Genetic Analysis of the Caenorhabditis Elegans GLH Family of P-Granule Proteins

Genetic Analysis of the Caenorhabditis elegans GLH Family of P-Granule Proteins

Caroline Spike,*,1,2 Nicole Meyer,*,1 Erica Racen,† April Orsborn,†,3 Jay Kirchner,*,4 Kathleen Kuznicki,†,5 Christopher Yee,†,6 Karen Bennett† and Susan Strome*,7 *Department of Biology, Indiana University, Bloomington, Indiana 47405 and †Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 65212 Manuscript received October 17, 2007 Accepted for publication February 1, 2008

ABSTRACT The Vasa DEAD-box helicases are widespread markers of germ cells across species, and in some organisms have been shown to be essential for germ-cell formation and development. In contrast to the single Vasa gene in most systems analyzed, Caenorhabditis elegans has four Vasa family members, the germline helicases GLH-1, GLH-2, GLH-3, and GLH-4. Our analysis of deletion alleles of each glh gene demonstrates that GLH-1 is the key member of the family: loss of GLH-1 function causes sterility that is mainly maternal effect, is manifested predominantly at elevated temperature, and is due to reduced germ- cell proliferation and impaired formation of both sperm and oocytes. The other GLHs are not essential. However, GLH-4 serves redundant roles with GLH-1: loss of both genes’ function causes glh-1-like sterility at all temperatures. Molecular epistasis analysis demonstrates that GLH-1 and GLH-4 are required for proper association of the PGL family of proteins with P granules, suggesting a pathway of P-granule assembly in which the GLHs are upstream of the PGL proteins and the mRNA cap-binding protein IFE-1. While loss of some P-granule components causes worms to be defective in RNA interference, loss of GLH-1 and GLH-4 does not compromise RNAi. Thus, RNAi likely does not require intact P granules but instead relies on particular P-granule factors. We discuss the evolution of the Vasa/GLH genes and current views of their functions and the assembly and roles of germ granules among species.

OW germ cells are distinguished from somatic cells loaded into the embryo and segregated to the primor- H is a critical issue in developmental biology (e.g., dial germ cells (PGCs) during the initial embryonic see Strome and Lehmann 2007). One unique feature of divisions, and PGC formation and/or function relies on germ cells is their possession of specialized cytoplasm germ-plasm components (reviewed in references called germ plasm, which contains distinctive non- above). In mammals, PGC formation does not depend membrane-bound organelles generically called germ on segregated germ plasm; instead PGCs are formed by a granules (reviewed in Wylie 2000; Santos and Lehmann different mechanism, induction, at a later stage of 2004; Zhou and King 2004; Strome 2005; Seydoux and embryonic development (reviewed in Saffman and Braun 2006). Germ granules are widespread in nature. Lasko 1999; Hayashi et al. 2007). Nevertheless, In several model systems, including Caenorhabditis, mammalian embryos contain germ-granule compo- Drosophila, and Xenopus, germ granules are maternally nents, which display specific protein–protein interactions and subcellular localization in nascent germ cells, suggesting that even mammalian PGCs may rely on assembled germ plasm for proper development (Fox 1These authors contributed equally to this work. et al. 2007). 2Present address: Department of Genetics, Cell Biology and Develop- The composition and functions of germ granules are ment, University of Minnesota, Minneapolis, MN 55455. being actively investigated. Perhaps surprisingly, many 3Present address: Molecular Biology Department, University of California, germ-granule components appear to be species specific; Berkeley, CA 94720. for example, PGL-1 is unique to worm germ granules 4Present address: Biology Department, University of the Cumberlands, awasaki Williamsburg, KY 40769. (K et al. 1998), while Oskar is unique to flies phrussi phrussi ehmann 5Present address: Duquesne University School of Law, Pittsburgh, PA (E et al. 1991; E and L 1992). 15282. The best-studied germ-granule component that is 6Present address: Department of Internal Medicine, Washington Uni- shared among species is Vasa and its homologs. First versity School of Medicine, St. Louis, MO 63100. discovered in Drosophila, Vasa is a DEAD (Asp-Glu-Ala- 7Corresponding author: MCD Biology, Sinsheimer 328, University of California, 1156 High St., Santa Cruz, CA 95064. Asp) family RNA helicase that has an RGG (Arg-Gly-Gly)- E-mail: [email protected] rich domain (Hay et al. 1988a,b; Lasko and Ashburner

Genetics 178: 1973–1987 (April 2008) 1974 C. Spike et al.

1988). Vasa is maternally supplied and required to Deletion alleles of glh-2, glh-3, and glh-4 do not cause assemble germ plasm at the posterior pole of the fly dramatic phenotypes on their own, but a glh-4 mutation oocyte; embryos lacking germ plasm fail to bud PGCs strongly enhances the glh-1 mutant phenotype, by abol- from the posterior end of the embryo and develop into ishing the sensitivity to temperature. Several mutant sterile adults (Lasko and Ashburner 1988; Hay et al. forms of GLH-1 protein do not assemble into P gran- 1990; Lehmann 1992; Liang et al. 1994). Strong Vasa ules; probably as a result, the PGL proteins also show alleles result in defects in oocyte growth, patterning, and defects in their association with P granules. Although differentiation (Lasko and Ashburner 1988; Liang pgl-1 mutants are defective in RNAi, glh-1 mutants and et al. 1994). Among the targets of Vasa translational reg- glh-4 glh-1 double mutants are not, revealing that dif- ulation are gurken, oskar, and nanos mRNA (Dahanukar ferent P-granule components serve different roles in and Wharton 1996; Markussen et al. 1997; Styhler RNAi. et al. 1998). Recent reports implicate Vasa and germ granules in microRNA control of gene expression in flies as well as in mice (Kotaja et al. 2006; Megosh et al. MATERIALS AND METHODS 2006). Strains: C. elegans strains were maintained as described in The reported Caenorhabditis elegans homologs of Vasa Brenner (1974). Strains used were wild-type variety Bristol are the four GLH germline helicases (Roussell and strain N2, LGI glh-1(bn103, bn125, gk100, ok439), glh-2(um2), Bennett 1993; Gruidl et al. 1996; Kuznicki et al. 2000). glh-3(um1), glh-4(gk225), dpy5(e61), unc-13(e1091), hDf8, the All four GLH proteins associate with C. elegans germ GFP-tagged balancer hT2[bli-4(e937) let-?(q782) qIs48], LGIII pgl-2(bn123), LGIV pgl-1(ct131, bn102), unc-24(e138), LGV pgl- granules or ‘‘P granules,’’named for their segregation to 3(bn104), and dpy-11(e224). Strains were usually kept at 20° as the germline P blastomeres P1,P2,P3, and P4. Like Vasa, the permissive temperature unless otherwise noted. each of the four GLHs contains a DEAD box helicase Isolation and sequencing of glh mutant alleles: glh- domain. Also like Vasa, GLH-1, GLH-2, and GLH-4 con- 1(gk100), glh-1(ok439), and glh-4(gk225) were isolated by the tain a Gly-rich domain. However, while Vasa contains C. elegans Gene Knockout Consortium. Worm libraries that had been mutagenized with trimethylpsoralen and UV irradi- RGG repeats, these three GLHs contain FGG repeats. ation were screened by PCR for deletions in glh-1 (T21G5.3) or This repeat domain of the GLHs differs in overall glh-4 (T12F5.3) (http://celeganskoconsortium.omrf.org/). A charge from that in Vasa and may serve a different role; similar approach was used to isolate glh-2(um2) and glh-3(um1) RGG repeats are an established RNA-binding domain (this work and Kuznicki et al. 2000). Deletion mutants were (Kiledjian and Dreyfuss 1992; Zanotti et al. 2006), outcrossed 6–10 times before analysis. The Tc1 insertion allele glh-1(bn103) was isolated from an rde-3 (formerly called mut-2) whereas FG repeats are common to nuclear pore pro- mutator background. The rde-3/mut-2 mutation r459 was teins (Suntharalingam and Wente 2003). In fact, P subsequently shown to map ,1 cM from glh-1 (Chen et al. granules are perinuclearly localized during most of the 2005) and is present in glh-1(bn103). glh-1(bn125) was isolated worm’s life cycle (Strome and Wood 1982), and they in a screen of EMS-mutagenized worms for those with T pike overlie nuclear pores (Pitt et al. 2000). All four GLH diffuse GFP PGL-1 (as described in detail by S et al. 2008). Sequencing was performed using ABI Prism Big proteins additionally contain CCHC-type zinc-finger mo- Dye mix and an ABI 3700 Prism automated fluorescent tifs not shared by Vasa. Previous RNAi analysis (Kuznicki sequencer. et al. 2000) suggested that among the GLHs, GLH-1 and Generation of glh-1/deficiency and glh-1 trans-heterozygous GLH-2 are the most important GLH family members; worms: To generate glh-1(gk100)/hDf8 and glh-1(bn125)/hDf8, depletion of either caused sterility at elevated tempera- homozygous glh-1 males were crossed to dpy-5 unc-13/hDf8 hermaphrodites. Among F1 progeny, L4 stage non-Dpy non- ture. Concomitant RNAi depletion of GLH-1 and GLH-4 Unc worms were picked to individual plates at 25°, allowed to caused sterility even at lower temperature, suggesting lay progeny for 24 hr, and assayed by single-worm PCR for the that GLH-1 and GLH-4 function redundantly. RNAi presence of each glh-1 allele. Among F2 progeny lacking Dpys depletion of GLH-3 did not cause notable defects and and Uncs, worms were scored for sterility as described below. did not enhance the glh-1(RNAi) or glh-2(RNAi) sterile To generate glh-1(bn125)/glh-1(gk100) and glh-1(bn125)/glh- 1(ok439), glh-1(gk100)/hT2 or glh-1(ok439)/hT2 males were phenotype, suggesting that GLH-3 serves only minor crossed to glh-1(bn125) hermaphrodites. Among F1 progeny, roles in the germline. Because of the uncertain effec- L4-stage worms lacking the balancer (GFP) were picked to tiveness of RNAi depletion of gene product, the poten- individual plates, allowed to lay progeny for 24 hr at 25°, and tial for dsRNAs to cross-hybridize and reduce expression analyzed by PCR for the presence of the deletion allele. F2 of multiple glh genes, and the difficulty discriminating progeny were scored for sterility. Generation of a glh-4(gk225) glh-1(ok439) double mutant: between maternal-effect and zygotic sterility by RNAi, we glh-1(ok439)/hT2-GFP hermaphrodites were crossed with glh- set out to isolate and study bona fide mutations in the four 4(gk225) homozygous males. Non-GFP F1 hermaphrodites glh genes. were individually mated with GFP1 F1 males. One hundred This article describes detailed genetic analysis of glh twenty-five GFP1 F2 hermaphrodites were individually plated mutants. Two severe deletion alleles of glh-1 reveal that and tested by PCR. The progeny from two plates were verified for deletions in both glh-1 and glh-4 (which are 4.2 map units the sterile phenotype is mainly maternal effect, is very apart) and for the ability to produce non-GFP double sensitive to temperature, and results from defects in homozygotes. The double glh-4(gk225) glh-1(ok439) is main- germline proliferation and in formation of gametes. tained over the hT2-GFP balancer. GLHs and Germ Cells 1975

Analysis of sterility: L4-stage glh-1/hT2-GFP or glh-4 glh-1/ Secondary antibodies used were FITC, TRITC, and Alexa488- hT2-GFP hermaphrodites were picked to individual plates and conjugated goat anti-rabbit, anti-chicken, and anti-mouse at incubated at 16°,20°, 24.5°,or26°.F1 M1Z homozygous glh 1:250 (Jackson ImmunoResearch, West Grove, PA; Molecular adult hermaphrodite progeny were scored for sterility at each Probes). Images were collected as single sections or Z-series temperature. Worms that lacked embryos in their uterus were projections using a Perkin Elmer Ultraview LC130E spinning- visually scored as sterile. Worms that contained material in disc confocal microscope and either Ultraview software their uterus were transferred to individual plates, to assess (Perkin Elmer Life Sciences, Downers Grove, IL) or Meta- whether they produced viable offspring. Fertile F1 M1Z Morph 7 software (Molecular Devices, Sunnyvale, CA). Images hermaphrodites were picked to new plates and placed back at were processed using Photoshop 7.0 (Adobe Systems, Palo each temperature to lay progeny. F2 MZ hermaphrodites Alto, CA) or ImageJ 1.38x (Wayne Rasband, NIH, http:// were scored for sterility at each temperature as described for rsb.info.nih.gov/ij/). the F1 worms. Germ-cell counts and analysis of gametogenesis: glh-1 MZ and glh-4 glh-1 M1Z animals were shifted to 26° RESULTS and their progeny fixed and stained with the DNA dye Hoechst Molecular characterization of glh-1 mutant alleles: (Shim et al. 2002) as adults, 24 hr after they were identified as midstage L4s. All germlines were scored for the presence of Previous RNAi depletion of each of the four GLH pro- sperm and oocyte nuclei; animals used for precise germ-cell teins suggested a hierarchy of GLH function, with GLH-1 counts are a subset of the total and were selected at random. being most important for germline development All identifiable germ cells other than sperm were counted. (Kuznicki et al. 2000). To enable genetic analysis of DAPI staining of dissected germlines: glh/hT2-GFP mothers the roles of GLH-1, we isolated mutant alleles (Figure 1) were shifted from 20° to 26°, their homozygous glh/glh offspring worms (M1Z) picked to individual plates at 26°, by a combination of approaches. The C. elegans Gene and their MZ hermaphrodite progeny analyzed as young Knockout Consortium isolated two deletion alleles, adults, 1 day beyond the L4 stage. Worms were picked into gk100 and ok439, by performing gene-specific PCR on 10 ml of M9 containing 5 mm levamisole on a slide and cut, and DNA from collections of UV-TMP-mutagenized worms. their gonads extruded using a 15-gauge needle. Samples were The Tc1 insertion allele, bn103, was obtained by overlaid with a coverslip, frozen on dry ice, fixed in formalde- screening through a collection of rde-3 (formerly called hyde fixation solution (3.73 ml 1 m K2HPO4, 1.47 ml 1 m mut-2) worms in which transposons had mobilized in KH2PO4, 10 ml 16% formaldehyde, 36.8 ml ddH2O) for 1 hr at room temperature, rinsed, fixed in methanol at 20° for the germline. The missense allele, bn125, was isolated in 5 min, washed, stained with 0.1 mg/ml DAPI, mounted in a screen of EMS-mutagenized worms (8500 haploid gelutol (Dupont, Wilmington, DE), and observed with a Zeiss genomes) for mutants that cause GFPTPGL-1 to lose its Axioplan microscope and a Spot CCD camera. P-granule association and be distributed diffusely in the RNAi analysis: The glh-1 RNAi construct was generated by pike cloning a 1.3-kb XmaI-NcoI genomic fragment from glh-1 into cytoplasm (S et al. 2008). Molecular analysis of the L4440 (Timmonsand Fire 1998). Other RNAi clones (Kamath genetic lesions and their effects on protein production et al. 2003) were purchased (Geneservice, Cambridge, UK). are shown in Figure 1. amath RNAi was essentially as described (K et al. 2001) but gk100: This allele deletes 582 nucleotides from intron used plates containing 0.2% lactose to induce dsRNA expres- 3 and exon 4 of the glh-1 gene. RT–PCR analysis of sion (E. Lambie, personal communication). Western blot analysis: Twenty-five glh-1(bn103), glh-1(bn125), mRNA products revealed that the remaining 15 nucleo- glh-1(gk100), glh-1(ok439), and wild-type adult hermaphrodites tides in intron 3 are not spliced out. The predicted were picked into SDS sample buffer containing protease inhib- protein product contains a 6-amino-acid insertion and itors (2 mm PMSF, 20 mg/ml pepstatin A, 20 mg/ml leupeptin, lacks 184 central amino acids (Val184–Ile367), which m and 5 m aprotinin), boiled, electrophoresed on a 10% SDS– comprise three CCHC zinc fingers and a Gly-rich PAGE gel, and transferred to nitrocellulose membrane. After blocking in 3% nonfat dry milk, 0.1% Triton X-100 in PBS, the segment. Indeed, by Western blot analysis, mutant membrane was incubated with rabbit anti-GLH-1 at 1:5000 and protein migrates at 62 kDa, consistent with its pre- with JLA20 mouse monoclonal anti-actin at 1:1000 as a loading dicted size of 585 amino acids. control. After washing, the membrane was incubated with ok439: In this allele, a 1594-nucleotide deletion re- horseradish peroxidase-conjugated goat anti-rabbit and goat moves the 39 segment of coding sequence, starting in anti-mouse each at 1:10,000. After washing, signal was detected using an enhanced chemiluminescence (ECL) kit (Amersham exon 4, and extends 39 of the gene. By RT–PCR, the Pharmacia Biotech, Piscataway, NJ). resulting mRNA would encode a protein product that Immunofluorescence microscopy: Cut worms and embryos lacks the last 240 amino acids of the protein (Pro524 were fixed and stained as described in Strome and Wood onward), and thus lacks much of the DEAD-box helicase (1983). Primary antibodies used were rabbit anti-GLH-1 at domain, and contains a novel 10-amino acid C-terminal 1:1000 (Gruidl et al. 1996; Kawasaki et al. 2004), chicken anti- GLH-2 at 1:100 (Gruidl et al. 1996), rabbit anti-GLH-3 at 1:50 extension. On Western blots, mutant protein migrates (Kuznicki et al. 2000), rabbit anti-GLH-4 at 1:50 (Kuznicki at 57 kDa, close to its predicted size of 533 amino acids. et al. 2000), rabbit anti-PGL-1 at 1:10,000 (Kawasaki et al. 1998), bn103: This allele is an insertion of a 1.6-kb Tc1 trans- rabbit anti-PGL-2 at 1:500 (Kawasaki et al. 2004), rat anti-PGL-3 poson after nucleotide 1808 in exon 4. RT–PCR analysis awasaki at 1:5000 (K et al. 2004), rabbit anti-histone H3 phos- revealed several different mRNA products, all of which phorylated on Ser10 at 1:200 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-hyperacetylated histone H4 at 1:500 lacked the transposon and would be predicted to pro- (Molecular Probes, Eugene, OR), and mouse PA3 anti-chromatin duce protein products with small deletions or insertions antibody at 1:500 (gift from M. Monestier; Monestier et al. 1994). in the middle (near Tyr554) of the DEAD-box helicase 1976 C. Spike et al.

Figure 1.—Mutant alleles of glh-1. (A) Schematic of the glh-1 gene, showing the positions and molecular lesions of the four mutant alleles. Coding regions are shown as boxes and introns as lines. (B) Graph of the GLH-1 polypeptide predicted to be produced by each mutant. Box with light shading is a Gly-rich region. Solid box is a CCHC zinc finger. Dark shading is a DEAD-box helicase domain. The arrows in gk100 and ok439 indicate the positions of missing amino acids. The ok439 lesion is predicted to fuse a novel 10-amino-acid stretch to the carboxy terminus of GLH-1. The asterisk in bn103 shows the position of the Tc1 insertion. bn125 removes the last 28 amino acids. (C) Western blot analysis of the GLH-1 polypeptide produced by each mutant. The blot was reacted with affinity-purified rabbit anti-GLH-1 and mouse anti-actin as a loading control (shown by an asterisk). Values beneath the lanes are the levels of GLH-1, normalized to the actin level and expressed relative to wild type.

domain. By Western blot analysis, this allele produces a glh-1 mutations cause temperature-sensitive sterility: reduced level (10% of wild type) of approximately All four mutations in glh-1 cause the same spectrum of normal-sized GLH-1 protein. phenotypes (Figure 2 and Table 1): temperature-sensi- bn125: This allele is a base-pair substitution that tive embryonic lethality and sterility that display a strong changes Gln736 to a premature stop codon. This would maternal effect. The mutations form an allelic series: delete 28 amino acids from the carboxyl terminus of the strongest–gk100, ok439, bn125–weakest. We did not protein. On Western blots, protein from this allele is include bn103 in this series and in fact discontinued reduced in level (50% of wild type) and migrates at a detailed analysis when sequencing revealed that the slightly smaller size than wild-type GLH-1. bn103-bearing chromosome contains a closely linked

Figure 2.—glh-1 mutations result in sterility that is sensitive to maternal genotype and temperature and is enhanced by a glh-4 mutation. (A) Analysis of zygotic (M1Z) and maternal-effect (MZ) sterility at different temperatures. The number of worms analyzed ranged from 10 to .200. (B) Graph showing the generation of M1Z and MZ worms. (C) Graphical display of sterility as a function of temperature for N2 (wild-type) and MZ glh-1, glh-4, and glh-4 glh-1 double mutants. glh-1 sterility is rela- tively low at 16°,20°, and 24.5° and high at 26°. glh- 4 glh-1 sterility is high at all temperatures. GLHs and Germ Cells 1977

TABLE 1 TABLE 2 Analysis of embryos and broods produced by fertile Phenotypic analysis of glh-1/hDf8 and glh-1 M1Z glh hermaphrodites at 26° heteroallelic combinations

Average % Average Maternal genotype % sterile progeny (n) Maternal embryonic brood genotype lethality (range) size (range) N2 0.5 (1216) glh-1(gk100) 100 (144) N2 0 168 (109–211) glh-1(ok439) 100 (216) glh-1(ok439) 17 (0–83) 48 (1–114) glh-1(bn125) 33 (601) glh-1(bn125) 15 (0–94) 49 (1–132) glh-1(gk100)/hDf8 100 (160) glh-4(gk225)a 8 (0–40) 45 (9–80) glh-1(bn125)/hDf8 63 (236) glh-4(gk225) glh-1(ok439) 53 (0–98) 21 (1–93) glh-1(bn125)/glh-1(gk100) 72 (253) glh-1(bn125)/glh-1(ok439) 57 (443) glh/balancer mothers were shifted from 20° to 26°, and 20 of their glh/glh offspring worms (M1Z) were picked for L4-stage M1Z hermaphrodites were picked to individual analysis. M1Z worms that produced live progeny were ana- plates at 25° and allowed to produce progeny for 24 hr. MZ lyzed for percentage of embryos laid that did not hatch (% progeny were scored as fertile or sterile by visual examination. embryonic lethality) and number of live progeny produced (brood size). gk100 was not included, as M1Z worms did not produce live progeny. a glh-4 worms are maintained as homozygotes. glh-4/glh-4 M1Z and MZ glh-1 mutants are fertile, whereas just mothers were shifted from 20° to 26°, and their glh-4/glh-4 off- 1.5° higher, the majority of M1Z gk100 and of MZ spring worms (MZ) were analyzed as described above. ok439 and bn125 are sterile (Figure 2, A and C). Twenty- five degrees, a typically used ‘‘restrictive temperature’’ (,1 cM) rde-3/mut-2 mutation (r459;Chen et al. 2005) for many temperature-sensitive mutations in C. elegans, that was present in the parental strain in which we yielded variable results (but mainly sterile worms) from screened for a transposon insertion into glh-1. The rde- experiment to experiment. The basis for the tempera- 3/mut-2 mutation on its own causes 100% sterility at 26° ture sensitivity of all glh-1 alleles is unknown but is (Q. Boese and J. Collins, personal communication; C. shared by mutations in another P-granule component, Spike, unpublished results), making it difficult to assess PGL-1 (Kawasaki et al. 1998, 2004; see discussion). the impact of bn103 on germline development. One At elevated temperature, even ‘‘fertile’’ glh-1 worms common feature of all glh-1 alleles is the variable severity have severely reduced fertility. A significant percentage of germline defects among worms and sometimes of their embryos fail to hatch and the number of viable between experiments. As discussed later, we think that offspring is small (Table 1). For example, at 26° fertile this is due to the sensitivity of glh-1 mutants to even slight M1Z ok439 hermaphrodites produced an average of variations in temperature and perhaps to the involve- 17% (range of 0–83%) dead embryos and an average of ment of GLH-1 in regulation of stochastic events. 48 (range of 1–114) viable progeny. Values for fertile Detailed phenotypic analysis is presented below. M1Z bn125 hermaphrodites were similar. For com- The glh-1 sterile phenotype shows a strong depen- parison, values for wild type were 0% dead embryos and dence on maternal genotype and on temperature. glh-1 168 (range of 109–211) viable progeny. These results mutants produced by heterozygous mothers inherit a suggest that 100% of M1Z glh-1 mutants have de- maternal load of glh-1(1) product but do not synthesize fective germlines at 26°. Many of those animals do not zygotic product (called M1Z, see Figure 2B). Such produce any viable progeny and hence are scored as M1Z hermaphrodites appear healthy but display sterile. The others (approximately half of the animals) temperature-sensitive sterility: 6–15% are sterile at 16°– produce some dead embryos and some viable embryos 20°, and 29–84% are sterile at 26° (Figure 2A). glh-1 that develop into sterile adults. mutants produced by homozygous mutant mothers, All of the glh-1 alleles produce some protein product which lack both maternal and zygotic glh-1(1) product (Figure 1C). To address whether their phenotypes (MZ), display a similar but more severe temperature- represent the null phenotype for the locus, we placed sensitive phenotype: 6–14% are sterile at 16°–20°, and two of the glh-1 alleles over a deficiency for the region 94–100% are sterile at 26° (Figure 2, A and C). Thus, loss (hDf8) and placed a weak allele over the two stronger of maternal and zygotic glh-1(1) function results in alleles and analyzed fertility/sterility among their prog- high-penetrance (100%) sterility at elevated temperature eny (MZ generation) at a borderline temperature and low-penetrance (10%) sterility at lower temper- (25°) (Table 2). gk100/hDf8 hermaphrodites, like atures. A maternal load of glh-1(1) product can rescue gk100/gk100 homozygotes, displayed 100% sterility. the fertility of a substantial fraction of ok439 and bn125 bn125/hDf8 hermaphrodites displayed higher sterility M1Z mutants even at elevated temperature. than bn125/bn125, consistent with bn125 being a weak The transition temperature for worms developing allele. bn125/gk100 and bn125/ok439 resembled bn125/ into fertile vs. sterile is 24°–25°. At 24.5°, the majority of hDf8 in displaying a higher percentage of sterility than 1978 C. Spike et al.

TABLE 3 Germline development in glh mutant hermaphrodites

Average no. of germ % nearly % gonad % gonad % worms nuclei per gonad empty arms lacking arms lacking lacking Maternal genotype Temperature arm (range)a gonad arms oocytesb spermb embryos N2 26° 787 (691–872) 0 2 6 0 glh-1(gk100) 26° 193 (0–407)c 38c 70 98 100 glh-1(ok439) 26° 287 (0–525)c 20c 43 68 86 glh-1(bn125) 26° NDd 14 43 33 82 glh-4(gk225) 26° NDd 0470 glh-4(gk225) glh-1(ok439) 26° 127 (0–667)c,e 68c,e 74e 80e 92 glh-4(gk225) glh-1(ok439) 20° NDd 39e 50e 42e 63 L4-stage MZ worms from glh-1/balancer grandmothers were picked to individual plates and shifted from 20° to 26°. Their adult progeny were analyzed (24 hr after L4). a Number of gonad arms examined was 8–30. Germ-cell counts included oocytes. b Number of gonad arms examined was 42–98 for mutants, 184 for N2. c In gk100 worms, 13% of gonad arms examined had 0 germ nuclei; 31% had .250 germ nuclei. In ok439 worms, 14% of gonad arms examined had 0 germ nuclei; 64% had .250 germ nuclei. In glh-4 glh-1 doubles, 63% of gonad arms examined had 0 germ nuclei; 27% had .250 germ nuclei. d Not done. e L4-stage M1Z progeny from glh-4 glh-1/balancer mothers were picked to individual plates and shifted from 20° to 26°. Their adult progeny were analyzed (24 hr after L4). bn125/bn125. Thus, gk100 and ok439 both behave as produces protein that is detectable by Western blot strong loss-of-function alleles. analysis, we investigated the distributions of the various Causes of sterility in glh-1 mutants: To investigate why mutant forms of protein. In wild-type worms, GLH-1 is MZ animals develop into sterile adults at elevated concentrated on P granules in larval and adult germlines temperature (26°), we Hoechst stained DNA in intact and in embryos (Gruidl et al. 1996). In contrast, in hermaphrodites, counted germ nuclei, and examined gk100, ok439, and bn103 mutant hermaphrodites, the germlines for the presence of gametes. We observed majority of GLH-1 is distributed uniformly in the dramatic decreases in both germ-cell counts and pres- cytoplasm in germlines and embryos (Figure 4 and data ence of gametes and also considerable variation from not shown). The weak allele bn125 causes a mild worm to worm (Table 3). In contrast to the 800 germ elevation in the level of diffuse cytoplasmic GLH-1, but nuclei observed in wild-type gonad arms, gonad arms in much of the mutant protein remains associated with P glh-1(gk100) hermaphrodites contained from 0 to 400 granules. The altered distributions of mutant forms of germ nuclei (mean ¼ 193). The extreme variation in GLH-1 are observed at low as well as high temperatures severity of germline proliferation defects is exempliﬁed (Figure 4 shows MZ worms grown at 20°). As de- by the fact that 38% of gonad arms contained 0 or very scribed above, low-temperature-grown animals gener- few germ nuclei, and 31% contained .250 germ nuclei. ally develop into fertile worms. Thus, dissociation of the Thirty percent of gonad arms contained oocytes, but majority of GLH-1 from P granules does not necessarily only 2% contained sperm, and none of the worms lead to sterility, although it may be a contributing factor. examined contained embryos. ok439 and bn125 mutants To determine the impact of glh-1 mutations on displayed similar but slightly milder defects. These P-granule integrity, we investigated the distributions of results demonstrate that glh-1 germlines are defective several other P-granule proteins in glh-1 MZ mutants in germ-cell proliferation and in production of gametes. grown at 20° (Figures 5 and 6). GLH-4 is concentrated Figure 3 shows representative images of glh mutant in granules in glh-1 mutants. However, in gk100 and germlines stained with DAPI. Table 4 documents that, ok439 mutant germlines, many of the GLH-4-containing while 100% of wild-type gonads contained mitotically granules have lost their perinuclear association (Figure dividing cells ( judged by staining with antibody to 5). The distributions of PGL-1, PGL-2, and PGL-3 are phosphorylated histone H3 Ser10; Hendzel et al. more dramatically altered in glh-1 mutants. PGL-1 and 1997) in their distal stem-cell region, many glh-1 mutant PGL-3 were studied in detail in gk100 and ok439 mu- gonads with reasonable numbers of germ nuclei lacked tants. Both PGL-1 and PGL-3 appear to be completely phosphorylated H3 Ser10-marked mitotic cells. This dispersed in the cytoplasm in some regions of the germ- supports the notion that glh-1 mutant germ nuclei are line (e.g., the pachytene region) but partially concentrated impaired in their stem-cell division capacity. on P granules in other regions of the germline (e.g.,mi- glh-1 mutants show defects in assembly or stability of totic region and diplotene) (Figure 6 and data not shown). P granules: Since each of the glh-1 mutant alleles In early-stage mutant embryos, a fraction of PGL-1 and GLHs and Germ Cells 1979

Figure 3.—glh-1 mutants display a range of germline sizes and gamete defects. glh/hT2Tgfp mothers were shifted from 20° to 26°. Homozygous glh/glh MZ hermaphrodites were DAPI stained 1 day beyond the L4 stage. Images represent a common phenotype for each strain. (A) Wild type. (B) glh-1(bn125) large germline. (C) glh-1(ok439) large germline containing endomitoti- cally replicating oocytes (arrows). (D) glh-1(ok439) small germline. (E) glh-1(gk100) small germline. (F) glh-4(gk225) ‘‘Y’’-shaped gonad observed in 1–15% of gonad arms. (G) glh-4(gk225) glh-1(ok439) very small germline. Bars for A–F and G, 50 mm.

PGL-3 is associated with P granules while a higher-than- expresses GFPTPGL-1 in the maternal germline and in normal fraction is dispersed in the cytoplasm. Later-stage early embryos (using pie-1 regulatory sequences) embryos show diminished staining of PGL-1 and PGL-3, (Cheeks et al. 2004). The distribution of GFP, which is but display clear concentration of both proteins on P bright in worms grown at $24.5°, recapitulates the granules (Figure 6 and data not shown). bn125 displays subcellular distribution of PGL-1 observed by fixing and some dissociation of PGL-1 and PGL-3 from P granules, staining samples with anti-PGL-1 antibody: concen- but generally resembles wild type more than the other trated on P granules at all stages examined. Screening three alleles. Thus, all alleles of glh-1 cause PGL-1 and in this strain for mutants with dispersed GFPTPGL-1 was PGL-3 to be dissociated from P granules, to a greater or the method used to isolate bn125 (Figure 7). After lesser extent depending on the stage of development. introducing the ok439 and bn103 mutations into the To assess PGL-1 association with P granules in living GFPTPGL-1 line, we observed GFPTPGL-1 mainly dis- glh-1 mutant worms, we used a transgenic strain that persed in the cytoplasm (Figure 7 and data not shown). To investigate the temporal requirement for GLH-1, we T TABLE 4 used RNAi to deplete GLH-1 from GFP PGL-1-expressing worms. RNAi was performed by feeding worms bacteria Analysis of mitotic germ cells in glh mutants at 25° expressing glh-1 dsRNA starting at the L4 stage. Within 24 hr, GFPTPGL-1 was notably dispersed in the cytoplasm in % gonad arms lacking phospho-H3 Ser10 adult germline tissue and in embryos (not shown). This Maternal genotype staining (n ¼ 40) suggests an ongoing requirement for the presence of adequate levels of GLH-1 in P granules for PGL-1 to be N2 0 efficiently recruited and/or retained on granules. glh-1(gk100) 36 glh-1(ok439) 70 glh-2 and glh-3 mutants do not display dramatic glh-1(bn125) 18 phenotypes on their own, while glh-4 mutants show glh-4(gk225) glh-1(ok439) 92 striking pleiotropic effects at high temperature: In previous RNAi studies, depletion of GLH-2 caused L4-stage N2 and glh-1 MZ mutants were picked to individual plates and shifted from 20° to 25°. The germlines of significant levels of sterility while depletion of GLH-3 their adult progeny were analyzed. N2 germlines contained or GLH-4 did not cause notable germline defects an average of six stained nuclei. (Kuznicki et al. 2000). Because of the uncertainties 1980 C. Spike et al.

Figure 4.—Two mutant forms of GLH-1 are not detectably associated with P granules. glh MZ hermaphrodites derived from glh/ hT2Tgfp grandmothers at 20° were cut, ﬁxed, and stained with mouse PA3 to stain DNA (A, C, E, G, and I) and with rabbit anti-GLH-1 (B, D, F, H, and J) and imaged by confocal microscopy. Each panel shows a single 0.5-mm optical section in the pachytene region of the germline. (A and B) Wild type. (C and D) glh-1(gk100).(E and F) glh-1(ok439). (G and H) glh-1(bn125).(I and J) glh-4(gk225) glh-1(ok439). The alleles gk100 and ok439 cause dispersal of GLH-1 from P granules (D, F, and J). The bn125 allele causes only modest dispersal (H). Bar, 10 mm.

associated with RNAi, we isolated mutant alleles of each blot analysis (supplemental Figure 1 and data not shown). gene (Figure 8), to enable genetic analysis. Western blots with the anti-GLH-4 antibody also verify glh-2(um2): This 2873-nucleotide deletion extends that the glh-4-like gene on cosmid T08D2 (Kuznicki et al. from exon 2 through exon 8. The sequence remains 2000) does not produce detectable protein, as predicted in frame and if translated would produce a small pro- if T08D2.3 is a pseudogene. tein of 130 amino acids. In fact, a chicken antibody All three of these strains were examined for sterility generated against an N-terminal GLH-2 peptide does and brood size after being raised at 15°,20°, and 26°. show some residual P-granule staining in fixed glh-2 None of the three mutants displayed significant sterility mutant worms (not shown). The deletion removes all at any temperature. glh-2(um2) and glh-3(um1) worms six CCHC zinc fingers and the DEAD-box helicase produced modestly reduced broods at 15° (25% below domain, which are likely essential for protein function. wild type for glh-2 and 30% below wild type for glh-3) and glh-3(um1): This 1811-nucleotide deletion extends close to wild-type broods at 20° and 26°. glh-4(gk225) from exon 2 through exon 4. If translated, the resultant worms grown at 26° for two generations have reduced protein would contain only the first 90 amino acids and broods (Table 1) and consistently show a remarkable would lack both the two CCHC zinc fingers and the phenotype in a small percentage (1–15%) of the worms DEAD-box helicase domain. An affinity-purified rabbit examined: the distal gonad is bifurcated, either slightly antibody raised against a C-terminal peptide does not or completely (Figure 3F). Bifurcated gonad arms were recognize any GLH-3 protein in the mutant background observed to have a distal tip cell on each tip (two of seven (Kuznicki et al. 2000). gonads) or only on one tip (five of seven gonads). The glh-4(gk225): This allele deletes 590 nucleotides, in- mechanism by which bifurcations arise is being including the first 280 nucleotides of coding sequence in vestigated. Bifurcations are occasionally seen in glh-1 exon 1. A rabbit anti-GLH-4 antibody raised against (RNAi) worms. Thus, gonad bifurcation appears to be a both an N-terminal peptide and a full-length GLH-4 mutant phenotype shared by multiple glh genes. fusion protein does not recognize GLH-4 protein in the glh-1 and glh-4 function redundantly: In previous mutant strain by immunocytochemistry or by Western RNAi studies, simultaneous depletion of GLH-1 and GLHs and Germ Cells 1981

GLH-4(1) is not sufficient for fertility at 26°. We note that glh-1(ok439) mutant worms containing a gfpTglh-4 transgene were easily propagated at 26°. This result suggests that boosting the level of GLH-4 can compensate for the loss of GLH-1 function at 26° and is consistent with the two GLH proteins being functionally redundant. The distributions of PGL-1 and PGL-3 were examined in the germlines of sterile MZ glh-4 glh-1 double mutants. Both proteins were completely dispersed in the cytoplasm (Figure 6 and data not shown). Thus, loss of GLH-4 enhances the P-granule phenotype observed in glh-1 single mutants. It is likely that dispersal of P-granule proteins is a primary defect in double mutants and not a secondary consequence of other defects in those sterile germlines (see discussion). glh mutants are not defective in RNAi: pgl-1 mutant worms are defective in RNAi (Robert et al. 2005; Figure 9). This finding raised the question whether it is PGL-1 Figure 5.—GLH-4 granules are not properly concentrated in particular or P granules in general that are critical for around nuclei in fixed glh-1 mutants. Worms were prepared as efficient RNAi. To address that, we tested whether glh-1 for Figure 4, but stained with mouse PA3 to stain DNA (A, C, and E) and with rabbit anti-GLH-4 (B, D, and F). Each panel mutant worms are also defective in RNAi. Neither gk100 shows a single 0.5-mm confocal section of the pachytene re- nor ok439 mutants displayed resistance to pos-1 RNAi gion of the germline. (A and B) Wild type. (C and D) glh- (Figure 9) or skn-1 RNAi (data not shown). Similarly, 1(gk100). (E and F) glh-1(ok439). The alleles gk100 and glh-4 single mutants and glh-4 glh-1 double mutants did ok439 cause some GLH-4 granules to lose their perinuclear not display resistance to RNAi (Figure 9). These results localization (D and F). Bar, 10 mm. suggest that particular components of P granules are critical for RNAi and others are not. Furthermore, loss GLH-4 caused enhanced sterility compared to depletion of PGL-1 (in pgl-1 mutants) produces much more severe of GLH-1 alone (Kuznicki et al. 2000). Analysis of effects on RNAi than dissociation of the majority of whether sterility was zygotic or maternal effect was not PGL-1 from P granules (in glh-1 mutants). done. To further investigate a potential genetic interaction between glh-1 and glh-4, we generated a glh-4(gk225) DISCUSSION glh-1(ok439) double-mutant strain. Compared to glh- 1(ok439) alone, the double mutant showed enhanced glh mutant phenotypes: Genetic analysis of the GLH zygotic and maternal-effect sterility at all temperatures family of genes has revealed a hierarchy of gene im- tested (Figure 2). The enhancement was most dramatic portance. GLH-1 is critical for fertility in worms grown at at low temperatures. For example, at 16° and 20° zygotic elevated temperature (26°): loss of zygotically encoded sterility was elevated from 11–15% in glh-1 alone to 25– GLH-1 function results in significant sterility, and loss 38% in glh-4 glh-1 double mutants. More dramatically, of both maternally supplied and zygotically encoded maternal-effect sterility was elevated from 10–11% in GLH-1 results in 100% sterility. Lower-temperature growth glh-1 alone to 97–100% in glh-4 glh-1 double mutants. (16°–24.5°) rescues the fertility of most glh-1 mutant At high temperature (26°)M1Z glh-4 glh-1 double- animals. Worms lacking GLH-2, GLH-3, or GLH-4 func- mutant hermaphrodites produced a higher percentage tion are generally fertile at all temperatures. Concom- of dead embryos and smaller broods than glh-1 alone itant loss of both GLH-1 and GLH-4 function causes the (Table 1), and MZ glh-4 glh-1 double-mutant her- same strong maternal, weaker zygotic sterility as de- maphrodites displayed more severe germline defects scribed for GLH-1, but independent of growth temper- than glh-1 alone (Figure 3, Tables 3 and 4). For example, ature. Thus, in the absence of GLH-1 function, GLH-4 63% of MZ glh-4 glh-1 double mutants lacked germ promotes healthy germline development in worms grown cells altogether, compared to 14% of MZ glh-1 alone at lower temperatures. GLH-2 and GLH-3 may contrib- (Table 3 footnote). Thus, on the basis of analyzing both ute as well but are not sufficient to confer fertility in the percentage of sterile worms and germline development absence of both GLH-1 and GLH-4. in sterile worms, glh-4 glh-1 double mutants show more A hierarchy of gene importance is also observed for severe defects than glh-1 alone. This finding suggests the PGL family of P-granule proteins. PGL-1 is the most that GLH-1 and GLH-4 function redundantly. In the important. pgl-1 mutants display phenotypes very similar absence of GLH-1, GLH-4(1) provides sufficient GLH to glh-1 mutants: strong maternal and weaker zygotic function to render most worms fertile at 16°–24.5°; sterility, predominantly at elevated temperature. Worms 1982 C. Spike et al.

Figure 6.—PGL-1 is not properly concentrated on P granules in ﬁxed glh-1 and glh-4 glh-1 mutants. Worms were prepared as for Figure 4, but stained with mouse PA3 to stain DNA (A, C, E, and G) and with rabbit anti-PGL-1 (B, D, F, and H). Each panel shows a single 0.5-mm confocal section of the distal tip plus pachytene region of the germline, and two panels include a 100- to 200-cell embryo as well. (A and B) Wild type. (C and D) glh-1(gk100). (E and F) glh-1(ok439). (G and H) glh-4(gk225) glh-1(ok439). The alleles gk100 and ok439 cause some PGL-1 to become dispersed in the cytoplasm, especially in the pachytene region (D and F). PGL-1 is completely dispersed in glh-4 glh-1 double mutants (H). Bar, 10 mm.

lacking the related proteins PGL-2 or PGL-3 are fertile gradual loss of wild-type protein in ﬁrst-generation at all temperatures. PGL-3 is the key redundant PGL homozygous mutants, it would be useful to have a factor: concomitant loss of both PGL-1 and PGL-3 thermolabile version of GLH-1 that would allow rapid function causes strong maternal and weaker zygotic protein inactivation. Such an allele of dynein heavy- sterility independent of growth temperature (Kawasaki chain DHC-1 was valuable in dissecting the requirement et al. 2004). for dynein function at different stages of development One noteworthy aspect of the glh-1 (and pgl-1) mutant (Schmidt et al. 2005). phenotypes is the extreme variability. For example, glh- Another noteworthy feature of the glh-1 and pgl-1 1(ok439) M1Z hermaphrodites grown at 26° produce mutant phenotypes is the sensitivity to temperature. broods that display from 0 to 83% embryo lethality, and Instead of being due to temperature-sensitive lesions, it the number of viable offspring ranges from 1 to .100. appears that strong loss-of-function and null alleles Although all of the viable MZ offspring develop into cause mild phenotypes at low temperatures and much sterile adults, their germlines contain widely varying more severe phenotypes at elevated temperature. For numbers of germ cells, from 0 to .500. Such variability is glh-1 and pgl-1, the mild-to-severe transition temperature observed even in glh-4 glh-1 double mutants. We envision is 24°–25°. Although wild-type animals are fully fertile at that this variability is due to variable accumulation of 26°, that temperature is likely to stress worms’ ability to defects caused by gradual loss of P-granule function in reproduce, such that worms are especially sensitive to M1Z and then in MZ worms. In the absence of loss of important factors such as GLH-1 and PGL-1. At zygotically expressed GLH-1, which normally commen- lower temperatures, other GLH and PGL family mem- ces in late embryogenesis (Y. Kohara, personal commu- bers provide sufﬁcient function for good fertility. nication), the maternal load of P granules must retain However, loss of both GLH-1 and GLH-4 or loss of both enough function to enable M1Z worms to develop PGL-1 and PGL-3 at low temperatures recapitulates the well-proliferated germlines, but not enough function to defects observed in GLH-1 and PGL-1 single mutants at ensure production of uniformly good gametes and em- high temperature. In fact, elevated temperature enhan- bryos. In the next generation of worms that lack both ces the phenotypes of many germline-required genes, maternal and zygotic GLH-1, germ-cell proliferation is such as him-17 (Reddy and Villeneuve 2004), hpl-2 also compromised. To get around the complications of (Couteau et al. 2002), rha-1 (Walstrom et al. 2005), mes-1 GLHs and Germ Cells 1983

The defects observed in MZ glh-1 mutants at elevated temperature and in glh-4 glh-1 double mutants at all temperatures suggest that the GLHs and probably germ granules in general serve diverse roles in the germline: promotion of stem cell divisions, production of adequate numbers of gametes, and production of high-quality gametes capable of successful embryogenesis after fertilization. RNAi depletion of GLH family members caused a similar range of defects but with a different gamete bias than observed in mutants. While RNAi-induced sterile worms usually contained sperm and only rarely contained oocytes (Kuznicki et al. 2000), MZ glh-1(gk100) and glh-1(ok439) single mutants and glh-4 glh-1(ok439) double mutants infrequently (2–32%) contained sperm, and a significant percentage (26–57%) contained oocytes (Table 3). The reason for this difference is not known. Now that glh-4 glh-1 double mutants exist, in-depth analysis and efforts to identify specific RNA targets of GLH regulation can be un- dertaken (see below). Figure 7.—GFPTPGL-1 is not properly concentrated on P Vasa regulates the translation of several mRNAs granules in living glh-1 mutants. Live worms containing a (Dahanukar and Wharton 1996; Markussen et al. GFPTPGL-1 transgene were imaged by confocal microscopy. 1997; Styhler et al. 1998) and is implicated in miRNA Images represent single 0.5-mm optical sections. (A and B) control of gene expression (Kotaja et al. 2006). Evidence Wild type. (C and D) glh-1(bn103). (E and F) glh-1(bn125). The from C. elegans suggests that P-granule components majority of GFPTPGL-1 is dispersed in the cytoplasm of glh- 1(bn103) embryos (C) and germlines (D). glh-1(bn125) worms could be involved in mRNA trafficking, translation, sta- display less dramatic dispersal of GFPTPGL-1. Bars, 10 mm. bility and RNAi-related processes (Schisa et al. 2001; Seydoux and Braun 2006; Spike et al. 2008). Since glh-1(ok439) germlines have P-granule assembly defects (Strome et al. 1995), deps-1 (Spike et al. 2008), prg-1 even at low temperatures (this work), we performed a (Yigit et al. 2006), several eri genes (Duchaine et al. genomewide microarray analysis to look at mRNA 2006), kgb-1 (Smith et al. 2002; Orsborn et al. 2007), vbh-1 accumulation patterns in glh-1(ok439) MZ germlines (Salinas et al. 2007), and mex-3 gld-1 double mutants at 20° (C. Spike and V. Reinke, unpublished results). (Ciosk et al. 2006; R. Ciosk, personal communication), Our preliminary analysis of those data indicates that few and enhances the expression of transgenes in the germ- mRNAs are misregulated in glh-1 mutant germlines line (Strome et al. 2001). Performing screens for germ- (only eight genes were misregulated $1.8-fold relative line mutants at high temperature may be analogous to to wild type), suggesting that GLH-1 and normally screening in a sensitized background and may identify assembled P granules do not generally regulate mRNA genes and gene families that would have been missed at stability. These conclusions are consistent with a sim- lower temperatures. ilar analysis performed on the MZ germlines of

Figure 8.—Mutant alleles of glh-2, glh-3, and glh-4. A schematic of each glh gene shows the position and extent of each deletion allele. Coding regions are shown as boxes and introns as lines. 1984 C. Spike et al.

sociated with granules in the distal region but not in the pachytene region (this study). In the future, electron microscopy may shed light on stage-specific variations in P-granule structure and perhaps function in the germline. As suggested above, GLH-1 and GLH-4 may participate directly or indirectly in the recruitment or retention of PGL proteins on P granules. Another possibility is that only functional GLH and PGL proteins associate with P granules. Mutant forms of GLH-1 may be nonfunctional and therefore dispersed in the cytoplasm. Our analysis of glh-1 mutants indicates that two different regions of GLH-1 are required for its association with P granules: the DEAD-box helicase domain and a Gly-rich region containing 3 CCHC zinc fingers. DEAD-box helicase and CCHC domains are able to bind Figure 9.—glh-1 mutants and glh-4 glh-1 double mutants are RNA (Dannull et al. 1994; Sengoku et al. 2006), sug- not resistant to RNAi. Worms of the genotypes shown were fed gesting that the association of GLH-1 with RNA might be bacteria expressing dsRNA against pos-1 and allowed to produce embryos at 20°. The following strains and generations critical for its association with RNA-rich P granules. were used: glh-1(ok439), glh-4(gk225), and pgl-1(bn101),MZ Alternatively, GLH-1 proteins lacking the helicase do- generation; glh-1(gk100) and glh-4(gk225) glh-1(ok439),M1Z main or zinc fingers might fail to localize to P granules generation. pgl-1 mutants were resistant to pos-1 RNAi, as re- because they are misfolded. If GLH-1 is required for obert ported by R et al. (2005). The other strains were sensi- proper PGL protein function, then the dispersal of PGL tive. glh-1(gk100) MZ animals also appear to be sensitive to pos-1(RNAi) (95% lethality), but glh-1(gk100) MZ animals proteins in glh-1 mutant germlines may be a result of loss not exposed to pos-1 dsRNA also laid a significant percentage of PGL function. This scenario resembles one model of eggs that died (51%). emerging for P-body assembly and function (Eulalio et al. 2007b). P bodies are cytoplasmic RNP particles that are the probable sites of mRNA decapping and another mutant required for P-granule assembly (deps-1; decay; they were first described in yeast and subse- Spike et al. 2008). We note, however, that microarray quently observed in mammals, flies, and worms (re- analysis of glh-1(ok439) germlines at low temperature viewed in Eulalio et al. 2007a). In Drosophila tissue would not be expected to reveal mRNAs/genes that are culture cells, the formation of P bodies appears to be the redundantly regulated by both GLH-1 and GLH-4. Such consequence of active processes, such as RNAi, in which targets remain to be identified and may shed light on the the components of P bodies participate (Eulalio et al. diverse roles of the GLHs and germ granules. 2007b). P-granule assembly and roles: Molecular epistasis Intriguingly, mutations in pgl-1 and in another results place the GLH proteins ‘‘upstream’’ of the PGL P-granule gene deps-1 lead to a germline RNAi-defective proteins in a P-granule assembly pathway. pgl-1; pgl-2; pgl-3 (Rde) phenotype (Robert et al. 2005; Spike et al. triple mutants display an apparently normal concentra- 2008; this study), suggesting potential links between tion of GLH-1 in perinuclear P granules (Kawasaki et al. P granules and RNAi. However, since (1) glh-1 mutants 2004), revealing that the PGL proteins are not required and glh-4 glh-1 double mutants do not show defects in for GLH-1 recruitment or retention in P granules. In RNAi and (2) PGL-1 and PGL-3 are completely dis- contrast, glh-1 mutants display partial dispersal of the persed from perinuclear granules in glh-4 glh-1 double PGL proteins, and glh-4 glh-1 double mutants display full mutants, localized PGL-1 and intact P granules do not dispersal. We think the latter is probably a direct effect appear to be essential for RNAi. This scenario also of loss of both GLH-1 and GLH-4 function and not resembles what is currently known about the relation- an indirect effect of germline sickliness, as analysis of ship between RNAi and P bodies. Although P bodies many sterile mutants, including pgl-1; pgl-2; pgl-3 triple require active processes such as RNAi to form, intact P mutants, demonstrates that P granules retain their bodies in Drosophila tissue culture cells are not re- granular nature even in severely compromised germ- quired for effective RNAi (Eulalio et al. 2007b). lines (Kawasaki et al. 2004; S. Strome, unpublished Because P granules share many components with P results). The finding that RNAi depletion of GLH-1 from bodies, including the C. elegans proteins CGH-1 and L4-stage worms results in dispersed GFPTPGL-1 within CAR-1, it is not surprising that several reports have 24 hr (C. Spike, unpublished results) suggests that P suggested that C. elegans P granules are related to P granules are likely to be dynamic structures and that bodies (Wickens and Goldstrohm 2003; Seydoux and GLH-1 is continuously required for PGL-1 retention. In Braun 2006; Strome and Lehmann 2007). P bodies can glh-1 single mutants, at least some PGL-1 remains as- store mRNAs that are silenced by microRNAs, and they GLHs and Germ Cells 1985 contain RNA-induced silencing complex (RISC) pro- Dreyfuss 1992). Second, all of the Caenorhabditis teins, including ALG-1/PIWI/Argonaute (Chan and GLH proteins possess several (two to six) CCHC-type Slack 2006; Jakymiw et al. 2007). Another P-body- zinc fingers not found in Drosophila and vertebrate associated protein in fission yeast is Dicer, the RNAseIII Vasa homologs (see phylogenetic analysis in Salinas enzyme essential for creating siRNAs used for RNAi and et al. 2007). Perhaps the CCHC zinc fingers participate for processing microRNAs, the noncoding, small RNAs in RNA binding and compensate for the lack of an RGG important in development (Carmichael et al. 2006). domain in GLH family members. Interestingly, Ciona Dicer is also in the chromatoid bodies of mouse male Vasa possesses a mix of FG and RG repeats, and the Vasa germ cells and physically associates with the mouse Vasa homologs in Ciona, Daphnia, Hydra, Artemia, and homolog MVH (Kotaja et al. 2006). Another study, several other marine invertebrates contain CCHC zinc using Drosophila protein lysates enriched for polar fingers (Fujimura and Takamura 2000; Sagawa et al. granules, recently reported that PIWI, a fly RISC pro- 2005). tein, forms a complex with DICER-1 to regulate levels of The closely related nematode C. briggsae contains a Vasa (Megosh et al. 2006). Thus, RISC proteins with glh-1-related gene and a glh-4-related gene (Stein et al. microRNA functions are associated with germ granules 2003). The predicted GLH-1 protein in C. briggsae shows and interact with Vasa homologs in flies and mice. GLH-1 highest sequence identity in the helicase domain to also has recently been found to have both a physical CeGLH-1 (82%), followed by CeGLH-2 (81%), CeGLH- and a genetic relationship to DCR-1 (E. Racen and K. 3 (71%), and CeGLH-4 (36%). CbGLH-1 and CeGLH-1 Bennett, unpublished results). Since glh-1 and glh-4 glh-1 also share a 240- to 270-aa N-terminal FG-rich domain. mutants are not RNAi resistant (this work), these GLHs We speculate that C. elegans glh-2 and glh-3 arose from could have a relationship related to microRNAs rather glh-1 by gene duplication events followed by expansion than to RNAi. of the FG domain in the case of glh-2 and loss of the FG Germline-required DEAD-box helicases: Drosophila domain in the case of glh-3. C. elegans and perhaps C. Vasa was the first protein component of germ plasm to briggsae rely on a division of labor between the two most be molecularly identified (Hay et al. 1988b). Vasa divergent GLHs, GLH-1 and GLH-4, for normal germ- homologs were subsequently identified in numerous granule assembly and germ-cell proliferation (this work). species, including worms, mice, chickens, frogs, zebra- It is not clear what roles the other C. elegans GLHs play fish, leeches, sea urchins, brine shrimp, and water fleas during germ-cell development, but GLH-2 was recently and, in fact, are widely used markers of germ cells identified as a factor that associates with sperm chro- (reviewed in Raz 2000). Many of these homologs have matin (Chu et al. 2006), suggesting that one of the GLHs been shown to be enriched in germ plasm and impor- may function in the nucleus as well as on P granules. xtavour kam tant for germline development (E and A We thank Emily Gressman-Coberly for helping isolate the glh-2(um2) 2003). While Vasa is a single-copy gene in Drosophila and glh-3(um1) alleles and Steve Dunkelbarger for generating the glh- and in numerous other systems, in C. elegans Vasa-related 1(RNAi) construct. This work was supported by Ruth Kirchstein proteins are encoded by the four glh genes described National Research Service Award postdoctoral fellowship GM69084 here and a second family of genes (vbh-1, Y71H2AM.19) (C.S.), American Cancer Society postdoctoral fellowship PF-04-034-01- DDC (C.S.), a University of Missouri Life Science Fellowship (A.O.), related both to Vasa and to the DEAD-box RNA helicase National Institutes of Health (NIH) training grant GM008396 (A.O. known as Belle (Johnstone et al. 2005; Salinas et al. and E.R.), NIH grant GM34059 (S.S.), and National Science Founda- 2007). Both gene families are required for normal germ- tion grants IBN 0078169 and IBN 0415699 (K.B.). Some nematode cell function and have members that localize to P gran- strains used in this work were provided by the Caenorhabditis Genetics ules (Gruidl et al. 1996; Kuznicki et al. 2000; Salinas Center, which is funded by the NIH National Center for Research Resources. et al. 2007). 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