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.
Citation: Dutt A, Canevascini S, Froehli-Hoier E, Hajnal A (2004) EGF Signal Propagation during C. elegans Vulval Development Mediated by ROM-1 Rhomboid. PLoS Biol 2(11): e334. doi:10.1371/journal.pbio.0020334
Academic Editor: Julie Ahringer, University of Cambridge
Received: April 27, 2004; Accepted: August 3, 2004; Published: September 28, 2004
Copyright: © 2004 Dutt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Competing interests: The authors have declared that no conflicts of interest exist.
Abbreviations: AC, anchor cell; ACEL, AC-specific enhancer; ADAM, a disintegrin and metalloprotease; EGF, epidermal growth factor; EGFR, EGF receptor; Egl, egg-laying defective; GFP, green fluorescent protein; hyp7, hypodermal syncytium; L[N], larval stage [N]; MAPK, mitogen-activated protein kinase; Muv, multivulva; NLS, nuclear localization signal; ORF, open reading frame; 1°, primary; RNAi, RNA interference; RTK, receptor tyrosine kinase; 2°, secondary; 3°, tertiary; TGF, transforming growth factor; VPC, vulval precursor cell; Vul, vulvaless
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 signal-receiving 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-α (TGF-α) 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 (Urban et al. 2001). Site-specific cleavage of Spitz by Rhomboid-1 in the Golgi apparatus allows the secretion of the extracellular portion of Spitz by the signal-sending cell (Lee et al. 2001; 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; Urban et al. 2002). 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 Zn2+ metalloprotease S2P (Urban and Freeman 2002). On the other hand, the secretion of vertebrate TGF-α involves processing by a disintegrin metalloprotease (ADAM), TNF-α-converting enzyme (Peschon et al. 1998). Whether a Rhomboid protease is also involved in the processing of TGF-α 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-α (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 (1°) cell fate characterized by a symmetrical cell lineage leading to eight 1° vulval cells (Sternberg and Horvitz 1986). The neighbors of P6.p, P5.p and P7.p, adopt the secondary (2°) cell fate, which is characterized by an asymmetrical lineage leading to seven 2° vulval cells. The more distally located VPCs, P3.p, P4.p, and P8.p, adopt the uninduced tertiary (3°) 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 (Katz et al. 1995). According to this model, P6.p receives the highest amount of the inductive signal and thus adopts the 1° fate, while an intermediate level of the LIN-3 signal specifies the 2° 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 2° 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 2° 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.
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 (Urban et al. 2001). 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.
Figure 1. The C. elegans Rhomboid Genes
(A) Dendogram showing the relation between the seven-pass transmembrane domains of Rhomboids from C. elegans (C.e.), Drosophila melanogaster (D.m.), 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 (Urban et al. 2001). 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
In order to confirm the predicted intron-exon structure of rom-1, we isolated rom-1 cDNA by RT-PCR. An SL1 trans-spliced leader sequence was identified at the 5′ 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 boundaries 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) (Urban et al. 2001). 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.
Table 1. Suppression of Multivulva Mutants by rom-1(0)doi:10.1371/journal.pbio.0020334.t001
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(+)] (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 (Beitel et al. 1990; 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-1α promoter (Lackner and Kim 1998) (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 heat-shock promoter (hs::lin-3extra) (Katz et al. 1995). Unlike full-length 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-3-independent 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 2° 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-of-function mutation in pry-1 axin or by overexpression of a N-terminally truncated BAR-1 β-catenin protein was not suppressed by the rom-1(0) mutation (Table 1, rows 22–25) (Gleason et al. 2002).
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 (Yoo et al. 2004). 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 (Yoo et al. 2004), 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).
Figure 2. Expression of the egl-17::cfp Reporter in rom-1(0) and lin-3(rf) Mutants
Photographic images on the left (A, C, E, G, and I) show the expression of the arIs92[egl-17::cfp] reporter in the VPCs of mid-L2 larvae of the different genotypes indicated.
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 Chi2 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 μm.doi:10.1371/journal.pbio.0020334.g002
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 5′ 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/μl), suggesting that elevated levels of ROM-1 are toxic to the animals.
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 3° uninduced fate and persisted in the proximal VPCs adopting induced vulval fates (Figure 3C and 3D). In 60% of zhIs5 animals, we observed an up-regulation 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 1° and 2° descendants of P5.p, P6.p and P7.p, while the 3° 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. 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 3° 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 up-regulates rom-1::nls::gfp expression in the VPCs that adopt vulval cell fates.
Figure 3. Expression Pattern of rom-1::nls::gfp
Expression pattern of the zhIs5[rom-1::nls::gfprom-1::] transcriptional reporter during vulval development. Images on the left (A, C, E, G, and I) show the corresponding Nomarski pictures with the arrows pointing at the Pn.p cell nuclei and the arrowhead indicating the position of the AC nucleus.
(B) A mid L2 larva before vulval induction with uniform rom-1::nls::gfp expression in all the Pn.p cells.
(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 3° 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 1° and 2° 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 μm.doi:10.1371/journal.pbio.0020334.g003
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(+) 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) (Beitel et al. 1990; Lackner and Kim 1998; Chang et al. 2000). In addition, we examined lin-3(+) animals because, as reported previously by Hill and Sternberg (1992), in animals that overexpress wild-type 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(+), 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).
Table 2. Gonad-Independent Function of rom-1 and lin-3doi:10.1371/journal.pbio.0020334.t002
Analogous results were obtained by examining the egl-17::cfp expression pattern after removal of the AC. In gonad-ablated 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 gonad-ablated 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(+); 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.
ROM-1 Can Act in the Pn.p Cells
The absence of detectable rom-1::nls::gfp expression in the AC around the time of vulval induction and the AC-independent function of rom-1 and lin-3 suggested that rom-1 may act cell-autonomously in the VPCs. To test this hypothesis, we expressed rom-1 under control of the Pn.p cell-specific lin-31 promoter (lin-31::rom-1) (Tan et al. 1998). The lin-31::rom-1 transgene restored vulval induction in rom-1(0); let-60(gf) and rom-1(0); hs::mpk-1 double mutants to levels comparable to those found in let-60(gf) and hs::mpk-1 single mutants (Table 3, rows 1–3 and 8–10). A transgene encoding bacterial Cre recombinase under control of the lin-31 promoter (lin-31::cre) that was used as a negative control had no effect on vulval induction (Table 3, rows 4 and 11) (Hoier et al. 2000). Consistent with a function of rom-1 in an AC-independent pathway, the lin-31::rom-1 transgene also increased induction in gonad-ablated rom-1(0); let-60(gf) animals (Table 3, rows 5–7). Finally, we expressed rom-1 in the AC under control of the AC-specific enhancer (ACEL) (ACEL::rom-1), which is located in the third intron of the lin-3 locus (Hwang and Sternberg 2003). In contrast to lin-31::rom-1, the ACEL::rom-1 transgene did not rescue the suppression of the let-60(gf) Muv phenotype by rom-1(0) (Table 3, rows 12 and 13). Thus, the tissue-specific expression of ROM-1 in the Pn.p cells efficiently rescues a loss of rom-1 function.
Table 3. Expression of rom-1 in the Pn.p Cells but Not in the AC Rescues the rom-1(0) Phenotypedoi:10.1371/journal.pbio.0020334.t003
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 (Tan et al. 1998). 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 1° 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).
Figure 4. Alternative Splicing of lin-3 mRNA
(A) RT-PCR amplification of lin-3 mRNA from mixed-stage N2 cDNA before (left) and after (right) size fractionation by preparative agarose gel electrophoresis. The lowest band corresponding to LIN-3S is most prominent, and the two upper bands correspond to LIN-3L and LIN-3XL.
(B) Intron-exon structure of the lin-3 locus. The lin-3L splice variant is generated by the usage of an alternative (more 3′ located) splice donor in exon 6a. The lin-3XL variant contains the additional exon 6b inserted between exons 6a and 7. The regions encoding the EGF repeat in exon 5 and part of 6a and the transmembrane domain in exon 7 are outlined, and the positions of the PstI sites used for the construction of the minigenes are indicated (see Materials and Methods). The structure of the lin-3S and lin-3L minigenes is shown in the lower part of the graphic.
(C) Sequence alignment of the alternatively spliced region in LIN-3 with the corresponding region in Drosophila Spitz. The 15 and 41 amino acids in LIN-3L and LIN-3XL, respectively, in the juxtamembrane region break the alignment of LIN-3 with Spitz. The C-terminal end of the EGF domain is underlined with a horizontally hatched bar, and the beginning of the transmembrane domain is underlined by a diagonally hatched line.doi:10.1371/journal.pbio.0020334.g004
Table 4. Pn.p Cell-Specific Function of lin-3doi:10.1371/journal.pbio.0020334.t004
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 1° 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/μl) or high (100 ng/μl) 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/μl exhibited a Muv phenotype, while all nine lines obtained by injecting 100 ng/μl 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).
Table 5. The lin-3L Splice Form Depends on rom-1 Activitydoi:10.1371/journal.pbio.0020334.t005
Table 6. Rescue of the Larval Lethality in lin-3(0) Mutants by the lin-3S and lin-3L Minigenesdoi:10.1371/journal.pbio.0020334.t006
To specifically test the function of the LIN-3 splice variants in the Pn.p cells, we cloned full-length cDNAs encoding the LIN-3S and LIN-3L splice variants under control of the Pn.p cell-specific lin-31 promoter (lin-31::lin-3S and lin-31::lin-3L). The lin-31::lin-3S and lin-31::lin-3L transgenes both caused a strong Muv phenotype in the presence and absence of the AC (Table 5, rows 7, 9, 11, and 13). Loss of rom-1 function did not change the phenotype of lin-31::lin-3S animals (Table 5, rows 8–10), but it strongly suppressed the Muv phenotype of lin-31::lin-3L animals (Table 5, rows 12 and 14). Thus, ROM-1 is required for the activity of the LIN-3L splice variant in the Pn.p cells, while LIN-3S functions independently of ROM-1.
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 (Urban et al. 2001). 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-α, and unlike Drosophila Spitz, which absolutely depends on Rhomboid function, membrane-bound 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 1° fate in the adjacent VPC P6.p through juxtacrine signaling (Anklesaria et al. 1990). Once the AC has induced the 1° cell fate in P6.p, the 2° 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 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 cell-autonomously 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 tissue-specific 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 1° 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 1° and 2° 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 1° cell can specify the 2° 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 2° cell fate in the absence of lateral signaling (Katz et al. 1995). It is therefore possible that the relay signal generated by ROM-1 and LIN-3 in P6.p contributes to the specification of the 2° 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 1° fate of P6.p and the 2° 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 1° 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.
Figure 5. A Relay Model for Vulval Induction
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
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 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 (Lee et al. 2001; 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.
Materials and Methods
General methods and strains used.
Standard methods were used for maintaining and manipulating Caenorhabditis elegans (Brenner 1974). The C. elegans Bristol strain, variety N2, was used as the wild-type reference strain in all experiments. Unless noted otherwise, the mutations used have been described in Riddle and National Center for Biotechnology Information (U.S.) (2001) and are listed below by their linkage group: LGI: pry-1(mu38) (Gleason et al. 2002); LGIII: dpy-19(e1259), lin-12(n137gf), rom-1(zh18) (this study), rom-2(ok966) (C. elegans Gene Knockout Consortium), and unc-119(e2498); LGIV: let-60(n1046) (Beitel et al. 1990), lin-3(n1049), unc-5(e53), unc-44(362), lin-45(sy96), unc-24(e138), mec-3(e1338), dpy-20(e1282), egl-38(n578), and mec-3(n3197); LGX: sem-5(n2019) and lin-15(n309); extrachromosomal and integrated arrays: zhEx22[lin-3(+), sur-5::gfp, unc-119(+)], zhE66[lin-31::rom-1, unc-119(+), sur-5::gfp], zhEx72[lin-31::lin-3S, unc-119(+), sur-5::gfp], zhEx68[lin-3S, unc-119(+), sur-5::gfp], zhEx69[lin-3L, sur-5::gfp, unc-119(+)],zhEx73[lin-31::lin- zhEx73[lin-31::lin-3L, unc-119(+) sur-5::gfp], zhEx78[ACEL::Δpes-10::nls::gfp, unc-119(+)], zhEx81[lin-31::cre, unc-119(+), myo-3::gfp], zhE88[lin-31::lin-3i, unc-119(+), myo-3::gfp], zhEx89[ACEL::rom-1, sur-5::gfp], syIs12[hs::lin-3extra] (Katz et al. 1995), zhIs5[rom-1::nls::gfp, unc-119(+)], huIs7[hs::bar-1ΔNT, dpy-20(+)] (Gleason et al. 2002), gaIs36[hs-mpk-1, IF1alpha-Dmek-2] (Lackner and Kim 1998), and arIs92[egl-17::cfp] (Yoo et al. 2004). Unless noted in the table legends, all experiments were conducted at 20 °C. Transgenic lines were generated by injecting the experimental DNA at a concentration of 100 ng/μl or at the concentrations indicated in the text into both arms of the syncytial gonad as described (Mello et al. 1991).The constructs pUnc-119 (20 ng/μl), pPD93.97 (myo-3::gfp, 40 ng/μl), and pTG96 (sur-5::gfp, 100 ng/μl) were used as a cotransformation markers (Maduro and Pilgrim 1995; Yochem et al. 1997). The extrachromosomal array zhEx[rom-1::nls::gfprom-1::gfp; unc-119(+)] was integrated in the genome animals following γ-irradiation with 3,000 Rad to generate the array zhIs5 and backcrossed six times before analysis. Double and triple mutants were constructed using standard genetic methods. Where cis-linked markers were used they are indicated in the table legends.
The transcriptional rom-1::nls::gfp reporter construct (pRH2) was generated by ligating a HindIII-NheI–restricted 6,998-bp genomic fragment spanning the entire 5′ upstream region of F26F4.3 isolated by PCR amplification with the primers OAD49 (5′-GGAAGCTTGCATGCCCAACGAAATCGATA-3′) and OAD59 (5′-GGGCTAGCCATGTTGTGGAGAAGGAGAAC-3′) into the HindIII-XbaI site of pPD96.04. The rom-1::gfp translational reporter construct (pAD31) was generated by PCR amplification of a 3,146-bp genomic fragment containing 1,849 bp of 5′ sequences and the entire rom-1 ORF using the primers OAD47 (5′-GACTCTAGAGTTGTCAAAAGGTCACGGG-3′) and OAD51 (5′-ATCCTCTAGAGTTGAGCAATTTTCGTTGTTCCAC-3′')followed by XbaI restriction and ligation to XbaI-digested vector pPD95.75. The upstream promoter region of this construct was further extended by replacing a 420-bp PstI fragment with a 2,099-bp PstI genomic fragment corresponding to positions –1,432 and –3,531 relative to the predicted translation start codon of F26F4.3. The lin-31::rom-1 construct (pAD16) was generated by ligating a 1,601-bp SalI-NotI fragment spanning the entire rom-1 coding sequence amplified with the primers OAD44 (5′-TTTTGGTCGACCTCCTTCTCCACAAC-3′) and OAD45 (5′-TTTGGCGGCCGCCTATGAGCAATTTTCG-3′) into the SalI-NotI site of the pB253 vector (Tan et al. 1998). To generate the ACEL::rom-1 construct, a 2.3-kb SalI genomic lin-3 fragment encompassing the third intron, which contains the ACEL (Hwang and Sternberg 2003), was cloned into the SalI site of the pTB11 plasmid, which consists of an nls::gfp reporter cassette under control of the truncated pes-10 minimal promoter (Berset et al. 2001). Transgenic animals carrying the resulting ACEL::Δpes-10::nls::gfp construct (pAH67) showed strong and specific GFP expression in the AC beginning in the mid L2 stage as reported (Hwang and Sternberg 2003). The KpnI-EcoRI fragment encoding the nls::gfp cassette was then replaced with a 1.6-kb KpnI-NotI rom-1 fragment isolated from the lin-31::rom-1 plasmid described above to yield the ACEL::rom-1 construct. For the lin-31::lin-3 splice variant constructs, partial cDNAs covering the differentially spliced region in the lin-3 mRNA were amplified with the primers OAD31 (5′-CCCTTCGTGGTTTCGTCAAGAACGTAGTGC-3′) and OAD32 (5′-CGTATCTGCAGAATCCAACTCGATATTAATTAC-3′)using first-strand cDNA synthesized from mixed-stage total RNA as template. The PCR-amplified products were size-fractionated by agarose gel electrophoresis, cloned into the pGEMT vector (Promega), and sequenced to identify clones encoding individual splice variants. To obtain full-length lin-3 cDNA construct (pAD27), a 1,996-bp XhoI fragment from the EST clone yk1053b07 (confirmed to encode full-length lin-3XL cDNA by DNA sequencing) was first subcloned into the XhoI site of a modified pBluescriptSK (Stratagene, La Jolla, California, United States) vector (pAD23) in which the PstI site had been destroyed by restriction with EcoRV and SmaI and religation of the resulting blunt ends. To generate full-length lin-3S and lin-3L cDNA constructs (pAD25 and pAD26, respectively), 1,065-bp and 1,110-bp PstI fragments specific for each splice variant isolated from the partial cDNA clones described above were used to replace the 1,188-bp PstI fragment in the full-length lin-3XL cDNA construct (pAD27). The lin-31::lin-3S and lin-31::lin-3L constructs were generated by cloning the 1,133-bp and 1,178-bp XhoI cDNA fragments of the S and L splice variants into the SalI site of pB253 (Tan et al. 1998). The lin-3 splice variant minigene constructs (pAH63, pAH64, and pAH65 for lin-3S, lin-3L, and lin-3XL, respectively) were generated by cloning a 6.1-kb genomic fragment spanning the entire ORF of lin-3 and 574 bp of 5′ and 236 bp of 3′ sequences amplified with the primers OAH137 (5′-CCAGAAAGTTCATGTGAATCAT-3′) and OAH138 (5′-TCACAGGAACTGAGAGGGAGAGTG-3′) into the pGEMT vector. From this construct, a 6,206-bp ApaI-SacI fragment was subcloned into pAD23 to obtain pAH62. The minigenes encoding each of the splice variants were obtained by replacing the 2,728-bp lin-3 genomic PstI fragment with 1,065-, 1,110-, and 1,188-bp PstI fragments isolated from cDNAs of the different splice variants. To construct the lin-31::lin-3 hairpin plasmid (pAD35), 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.
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 °C. RNAi of rom-2 and rom-3 was done by feeding the animals dsRNA-producing E. coli at 20 °C as described (Kamath et al. 2001).
Isolation of the rom-1(zh18) deletion allele.
The rom-1(zh18) deletion mutant was isolated from an ethyl methanesulfonate–mutagenized library consisting of approximately 106 haploid genomes as previously described (Jansen et al. 1997; Berset et al. 2001). DNA pools were screened by nested PCR with primers Rho13 (5′-GAGACCGGGGACCGTATTCTGGCAC-3′) and Rho10 (5′-GAGAGCATAAACTCCTGCGGAAGCACC-3′) in a first PCR reaction, and Rho35 (5′-GGGAATCCGACGGTGGTAGAAGC-3′) and Rho10 (5′-GAGAGCATAAACTCCTGCGGAAGCACC-3′) in a second PCR reaction. The zh18 deletion removes 1,556 bp including 384 bp of 5′ upstream sequences and 618 bp of the rom-1 ORF (positions 4906804–4908360 in the cosmid F26F4). The mutant strain was backcrossed six times against N2 before further experiments were done.
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 1° or 2° vulval fate was counted for each animal as described (Sternberg and Horvitz 1986), and the induction index was calculated by dividing the number of 1° or 2° 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.
The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers of the Rhomboid genes discussed in this paper are C.e. ROM-1 (AAA91218), C.e. ROM-2 (CAA82377), C.e. ROM-3 (CAB55154), C.e. ROM-4 (CAB55122), C.e. ROM-5 (AAF60768), D.m. Rho-1 (CAA36692), D.m. Rho-2 (AAK06752), D.m. Rho-3 (AAK06753), D.m. Rho-4 (AAK06754), D.m. Rho-6 (NP_523557), H.s. Rho-1 (CAA76629).
We wish to thank all the members of our group for fruitful discussion and input into this work. We are grateful to Ernst Hafen and Michael Hengartner for their critical comments on the manuscript, to Andy Fire for GFP vectors (including pPD 95.75 and pPD96.04), and to the C. elegans Genetics Center, Stuart Kim, Iva Greenwald, and Paul Sternberg for providing some of the strains used. The yk1053b07EST clone was a gift of Y. Kohara. The rom-2 deletion mutant was kindly provided by the C. elegans Gene Knockout Consortium. This research was supported by the Swiss National Science Foundation and the Kanton of Zürich.
AD and AH conceived and designed the experiments. AD performed the experiments. AD and AH analyzed the data. SC and EFH contributed reagents/materials/analysis tools. AD and AH wrote the paper.
- 1. Anklesaria P, Teixido J, Laiho M, Pierce JH, Greenberger JS, et al. (1990) Cell-cell adhesion mediated by binding of membrane-anchored transforming growth factor alpha to epidermal growth factor receptors promotes cell proliferation. Proc Natl Acad Sci U S A 87: 3289–3293.
- 2. Arribas J, Coodly L, Vollmer P, Kishimoto TK, Rose-John S, et al. (1996) Diverse cell surface protein ectodomains are shed by a system sensitive to metalloprotease inhibitors. J Biol Chem 271: 11376–11382.
- 3. Bang AG, Kintner C (2000) Rhomboid and Star facilitate presentation and processing of the Drosophila TGF-alpha homolog Spitz. Genes Dev 14: 177–186.
- 4. Beitel GJ, Clark SG, Horvitz HR (1990) Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348: 503–509.
- 5. Berset T, Hoier EF, Battu G, Canevascini S, Hajnal A (2001) Notch inhibition of RAS signaling through MAP kinase phosphatase LIP-1 during C. elegans vulval development. Science 291: 1055–1058.
- 6. Bier E, Jan LY, Jan YN (1990) Rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogaster. Genes Dev 4: 190–203.
- 7. Bogdan S, Klambt C (2001) Epidermal growth factor receptor signaling. Curr Biol 11: 292–295.
- 8. Bosenberg MW, Pandiella A, Massague J (1993) Activated release of membrane-anchored TGF-alpha in the absence of cytosol. J Cell Biol 122: 95–101.
- 9. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94.
- 10. Chang C, Newman AP, Sternberg PW (1999) Reciprocal EGF signaling back to the uterus from the induced C. elegans vulva coordinates morphogenesis of epithelia. Curr Biol 9: 237–246.
- 11. Chang C, Hopper NA, Sternberg PW (2000) Caenorhabditis elegans SOS-1 is necessary for multiple RAS-mediated developmental signals. EMBO J 19: 3283–3294.
- 12. Chang H, Riese DJ 2nd, Gilbert W, Stern DF, McMahan UJ (1997) Ligands for ErbB-family receptors encoded by a neuregulin-like gene. Nature 387: 509–512.
- 13. Clandinin TR, DeModena JA, Sternberg PW (1998) Inositol trisphosphate mediates a RAS-independent response to LET-23 receptor tyrosine kinase activation in C. elegans. Cell 92: 523–533.
- 14. Clark SG, Lu X, Horvitz HR (1994) The Caenorhabditis elegans locus lin-15, a negative regulator of a tyrosine kinase signaling pathway, encodes two different proteins. Genetics 137: 987–997.
- 15. Felix MA, De Ley P, Sommer RJ, Frisse L, Nadler SA, et al. (2000) Evolution of vulva development in the Cephalobina (Nematoda). Dev Biol 221: 68–86.
- 16. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811.
- 17. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, et al. (2000) Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408: 325–330.
- 18. Freeman M, Gurdon JB (2002) Regulatory principles of developmental signaling. Annu Rev Cell Dev Biol 18: 515–539.
- 19. Freeman M, Klambt C, Goodman CS, Rubin GM (1992) The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye. Cell 69: 963–975.
- 20. Gallio M, Sturgill G, Rather P, Kylsten P (2002) A conserved mechanism for extracellular signaling in eukaryotes and prokaryotes. Proc Natl Acad Sci U S A 99: 12208–12213.
- 21. Gleason JE, Korswagen HC, Eisenmann DM (2002) Activation of Wnt signaling bypasses the requirement for RTK/Ras signaling during C. elegans vulval induction. Genes Dev 16: 1281–1290.
- 22. Golembo M, Raz E, Shilo BZ (1996) The Drosophila embryonic midline is the site of Spitz processing, and induces activation of the EGF receptor in the ventral ectoderm. Development 122: 3363–3370.
- 23. Greenwald IS, Sternberg PW, Horvitz HR (1983) The lin-12 locus specifies cell fates in Caenorhabditis elegans. Cell 34: 435–444.
- 24. Guichard A, Roark M, Ronshaugen M, Bier E (2000) Brother of rhomboid, a rhomboid-related gene expressed during early Drosophila oogenesis, promotes EGF-R/MAPK signaling. Dev Biol 226: 255–266.
- 25. Hill RJ, Sternberg PW (1992) The gene lin-3 encodes an inductive signal for vulval development in C. elegans. Nature 358: 470–476.
- 26. Hoier EF, Mohler WA, Kim SK, Hajnal A (2000) The Caenorhabditis elegans APC-related gene apr-1 is required for epithelial cell migration and Hox gene expression. Genes Dev 14: 874–886.
- 27. Huang LS, Tzou P, Sternberg PW (1994) The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development. Mol Biol Cell 5: 395–411.
- 28. Hwang BJ, Sternberg PW (2003) A cell-specific enhancer that specifies lin-3 expression in the C. elegans anchor cell for vulval development. Development 131: 143–151.
- 29. Jansen G, Hazendonk E, Thijssen KL, Plasterk RH (1997) Reverse genetics by chemical mutagenesis in Caenorhabditis elegans. Nat Genet 17: 119–121.
- 30. Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2: RESEARCH0002.
- 31. Katz WS, Hill RJ, Clandinin TR, Sternberg PW (1995) Different levels of the C. elegans growth factor LIN-3 promote distinct vulval precursor fates. Cell 82: 297–307.
- 32. Kenyon C (1995) A perfect vulva every time: Gradients and signaling cascades in C. elegans. Cell 82: 171–174.
- 33. Kimble J (1981) Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans. Dev Biol 87: 286–300.
- 34. Kornfeld K (1997) Vulval development in Caenorhabditis elegans. Trends Genet 13: 55–61.
- 35. Lackner MR, Kim SK (1998) Genetic analysis of the Caenorhabditis elegans MAP kinase gene mpk-1. Genetics 150: 103–117.
- 36. Lee JR, Urban S, Garvey CF, Freeman M (2001) Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila. Cell 107: 161–171.
- 37. Maduro M, Pilgrim D (1995) Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics 141: 977–988.
- 38. McQuibban GA, Saurya S, Freeman M (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423: 537–541.
- 39. Mello CC, Kramer JM, Stinchcomb D, Ambros V (1991) Efficient gene transfer in C.elegans: Extrachromosomal maintenance and integration of transforming sequences. EMBO J 10: 3959–3970.
- 40. Meyer D, Yamaai T, Garratt A, Riethmacher-Sonnenberg E, Kane D, et al. (1997) Isoform-specific expression and function of neuregulin. Development 124: 3575–3586.
- 41. Newman AP, White JG, Sternberg PW (1996) Morphogenesis of the C. elegans hermaphrodite uterus. Development 122: 3617–3626.
- 42. Pandiella A, Massague J (1991) Cleavage of the membrane precursor for transforming growth factor alpha is a regulated process. Proc Natl Acad Sci U S A 88: 1726–1730.
- 43. Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, et al. (1998) An essential role for ectodomain shedding in mammalian development. Science 282: 1281–1284.
- 44. Riddle DL, National Center for Biotechnology Information (2001) C. elegans II. Plainview (New York): Cold Spring Harbor Laboratory Press. 1222 p.
- 45. Rutledge BJ, Zhang K, Bier E, Jan YN, Perrimon N (1992) The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis. Genes Dev 6: 1503–1517.
- 46. Sherwood DR, Sternberg PW (2003) Anchor cell invasion into the vulval epithelium in C. elegans. Dev Cell 5: 21–31.
- 47. Simske JS, Kim SK (1995) Sequential signalling during Caenorhabditis elegans vulval induction. Nature 375: 142–146.
- 48. Simske JS, Kaech SM, Harp SA, Kim SK (1996) LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction. Cell 85: 195–204.
- 49. Sommer RJ, Sternberg PW (1994) Changes of induction and competence during the evolution of vulva development in nematodes. Science 265: 114–118.
- 50. Sternberg PW (1988) Lateral inhibition during vulval induction in Caenorhabditis elegans. Nature 335: 551–554.
- 51. Sternberg PW, Han M (1998) Genetics of RAS signaling in C. elegans. Trends Genet 14: 466–472.
- 52. Sternberg PW, Horvitz HR (1986) Pattern formation during vulval development in C. elegans. Cell 44: 761–772.
- 53. Sternberg PW, Horvitz HR (1989) The combined action of two intercellular signaling pathways specifies three cell fates during vulval induction in C. elegans. Cell 58: 679–693.
- 54. Sulston JE, Horvitz HR (1977) Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol 56: 110–156.
- 55. Sulston JE, White JG (1980) Regulation and cell autonomy during postembryonic development of Caenorhabditis elegans. Dev Biol 78: 577–597.
- 56. Tan PB, Lackner MR, Kim SK (1998) MAP kinase signaling specificity mediated by the LIN-1 Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction. Cell 93: 569–580.
- 57. Thomas JH, Stern MJ, Horvitz HR (1990) Cell interactions coordinate the development of the C. elegans egg-laying system. Cell 62: 1041–1052.
- 58. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.
- 59. Timmons L, Tabara H, Mello CC, Fire AZ (2003) Inducible systemic RNA silencing in Caenorhabditis elegans. Mol Biol Cell 14: 2972–2983.
- 60. Tsruya R, Schlesinger A, Reich A, Gabay L, Sapir A, et al. (2002) Intracellular trafficking by Star regulates cleavage of the Drosophila EGF receptor ligand Spitz. Genes Dev 16: 222–234.
- 61. Urban S, Freeman M (2002) Intramembrane proteolysis controls diverse signalling pathways throughout evolution. Curr Opin Genet Dev 12: 512–518.
- 62. Urban S, Freeman M (2003) Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain. Mol Cell 11: 1425–1434.
- 63. Urban S, Lee JR, Freeman M (2001) Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 107: 173–182.
- 64. Urban S, Lee JR, Freeman M (2002) A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands. Embo J 21: 4277–4286.
- 65. Wang M, Sternberg PW (2001) Pattern formation during C. elegans vulval induction. Curr Top Dev Biol 51: 189–220.
- 66. Wasserman JD, Freeman M (1998) An autoregulatory cascade of EGF receptor signaling patterns the Drosophila egg. Cell 95: 355–364.
- 67. Wasserman JD, Urban S, Freeman M (2000) A family of rhomboid-like genes: Drosophila rhomboid-1 and roughoid/rhomboid-3 cooperate to activate EGF receptor signaling. Genes Dev 14: 1651–1663.
- 68. Whitfield CW, Benard C, Barnes T, Hekimi S, Kim SK (1999) Basolateral localization of the Caenorhabditis elegans epidermal growth factor receptor in epithelial cells by the PDZ protein LIN-10. Mol Biol Cell 10: 2087–2100.
- 69. Wolfe MS, De Los Angeles J, Miller DD, Xia W, Selkoe DJ (1999) Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease. Biochemistry 38: 11223–11230.
- 70. Yochem J, Sundaram M, Han M (1997) Ras is required for a limited number of cell fates and not for general proliferation in Caenorhabditis elegans. Mol Cell Biol 17: 2716–2722.
- 71. Yoo AS, Bais C, Greenwald I (2004) Crosstalk between the EGFR and LIN-12/Notch pathways in C. elegans vulval development. Science 303: 663–666.