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Genetics, Vol. 174, 1933-1945, December 2006, Copyright © 2006
doi:10.1534/genetics.105.052621
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* Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, ** Howard Hughes Medical Institute, Seattle, Washington 98109, Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195, Children's Hospital and Regional Medical Center, Department of Hematology and Oncology, Seattle, Washington 98105 and Department of Biology, Central Michigan University, Mount Pleasant, Michigan 48859
2 Corresponding author: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, Mailstop c3-168, Seattle, WA 98109.
E-mail: bdpage{at}gmail.com
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The PAR proteins, in particular the Ser/Thr kinase PAR-1, are thought to act on members of the CCCH zinc-finger protein family to regulate cell fate. PAR-1 restricts two CCCH proteins, MEX-5 and MEX-6, to the anterior pole of the one-cell embryo. The function of both MEX-5 and MEX-6 proteins is required to limit the accumulation of three other CCCH proteins, PIE-1, POS-1 and MEX-1, such that they are enriched in the posterior of the embryo (SCHUBERT et al. 2000; CUENCA et al. 2003). PIE-1 acts in the nucleus of germline precursors to repress transcription and in the cytoplasm where it prevents degradation of at least one maternal mRNA and promotes expression of that message (MELLO et al. 1996; SEYDOUX et al. 1996; BATCHELDER et al. 1999; TENENHAUS et al. 2001). POS-1 is critical for both somatic and germline fates (TABARA et al. 1999; D'AGOSTINO et al. 2006) and acts as a translational repressor by directly binding the 3'-UTR of its target gene (OGURA et al. 2003), and MEX-1 affects the accumulation of several proteins (MELLO et al. 1992; GUEDES and PRIESS 1997).
Each member of the above C. elegans family of CCCH zinc-finger proteins controls cell fate by its ability to regulate the accumulation and/or expression of other proteins. The molecular mechanism(s) by which this regulation is achieved is unknown for MEX-5, MEX-6, MEX-1, and for the cytoplasmic function of PIE-1. Proteins with CCCH motifs in mammals, flies, and yeast have been associated with diverse aspects of RNA regulation, including processing, localization, and destabilization (BEGEMANN et al. 1997; BLACKSHEAR 2002; ADERETH et al. 2005; LADD et al. 2005; PUIG et al. 2005). For example, the mammalian TTP and the yeast Cth2 CCCH proteins have been shown to regulate expression by binding and targeting mRNAs for degradation (BLACKSHEAR 2002; PUIG et al. 2005). Possibly, the C. elegans CCCH proteins use similar mechanisms to control cell fate in the early embryo.
Both CCCH proteins MEX-5 and POS-1 affect SKN-1 via its accumulation and/or its activity (TABARA et al. 1999; SCHUBERT et al. 2000). SKN-1 is a transcription factor that acts in descendants of the posterior blastomere of the two-cell embryo and is important for the specification of endoderm and a subset of mesodermal tissues (BOWERMAN et al. 1992). In wild-type two-cell embryos, SKN-1 is present at a high level in the posterior cell, and is detected at a lower level in its anterior sister (BOWERMAN et al. 1993). In mex-5 mutant embryos, SKN-1 protein accumulates to a high level in the anterior blastomere, similar to the level seen in its posterior sister. This misexpression of SKN-1 results in a Mex (muscle excess) phenotype. In these mutant embryos, the anterior blastomere of the two-cell embryo produces ectopic mesodermal tissues, body-wall muscle, and pharyngeal cells (SCHUBERT et al. 2000). Loss of pos-1 function has a very different effect on SKN-1. Although SKN-1 expression appears wild-type in pos-1(–) embryos, loss of pos-1 appears to reduce SKN-1 activity. Terminally developed pos-1 mutant embryos lack endoderm and a subset of mesoderm, a phenotype similar to that of skn-1(–) embryos (TABARA et al. 1999). Thus, the effects of mex-5(–) and pos-1(–) are very different; loss of mex-5 causes ectopic accumulation and activity of SKN-1, whereas loss of pos-1 reduces SKN-1 activity.
In addition to mex-5, mutations in three other genes, mex-1, efl-1, or dpl-1, disrupt SKN-1 asymmetry and cause a Mex phenotype (MELLO et al. 1992; BOWERMAN et al. 1993; PAGE et al. 2001). MEX-1 is a CCCH protein (GUEDES and PRIESS 1997). efl-1 and dpl-1 encode proteins similar to the mammalian transcription factors E2F and DP1, respectively (PAGE et al. 2001). These two proteins function in the maternal germline where they upregulate the transcription of genes involved in oogenesis and early embryogenesis. Their targets include mex-6, mex-5, and mex-1 (CHI and REINKE 2006); thus, the Mex phenotype of efl-1(–) or dpl-1(–) embryos is most likely caused by reduced transcription of these CCCH-encoding genes.
We are interested in the interactions among the various proteins that control cell fate in the early embryo. To achieve this objective, we screened for dominant mutations that suppress the temperature-sensitive Mex phenotype of efl-1(se1). We isolated a loss-of-function mutation in the pos-1 gene. Our analysis indicates that SKN-1 activity is very sensitive to the dosage level of pos-1(+), but that pos-1 is not essential for SKN-1 to specify mesoderm or endoderm. We propose that POS-1 can indirectly affect SKN-1 in the anterior blastomere, possibly by repressing mex-6 translation. In addition, our analysis reveals that mex-5 is required for the asymmetric pattern of two class II messages, pos-1 and mex-1. Taken together, these data indicate mutually restrictive interactions among the anterior and posterior CCCH finger proteins that may contribute to their complementary expression patterns.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Screen for suppressors of efl-1(se1):
We screened for modifiers of efl-1(se1) using the strain efl-1(se1);lin-2(e1309). This strain was mutagenized with EMS, and the F1 were shifted to 26° at the L4 stage and screened as adults. We recovered those worms that produced viable progeny (a bag of viable worms). We screened 36,000 F1 hermaphrodites and isolated 12 dominant suppressors of the efl-1(se1) temperature-sensitive phenotype. Upon further examination one of these suppressors was discovered to be allele zu454 of pos-1.
Antibodies and fluorescence:
We detected SKN-1, POS-1, and MEX-5 proteins using antibodies and procedures that have been previously described (BOWERMAN et al. 1993; TABARA et al. 1999; SCHUBERT et al. 2000). We used the monoclonal antibody mAb3NB12 to detect pharyngeal muscle cells (PRIESS and THOMSON 1987), and the expression of med-1 was detected by the fusion construct MED-1:GFP created and integrated by MADURO et al. (2001). The presence of endoderm was scored using polarizing optics for the intestinal-cell-specific gut granules (BOWERMAN et al. 1992).
RNA-mediated interference:
To direct RNA-mediated interference against pos-1 and mex-1, we used a pair of nested primers to PCR amplify part of the coding region of these genes from genomic DNA. Each set of internal primers contains the T7 sequence at the 5'-end to use T7 RNA polymerase to generate double-strand RNA (dsRNA). To specifically remove mex-5 or mex-6, we targeted the region described by SCHUBERT et al. (2000). To direct RNA-mediated interference against skn-1, we used the cDNA yk2d12 in which the skn-1 insert is flanked by T7 and T3 RNA polymerase binding sites. L4 worms were soaked in dsRNA for 
15 hr at the temperature specified. In the case of efl-1(se1) pos-1(zu148)/efl-1(se1) pos-1(+) heterozygous worms treated with mex-6(dsRNA), we were unable to distinguish heterozygotes from the homozygous efl-1(se1) pos-1(+) worms at the L4 stage. Since soaking causes only a transient removal of mex-6, we scored the phenotypic outcome for the progeny of each treated worm and later determined the corresponding genotype of each worm on the basis of its viable progeny.
In situ hybridization:
cDNA probes for pos-1 and mex-1 were generated using asymmetric PCR, digoxigenin-labeled nucleotides, and the cDNAs yk117h11 and pJPSG9, respectively. Fixation and hybridization procedures were essentially as described in SCHISA et al. (2001). Briefly, adults were dissected in M9 on a glass coverslip to remove embryos. The coverslips were inverted on a 0.1% polylysine-coated slide and frozen on dry ice. After removal of the coverslip, the slide was immersed in 100% methanol at –20° (5 min), 100% methanol at room temperature (5 min), 90% methanol (1 min), 70% methanol (1 min), and 50% methanol (1 min) and washed twice in PTw (5 min; 1x PBS, 0.1% Tween 20). Embryos were treated with proteinase K (20 µg/ml; 15 min) at 37° and washed in 2 mg/ml glycine in PTw (2 min) and PTw (5 min). Embryos were fixed for 20 min at room temperature in 4% formaldehyde in PBS and then washed in PTw (5 min), 2 mg/ml glycine in PTw (5 min), PTw (5 min), and 2x SSC (5 min).
Hybridization buffer consisted of 100 µg/ml salmon sperm DNA, 50 µg/ml heparin, 0.1% Tween 20, 50% formamide, and 5x SSC. Embryos were prehybridized for 10 min at 48°. Probes were boiled in hybridization buffer for 10 min and put on ice prior to applying to the sample tissue on a microscope slide. The tissue was covered with a glass coverslip, sealed with rubber cement, and incubated at 37°or 48° for 12–18 hr. After hybridization, the slide was washed at 48° with hybridization buffer (15 min, 30 min). The slide was then washed twice in 2x SSC (10 min). For alkaline phosphatase detection, the slide was washed in PBT (1x PBS, 0.1% BSA, 0.1% Triton X-110) twice for 5 min at room temperature and 5-bromo-4-chloro-3-indolyl phosphate and 4-nitro blue tetrazolium chloride substrates were added. To stop the detection reaction, embryos were washed twice in 150 mM NaCl, 50 mM Tris–HCl pH 7.8, 0.1% BSA, 0.1% Tween-20. Embryos were placed in PBS containing 0.08 µg/ml DAPI and covered with mounting media.
Yeast trihybrid:
We used the yeast trihybrid system designed by PUTZ et al. (1996). This assay for RNA–protein interactions was also used by OGURA et al. (2003) to demonstrate that POS-1 binds the glp-1 3'-UTR. Therefore, we used their pos-1 fusion construct in our experiments and tested its interaction with the glp-1 3'-UTR as a positive control. We also used their mutant pos-1(ne51) construct as a negative control. The pos-1(ne51) mutant allele has a missense mutation that alters the second zinc finger in the POS-1 protein (OGURA et al. 2003). This mutation most likely disrupts pos-1 function, and OGURA et al. (2003) did not detect binding of the glp-1 3'-UTR with this mutant pos-1. We constructed the fusion RNA between Rev response element (RRE) and the mex-6 3'-UTR by using a mex-6 PCR product. Our PCR primers were 5'-cgcgacgcgtccatttttgatttacccactgagagtcc-3' and 5'-agaatgcggccgcaggcgcagggtattttggaatgg-3'. The 5'-end of each primer contains a restriction enzyme site, MluI and NotI, respectively, allowing for unidirectional cloning. The PCR product was digested and cloned into the pRevRX vector. The wild-type 226-bp sequence of the mex-6 3'-UTR was confirmed by sequencing.
The yeast strain PJ69-4A was cotransformed with plasmids containing the pos-1 fusion and the mex-6 3'-UTR fusion. Transformants were selected on synthetic complete media lacking leucine and tryptophan. The RNA–protein interactions were detected by growth on synthetic complete plates lacking tryptophan, leucine, and histidine and supplemented with 5 mM 3-amino-1,2,4-triazole, a His3p inhibitor.
Construction and integration of gfp:mex-6 fusion:
Standard techniques were used to manipulate and amplify DNA. A pie-1promoter::gfp::mex-6 transgene, pJT78, was created by modification of a previously described pie-1::gfp expression vector (STROME et al. 2001). The mex-6-coding sequence was PCR amplified from mex-6 cDNA yk733b2 using primers with SpeI adapters at the 5'-ends. (Complete primer sequences are available upon request.) The mex-6 PCR product was cloned downstream of gfp into the SpeI site of the pie-1 promoter::gfp plasmid. Sequencing was performed to confirm that the mex-6 insert was in the correct orientation and in-frame with the gfp sequence. A unique NotI site was created in the plasmid by mutagenesis of one of two NotI sites. The unc-119(+) genomic fragment was inserted into this NotI site (MADURO and PILGRIM 1995). A pie-1promoter::gfp::mex-6 (mex-6 3'-UTR) transgene, pJT79, was created by modification of pJT78. A KpnI fragment of pJT78, containing gfp::mex-6 and pie-1 3'-UTR, was removed and used as a template for PCR amplification of gfp::mex-6. Genomic DNA was used as a template to amplify 939 bp of mex-6 3'-UTR immediately downstream of the TAG codon. A two-step fusion PCR method was used to fuse gfp::mex-6 and mex-6 3'-UTR sequences (HOBERT 2002). The final fusion PCR product included KpnI sites at both the 5'- and 3'-ends, and was cloned into the KpnI site of pJT78.
Strains expressing pie-1promoter::gfp::mex-6 (mex-6 3'-UTR) were obtained by microparticle bombardment of unc-119(ed3) worms with the pie-1 promoter::gfp::mex-6 (mex-6 3'-UTR) plasmid described above (PRAITIS et al. 2001).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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We sequenced the newly isolated pos-1 allele, zu454, and discovered a transition of a C to T that conceptually results in a nonsense codon at codon 92. Thus, in this mutant the POS-1 protein would be truncated to a length of 91 amino acids instead of its normal length of 264 amino acids. The wild-type POS-1 protein contains two CCCH zinc-finger motifs, each of which is required for POS-1 function (TABARA et al. 1999; OGURA et al. 2003). Both domains would be absent in the zu454 truncated POS-1 protein, suggesting that the suppressing mutation of pos-1 results in a nonfunctional protein.
To confirm that a reduction of pos-1(+) function can suppress the efl-1(se1) phenotype, we tested whether the null pos-1 allele zu148 (TABARA et al. 1999) suppressed the efl-1(se1) Mex phenotype. This mutant pos-1 allele strongly suppressed the efl-1(se1) temperature-sensitive Mex phenotype (Table 1; Figure 1), equal to the levels observed with the newly isolated pos-1 allele. Thus, reduction of pos-1(+) gene dosage appears to be sufficient for suppression of the efl-1(se1) Mex phenotype.
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We also tested whether reducing pos-1(+) dosage could dominantly suppress mex-1 mutant embryos. mex-1 mutant embryos resemble mex-5, dpl-1, and efl-1 single-mutant embryos with respect to their terminally differentiated phenotype, and all four mutant embryos accumulate high levels of SKN-1 in the anterior blastomere and its daughters, compared to wild-type embryos (MELLO et al. 1992; BOWERMAN et al. 1993; SCHUBERT et al. 2000; PAGE et al. 2001). However, unlike mex-5, dpl-1, and efl-1 mutants, mex-1 is not suppressed by reducing Ras/MAPK signaling (PAGE et al. 2001). mex-1(–) was also not suppressed by reducing pos-1(+) dosage as mex-1;pos-1(–)/pos-1(+) mothers produced all dead progeny with the Mex phenotype (Table 1), consistent with the previous distinction between mex-1(–) embryos and those Mex embryos caused by mutations in mex-5, dpl-1, or efl-1.
Reduction of pos-1(+) dosage does not alter the terminal phenotype of mex-5;mex-6 double-mutant embryos:
MEX-5 and MEX-6 encode highly similar proteins that function redundantly in the embryo (SCHUBERT et al. 2000; HUANG et al. 2002). In a two-cell mex-5(–) embryo, SKN-1 is the only protein whose asymmetry is consistently disrupted, and the resulting embryos are inviable. In contrast, no disruption of embryonic asymmetry has been detected in mex-6 mutant embryos, and these embryos are viable. mex-5;mex-6 double-mutant embryos show a severe disruption in the asymmetry of multiple proteins. At the two-cell stage, proteins such as MEX-3 and GLP-1, that are expressed anteriorly in both wild-type and mex-5(–) embryos, are absent in mex-5(–);mex-6(–) embryos. Conversely, the proteins PIE-1, MEX-1, and POS-1, that accumulate posteriorly in wild-type and mex-5(–) two-cell embryos, are detected in both blastomeres in the double mutant (SCHUBERT et al. 2000).
Since reduced dosage of pos-1(+) can make mex-5(–) embryos viable, we tested whether reduction or loss of pos-1(+) function could affect the terminal phenotype of mex-5;mex-6 double-mutant embryos. A terminal mex-5(–);mex-6(–) embryo possesses ectopic body-wall muscle and hypodermal cells but lacks pharyngeal muscle and endoderm (SCHUBERT et al. 2000). An additional characteristic of this double-mutant embryo is that it generates a small number of neurons (10/embryo), whereas a wild-type embryo possesses 269 neurons. To determine if reduction of pos-1(+) dosage could affect the mex-5(–);mex-6(–) phenotype, we examined terminally developed embryos from pos-1(zu454)/pos-1(+);mex-5(RNAi);mex-6(RNAi) mothers. We detected no difference between these embryos and those from mex-5(–);mex-6(–) double-mutant mothers (Table 2). To test whether a complete loss of pos-1 function could affect the mex-5(–);mex-6(–) terminal phenotype, we also examined pos-1(zu454);mex-5(RNAi);mex-6(RNAi) embryos. Scoring for both the presence of endoderm and the number of neurons produced, we detected no difference in the terminal phenotype of pos-1;mex-5;mex-6 triple-mutant embryos compared to mex-5;mex-6 double-mutant embryos (Table 2). Thus, reduction of pos-1(+) did not suppress or modify the mex-5(–);mex-6(–) terminal phenotype.
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In mex-5, efl-1, and dpl-1 single-mutant embryos, SKN-1 is present at high levels in the anterior blastomere and its daughters, compared to the lower levels observed in these cells in wild-type embryos. In Mex mutant embryos suppressed via reduced Ras/MAPK signaling, SKN-1 accumulation is restored to the wild-type pattern, indicating that the Ras/MAPK pathway functions upstream of SKN-1 (PAGE et al. 2001). To determine if reduction of pos-1(+) also suppresses the misexpression of SKN-1 in Mex mutants, we stained embryos from pos-1(zu148)/pos-1(+);mex-5(RNAi) mothers with anti-SKN-1 serum. In all two-cell and four-cell embryos examined (n = 42), we detected a high level of SKN-1 in the anterior blastomere(s); this pattern appeared identical to that seen in mex-5(RNAi) control embryos (Figure 2). The majority of embryos from pos-1(zu148)/pos-1(+);mex-5(RNAi) mothers that were allowed to terminally differentiate had normal morphology (67%, n = 209; Table 1). These results suggest that reducing pos-1(+) does not restore the wild-type pattern of SKN-1 accumulation and indicate that reducing pos-1(+) affects SKN-1 activity. This result is consistent with analysis of SKN-1 in pos-1(–) single-mutant embryos. In these mutant embryos, SKN-1 accumulation appears wild type even though SKN-1 activity is decreased (TABARA et al. 1999; MADURO et al. 2001).
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To confirm this interpretation of the pos-1;mex-1 double mutant, we constructed a pos-1;mex-1 mutant in the background of glp-1(e2142) and stained for pharyngeal muscle in those embryos produced at the restrictive temperature for glp-1(e2142). In glp-1 mutant embryos, the only pharyngeal muscle produced is dependent on the autonomous function of SKN-1 (MELLO et al. 1992; BOWERMAN et al. 1997). Previous analysis of pos-1 mutant embryos suggested that the presence of pharyngeal muscle is dependent on glp-1 (TABARA et al. 1999). We confirmed that glp-1(e2142);pos-1(zu148) double-mutant embryos did not produce pharyngeal muscle (n = 13; Figure 3). In contrast, all glp-1(e2142);pos-1(zu148);mex-1(RNAi) triple-mutant embryos produced pharyngeal muscle, demonstrating that SKN-1 specifies mesoderm without POS-1 (n = 18) (Figure 3).
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Since efl-1(se1) suppressed the loss of endoderm in pos-1 mutant embryos, we determined whether efl-1(se1) also suppressed the loss of mesoderm. To score this suppression, we examined the presence of pharyngeal muscle in glp-1(e2142);efl-1(se1) pos-1(zu148) triple-mutant embryos produced at the restrictive temperature for glp-1(e2142) and efl-1(se1). As stated above, no glp-1(e2142);pos-1(zu148) double-mutant embryos produced pharyngeal muscle (n = 13); however, 64% of glp-1(e2142);efl-1(se1) pos-1(zu148) embryos produced pharyngeal muscle (n = 150) (Figure 3). Thus, in the efl-1(se1) mutant background, SKN-1 can specify both endodermal and mesodermal tissues without pos-1(+) function. To determine whether SKN-1 was functioning at its normal site of action to specify these tissues, we looked at the expression of MED-1:GFP in efl-1 pos-1 double-mutant embryos. We detected the wild-type pattern of MED-1:GFP expression in all efl-1(se1) pos-1(zu148) embryos examined (n = 22).
At this point, the data indicated that the level of pos-1(+) is important for SKN-1 to specify mesoderm and endoderm in certain backgrounds, but that pos-1(+) was not necessary for this SKN-1 activity. These conclusions lead us to examine two hypotheses that might explain why reduction in pos-1(+) dosage suppresses Mex mutants. These hypotheses are not mutually exclusive: (1) POS-1 is misexpressed in the above Mex mutants, and this ectopic POS-1 is important for ectopic SKN-1 activity and (2) POS-1(+) affects SKN-1 activity by acting as a translational repressor; i.e., POS-1 represses translation of a SKN-1 inhibitor.
efl-1 and mex-5 mutant embryos have an asymmetric POS-1 expression pattern but a symmetric pos-1 mRNA distribution:
In wild-type two-cell embryos, POS-1 and SKN-1 are more concentrated in the posterior blastomere than in the anterior; however, both proteins are detected at a low level in the anterior blastomere (BOWERMAN et al. 1993; TABARA et al. 1999). Our genetic analysis demonstrates that the ectopic activity of SKN-1 is very sensitive to pos-1(+) levels. Could the ectopic activity of SKN-1 in the efl-1, dpl-1 and mex-5 single mutants be due in part to POS-1 misexpression?
To test this possibility, we examined the localization of the POS-1 protein and pos-1 mRNA in Mex mutant embryos. In early efl-1 and mex-5 mutant embryos, POS-1 accumulation appeared asymmetric, similar to its pattern in wild-type two-cell and four-cell embryos (Figure 4). This observation suggests that ectopic SKN-1 activity in efl-1 and mex-5 mutant embryos is not due to ectopic POS-1 accumulation.
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POS-1 binds the 3'-UTR of mex-6:
Since POS-1 has been demonstrated to be a translational repressor (OGURA et al. 2003), we reasoned that reduction of this function might explain the suppression of the Mex phenotype, i.e., reduction in pos-1(+) levels leads to increased expression of its target. We would expect such a target to be more highly expressed in the anterior of the embryo, an expression pattern complementary to that of POS-1, and possibly to possess similarity to the glp-1 spatial control region (SCR), a sequence within the glp-1 3'-UTR that POS-1 directly binds (EVANS et al. 1994; OGURA et al. 2003). An attractive candidate for such a target is mex-6. A MEX-6:GFP fusion protein is detected in the anterior blastomeres of the two-cell and the early four-cell embryo (CUENCA et al. 2003), and we identified a region in the mex-6 3'-UTR that is very similar to the conserved SCR of glp-1 (Figure 6A). In this region of the mex-6 3'-UTR, 27 nucleotides are identical to the 34 nucleotides shown to be sufficient for glp-1 repression in the embryo (MARIN and EVANS 2003). Additionally, mex-5 and mex-6 encode very similar proteins that function redundantly during early embryogenesis (SCHUBERT et al. 2000); thus, an increase in mex-6 gene function might compensate for a loss of mex-5.
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mex-6 is required for the suppression of the Mex phenotype via reduction of pos-1(+):
If reducing the dosage of pos-1(+) relieves repression of MEX-6 translation, then the ability of pos-1(–) to dominantly suppress the Mex phenotype of efl-1 should require mex-6(+). To test this idea, we removed mex-6 by RNAi in the efl-1(se1) pos-1(–)/efl-1(se1) pos-1(+) background. When efl-1(se1) pos-1(zu148)/efl-1(se1) pos-1(+) mothers were grown at 26°, 63% of their progeny appeared wild type (Table 1; Figure 1). In contrast, only 3% of the progeny from efl-1(se1) pos-1(zu148)/efl-1(se1) pos-1(+);mex-6(RNAi) mothers appeared wild type and 92% of the progeny were Mex (n = 88) (Figure 1). This result is consistent with the hypothesis that reducing pos-1(+) dosage suppresses the Mex phenotype by increasing MEX-6 expression.
pos-1 does not spatially restrict GFP:MEX-6 expression:
To examine MEX-6 expression in a pos-1 mutant embryo, we generated a C. elegans strain that expressed a gfp:mex-6 fusion containing the endogenous mex-6 3'-UTR (MATERIALS AND METHODS). In a wild-type background, the GFP:MEX-6 signal was enriched in the anterior blastomere of the two-cell embryo and in this blastomere's daughters at the early four-cell stage. We could not compare this pattern to that of endogenous MEX-6 because no antibodies presently exist that specifically recognize MEX-6. However, the GFP:MEX-6 expression pattern was very similar to that of MEX-5. When we removed pos-1 by RNAi, we detected no difference in the GFP:MEX-6 expression pattern (n = 13 for pos-1(–) embryos and n = 7 for wild-type embryos).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In addition to showing that reduced dosage of pos-1(+) can suppress Mex mutants, we made three novel discoveries: (1) Although SKN-1 activity is exquisitely sensitive to the level of pos-1(+), pos-1 is not required for SKN-1 to specify mesodermal or endodermal tissues; (2) POS-1, a demonstrated translational repressor, can bind the 3'-UTR of the mex-6 message; and (3) the asymmetry of two class II messages is disrupted in mex-5 mutant embryos.
How does reducing pos-1(+) levels suppress Mex mutants?
If pos-1(+) is not required for SKN-1 to specify mesoderm, then how does reduction of pos-1(+) suppress this SKN-1 ectopic activity? We propose that the low level of POS-1 detected in the anterior blastomere is functionally significant, and since POS-1 has been demonstrated to act as a translational repressor (OGURA et al. 2003), it is a decrease in this function that suppresses the Mex phenotype. Thus, we searched for a potential target of POS-1 with the expectations that such a target would have an accumulation pattern complementary to that of POS-1 and might have a 3'-UTR with similarity to the known POS-1 target, glp-1 (OGURA et al. 2003). We determined that the 3'-UTR of mex-6 contains similarity to the glp-1 3'-UTR and, using the yeast trihybrid assay, we showed that POS-1 binds this region of the mex-6 message (Figure 6). Consistent with our hypothesis, suppression of the Mex phenotype by reduced pos-1(+) dosage requires mex-6. Together, these results suggest that reducing the level of pos-1(+) suppresses the Mex phenotype by decreasing repression of mex-6 translation (Figure 7).
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POS-1 does not exclusively restrict MEX-6 expression:
Although we have shown that POS-1 binds the 3'-UTR of mex-6, loss of pos-1 function did not result in ectopic MEX-6 expression. The GLP-1 protein, a previously identified target of POS-1 repression, is ectopically expressed in the posterior blastomere of pos-1(–) mutant embryos (OGURA et al. 2003; B. D. PAGE, unpublished observation). If POS-1 also represses translation of mex-6, then why is MEX-6 not misexpressed in the posterior blastomere of pos-1 mutant embryos? Two possibilities could explain this result. First, other CCCH proteins in the posterior blastomere may act redundantly with POS-1 to repress mex-6 translation. Second, MEX-6 protein asymmetry may also be regulated post-translationally. We favor the latter possibility since CUENCA et al. (2003) observed that after replacing the mex-6 3'-UTR with that of another gene, MEX-6 protein was still enriched in the anterior. The asymmetric distribution of several CCCH finger proteins is controlled by protein stability (REESE et al. 2000; DERENZO et al. 2003). In the cases where this has been demonstrated, these proteins are targeted for degradation in anterior blastomeres (DERENZO et al. 2003). If the MEX-6 protein is selectively degraded in the posterior blastomere, then POS-1 would function as an additional level of regulation in restricting MEX-6 accumulation, and MEX-6 misexpression might be detected only in an embryo defective in both MEX-6 degradation and POS-1 activity.
An example of a protein whose expression is under such dual regulation is the Drosophila transcription factor Tramtrack. Tramtrack is regulated both by its protein stability and by translational repression (HIROTA et al. 1999; OKABE et al. 2001). Reduced function of the translational repressor alone has phenotypic consequences in the developing eye; however, under this circumstance, misexpression of Tramtrack is not detected. Extensive misexpression of Tramtrack is seen when the function of both pathways is reduced (HIROTA et al. 1999). From our analysis, we suspect that MEX-6 accumulation is under similar dual regulation in the C. elegans embryo.
Mutations in efl-1 and mex-5 disrupt the asymmetric pattern of class II messages:
Many markers that are asymmetrically expressed or localized in the two-cell embryo have been identified (SEYDOUX and FIRE 1994; ROSE and KEMPHUES 1998; BOWERMAN 2000; PELLETTIERI and SEYDOUX 2002). In previous studies of efl-1 and mex-5 mutant embryos, the only marker consistently detected as altered at the two-cell stage was the SKN-1 protein (SCHUBERT et al. 2000; PAGE et al. 2001). In our current analysis of mex-5 and efl-1 single-mutant embryos, we observed that the asymmetry of two class II mRNAs is disrupted (Table 3). In the maternal germline of wild-type hermaphrodites, both pos-1 and mex-1 messages are detected on P granules and uniformly throughout the egg cytoplasm (GUEDES and PRIESS 1997; TABARA et al. 1999; SCHISA et al. 2001). Like other class II mRNAs in the early embryo, these messages become asymmetrically localized to the posterior cytoplasm and/or degraded from the anterior (SEYDOUX and FIRE 1994; GUEDES and PRIESS 1997; TABARA et al. 1999). In mex-5 and efl-1 mutant embryos, the mechanism(s) that control this asymmetry appear defective.
The idea that mex-5 and efl-1 influences more than SKN-1 asymmetry is not novel. Certain double-mutant combinations with mex-5 or efl-1 have extensive effects on embryonic polarity. In efl-1;mex-5 and mex-5;mex-6 double-mutant embryos, the normal asymmetric pattern of many markers is altered. In such double-mutant embryos, wild-type anterior markers are absent, and proteins that are normally restricted to the posterior and/or germline progenitor are expressed throughout the two-cell and four-cell embryo. Not surprisingly, we observed a symmetric distribution of a class II message in mex-5;mex-6 double-mutant embryos (Table 3). This is consistent with the recent report that the class II message nos-2 is symmetrically distributed in mex-5(RNAi);mex-6 (RNAi) embryos at the 16-cell stage (D'AGOSTINO et al. 2006). Since many posterior proteins are misexpressed in these double-mutant embryos, the disruption of message asymmetry could be due to mislocalization of the posterior proteins. In early mex-5 single-mutant embryos, the majority of posteriorly expressed proteins are properly localized (SCHUBERT et al. 2000); yet, we still detected uniformly distributed messages in this mutant embryo. Thus, we propose that a primary function of mex-5 involves message localization and/or stability. This function could be similar to that of the CCCH finger proteins TTP and Cth2. Each of these proteins targets mRNAs for destabilization, often by binding an AU-rich element (ARE) in the 3'-UTR of the mRNAs (LAI et al. 1999; PUIG et al. 2005). Consistent with this idea, the 3'UTR of mex-1, contains an exact match to the ARE sequence 5'-UAUUUAUU-3'.
Interestingly, the dynamic pattern of MEX-5 expression is consistent with a function in mRNA degradation. At each early cell division of the germline lineage, a somatic sister cell and a germline progenitor are produced. At each of these divisions, MEX-5 becomes enriched in the somatic sister cell (SCHUBERT et al. 2000), and maternal mRNAs disappear from this cell (SEYDOUX and FIRE 1994).
Do the anterior and posterior CCCH proteins mutually restrict one another's expression?
The anterior CCCH protein MEX-5 and the posterior CCCH protein POS-1 have complementary roles in regulating protein expression in the early embryo. MEX-5 limits the expression of a posterior protein, whereas POS-1 restricts the expression of at least one anterior protein (SCHUBERT et al. 2000; OGURA et al. 2003). However, neither of these proteins exclusively restricts the expression of the other in the two-cell embryo. In mex-5 mutant embryos, POS-1 was more highly enriched in the posterior, identical to its wild-type pattern, and in pos-1 mutant embryos, MEX-5 was more concentrated in the anterior. Yet, our analyses of these two proteins suggest that they exert a subtle influence on one another. In mex-5 mutant embryos, the asymmetry of the pos-1 message was affected, such that pos-1 mRNA was detected at high levels in both posterior and anterior cells. This disruption of pos-1 message asymmetry did not affect POS-1 protein asymmetry, but it may affect the level of POS-1 present in the anterior. We have also demonstrated that POS-1 can bind the 3'-UTR of mex-6, a gene redundant in function to mex-5, and provided genetic data suggesting that POS-1 influences the amount of MEX-6 in the anterior blastomere. This intricate regulation between the MEX-5/6 and POS-1 proteins may be important for maintaining their strong complementary patterns. These complementary patterns not only are present at the two-cell stage but also are repeated at each subsequent asymmetric division of the germline lineage (TABARA et al. 1999; SCHUBERT et al. 2000). Although their asymmetric patterns were not altered at the two-cell stage in pos-1 or mex-5 mutant embryos, the expression of MEX-5 or POS-1 is more likely to become symmetric with each subsequent germline division in the respective mutant embryo (J. A. SCHISA and B. D. PAGE, unpublished observations). Thus, the interactions of these proteins may initiate a feedback loop that reestablishes and refines their expression in later asymmetric cell divisions.
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