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Genome Res. 15:250-259, 2005 ©2005 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/05 $5.00 Letter New genes with roles in the C. elegans embryo revealed using RNAi of ovary-enriched ORFeome clones1 Department of Biology, New York University, New York, New York 10003, USA 2 Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA 3 Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA
Several RNA interference (RNAi)-based functional genomic projects have been performed in Caenorhabditis elegans to identify genes required during embryogenesis. These studies have demonstrated that the ovary is enriched for transcripts essential for the first cell divisions. However, comparing RNAi results suggests that many genes involved in embryogenesis have yet to be identified, especially those eliciting partially penetrant phenotypes. To discover additional genes required for C. elegans embryonic development, we tested by RNAi 1123 ORFeome clones selected to represent ovary-enriched genes not associated with an embryonic phenotype. We discovered 155 new ovary-enriched genes with roles during embryogenesis, of which 69% show partial penetrance lethality. Time-lapse microscopy revealed specific phenotypes during early embryogenesis for genes giving rise to high penetrance lethality. Together with previous studies, we now have evidence that 1843 C. elegans genes have roles in embryogenesis, and that many more remain to be found. Using all available RNAi phenotypic data for the ovary-enriched genes, we re-examined the distribution of genes by chromosomal location, functional class, ovary enrichment, and conservation and found that trends are driven almost exclusively by genes eliciting high-penetrance phenotypes. Furthermore, we discovered a striking direct relationship between phylogenetic distribution and the penetrance level of embryonic lethality elicited by RNAi.
A continuing challenge in contemporary biology is to extract meaningful functional data from the genome sequence. High-throughput functional genomics projects are currently ongoing in several model systems to characterize gene function on multiple levels, including gene expression patterns (Spellman et al. 1998  ; Reinke et al. 2000, 2004; Jiang et al. 2001; Arbeitman et al. 2002; Baugh et al. 2003), protein-protein interactions (Uetz et al. 2000; Walhout et al. 2000; Ito et al. 2001; Giot et al. 2003; Li et al. 2004), and protein structures (Chance et al. 2002; Goh et al. 2003). Phenotypic data complement these studies and are necessary to make the connection between predicted biochemical function and biological role in living cells. The development of RNA interference (RNAi) (Guo and Kemphues 1995; Fire et al. 1998) has made possible large-scale in vivo gene analyses in a functional genomic context in Caenorhabditis elegans (Gönczy et al. 2000; Piano et al. 2000, 2002; Hanazawa et al. 2001; Maeda et al. 2001; Kamath et al. 2003; Simmer et al. 2003; Rual et al. 2004) and in cell culture systems from Drosophila melanogaster and mammals (Somma et al. 2002; Kiger et al. 2003; Berns et al. 2004; Boutros et al. 2004; Paddison et al. 2004). RNAi studies enable loss-of-function analyses to be performed on genes that had not been uncovered previously by mutagenesis, thus potentially allowing phenotypic characterization of every gene in the genome. Data from different types of functional genomics projects can be combined to make inferences about gene function and protein networks on a large scale (Marcotte et al. 1999; Ge et al. 2001; Boulton et al. 2002; von Mering et al. 2002; Walhout et al. 2002).
C. elegans embryogenesis is a powerful in vivo model to study gene function in the context of animal development. Its germline is highly sensitive to RNAi, facilitating the detection of genes with essential functions in the embryo. The set of genes known to be required during embryogenesis is enriched for components of pathways that drive cell proliferation and other fundamental processes, and these genes tend to be highly conserved across species (Gönczy et al. 2000
Despite the unprecedented scale of in vivo functional data obtained using RNAi, there are technical challenges to RNAi-based phenotypic analysis. In C. elegans, variables that can affect RNAi include the double-stranded (ds)RNA template source, the delivery method, and scoring criteria (for review, see Piano and Gunsalus 2002
Previous RNAi studies in C. elegans have shown that the set of genes expressed at higher levels in the ovary, relative to the soma or male germline, is enriched for genes required for embryogenesis (Piano et al. 2002 To identify new genes required for embryogenesis, we analyzed 1011 ovary-enriched genes represented in the ORFeome. Over 85% of these had been assayed by RNAi in prior studies and not shown embryonic lethality. This group of genes may thus be enriched for genes whose embryonic requirement, if any, is more difficult to detect by RNAi. We used a combination of repeated RNAi by soaking and injection, and here report the identification of 192 genes involved in egg production or embryogenesis, 155 of which have no previously reported requirement in these stages. A majority of the genes in the ovary-enriched set have now been studied by RNAi by at least three independent studies, making it the most deeply studied set of genes available in C. elegans. Using the combined data from all previous studies of this set, we now include the dimension of penetrance in our analyses of phenotypic trends among the ovary-enriched genes. We observe that trends with regard to sequence similarity, expression level, and chromosomal location are predominantly due to genes with highly penetrant embryonic lethality or sterility. We also observe a relationship between the pattern of conservation across phyla and the penetrance level of embryonic lethality elicited by RNAi.
Evaluation of RNAi results To explore the large-scale RNAi data, we evaluated results from five RNAi studies in C. elegans (Gönczy et al. 2000 ; Piano et al. 2002; Maeda et al. 2001; Kamath et al. 2003; Simmer et al. 2003) with respect to their ability to detect sterile or embryonic lethal phenotypes. Comparisons of RNAi-induced phenotypes with phenotypes arising from genetic mutation suggest that large-scale RNAi studies performed in the wild-type genetic background have a detection rate of  75%, or a false-negative rate of 25%, for maternal sterility or embryonic lethality (Fig. 1). False positive rates, estimated from the number of genes with RNAi-induced phenotypes that are not reported by genetics, were negligible in RNAi studies that used feeding (Kamath et al. 2003; Simmer et al. 2003) or injection (Gönczy et al. 2000; Piano et al. 2002) as a delivery method. In contrast, of the 32 embryonic lethal or sterile genes represented by mutant alleles from the large-scale soaking study (Maeda et al. 2001), six were not reported to be embryonic lethal or sterile by genetics (Kamath et al. 2003). One interpretation of this observation is that RNAi by soaking gives rise to more false positives than does feeding or injection. Another possibility is that the soaking protocol causes physiological changes that influence the effects of reducing gene function on fecundity or embryonic development. However, our own data (see below) suggest a third possibility: that comparisons to genetic analyses include cases where the genetic results were not performed using null alleles or did not report on embryonic lethality simply because the focus of the study was not the germline or embryo. This is more likely to be the case when the genetic null allele shows only partially penetrant sterility (reduced brood size) or embryonic lethality (few unhatched embryos).
An alternative way to examine the efficacy of various RNAi approaches is to compare results from different large-scale RNAi studies directly with each other (Fig. 2). Among genes that give rise to embryonic lethality, studies agreed more often when the penetrance level was high (Class I, 80%-100% embryonic lethality or maternal sterile) than when the penetrance was partial (Class II, 6%-79% embryonic lethality), as judged by the relative number of genes placed into the same versus different penetrance bins. Studies thus appeared more closely matched in their ability to detect genes giving rise to highly penetrant phenotypes and exhibited more variability in reporting partially penetrant lethality, which can be partly explained by scoring protocols (for review, see Sugimoto 2004 ; Piano and Gunsalus 2002). Focusing on the three studies with the most similar scoring methods and conducted in the same genetic background (Piano et al. 2002, "injection"; Maeda et al. 2001, "soaking"; and Kamath et al. 2003, "feeding"), we observe trends not revealed by comparisons with genetics. In direct comparisons between RNAi studies, injection and soaking show similar levels of detection among genes tested by both methods; however when compared to feeding, soaking and injection show more than twice the number of genes required for embryogenesis or oogenesis (Fig. 2). The magnitude of the differences cannot be fully explained by false positives in both the soaking and injection studies, since in comparisons with genetics the injection study had no detectable false positives and soaking had at most 19%. These differences are most parsimoniously explained by a higher level of false negative results in the feeding study that are not revealed by the comparisons with genetics because genes with partially penetrant phenotypes are likely underrepresented among the characterized genetic loci.
Since the comparisons suggest a difference in the strength of the RNAi effect elicited by the different methods, we might expect any such differences to be reflected in the penetrance levels of phenotypes reported. Indeed, when the same gene is analyzed by different studies, we observe a consistent trend in which soaking and injection elicit equivalent or stronger phenotypes than feeding, as judged by the relative penetrance of embryonic lethal phenotypes. For example, 67% of the 46 genes reported as Class II by feeding are classified in either the same or higher penetrance category based on soaking, whereas 22% of the 168 Class II genes reported by soaking are in an equal or higher class based on feeding (Fig. 2). Similarly, 93% of the 28 genes in Class II by feeding are in the same or higher penetrance category in the injection results, whereas 14% of the 164 Class II genes by injection are in an equivalent or higher penetrance category by feeding. A stronger RNAi effect might also be expected to elicit an earlier defect relative to the time of dsRNA delivery. In all studies analyzed, the RNAi treatment was initiated at the young adult stage, so the earliest observable defect in all studies was sterility of the treated animal, followed by embryonic defects among the progeny, and finally postembryonic phenotypes in the progeny. We therefore asked whether any genes displayed sterility or embryonic lethality in one study but only postembryonic defects that were not also accompanied by earlier defects in another study (Supplemental Fig. S1). Comparing genes studied by both feeding and soaking, the total number of genes showing only post-embryonic defects is similar: 128 by feeding and 125 by soaking. However, of the 128 genes that showed only post-embryonic phenotypes in the feeding study, the soaking study found 38 (30%) to be embryonic lethal or sterile. In contrast, of the 125 genes found to have post-embryonic defects alone from the soaking study, only 10 (8%) were found to show embryonic lethality or sterility when tested by feeding. The trend is even more pronounced when comparing feeding and injection (Supplemental Fig. S1). In addition to revealing technical aspects of RNAi analyses, these comparisons indicate that pleiotropy, or the reuse of the same cellular components in different developmental contexts, is a prevalent theme in the biology of this organism. Since the largest studies in C. elegans have been carried out using feeding, the combined data indicate that many genes required for embryogenesis remain to be found, and using RNAi by either soaking or injection could enhance the ability to find them. In addition, the data suggest that comparing phenotypes arising from genetic mutation with those elicited by RNAi does not realistically assess RNAi hit rates among genes with partially penetrant embryonic lethality. We conclude from this analysis that all RNAi delivery methods can detect high-penetrance embryonic lethal or maternal sterile genes with similar efficiency, and that RNAi by soaking or injection appear to be more efficacious in discovering genes that give rise to partial-penetrance embryonic lethality.
Selection of the target gene set and RNAi results
We optimized the RNAi by soaking protocol and validated that we could obtain results comparable to those from singlegene injections using a control gene set of 83 genes (data not shown). In addition we tested our RNAi protocol using GFP-specific dsRNA and found negligible levels of embryonic lethality (1.2%, n = 1572). In a first round of RNAi assays applied to the test set, we found a total of 569 clones that gave rise to sterility of the RNAi-treated animal (Ste) and/or embryonic lethality (Emb); 241 of these were retained as Emb or Ste upon retesting. Sequencing verified the expected identity of 201 of the 241 clones; the remaining 40 were discarded from further analysis. By mapping these clones to genes we identified 195 independent genes that gave rise to high-confidence Emb and/or Ste phenotypes, of which 158 are identified here for the first time (Fig. 3, Supplemental Table S1). Three of these were removed from the final analyses because they currently map to genes not in the ovary-enriched list (Supplemental Table S1). We estimated the likelihood of false positive results by comparing our phenotypes to those derived from genetic analyses (Fig. 3B, Supplemental Table S2). Nineteen genes that we found to be maternal sterile or embryonic lethal (cdk-5, mex-5, mdt-6, air-2, mom-5, hrp-1, aph-1, egl-18, dpy-30, ife-3, mom-4, aph-2, sqv-7, sqv-5, ooc-3, spn-4, lrs-2, csc-1, and lin-3) were represented by characterized mutant alleles. For this analysis we excluded genes represented by mutant alleles that were not well characterized (listed in Supplemental Table S2). Null or severe hypomorphic alleles of all but one of these genes also give rise to embryonic lethality or maternal sterility. Our single putative false positive is cdk-5. We conclude that our false positive rate is low (1/19) and that the majority of the genes we uncovered here have a role in embryogenesis. To gauge our false-negative rate we also performed comparisons with published reports from genetic analyses. Of the 25 total genes we studied that also give rise to embryonic lethal or maternal sterile phenotypes upon genetic mutation, we identified 18 (72%) and failed to report seven (hus-1, mom-1, inx-3, cup-5, puf-8, sqv-1, and lin-41). Notably, four of the genes we missed showed lethality in one round of assays only, and were thus classified as low-confidence lethals (Supplemental Table S2). Thus, although our selection criteria might be expected to preferentially include genes that are harder to identify using RNAi, our false-negative rate based on genetic comparisons was comparable to those of previous studies (Fig. 1). Independent RNAi studies found that 25 of the genes we tested elicit embryonic lethal or maternal sterile phenotypes upon RNAi. We detected 17 of these, of which 14 were detected reproducibly. In order to minimize false positives, we chose to be conservative in our assignment of phenotypes; thus we did not report embryonic lethality in our final analysis for any of the low-confidence lethal results. Our comparisons suggest that this practice elevated our false-negative rate and also indicate that we have not yet identified all the genes required for embryogenesis in our sample set. Based on our comparisons of published RNAi studies and the gene selection strategy we used in this study, we would expect to recover a nonrandom distribution of Emb or Ste genes among the different subsets of genes we assayed by RNAi. The distribution of phenotypes among the different subsets is consistent with this expectation (Fig. 4). First, a significantly higher overall rate of Emb and Ste phenotypes was detected in the gene sets that either had never been tested by RNAi (19/69 or 28%; Fig. 4B) or had been tested and found to give rise to post-embryonic phenotypes only (52/151 or 34%; Fig. 4C), versus the set of genes that previously failed to give rise to any detectable phenotype (107/766 or 14%; Fig. 4D). Furthermore, 58% (11/19) of the Emb and Ste genes found in the set that had never been tested were in the highest penetrance classes (MS and EL), whereas only 30% (47/159) of Emb or Ste genes from among those that had been tested before but had not previously shown embryonic lethality fell into these classes. These results are consistent with the idea that many of the highest-penetrance embryonic lethal genes had already been removed from the set we analyzed. In addition, the data show that genes with known post-embryonic RNAi phenotypes are much more likely to display embryonic lethality upon retesting than genes with no reported RNAi phenotypes from prior assays.
The 158 new genes we have identified with roles in embryogenesis or egg production augment by 9% our knowledge of the genes required for these stages of development in C. elegans. The early embryo is particularly well suited to study cell biological defects generated by the RNAi treatments and can help reveal specific roles in cell division. To further characterize the effects on embryogenesis, we repeated RNAi by injection or soaking for the 39 genes whose RNAi analysis elicited the highest-penetrance embryonic lethality. We studied the early embryonic defects by time-lapse digital Nomarski microscopy from the one-cell stage to the eight-cell stage (we obtained, on average, eight time-lapse recordings per gene, available as Quicktime movies though www.RNAi.org). Among these are five genes (csc-1, mom-5, mex-5, air-2, spn-4) whose role in early embryogenesis was already known, and we recovered the known phenotypes in our assays. For the 34 genes that were newly identified here or were not previously studied in the early embryo, we analyzed detailed phenotypes during the first 50 min of development and scored them as previously described (Piano et al. 2002; Supplemental Table S4). The range of defects observed is similar to that of previous reports (Gönczy et al. 2000, Piano et al. 2000, 2002; Zipperlen et al. 2001), and results in the identification of new genes required for specific early embryonic events such as completion of meiosis, nuclear movements, and mitosis.
Among the genes identified as being required for embryogenesis, we identified four genes (C47D12.8, F14B4.2, F54F2.8, and F35F11.1) whose human homologs have roles in disease (ERCC4, HK2, PXF, and HRPT2, respectively). We recorded early embryonic phenotypes for C47D12.8 and F35F11.1 (the others showed partial-penetrance lethality). Both of these genes are homologs of human genes whose mutations are associated with tumors. Interestingly, RNAi of F35F11.1 (the HPRT2 homolog) causes defects in the proper formation of polar bodies, pronuclei, and mitotic nuclei, indicating that this gene is important for the proper completion of both meiosis and mitosis (see Supplemental Table S4 and associated supplementary recordings). Although HRPT2 was demonstrated to function as a tumor suppressor in parathyroid carcinoma (Carpten et al. 2002
How many genes are required for embryogenesis in C. elegans?
Functional trends for the ovary-enriched genes
When we examined the combined data with respect to penetrance, we found that functional trends are driven mostly by genes giving rise to the highest-penetrance phenotypes. The ovary-enriched gene set displays a positive and statistically significant correlation between essential gene function in the embryo and both sequence conservation (Fig. 5A) and fold enrichment in the ovary (Fig. 5B); however, these trends are dominated by the highest-penetrance phenotypic class ("MS-EL"). Previous studies have observed that the X chromosome contains significantly fewer than expected genes required for oogenesis or embryogenesis (Piano et al. 2000
We also examined patterns of functional annotations with respect to penetrance (Fig. 5D). In the highest penetrance class, we observed a highly significant (P < 0.001) overrepresentation of genes involved in fundamental cellular processes (RNA and protein synthesis; DNA synthesis and repair/cell cycle, and cellular architecture) that was mirrored by a significant depletion among genes with no known requirement in the embryo. Overrepresentation of these functional classes was also observed among genes with nonviable phenotypes in a genome-wide RNAi survey (Kamath et al. 2003 ). However, including the new dimension of penetrance here offers a level of resolution that reveals a more subtle pattern in the data: the proportion of genes involved in these processes rises as the penetrance of embryonic lethality increases. Thus there is a positive correlation between penetrance and enrichment in these functional classes. The only other statistically significant deviation is an overrepresentation of partially penetrant genes with metabolic functions, potentially reflecting some level of redundancy in embryonic metabolic pathways. Among genes that display only post-embryonic phenotypes, no functional classes are significantly over- or underrepresented (P < 0.01 for all classes, data not shown). One way to interpret these trends is that genes classified as playing a role in fundamental cellular processes are more likely to be indispensable for embryogenesis than genes classified in more regulatory roles.
Penetrance level and conservation across phyla
A priori, we suspected that penetrance designations derived from RNAi assays may not be particularly informative, as RNAi does not always deplete gene function completely and thus may not necessarily phenocopy null phenotypes. However, our analyses revealed strong trends among the different penetrance classes. This suggests that penetrance reported by RNAi can genuinely reflect differences in gene function when animals are observed under laboratory conditions, particularly when a gene has been assayed multiple times by different groups. The group of genes showing the strongest penetrance not only contains a higher proportion of genes involved in a few critical cellular processes, but is also significantly enriched for genes that are highly conserved in other species—a trend that is not observed among other, less penetrant phenotypic categories. Together these observations suggest that highly conserved genes carrying out indispensable functions occupy critical positions in gene networks that are essential to cell survival at any stage of development, whereas less-conserved genes may contribute more peripheral, or modulatory, functions that serve to adapt these core modules to specific cellular contexts and provide plasticity to genetic networks. The observation that the proportion of eukaryotic genes (the most ancient phylogenetic class) increases with increasing penetrance and that the proportion of worm-specific genes (the most recently evolved phylogenetic class) decreases with increasing penetrance suggests that the importance of a given gene's function can change over time. We speculate that as new genes arose over history, some have gradually become fixed in more crucial roles in the networks underlying biological processes. Another way to express this is to say that those genes that are retained over very long evolutionary times are those that have taken on indispensable roles. This suggests the possibility of a general architectural motif in genetic networks, in which genes entrenched in critical positions consist largely of older components, whereas newer functions added later provide additional layers of regulation that lend robustness or adaptive functions to these core networks. Combined analysis of phenotypic, gene expression, and protein interaction data from C. elegans will help resolve questions about how gene networks are adapted to different cellular contexts, and will reveal underlying architectural principles of metazoan development.
Generation of dsRNA ORFeome clones were amplified by PCR using T7 primers. Amplified products were obtained for 1123 of the 1245 ORFeome clones successfully cherry-picked for this study. PCR products were used as templates for in vitro transcription by T7 RNA polymerase in a 50-µL reaction. All RNA products were verified on 1% agarose gels, then precipitated by adding 5 µL sodium acetate (pH 5.2), 2 µL Pellet Paint (Novagen), and 100 µL ethanol. The dsRNA was stored in this manner at -20°C for a maximum of 1 wk. All molecular biology procedures and soaking were performed in 96-well plates.
RNAi by soaking
Penetrance designations
Selection of ORFeome clones and mapping to C. elegans gene products After completing the RNAi analysis, we remapped this set of ORFeome clones and the PCR products used to define the ovary-enriched gene set to the latest WormBase freeze (WS110). The set of 1083 ORFeome clones for which we reported RNAi results currently maps to 1011 ovary-enriched and 16 non-ovary-enriched genes (not including alternative splice forms). Only the ovary-enriched fraction was used in global analyses of ovary-enriched gene function. A total of 53 genes were independently assayed by two or more different ORFeome clones; for 14 of these, replicate assays yielded results in different penetrance classes. RNAi results for all clones assayed are reported in Supplemental Table S1. Some ORFeome clones overlap two adjacent genes. This indicates a discrepancy between the current gene models in WormBase and sequence evidence from these ORFeome clones; each of these cases was reviewed at WormBase, and the gene models were left unchanged due to discrepancies between EST data from cDNA clones and evidence based on the ORFeome clones. For clones that extensively overlapped two open reading frames, when the RNAi assay was negative (LCL or NELD), we reported this result for each gene. In the few rare cases where a clone overlapped one ORF extensively and another by <200 base pairs, we considered only the former gene to have been reliably assayed and conservatively reported no RNAi result for that clone in association with the second gene. Wherever we had independent confirmation of such assays from clones that spanned only one of the two adjacent transcription units, the results were always consistent with these policies.
Time-lapse imaging
Bioinformatic analysis
Reciprocal whole-proteome BLASTP analysis (Altschul et al. 1997
For comparative analyses and characterization of phenotypic trends among ovary-enriched genes, all RNAi results were mapped to genes in WormBase (WS110) using the chromosome coordinates of the reagents used for each assay. Genes were placed into exclusive categories according to the most severe phenotype observed by any group. Our comparative analyses included only those phenotypic results from Simmer et al. (2003
We thank Casey Roehrig, Danny Shapiro, Tony Tao, and Krishna Vijayendran for technical assistance; Wanchen Yueh, Tong Hao, and Philippe Lamesch for help with data processing; WormBase curators for reviewing gene models; Dr. Julie Ahringer for critically reviewing our manuscript; Dr. Jane Hubbard for useful insights; and anonymous reviewers who provided useful comments that improved our manuscript. This work was supported by grants from NSF (DBI-0137617, to K.C.G.) and NIH(HG003019-01, to F.P.). A.J.S. is currently funded by the NCI CPFP. The ORFeome project is supported by a grant from NCI (7R33CA81658-02, to M.V.).
4 These authors contributed equally to this work. 
5 Corresponding author. [Supplemental material is available online at www.genome.org and RNAiDB (www.RNAi.org). Results from this study are also available through WormBase (www.wormbase.org).] Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.3194805.
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ABSTRACT
Results and Discussion
80% embryonic lethal); II, Class II penetrance (6%-79% embryonic lethal); III, Class III (no sterility or embryonic lethality detected). Numbers in green indicate similar results by both studies; purple and blue numbers represent genes with sterility or lethality according to one study but not the other (blue: detected only by the study listed at the head of the panel column; purple: detected only by the study at the head of the panel row). For each study the total number of unique genes (excluding alternatively spliced forms) assayed (N) is indicated. Venn diagrams summarize the data in adjacent tables and show the total numbers of genes with sterile or embryonic lethal phenotypes detected by both (green) or only one study (blue, purple). The total number of overlapping genes analyzed by both studies (n) is indicated below each Venn diagram. Results from Simmer et al. (2003
10 progeny; EL, embryonic lethal, 80%-100% embryonic lethality; HPEL, high-penetrance embryonic lethality, 50%-80%; LPEL, low-penetrance embryonic lethality, 20%-50%; VLL, very low lethality, 5%-20%; LCL, low-confidence lethality; NELD, no embryonic lethality detected.
