; oma-2-compromised 4-cell embryos (Fig. 7C-d). In mutant embryos in which PIE-1 was abnormally present in more than one cell, Tenenhaus et al. (1998) reported that the majority of those PIE-1-containing cells were not negative but actually positive for RNAPII-H5, a marker for active transcription. This finding indicates that transcription can become activated in the germ lineage of mutants that localize PIE-1 to more than one cell (Mello et al. 1996; Seydoux et al. 1996; Tenenhaus et al. 1998, 2001). Tenenhaus et al. (1998) suggested that there is a certain threshold of PIE-1 that is required to completely repress RNAPII-H5 expression and that this threshold is rarely met in mutants in which PIE-1 is present in more than one cell. Although we could not directly investigate the expression of RNAPII-H5, it is highly probable that such misexpression of soma-specific genes occurred in germline blastomere in oma-1; oma-2 mutant embryos, because PIE-1 is present in more than one cell as reported by Tenenhaus et al. (1998). Thus, oma-1 and oma-2 suppression seems to abolish germline-specific events, the specific association of P granules and the absence of embryonically transcribed mRNAs in early stage C. elegans embryos. We speculate that OMA-1 functions upstream of the proper segregation mechanism of P granules, and our findings demonstrate that oma genes are essential for germline specification. It is also possible that oma-1; oma-2 may be required for the specification of the sister blastomeres, such as EMS, C, or D, and these cells might have been transformed into P4 fate instead of somatic fates. Although the P-granule positive embryonic cells in the oma-1; oma-2 depleted embryos look like germ cells, they may not be fully differentiated as P4 or Z2/Z3 and actually contain C or D-like characters, as is the case in mes-1 mutant, in which the P-granule positive embryonic cells frequently differentiate as muscle cells.
Switch from maturation to cell fate determination
Another interesting finding in this study is the rapid disappearance of OMA-1 protein from the cytoplasm at the time of first mitosis (Fig. 2B). We found that rpn-1 (subunit of the 26S proteasome) gene products are required for timely degradation of OMA-1 and OMA-2 (Shimada et al. 2002 and our unpublished observation). In addition, it was reported that MBK-2, CDK-1 and GSK-3 kinases are involved in the elimination of maternal protein, including OMA-1, in C. elegans (Pellettieri et al. 2003; DeRenzo & Seydoux 2004; Nishi & Lin 2005; Shirayama et al. 2006; Stitzel et al. 2006). These observations imply that OMA-1 as well as OMA-2 is a target of ubiquitin and the 26S proteasome-mediated pathway (Kawahara et al. 2000a,b). Since OMA family proteins are essential for two vital events, oocyte maturation (Detwiler et al. 2001; Shimada et al. 2002) and proper germline determination (this study), it is possible that cytoplasmic accumulation of OMA-1 and OMA-2 proteins is necessary for oocyte maturation but that these proteins need to be removed from the cytosol before the onset of embryogenesis. If this is a case, the removal of cytoplasmic OMA proteins is a molecular switch triggered by fertilization, changing from a maturation-regulating mode to a developmental-regulation mode. OMA proteins in the cytoplasm regulate oocyte maturation processes, while OMA proteins on P granules might be involved in events determining cell fate after fertilization. Interestingly, Lin (2003) reported that a gain-of-function mutant of OMA-1, zu405, results in improper degradation of the OMA-1 protein in embryos. In zu405 mutant embryos, the C blastomere is transformed to EMS blastomere fate, resulting in embryonic lethality. These results suggest that correct temporal regulation of OMA-1 protein degradation is indeed critical for proper embryo development.
Some of the CCCH-type zinc-finger proteins are known to regulate RNA metabolism (Bai & Tolias 1996; Batchelder et al. 1999; Lai et al. 1999; Lai & Blackshear 2001). Although the significance of the presence of OMA proteins in the cytoplasm is not known, it is possible that cytoplasmic OMA-1 is necessary to suppress the translation of maternal mRNA until the developmental program resumes (Evans et al. 1994). If this is the case, cytoplasmic degradation might be necessary to start appropriate translational events after fertilization. However, complete loss of OMA proteins is inappropriate for proper developmental processes, because RNAi treatment of oma genes resulted in an abnormal distribution of P granules. Cytoplasm-specific removal or P granule-oriented re-distribution of OMA proteins might be required for proper development in wild-type embryos. Experimental control of the OMA degradation pathway is needed to determine the significance of cytoplasmic removal of OMA proteins.
In summary, we found that oma gene products are novel multifunctional proteins collectively required for germline specification. Fertilization-induced cytoplasmic degradation, germ granules-specific inheritance, and genetic properties of OMA-1; OMA-2 elimination provide novel examples of a regulatory pathway for proper embryonic development in C. elegans. Determination of the relationships with components of known P granules as well as with the factor(s) that regulates cell fate determination is a challenge for our future studies.
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Experimental procedures |
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C. elegans strains were cultured using standard techniques (Brenner 1974). C. elegans N2 variety Bristol was used for RNAi analysis and for wild-type observations. par-2 (it5), par-3 (e2074), mex-1 (zu121) and mex-5 (zu199) mutant alleles obtained from the Caenorhabditis elegans Genomic Center (CGC) (University of Minnesota) are temperature-sensitive. They were maintained at a permissive temperature (16 °C) and then later kept at restrictive temperature (25 °C). After the temperature shift, arrested embryos were observed as previously described (Kemphues et al. 1988; Mello et al. 1992; Etemad-Moghadam et al. 1995; Boyd et al. 1996; Schnabel et al. 1996; Guedes & Priess 1997; Schubert et al. 2000). In this report, "Embryo" is also used to refer to a fertilized oocyte irrespective of whether the oocyte completes the meiotic divisions.
Antibodies and immunological analysis
The anti-OMA-1a and anti-OMA-1b specific peptide antibodies were produced and verified as described in the Results section. The peptide used for production of an anti-MEX-1 antibody corresponds to MEX-1 amino acids 270–289 (FNNFHDMSDSGYSAPRRRLH), which is the region specific to MEX-1 (Guedes & Priess 1997). Similarly, the region of PIE-1 N-terminal amino acids 54–73 (DSLSSGYSGKWLRPKREALK) in the predicted PIE-1 amino acid sequence was used for production of an anti-PIE-1 antibody (Mello et al. 1996). All of the rabbit antisera obtained were successively purified with Protein A-Sepharose and corresponding antigenic peptides affinity chromatography. Purified immunoglobulins were used for Western blot analysis and immunocytochemistry.
Samples for Western blot analysis were prepared by sonicating mixed-stage C. elegans. Primary antibodies for anti-OMA-1a, anti-OMA-1b and anti-OMA-2a were used at concentrations of 3.3 nM, 6.3 nM and 1.8 nM, respectively. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Tago) was used as a secondary antibody, and ECL signals were detected. For peptide absorption experiments, an excessive amount of an antigenic peptide was added to the primary antibody reaction solution; OMA-1a, OMA-1b and OMA-2a antigenic peptides were used at concentrations of 6.7 µM, 12.6 µM and 3.5 µM, respectively.
For immunocytochemistry of embryos, adult hermaphrodites were cut open to release gonads and embryos at the indicated time, and freeze/cracked samples in M9 buffer were fixed by incubating in cold methanol and then in cold acetone. Developmental stage was analyzed by DIC microscopy (Sulston et al. 1983).
Double staining of P granules and OMA-1 was performed using anti-OMA-1 antibody at a concentration of 33 nM and anti-P granules antibodies (OIC1D4 or K76) at a 1 : 100 dilution. AlexaTM 488-conjugated anti-rabbit IgG antibody and AlexaTM 594-conjugated anti-mouse IgG antibody were used as secondary antibodies at dilutions of 1 : 800. For peptide absorption experiments, antigenic peptides were used at concentrations of 125.2 µM (for anti-OMA-1a, b). Anti-PGL-1 antibody was a generous gift from Dr I. Kawasaki and used as previously described (Kawasaki et al. 1998). A mouse monoclonal anti-
-tubulin antibody (DM1A) (Sigma) at a 1 : 800 dilution was used for -tubulin staining. To see the state of nucleus, embryos were stained with 2.5 µg/mL Hoechst 33342 in PBS at the time of antibody staining. Immunofluorescent images of embryos were obtained in Axioplan II using x 40 objective and LSM510 confocal microscopy system (Carl Zeiss).
RNA interference (RNAi)
RNAi experiments were mainly performed using the feeding methods (Timmons & Fire 1998; Timmons et al. 2001). oma-1 (521–878 bp), oma-2 (40–457 bp) and other cloned genes were amplified by PCR from a C. elegans mixed-stage cDNA library. Primer sequences are available upon request. The amplified PCR products were subcloned into the pPD129.36 vector, and transfected to E. coli HT115(DE3) strain. RNA transcription from pPD129.36 vector plasmids was induced by adding 0.4 mM IPTG to E. coli culture medium (at the optical density of 0.4) and subsequently incubated for 4 h at 37 °C. Harvested E. coli were spread on NGM plates (containing 50 µg/mL ampicillin, 12.5 µg/mL tetracycline, 0.4 mM IPTG), and was used for feeding experiments. Feeding was started from the L4 stage and allowed to lay embryos at 25 °C for indicated periods unless otherwise noted. The findings were confirmed by using the microinjection method (Fire et al. 1998; Shimada et al. 2002).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: kawahara{at}pharm.hokudai.ac.jp
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References |
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Received: 7 December 2005
Accepted: 25 December 2005
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