Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi

Brief Communication 171

Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi Ikuma Maeda*, Yuji Kohara†‡, Masayuki Yamamoto* and Asako Sugimoto*§ Genome-wide analysis of gene function is essential for the post-genome era, and development of efficient and economical technology suitable for it has been in demand. Here we report a large-scale inactivation of the expressed genes in the nematode Caenorhabditis elegans. For this purpose, we have established a high-throughput “RNAi-by-soaking” methodology by modifying the conventional RNAi method [1, 2]. A set of tag-sequenced, nonredundant cDNAs corresponding to approximately 10,000 genes [3] (representing half of the predicted genes [4]) was used for the systematic RNAi analysis. We have processed approximately 2500 genes to date. In development, 27% of them showed detectable phenotypes, such as embryonic lethality, postembryonic lethality, sterility, and morphological abnormality. Of these, we analyzed the phenotypes of F1 sterility in detail, and we have identified 24 genes that might play important roles in germline development. Combined with the ongoing analysis of expression patterns of these cDNAs [3, 5], the functional information obtained in this work will provide a starting point for the further analysis of each gene. Another finding from this screening is that the incidence of essential genes is significantly lower in the X chromosome than in the autosomes. *Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. †Genome Biology Laboratory, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan. ‡Core Research for Evolutional Science and Technology and §Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan.

C. elegans [1, 2, 6]. In this method, double-stranded RNA that corresponds to a particular gene is introduced into the worm, which results in specific degradation of the targeted mRNA and thus suppresses the expression of the gene [2, 7]. Although RNAi is less time consuming and easier to perform compared to the isolation of chromosomal mutations, microinjection of dsRNA into individual worms is a labor-intensive process and is not adequate for high-throughput reverse genetics. To circumvent this, we chose to use the “RNAi by soaking” method, in which worms are soaked in dsRNA solutions for RNA delivery. Soaking had been considered to be less potent than conventional microinjection for disrupting gene functions [1], but we found that the addition of spermidine significantly improved the efficiency and reproducibility of RNAi (Figure 1). We applied this RNAi-by-soaking method to several control genes whose loss-of-function phenotypes were well characterized. In all genes examined, F1 progeny from the worms subjected to RNAi by soaking phenocopied respective mutant phenotypes with high penetrance (Figure 1). It was equally effective for genes involved in early embryogenesis (glp-1 [8] and mei-1 [9]), germline development (mes-3 [10], fem-1 [11], gld-1 [12], and daz-1 [13]), and muscle function (unc-22 [14]). Thus, this method appeared to be effective throughout the F1 generation. For some genes (unc-22, gld-1, and daz-1), mutant phenotypes were also observed in the P0 animals that were soaked in the RNA solution. Figure 1

Correspondence: Asako Sugimoto E-mail: [email protected] Received: 2 November 2000 Revised: 20 November 2000 Accepted: 11 December 2000 Published: 6 February 2001 Current Biology 2001, 11:171–176 0960-9822/01/$ – see front matter  2001 Elsevier Science Ltd. All rights reserved.

Results and discussion A few years ago, a new reverse genetic method called RNA-mediated interference (RNAi) was established in

Efficiency of RNAi by soaking. The addition of spermidine increased the penetrance of the phenotype. The percentage of the F1 progeny from four P0 animals that showed the expected phenotype is indicated. The addition of spermidine increased the penetrance of the phenotype (compare the penetrance of the unc-22 phenotype without spermidine [⫺Sp] to that with spermidine [⫹Sp]). Spermidine is included in the rest of the reactions. unc-22 shows the “twitcher” phenotype. glp-1 and mei-1 show the embryonic lethal phenotype. mes-3, fem-1, gld-1, and daz-1 show the sterile phenotype.

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Figure 2

Strategy of the RNAi screening. (a) RNA preparation for RNAi by soaking. cDNAs that had been cloned into lambda ZAPII vectors were PCR amplified with T3 and T7 primers. Each strand of RNA was synthesized by T3 and T7 RNA polymerases. All steps were performed in a multi-well format. (b) Diagram of RNAi-by-soaking

screening. Four L4 hermaphrodites were soaked in each RNA solution derived from a single cDNA clone and were then incubated for 24 hr. The worms were then recovered to a new plate, and the phenotypes of both P0s and F1 progeny were observed. The soaking of worms was also performed in a multi-well format.

In the C. elegans cDNA project led by Y. K., 5⬘- and 3⬘ends of approximately 60,000 cDNA clones from various stages were sequenced and categorized into approximately 10,000 cDNA groups corresponding to independent genes [3]. We established a nonredundant cDNA set by choosing one clone per cDNA group, and we used it as the source of RNA synthesis templates to be used in the large-scale RNAi experiment. The nonredundant cDNA set represented half of the approximately 19,000 genes predicted by the genome project of C. elegans. The use of this nonredundant cDNA set was thought to have the following advantages: (i) Because the target of RNAi is mature mRNA [2, 7], cDNA is a better template to synthesize RNA molecules for RNAi than is genomic DNA, which may include introns. (ii) The use of cDNA guarantees that only expressed genes are targeted. (The prediction of genes from genomic sequences alone may have mistakes). (iii) Each cDNA is flanked by T3 and T7 primers in a phagemid vector and is thus convenient for RNA synthesis by the T3 and T7 RNA polymerases.

worms were then recovered to a new plate, and the phenotypes of both P0 worms and F1 progeny were observed. All phenotypes visible under the dissection microscope were recorded. Such phenotypes included embryonic lethality, larval lethality, sterility, and abnormality in morphology or locomotion. In this high-throughput RNAi analysis, we used a 96-well plate format for PCR amplification of the cDNA templates, dsRNA synthesis, and soaking of the worms in RNA solution, which may be suitable for robotization in the future (Figure 2).

The procedure for the large-scale RNAi by soaking is schematically shown in Figure 2. Each cDNA clone was amplified by PCR with the T3 and T7 primers, and amplified products were used as templates for in vitro transcription by the T3 and T7 RNA polymerases. Four hermaphrodite animals in the L4 stage were soaked into each purified dsRNA solution and incubated for 24 hr. The

To date, we have examined 2479 nonredundant cDNA clones. This number corresponds to approximately 13% of the predicted genes in C. elegans ([4];Table 1). Of these, approximately 27% exhibited detectable phenotypes. (See Table S1 available with this article on the internet for the complete list of the cDNAs used and their phenotypes.) The most frequently observed phenotype was embryonic lethality (14% of the examined clones, Table 1). These clones are regarded as genes essential for various aspects of embryogenesis. The second-most frequent phenotype was sterility in the soaked P0 worms (6.7%). The clones that caused P0 sterility should play some role in germline development/differentiation in adults (e.g., yk201d3 in Figure 3g, see below). Many clones that caused P0 sterility also resulted in additional phenotypes, such as F1 embryonic or larval lethality. These clones are likely to have, in addition to roles in the germline, other

Brief Communication 173

Table 1 Summary of RNAi phenotypes

Phenotype (Phenotype of escapers) P0 Sterile P0 Other phenotypes F1 Embryonic lethal (No other phenotype) (⫹ Larval arrest/lethal) (⫹ Sterile) (⫹ Other phenotype) F1 Larval arrest/lethal F1 Sterile F1 Other phenotypes † Clones that showed phenotypes No phenotype Total

Number of cDNA clones per each chromosome (%)

Number of cDNA clones (%) 167 10 344 227 97 18 2 112 24 18 675 1804 2479

(6.7) (0.4) (13.9) (9.2) (3.9) (0.7) (0.1) (4.5) (1.0) (0.7) (27.2) (72.8) (100)

I 34 1 82 55 22 5 0 20 4 4 145 282 427

II

(8.0) 32 (7.8) 28 (0.2) 2 (0.5) 1 (19.2) 58 (14.2) 54 (12.9) 38 (9.3) 38 (5.2) 16 (3.9) 13 (1.2) 3 (0.7) 3 (0.0) 1 (0.2) 0 (4.7) 20 (4.9) 21 (0.9) 0 (0.0) 7 (0.9) 2 (0.5) 4 (34.0) 114 (27.9) 115 (66.0) 294 (72.1) 261 (100) 408 (100) 376

III

IV

(7.4) 32 (7.8) 27 (0.3) 2 (0.5) 3 (14.4) 57 (13.9) 58 (10.1) 33 (8.1) 38 (3.5) 21 (5.1) 15 (0.8) 2 (0.5) 5 (0.0) 1 (0.2) 0 (5.6) 16 (3.9) 18 (1.9) 7 (1.7) 4 (1.1) 3 (0.7) 3 (30.6) 117 (28.6) 113 (69.4) 292 (71.4) 329 (100) 409 (100) 442

V

All autosomes

(6.1) (0.7) (13.1) (8.6) (3.4) (1.1) (0.0) (4.1) (0.9) (0.7) (25.6) (74.4) (100)

153 9 309 202 87 18 2 95 22 16 604 1458 2062

(7.4) (0.4) (15.0) (9.8) (4.2) (0.9) (0.1) (4.6) (1.1) (0.8) (29.3) (70.7) (100)

X 14 (3.8) 0 (0.0) 29 (7.8) 20 (5.4) 9 (2.4) 0 (0.0) 0 (0.0) 15 (4.0) 1 (0.3) 2 (0.5) 61 (16.4) 311 (83.6) 372 (100)

No match 0 1 6 5 1 0 0 2 1 0 10 35 45

(0.0) (2.2) (13.3) (11.1) (2.2) (0.0) (0.0) (4.4) (2.2) (0.0) (22.2) (77.8) (100)

† Morphological abnormality, such as multivulva, dumpy, or locomotion defect. RNA species that caused multiple phenotypes are categorized into the most severe phenotype, without regard to the frequency of each phenotype. “F1 Embryonic lethal” is sub-categorized with the secondary phenotypes observed among the F1 progeny. Penetrance of phenotypes varies for each gene.

functions in different tissues. Some of them may be “house-keeping” genes whose function is required in all cells, including germ cells. Because the germline is the only proliferating tissue in adults, it may require high metabolic activity and may thus be more susceptible to the loss of function of house-keeping genes than are somatic tissues. Alternatively, they may play distinct roles in different developmental stages. The genes that caused F1 sterility with no apparent soFigure 3 Examples of sterile phenotypes. Nomarski DIC images of hermaphrodite adult gonads are shown. (a) Wild-type hermaphrodite. (b–g) RNAi phenotypes of sterile animals. (b) yk187a3 (F1). Few germ cells were detected in the gonads. No gametogenesis was observed. (c) yk260b7 (F1). Some F1 animals showed over-proliferation of germ cells, and others had fewer germ cells (not shown). (d) yk213d3 (F1). Abnormal gonad morphology. No gametes were observed. (e) yk218f9 (F1). Unfertilized eggs were accumulated in the uterus. (f) yk267a9 (F1). Oocytes were vacuolated in the proximal region of the gonad. (g) yk201d3 (P0). Ovulation was not observed, and oocytes had accumulated in the ovary. Abbreviations are as follows: Vu, vulva; DT, distal tip of the gonad; Oo, oocytes; Sp, Sperm; E, fertilized eggs; UG, undifferentiated germ cells; UE, unfertilized eggs; V, vacuolated oocytes; A, accumulated oocytes. The scale bar represents 10 ␮m.

matic phenotypes (1%, 24 clones) are likely to play a germline-specific function (see Table S2). (Note that the germline-specific genes (mes-3, gld-1, fem-3, and daz-1) used in Figure 1 all resulted in F1 sterility by RNAi by soaking). Two of the cDNA clones in this category were identical to known genes, mes-1 and mes-3, whose loss of function results in maternal-effect sterility due to the defect in post-embryonic germline development. We further characterized the sterile phenotypes of the remaining genes in this category, as well as some genes that caused

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P0 sterility, with DIC microscopy for cellular morphology (see examples in Figure 3) and with DAPI staining for nuclear morphology (data not shown). Each clone caused a distinct germline aberration, such as a defect in the number of germ cells (e.g., yk187a3 [Figure 3b]; yk260b7 [Figure 3c]), germline sex determination (yk267a9), gonadogenesis (yk213d3 [Figure 3d], and yk224b11), and gametogenesis (e.g., yk218f9 [Figure 3e]; yk267a9 [Figure 3f]; and yk201d3 [Figure 3g]). Thus, the RNAi screening proved to be useful for isolating genes involved in various aspects of germline development.

Figure 4

Our screen marked multiple genes that were originally isolated by forward genetics, and this result demonstrates the efficacy of the method. In most cases, the phenotypes resulting from chromosomal mutations and those by RNAi agreed with each other (e.g., emb-5 [15], pat-3 [16] [embryonic lethal]; mes-1 [17], mes-3 [10] [F1 sterile]; unc-22 [14] [locomotion defect]; lin-1 [18] [defect in vulval development]; and dpy-13 [19] [morphology defect]). In a rare case, however, RNAi resulted in more severe phenotypes than those of existing null mutants. For example, ama1(null) is L1 lethal [20], but ama-1(RNAi) was 100% embryonic lethal. This is probably because RNAi suppresses both maternal and zygotic gene functions, while the null mutant contains intact maternal products. Genes that caused lethality and/or sterility added up to 26% of the total examined. This frequency is consistent with the predicted ratio of essential genes (15%–30% of the total genes) estimated from the accumulated data of forward genetics [21, 22]. This suggests that our RNAi screen is sufficiently effective to identify most essential genes. However, the frequency of appearance of the Unc (uncoordinated) phenotype was low (only the unc-22 gene, whose loss of function causes muscle disorganization, has been identified in our screen to date), despite the fact that forward genetics has so far identified more than 130 unc (uncoordinated) genes. Considering that neuronal defects cause the majority of Unc phenotypes, the low frequency of the Unc phenotype detected in our screen may be due to the poor susceptibility of neuronal cells to RNAi for an unknown reason [6]. We performed the RNAi on cDNA clones randomly sampled from the nonredundant cDNA set. Thus, the corresponding genes distribute almost evenly to all chromosomes (Table 1 and Figure 4). Assignment of the chromosomal locations of the clones that caused phenotypes revealed that the X chromosome is much less populated by essential genes than are the autosomes. The incidence of the clones that caused detectable phenotypes was nearly the same among the autosomes; it averaged 29.3% (ranging from 25.6% of chromosome V to 34.0% of chromosome I; see Table 1). In contrast, the incidence on the X chromosome was 16.4%, which is significantly lower

Distribution of the cDNA clones used on each chromosome. The black vertical lines each represent one chromosome. 1910 cDNA clones and their phenotypes are shown. Black lateral lines indicate the location of each clone used in this study. Four columns of lateral lines correspond to phenotypes by RNAi, as follows: first column, (red) P0 sterile and (gray) P0 others; second column, (green) embryonic lethal; third column, (blue) larval arrest/larval lethal; fourth column, (orange) F1 sterile and (gray) F1 other phenotypes. In the case of clones that showed multiple phenotypes (such as embryonic lethal and sterile), both phenotypes are indicated. cDNA clones whose sequencing is not finished and those that did not match to the available genome sequence are not shown in this figure.

than that of autosomes (chi-square test: p ⫽ 4.6 ⫻ 10⫺8). When one considers that lethality and sterility accounted for most of the phenotypes that we observed, this result suggests that essential genes are underrepresented on the X chromosome. Because the mode of sex determination in C. elegans is via XX hermaphrodites and XO males, mutations in the essential genes on the X chromosome directly leads to the lethality/sterility of males. This may explain why essential genes tend to be removed from the X chromosome. In this study, we subjected L4 larvae to RNAi by soaking in order to detect the earliest phenotypes in the F1 generation. However, one can apply the RNAi-by-soaking

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method to characterize postembryonic gene function. When L1 animals are soaked in the RNA solution, postembryonic phenotypes can be specifically observed in the P0 animals ([23]; our unpublished data). L1 soaking will be particularly useful for the characterization of genes essential for both embryogenesis and postembryonic development because RNAi by injection or L4 soaking results in embryonic lethality and thus prevents the appearance of postembryonic phenotypes.

plate after 24 hr. They were allowed to lay eggs for up to 48 hr. P0s and F1 progeny were reared at 25⬚C so that temperature-sensitive phenotypes could be detected.

Observation of phenotypes Phenotypes of P0 and F1 were observed under a dissection microscope (Zeiss Stemi 2000). For further characterization by DIC optics and DAPI staining, Zeiss Axiophot microscope was used. Pictures were taken digitally with the chilled CCD camera C5985 with ARGUS 20 Image Processor (Hamamatsu Photonics).

Distribution of cDNA clones

Although we used the RNAi screen method for the general characterization of the gene function of the genome, it may be readily applied to other types of gene screening. For example, by using a particular mutant as the host strain, one can identify suppressor genes whose RNAi suppresses the mutant phenotype. The use of strains with tissue-specific GFP markers will help to screen for genes involved in organogenesis. It can also be used to screen for genes that cause resistance or sensitivity to drugs. As a final note, comprehensive expression analyses by whole-mount in situ hybridization on the cDNA set used in this paper is currently in progress ([3, 5]; Y. K., unpublished data). The phenotypic data for each cDNA clone obtained in this study have been compiled with the currently available expression data set in the NEXTDB (The Nematode Expression Pattern DataBase; http://watson. genes.nig.ac.jp/db/index.html). Once both the expression analysis and the RNAi characterization for the cDNA set are complete, researchers will be able to use the data set as the starting point for the detailed analysis of genes of their own interest.

Materials and methods RNA preparation All cDNA clones in lambda ZAPII vectors were derived from Y. K.’s stock from the Expressed Sequence Tag(s) (EST) project. cDNA inserts were PCR amplified with primers BS619 (5⬘-TGAATTGTAATACGACTCAC-3⬘) and BS793 (5⬘-GAAATTAACCCTCACTAAAG-3⬘). Each cDNA fragment was reamplified with T3 Primer (5⬘-AATTAACCCTCACTAAAGGG AACAAAAGCTGG-3⬘) and T7 Primer (5⬘-GTAATACGACTCACTATAG GGCGAATTGGGTA-3⬘) in 20 ␮l scale. PCR products (7.5 ␮l) were directly used for in vitro transcription with either T3 or T7 RNA polymerases (Stratagene) in 25 ␮l scale, per the manufacturer’s instruction. Template DNA was not removed after RNA synthesis. Equal volumes of each strand of RNA were mixed and extracted with phenol, phenol/ chloroform, and chloroform. The RNA molecules were precipitated with isopropanol and were dissolved in 4 ␮l of soaking buffer (10.9 mM Na2HPO4, 5.5 mM KH2PO4, 2.1 mM NaCl, 4.7 mM NH4Cl, 3 mM spermidine, and 0.05% gelatin). The final RNA concentration varied from 1 to 5 ␮g/␮l.

RNAi by soaking L4-stage hermaphrodites were washed well in M9 solution (43.6 mM Na2HPO4, 22.0 mM KH2PO4, 8.6 mM NaCl, and 18.7 mM NH4Cl). Then they were transferred to a fresh NGM plate [24] without bacterial loan and were allowed to crawl for several minutes to remove bacteria completely. Four worms were soaked in 4 ␮l of dsRNA solution in a 48-well PCR plate (half of the 96-well plate) and incubated at 20⬚C for 24 hr. The worms were recovered to a new ENG plate (http://eatworms.swmed. edu/ⵑleon/revgen/protocol.html), and P0s were transferred to a new

Each cDNA clone (“yk clone”) and the cDNA set (as PCR-amplified DNA fragments) used in this study are available upon request. Requests should be addressed to Y. K. ([email protected]).

Supplementary material Two supplementary tables showing the full phenotypic description for each cDNA can be found with this article on the internet at http://currentbiology.com/supmatin.htm.

Acknowledgements We thank Tim Schedl for helpful discussion. We are grateful to Tadasu Shin-i for the clone data, to Yohei Minakuchi for preparing the figure, and to all technicians of the Kohara lab for preparing the clone set. This work was supported by grants from Japan Science and Technology Corporation (A. S. and Y. K.), and grants-in-aid for Scientific Research on Priority Areas (A. S. and Y. K.) and for Specially Promoted Research (M. Y.) from the Ministry of Education, Science, Sports, and Culture of Japan.

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