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Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development

Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development CORRIGENDUM Development 134, 4503-4505 (2007) doi:10.1242/dev.017475 Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development Claudia B. Walser, Gopal Battu, Erika Fröhli Hoier and Alex Hajnal There were errors published in Development 133, 3461-3471. We have discovered that the expression pattern of the PUF-8::GFP reporter zhEx61 shown in Fig. 2B-L on p. 3466 of this article, is based on an incorrect reporter construct that carries the insert in the reverse orientation. The corrected Fig. 2 below shows the PUF-8::GFP expression pattern that is observed with the correct PUF-8::GFP reporter zhEx274.1, which carries the insert in the correct orientation. Also shown is a corrected supplementary Fig. S1, in which a quantification of the expression pattern of zhEx274.1 is shown (A-C). Although the overall expression pattern observed with the PUF-8::GFP reporter zhEx274.1 is similar to that obtained with the reverse reporter zhEx61 shown in Fig. 2B-L of Walser et al. (2006), there are four differences in its expression, which are accounted for in the text changes detailed below. Also provided are new methods for the generation of zhEx274.1. These corrections do not change the overall conclusions of this paper. The page, paragraph and line numbers below refer to the PDF version of the article. Fig. 2. PUF-8::GFP and FBF-2::GFP expression during vulval development. (A) Structure of the translational puf-8::gfp and fbf-2::gfp reporters. (B,D,F,H) Time-course analysis of PUF-8::GFP expression in the vulval cells from the L2 until the L4 stage with (C,E,G,J) the corresponding Nomarski images. For a semi-quantitative analysis of the expression patterns, see Fig. S1 in the supplementary material. (K,L) PUF-8::GFP expression in gonad-ablated eff-1(hy21) animals, and the corresponding Nomarski image. All VPC descendants showed PUF-8::GFP expression with a strong increase in the descendants of P6.p. Note that despite the extra round of cell divisions in P5.p and P6.p descendants of gonad-ablated eff-1 mutants, no vulval differentiation was observed. (M-R) FBF-2::GFP expression, and the corresponding Nomarski images, from the early L3 until the L4 stage. In all panels, anterior is to the left and ventral is to the bottom. Scale bars: 10 m. 4504 CORRIGENDUM Fig. S1. PUF-8::GFP and FBF-2::GFP expression analysis. (A) Semi-quantitative time-course analysis of PUF-8::GFP expression in wild-type animals. The two daughter cells after the first cell division are termed Pn.px for all VPCs. The descendants of the second cell divisions of induced VPCs are termed Pn.pxx, and after the third round of cell divisions Pn.pxxx cells. Gray areas indicate the proportion of PUF-8::GFP-positive vulval cells, white areas the proportion of PUF-8::GFP-negative cells. (B) Analysis of PUF-8::GFP expression pattern in (top row) eff-1(hy21) mutants at the Pn.pxxx stage without gonad ablation and (bottom row) gonad-ablated eff-1(hy21) mutants at the Pn.px stage. Both conditions were analyzed in L4 larvae, but since VPCs in gonad-ablated animals are not induced to adopt vulval cell fates, they divide once and arrest at the Pn.px stage or occasionally divide a second time, as shown in Fig. 2K,L. (C) Analysis of the PUF-8::GFP expression pattern in let-60(n1046gf) L4 larvae at the Pn.pxxx stage. Since let-60(n1046gf); zhEx274.1[puf-8::gfp] animals developed into sterile adults for unknown reasons, the let-60(n1046); zhEx274.1[puf-8::gfp] animals were maintained as heterozygotes, and their multivulva progeny homo- or heterozygous for let-60(n1046gf) were scored at the Pn.pxxx stage. (D) Semi-quantitative time-course analysis of FBF-2::GFP expression in wild-type animals. Only animals showing bright FBF-2::GFP expression in somatic tissues were used for the analysis. White indicates no FBF-2::GFP expression, grey low expression and black high expression. Correction to the text on p. 3462, paragraph 6 Extrachromosomal transgenic arrays [transgenes; co-transformation marker; pBS: Bluescript (concentration in ng/l)] were generated by microinjection of DNA into young adult worms (Mello et al., 1991): Correction to the text on p. 3462, paragraph 7, line 6 … zhEx220[fbf-2::gfp; lin-48::gfp (100;50)], zhEx274.1[puf-8::gfp; lin-48::gfp (80;50)]. Correction to the text on p. 3464, paragraph 2, from line 4 PUF-8::GFP was expressed in various tissues including the hypodermis, the ventral cord motor neurons (not shown) and the vulval cells (Fig. 2B-J and see Fig. S1A in the supplementary material). Before vulval induction in L2 larvae, PUF-8::GFP was expressed in all six vulval precursor cells, although expression was more frequently observed in the distal VPCs (P3.p, P4.p and P8.p) than in the proximal VPCs (P5.p, P6.p and P7.p, Fig. 2B,C, and row with Pn.p cells in Fig. S1A in the supplementary material). After vulval induction in early L3 larvae, PUF-8::GFP expression persisted in the descendants of the 3° distal VPCs (P3.p, P4.p and P8.p), while expression faded in the 1° and 2° descendants of the proximal VPCs (P5.p, P6.p and P7.p, Fig. 2D-J, Fig. S1A in the supplementary material, rows Pn.px to Pn.pxxx). Correction to the text on p. 3464, paragraph 3, from line 1 We hypothesized that PUF-8::GFP expression in the descendants of the distal 3° VPCs might persist because these cells fuse with the hyp7 hypodermis that also expresses PUF-8::GFP. To test if the expression of PUF-8::GFP in the descendants of the 3° VPCs is a consequence of their fusion with hyp7, we examined PUF-8::GFP expression in an eff-1(hy21) background, in which no cell fusions occur (Mohler et al., 2002). CORRIGENDUM 4505 Correction to the text on p. 3464, paragraph 3, from line 10 In most gonad-ablated eff-1(hy21) animals, PUF-8::GFP expression was observed in the VPCs and their descendants (Fig. 2K,L and see Fig. S1B in the supplementary material). Moreover, in let-60 ras(gf) animals, in which the distal VPCs frequently adopt the 1° or 2° induced cell fates, PUF-8::GFP expression was often absent in the distal VPCs and their descendants (see Fig. S1C in the supplementary material) (Beitel et al., 1990; Greenwald et al., 1983). We conclude that PUF-8::GFP is expressed in the descendants of VPCs that have adopted the uninduced 3° cell fate independently of their fusion with hyp7. Correction to the text on p. 3466, paragraph 2, line 1 The expression of PUF-8::GFP in the distal 3° vulval cells raises the possibility that PUF-8 might regulate the competence of the distal vulval cells to respond to the inductive signal. Correction to the text on p. 3469, paragraph 2, line 7 A PUF-8::GFP reporter transgene is expressed predominantly in the distal VPCs (P3.p, P4.p and P8.p) and their descendants that have adopted the 3° fate. The authors apologise to readers for these mistakes and are grateful to Dave Hansen for discovering the error in the plasmid used to generate zhEx61. Publisher’s note: Although the mistake reported in this corrigendum has resulted in several corrections being made to Walser et al. (2006) and in an unusually lengthy corrigendum, we would like to reassure readers that expert opinion has confirmed that the minor changes in expression that are seen between the incorrect reporter zhEx61 and the correct reporter zhEx274.1 do not alter or affect the conclusions drawn by this paper. RESEARCH ARTICLE 3461 Development 133, 3461-3471 (2006) doi:10.1242/dev.02496 Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development 1,2, 1, 1 1,† Claudia B. Walser *, Gopal Battu *, Erika Fröhli Hoier and Alex Hajnal The C. elegans PUF and FBF proteins regulate various aspects of germline development by selectively binding to the 3 untranslated region of their target mRNAs and repressing translation. Here, we show that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulvaI development. Loss-of-function mutations in puf-8 cause ectopic vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway and suppress the vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signalling. PUF-8 acts cell-autonomously in the vulval cells to limit their temporal competence to respond to the extrinsic patterning signals. fbf-1 and fbf-2, however, redundantly inhibit primary vulval cell fate specification in two distinct pathways acting in the soma and in the germline. The FBFs thereby ensure that the inductive signal selects only one vulval precursor cell for the primary cell fate. Thus, translational repressors regulate various aspects of vulval cell fate specification, and they may play a conserved role in modulating signal transduction during animal development. KEY WORDS: Caenorhabditis elegans, Vulva, Pumilio, Translational control, Signal transduction INTRODUCTION additional flanking nucleotides (Murata and Wharton, 1995; The spatial and temporal regulation of gene expression can occur Tadauchi et al., 2001; Wharton et al., 1998; Zamore et al., 1997; either at the level of gene transcription or at the level of mRNA export, Zhang et al., 1997). stability or translation through RNA-binding proteins or micro RNAs Pumilio, the only PUF protein in Drosophila melanogaster, (de Moor et al., 2005; Kuersten and Goodwin, 2003). Work on model controls, together with Nanos, the establishment of the anterior- organisms such as Drosophila melanogaster and Caenorhabditis posterior axis of the embryo by repressing the translation of maternal elegans has contributed much to our current understanding of post- hunchback mRNA (Barker et al., 1992; Murata and Wharton, 1995). transcriptional gene regulation during development. Translational Pumilio and Nanos also inhibit cyclin B translation in migrating pole control by RNA binding proteins is frequently used in the C. elegans cells allowing them to arrest in G2 until they reach the gonads germline and early embryo, but translational regulation has also been (Asaoka-Taguchi et al., 1999). In addition to its roles during observed during larval development (Kuersten and Goodwin, 2003; development, Drosophila Pumilio was recently shown to be Rougvie, 2001). Many mRNAs contain sequence motifs in their 5 or necessary for the activity-dependent expression of the voltage-gated 3 untranslated regions (5UTRs or 3UTRs) that serve as binding sites sodium channel Paralytic in the central nervous system (Mee et al., for regulatory proteins controlling different aspects of mRNA 2004). The human and mouse genomes each encode two PUF localization, translation or stability. proteins with unknown functions (Spassov and Jurecic, 2002; The PUF gene family is conserved from yeast to humans. PUF Spassov and Jurecic, 2003). proteins function as translational repressors that bind to specific The C. elegans genome contains the surprisingly high number of elements in the 3UTRs of their target mRNAs (reviewed by eleven PUF genes (fbf-1 and fbf-2, puf-3 to puf-11). PUF-8 forms, Wickens et al., 2002). The first characterized members of this family together with PUF-9, a distinct subgroup among the C. elegans PUF were Drosophila Pumilio and the two C. elegans FBF proteins. proteins, as PUF-8 and PUF-9 are more similar to the Drosophila Hence, this family is referred to as PUF for Pumilio and FBF repeat and to the two vertebrate pumilio proteins than to the other C. proteins (Zhang et al., 1997). Typical PUF proteins contain eight elegans PUF proteins (Wickens et al., 2002). FBF-1 and FBF-2 PUF repeats of approximately 40 amino acids with a core consensus (fem-3-binding factor-1 and -2) are two closely related proteins that sequence containing aromatic and basic residues. The PUF repeats regulate the sperm/oocyte switch in the hermaphrodite germline by directly bind to the target mRNAs and recruit additional proteins binding to the PME (point mutation element) in the 3 UTR of fem- such as Nanos, Brain tumor and CPEB (Kraemer et al., 1999; 3 mRNA (Ahringer and Kimble, 1991; Kraemer et al., 1999; Zhang Luitjens et al., 2000; Sonoda and Wharton, 1999; Sonoda and et al., 1997). In addition, FBF-1 and FBF-2 both regulate the mitosis Wharton, 2001). The cis-regulatory elements in the 3 UTRs of their versus meiosis decision in the distal region of the germline by target mRNAs contain a UGUR tetra nucleotide sequence motif repressing gld-1 translation in the mitotic region to prevent the stem termed a Nanos response element (NRE). The binding specificity cells from entering meiosis (Crittenden et al., 2002; Kadyk and of the individual PUF proteins is thought to be determined by Kimble, 1998). Furthermore, FBF and PUF proteins are required for germ cell survival, germ cell migration and the mitotic arrest of germ cells during embryogenesis (Kraemer et al., 1999; Subramaniam and Zoologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Seydoux, 1999). PUF-8 is necessary for the meiotic division of the Switzerland. Molecular Life Science PhD Program, Molekular biologisches Institut, primary spermatocytes in hermaphrodites and males (Subramaniam Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland. and Seydoux, 2003). *These authors contributed equally to this work Here, we show that the same PUF proteins that control germline Author for correspondence (e-mail: [email protected]) development also act in the soma during vulval induction. During larval development, the hermaphrodite vulva is formed out of 22 Accepted 15 June 2006 DEVELOPMENT 3462 RESEARCH ARTICLE Development 133 (17) cells that are generated by three out of six equivalent vulval (10;20;120)], zhEx172.1-2[P ::puf-8; sur-5::gfp; pBS (50;50;50)], dpy-7 ::fbf-2; sur-5::gfp; pBS (50;50;50)], zhEx220[fbf-2::gfp; zhEx176.1-3[P precursor cells (VPCs; P3.p through P8.p) (Greenwald, 1997). To dpy-7 lin-48::gfp (100;50)]. induce vulval differentiation, the anchor cell (AC) in the somatic GFP and YFP expression was observed under fluorescent light gonad sends an epidermal growth factor signal (LIN-3) to the illumination with a Leica DMRA microscope equipped with a cooled CCD adjacent VPCs (Hill and Sternberg, 1992). This inductive AC signal camera (Hamamatsu ORCA-ER) controlled by the Openlab 3.0 software activates the LET-23 EGFR signalling pathway in the nearest VPC (Improvision). Animals were mounted on 3% agarose pads in M9 solution (P6.p) to specify the primary (1°) cell fate. P6.p then sends a lateral . Larvae were first inspected using Nomarski optics containing 15 mM NaN signal to the neighbouring VPCs, P5.p and P7.p, via the LIN-12 to identify the position of the Pn.p cells or their descendants, and GFP or NOTCH pathway (Greenwald et al., 1983; Sternberg, 1988). LIN- YFP expression was then scored under fluorescent light illumination using 12 signalling inhibits the 1° fate specification in P5.p and P7.p and the same exposure settings for a particular transgene in all different genetic instead instructs the secondary (2°) fate in these cells (Ambros, backgrounds. For the PUF-8::GFP FBF-2::GFP and the EGL-17::YFP experiments, three semi-quantitative classes were made: no expression if the 1999; Sternberg, 1988). Multiple inhibitory signalling pathways fluorescence was not distinguishable from the background staining, low antagonize the EGFR/RAS/MAPK pathway to control the cell fate expression if there was a weak but clearly visible signal, and high expression choice in the VPCs (reviewed by Fay and Han, 2000). These if the fluorescence signal was strong. The images of PUF-8::GFP and inhibitors ensure that the distal VPCs (P3.p, P4.p and P8.p), which FBF-2::GFP at the L4 stages needed a correction to prevent overexposure. receive little or no inductive and lateral signals, adopt the tertiary The induction index of the VPCs was scored under Nomarski optics and (3°) non-vulval cell fate. After the vulval cell fates have been the average number of 1° or 2° induced VPCs per animal was calculated as specified, the VPCs undergo stereotypic patterns of cell divisions described previously (Dutt et al., 2004). before they differentiate and form the mature organ. Three rounds Laser ablation of the somatic gonad precursors Z1 and Z4 and germline of symmetric cell divisions generate eight 1° descendants, of which precursors Z2 and Z3 were done as described by Kimble (Kimble, 1981), four adopt the VulE and four the VulF subfate. The last of the three and induction was scored in L4 larvae. cell divisions in the 2° lineage generates only seven descendants that Genetic screens and positional molecular cloning of puf-8 further differentiate into the VulA, VulB, VulC and VulD subfates gap-1 enhancer screen to isolate puf-8(ga145): young adult gap-1(ga133) (Inoue et al., 2002; Sternberg and Horvitz, 1986). The 3° cells divide hermaphrodites were mutagenized with 50 mM ethyl-methanesulfonate only once and then fuse with the surrounding hypodermal syncytium (EMS) for 4 hours at room temperature, and the F2 generation was (hyp7). screened for mutants displaying a multivulva (Muv) phenotype. Our analysis indicates that puf-8, fbf-1 and fbf-2 negatively Approximately 30,000 haploid genomes were screened (Canevascini et regulate vulval induction in parallel with the known inhibitors of al., 2005). Non-complementation screen to isolate puf-8(zh17): gap-1(ga133) males the EGFR/RAS/MAPK pathway. puf-8 restricts the temporal were mutagenized with EMS as described above, mated with unc-4(e120) competence of the vulval cells by promoting the fusion of the puf-8(ga145); gap-1(ga133) hermaphrodites and the nonUnc F1 progeny uninduced 3° cells with hyp7, while fbf-1 and fbf-2 control the 1° was screened for Muv animals. After screening 2,000 haploid genomes one versus 2°/3° cell fate decision. Muv non-Unc animal was identified and propagated. ga145 was mapped with three-factor mapping between dpy-10 and unc-4 on LGII and further MATERIALS AND METHODS narrowed down by transformation rescue experiments using YACs and Nematode strains and general methods cosmids to the cosmid clone C30G12. RNAi analysis of the genes encoded All strains were derivatives of Bristol strain N2 of Caenorhabditis elegans by C30G12 in a gap-1(ga133) background identified the puf-8 gene as and grown under standard conditions at 20°C (Brenner, 1974) or at the candidate, and DNA sequencing of the puf-8 coding region in the ga145 and temperature indicated in the table footnotes. Unless noted otherwise, the zh17 alleles identified the molecular lesions. mutations used have been described previously (Riddle et al., 1997) and are listed below by their linkage group. RNA interference analysis LGI: lin-10(e1438), unc-13(e1091) to cis-link lin-10(e1438). LGII: fbf- RNA interference analysis (RNAi) was performed by feeding animals 1(ok91) (Crittenden et al., 2002), fbf-2(q738) (Lamont et al., 2004), fbf- dsRNA-producing bacteria as described previously (Kamath and Ahringer, 2(q704) (Crittenden et al., 2002), puf-8(zh17) (this work), puf-8(ga145) (this 2003) with the following modifications. During the cloning of puf-8, work), puf-8(ok302) (Subramaniam and Seydoux, 2003), rrf-3(pk1426) dsRNA-producing bacteria were grown on plates containing 1 mM IPTG (Sijen et al., 2001), eff-1(hy21) (Mohler et al., 2002), lin-7(e1413), unc- and 5-10 adult P0 gap-1(ga133) animals were put on each plate. For the 4(e120) to cis-link puf-8 alleles, puf-8(ok302) and the fbf mutations were syIs90; gap-1(ga133) strain, bacteria were induced with 6 mM IPTG, and balanced with mIn1(mIs14 dpy-10(e128)) (Edgley and Riddle, 2001). LGIII: for all other RNAi experiments, 5-15 P0 animals were put, as L1 larvae or unc-119(e2498), unc-119(ed4) for syIs90. LGIV: ark-1(sy247) (Hopper et as adults, on plates containing bacteria grown on 3 mM IPTG. Vulval al., 2000), dpy-20(e1282) to cis-link ark-1, let-60(n1046gf), let-60(n2021). induction was scored in the F1 progeny at the L4 larval stage to count the LGX: gap-1(ga133) (Hajnal et al., 1997), lin-2(n105ts), lin-15(n765ts), sli- number of induced VPCs or in adults to count the percentage of Muv 1(sy143). animals (indicated in the table footnotes). All dsRNA-producing bacteria Integrated transgenic arrays (transgenes; co-transformation marker): were from the Ahringer library (Kamath and Ahringer, 2003), except for the syIs90[egl-17::yfp + unc-119(+)] III (Inoue et al., 2002), swIs79[ajm- fem-3 RNAi bacteria, which were a gift from C. Eckmann. 1::gfp, unc-119(+)] IV (Mohler et al., 1998). Extrachromosomal transgenic arrays [transgenes; co- Plasmids and PCR fusion constructs transformation marker; pBS: Bluescript (concentration in ng/l)] were For the puf-8::gfp translational reporter, a 3.3 kb SalI genomic fragment generated by microinjection of DNA into young adult worms (Mello containing a 1.3 kb upstream promoter fragment and the entire C30G12.7 et al., 1991), except for the zhEx61[puf-8::gfp; unc-119(+)] open reading frame was cloned into the SalI site of plasmid pPD95.75 (a gift extrachromosomal line, which was generated by microparticle from A. Fire). For the fbf-2::gfp translational reporter, a 3.73 kb BamHI bombardment using 0.1 mg of 1 m gold beads coated with 16 g puf- genomic fragment containing a 1.5 kb upstream promoter fragment and the 8::gfp and 8 g unc-119(+) plasmids as described previously (Praitis et entire fbf-2 open reading frame was cloned into the BamHI site of plasmid al., 2001): pPD95.75. All the dpy-7 and bar-1 promoter fusions were generated by the zhEx173.1-3[P ::puf-8; sur-5::gfp; pBS (50;50;50)], zhEx175.1- PCR fusion method (Hobert, 2002). Details on the primers used and bar-1 3[P ::fbf-1; sur-5::gfp; pBS (50;50;50)], zhEx174.1-3[P ::fbf-2; sur- constructions of the gfp reporters and promoter fusions are available on bar-1 bar-1 5::gfp; pBS (50;50;50)], zhEx170.1[P ::puf-8; sur-5::gfp; pBS request. dpy-7 DEVELOPMENT C. elegans PUF proteins in vulval development RESEARCH ARTICLE 3463 RESULTS and Table 2, row 7). This gene has previously been named puf-8, as Identification of puf-8 as a negative regulator of it encodes one of the two C. elegans Pumilio homologues (Wickens vulval development et al., 2002). Sequencing the puf-8 coding region revealed a stop Single mutants in negative regulators of vulval induction often mutation at position 485 of the ORF (CAA to TAA) before the PUF exhibit a wild-type vulval phenotype because these genes are mostly repeats in zh17, and a G to A (GGA to AGA) transition at position genetically redundant. We therefore performed a forward genetic 1174, replacing glycine 317 with arginine in the fourth PUF repeat screen in a gap-1(ga133) loss-of-function background to identify in ga145 animals (Fig. 1A). The glycine mutated in ga145 is synthetic mutations in additional inhibitors of vulval induction conserved in PUF-9, Drosophila Pumilio and the vertebrate PUF (Canevascini et al., 2005). gap-1 encodes a GTPase-activating proteins. This glycine is adjacent to an asparagine residue that is protein that stimulates the intrinsic GTPase activity of LET-60 RAS directly involved in binding to the target mRNA (Opperman et al., and thus inhibits the transduction of the inductive signal (Hajnal et 2005). In addition to the vulval phenotype, both puf-8 alleles we al., 1997). gap-1(ga133) single mutants exhibit an elevated activity isolated showed the same partially penetrant sterile phenotype at of the EGFR/RAS/MAPK signalling pathway, yet they develop 20°C as the puf-8(ok302) deletion strain (Fig. 1A) (Subramaniam a wild-type vulva (Fig. 1B and Table 1, row 2). After screening and Seydoux, 2003), and the puf-8(ok302) deletion caused a Muv approximately 30,000 haploid genomes, we isolated 27 mutants that phenotype in a gap-1(ga133) background of similar penetrance to displayed a synthetic multivulva (Muv) phenotype in a gap- zh17 or ga145 (Table 1, row 7). Thus, zh17 and ga145 are strong 1(ga133) background and defined at least four complementation reduction-of-function or null alleles of puf-8. groups. The ga145 mutation found in this screen caused a 60% penetrant Muv phenotype in the gap-1(ga133) background, but no Genetic interaction of puf-8 with the obvious vulval phenotype as a single mutant (Table 1, rows 3 and 5). EGFR/RAS/MAPK pathway To identify additional alleles of this complementation group, we We examined the genetic interaction of puf-8(zh17) with mutations performed a non-complementation screen (for details see Materials that either reduce or increase the activity of the EGFR/RAS/MAPK and methods) that yielded a new allele (zh17) displaying an equally signalling pathway. puf-8(zh17) partially suppressed the vulvaless penetrant synthetic Muv phenotype (Table 1, rows 4 and 6 and Fig. (Vul) phenotype caused by mutations in lin-2, lin-7, lin-10 and let- 1C). The corresponding gene was mapped to LGII between dpy-10 60, which reduce but do not inactivate the inductive signal (Table 1, and unc-4 and further narrowed down by transformation rescue rows 9-16) (Kaech et al., 1998). We also combined puf-8(zh17) with experiments to the cosmid C30G12. The six genes on this cosmid mutations in inhibitors of the EGFR/RAS/MAPK pathway such as were tested by RNAi analysis. Feeding gap-1(ga133) animals with ark-1, sli-1 or lin-15 that exhibit a wild-type or only a very weak bacteria producing dsRNA derived from the C30G12.7 open reading Muv phenotype as single mutants (Herman and Hedgecock, 1990; frame caused a Muv phenotype of 80% penetrance (Table 1, row 8 Hopper et al., 2000; Jongeward et al., 1995; Yoon et al., 1995). With Fig. 1. PUF proteins that negatively regulate vulval development. (A) Intron-exon structure and alleles of puf-8, fbf-1 and fbf-2. White boxes indicate the 5UTRs, white boxes with arrowheads the 3UTRs, grey boxes the coding regions and black boxes the PUF repeats. (B-E) Nomarski images of the vulval cells in L4 larvae of (B) gap-1(ga133), (C) puf-8(zh17); gap-1(ga133), and of (D,E) fbf-1(ok91) fbf-2(q704); gap-1(ga133) animals. In all panels, anterior is to the left and ventral is to the bottom. Note the ectopic induction of P4.p and P8.p (arrows in C,D,E). Arrowhead in E indicates an example of defects in the 2° cell lineage generated by P5.p resulting in the detachment of the P5.p descendants from the cuticle in a fbf-1(ok91) fbf-2(q704); gap-1(ga133) larva. Scale bar: 10 m. DEVELOPMENT 3464 RESEARCH ARTICLE Development 133 (17) Table 1. puf-8 negatively regulates vulval development † ‡ § Row Genotype* % Muv % Vul Induction n 1 Wild-type 0 0 3.0 many 2 gap-1(ga133) 0 0 3.0 30 3 puf-8(ga145) 0 0 – 176 4 puf-8(zh17) 0 0 3.0 36 5 puf-8(ga145); gap-1(ga133) 60 0 4.0 28 6 puf-8(zh17); gap-1(ga133) 59 0 3.9 46 7 puf-8(ok302); gap-1(ga133) 80 0 4.2 24 8 gap-1(ga133); puf-8 RNAi 83 0 4.2 30 9 lin-2(n105) 0 56 1.6 18 10 puf-8(zh17); lin-2(n105) 0 18 2.5 22 11 lin-7(e1413) 0 95 0.6 38 12 puf-8(zh17); lin-7(e1413) 0 58 1.7 36 13 lin-10(e1438)** 2 83 1.3 42 14 lin-10(e1438); puf-8(zh17)** 0 41 2.1 29 15 let-60(n2021) 0 44 2.6 133 16 puf-8(zh17); let-60(n2021) 0 16 2.8 224 †† 17 ark-1(sy247) 0 0 3.0 33 †† 18 puf-8(zh17); ark-1(sy247) 55 0 3.6 42 19 sli-1(sy143) 0 0 3.0 37 20 puf-8(zh17); sli-1(sy143) 11 0 3.1 38 ‡‡ 21 lin-15(n765ts) 0 0 3.0 32 ‡‡ 22 puf-8(zh17); lin-15(n765ts) 23 0 3.4 30 23 eff-1(hy21) 29 0 3.3 21 24 eff-1(hy21); gap-1(ga133) 40 0 3.4 20 *All the strains carrying the puf-8 mutations ga145, zh17 or ok302 carried the cis-linked marker unc-4(e120). % Muv indicates the fraction of animals with more than three induced VPCs. % Vul indicates the fraction of animals with less than three induced VPCs. Induction indicates the average number of induced VPCs per animal, puf-8(ga145) was scored under a dissection microscope. These strains were grown at 25°C. **lin-10(e1438) was cis-linked with unc-13(e1091). †† These strains had ark-1(sy247) cis-linked with dpy-20(e1282). ‡‡ These strains were kept at 14°C before scoring. each of these mutations, puf-8(zh17) caused a synthetic Muv PUF-8::GFP expression in an eff-1(hy21) background, in which no phenotype as described above for gap-1(ga133) (Table 1, row 6 and cell fusions occur (Mohler et al., 2002). Since eff-1(hy21) animals rows 17-22). Thus, puf-8 either encodes a negative regulator of the exhibit excess vulval induction (Table 1, row 23), we additionally EGFR/RAS/MAPK pathway, or alternatively, puf-8 regulates the ablated the somatic gonad precursors Z1 and Z4 to prevent induction competence of the VPCs to respond to the inductive signal. by the anchor cell. In most gonad-ablated eff-1(hy21) animals, PUF- 8::GFP expression was upregulated in all VPCs and their PUF-8::GFP is expressed in vulval cells and the descendants, except for the P8.p descendants (Fig. 2K,L and Fig. surrounding epidermis S1B in the supplementary material). Moreover, in let-60 ras(gf) To analyze the expression pattern of PUF-8, we constructed a animals, in which the distal VPCs frequently adopt the 1° or 2° translational puf-8::gfp reporter by fusing a genomic DNA fragment induced cell fates, PUF-8::GFP expression often remained low covering 1.3 kb of 5 regulatory sequences up to the next gene and in the distal VPCs and their descendants (Fig. S1C in the the entire puf-8 coding sequence to a GFP cassette (Fig. 2A). PUF- supplementary material) (Beitel et al., 1990). We conclude that PUF- 8::GFP was expressed in various tissues including the pharyngeal 8::GFP is upregulated in the descendants of VPCs that have adopted muscles, the hypodermis, the ventral cord motor neurons (not the uninduced 3° cell fate independently of their fusion with hyp7. shown) and the vulval cells (Fig. 2B-J and Fig. S1A in the supplementary material). Before vulval induction in L2 larvae, PUF- fbf-1 and fbf-2 negatively regulate vulval 8::GFP was expressed in all six vulval precursor cells at equal levels development (Fig. 2B,C and row with Pn.p cells in Fig. S1A in the supplementary To examine whether additional C. elegans PUF proteins besides material). After vulval induction in early L3 larvae, PUF-8::GFP PUF-8 play a role in regulating vulval development, we performed was upregulated in the descendants of the 3° distal VPCs (P3.p, P4.p an RNA interference (RNAi) analysis by feeding rrf-3(pk1426); gap- and P8.p), while expression faded in the 1° and 2° descendants of 1(ga133) animals with dsRNA-producing bacteria derived from the the proximal VPCs (P5.p, P6.p and P7.p, Fig. 2D-J, Fig. S1A in the other puf genes (Kamath and Ahringer, 2003). The rrf-3(pk1426) supplementary material, rows Pn.px to Pn.pxxx). In addition, PUF- mutation was used to increase the sensitivity for RNAi (Simmer et 8::GFP expression was detected in the VulC sublineage of the 2° al., 2002). Of the six other PUF proteins that were tested, RNAi cells at the Pn.pxxx stage (inset in Fig. 2H,J and Fig. S1A in the against fbf-1 and fbf-2 induced a penetrant Muv phenotype, whereas supplementary material). RNAi against puf-9, which is most similar to puf-8, did not cause a We hypothesized that the increase in PUF-8::GFP expression in Muv phenotype (Table 2, rows 1-8). Because of the high degree of the descendants of the distal 3° VPCs might occur because these sequence similarity between the two fbf genes (over 90% identity at cells fuse with the hyp7 hypodermis that also expresses PUF-8::GFP. the nucleotide level), RNAi against either fbf gene most likely reduces To test if the upregulation of PUF-8::GFP in the descendants of the both fbf-1 and fbf-2 expression. We therefore tested whether fbf-1 or 3° VPCs is a consequence of their fusion with hyp7, we examined fbf-2 single mutants or only the fbf-1 fbf-2 double mutant show a Muv DEVELOPMENT C. elegans PUF proteins in vulval development RESEARCH ARTICLE 3465 Table 2. fbf-1 and fbf-2 redundantly regulate vulval induction Row Genotype RNAi % Muv* n Induction n 1 gap-1(ga133) gfp 0 50 3.0 40 2 gap-1(ga133) fbf-1 85 110 4.1 21 3 gap-1(ga133) fbf-2 56 100 3.6 21 4 gap-1(ga133) puf-3 031 –– 5 gap-1(ga133) puf-5 332 –– 6 gap-1(ga133) puf-7 041 –– 7 gap-1(ga133) puf-8 79 104 3.9 24 8 gap-1(ga133) puf-9 040 –– 9 fbf-1(ok91) – 0 200 3.0 27 10 fbf-1(ok91); gap-1(ga133) – 1 157 3.0 55 11 fbf-2(q738) – 0 80 3.0 25 12 fbf-2(q738); gap-1(ga133) – 0 63 3.0 24 13 fbf-1(ok91) fbf-2(q704) – 28 74 3.6 26 14 fbf-1(ok91) fbf-2(q704); gap-1(ga133) – 94 282 4.9 282 15 fbf-1(ok91) fbf-2(q704); gap-1(ga133) gfp 94 154 4.8 36 16 fbf-1(ok91) fbf-2(q704); gap-1(ga133) gld-1 19 168 3.3 29 17 fbf-1(ok91) fbf-2(q704); gap-1(ga133) fem-3 91 80 –– 18 puf-8(zh17); gap-1(ga133) gfp 76 162 3.6 27 § ¶ 19 puf-8(zh17); gap-1(ga133) gld-1 82 232 4.0 22 § ¶ 20 puf-8(zh17); gap-1(ga133) fem-3 69 108 –– *% Muv indicates the fraction of animals showing ectopic vulval induction under a dissection microscope. Induction indicates the average number of induced VPCs per animal. These strains carried the rrf-3(pk1426) mutation, which made them more sensitive to RNAi (Simmer et al., 2002). These strains were cis-linked with unc-4(e120). RNAi against fem-3 and gld-1 was additionally checked for presence of the germline phenotype. phenotype when combined with gap-1(ga133). fbf-1(ok91); gap- expression in the proximal VPC descendants compared to gap- 1(ga133) and fbf-2(q738); gap-1(ga133) animals both developed a 1(ga133) single mutants (Fig. 3J,K). Moreover, the descendants of wild-type vulva, but fbf-1(ok91) fbf-2(q704); gap-1(ga133) triple P5.p and P7.p adopted a proper 2° cell fate, as they generated seven mutants showed a strong Muv phenotype (Fig. 1D,E and Table 2 descendants that exhibited a normal morphology and a normal EGL- rows 9-14). Interestingly, even in a gap-1(+) background fbf-1(ok91) 17::YFP expression pattern in the VulC and VulD subfates (compare fbf-2(q704) double mutants were weakly Muv (Table 2, row 13). Fig. 3G with L). In the distal cells (the P3.p, P4.p and P8.p Finally, we tested for a possible redundancy among the puf genes by descendants) we observed only a very mild increase in the early, 1°- performing puf-3, puf-5, puf-7, puf-8 and puf-9 RNAi in the puf- specific or the late, 2°-specific EGL-17::YFP expression that did not 8(zh17) and fbf-1(ok91) fbf-2(q704) backgrounds, but observed no match the frequency of ectopic vulval induction observed in this synthetic Muv phenotypes among the other PUF genes (data not background (Fig. 3J-M). However, it should be noted that also in shown). Thus, besides puf-8 the two fbf genes encode functionally other mutant backgrounds such as let-60(n1046gf) the frequency and redundant negative regulators of vulval development. strength of ectopic EGL-17::YFP expression does not mirror the level of ectopic vulval induction (Burdine et al., 1998). fbf-1 and fbf-2 inhibit specification of the 1° In contrast to puf-8 mutants, fbf-1(ok91) fbf-2(q704); gap- vulval cell fate 1(ga133) triple mutants displayed a clear upregulation of the early, We next determined whether PUF-8 or the FBF proteins regulate the 1°-specific EGL-17::YFP expression in all VPCs and their specification of the 1° vulval cell fate using the egl-17::yfp reporter descendants (Fig. 3N,O). Especially in the descendants of P5.p and as a marker for the 1° cell fate (Inoue et al., 2002). egl-17 encodes a P7.p, the 1°-specific EGL-17::YFP expression was much stronger fibroblast growth factor (FGF) homolog that is normally expressed than in gap-1(ga133) single mutants. In addition to the late EGL- in P6.p and its descendants from the time of induction until the 17::YFP expression in the ectopically induced pseudovulvae, fbf- Pn.pxx stage (Fig. 3A,B) (Burdine et al., 1998; Inoue et al., 2002). 1(ok91) fbf-2(q704); gap-1(ga133) mutants also exhibited an In L4 larvae at the Pn.pxxx stage, EGL-17::YFP expression expansion of the 2°-specific EGL-17::YFP expression to 2° subfates disappears in the 1° cells and appears in the VulC and VulD cells of that normally do not express the marker (e.g. VulA and VulB in Fig. the 2° lineage (Fig. 3C,D) (Burdine et al., 1998; Inoue et al., 2002). 3P,Q). This aberrant EGL-17::YFP expression pattern within the 2° Both the early (1° fate-specific) and late (2° subfate-specific) EGL- lineage was accompanied by morphological changes of the P5.p and 17::YFP expression depend on inductive signalling (Burdine et al., P7.p descendants that are characteristic of a partial transformation 1998). towards the 1° fate (note the detachment of the P5.p descendants in We observed a slight expansion of the early, 1°-specific EGL- Fig. 1E and Fig. 3P) (Berset et al., 2005). Such defects in the 2° cell 17::YFP expression in gap-1(ga133) animals causing the lineage were only rarely observed in puf-8(zh17); gap-1(ga133) descendants of P5.p and P7.p and occasionally also of P8.p to animals (Fig. 3M). express EGL-17::YFP (Fig. 3E,F), although, gap-1(ga133) mutants Thus, PUF-8 and the FBF proteins perform clearly distinct roles exhibit normal vulval induction and correct 2° cell fate specification during vulval cell fate specification. FBF-1 and FBF-2 inhibit 1° in P5.p and P7.p (Fig. 3G,H). fate-specific gene expression and are required for proper 2° fate Surprisingly, in puf-8(zh17); gap-1(ga133) double mutants or puf- execution in P5.p and P7.p, whereas PUF-8 does not regulate 1°- 8 RNAi-treated gap-1(ga133) animals we observed no increase – specific gene expression and appears to regulate vulval induction and sometimes even a reduction – in the 1°-specific EGL-17::YFP through a different mechanism. DEVELOPMENT 3466 RESEARCH ARTICLE Development 133 (17) Fig. 2. PUF-8::GFP and FBF-2::GFP expression during vulval development. (A) Structure of the translational puf-8::gfp and fbf-2::gfp reporters. (B,D,F,H) Time-course analysis of PUF-8::GFP expression in the vulval cells from the L2 until the L4 stage with (C,E,G,J) the corresponding Nomarski images. For a semi-quantitative analysis of the expression patterns, see Fig. S1 in the supplementary material. (K,L) PUF-8::GFP expression in gonad- ablated eff-1(hy21) animals, and the corresponding Nomarski image. Note that despite the extra round of cell divisions in P4.p and P5.p descendants of gonad-ablated eff-1 mutants no vulval differentiation was observed. (M-R) FBF-2::GFP expression, and the corresponding Nomarski images, from the early L3 until the L4 stage. In all panels, anterior is to the left and ventral is to the bottom. Scale bars: in C,L,N and in the inset of J, 10 m. gld-1 is an FBF target during vulval development fate is only sealed after the Pn.px cells have fused with hyp7 (Wang Since PUF proteins function as translational repressors, the Muv and Sternberg, 1999), the puf-8(lf) mutations might allow distal phenotype caused by puf-8 and fbf-1 and fbf-2 mutations is probably vulval cells to stay unfused and hence receive the inductive signal caused by enhanced translation of their target mRNAs. Thus, RNAi over a longer time period, which in combination with a second against a target mRNA that encodes a positive regulator of vulval mutation in a negative regulator of the EGFR/RAS/MAPK pathway development should suppress the Muv phenotype of puf-8(zh17); would result in excess vulval induction. gap-1(ga133) and/or fbf-1(ok91) fbf-2(q704); gap-1(ga133) To observe the timing of vulval cell fusions, we used the ajm-1::gfp mutants. In the germline, gld-1 and fem-3 are direct FBF targets that reporter, which labels the adherens junctions of the VPCs and their function in mitosis/meiosis and sperm/oocyte decision, respectively descendants as long as they have not fused with hyp7 (Mohler et al., (Crittenden et al., 2002; Zhang et al., 1997). No targets of PUF-8 1998). In wild-type animals, the uninduced distal VPCs divide once have so far been found. RNAi against gld-1 suppressed the fbf- and then rapidly fuse with hyp7. Therefore, in the majority of wild- 1(ok91) fbf-2(q704); gap-1(ga133) but not the puf-8(zh17); gap- type larvae we analyzed at the Pn.px stage, the descendants of P3.p, 1(ga133) Muv phenotype, whereas RNAi against fem-3 had no P4.p and P8.p had already fused with hyp7 as demonstrated by the loss effect on the Muv phenotype of either strain (Table 2, rows 15-20). of AJM-1::GFP staining (Fig. 4A-C). In puf-8(zh17) mutants, Thus, the FBF proteins negatively regulate vulval induction by however, the fusion of P4.p and P8.p descendants was significantly repressing, among others, gld-1 expression. PUF-8, however, delayed, as in approximately 50% of the animals AJM-1::GFP appears to act through a distinct set of yet unknown target genes. staining was still present in P4.px and P8.px (Fig. 4D-F). Note that despite the delay in cell fusion puf-8(zh17) single mutants never puf-8 controls the timing of 3° cell fusions showed ectopic induction of the distal VPCs (Table 1, row 4). In fbf- The upregulation of PUF-8::GFP in the distal 3° vulval cells raises 1(ok91) fbf-2(q704) mutants, P4.p and P8.p descendants were unfused the possibility that PUF-8 might regulate the competence of the in approximately 20% of the cases (Fig. 4G-J). Since 28% of fbf- distal vulval cells to respond to the inductive signal. Since the 3° cell 1(ok91) fbf-2(q704) double mutants exhibit a Muv phenotype in a DEVELOPMENT C. elegans PUF proteins in vulval development RESEARCH ARTICLE 3467 Fig. 3. fbf-1 and fbf-2 inhibit 1° cell fate specification. Analysis of EGL-17::YFP expression in mid-L3 larvae at the Pn.px or Pn.pxx stage (left side) and in L4 larvae at the Pn.pxxx stage (right side). (A-D) Wild-type, (E-H) gap-1(ga133), (J-M) gap-1(ga133); puf-8 RNAi and (N-Q) fbf-1(ok91) fbf- 2(q704); gap-1(ga133) larvae. In all panels, anterior is to the left and ventral is to the bottom. In the graphs, white indicates no EGL-17::YFP expression, grey low expression and black high expression. The arrows in L and P indicate ectopic induction of distal vulval cells; the arrowhead in P indicates an example with expanded EGL-17::YFP expression in VulA and VulB, and the resulting defect in the 2° fate execution. Scale bars: in A,C, 10 m. gap-1(+) background (Table 2, row 13), the distal cells were probably row 3 and Fig. S2B in the supplementary material), and ablation of the unfused because they had adopted a 1° or 2° vulval cell fate in these somatic gonad precursors Z1 and Z4, which give raise to the AC, animals. PUF-8 therefore inhibits vulval development by promoting resulted in a suppression of the Muv phenotype to nearly wild-type the fusion of the 3° cells with the surrounding hyp7 hypodermis. levels of vulval induction (Table 3, row 4 and Fig. S2C in the Similar to puf-8(lf), a mutation in the effector of cell fusion eff- supplementary material). Even after ablation of all four gonad 1 that blocks all cell fusions causes a weak Muv phenotype that precursor cells (Z1 to Z4), we observed gonad-independent vulval was further enhanced by the gap-1(ga133) background (Table 1, induction in 19% of the animals (Table 3, row 5 and Fig. S2D in the rows 23 and 24) (Mohler et al., 2002). However, it should be noted supplementary material). Since the gap-1(ga133) mutation alone does that eff-1(hy21); gap-1(ga133) double mutants display a weaker not cause any gonad-independent vulval induction (Hajnal et al., Muv phenotype than puf-8(zh17); gap-1(ga133) animals (Table 1, 1997), fbf-1 and fbf-2 inhibit vulval differentiation not only by compare rows 6 and 24), indicating that puf-8 is likely to have repressing specific target genes in the germ cells but also in somatic additional functions besides controlling the timing of 3° cell cells outside of the gonad. Supporting this hypothesis, a translational fusions. FBF-2::GFP reporter showed an expression pattern similar to the PUF-8::GFP pattern described above. Expression of FBF-2::GFP was fbf-1 and fbf-2 act in the germline and in the soma first observed at the Pn.px stage in the 3° descendants of the distal Thompson et al. (Thompson et al., 2006) recently reported that VPCs, and it persisted throughout the L4 stage (Fig. 2A,M-R and Fig. feminized fbf-1 fbf-2 mutants (i.e. fbf-1 fbf-2; fog-1 or fbf-1 fbf-2; fog- S1D in the supplementary material). 3 triple mutants) display a strong Muv phenotype that is completely suppressed by ablation of the germ cell precursors Z2 and Z3. This puf-8, fbf-1 and fbf-2 act in the vulval cells observation indicated that fbf-1 and fbf-2 inhibit vulval induction in a We next sought to identify the somatic tissue in which puf-8 and fbf- non cell-autonomous manner, probably by repressing the translation 1 and fbf-2 act. Since puf-8::gfp and fbf-2::gfp are both expressed of a positive regulator of vulval development in the germ cells. We in the vulval cells as well as in the hyp7 hypodermis, we tested performed similar gonad precursor cell ablations, but used the fbf- whether puf-8, fbf-1 and fbf-2 act cell-autonomously in the VPCs 1(ok91) fbf-2(q704); gap-1(ga133) background. Ablation of Z2 and and their descendants or non cell-autonomously in hyp7. To Z3 resulted in a partial suppression of the Muv phenotype (Table 3, distinguish between these two possibilities, we expressed puf-8 and DEVELOPMENT 3468 RESEARCH ARTICLE Development 133 (17) Fig. 4. puf-8 regulates the fusion of the distal vulval cells. Vulval cell fusion was analyzed at the Pn.px stage using AJM-1::GFP as a cell junction marker for unfused cells. (A-C) Wild-type, (D-F) puf-8(zh17) single mutants and (G-J) fbf-1(ok91) fbf-2(q704) double mutants. In all panels, anterior is to the left and ventral is to the bottom. In the graphs, white represents fused Pn.px cells, grey indicates fusing Pn.px cells that have started to dissolve their junctions as can be seen for the P8.px cells in G, and black indicates unfused cells with intact AJM-1::GFP-positive junctions. Note that the fraction of unfused cells in fbf-1(ok91) fbf-2(q704) double mutants matches the frequency of ectopically induced distal cells that give rise to the 28% penetrant Muv phenotype (see Table 2, row 13). Scale bar in B: 10 m. fbf-2 under the control of the hypodermal dpy-7 promoter (e.g. well as puf-8 have an additional focus in the germline, since the P ::puf-8) (Gilleard et al., 1997), and each of the three genes multicopy extrachromosomal arrays we used for these experiments dpy-7 under control of a 3.1 kb bar-1 promoter fragment that drives are normally silenced in the germ cells. Thus, puf-8, fbf-1 and fbf-2 expression in the vulval cells, the gonadal sheath cells and in the negatively regulate vulval development at least partly in the VPCs adult seam cells (e.g. P ::puf-8) (Natarajan et al., 2004). Neither or their descendants. bar-1 the sheath cells nor the seam cells are in contact with the vulval cells, making it very unlikely that expression of a gene in these DISCUSSION tissues could affect vulval induction. None of the three P ::puf- PUF proteins control somatic development dpy-7 8 transgenes tested caused a significant rescue of puf-8(ok302); Translational repressors of the Pumilio/FBF (PUF) family regulate gap-1(ga133) Muv phenotype, but two out of three P ::puf-8 various aspects of germ cell development in C. elegans by bar-1 lines exhibited partial rescue, and the third line showed a weak controlling the translation of maternally provided mRNAs reduction of the Muv phenotype (Table 4, rows 5-11). It should be (Crittenden et al., 2002; Zhang et al., 1997). Here, we show that noted that even injection of a cosmid spanning the entire puf-8 locus three of the eleven C. elegans PUF genes also function in the soma never gave complete rescue of the Muv phenotype (Table 4, rows to control cell fate specifications during larval development. In 1-4). Moreover, co-injection of P ::puf-8 with P ::puf-8 did particular, we have found that PUF-8, FBF-1 and FBF-2 negatively bar-1 dpy-7 not cause a stronger rescue than injection of P ::puf-8 alone regulate vulval development in the hermaphrodite. Like most bar-1 (data not shown). previously identified inhibitors of vulval development, single Similarly, all but one of the P ::fbf-1 and P ::fbf-2 mutants in one of these three puf genes do not change the normal bar-1 bar-1 transgenes reduced the penetrance of the fbf-1(ok91) fbf-2(q704); pattern of vulval cell fates. However, when combined with another gap-1(ga133) Muv phenotype from 90% down to 55-60%, and only mutation in an inhibitor of the inductive EGFR/RAS/MAPK one of the three P ::fbf-2 transgenes had a slightly significant pathway, puf-8 or fbf mutants exhibit a hyperinduced multivulva dpy-7 effect (Table 4, rows 12-21). The incomplete rescue with the phenotype. Genetic epistasis analysis indicates that fbf-1 and fbf-2 different constructs is consistent with the model that fbf-1, fbf-2 as perform a redundant function to inhibit 1° vulval fate specification, DEVELOPMENT C. elegans PUF proteins in vulval development RESEARCH ARTICLE 3469 Table 3. fbf-1 and fbf-2 act in the soma and the germline † ‡ Row Genotype Ablation % Muv* % Vul Induction n 1 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Unablated 84 0 4.1 48 2 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Mock ablated 74 0 4.0 31 3 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Z2/Z3 (germ line) 27 0 3.3 22 4 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Z1/Z4 (somatic gonad) 8 8 3.0 12 5 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Z1-Z4 (somatic gonad + germline) 0 81 0.6 21 *% Muv indicates the fraction of animals with more than three induced VPCs. % Vul indicates the fraction of animals with less than three induced VPCs. Induction indicates the average number of induced VPCs per animal. whereas puf-8 plays a distinct role in regulating the temporal 8 limits the time period during which the vulval cells can receive and competence of the vulval cells to respond to the inductive and lateral integrate the vulval patterning signals. In the absence of PUF-8, the signals. vulval cells can receive the inductive signal over a longer time period, which may result in the accumulation of higher levels of PUF-8 regulates the temporal competence of the activated MAPK in the distal vulval cells. When combined with a vulval cells mutation in a direct inhibitor of the EGFR/RAS/MAPK pathway Loss-of-function mutations in puf-8 partially suppress the Vul such as gap-1, this results in the ectopic vulval differentiation and a phenotype caused by mutations that reduce but do not inactivate the Muv phenotype. Supporting this idea, a mutation in the effector of EGFR/RAS/MAPK signalling pathway. Although this observation cell fusion eff-1, which blocks all cell fusions, caused a weak Muv does not prove a direct involvement of PUF-8 in regulating the phenotype (Mohler et al., 2002). However, puf-8 mutants exhibit inductive EGFR/RAS/MAPK signalling pathway, it indicates that more ectopic vulval induction in the gap-1 background than eff-1 in the absence of PUF-8 lower levels of inductive signal are mutants, which points to additional functions of PUF-8 besides sufficient to induce vulval differentiation. A PUF-8::GFP reporter controlling the timing of cell fusions. transgene is initially expressed in all VPCs at equal levels, but after The distal VPC descendants fuse with hyp7 shortly after they vulval induction PUF-8::GFP expression increases in the have been born, suggesting that they exit from the cell cycle as descendants of the distal VPCs (P3.p, P4.p and P8.p) that have they lose their competence (Wang and Sternberg, 1999). The adopted the 3° fate. This expression pattern correlates well with the proximal vulval cells, on the other hand, go on to divide two more observed delay in the fusion of the distal 3° cells with the hyp7 times before undergoing terminal differentiation and forming a hypodermis in puf-8 mutants. All vulval cells are competent to functional vulva. It is therefore possible that PUF-8 ensures that respond to the inductive AC and lateral Notch signals until they fuse the distal vulval cells exit from the cell cycle immediately after with hyp7 (Wang and Sternberg, 1999). Even after the first round of they have been generated and then fuse with hyp7. A somewhat vulval cell divisions, a single pulse of MAPK activity can reprogram similar function has been proposed for the Drosophila PUF-8 a 2° or 3° cell to adopt the 1° cell fate (Berset et al., 2005). It thus orthologue Pumilio, which blocks the cell cycle progression of the appears that by promoting the fusion of the 3° cells with hyp7, PUF- migrating pole cells during embryogenesis by repressing cyclin B Table 4. puf-8, fbf-1 and fbf-2 act in part in the vulval cells † 2 ‡ Row Genotype Transgene % Muv* Induction  -test n 1 puf-8(ga145); gap-1(ga133) – 85±3 – 477 2 puf-8(ga145); gap-1(ga133) Cosmid C30G12 line 1 57±10 – x 104 3 puf-8(ga145); gap-1(ga133) Cosmid C30G12 line 2 24±8 – x 116 4 puf-8(ga145); gap-1(ga133) Cosmid C30G12 line 3 35±9 – x 121 5 puf-8(ok302); gap-1(ga133) – 68±6 4.0 260 6 puf-8(ok302); gap-1(ga133) zhEx173.1 [P ::puf-8] 30±13 3.3 x 46 bar-1 7 puf-8(ok302); gap-1(ga133) zhEx173.2 [P ::puf-8] 38±19 3.5 y 26 bar-1 8 puf-8(ok302); gap-1(ga133) zhEx173.3 [P ::puf-8] 52±20 3.7 23 bar-1 9 puf-8(ok302); gap-1(ga133) zhEx170.1 [P ::puf-8] 71±18 4.2 24 dpy-7 10 puf-8(ok302); gap-1(ga133) zhEx172.1 [P ::puf-8] 77±12 4.0 51 dpy-7 11 puf-8(ok302); gap-1(ga133) zhEx172.2 [P ::puf-8] 63±18 3.9 27 dpy-7 12 fbf-1(ok91) fbf-2(q704); gap-1(ga133) – 96±2 4.9 441 13 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx175.1 [P 1::fbf-1] 57±18 3.9 x 30 bar- 14 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx175.2 [P ::fbf-1] 58±20 3.8 x 24 bar-1 15 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx175.3 [P ::fbf-1] 73±16 4.1 x 30 bar-1 16 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx174.1 [P ::fbf-2] 60±21 3.8 20 bar-1 17 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx174.2 [P ::fbf-2] 59±19 3.8 x 27 bar-1 18 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx174.3 [P ::fbf-2] 52±18 3.6 x 29 bar-1 19 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx176.1 [P ::fbf-2] 81±14 4.5 32 dpy-7 20 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx176.2 [P ::fbf-2] 83±12 4.3 y 35 dpy-7 21 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx176.3 [P ::fbf-2] 94±8 4.7 35 dpy-7 Rows 1, 5 and 12 show the average of animals without the transgene that were counted for each genotype in parallel. *% Muv indicates the fraction of animals with more than three induced VPCs, and the 95% confidence intervals are indicated. Induction indicates the average number of induced VPCs per animal. ‡ 2 For each line the  test was performed comparing the animals with and without the array from the same plate. x indicates a P value <0.01 and y indicates a P value <0.05. puf-8(ga145) and puf-8(ok302) were cis-linked with unc-4(e120). These strains were maintained balanced with mIn1 and their homozygous fbf-1 fbf-2 double mutant F1 progeny was scored. DEVELOPMENT 3470 RESEARCH ARTICLE Development 133 (17) translation to prevent their premature differentiation (Asaoka- Supplementary material Supplementary material for this article is available at Taguchi et al., 1999). One could, for example, imagine that the http://dev.biologists.org/cgi/content/full/133/17/3461/DC1 cell cycle state of the vulval cells and the hyp7 hypodermis needs References to be coordinated to allow the fusion between these two different Ahringer, J. and Kimble, J. (1991). 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Rev. fates in the C. elegans hermaphrodite germline. Nature 390, 477-484. Genet. 2, 690-701. Sijen, T., Fleenor, J., Simmer, F., Thijssen, K. L., Parrish, S., Timmons, L., DEVELOPMENT http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Development The Company of Biologists

Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development

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CORRIGENDUM Development 134, 4503-4505 (2007) doi:10.1242/dev.017475 Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development Claudia B. Walser, Gopal Battu, Erika Fröhli Hoier and Alex Hajnal There were errors published in Development 133, 3461-3471. We have discovered that the expression pattern of the PUF-8::GFP reporter zhEx61 shown in Fig. 2B-L on p. 3466 of this article, is based on an incorrect reporter construct that carries the insert in the reverse orientation. The corrected Fig. 2 below shows the PUF-8::GFP expression pattern that is observed with the correct PUF-8::GFP reporter zhEx274.1, which carries the insert in the correct orientation. Also shown is a corrected supplementary Fig. S1, in which a quantification of the expression pattern of zhEx274.1 is shown (A-C). Although the overall expression pattern observed with the PUF-8::GFP reporter zhEx274.1 is similar to that obtained with the reverse reporter zhEx61 shown in Fig. 2B-L of Walser et al. (2006), there are four differences in its expression, which are accounted for in the text changes detailed below. Also provided are new methods for the generation of zhEx274.1. These corrections do not change the overall conclusions of this paper. The page, paragraph and line numbers below refer to the PDF version of the article. Fig. 2. PUF-8::GFP and FBF-2::GFP expression during vulval development. (A) Structure of the translational puf-8::gfp and fbf-2::gfp reporters. (B,D,F,H) Time-course analysis of PUF-8::GFP expression in the vulval cells from the L2 until the L4 stage with (C,E,G,J) the corresponding Nomarski images. For a semi-quantitative analysis of the expression patterns, see Fig. S1 in the supplementary material. (K,L) PUF-8::GFP expression in gonad-ablated eff-1(hy21) animals, and the corresponding Nomarski image. All VPC descendants showed PUF-8::GFP expression with a strong increase in the descendants of P6.p. Note that despite the extra round of cell divisions in P5.p and P6.p descendants of gonad-ablated eff-1 mutants, no vulval differentiation was observed. (M-R) FBF-2::GFP expression, and the corresponding Nomarski images, from the early L3 until the L4 stage. In all panels, anterior is to the left and ventral is to the bottom. Scale bars: 10 m. 4504 CORRIGENDUM Fig. S1. PUF-8::GFP and FBF-2::GFP expression analysis. (A) Semi-quantitative time-course analysis of PUF-8::GFP expression in wild-type animals. The two daughter cells after the first cell division are termed Pn.px for all VPCs. The descendants of the second cell divisions of induced VPCs are termed Pn.pxx, and after the third round of cell divisions Pn.pxxx cells. Gray areas indicate the proportion of PUF-8::GFP-positive vulval cells, white areas the proportion of PUF-8::GFP-negative cells. (B) Analysis of PUF-8::GFP expression pattern in (top row) eff-1(hy21) mutants at the Pn.pxxx stage without gonad ablation and (bottom row) gonad-ablated eff-1(hy21) mutants at the Pn.px stage. Both conditions were analyzed in L4 larvae, but since VPCs in gonad-ablated animals are not induced to adopt vulval cell fates, they divide once and arrest at the Pn.px stage or occasionally divide a second time, as shown in Fig. 2K,L. (C) Analysis of the PUF-8::GFP expression pattern in let-60(n1046gf) L4 larvae at the Pn.pxxx stage. Since let-60(n1046gf); zhEx274.1[puf-8::gfp] animals developed into sterile adults for unknown reasons, the let-60(n1046); zhEx274.1[puf-8::gfp] animals were maintained as heterozygotes, and their multivulva progeny homo- or heterozygous for let-60(n1046gf) were scored at the Pn.pxxx stage. (D) Semi-quantitative time-course analysis of FBF-2::GFP expression in wild-type animals. Only animals showing bright FBF-2::GFP expression in somatic tissues were used for the analysis. White indicates no FBF-2::GFP expression, grey low expression and black high expression. Correction to the text on p. 3462, paragraph 6 Extrachromosomal transgenic arrays [transgenes; co-transformation marker; pBS: Bluescript (concentration in ng/l)] were generated by microinjection of DNA into young adult worms (Mello et al., 1991): Correction to the text on p. 3462, paragraph 7, line 6 … zhEx220[fbf-2::gfp; lin-48::gfp (100;50)], zhEx274.1[puf-8::gfp; lin-48::gfp (80;50)]. Correction to the text on p. 3464, paragraph 2, from line 4 PUF-8::GFP was expressed in various tissues including the hypodermis, the ventral cord motor neurons (not shown) and the vulval cells (Fig. 2B-J and see Fig. S1A in the supplementary material). Before vulval induction in L2 larvae, PUF-8::GFP was expressed in all six vulval precursor cells, although expression was more frequently observed in the distal VPCs (P3.p, P4.p and P8.p) than in the proximal VPCs (P5.p, P6.p and P7.p, Fig. 2B,C, and row with Pn.p cells in Fig. S1A in the supplementary material). After vulval induction in early L3 larvae, PUF-8::GFP expression persisted in the descendants of the 3° distal VPCs (P3.p, P4.p and P8.p), while expression faded in the 1° and 2° descendants of the proximal VPCs (P5.p, P6.p and P7.p, Fig. 2D-J, Fig. S1A in the supplementary material, rows Pn.px to Pn.pxxx). Correction to the text on p. 3464, paragraph 3, from line 1 We hypothesized that PUF-8::GFP expression in the descendants of the distal 3° VPCs might persist because these cells fuse with the hyp7 hypodermis that also expresses PUF-8::GFP. To test if the expression of PUF-8::GFP in the descendants of the 3° VPCs is a consequence of their fusion with hyp7, we examined PUF-8::GFP expression in an eff-1(hy21) background, in which no cell fusions occur (Mohler et al., 2002). CORRIGENDUM 4505 Correction to the text on p. 3464, paragraph 3, from line 10 In most gonad-ablated eff-1(hy21) animals, PUF-8::GFP expression was observed in the VPCs and their descendants (Fig. 2K,L and see Fig. S1B in the supplementary material). Moreover, in let-60 ras(gf) animals, in which the distal VPCs frequently adopt the 1° or 2° induced cell fates, PUF-8::GFP expression was often absent in the distal VPCs and their descendants (see Fig. S1C in the supplementary material) (Beitel et al., 1990; Greenwald et al., 1983). We conclude that PUF-8::GFP is expressed in the descendants of VPCs that have adopted the uninduced 3° cell fate independently of their fusion with hyp7. Correction to the text on p. 3466, paragraph 2, line 1 The expression of PUF-8::GFP in the distal 3° vulval cells raises the possibility that PUF-8 might regulate the competence of the distal vulval cells to respond to the inductive signal. Correction to the text on p. 3469, paragraph 2, line 7 A PUF-8::GFP reporter transgene is expressed predominantly in the distal VPCs (P3.p, P4.p and P8.p) and their descendants that have adopted the 3° fate. The authors apologise to readers for these mistakes and are grateful to Dave Hansen for discovering the error in the plasmid used to generate zhEx61. Publisher’s note: Although the mistake reported in this corrigendum has resulted in several corrections being made to Walser et al. (2006) and in an unusually lengthy corrigendum, we would like to reassure readers that expert opinion has confirmed that the minor changes in expression that are seen between the incorrect reporter zhEx61 and the correct reporter zhEx274.1 do not alter or affect the conclusions drawn by this paper. RESEARCH ARTICLE 3461 Development 133, 3461-3471 (2006) doi:10.1242/dev.02496 Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development 1,2, 1, 1 1,† Claudia B. Walser *, Gopal Battu *, Erika Fröhli Hoier and Alex Hajnal The C. elegans PUF and FBF proteins regulate various aspects of germline development by selectively binding to the 3 untranslated region of their target mRNAs and repressing translation. Here, we show that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulvaI development. Loss-of-function mutations in puf-8 cause ectopic vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway and suppress the vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signalling. PUF-8 acts cell-autonomously in the vulval cells to limit their temporal competence to respond to the extrinsic patterning signals. fbf-1 and fbf-2, however, redundantly inhibit primary vulval cell fate specification in two distinct pathways acting in the soma and in the germline. The FBFs thereby ensure that the inductive signal selects only one vulval precursor cell for the primary cell fate. Thus, translational repressors regulate various aspects of vulval cell fate specification, and they may play a conserved role in modulating signal transduction during animal development. KEY WORDS: Caenorhabditis elegans, Vulva, Pumilio, Translational control, Signal transduction INTRODUCTION additional flanking nucleotides (Murata and Wharton, 1995; The spatial and temporal regulation of gene expression can occur Tadauchi et al., 2001; Wharton et al., 1998; Zamore et al., 1997; either at the level of gene transcription or at the level of mRNA export, Zhang et al., 1997). stability or translation through RNA-binding proteins or micro RNAs Pumilio, the only PUF protein in Drosophila melanogaster, (de Moor et al., 2005; Kuersten and Goodwin, 2003). Work on model controls, together with Nanos, the establishment of the anterior- organisms such as Drosophila melanogaster and Caenorhabditis posterior axis of the embryo by repressing the translation of maternal elegans has contributed much to our current understanding of post- hunchback mRNA (Barker et al., 1992; Murata and Wharton, 1995). transcriptional gene regulation during development. Translational Pumilio and Nanos also inhibit cyclin B translation in migrating pole control by RNA binding proteins is frequently used in the C. elegans cells allowing them to arrest in G2 until they reach the gonads germline and early embryo, but translational regulation has also been (Asaoka-Taguchi et al., 1999). In addition to its roles during observed during larval development (Kuersten and Goodwin, 2003; development, Drosophila Pumilio was recently shown to be Rougvie, 2001). Many mRNAs contain sequence motifs in their 5 or necessary for the activity-dependent expression of the voltage-gated 3 untranslated regions (5UTRs or 3UTRs) that serve as binding sites sodium channel Paralytic in the central nervous system (Mee et al., for regulatory proteins controlling different aspects of mRNA 2004). The human and mouse genomes each encode two PUF localization, translation or stability. proteins with unknown functions (Spassov and Jurecic, 2002; The PUF gene family is conserved from yeast to humans. PUF Spassov and Jurecic, 2003). proteins function as translational repressors that bind to specific The C. elegans genome contains the surprisingly high number of elements in the 3UTRs of their target mRNAs (reviewed by eleven PUF genes (fbf-1 and fbf-2, puf-3 to puf-11). PUF-8 forms, Wickens et al., 2002). The first characterized members of this family together with PUF-9, a distinct subgroup among the C. elegans PUF were Drosophila Pumilio and the two C. elegans FBF proteins. proteins, as PUF-8 and PUF-9 are more similar to the Drosophila Hence, this family is referred to as PUF for Pumilio and FBF repeat and to the two vertebrate pumilio proteins than to the other C. proteins (Zhang et al., 1997). Typical PUF proteins contain eight elegans PUF proteins (Wickens et al., 2002). FBF-1 and FBF-2 PUF repeats of approximately 40 amino acids with a core consensus (fem-3-binding factor-1 and -2) are two closely related proteins that sequence containing aromatic and basic residues. The PUF repeats regulate the sperm/oocyte switch in the hermaphrodite germline by directly bind to the target mRNAs and recruit additional proteins binding to the PME (point mutation element) in the 3 UTR of fem- such as Nanos, Brain tumor and CPEB (Kraemer et al., 1999; 3 mRNA (Ahringer and Kimble, 1991; Kraemer et al., 1999; Zhang Luitjens et al., 2000; Sonoda and Wharton, 1999; Sonoda and et al., 1997). In addition, FBF-1 and FBF-2 both regulate the mitosis Wharton, 2001). The cis-regulatory elements in the 3 UTRs of their versus meiosis decision in the distal region of the germline by target mRNAs contain a UGUR tetra nucleotide sequence motif repressing gld-1 translation in the mitotic region to prevent the stem termed a Nanos response element (NRE). The binding specificity cells from entering meiosis (Crittenden et al., 2002; Kadyk and of the individual PUF proteins is thought to be determined by Kimble, 1998). Furthermore, FBF and PUF proteins are required for germ cell survival, germ cell migration and the mitotic arrest of germ cells during embryogenesis (Kraemer et al., 1999; Subramaniam and Zoologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Seydoux, 1999). PUF-8 is necessary for the meiotic division of the Switzerland. Molecular Life Science PhD Program, Molekular biologisches Institut, primary spermatocytes in hermaphrodites and males (Subramaniam Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland. and Seydoux, 2003). *These authors contributed equally to this work Here, we show that the same PUF proteins that control germline Author for correspondence (e-mail: [email protected]) development also act in the soma during vulval induction. During larval development, the hermaphrodite vulva is formed out of 22 Accepted 15 June 2006 DEVELOPMENT 3462 RESEARCH ARTICLE Development 133 (17) cells that are generated by three out of six equivalent vulval (10;20;120)], zhEx172.1-2[P ::puf-8; sur-5::gfp; pBS (50;50;50)], dpy-7 ::fbf-2; sur-5::gfp; pBS (50;50;50)], zhEx220[fbf-2::gfp; zhEx176.1-3[P precursor cells (VPCs; P3.p through P8.p) (Greenwald, 1997). To dpy-7 lin-48::gfp (100;50)]. induce vulval differentiation, the anchor cell (AC) in the somatic GFP and YFP expression was observed under fluorescent light gonad sends an epidermal growth factor signal (LIN-3) to the illumination with a Leica DMRA microscope equipped with a cooled CCD adjacent VPCs (Hill and Sternberg, 1992). This inductive AC signal camera (Hamamatsu ORCA-ER) controlled by the Openlab 3.0 software activates the LET-23 EGFR signalling pathway in the nearest VPC (Improvision). Animals were mounted on 3% agarose pads in M9 solution (P6.p) to specify the primary (1°) cell fate. P6.p then sends a lateral . Larvae were first inspected using Nomarski optics containing 15 mM NaN signal to the neighbouring VPCs, P5.p and P7.p, via the LIN-12 to identify the position of the Pn.p cells or their descendants, and GFP or NOTCH pathway (Greenwald et al., 1983; Sternberg, 1988). LIN- YFP expression was then scored under fluorescent light illumination using 12 signalling inhibits the 1° fate specification in P5.p and P7.p and the same exposure settings for a particular transgene in all different genetic instead instructs the secondary (2°) fate in these cells (Ambros, backgrounds. For the PUF-8::GFP FBF-2::GFP and the EGL-17::YFP experiments, three semi-quantitative classes were made: no expression if the 1999; Sternberg, 1988). Multiple inhibitory signalling pathways fluorescence was not distinguishable from the background staining, low antagonize the EGFR/RAS/MAPK pathway to control the cell fate expression if there was a weak but clearly visible signal, and high expression choice in the VPCs (reviewed by Fay and Han, 2000). These if the fluorescence signal was strong. The images of PUF-8::GFP and inhibitors ensure that the distal VPCs (P3.p, P4.p and P8.p), which FBF-2::GFP at the L4 stages needed a correction to prevent overexposure. receive little or no inductive and lateral signals, adopt the tertiary The induction index of the VPCs was scored under Nomarski optics and (3°) non-vulval cell fate. After the vulval cell fates have been the average number of 1° or 2° induced VPCs per animal was calculated as specified, the VPCs undergo stereotypic patterns of cell divisions described previously (Dutt et al., 2004). before they differentiate and form the mature organ. Three rounds Laser ablation of the somatic gonad precursors Z1 and Z4 and germline of symmetric cell divisions generate eight 1° descendants, of which precursors Z2 and Z3 were done as described by Kimble (Kimble, 1981), four adopt the VulE and four the VulF subfate. The last of the three and induction was scored in L4 larvae. cell divisions in the 2° lineage generates only seven descendants that Genetic screens and positional molecular cloning of puf-8 further differentiate into the VulA, VulB, VulC and VulD subfates gap-1 enhancer screen to isolate puf-8(ga145): young adult gap-1(ga133) (Inoue et al., 2002; Sternberg and Horvitz, 1986). The 3° cells divide hermaphrodites were mutagenized with 50 mM ethyl-methanesulfonate only once and then fuse with the surrounding hypodermal syncytium (EMS) for 4 hours at room temperature, and the F2 generation was (hyp7). screened for mutants displaying a multivulva (Muv) phenotype. Our analysis indicates that puf-8, fbf-1 and fbf-2 negatively Approximately 30,000 haploid genomes were screened (Canevascini et regulate vulval induction in parallel with the known inhibitors of al., 2005). Non-complementation screen to isolate puf-8(zh17): gap-1(ga133) males the EGFR/RAS/MAPK pathway. puf-8 restricts the temporal were mutagenized with EMS as described above, mated with unc-4(e120) competence of the vulval cells by promoting the fusion of the puf-8(ga145); gap-1(ga133) hermaphrodites and the nonUnc F1 progeny uninduced 3° cells with hyp7, while fbf-1 and fbf-2 control the 1° was screened for Muv animals. After screening 2,000 haploid genomes one versus 2°/3° cell fate decision. Muv non-Unc animal was identified and propagated. ga145 was mapped with three-factor mapping between dpy-10 and unc-4 on LGII and further MATERIALS AND METHODS narrowed down by transformation rescue experiments using YACs and Nematode strains and general methods cosmids to the cosmid clone C30G12. RNAi analysis of the genes encoded All strains were derivatives of Bristol strain N2 of Caenorhabditis elegans by C30G12 in a gap-1(ga133) background identified the puf-8 gene as and grown under standard conditions at 20°C (Brenner, 1974) or at the candidate, and DNA sequencing of the puf-8 coding region in the ga145 and temperature indicated in the table footnotes. Unless noted otherwise, the zh17 alleles identified the molecular lesions. mutations used have been described previously (Riddle et al., 1997) and are listed below by their linkage group. RNA interference analysis LGI: lin-10(e1438), unc-13(e1091) to cis-link lin-10(e1438). LGII: fbf- RNA interference analysis (RNAi) was performed by feeding animals 1(ok91) (Crittenden et al., 2002), fbf-2(q738) (Lamont et al., 2004), fbf- dsRNA-producing bacteria as described previously (Kamath and Ahringer, 2(q704) (Crittenden et al., 2002), puf-8(zh17) (this work), puf-8(ga145) (this 2003) with the following modifications. During the cloning of puf-8, work), puf-8(ok302) (Subramaniam and Seydoux, 2003), rrf-3(pk1426) dsRNA-producing bacteria were grown on plates containing 1 mM IPTG (Sijen et al., 2001), eff-1(hy21) (Mohler et al., 2002), lin-7(e1413), unc- and 5-10 adult P0 gap-1(ga133) animals were put on each plate. For the 4(e120) to cis-link puf-8 alleles, puf-8(ok302) and the fbf mutations were syIs90; gap-1(ga133) strain, bacteria were induced with 6 mM IPTG, and balanced with mIn1(mIs14 dpy-10(e128)) (Edgley and Riddle, 2001). LGIII: for all other RNAi experiments, 5-15 P0 animals were put, as L1 larvae or unc-119(e2498), unc-119(ed4) for syIs90. LGIV: ark-1(sy247) (Hopper et as adults, on plates containing bacteria grown on 3 mM IPTG. Vulval al., 2000), dpy-20(e1282) to cis-link ark-1, let-60(n1046gf), let-60(n2021). induction was scored in the F1 progeny at the L4 larval stage to count the LGX: gap-1(ga133) (Hajnal et al., 1997), lin-2(n105ts), lin-15(n765ts), sli- number of induced VPCs or in adults to count the percentage of Muv 1(sy143). animals (indicated in the table footnotes). All dsRNA-producing bacteria Integrated transgenic arrays (transgenes; co-transformation marker): were from the Ahringer library (Kamath and Ahringer, 2003), except for the syIs90[egl-17::yfp + unc-119(+)] III (Inoue et al., 2002), swIs79[ajm- fem-3 RNAi bacteria, which were a gift from C. Eckmann. 1::gfp, unc-119(+)] IV (Mohler et al., 1998). Extrachromosomal transgenic arrays [transgenes; co- Plasmids and PCR fusion constructs transformation marker; pBS: Bluescript (concentration in ng/l)] were For the puf-8::gfp translational reporter, a 3.3 kb SalI genomic fragment generated by microinjection of DNA into young adult worms (Mello containing a 1.3 kb upstream promoter fragment and the entire C30G12.7 et al., 1991), except for the zhEx61[puf-8::gfp; unc-119(+)] open reading frame was cloned into the SalI site of plasmid pPD95.75 (a gift extrachromosomal line, which was generated by microparticle from A. Fire). For the fbf-2::gfp translational reporter, a 3.73 kb BamHI bombardment using 0.1 mg of 1 m gold beads coated with 16 g puf- genomic fragment containing a 1.5 kb upstream promoter fragment and the 8::gfp and 8 g unc-119(+) plasmids as described previously (Praitis et entire fbf-2 open reading frame was cloned into the BamHI site of plasmid al., 2001): pPD95.75. All the dpy-7 and bar-1 promoter fusions were generated by the zhEx173.1-3[P ::puf-8; sur-5::gfp; pBS (50;50;50)], zhEx175.1- PCR fusion method (Hobert, 2002). Details on the primers used and bar-1 3[P ::fbf-1; sur-5::gfp; pBS (50;50;50)], zhEx174.1-3[P ::fbf-2; sur- constructions of the gfp reporters and promoter fusions are available on bar-1 bar-1 5::gfp; pBS (50;50;50)], zhEx170.1[P ::puf-8; sur-5::gfp; pBS request. dpy-7 DEVELOPMENT C. elegans PUF proteins in vulval development RESEARCH ARTICLE 3463 RESULTS and Table 2, row 7). This gene has previously been named puf-8, as Identification of puf-8 as a negative regulator of it encodes one of the two C. elegans Pumilio homologues (Wickens vulval development et al., 2002). Sequencing the puf-8 coding region revealed a stop Single mutants in negative regulators of vulval induction often mutation at position 485 of the ORF (CAA to TAA) before the PUF exhibit a wild-type vulval phenotype because these genes are mostly repeats in zh17, and a G to A (GGA to AGA) transition at position genetically redundant. We therefore performed a forward genetic 1174, replacing glycine 317 with arginine in the fourth PUF repeat screen in a gap-1(ga133) loss-of-function background to identify in ga145 animals (Fig. 1A). The glycine mutated in ga145 is synthetic mutations in additional inhibitors of vulval induction conserved in PUF-9, Drosophila Pumilio and the vertebrate PUF (Canevascini et al., 2005). gap-1 encodes a GTPase-activating proteins. This glycine is adjacent to an asparagine residue that is protein that stimulates the intrinsic GTPase activity of LET-60 RAS directly involved in binding to the target mRNA (Opperman et al., and thus inhibits the transduction of the inductive signal (Hajnal et 2005). In addition to the vulval phenotype, both puf-8 alleles we al., 1997). gap-1(ga133) single mutants exhibit an elevated activity isolated showed the same partially penetrant sterile phenotype at of the EGFR/RAS/MAPK signalling pathway, yet they develop 20°C as the puf-8(ok302) deletion strain (Fig. 1A) (Subramaniam a wild-type vulva (Fig. 1B and Table 1, row 2). After screening and Seydoux, 2003), and the puf-8(ok302) deletion caused a Muv approximately 30,000 haploid genomes, we isolated 27 mutants that phenotype in a gap-1(ga133) background of similar penetrance to displayed a synthetic multivulva (Muv) phenotype in a gap- zh17 or ga145 (Table 1, row 7). Thus, zh17 and ga145 are strong 1(ga133) background and defined at least four complementation reduction-of-function or null alleles of puf-8. groups. The ga145 mutation found in this screen caused a 60% penetrant Muv phenotype in the gap-1(ga133) background, but no Genetic interaction of puf-8 with the obvious vulval phenotype as a single mutant (Table 1, rows 3 and 5). EGFR/RAS/MAPK pathway To identify additional alleles of this complementation group, we We examined the genetic interaction of puf-8(zh17) with mutations performed a non-complementation screen (for details see Materials that either reduce or increase the activity of the EGFR/RAS/MAPK and methods) that yielded a new allele (zh17) displaying an equally signalling pathway. puf-8(zh17) partially suppressed the vulvaless penetrant synthetic Muv phenotype (Table 1, rows 4 and 6 and Fig. (Vul) phenotype caused by mutations in lin-2, lin-7, lin-10 and let- 1C). The corresponding gene was mapped to LGII between dpy-10 60, which reduce but do not inactivate the inductive signal (Table 1, and unc-4 and further narrowed down by transformation rescue rows 9-16) (Kaech et al., 1998). We also combined puf-8(zh17) with experiments to the cosmid C30G12. The six genes on this cosmid mutations in inhibitors of the EGFR/RAS/MAPK pathway such as were tested by RNAi analysis. Feeding gap-1(ga133) animals with ark-1, sli-1 or lin-15 that exhibit a wild-type or only a very weak bacteria producing dsRNA derived from the C30G12.7 open reading Muv phenotype as single mutants (Herman and Hedgecock, 1990; frame caused a Muv phenotype of 80% penetrance (Table 1, row 8 Hopper et al., 2000; Jongeward et al., 1995; Yoon et al., 1995). With Fig. 1. PUF proteins that negatively regulate vulval development. (A) Intron-exon structure and alleles of puf-8, fbf-1 and fbf-2. White boxes indicate the 5UTRs, white boxes with arrowheads the 3UTRs, grey boxes the coding regions and black boxes the PUF repeats. (B-E) Nomarski images of the vulval cells in L4 larvae of (B) gap-1(ga133), (C) puf-8(zh17); gap-1(ga133), and of (D,E) fbf-1(ok91) fbf-2(q704); gap-1(ga133) animals. In all panels, anterior is to the left and ventral is to the bottom. Note the ectopic induction of P4.p and P8.p (arrows in C,D,E). Arrowhead in E indicates an example of defects in the 2° cell lineage generated by P5.p resulting in the detachment of the P5.p descendants from the cuticle in a fbf-1(ok91) fbf-2(q704); gap-1(ga133) larva. Scale bar: 10 m. DEVELOPMENT 3464 RESEARCH ARTICLE Development 133 (17) Table 1. puf-8 negatively regulates vulval development † ‡ § Row Genotype* % Muv % Vul Induction n 1 Wild-type 0 0 3.0 many 2 gap-1(ga133) 0 0 3.0 30 3 puf-8(ga145) 0 0 – 176 4 puf-8(zh17) 0 0 3.0 36 5 puf-8(ga145); gap-1(ga133) 60 0 4.0 28 6 puf-8(zh17); gap-1(ga133) 59 0 3.9 46 7 puf-8(ok302); gap-1(ga133) 80 0 4.2 24 8 gap-1(ga133); puf-8 RNAi 83 0 4.2 30 9 lin-2(n105) 0 56 1.6 18 10 puf-8(zh17); lin-2(n105) 0 18 2.5 22 11 lin-7(e1413) 0 95 0.6 38 12 puf-8(zh17); lin-7(e1413) 0 58 1.7 36 13 lin-10(e1438)** 2 83 1.3 42 14 lin-10(e1438); puf-8(zh17)** 0 41 2.1 29 15 let-60(n2021) 0 44 2.6 133 16 puf-8(zh17); let-60(n2021) 0 16 2.8 224 †† 17 ark-1(sy247) 0 0 3.0 33 †† 18 puf-8(zh17); ark-1(sy247) 55 0 3.6 42 19 sli-1(sy143) 0 0 3.0 37 20 puf-8(zh17); sli-1(sy143) 11 0 3.1 38 ‡‡ 21 lin-15(n765ts) 0 0 3.0 32 ‡‡ 22 puf-8(zh17); lin-15(n765ts) 23 0 3.4 30 23 eff-1(hy21) 29 0 3.3 21 24 eff-1(hy21); gap-1(ga133) 40 0 3.4 20 *All the strains carrying the puf-8 mutations ga145, zh17 or ok302 carried the cis-linked marker unc-4(e120). % Muv indicates the fraction of animals with more than three induced VPCs. % Vul indicates the fraction of animals with less than three induced VPCs. Induction indicates the average number of induced VPCs per animal, puf-8(ga145) was scored under a dissection microscope. These strains were grown at 25°C. **lin-10(e1438) was cis-linked with unc-13(e1091). †† These strains had ark-1(sy247) cis-linked with dpy-20(e1282). ‡‡ These strains were kept at 14°C before scoring. each of these mutations, puf-8(zh17) caused a synthetic Muv PUF-8::GFP expression in an eff-1(hy21) background, in which no phenotype as described above for gap-1(ga133) (Table 1, row 6 and cell fusions occur (Mohler et al., 2002). Since eff-1(hy21) animals rows 17-22). Thus, puf-8 either encodes a negative regulator of the exhibit excess vulval induction (Table 1, row 23), we additionally EGFR/RAS/MAPK pathway, or alternatively, puf-8 regulates the ablated the somatic gonad precursors Z1 and Z4 to prevent induction competence of the VPCs to respond to the inductive signal. by the anchor cell. In most gonad-ablated eff-1(hy21) animals, PUF- 8::GFP expression was upregulated in all VPCs and their PUF-8::GFP is expressed in vulval cells and the descendants, except for the P8.p descendants (Fig. 2K,L and Fig. surrounding epidermis S1B in the supplementary material). Moreover, in let-60 ras(gf) To analyze the expression pattern of PUF-8, we constructed a animals, in which the distal VPCs frequently adopt the 1° or 2° translational puf-8::gfp reporter by fusing a genomic DNA fragment induced cell fates, PUF-8::GFP expression often remained low covering 1.3 kb of 5 regulatory sequences up to the next gene and in the distal VPCs and their descendants (Fig. S1C in the the entire puf-8 coding sequence to a GFP cassette (Fig. 2A). PUF- supplementary material) (Beitel et al., 1990). We conclude that PUF- 8::GFP was expressed in various tissues including the pharyngeal 8::GFP is upregulated in the descendants of VPCs that have adopted muscles, the hypodermis, the ventral cord motor neurons (not the uninduced 3° cell fate independently of their fusion with hyp7. shown) and the vulval cells (Fig. 2B-J and Fig. S1A in the supplementary material). Before vulval induction in L2 larvae, PUF- fbf-1 and fbf-2 negatively regulate vulval 8::GFP was expressed in all six vulval precursor cells at equal levels development (Fig. 2B,C and row with Pn.p cells in Fig. S1A in the supplementary To examine whether additional C. elegans PUF proteins besides material). After vulval induction in early L3 larvae, PUF-8::GFP PUF-8 play a role in regulating vulval development, we performed was upregulated in the descendants of the 3° distal VPCs (P3.p, P4.p an RNA interference (RNAi) analysis by feeding rrf-3(pk1426); gap- and P8.p), while expression faded in the 1° and 2° descendants of 1(ga133) animals with dsRNA-producing bacteria derived from the the proximal VPCs (P5.p, P6.p and P7.p, Fig. 2D-J, Fig. S1A in the other puf genes (Kamath and Ahringer, 2003). The rrf-3(pk1426) supplementary material, rows Pn.px to Pn.pxxx). In addition, PUF- mutation was used to increase the sensitivity for RNAi (Simmer et 8::GFP expression was detected in the VulC sublineage of the 2° al., 2002). Of the six other PUF proteins that were tested, RNAi cells at the Pn.pxxx stage (inset in Fig. 2H,J and Fig. S1A in the against fbf-1 and fbf-2 induced a penetrant Muv phenotype, whereas supplementary material). RNAi against puf-9, which is most similar to puf-8, did not cause a We hypothesized that the increase in PUF-8::GFP expression in Muv phenotype (Table 2, rows 1-8). Because of the high degree of the descendants of the distal 3° VPCs might occur because these sequence similarity between the two fbf genes (over 90% identity at cells fuse with the hyp7 hypodermis that also expresses PUF-8::GFP. the nucleotide level), RNAi against either fbf gene most likely reduces To test if the upregulation of PUF-8::GFP in the descendants of the both fbf-1 and fbf-2 expression. We therefore tested whether fbf-1 or 3° VPCs is a consequence of their fusion with hyp7, we examined fbf-2 single mutants or only the fbf-1 fbf-2 double mutant show a Muv DEVELOPMENT C. elegans PUF proteins in vulval development RESEARCH ARTICLE 3465 Table 2. fbf-1 and fbf-2 redundantly regulate vulval induction Row Genotype RNAi % Muv* n Induction n 1 gap-1(ga133) gfp 0 50 3.0 40 2 gap-1(ga133) fbf-1 85 110 4.1 21 3 gap-1(ga133) fbf-2 56 100 3.6 21 4 gap-1(ga133) puf-3 031 –– 5 gap-1(ga133) puf-5 332 –– 6 gap-1(ga133) puf-7 041 –– 7 gap-1(ga133) puf-8 79 104 3.9 24 8 gap-1(ga133) puf-9 040 –– 9 fbf-1(ok91) – 0 200 3.0 27 10 fbf-1(ok91); gap-1(ga133) – 1 157 3.0 55 11 fbf-2(q738) – 0 80 3.0 25 12 fbf-2(q738); gap-1(ga133) – 0 63 3.0 24 13 fbf-1(ok91) fbf-2(q704) – 28 74 3.6 26 14 fbf-1(ok91) fbf-2(q704); gap-1(ga133) – 94 282 4.9 282 15 fbf-1(ok91) fbf-2(q704); gap-1(ga133) gfp 94 154 4.8 36 16 fbf-1(ok91) fbf-2(q704); gap-1(ga133) gld-1 19 168 3.3 29 17 fbf-1(ok91) fbf-2(q704); gap-1(ga133) fem-3 91 80 –– 18 puf-8(zh17); gap-1(ga133) gfp 76 162 3.6 27 § ¶ 19 puf-8(zh17); gap-1(ga133) gld-1 82 232 4.0 22 § ¶ 20 puf-8(zh17); gap-1(ga133) fem-3 69 108 –– *% Muv indicates the fraction of animals showing ectopic vulval induction under a dissection microscope. Induction indicates the average number of induced VPCs per animal. These strains carried the rrf-3(pk1426) mutation, which made them more sensitive to RNAi (Simmer et al., 2002). These strains were cis-linked with unc-4(e120). RNAi against fem-3 and gld-1 was additionally checked for presence of the germline phenotype. phenotype when combined with gap-1(ga133). fbf-1(ok91); gap- expression in the proximal VPC descendants compared to gap- 1(ga133) and fbf-2(q738); gap-1(ga133) animals both developed a 1(ga133) single mutants (Fig. 3J,K). Moreover, the descendants of wild-type vulva, but fbf-1(ok91) fbf-2(q704); gap-1(ga133) triple P5.p and P7.p adopted a proper 2° cell fate, as they generated seven mutants showed a strong Muv phenotype (Fig. 1D,E and Table 2 descendants that exhibited a normal morphology and a normal EGL- rows 9-14). Interestingly, even in a gap-1(+) background fbf-1(ok91) 17::YFP expression pattern in the VulC and VulD subfates (compare fbf-2(q704) double mutants were weakly Muv (Table 2, row 13). Fig. 3G with L). In the distal cells (the P3.p, P4.p and P8.p Finally, we tested for a possible redundancy among the puf genes by descendants) we observed only a very mild increase in the early, 1°- performing puf-3, puf-5, puf-7, puf-8 and puf-9 RNAi in the puf- specific or the late, 2°-specific EGL-17::YFP expression that did not 8(zh17) and fbf-1(ok91) fbf-2(q704) backgrounds, but observed no match the frequency of ectopic vulval induction observed in this synthetic Muv phenotypes among the other PUF genes (data not background (Fig. 3J-M). However, it should be noted that also in shown). Thus, besides puf-8 the two fbf genes encode functionally other mutant backgrounds such as let-60(n1046gf) the frequency and redundant negative regulators of vulval development. strength of ectopic EGL-17::YFP expression does not mirror the level of ectopic vulval induction (Burdine et al., 1998). fbf-1 and fbf-2 inhibit specification of the 1° In contrast to puf-8 mutants, fbf-1(ok91) fbf-2(q704); gap- vulval cell fate 1(ga133) triple mutants displayed a clear upregulation of the early, We next determined whether PUF-8 or the FBF proteins regulate the 1°-specific EGL-17::YFP expression in all VPCs and their specification of the 1° vulval cell fate using the egl-17::yfp reporter descendants (Fig. 3N,O). Especially in the descendants of P5.p and as a marker for the 1° cell fate (Inoue et al., 2002). egl-17 encodes a P7.p, the 1°-specific EGL-17::YFP expression was much stronger fibroblast growth factor (FGF) homolog that is normally expressed than in gap-1(ga133) single mutants. In addition to the late EGL- in P6.p and its descendants from the time of induction until the 17::YFP expression in the ectopically induced pseudovulvae, fbf- Pn.pxx stage (Fig. 3A,B) (Burdine et al., 1998; Inoue et al., 2002). 1(ok91) fbf-2(q704); gap-1(ga133) mutants also exhibited an In L4 larvae at the Pn.pxxx stage, EGL-17::YFP expression expansion of the 2°-specific EGL-17::YFP expression to 2° subfates disappears in the 1° cells and appears in the VulC and VulD cells of that normally do not express the marker (e.g. VulA and VulB in Fig. the 2° lineage (Fig. 3C,D) (Burdine et al., 1998; Inoue et al., 2002). 3P,Q). This aberrant EGL-17::YFP expression pattern within the 2° Both the early (1° fate-specific) and late (2° subfate-specific) EGL- lineage was accompanied by morphological changes of the P5.p and 17::YFP expression depend on inductive signalling (Burdine et al., P7.p descendants that are characteristic of a partial transformation 1998). towards the 1° fate (note the detachment of the P5.p descendants in We observed a slight expansion of the early, 1°-specific EGL- Fig. 1E and Fig. 3P) (Berset et al., 2005). Such defects in the 2° cell 17::YFP expression in gap-1(ga133) animals causing the lineage were only rarely observed in puf-8(zh17); gap-1(ga133) descendants of P5.p and P7.p and occasionally also of P8.p to animals (Fig. 3M). express EGL-17::YFP (Fig. 3E,F), although, gap-1(ga133) mutants Thus, PUF-8 and the FBF proteins perform clearly distinct roles exhibit normal vulval induction and correct 2° cell fate specification during vulval cell fate specification. FBF-1 and FBF-2 inhibit 1° in P5.p and P7.p (Fig. 3G,H). fate-specific gene expression and are required for proper 2° fate Surprisingly, in puf-8(zh17); gap-1(ga133) double mutants or puf- execution in P5.p and P7.p, whereas PUF-8 does not regulate 1°- 8 RNAi-treated gap-1(ga133) animals we observed no increase – specific gene expression and appears to regulate vulval induction and sometimes even a reduction – in the 1°-specific EGL-17::YFP through a different mechanism. DEVELOPMENT 3466 RESEARCH ARTICLE Development 133 (17) Fig. 2. PUF-8::GFP and FBF-2::GFP expression during vulval development. (A) Structure of the translational puf-8::gfp and fbf-2::gfp reporters. (B,D,F,H) Time-course analysis of PUF-8::GFP expression in the vulval cells from the L2 until the L4 stage with (C,E,G,J) the corresponding Nomarski images. For a semi-quantitative analysis of the expression patterns, see Fig. S1 in the supplementary material. (K,L) PUF-8::GFP expression in gonad- ablated eff-1(hy21) animals, and the corresponding Nomarski image. Note that despite the extra round of cell divisions in P4.p and P5.p descendants of gonad-ablated eff-1 mutants no vulval differentiation was observed. (M-R) FBF-2::GFP expression, and the corresponding Nomarski images, from the early L3 until the L4 stage. In all panels, anterior is to the left and ventral is to the bottom. Scale bars: in C,L,N and in the inset of J, 10 m. gld-1 is an FBF target during vulval development fate is only sealed after the Pn.px cells have fused with hyp7 (Wang Since PUF proteins function as translational repressors, the Muv and Sternberg, 1999), the puf-8(lf) mutations might allow distal phenotype caused by puf-8 and fbf-1 and fbf-2 mutations is probably vulval cells to stay unfused and hence receive the inductive signal caused by enhanced translation of their target mRNAs. Thus, RNAi over a longer time period, which in combination with a second against a target mRNA that encodes a positive regulator of vulval mutation in a negative regulator of the EGFR/RAS/MAPK pathway development should suppress the Muv phenotype of puf-8(zh17); would result in excess vulval induction. gap-1(ga133) and/or fbf-1(ok91) fbf-2(q704); gap-1(ga133) To observe the timing of vulval cell fusions, we used the ajm-1::gfp mutants. In the germline, gld-1 and fem-3 are direct FBF targets that reporter, which labels the adherens junctions of the VPCs and their function in mitosis/meiosis and sperm/oocyte decision, respectively descendants as long as they have not fused with hyp7 (Mohler et al., (Crittenden et al., 2002; Zhang et al., 1997). No targets of PUF-8 1998). In wild-type animals, the uninduced distal VPCs divide once have so far been found. RNAi against gld-1 suppressed the fbf- and then rapidly fuse with hyp7. Therefore, in the majority of wild- 1(ok91) fbf-2(q704); gap-1(ga133) but not the puf-8(zh17); gap- type larvae we analyzed at the Pn.px stage, the descendants of P3.p, 1(ga133) Muv phenotype, whereas RNAi against fem-3 had no P4.p and P8.p had already fused with hyp7 as demonstrated by the loss effect on the Muv phenotype of either strain (Table 2, rows 15-20). of AJM-1::GFP staining (Fig. 4A-C). In puf-8(zh17) mutants, Thus, the FBF proteins negatively regulate vulval induction by however, the fusion of P4.p and P8.p descendants was significantly repressing, among others, gld-1 expression. PUF-8, however, delayed, as in approximately 50% of the animals AJM-1::GFP appears to act through a distinct set of yet unknown target genes. staining was still present in P4.px and P8.px (Fig. 4D-F). Note that despite the delay in cell fusion puf-8(zh17) single mutants never puf-8 controls the timing of 3° cell fusions showed ectopic induction of the distal VPCs (Table 1, row 4). In fbf- The upregulation of PUF-8::GFP in the distal 3° vulval cells raises 1(ok91) fbf-2(q704) mutants, P4.p and P8.p descendants were unfused the possibility that PUF-8 might regulate the competence of the in approximately 20% of the cases (Fig. 4G-J). Since 28% of fbf- distal vulval cells to respond to the inductive signal. Since the 3° cell 1(ok91) fbf-2(q704) double mutants exhibit a Muv phenotype in a DEVELOPMENT C. elegans PUF proteins in vulval development RESEARCH ARTICLE 3467 Fig. 3. fbf-1 and fbf-2 inhibit 1° cell fate specification. Analysis of EGL-17::YFP expression in mid-L3 larvae at the Pn.px or Pn.pxx stage (left side) and in L4 larvae at the Pn.pxxx stage (right side). (A-D) Wild-type, (E-H) gap-1(ga133), (J-M) gap-1(ga133); puf-8 RNAi and (N-Q) fbf-1(ok91) fbf- 2(q704); gap-1(ga133) larvae. In all panels, anterior is to the left and ventral is to the bottom. In the graphs, white indicates no EGL-17::YFP expression, grey low expression and black high expression. The arrows in L and P indicate ectopic induction of distal vulval cells; the arrowhead in P indicates an example with expanded EGL-17::YFP expression in VulA and VulB, and the resulting defect in the 2° fate execution. Scale bars: in A,C, 10 m. gap-1(+) background (Table 2, row 13), the distal cells were probably row 3 and Fig. S2B in the supplementary material), and ablation of the unfused because they had adopted a 1° or 2° vulval cell fate in these somatic gonad precursors Z1 and Z4, which give raise to the AC, animals. PUF-8 therefore inhibits vulval development by promoting resulted in a suppression of the Muv phenotype to nearly wild-type the fusion of the 3° cells with the surrounding hyp7 hypodermis. levels of vulval induction (Table 3, row 4 and Fig. S2C in the Similar to puf-8(lf), a mutation in the effector of cell fusion eff- supplementary material). Even after ablation of all four gonad 1 that blocks all cell fusions causes a weak Muv phenotype that precursor cells (Z1 to Z4), we observed gonad-independent vulval was further enhanced by the gap-1(ga133) background (Table 1, induction in 19% of the animals (Table 3, row 5 and Fig. S2D in the rows 23 and 24) (Mohler et al., 2002). However, it should be noted supplementary material). Since the gap-1(ga133) mutation alone does that eff-1(hy21); gap-1(ga133) double mutants display a weaker not cause any gonad-independent vulval induction (Hajnal et al., Muv phenotype than puf-8(zh17); gap-1(ga133) animals (Table 1, 1997), fbf-1 and fbf-2 inhibit vulval differentiation not only by compare rows 6 and 24), indicating that puf-8 is likely to have repressing specific target genes in the germ cells but also in somatic additional functions besides controlling the timing of 3° cell cells outside of the gonad. Supporting this hypothesis, a translational fusions. FBF-2::GFP reporter showed an expression pattern similar to the PUF-8::GFP pattern described above. Expression of FBF-2::GFP was fbf-1 and fbf-2 act in the germline and in the soma first observed at the Pn.px stage in the 3° descendants of the distal Thompson et al. (Thompson et al., 2006) recently reported that VPCs, and it persisted throughout the L4 stage (Fig. 2A,M-R and Fig. feminized fbf-1 fbf-2 mutants (i.e. fbf-1 fbf-2; fog-1 or fbf-1 fbf-2; fog- S1D in the supplementary material). 3 triple mutants) display a strong Muv phenotype that is completely suppressed by ablation of the germ cell precursors Z2 and Z3. This puf-8, fbf-1 and fbf-2 act in the vulval cells observation indicated that fbf-1 and fbf-2 inhibit vulval induction in a We next sought to identify the somatic tissue in which puf-8 and fbf- non cell-autonomous manner, probably by repressing the translation 1 and fbf-2 act. Since puf-8::gfp and fbf-2::gfp are both expressed of a positive regulator of vulval development in the germ cells. We in the vulval cells as well as in the hyp7 hypodermis, we tested performed similar gonad precursor cell ablations, but used the fbf- whether puf-8, fbf-1 and fbf-2 act cell-autonomously in the VPCs 1(ok91) fbf-2(q704); gap-1(ga133) background. Ablation of Z2 and and their descendants or non cell-autonomously in hyp7. To Z3 resulted in a partial suppression of the Muv phenotype (Table 3, distinguish between these two possibilities, we expressed puf-8 and DEVELOPMENT 3468 RESEARCH ARTICLE Development 133 (17) Fig. 4. puf-8 regulates the fusion of the distal vulval cells. Vulval cell fusion was analyzed at the Pn.px stage using AJM-1::GFP as a cell junction marker for unfused cells. (A-C) Wild-type, (D-F) puf-8(zh17) single mutants and (G-J) fbf-1(ok91) fbf-2(q704) double mutants. In all panels, anterior is to the left and ventral is to the bottom. In the graphs, white represents fused Pn.px cells, grey indicates fusing Pn.px cells that have started to dissolve their junctions as can be seen for the P8.px cells in G, and black indicates unfused cells with intact AJM-1::GFP-positive junctions. Note that the fraction of unfused cells in fbf-1(ok91) fbf-2(q704) double mutants matches the frequency of ectopically induced distal cells that give rise to the 28% penetrant Muv phenotype (see Table 2, row 13). Scale bar in B: 10 m. fbf-2 under the control of the hypodermal dpy-7 promoter (e.g. well as puf-8 have an additional focus in the germline, since the P ::puf-8) (Gilleard et al., 1997), and each of the three genes multicopy extrachromosomal arrays we used for these experiments dpy-7 under control of a 3.1 kb bar-1 promoter fragment that drives are normally silenced in the germ cells. Thus, puf-8, fbf-1 and fbf-2 expression in the vulval cells, the gonadal sheath cells and in the negatively regulate vulval development at least partly in the VPCs adult seam cells (e.g. P ::puf-8) (Natarajan et al., 2004). Neither or their descendants. bar-1 the sheath cells nor the seam cells are in contact with the vulval cells, making it very unlikely that expression of a gene in these DISCUSSION tissues could affect vulval induction. None of the three P ::puf- PUF proteins control somatic development dpy-7 8 transgenes tested caused a significant rescue of puf-8(ok302); Translational repressors of the Pumilio/FBF (PUF) family regulate gap-1(ga133) Muv phenotype, but two out of three P ::puf-8 various aspects of germ cell development in C. elegans by bar-1 lines exhibited partial rescue, and the third line showed a weak controlling the translation of maternally provided mRNAs reduction of the Muv phenotype (Table 4, rows 5-11). It should be (Crittenden et al., 2002; Zhang et al., 1997). Here, we show that noted that even injection of a cosmid spanning the entire puf-8 locus three of the eleven C. elegans PUF genes also function in the soma never gave complete rescue of the Muv phenotype (Table 4, rows to control cell fate specifications during larval development. In 1-4). Moreover, co-injection of P ::puf-8 with P ::puf-8 did particular, we have found that PUF-8, FBF-1 and FBF-2 negatively bar-1 dpy-7 not cause a stronger rescue than injection of P ::puf-8 alone regulate vulval development in the hermaphrodite. Like most bar-1 (data not shown). previously identified inhibitors of vulval development, single Similarly, all but one of the P ::fbf-1 and P ::fbf-2 mutants in one of these three puf genes do not change the normal bar-1 bar-1 transgenes reduced the penetrance of the fbf-1(ok91) fbf-2(q704); pattern of vulval cell fates. However, when combined with another gap-1(ga133) Muv phenotype from 90% down to 55-60%, and only mutation in an inhibitor of the inductive EGFR/RAS/MAPK one of the three P ::fbf-2 transgenes had a slightly significant pathway, puf-8 or fbf mutants exhibit a hyperinduced multivulva dpy-7 effect (Table 4, rows 12-21). The incomplete rescue with the phenotype. Genetic epistasis analysis indicates that fbf-1 and fbf-2 different constructs is consistent with the model that fbf-1, fbf-2 as perform a redundant function to inhibit 1° vulval fate specification, DEVELOPMENT C. elegans PUF proteins in vulval development RESEARCH ARTICLE 3469 Table 3. fbf-1 and fbf-2 act in the soma and the germline † ‡ Row Genotype Ablation % Muv* % Vul Induction n 1 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Unablated 84 0 4.1 48 2 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Mock ablated 74 0 4.0 31 3 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Z2/Z3 (germ line) 27 0 3.3 22 4 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Z1/Z4 (somatic gonad) 8 8 3.0 12 5 fbf-1(ok91) fbf-2(q704); gap-1(ga133) Z1-Z4 (somatic gonad + germline) 0 81 0.6 21 *% Muv indicates the fraction of animals with more than three induced VPCs. % Vul indicates the fraction of animals with less than three induced VPCs. Induction indicates the average number of induced VPCs per animal. whereas puf-8 plays a distinct role in regulating the temporal 8 limits the time period during which the vulval cells can receive and competence of the vulval cells to respond to the inductive and lateral integrate the vulval patterning signals. In the absence of PUF-8, the signals. vulval cells can receive the inductive signal over a longer time period, which may result in the accumulation of higher levels of PUF-8 regulates the temporal competence of the activated MAPK in the distal vulval cells. When combined with a vulval cells mutation in a direct inhibitor of the EGFR/RAS/MAPK pathway Loss-of-function mutations in puf-8 partially suppress the Vul such as gap-1, this results in the ectopic vulval differentiation and a phenotype caused by mutations that reduce but do not inactivate the Muv phenotype. Supporting this idea, a mutation in the effector of EGFR/RAS/MAPK signalling pathway. Although this observation cell fusion eff-1, which blocks all cell fusions, caused a weak Muv does not prove a direct involvement of PUF-8 in regulating the phenotype (Mohler et al., 2002). However, puf-8 mutants exhibit inductive EGFR/RAS/MAPK signalling pathway, it indicates that more ectopic vulval induction in the gap-1 background than eff-1 in the absence of PUF-8 lower levels of inductive signal are mutants, which points to additional functions of PUF-8 besides sufficient to induce vulval differentiation. A PUF-8::GFP reporter controlling the timing of cell fusions. transgene is initially expressed in all VPCs at equal levels, but after The distal VPC descendants fuse with hyp7 shortly after they vulval induction PUF-8::GFP expression increases in the have been born, suggesting that they exit from the cell cycle as descendants of the distal VPCs (P3.p, P4.p and P8.p) that have they lose their competence (Wang and Sternberg, 1999). The adopted the 3° fate. This expression pattern correlates well with the proximal vulval cells, on the other hand, go on to divide two more observed delay in the fusion of the distal 3° cells with the hyp7 times before undergoing terminal differentiation and forming a hypodermis in puf-8 mutants. All vulval cells are competent to functional vulva. It is therefore possible that PUF-8 ensures that respond to the inductive AC and lateral Notch signals until they fuse the distal vulval cells exit from the cell cycle immediately after with hyp7 (Wang and Sternberg, 1999). Even after the first round of they have been generated and then fuse with hyp7. A somewhat vulval cell divisions, a single pulse of MAPK activity can reprogram similar function has been proposed for the Drosophila PUF-8 a 2° or 3° cell to adopt the 1° cell fate (Berset et al., 2005). It thus orthologue Pumilio, which blocks the cell cycle progression of the appears that by promoting the fusion of the 3° cells with hyp7, PUF- migrating pole cells during embryogenesis by repressing cyclin B Table 4. puf-8, fbf-1 and fbf-2 act in part in the vulval cells † 2 ‡ Row Genotype Transgene % Muv* Induction  -test n 1 puf-8(ga145); gap-1(ga133) – 85±3 – 477 2 puf-8(ga145); gap-1(ga133) Cosmid C30G12 line 1 57±10 – x 104 3 puf-8(ga145); gap-1(ga133) Cosmid C30G12 line 2 24±8 – x 116 4 puf-8(ga145); gap-1(ga133) Cosmid C30G12 line 3 35±9 – x 121 5 puf-8(ok302); gap-1(ga133) – 68±6 4.0 260 6 puf-8(ok302); gap-1(ga133) zhEx173.1 [P ::puf-8] 30±13 3.3 x 46 bar-1 7 puf-8(ok302); gap-1(ga133) zhEx173.2 [P ::puf-8] 38±19 3.5 y 26 bar-1 8 puf-8(ok302); gap-1(ga133) zhEx173.3 [P ::puf-8] 52±20 3.7 23 bar-1 9 puf-8(ok302); gap-1(ga133) zhEx170.1 [P ::puf-8] 71±18 4.2 24 dpy-7 10 puf-8(ok302); gap-1(ga133) zhEx172.1 [P ::puf-8] 77±12 4.0 51 dpy-7 11 puf-8(ok302); gap-1(ga133) zhEx172.2 [P ::puf-8] 63±18 3.9 27 dpy-7 12 fbf-1(ok91) fbf-2(q704); gap-1(ga133) – 96±2 4.9 441 13 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx175.1 [P 1::fbf-1] 57±18 3.9 x 30 bar- 14 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx175.2 [P ::fbf-1] 58±20 3.8 x 24 bar-1 15 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx175.3 [P ::fbf-1] 73±16 4.1 x 30 bar-1 16 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx174.1 [P ::fbf-2] 60±21 3.8 20 bar-1 17 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx174.2 [P ::fbf-2] 59±19 3.8 x 27 bar-1 18 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx174.3 [P ::fbf-2] 52±18 3.6 x 29 bar-1 19 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx176.1 [P ::fbf-2] 81±14 4.5 32 dpy-7 20 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx176.2 [P ::fbf-2] 83±12 4.3 y 35 dpy-7 21 fbf-1(ok91) fbf-2(q704); gap-1(ga133) zhEx176.3 [P ::fbf-2] 94±8 4.7 35 dpy-7 Rows 1, 5 and 12 show the average of animals without the transgene that were counted for each genotype in parallel. *% Muv indicates the fraction of animals with more than three induced VPCs, and the 95% confidence intervals are indicated. Induction indicates the average number of induced VPCs per animal. ‡ 2 For each line the  test was performed comparing the animals with and without the array from the same plate. x indicates a P value <0.01 and y indicates a P value <0.05. puf-8(ga145) and puf-8(ok302) were cis-linked with unc-4(e120). These strains were maintained balanced with mIn1 and their homozygous fbf-1 fbf-2 double mutant F1 progeny was scored. DEVELOPMENT 3470 RESEARCH ARTICLE Development 133 (17) translation to prevent their premature differentiation (Asaoka- Supplementary material Supplementary material for this article is available at Taguchi et al., 1999). One could, for example, imagine that the http://dev.biologists.org/cgi/content/full/133/17/3461/DC1 cell cycle state of the vulval cells and the hyp7 hypodermis needs References to be coordinated to allow the fusion between these two different Ahringer, J. and Kimble, J. (1991). 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