Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA
*Author for correspondence (e-mail: mhan{at}colorado.edu)
Accepted September 10, 2001
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SUMMARY |
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Key words: lin-40 MTA, lin-39 Hox, VPCs competence, Vulval induction, Morphogenesis, Division orientation, C. elegans
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INTRODUCTION |
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Vulval differentiation in C. elegans is also controlled by several other regulatory pathways, including the RTK/Ras/MAPK (Kornfeld, 1997; Sternberg and Han, 1998), lin-39 Hox (Wang et al., 1993; Clark et al., 1993) and LIN-12/Notch signaling pathways (Greenwald, 1998). During the first and second larval stages (L1 and L2), six out of the twelve ectodermal Pn.p cells, P3.p to P8.p, have the potential to adopt the vulval fate, whereas the other Pn.p cells fuse with the surrounding hypodermal syncytium and lose their competence for vulval induction (Fig. 1). The lin-39 Hox gene, which is expressed in P(3-8).p (referred to as the vulval precursor cells or VPCs), is essential for preventing them from fusing with the hypodermis and maintaining their identity as the VPCs (Fig. 1) (Clark et al., 1993; Wang et al., 1993). Later during the third larval stage (L3), the vulval fate is induced in three (P5.p, P6.p, and P7.p) of the VPCs, while the remaining three fuse with the hypodermal syncytium, hyp7 (Fig. 1). lin-39 activity is also required at this stage of vulval development (Clandinin et al., 1997; Maloof and Kenyon, 1998), possibly to prevent the progeny of the VPCs from fusing to hyp7 and keep them responsive to other signaling events. The RTK/Ras/mitogen-activated protein kinase (MAPK) pathway is activated during L3 in P(5-7).p by an inductive signal from the anchor cell in the neighboring gonad and actively promotes the vulval fate in the VPCs (Sternberg and Han, 1998).
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We have identified lin-40 MTA as a regulator of the cell-specific division pattern during vulval morphogenesis, and further studies reveal its involvement in vulval fate specification. Our genetic analyses of lin-40 mutations disagree with a previous conclusion that lin-40 (also known as egr-1) acts as a class A synMuv gene in repressing vulval induction (Solari and Ahringer, 2000). We report our studies of the genetic interactions between lin-40 and the synMuv genes, the Ras/MAPK pathway and the lin-39 Hox gene.
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MATERIALS AND METHODS |
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The lin-40 alleles used in the non-complementation tests with ku285 were e2173, s1053, s1345, s1351, s1352, s1358, s1360, s1373, s1506, s1593, s1611, s1634, s1669 and s1675 (Clark et al., 1990; Johnsen and Baillie, 1991). These lin-40 alleles are associated with a lethal or sterile phenotype and thus were maintained in heterozygous strains (dpy-18(e364)/eT1 III; lin-40 unc-46(e177)/eT1 V) (Clark et al., 1990; Johnsen and Baillie, 1991; Rosenbluth and Baillie, 1981). ku285 failed to complement 10 of these alleles, s1053, s1345, s1351, s1352, s1358, s1360, s1373, s1593, s1669 and s1675.
In double mutants between ku285 and the synMuv alleles, the actual genotypes of the synMuv mutations (Riddle et al., 1997) were dpy-10(e128) lin-8(n111) II, rol-1(e91) lin-38(n751) unc-52(e444) II, lin15(n433) X (Ferguson and Horvitz, 1989), unc-3(e151) lin-15(n767) X, dpy-17(e164) lin-9(n112) III, unc-13(e1091) lin-35(n745) I, unc-32(e189) lin-36(n766) III, and lon-1(e185) lin-37(n758) III. Some class A synMuv mutations, such as lin-8(n111), lin-38(n751) and lin-15(n433), were linked with no or remote phenotypic markers. To confirm the presence of these class A mutations in the double mutants between them and ku285, a class B synMuv mutation was introduced into the double mutants to test the segregation of a Muv phenotype.
Other alleles used in this study (Riddle et al., 1997) included egl-27(n170) II, lin-39(n1760) III (Clark et al., 1993), let-60(n1046) IV, unc-60(m35) V (McKim et al., 1994), lag-2(q393) V (Henderson et al., 1994), unc-46(e177) V and sDf27 V (Rosenbluth et al., 1985).
Cloning and sequencing of lin-40
Standard three-point mapping techniques were used to locate the mutation in the ku285 allele of lin-40. Among 27 Unc non-Egl recombinant progeny from unc-60 ku285/lag-2 heterozygotes, 21 animals segregated the Let phenotype of lag-2. Among 17 Unc non-Let progeny from lag-2 unc-46/ku285 heterozygous animals, 13 recombinants segregated the Egl phenotype of ku285. We thus mapped ku285 to a genetic location of 8.0 map unit between lag-2 and unc-46 on chromosome V.
In DNA-mediated germline transformation experiments (Mello and Fire, 1995) using a sur-5::gfp reporter pTG96 (Gu et al., 1998), two overlapping cosmids, W08A12 and T27C4, fully rescued the mutant phenotypes of ku285, including late larval lethality, sterility, Muv and abnormal vulval morphogenesis. A EcoRI to KpnI subclone of these two cosmids, pZC78, retains the rescuing activity of ku285 and three other lin-40 alleles, s1593, s1669 and s1675. pZC78 contains a single open reading frame, T27C4.4, as predicted by the Genome Sequencing Consortium, as well as 5 kb of upstream sequence and 1.5 kb of sequence downstream of the stop codon. However, the early-to-mid larval lethality associated with several other lin-40 alleles was not rescued by this genomic fragment. As three of the rescued lin-40 alleles are likely to be null mutations and most of the other non-rescuable alleles were generated from Tc1 transposon mutagenesis, it is likely that the non-rescuable alleles contain lesions in other loci besides lin-40. The molecular lesions in these four rescuable alleles were determined by sequencing genomic DNA from mutant animals. s1669 is a 128 bp deletion from nucleotide 1383 to 1510 after the first ATG in lin-40 cDNA sequence. s1675 is a 5bp deletion from nucleotide 458 to 462 after the first ATG. Lesions of ku285 and s1593 are indicated in Fig. 2.
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An independent study by Solari and Ahringer (Solari and Ahringer, 2000) also identified the lin-40b transcript and lesions in two lin-40 alleles, s1593 and s1669. The lin-40 gene has also been named egr-1 (for egl-27-related gene) in previous reports (Solari and Ahringer, 2000; Solari et al., 1999). As egr-1 is the previously published lin-40 locus and the name egr does not directly reflect the mutant phenotype or protein property, we thus refer to the gene as lin-40 or lin-40 MTA.
Double-stranded RNA interference (RNAi)
PCR primers that each contain a T7 promoter sequence were used to generate a cDNA fragment that corresponds to the first 1.4 kb of the lin-40 coding sequence. Double-stranded RNA (dsRNA) was generated from the 1.4 kb template using a large-scale T7 transcription kit (Novagen, Madison, WI). RNAi was carried out as described previously (Fire et al., 1998). The dsRNA was injected into worms at 25 ng/µl. Injected animals were transferred to individual fresh plates after 16 hours and their progeny were scored for mutant phenotype. A higher concentration of dsRNA (100 ng/µl) was also tested and it did not cause additional or more severe defects.
GFP reporter constructs
The coding sequence of the gfp reporter gene (Chalfie et al., 1994) was excised from the plasmid pPD102.33 (a gift from A. Fire, S. Xu, J. Ahnn and G. Seydoux) and was inserted in-frame after the first methionine residue of the predicted lin-40-coding sequence at a SacI site. Three different GFP reporter constructs were generated. One of them contains the same genomic sequence as in pZC78. In the other two reporter constructs, the genomic sequence after intron II was replaced with either the lin-40a or lin-40b cDNA sequence. All three constructs were injected into unc-119(ed3) animals at 10 ng/µl, together with an unc-119(+) plasmid pDP#MM016B (Maduro and Pilgrim, 1995) at 40 ng/µl.
jam-1::gfp reporter
The jam-1 (junction associated molecule 1) gene product is present in cell adhesion junctions (Mohler et al., 1998). A jam-1::gfp reporter (a gift from J. Simske and J. Harding) was injected into unc-119(ed3) animals together with pDP#MM016B (Maduro and Pilgrim, 1995), and was integrated into the worm genome using gamma irradiation (W. Hanna-Rose, unpublished). A resulting integrated transgene kuIs46[jam-1::gfp+ u119(+)] X was used in this study.
Antibody staining
A lin-39::lacZ reporter integrated on chromosome IV, muIs6[lin-39::lacZ+pRF4(rol-6d)] (Wang et al., 1993), was introduced into lin-40(ku285) by mating. As other lin-40 alleles, all associated with lethality or sterility, were maintained as heterozygotes, and the maternal lin-40 gene product might interfere with the testing result, these alleles were not examined in this experiment. Animals were fixed according to the protocol of Bettinger et al. (Bettinger et al., 1996), and then stained overnight with an anti-ß-galactosidase antibody (1:500 dilution) from Promega (Madison, WI). After three washes in the buffer B (Bettinger et al., 1996) for several hours, animals were incubated with a Cy3-conjugated goat anti-mouse antibody (Jackson Immunoresearch, West Grove, PA) for several hours in the dark. All animals were also stained with DAPI and a monoclonal antibody MH27 (a gift from R. Waterston), which recognizes an epitope in the jam-1 gene product in cell adhesion junctions. The MH27 antibody served as a positive control for the antibody staining and the DAPI staining helped in identifying Pn.ps. Only animals that had positive MH27 antibody staining and had at least one Pn.p cell stained with anti-ß-galactosidase were counted in the experiment.
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RESULTS |
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We further investigated the effect of the lin-40(ku285) mutation on vulval induction in a let-60 Ras(n1046gf) background, where P3.p, P4.p and P8.p are often ectopically induced, resulting in a Multivulva (Muv) phenotype (Beitel et al., 1990; Han et al., 1990). Although lin-40(ku285) did not cause any vulval induction phenotype in an otherwise wild-type background, it led to an elevated level of vulval induction in combination with the let-60(n1046gf) mutation in P3.p, P4.p and P8.p (see Table 4). This is in support of the above conclusion that lin-40 represses the vulval fate in the VPCs.
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As it was also shown in a previous report (Solari and Ahringer, 2000), the lin-40 gene encodes a protein that is homologous to MTA1 and MTA2 in mammals, which were identified as components of the NuRD complex (Xue et al., 1998; Zhang et al., 1999). The similarity between MTA and its C. elegans homologs, LIN-40 and EGL-27 (Chng and Kenyon, 1999; Herman et al., 1999; Solari et al., 1999), is mostly restricted to several conserved peptide motifs, including a leucine zipper, a SANT domain (a DNA-binding domain first identified in the oncogene myb), and a zinc-finger motif (Fig. 2B) (Nawa et al., 2000; Solari et al., 1999; Toh et al., 2000). Another conserved domain, the SH3-binding domain, is only present in mammalian MTA1 (Fig. 2B) (Toh et al., 1994; Toh et al., 1995). The similarity between LIN-40, MTA1 and MTA2 suggests that lin-40 might function as a transcriptional regulator.
We have examined the expression pattern of lin-40 using a gfp translational fusion reporter, which contains the genomic sequence of lin-40 and fully rescued the four lin-40 alleles. The fusion protein was predominantly localized to the nuclei of most, if not all, somatic cells (data not shown). A similar observation was reported previously by Solari and Ahringer (Solari and Ahringer, 2000). We further analyzed the expression of lin-40a and lin-40b isoforms using gfp reporters fused to the respective cDNA sequences and revealed that LIN-40A was mostly localized to the nucleus (Fig. 2C,D), whereas LIN-40B was present at a lower level in both the cytoplasm and the nucleus (Fig. 2E,F).
lin-40 does not function as a typical class A or class B synMuv gene
Given that mammalian MTA1 and MTA2 were found to be associated with the NuRD complex and that several class B synMuv genes encode proteins similar to components of the complex, we hypothesized that lin-40 acts as a class B synMuv gene. To test this model, we constructed double mutants between lin-40(ku285) and several class A synMuv mutations, including lin-8(n111), lin-38(n751) and lin-15A(n433). None of these class A synMuv mutations exhibited a Muv phenotype in combination with lin-40(ku285) (Table 2). Replacing lin-40(ku285) with RNAi against lin-40 generated similar results in these class A mutants (Table 2), suggesting that lin-40 is not a class B synMuv gene. However, another class A mutation, lin-15(n767), showed an allele-specific interaction with lin-40(ku285) as it caused a Muv phenotype in the ku285 background (an average of 3.3 VPCs were induced in the lin-40(ku285); lin-15(n767) double mutant versus three VPCs being induced in wild-type animals) (Table 2). This allele also behaved differently from other class A synMuv mutations under other assay conditions (Z. C. and M. H., unpublished).
A previous study by Solari and Ahringer (Solari and Ahringer, 2000) led to the conclusion that lin-40 acts as a class A synMuv gene. They showed that in two class B synMuv mutants, lin-9(n112) and lin-37(n758), which were subjected to lin-40(RNAi), multiple vulva-like protrusions were observed in the animals (in an average of 61% of lin-37 mutants) under dissecting microscopes. This has led to the suggestion that P3.p, P4.p or P8.p. is ectopically induced into vulval cells. Such a defect was not seen in class A synMuv or wild-type animals subjected to lin-40(RNAi) (Solari and Ahringer, 2000). To test this possibility of lin-40 being a class A synMuv gene, we constructed double mutants between lin-40(ku285) and four class B synMuv mutations, lin-9(n112), lin-35(n745), lin-36(n766) and lin-37(n758). Examination of vulval induction under Nomarski optics showed that the average number of VPCs being induced in these double mutants was not significantly higher than that in wild-type animals (Table 2). Interestingly, in these double mutants, P(5-7).p sometimes failed to be induced to adopt the vulval fate, whereas the other three VPCs were, at a low frequency, ectopically induced (Table 3). In addition, an interesting vulval morphogenetic defect was often observed. The induced vulval cells sometimes did not migrate or integrate properly with other vulval cells so that the progeny of P(5-7).p often formed individual vulval invaginations (Fig. 3B). In these lin-40(ku285); synMuvB double mutants, up to three vulva-like protrusions could be formed from the progeny of P(5-7).p, whereas in wild-type animals, these cells form only a single vulval invagination. We also applied RNAi against lin-40 to the same class B synMuv mutants as did Solari and Ahringer (Solari and Ahringer, 2000) and observed a similar morphogenetic defect as in lin-40(ku285); synMuvB animals. The phenotype of underinduction in P(5-7).p and overinduction in P(3,4,8).p was also seen in lin-40(RNAi); synMuvB double mutants (Table 3). Such a phenotype in vulval induction is clearly different from the synthetic Muv phenotype caused by a combination between class A and class B genes (Ferguson and Horvitz, 1989; Lu and Horvitz, 1998). Overall, these results indicate that lin-40 does not function as a typical class A synMuv gene and that lin-40 and class B genes have weak synergistic effect on vulval induction in both negative and positive directions. Although we observed a similar percentage of animals showing the multiprotrusion phenotype in lin-40; synMuvB animals (62% of lin-37(n745); lin-40(RNAi) animals, n=21 and 36% of lin-37(n745); lin-40(ku285) animals, n=56) as did Solari and Ahringer (Solari and Ahringer, 2000), we concluded that such a phenotype was mostly due to the defect in vulval morphogenesis. instead of ectopic vulval induction.
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lin-40 promotes the fusion of the Pn.p cells with hyp7
The fusion between Pn.ps and the hypodermal syncytium, hyp7, directly affects vulval induction. Blocking this fusion in P(3-8).p during L1/L2 is essential to maintain their responsiveness to the vulval inductive signal at the later stage. lin-39 Hox activity is required to prevent this fusion as lin-39 loss-of-function mutations cause abnormal fusion of P(3-8).p with hyp7 and eliminate vulval induction in the fused cells (Clark et al., 1993; Wang et al., 1993). It is thus conceivable that lin-40 may negatively regulate vulval induction by acting early in the fusion process to promote cell fusion and thus repress the competence of the VPCs for vulval induction.
In wild-type hermaphrodites, P4.p to P8.p remain unfused with hyp7 100% of the time at the L2 stage, whereas P3.p fuses with hyp7 and loses its competence for vulval induction about 50% of the time (Sternberg and Horvitz, 1986; Sulston and White, 1980) (Fig. 4A). Such variability in the P3.p cell fusion provides us a sensitive assay to test if lin-40 affects the cell fusion process. In this test, the frequency of the P3.p cell fusion served as an indicator of the competence status of the VPCs. A JAM-1::GFP fusion protein, which is present in cell adhesion junctions (Mohler et al., 1998), was used to score the fusion event (Fig. 3C-F; Materials and Methods). In a VPC that has fused with hyp7, this fusion protein can no longer be seen at the apical surface of the cell (Fig. 3E). We observed that lin-40(ku285) significantly increased the frequency of P3.p being unfused to about 91% (Fig. 4A), suggesting that the normal function of lin-40 is to promote the fusion between the VPCs, at least P3.p, and the hyp7 cell.
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We also tested if the other MTA1 homolog in C. elegans, egl-27, is also involved in controlling the competence of the VPCs. We observed that P3.p cells remained unfused at a higher frequency in egl-27(n170) null mutants (Fig. 4A). In addition, although egl-27(n170) did not cause a Muv phenotype in an otherwise wild-type background (data not shown), it increased the vulval induction level in a let-60(n1046gf) mutant background (Table 4). These results indicate that egl-27 also promotes cell fusion and therefore represses the competence of the vulval precursor cells.
lin-40 also regulates the cell fusion decision in posterior Pn.ps
In egl-27 mutants, the posterior P9.p though P11.p cells sometimes fail to fuse with hyp7 (Chng and Kenyon, 1999). We found that lin-40(ku285) was not able to affect cell fusion in these posterior Pn.ps in otherwise wild-type animals (Fig. 4B). However, lin-40(ku285) significantly exacerbated the fusion defect in an egl-27(n170) mutant background; these posterior Pn.ps remained unfused at much higher frequencies in egl-27; lin-40 double mutants (Fig. 4B). In addition, lin-40(ku285) weakly induced the vulval fate in the unfused Pn.ps in egl-27(n170) mutants, so that these cells sometimes underwent cell divisions and other morphogenetic events to form pseudo-vulval structures (data not shown).
Although we were unable to test the competence of P(4-8).p using the JAM-1::GFP reporter, as these cells always remain unfused with hyp7 in wild-type animals, we speculate that lin-40 also promotes cell fusion in these Pn.ps, based on the fact it functions similarly in both P3.p and P(9-11).p. This hypothesis is supported by the observation that a lin-40 mutation increased the competence of not only P3.p, but also P4.p and P8.p, in a let-60(n1046) background (Table 4).
lin-40 negatively regulates lin-39 expression
Given the fact that lin-39 encodes a Hox family transcription factor, and the fact that LIN-40 is likely to be involved in transcriptional repression like mammalian MTA proteins, it is conceivable that lin-40 controls vulval cell competence partly by regulating the lin-39 gene, most probably in a negative manner. This inhibition of lin-39 might be accomplished by either downregulating lin-39 expression or acting as a transcriptional co-repressor to inhibit the downstream targets of lin-39. Because the vulva-specific targets of lin-39 remain unknown, we tested only the first hypothesis by examining lin-39 expression in a lin-40 mutant background.
To analyze the vulva-specific expression of lin-39, we made use of an integrated lin-39::lacZ reporter transgene (Wang et al., 1993) (Materials and Methods). This reporter is weakly but consistently expressed in P5.p through P8.p during the L2 stage, and is present at very low levels in P3.p and P4.p (Wang et al., 1993) (Fig. 3G). Using an anti-ß-galactosidase antibody, the fusion gene product was assayed in the VPCs during the L2 stage, as lin-39 expression is upregulated during the later L3 stage by the Ras/MAPK pathway (Maloof and Kenyon, 1998). The frequency of visible LIN-39::lacZ reporter in each VPC was used as a measure of lin-39 expression and a MH27 antibody was used as a positive control for antibody staining (see Materials and Methods). As indicated in Table 5, lin-39::lacZ expression was significantly increased in lin-40(ku285) mutants. For example, 67.1% of P5.p cells had visible lin-39::lacZ expression in lin-40(ku285) as opposed to 43.7% in wild-type animals. This result suggests that lin-40 negatively regulates lin-39 expression, which is consistent with the above genetic results showing that a lin-40 mutation increased the unfused cell fate in Pn.ps and enhanced vulval induction in the VPCs.
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DISCUSSION |
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Our result indicates that lin-40 has an opposite effect to that of the Ras/MAPK pathway in controlling lin-39 expression. Previously it has been shown that the Ras/MAPK pathway positively regulates lin-39 expression and that the level of LIN-39 correlates with the strength of the inductive signaling mediated by the Ras/MAPK pathway (Maloof and Kenyon, 1998). Constitutive activation of the Ras/MAPK pathway leads to an elevated level of LIN-39, whereas impairment of the activity of this pathway results in a reduced level of LIN-39 in all VPCs (Maloof and Kenyon, 1998). In contrast, we found that the lin-40(ku285) loss-of-function mutation caused an upregulation of lin-39 expression (Table 5), suggesting that lin-40 plays an inhibitory role in controlling lin-39 expression. Consistently, in this lin-40 mutant, P3.p remained unfused at a higher frequency during the L2 stage (Fig. 4A) and was induced by the Ras/MAPK pathway to adopt the vulval fate at a higher level during the L3/L4 stages (Table 4). Furthermore, reduction of lin-39 activity by a lin-39(n1760) mutation was able to overcome the effect caused by lin-40(ku285) (Fig. 4A). Taken together, these results indicate that lin-40 functions upstream of lin-39 to repress its expression and this inhibition of lin-39 allows the VPCs to fuse with hyp7 and therefore represses vulval induction (Fig. 5). Further analyses on synMuv genes in regulating this lin-39-mediated cell fusion process revealed a highly related, although somewhat different from that of lin-40, function of these genes (Z. C. and M. H., unpublished).
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The observation of a multiprotrusion phenotype in lin-40 and in lin-40; synMuvB double mutants, which mostly resulted from a failure in vulval cell migration or fusion during vulval morphogenesis (Fig. 3B), suggests a previously uncharacterized function for lin-40 and some class B synMuv genes in controlling these cellular processes. We further showed that lin-40 also regulates cell divisions during vulval morphogenesis, as the divisions along the transverse axis during the third round of vulval cell divisions were specifically affected by lin-40 mutations (Table 1). lin-40 appears to prevent the longitudinal division in cells where the division plane will be changed to an angle perpendicular to the previous one. This transition of division orientation requires a pause in the cell division cycle because the transverse divisions usually occur later than the longitudinal divisions. In lin-40 mutants, when cells abnormally divided longitudinally, the delay before the third cell division was also eliminated. Thus, it is possible that lin-40 functions to impose a brief break in the cell cycle to allow the rearrangement of the cytoskeleton for the next cytokinesis.
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ACKNOWLEDGMENTS |
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