EGF Signal Propagation during C. elegans Vulval Development Mediated by ROM-1 Rhomboid

During Caenorhabditis elegans vulval development, the anchor cell (AC) in the somatic gonad secretes an epidermal growth factor (EGF) to activate the EGF receptor (EGFR) signaling pathway in the adjacent vulval precursor cells (VPCs). The inductive AC signal specifies the vulval fates of the three proximal VPCs P5.p, P6.p, and P7.p. The C. elegans Rhomboid homolog ROM-1 increases the range of EGF, allowing the inductive signal to reach the distal VPCs P3.p, P4.p and P8.p, which are further away from the AC. Surprisingly, ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. This observation led to the discovery of an AC–independent activity of EGF in the VPCs that promotes vulval cell fate specification and depends on ROM-1. Of the two previously reported EGF splice variants, the longer one requires ROM-1 for its activity, while the shorter form acts independently of ROM-1. We present a model in which ROM-1 relays the inductive AC signal from the proximal to the distal VPCs by allowing the secretion of the LIN-3L splice variant. These results indicate that, in spite of their structural diversity, Rhomboid proteins play a conserved role in activating EGFR signaling in C. elegans, Drosophila, and possibly also in mammals.


Introduction
Intercellular signaling pathways control many diverse processes, such as cell proliferation, differentiation, survival, migration, shape changes, and responses to the environment. In most instances, the release of the signaling molecules by the signal-sending cells constitutes a rate-limiting step that determines the spatial distribution and temporal duration of the response (Freeman and Gurdon 2002). On the other hand, the binding sites on the receptors that are presented by the signal-receiving cells are usually in excess of the available ligands (Freeman and Gurdon 2002). Specificity is achieved by the tissue-specific expression of the signal or by the regulated activation of an inactive precursor molecule. For example, growth factor peptides are often produced as inactive precursors that need to be processed before they can be released and activate their cognate receptors on the signalreceiving cells (Arribas et al. 1996).
The epidermal growth factor (EGF) receptor (EGFR) acts in a highly conserved signal transduction pathway that controls various cell fate decisions in metazoans (Bogdan and Klambt 2001). EGFR ligands of the transforming growth factor-a (TGF-a) family are produced as membrane-tethered precursor proteins with a single extracellular EGF repeat that is cleaved off the membrane anchor (Pandiella and Massague 1991;Bosenberg et al. 1993). The best-studied example of EGF processing is probably the Drosophila growth factor Spitz, which activates the EGFR in multiple developmental processes (Rutledge et al. 1992;Golembo et al. 1996). Genetic analysis of Drosophila EGFR signaling has identified Rhomboid-1 as a protein necessary for Spitz activation in the signal-sending cell (Bier et al. 1990; Golembo et al. 1996;Guichard et al. 2000;Wasserman et al. 2000). Drosophila Rhomboid-1 is the founding member of a family of seven-pass transmembrane proteins that function as intramembrane serine proteases . Site-specific cleavage of Spitz by Rhomboid-1 in the Golgi apparatus allows the secretion of the extracellular portion of Spitz by the signalsending cell Urban and Freeman 2003). The Drosophila genome encodes a total of seven rhomboid genes with partially overlapping functions in different tissues that utilize the EGFR pathway (Wasserman et al. 2000;. There are four predicted Rhomboid homologs in humans and five in C. elegans (Wasserman et al. 2000). Rhomboid-like proteins are even found in yeast and bacteria (Gallio et al. 2002;McQuibban et al. 2003). Rhomboids are part of the larger family of I-Clip proteases that includes the aspartyl protease Presenilin (Wolfe et al. 1999) and the Zn 2þ metalloprotease S2P . On the other hand, the secretion of vertebrate TGF-a involves processing by a disintegrin metalloprotease (ADAM), TNF-aconverting enzyme (Peschon et al. 1998). Whether a Rhomboid protease is also involved in the processing of TGF-a is currently unknown.
The development of the C. elegans hermaphrodite vulva serves as a simple model by which to study signal transduction and cell fate determination during organogenesis (Kornfeld 1997;Sternberg and Han 1998). During C. elegans postembryonic development, the anchor cell (AC) in the somatic gonad induces three out of six equivalent vulval precursor cells (the VPCs, termed P3.p through P8.p) in the ventral hypodermis to adopt vulval cell fates (Sulston and White 1980;Kimble 1981). The AC produces the LIN-3 growth factor, which is similar to Drosophila Spitz and mammalian TGF-a (Hill and Sternberg 1992). The VPCs express the EGFR homolog LET-23 on the basolateral surface that faces the AC (Whitfield et al. 1999), and they are all competent to activate the RAS/mitogen-activated protein kinase (MAPK) signaling pathway in response to the inductive LIN-3 EGF signal. The VPC closest to the AC, P6.p, adopts the primary (18) cell fate characterized by a symmetrical cell lineage leading to eight 18 vulval cells (Sternberg and Horvitz 1986). The neighbors of P6.p, P5.p and P7.p, adopt the secondary (28) cell fate, which is characterized by an asymmetrical lineage leading to seven 28 vulval cells. The more distally located VPCs, P3.p, P4.p, and P8.p, adopt the uninduced tertiary (38) cell fate. After dividing once, they fuse with the surrounding hypodermal syncytium (hyp7). LIN-3 EGF dosage experiments have suggested that the inductive signal acts in a graded manner . According to this model, P6.p receives the highest amount of the inductive signal and thus adopts the 18 fate, while an intermediate level of the LIN-3 signal specifies the 28 fate in P5.p and P7.p. The distal VPCs, P3.p, P4.p, and P8.p, receive too little signal to adopt vulval cell fates. However, in response to the inductive signal, P6.p produces a lateral signal that activates the LIN-12 NOTCH signaling pathway in the neighboring VPCs, P5.p and P7.p (Greenwald et al. 1983). The LIN-12 NOTCH signal is both necessary and sufficient to induce the 28 cell fate (Sternberg 1988;Simske and Kim 1995). Thus, the graded LIN-3 EGF signal may act redundantly with the lateral LIN-12 NOTCH signal to specify the 28 vulval cell fate in the neighbors of P6.p (Kenyon 1995).
Like its Drosophila and vertebrate homologs, LIN-3 EGF is synthesized as a transmembrane precursor protein (Hill and Sternberg 1992). Experiments with dig-1 mutants in which the AC is dorsally displaced indicate that the AC is capable of inducing vulval cell fates from a distance, suggesting that proteolytic cleavage of membrane-bound LIN-3 occurs in the AC (Thomas et al. 1990). Here, we report the identification of the C. elegans Rhomboid homolog ROM-1 as a positive regulator of vulval induction. Surprisingly, we find that ROM-1 acts in the signal-receiving VPCs rather than in the signal-sending AC. Furthermore, we uncover an AC-independent function of LIN-3 EGF that depends on ROM-1 activity in the VPCs. Two LIN-3 splice variants, termed LIN-3S and LIN-3L, that differ by an insertion of 15 amino acids in the region in the juxtamembrane domain critical for processing, have been described (Hill and Sternberg 1992). Genetic epistasis experiments indicate that LIN-3L activity in the VPCs depends on ROM-1, while LIN-3S or a truncated form of LIN-3 lacking the transmembrane domain act independently of ROM-1. We propose a relay model in which ROM-1 is required for the activation of LIN-3L in the proximal VPCs to transmit the inductive AC signal to the distal VPCs.

Results
Five Rhomboid-Like Proteins in C. elegans Since the LIN-3 EGF growth factor is produced as a transmembrane precursor protein (Hill and Sternberg 1992), we asked whether an intramembrane serine protease of the Rhomboid family is involved in the proteolytic processing of LIN-3 EGF. Rhomboid proteins in metazoans share a characteristic secondary structure consisting of seven transmembrane domains (Bier et al. 1990;Urban et al. 2001). We searched the complete C. elegans genome sequence for genes with similarity to Drosophila rhomboid-1 and identified five rhomboid-like genes termed rom-1 (F26F4.3), rom-2 (C48B4.2), rom-3 (Y116A8C.14), rom-4 (Y116A8C.16), and rom-5 (Y54E10A.14) ( Figure 1A). All five C. elegans ROM proteins display the typical secondary structure of Rhomboids (Wasserman et al. 2000). The transmembrane domains show the highest degree of sequence conservation, while the hydrophilic N termini are more divergent ( Figure 1B). ROM-1 is most similar to Drosophila Rhomboid-1 (35% identity), followed by ROM-2 (29% identity) and the more diverged ROM-3 (24% identity), ROM-4 (26% identity), and ROM-5 (29% identity). Mutagenesis experiments with Drosophila Rhomboid-1 have identified a catalytic triad formed by conserved asparagine, serine, and histidine residues that are necessary for the serine protease activity . This catalytic triad is conserved only in ROM-1 (black triangles in Figure 1B), suggesting that the other four Rhomboid-like proteins do not function as serine proteases.
In order to confirm the predicted intron-exon structure of rom-1, we isolated rom-1 cDNA by RT-PCR. An SL1 transspliced leader sequence was identified at the 59 end of the message that was spliced to the second of the six exons predicted by the C. elegans genome project ( Figure 1C) (see http://www.wormbase.org). The remaining intron-exon boun- , and Homo sapiens (H.s.) calculated with the neighbor joining method using CLUSTAL X (Thompson et al. 1997). (B) Alignment of C. elegans (C.e.) ROM-1 and ROM-2 and Homo sapiens (H.s.) Rho-1 relative to Drosophila melanogaster (D.m.) Rho-1. Residues identical to those of Drosophila Rho-1 are highlighted in black, and similar residues are highlighted in grey. The thick black lines indicate the predicted seven-pass transmembrane domains. The three black triangles point at the residues forming a catalytic triad that forms a charge-relay system to activate the essential serine residue during peptide bond cleavage, and the three open triangles indicate other conserved residues necessary for the enzymatic activity as identified in D.m. Rho-1 ). The region underlined with a dotted line indicates the extent of deletion in the rom-1(zh18) allele. (C) Intron-exon structure of the rom-1 locus and extent of the deletion in the rom-1(zh18) strain. The numbers indicate the position of the deletion break-points relative to the A in the ATG start codon. DOI: 10.1371/journal.pbio.0020334.g001 daries were confirmed experimentally and corresponded exactly to the predicted boundaries. The conceptual translation of the 1,071-bp open reading frame (ORF) predicts a protein of 356 amino acids, with very short stretches of hydrophilic amino acids between the seven-pass transmembrane domains, except for a longer loop consisting of 43 amino acids between the first and second transmembrane domains (see Figure 1B).

ROM-1 and ROM-2 Are Not Essential for Normal Vulval Development
As a first step to examine the biological function of the rom genes, we used RNA interference (RNAi) to transiently knock down their expression (Fire et al. 1998;Fraser et al. 2000;Kamath et al. 2001). Double-stranded RNA derived from a 352-bp rom-1 or a 718-bp rom-2 cDNA fragment of the divergent N-terminal portion was injected into the hermaphrodite gonads, and vulval development was examined in the F1 progeny under Nomarski optics. No obvious vulval phenotype was observed when rom-1 or rom-2 RNAi was performed in a wild-type background. Also, feeding wild-type or let-60(n1046gf) animals with bacteria producing rom-3 dsRNA had no effect on vulval development (unpublished data). Due to the high degree of sequence similarity between rom-3 and rom-4 (69.8% identity), rom-3 RNAi is likely to simultaneously reduce rom-4 function.
Using a PCR-based assay to screen a library of mutagenized worms, we isolated a 1,556-bp deletion in the rom-1 gene (see Figure 1C) (Jansen et al. 1997;Berset et al. 2001). The zh18 deletion removes 206 amino acids from the N terminus, including the first three transmembrane domains and 384 bp of promoter sequences. Thus, the zh18 deletion probably results in a complete loss of rom-1 function and will be referred to as rom-1(0). The rom-1(0) single mutants exhibited no obvious phenotype; they were healthy and fertile. In addition, we obtained the rom-2(ok966) allele from the C. elegans Gene Knockout Consortium. The rom-2(ok966) animals carry a 530-bp deletion that removes the fifth exon, which contains the predicted catalytic center with the essential histidine residue (see Figure 1B) . Since this allele is predicted to inactivate any potential protease activity of ROM-2, we refer to it as rom-2(rf). Consistent with the RNAi experiments, both rom-1(0) and rom-2(rf) single mutants exhibited normal vulval development (Table 1, rows 2 and 3). Also, in rom-1(0) rom-2(rf) double mutants, no defects in vulval development were observed, ruling out a possible redundant function of the two genes (Table 1, row 4). Thus, neither ROM-1 nor ROM-2 are required for vulval induction under normal conditions.

ROM-1 Positively Regulates the EGFR/RAS/MAPK Pathway in Distal VPCs
Next, we examined whether loss of rom-1 or rom-2 function affects vulval induction in a sensitized genetic background by using mutations that hyperactivate the EGFR/RAS/MAPK pathway. The rom-1(0) mutation as well as rom-1 RNAi partially suppressed the multivulva (Muv) phenotype caused by overexpression of the LIN-3 EGF growth factor [lin-3(þ)] (Hill and Sternberg 1992) or by the n1046 gain-of-function (gf) mutation in the let-60 ras gene, which renders vulval development partially independent of upstream signaling Chang et al. 2000) (Table 1, rows 5-7 and 12-14). In addition, the rom-1(0) mutation suppressed the Muv phenotype of hs::mpk-1 animals that overexpress the wild-type MAPK MPK-1 under control of a heat-shock promoter together with Drosophila MEK-2 under control of the interferon-1a promoter   (Table 1, rows 16 and 17). In contrast to rom-1, neither the rom-2(rf) mutation nor rom-2 RNAi affected the Muv phenotype of let-60(gf) animals (Table 1, row 15; unpublished data).
The rom-1(0) mutation did not significantly enhance the vulvaless (Vul) phenotype caused by the lin-3(e1417), lin-2(n397), sem-5(n2019), or let-60(n2021) mutations that reduce the activity of the receptor tyrosine kinase (RTK)/RAS/MAPK pathway (unpublished data). Since these Vul mutants affect the cell fates of only the proximal VPCs (P5.p, P6.p, and P7.p), ROM-1 plays no role in the induction of the proximal VPCs by the AC. Thus, ROM-1 enhances the activity of the EGFR/ RAS/MAPK pathway to allow the induction of the distal VPCs P3.p, P4.p, and P8.p.

ROM-1 Regulates LIN-3 EGF Activity during Vulval Induction
A soluble form of LIN-3 that consists of the extracellular domain with the EGF repeat but lacks the transmembrane and intracellular domains is biologically active and causes a Muv phenotype when overexpressed under control of a heatshock promoter (hs::lin-3extra) . Unlike fulllength LIN-3, the Muv phenotype induced by a low or high dosage of LIN-3extra was not suppressed by rom-1(0) ( Table 1, rows 8-11). In lin-15(rf) mutants, all VPCs adopt vulval cell fates independently of the LIN-3 signal, though induction in lin-15(rf) mutants depends on the activity of LET-23 and the other components of the EGFR/RAS/MAPK pathway (Clark et al. 1994;Huang et al. 1994). The rom-1(0) mutation did not suppress the Muv phenotype of lin-15(rf) animals, suggesting that loss of rom-1 function affects the LIN-3-dependent induction of vulval cell fates rather than the LIN-3independent activity of the EGFR/RAS/MAPK pathway (Table  1, rows 18 and 19).
Finally, we examined the genetic interaction between rom-1 and the Notch and Wnt pathways, since both pathways control vulval cell fate specification in parallel with the RTK/ RAS/MAPK pathway (Wang and Sternberg 2001). In lin-12 notch(gf) animals, no AC is formed, and all VPCs adopt the 28 cell fate (Sternberg and Horvitz 1989). The same phenotype was observed in rom-1(0) lin-12(gf) double mutants (Table 1, rows 20 and 21). In addition, the Muv phenotype caused by hyperactivation of the Wnt pathway through a reduction-offunction mutation in pry-1 axin or by overexpression of a Nterminally truncated BAR-1 b-catenin protein was not suppressed by the rom-1(0) mutation (Table 1, rows 22-25) .
In summary, these experiments suggest that ROM-1 promotes the LIN-3-dependent activation of the EGFR/RAS/ MAPK signaling pathway. ROM-1 likely acts at the level or upstream of LIN-3 since full-length but not a soluble form of LIN-3 was sensitive to loss of rom-1 function.

ROM-1 Is Required to Transmit the Inductive Signal to the Distal VPCs
To assess how much inductive signal each VPC receives, we examined the expression pattern of the egl-17::cfp reporter, which is a transcriptional target of the EGFR/RAS/MAPK pathway ). In mid L2 larvae, before the LIN-12 NOTCH-mediated lateral inhibition becomes effective, egl-17::cfp is expressed in a graded manner with highest levels in P6.p, intermediate levels in P5.p and P7.p , and lower levels in P3.p, P4.p, and P8.p (Figure 2A and 2B). We therefore examined the effect of the rom-1(0) mutation on the egl-17::cfp expression pattern in mid L2 larvae. For this purpose, larvae were synchronized in the mid L1 stage at 13 h of development by letting them hatch in the absence of food, and then development was allowed to proceed by adding food for another 24 h until they reached the mid L2 stage (approximately 37 h of development). Loss of ROM-1 function had no effect on egl-17::cfp expression in the proximal VPCs (P5.p, P6.p, and P7.p), but significantly reduced egl-17::cfp expression in the distal VPCs when compared to wild-type animals ( Figure 2C and 2D). Thus, ROM-1 increases the range of the inductive LIN-3 signal, allowing the distal VPCs to activate the EGFR/RAS/MAPK pathway. The altered egl-17::cfp expression pattern in rom-1(0) animals is consistent with the epistasis data, which showed that loss of rom-1 function affects the induction of only the distal VPCs (see above).

ROM-1 Is Expressed in the VPCs but Not in the AC during Vulval Induction
To analyze the expression pattern of ROM-1, we generated a transcriptional rom-1 reporter by fusing 6.9 kb of the 59 rom-1 promoter/enhancer region to the green fluorescent protein (gfp) ORF carrying a nuclear localizing signal (zhIs5[rom-1::nls::gfp]). With a translational full-length rom-1::gfp fusion construct, we failed to obtain transgenic lines that consistently expressed ROM-1::GFP. Moreover, a genomic DNA fragment encompassing the entire rom-1 locus failed to produce stable transgenic lines even when injected at relatively low concentrations (1-10 ng/ll), suggesting that elevated levels of ROM-1 are toxic to the animals.
[hs::lin-3extra] low c 23 0 3.5 26 9 rom-1(0); Vulval induction was scored using Nomarski optics as described in Materials and Methods. % Vul indicates the fraction of animals with fewer than three induced VPCs, % Muv indicates the fraction of animals with more than three induced VPCs, and the induction index indicates the average number of VPCs per animal that had adopted 18 or 28 vulval fates. Number of animals scored is designated by n. Alleles used: pry-1(mu38), rom-1(zh18), rom-2(ok996), lin-12(n137gf), dpy-19(e1259), huIs7[hs::bar-1DNT], let-60(n1046gf), gaIs36[hs::mpk-1, D-mek-2(gf)], lin-15(n309), zhEx22[lin-3(þ), sur-5::gfp, unc-119(þ)], and syIs12[hs::lin-3extra]. Statistical analysis was done as described in Materials and Methods. a Five independent transgenic lines generated with this construct displayed induction indices ranging from 4.1 to 5.4, and one of the lines displaying a penetrant Muv phenotype (zhEx22) was used for the further analysis in the different backgrounds. b dsRNA was injected into the syncytial gonad of the parents, and vulval induction in the F1 progeny was scored for rows 4 and 11 by inspection under a dissecting microscope. c To provide a low dose of LIN-3extra, L1 larvae were heat-shocked for 5 min at 33 8C and grown at 25 8C until L4. d To provide a high dose of LIN-3extra, or MPK-1 or BAR-1DNT, respectively, early L21 larvae were heat-shocked for 30 min at 33 8C and grown at 25 8C until L4. e These strains carried the dpy-19(e1259) mutation in cis to lin-12(n137gf). ** p 0.001; *** p 0.0001; numbers in brackets next to asterisks indicate the row to which a dataset was compared. n.d., no data. DOI: 10.1371/journal.pbio.0020334.t001 The transcriptional rom-1::nls::gfp reporter was widely expressed in somatic cells throughout development. Surprisingly, we did not detect any rom-1::nls::gfp expression in the gonadal AC before the L4 stage, while consistent expression was observed in the Pn.p cells and the Pn.a-derived neurons from the L1 stage on. In early L2 zhIs5 larvae, the six VPCs expressed rom-1::nls::gfp at equal levels ( Figure 3A and 3B). The Pn.p cells that are not part of the vulval equivalence group and had fused to hyp7 at the end of the L1 stage showed relatively higher rom-1::nls::gfp expression than the VPCs (for example, P1.p, P2.p, and P9.p in Figure 3C and 3D). Toward the end of the L2 stage, rom-1::nls::gfp expression decreased in distal VPCs adopting the 38 uninduced fate and persisted in the proximal VPCs adopting induced vulval fates ( Figure 3C and 3D). In 60% of zhIs5 animals, we observed an upregulation of rom-1::nls::gfp in P6.p, and in 35% and 45% of the cases, rom-1::nls::gfp expression was higher in P5.p and P7.p, respectively (n = 20). After vulval induction, rom-1::nls::gfp was down-regulated in the 18 and 28 descendants of P5.p, P6.p and P7.p, while the 38 descendants of P3.p, P4.p, and P8.p again expressed high levels of rom-1::nls::gfp after they had fused with hyp7 ( Figure 3E and 3F). Expression of rom-1::nls::gfp was observed in the AC and other cells of the somatic gonad only beginning in the L4 stage, before the AC fused with the uterine seam cell and persisting after fusion ( Figure 3G and 3H) (Sulston and Horvitz 1977; Newman et al. Pie graphs on the right (B, D, F, H, and J) show semi-quantitative representations of the expression levels observed in individual VPCs in the different backgrounds. A solid black color indicates the strongest expression of EGL-17::CFP as it was observed in P6.p of many (59%) wild-type animals; dark grey indicates intermediate, light grey weak, and white undetectable expression. The numbers inside the pie charts are the corresponding percentage values, and n refers to the number of animals examined for each case. EGL-17::CFP expression in each VPC of rom-1(0) or lin-3(e1417rf) animals was compared against the same VPC in wild-type animals (considered as expected value) with a Chi 2 test for its independence; *** p 0.0001, ** p 0.001. The row to which a dataset was compared is indicated on the right. All photographs were taken with identical exposure and contrast settings. The scale bar in (I) is 20 lm. DOI: 10.1371/journal.pbio.0020334.g002 PLoS Biology | www.plosbiology.org (D) An early L3 larva in which rom-1::nls::gfp expression was decreased in all VPCs except P6.p (see text for a quantification of the expression pattern). Note that the nuclei of hyp7 and the Pn.p cells that had fused to hyp7 displayed strong rom-1::nls::gfp expression (P1.p, P2.p, P3.p and P9.p in the example shown). (F) A mid to late L3 larva in which P6.p had generated four descendants. Expression of rom-1::nls::gfp occurred only in the 38 descendants of P.4.p and P8.p after they fused to hyp7. (H) An L4 larva during vulval invagination. No rom-1::nls::gfp was detectable in the 18 and 28 descendants of P5.p, P6.p, and P7.p, but the AC and the surrounding uterine cells displayed strong rom-1::nls::gfp expression. (K) A late L2 to early L3 larva following the ablation of the precursors of the somatic gonad. No up-regulation of rom-1::nls::gfp in P5.p, P6.p, or P7.p was observed. The scale bar in (K) is 10 lm. DOI: 10.1371/journal.pbio.0020334.g003 1996). To test whether the inductive AC signal is required for the elevated rom-1::nls::gfp expression in the proximal VPCs, we ablated in zhIs5 animals the precursors of the somatic gonad Z1 and Z4 (Kimble 1981). Uniformly low rom-1::nls::gfp expression was found in all six VPCs of gonad-ablated zhIs5 animals at the late L2 to early L3 stage, before the descendants of the 38 VPCs had fused to hyp7 ( Figure 3J and 3K, n = 20). To test whether rom-1::nls::gfp expression depends on RTK/RAS/MAPK signaling in the VPCs, we introduced the zhIs5 transgene into lin-7(e1413) mutants that exhibit a penetrant Vul phenotype due to reduced LET-23 EGFR activity (Simske et al. 1996). In lin-7(e1413); zhIs5 animals, the up-regulation of rom-1::nls::gfp occurred less frequently (in 13%, 33%, and 7% of the cases in P5.p, P6.p, and P7.p, respectively, n = 15). Thus, the AC signal upregulates rom-1::nls::gfp expression in the VPCs that adopt vulval cell fates.

ROM-1 Acts in an AC-Independent Pathway that Promotes Vulval Induction
To examine whether ROM-1 acts in cells other than the AC (which is part of the somatic gonad), we tested the effect of loss of rom-1(þ) function on vulval induction in gonad-ablated animals. If ROM-1 acts exclusively in the AC, then the rom-1(0) mutation should not affect vulval induction in gonad-ablated animals. On the other hand, if ROM-1 acts in cells other than the AC, then the rom-1(0) mutation should suppress vulval induction even in the absence of the AC. Since the inductive AC signal is absolutely required to initiate vulval development (Kimble 1981), we performed the gonad ablation experiments in let-60(gf) or hs::mpk-1 animals that exhibit a hyperactive EGFR/RAS/MAPK signaling pathway causing AC-independent vulval induction (Table 2, rows 5 and 8) Lackner and Kim 1998;Chang et al. 2000). In addition, we examined lin-3(þ) animals because, as reported previously by Hill and Sternberg (1992), in animals that overexpress wildtype lin-3 under control of its own promoter, some vulval differentiation could still be observed in the absence of the AC, pointing at an additional source of LIN-3 from the transgene in cells outside of the gonad (Table 2, row 2). Loss of rom-1 function in gonad-ablated lin-3(þ), let-60(gf), or hs::mpk-1 animals caused a strong further reduction in vulval induction (Table 2, compare rows 2 with 3, 5 with 6, and 8 with 9). In contrast, vulval induction in gonad-ablated lin-15(rf) animals that exhibit lin-3 independent vulval differentiation was not affected by the rom-1(0) mutation (Table2, rows 13 and 14).
Analogous results were obtained by examining the egl-17::cfp expression pattern after removal of the AC. In gonadablated animals, residual egl-17::cfp expression was observed in all VPCs ( Figure 2E and 2F). In many cases, P6.p expressed higher levels of the reporter than did the other VPCs despite the absence of the AC. Loss of rom-1 function in gonadablated animals caused a further decrease in egl-17::cfp expression in all VPCs ( Figure 2G and 2H). Thus, ROM-1 acts in cells outside of the somatic gonad to promote vulval induction.
An AC-Independent Activity of LIN-3 EGF Next, we used an analogous strategy to test whether endogenous LIN-3 acts with ROM-1 in an AC-independent pathway. The decrease in vulval induction in hs::mpk-1 animals that was caused by the lin-3(n1049) loss-of-function mutation [lin-3(0)] was much stronger than the decrease observed in gonad-ablated lin-3(þ); hs::mpk-1 animals ( Table 2, compare rows 8 and 10; the L1 larval lethal phenotype caused by the lin-3(0) mutation was suppressed by the hs::mpk-1 transgene). Vulval induction in lin-3(0); hs::mpk-1 animals was not affected by gonad ablation since the lin-3(0) allele eliminated lin-3 function in the AC (Table 2, compare rows 10 and 11). Thus, a complete loss of lin-3 function had a more severe effect on vulval induction than did just the removal of the AC. Likewise, the lin-3(e1417) reduction-of-function mutation almost completely abolished the expression of the egl-17::cfp marker ( Figure 2I and 2J). Thus, LIN-3 is also necessary for the AC-independent egl-17::cfp expression in the VPCs. Loss of rom-1 function in a lin-3(0); hs::mpk-1 background caused no further decrease in vulval induction, suggesting that ROM-1 does not affect vulval development in the absence of LIN-3 (Table 2, compare rows 10 and 12). Taken together, these experiments indicate that not only the AC but also cells outside of the gonad produce LIN-3 to promote vulval fate specification. This AC-independent activity of LIN-3 requires ROM-1 function.

LIN-3 EGF from the Pn.p Cells Amplifies the AC Signal
To examine whether the VPCs or their descendants are the source of the AC-independent LIN-3 signal, we expressed lin-3 dsRNA in the Pn.p cells in order to down-regulate by RNAi any possible lin-3 expression in the VPCs (Timmons et al. 2003). For this purpose, a vector consisting of an inverted repeat of a 921-bp lin-3 cDNA fragment under control of the same Pn.p cell-specific lin-31 promoter used above (lin-31::lin-3i) was introduced into wild-type animals . Vulval induction occurred normally in lin-31::lin-3i animals ( Table 4, row 1), although the adult animals displayed an 80% penetrant egg-laying defective (Egl) phenotype due to a defect in vulval morphogenesis (n = 122). In wild-type L4 larvae, the 18 descendants of P6.p in the vulF toroid ring (P6.papl/r and P6.ppal/r,), secrete LIN-3 to specify the ventral uterine (uv1) cell fate in the somatic gonad (Chang et al. 1999). If LIN-3 expression in the F cells is blocked through a mutation in the egl-38 pax transcription factor, then the uv1 cell adopts a uterine seam fate, resulting in an Egl phenotype. Thus, lin-31::lin-3i appeared to efficiently reduce LIN-3 expression in the vulval F cells without reducing the activity of LIN-3 in the AC. To further authenticate the efficiency of this approach, we crossed animals carrying the lin-31::lin-3i transgene to animals expressing the short splice variant of lin-3 cDNA in the Pn.p cells under control of the lin-31 promoter (lin-31::lin-3S, see below). The lin-31::lin-3i transgene almost completely suppressed the Muv phenotype caused by the lin-31::lin-3S transgene, while the lin-31::cre transgene that was used as negative control had no effect (Table 4, rows 2-4). Furthermore, the lin-31::lin-3i transgene significantly reduced vulval induction in lin-3S animals that carry a lin-3 minigene encoding the short splice variant ( Figure 4A), as well as in let-60 ras(gf) and hs::mpk-1 animals (Table 4, rows 5-14). Consistent with an AC-independent function of LIN-3 in the VPCs, the lin-31::lin-3i transgene also affected vulval induction in let-60(gf) animals lacking a gonad (Table 4, rows 11 and 12).
As an independent test to determine if a reduction of LIN-3 expression in the vulval cell lineage affects induction, we used the n578 reduction-of-function mutation in the egl-38 pax transcription factor, because this egl-38 allele has been shown to eliminate LIN-3 expression in the 18 cell lineage (Chang et al. 1999). Although egl-38(rf) single mutants exhibited wild-type levels of vulval induction, the egl-38(rf) mutation reduced the Muv phenotype of hs::mpk-1 animals to a similar degree as the rom-1(0) mutation or the lin-31::lin-3i transgene (Table 4, rows 15 and 16). Thus, EGL-38 is necessary for the AC-independent function of LIN-3.
In summary, these experiments indicated that, during the process of vulval cell fate specification, some of the Pn.p cells (probably the VPCs) produce LIN-3 to amplify the inductive signal.

Three LIN-3 EGF Splice Variants that Differ in the Juxtamembrane Domain
The lin-3 locus encodes two splice variants termed LIN-3S (short) and LIN-3L (long) that are generated by the differential choice of the splice donor of exon 6 ( Figure 4B) (Hill and Sternberg 1992). While performing RT-PCR experiments using a primer pair flanking the differentially spliced exons, we discovered a third splice variant, termed LIN-3XL, that is generated by the insertion of an additional exon (6b) between exons 6 and 7 ( Figure 4A and 4B). The LIN-3XL splice variant was independently isolated from the yk1053b07EST clone. LIN-3XL contains a 41 amino acid insert, and LIN-3L contains a 15 amino acid insert, in the region between the EGF repeat and the transmembrane domain, when compared to LIN-3S ( Figure 4C). Since the analogous region in Drosophila Spitz EGF is required for the proteolytic processing of Spitz by Rhomboid (Bang and Kintner 2000;Lee et al. 2001;Urban and Freeman 2003), we sought to determine which of the LIN-3 splice variants depend on ROM-1 activity. To address this question, we first constructed lin-3 minigenes by replacing the differentially spliced exons with cDNA fragments encoding either of the splice forms (see Figure 4B). Both lin-3L and lin-3S minigenes were capable of inducing a Muv phenotype, but we observed a marked difference in the dosages required to elicit this phenotype. All (12 out of 12) transgenic lines generated by injection of a relatively low (1 ng/ll) or high (100 ng/ll) concentration of the lin-3S minigene exhibited a strong Muv phenotype (with induction indices ranging from 4.1 to 5.6). In contrast, the lin-3L construct caused a Muv phenotype only when injected at a high concentration. (None of the seven lin-3L lines obtained by injecting 1 ng/ll exhibited a Muv phenotype, while all nine lines obtained by injecting 100 ng/ll exhibited a Muv phenotype, with induction indices ranging from 4.2 to 5.0). For the lin-3XL construct, we obtained variable results; some lines exhibited a weak Muv and others no or even a Vul phenotype (unpublished data). Since we failed to observe a consistent phenotype with this minigene construct, we did not further pursue the analysis of the lin-3XL minigene.

ROM-1 Is Necessary for the Activation of the Long LIN-3 Splice Variant
To investigate the genetic interactions between rom-1 and the lin-3 splice variants, we compared one line for each of the lin-3S and lin-3L minigenes that displayed a similar degree of vulval induction (Table 5, rows 1 and 4). Since the presence of endogenous LIN-3 might mask a specific requirement of either LIN-3 splice variant, we introduced the two minigenes into a lin-3(0) background. The lin-3S and lin-3L transgenes both rescued the larval lethality of lin-3(0) mutants, yielding adult Muv animals (Table 5, rows 2 and 5 and Table 6, rows 1 and 3). Since we used multicopy arrays that are silenced in the germ cells and LIN-3 is required in the oocytes to induce ovulation (Clandinin et al. 1998), the rescued lin-3(0); lin-3S and lin-3(0); lin-3L animals were sterile. Loss of rom-1 function did not affect the viability or the Muv phenotype of lin-3(0); lin-3S animals (Table 5, rows 2 and 3 and Table 6, row 2). In contrast, the efficiency of the lin-3L transgene in rescuing the larval lethality of lin-3(0) mutants was reduced by loss of rom-1 function (Table 6, row 4). Moreover, the rare rom-1(0), lin-3(0); lin-3L animals that escaped the larval lethality exhibited a weaker Muv phenotype than lin-3(0); lin-3L animals, suggesting that the function of LIN-3L during vulval induction partially depends on ROM-1 activity (Table 5, rows 5 and 6).

ROM-1 Positively Regulates LIN-3 EGF Mediated Vulval Induction
Of the five Rhomboid proteins predicted by the complete C. elegans genome sequence, only ROM-1, the closest homolog of Drosophila Rhomboid-1, possesses the hallmarks of a serine protease with an intact catalytic center ). Here, we show that ROM-1 acts as a positive regulator of the EGFR/RAS/MAPK signaling pathway during vulval induction, as loss of rom-1 function partially suppresses ectopic vulval induction caused by hyperactivation of the EGFR/RAS/MAPK pathway. Our epistasis analysis points at a role of ROM-1 in activating LIN-3 EGF. The activity of a soluble form of LIN-3 lacking the transmembrane and intracellular domains is completely independent of ROM-1 activity, but the activity of full-length LIN-3 EGF is sensitive to loss of ROM-1 function. Moreover, a mutation in lin-15, which renders vulval induction independent of LIN-3 activity, is not suppressed by loss of rom-1 function, but mutations in LET-23 or downstream components of the EGFR/RAS/MAPK pathway efficiently suppress the lin-15 Muv phenotype (Clark et al. 1994;Huang et al. 1994). Although ROM-1 enhances the activity of the inductive LIN-3 EGF signal, ROM-1 is not required for vulval induction under normal growth conditions. Loss of rom-1 function does not enhance the Vul phenotype caused by mutations that reduce RTK/RAS/MAPK signaling in the proximal VPCs, indicating that ROM-1 is not required for the induction of the proximal VPCs by the AC. These observations point at the existence of another, yet unidentified protease that mediates the release of LIN-3 from the AC. Like vertebrate TGF-a, and unlike Drosophila Spitz, which absolutely depends on Rhomboid function, membranebound LIN-3 might be cleaved at the surface of the AC by an ADAM family metalloprotease (Peschon et al. 1998). Another possibility our experiments have not ruled out is that in rom-1(0) mutants an unprocessed, membrane-bound form of LIN-3 that is retained on the plasma membrane of the AC induces the 18 fate in the adjacent VPC P6.p through juxtacrine signaling (Anklesaria et al. 1990). Once the AC has induced the 18 cell fate in P6.p, the 28 cell fate specification in the neighboring VPCs P5.p and P7.p can occur exclusively through lateral LIN-12 NOTCH signaling, resulting in a wild-type vulva (Greenwald et al. 1983;Kenyon 1995;Simske and Kim 1995). However, the AC is separated from the VPCs by two adjacent basal laminas that dissolve only after the vulval cell fates have been induced (Sherwood and Sternberg 2003). It is therefore difficult to predict whether LIN-3 anchored in the plasma membrane of the AC could reach its receptor LET-23 EGFR on the basolateral surface of P6.p.

ROM-1 Is Required for LIN-3 EGF Activity in the Pn.p Cells
Three lines of evidence indicate that ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. First, a rom-1::nls::gfp transcriptional reporter is expressed in the VPCs but not in the AC around the time of vulval induction. The rom-1 gene appears to be a transcriptional  See Table 1 legend for key to abbreviations and terminology. To confirm that the viable Dpy nonUnc animals were the rescued lin-3(0) homozygotes and not recombinants between dpy-20 and lin-3, they were scored for fertility 4-5 d later.
All animals counted for this table developed into sterile adults due to the silencing of the rescuing transgenes in the germline. In contrast, all viable Dpy nonUnc progeny segregated by dpy-20(e1282) lin-3(n1049)/ unc-44(e362) unc24(e138) mothers lacking a lin-3 minigene developed into fertile adults, indicating recombination between dpy-20 and lin-3. (Seven recombinants were found among 1,000 F1 progeny animals.) Alleles used: rom-1(zh18), lin-3(n1049), dpy-20 (1282) target of the EGFR/RAS/MAPK pathway, as rom-1::nls::gfp expression is up-regulated in the induced VPCs in response to AC signaling. Second, the expression of rom-1 in the Pn.p cells rescues loss of rom-1 function. Third, loss of rom-1 function in animals lacking an AC results in a further suppression of vulval induction and reduction of egl-17::cfp expression, indicating that the main, if not the only, focus of rom-1 action is outside of the gonad. On the other hand, our epistasis analysis and the biochemical experiments done with Drosophila Rhomboid-1 (Bang and Kintner 2000;Lee et al. 2001;Urban and Freeman 2003) suggest that ROM-1 is required cellautonomously for the activation of a membrane-bound LIN-3 precursor. This apparent discrepancy can be explained by the previously published observation of residual lin-3 activity in the absence of the gonad (Hill and Sternberg 1992) and by the additional experiments presented in this paper that uncovered an AC-independent function of LIN-3 during vulval induction. The most likely source of LIN-3 besides the AC are the VPCs, as reducing lin-3 function in the Pn.p cells by tissuespecific RNAi or a mutation in the egl-38 gene, which is required for lin-3 expression in vulval cells (Chang et al. 1999), had essentially the same effect as loss of rom-1 function. Using transcriptional reporter constructs, lin-3 expression has been observed in vulval cells of the 18 lineage beginning in the early L4 stage (Chang et al. 1999), and occasionally we observed weak lin-3 expression in the VPCs or their daughter cells (unpublished data). It is possible that the reporter constructs used were lacking some of the regulatory sequences necessary to drive strong lin-3 expression in the VPC lineage. Other potential sources of LIN-3 may be the posterior ectoderm or the excretory system in the head. However, it seems unlikely that LIN-3 secreted from cells at the anterior or posterior end of the animal influences vulval induction, since we did not observe a bias favoring the induction of anterior or posterior VPCs in the absence of the AC.

A Relay Model for Vulval Induction
Expression levels of rom-1::nls::gfp are highest in the proximal VPCs (P5.p, P6.p, and P7.p) that adopt 18 and 28 vulval cell fates, suggesting that the proximal VPCs are competent to secrete LIN-3 in response to the inductive AC signal. LIN-3 from the proximal VPCs may facilitate the induction of the more distally located VPCs by paracrine signaling (Figure 5). Such a relay model is reminiscent of the EGF signaling during Drosophila oogenesis (Freeman et al. 1992;Wasserman and Freeman 1998). The Gurken growth factor produced by the Drosophila oocyte initially activates the EGFR/RAS/MAPK pathway in the adjacent epithelial follicle cells on the dorsal side of the oocyte independently of Rhomboid. In response to the Gurken signal, the dorsal follicle cells secrete Spitz in a Rhomboid-dependent manner and activate the EGFR in the neighboring follicle cells by paracrine signaling, allowing the signal to spread along the dorsal follicle cell layer. In contrast to Drosophila oogenesis, signal spreading is not necessary for the development of a wild-type C. elegans vulva. The AC needs to induce only the nearest VPC, P6.p, since a 18 cell can specify the 28 fate in the neighboring cells exclusively through lateral LIN-12 NOTCH signaling (Greenwald et al. 1983;Simske and Kim 1995). On the other hand, dosage experiments have indicated that low levels of inductive LIN-3 signal can directly specify the 28 cell fate in the absence of lateral signaling . It is therefore possible that the relay signal generated by ROM-1 and LIN-3 in P6.p contributes to the specification of the 28 cell fate in the neighboring VPCs in combination with the lateral LIN-12 NOTCH signal. LIN-3 secreted from the proximal VPCs could initially serve to maintain the competence of all VPCs, while at a later phase of induction the AC and lateral signals would seal the 18 fate of P6.p and the 28 fate of P5.p and P7.p, respectively. A similar two-step model of vulval induction has been proposed for other rhabditid nematode species such as Oscheius sp. (Felix et al. 2000). In C. elegans, ROM-1 is dispensable for the induction of the proximal VPCs, and the relay mechanism mediated by LIN-3 and ROM-1 only becomes apparent in a sensitized genetic background in which distal VPCs adopt induced cell fates. It is interesting to note in this context that in Mesorhabditis and Teratorhabditis, in which the vulva develops in the posterior body region, induction occurs without a signal from an AC or any other gonad cell (Sommer and Sternberg 1994). In these posterior-vulva Rhabditidae, the VPCs are not equivalent, because only P5.p and P6.p are competent to adopt the 18 fate. The mechanism that generates this intrinsic difference among the VPCs is unknown. It is possible that in these nematodes the specification of the VPC cell fates occurs in a cell-autonomous manner that could involve EGF signaling between the VPCs. The AC in C. elegans serves to position the vulva in the central body region, and the function of an AC appears to be absent in the posterior-vulva Rhabditidae.

Splice Variant-Specific Action of ROM-1
The two previously identified LIN-3 splice forms (LIN-3S and LIN-3L) as well as the newly identified longer variant (LIN-3XL) differ by 15 and 41 amino acid insertions in the juxtamembrane region just prior to the predicted Rhomboid cleavage site at the start of the transmembrane domain (Hill The AC initiates vulval development by secreting the LIN-3 growth factor independently of ROM-1. In response to the AC signal, the proximal VPCs up-regulate ROM-1 expression and start secreting LIN-3 in a ROM-1-dependent manner to relay the AC signal. DOI: 10.1371/journal.pbio.0020334.g005 and Sternberg 1992). Our experiments with LIN-3 minigenes indicate that the activity of the shortest splice form (LIN-3S) is completely independent of ROM-1 function. LIN-3S is expressed at all stages of development, and the LIN-3S minigene rescued all phenotypes caused by the lin-3(0) mutation, including the ovulation defects (unpublished data). This may explain why loss of rom-1 function causes neither the larval lethality nor the sterility observed in lin-3 mutants. Furthermore, our data indicate that LIN-3L function in the VPCs almost completely depends on ROM-1 activity. It seems improbable that the 15 amino acid insertion in LIN-3L could change the substrate specificity toward the ROM-1 protease. A more likely explanation for the inherent difference in the dependence of the LIN-3 splice variants on ROM-1 is suggested by the experiments performed with Drosophila Spitz Tsruya et al. 2002). When expressed in mammalian cells that lack Rhomboid activity, Spitz is retained in the Golgi apparatus. Introducing functional Rhomboid into these cells allows the cleavage and release of Spitz from the Golgi apparatus, resulting in the secretion of the extracellular portion of Spitz. In analogy to Spitz, the small insert in LIN-3L could cause the retention of this LIN-3 isoform in the Golgi apparatus, thus rendering LIN-3L dependent on ROM-1 mediated processing. The tissue distribution of the LIN-3 splice variants is unknown, although all three forms can be detected by RT-PCR in L2 and L3 larvae around the time of vulval induction (unpublished data). In view of the relay model discussed above (Figure 5), tissue-specific splicing may account for the distinct functions of LIN-3. The roles of the two Drosophila EGF-like growth factors Gurken and Spitz may be fulfilled in C. elegans by the splice variants LIN-3S and LIN-3L, respectively. In this model, the AC uses the ROM-1-independent isoform LIN-3S to induce vulval development, and the proximal VPCs relay the AC signal to the distal VPCs by secreting LIN-3L. Alternative splicing has also been reported for the Neuregulin family of EGF-like ligands in vertebrates (Chang et al. 1997). Different Neuregulin isoforms elicit distinct responses by activating different EGFRs (Meyer et al. 1997). The tissue-specific expression of Rhomboid family proteases could determine which isoforms a particular cell type can secrete, thus adding another level of regulation. a 964-bp NdeI-HindIII lin-3S cDNA fragment from pAD25 was cloned into NdeI-HindIII-digested pAD27 using the recA -E. coli SURE strain as host to obtain pAD32. The resulting 1,918-bp lin-3 hairpin fragment was excised with XhoI from pAD32 and subcloned in E. coli SURE into the SalI site of the pB253 vector to obtain pAD35.
RNA interference. The dsRNA to interfere with rom-1 function was generated by in vitro transcription using a 350-bp rom-1 cDNA fragment corresponding to the nucleotides -17 to 725 relative to the predicted start codon of the ORF inserted into pGEM-T (Stratagene) as template. Transcripts were prepared using T7 and Sp6 RNA polymerase and annealed prior to injection as described (Fire et al. 1998). Progeny of the injected animals were assayed at 20 8C. RNAi of rom-2 and rom-3 was done by feeding the animals dsRNA-producing E. coli at 20 8C as described (Kamath et al. 2001).
Vulval induction assay. Vulval induction was scored by examining worms at the L4 stage under Nomarski optics as described (Sternberg and Horvitz 1986). The number of VPCs that adopted a 18 or 28 vulval fate was counted for each animal as described (Sternberg and Horvitz 1986), and the induction index was calculated by dividing the number of 18 or 28 induced cells by the number of animals scored. Statistical analysis was performed using a t-test for independent samples. To remove the AC, the nuclei of the Z1 to Z4 gonadal precursor cells were ablated in early L1 larvae with a laser microbeam as described (Sulston and White 1980;Kimble 1981). The operated animals were allowed to develop until the L4 stage. Only those animals in which neither gonad arm developed and no residual gonadal cells survived were scored.