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Identification of genes expressed in the hermaphrodite germ line ofC. elegansusing SAGE Wang, Xin; Zhao, Yongjun; Wong, Kim; Ehlers, Peter; Kohara, Yuji; Jones, Steven J; Marra, Marco A; Holt, Robert A; Moerman, Donald G; Hansen, Dave May 9, 2009

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ralssBioMed CentBMC GenomicsOpen AcceResearch articleIdentification of genes expressed in the hermaphrodite germ line of C. elegans using SAGEXin Wang1, Yongjun Zhao2, Kim Wong2, Peter Ehlers3, Yuji Kohara4, Steven J Jones2, Marco A Marra2, Robert A Holt2, Donald G Moerman5 and Dave Hansen*1Address: 1Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada, 2Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 4S6, Canada, 3Department of Mathematics and Statistics, University of Calgary, Calgary, Alberta T2N 1N4, Canada, 4National Institute of Genetics, 1111 Yata, Mishima 411-8540, Japan and 5Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, CanadaEmail: Xin Wang - wangxin@ucalgary.ca; Yongjun Zhao - yjzhao@bcgsc.ca; Kim Wong - kimw@bcgsc.ca; Peter Ehlers - ehlers@math.ucalgary.ca; Yuji Kohara - ykohara@lab.nig.ac.jp; Steven J Jones - sjones@bcgsc.ca; Marco A Marra - mmarra@bcgsc.ca; Robert A Holt - rholt@bcgsc.ca; Donald G Moerman - moerman@zoology.ubc.ca; Dave Hansen* - dhansen@ucalgary.ca* Corresponding author    AbstractBackground: Germ cells must progress through elaborate developmental stages from anundifferentiated germ cell to a fully differentiated gamete. Some of these stages include exitingmitosis and entering meiosis, progressing through the various stages of meiotic prophase, adoptingeither a male (sperm) or female (oocyte) fate, and completing meiosis. Additionally, many of thefactors needed to drive embryogenesis are synthesized in the germ line. To increase ourunderstanding of the genes that might be necessary for the formation and function of the germ line,we have constructed a SAGE library from hand dissected C. elegans hermaphrodite gonads.Results: We found that 4699 genes, roughly 21% of all known C. elegans genes, are expressed inthe adult hermaphrodite germ line. Ribosomal genes are highly expressed in the germ line; roughlyfour fold above their expression levels in the soma. We further found that 1063 of the germline-expressed genes have enriched expression in the germ line as compared to the soma. Acomparison of these 1063 germline-enriched genes with a similar list of genes prepared usingmicroarrays revealed an overlap of 460 genes, mutually reinforcing the two lists. Additionally, weidentified 603 germline-enriched genes, supported by in situ expression data, which were notpreviously identified. We also found >4 fold enrichment for RNA binding proteins in the germ lineas compared to the soma.Conclusion: Using multiple technological platforms provides a more complete picture of globalgene expression patterns. Genes involved in RNA metabolism are expressed at a significantlyhigher level in the germ line than the soma, suggesting a stronger reliance on RNA metabolism forcontrol of the expression of genes in the germ line. Additionally, the number and expression levelof germ line expressed genes on the X chromosome is lower than expected based on a randomPublished: 9 May 2009BMC Genomics 2009, 10:213 doi:10.1186/1471-2164-10-213Received: 2 December 2008Accepted: 9 May 2009This article is available from: http://www.biomedcentral.com/1471-2164/10/213© 2009 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 19(page number not for citation purposes)distribution.BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213BackgroundGerm cells follow an elaborate developmental program toproduce fully differentiated gametes. In most animals,germ cells first proliferate to generate a pool of cells, someof which cease proliferating and enter into meioticprophase, progress through the many stages of meiosisand finally differentiate into sperm or oocytes. Through-out the entire process of gamete formation, genesinvolved in differentiation of the soma must be repressed,and genes necessary for progression through meioticprophase, gamete formation and germline sex determina-tion must be expressed [1]. Additionally, many of the fac-tors needed for embryogenesis are produced in the germline. Much of our understanding of the many stepsinvolved in germline development and gamete formationhas come from study of genetically tractable model organ-isms, such as Drosophila and C. elegans. The purpose of thisstudy is to identify many of the genes involved in theproper function of the C. elegans hermaphrodite germline.The gonad in the C. elegans hermaphrodite consists of twoU-shaped tubes that meet at a common uterus (Figure 1)[2]. The gonad is completely enclosed by a basementmembrane. Along the length of the gonad, and within thebasement membrane, are five pairs of somatic sheath cellsthat provide structure to the gonad, as well as providesome reproductive functions [2-4]. The end of each tubefurthest, or most distal, from the uterus is capped by asomatic cell called the distal tip cell (DTC). Most of thenuclei within the gonad are only partially enclosed bymembranes; i.e., they are syncitial. We will refer to thenuclei, their surrounding cytoplasm, and partially enclos-ing membranes as cells. Germ cells closest to the DTC areproliferative. As the proliferative germ cells divide, theymove away from the DTC, towards the uterus. Once theyare approximately 20 cell diameters from the DTC, theyshow the first signs of entering into meiotic prophase[5,6]. These early meiotic cells have a crescent shapednuclear morphology and are within a region of the gonadreferred to as the transition zone [5,7]. As cells continue tomove proximally, towards the uterus, they progressthrough the various stages of meiotic prophase. In the her-maphrodite, the first ~40 germ cells in each gonad armdifferentiate as ~160 spermatocytes, which are stored inthe spermatheca [2]. All subsequent germ cells differenti-ate as oocytes, which undergo ovulation and enter thespermatheca, where they are fertilized by the sperm. Thezygote then moves into the uterus and undergoes earlyembryonic development before being expelled into theenvironment through the vulva [2]. While there are only959 somatic cells in the adult hermaphrodite, each gonadarm generates about 1000 cells [8,9]. Somatic cells do notmuch of the adult life of the animal [2,9]. Therefore, asubstantial amount of energy and resources in the adulthermaphrodite are spent on producing gametes.Genetic screens, both forward and reverse, have identifiedmany of the genes and genetic pathways involved in gen-erating gametes [10]. However, we are still far from fullyuncovering the underlying biological complexity of gam-ete formation. Large-scale microarray studies have greatlyassisted in determining the genes involved in germlinedevelopment and gamete formation by identifying mRNAtranscripts that are enriched in the germ line [11,12]. Aspart of these studies, transcripts in intact animals werecompared to animals that lacked a germ line due togenetic mutation. In L4 and adult hermaphrodites, 3144genes were demonstrated to have germline-enrichedexpression. The generation of this data set, as well as datasets of male germ cells, female germ cells and staged lar-vae, have assisted in our general understanding of thegenes involved in gamete formation, as well as provided astarting point for more detailed analyses of the specificroles these genes play in the germ line [7,13-21]. How-ever, all genome analysis technological platforms haveinherent advantages and disadvantages [22]. Therefore,using multiple technological platforms provides a morecomplete view of the transcription profile of the tissue oranimal being studied, as well as provide increased confi-dence of the overlapping data. It also provides an oppor-tunity to analyze the strengths and limitations of eachplatform.Here we describe our analysis of germline transcription inthe adult hermaphrodite germ line using Serial Analysis ofGene Expression (SAGE) [23]. This analysis adds to previ-ous microarray analyses by identifying genes transcribedin the germ line independent of their level of expressionin the soma [11,12]. Furthermore, we use the SAGE dataas a measure of relative expression levels between genes toidentify the genes and gene classes whose mRNA is mostabundant in the germ line. Finally, by comparing our datawith previously published soma SAGE data [24], we areable to identify genes that are enriched in the germ line,which allows for a comparison of the SAGE and microar-ray platforms [11].ResultsProduction and overview of the germline SAGE libraryWe constructed a SAGE library from ~150 hand dissectedC. elegans hermaphrodite gonads to identify genes that aretranscribed in the germ line. The germline SAGE libraryidentified 92,007 tags, which was normalized to 100,000tags for further analysis. While important previous studieshave identified transcribed genes that are expressed at aPage 2 of 19(page number not for citation purposes)divide in the adult hermaphrodite; however, germ cellscontinue to proliferate and form gametes throughouthigher level in the germ line than the soma [11,12], ouranalysis identifies genes that are expressed in the germBMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213Page 3 of 19(page number not for citation purposes)The germ line of an adult C. elegans hermaphroditeFigur  1The germ line of an adult C. elegans hermaphrodite. (A) On the top is a drawing of an adult C. elegans hermaphrodite emphasizing the cells of the germ line. The gonad consists of two reflexed arms that meet at a common uterus. At the very dis-tal end of each arm is the somatic distal tip cell (DTC; yellow). Germ cells near the DTC are proliferative (green). As cells move proximally, towards the uterus, they enter meiotic prophase (red). The first cells to differentiate are sperm (blue), which are stored in the spermatheca. As oocytes (orange) pass through the spermatheca, they become fertilized and begin embryo-genesis (green). Below the diagram is a DIC image of one gonad arm in an adult hermaphrodite. (B) Illustrated is a summary of the tissues used to obtain mRNA for the germline microarray analysis [11], the soma SAGE library [24] and the germline SAGE library (this work). In the microarray analysis mRNA obtained from wild-type (N2) worms was compared to mRNA obtained from glp-4(bn2ts) worms. Germ cells in glp-4(bn2ts) animals grown at the restrictive temperature arrest in mitosis such that only ~12 germ cells are present [41]. The somatic gonad is still present, but the gonad arms do not reflex back. mRNA for the construction of the germline SAGE library was obtained from ~150 hand dissected gonad arms. The gonad arms were dis-sected away from the body of the animal at or near the spermatheca. The soma SAGE library was constructed from mRNA isolated from glp-4(bn2ts) animals grown at the restrictive temperature.BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213line irrespective of their level of soma expression. Thisprovides a broader picture of genes involved in germlinefunction, independent of the roles the genes may have inthe soma. Additionally, since the number of tags per geneis quantified in SAGE, we are able to consider the relativelevels of expression between genes.We dissected the gonad arms away from the soma of theanimals at, or close to, the spermatheca (see Methods)(Figure 1). Therefore, the cell types in the dissected gonadarms include the proliferative germ cells at the distal endof the gonad, germ cells at various stages of meioticprophase, oocytes, and perhaps some sperm. The dis-sected gonads also contain some cells of the somaticgonad, including the distal tip cell, sheath cells, and atleast some of the 24 cells that form the spermatheca.The analysis of the germline SAGE library identified a totalof 4699 genes with associated SAGE tags (Additional file1), suggesting that these genes are transcribed in the germline (see Methods for a description of the criteria used todetermine genes associated with SAGE tags). Theseexpressed genes correspond to approximately 21% of thetotal predicted genes in the C. elegans genome. The germ-line-expressed genes have a wide range of expression lev-els based on the number of tags per gene, with a low ofone tag per gene to a high of 916 tags per gene (Figure 2;Additional file 1). The number of genes within a range ofSAGE tags per gene obeys a power law; that is, that the log-arithm of the number of tags per gene has a linear rela-tionship to the logarithm of the number of genes thathave a similar number of tags (Figure 2a). Therefore, thenumber of genes with a given expression level decreases asthe level of expression increases (Figure 2). Other C. ele-gans SAGE libraries have also shown a power law relation-ship between the expression level of a group of genes, andthe number of genes within the group [24].Genes highly represented in SAGE libraryTo better understand the functions of the genes with themost abundant transcript levels in the C. elegans hermaph-rodite gonad, we analyzed the genes with the highest tagcounts. So as to analyze a manageable list of genes, weanalyzed genes with tag counts greater than 60, whichresulted in a list of 157 genes (Additional file 2). To firstcorroborate our identification of these genes as beingexpressed in the germ line, we categorized their expressionpatterns using the NEXTDB large scale in situ database,which provides pictures of mRNA in situ staining patternsat various stages of development for many genes in thegenome [25]. 124 of the 157 genes are included in theNEXTDB database and have discernable expression pat-terns (Additional file 2). All of the 124 genes showedexpression in the germ line, 18% with exclusive germlineexpression, 63% with enriched germline expression and19% with similar expression in the germ line and somaDistribution of transcripts in the germline SAGE library and the germline-specific/enriched dataset identifiedFigure 2Distribution of transcripts in the germline SAGE library and the germline-specific/enriched dataset identified. (A) The distribution of tag counts in the germline SAGE library obeys a power law. More genes were identified as being expressed at low transcript levels, whereas significantly fewer genes are expressed at modest to high transcript levels. How-ever, considerably higher numbers of tags are identified by the small subset of genes expressed at relatively high levels. Only 52 genes were identified to have a tag counts > 254, whereas they account for ~33% the total tags identified in the germline library. (B) The portion of the germline library identified to be germline-specific/enriched increases with increasing tag counts. Tag count ≥ 9 was chosen as a cut off to increase the confidence of the germline-specific/enriched gene set. With tag count ≥ Page 4 of 19(page number not for citation purposes)9, 1063 out of 1407 (~75%) of the germline library genes were identified to be germline-specific or germline-enriched.BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213(Additional file 2). Therefore, the NEXTDB mRNA in situexpression patterns are consistent with our identificationof these genes as being transcribed in the germ line. Thefunctions of most of these genes have been analyzed, at agross level, in large-scale RNA interference (RNAi) screens[26,27]. The majority of the genes (83%) have pheno-types consistent with a germline function, such as germcell proliferation abnormal (Gpro), gonad developmentabnormal (Gdb), sterile (Ste), sterile progeny (Stp) andabnormal embryogenesis (Emb) (Additional file 2) (Thisis statistically significant, p < 0.001, as when we analyzedall genes in the genome tested by RNAi, only 15.5% havethese germline phenotypes). Some of the genes not show-ing a germline phenotype may still have a germline func-tion, but the phenotype may have been too subtle to bedetected in large-scale screens, or they may functionredundantly with another gene(s). It is also possible thatsome genes that are transcribed in the germ line do nothave germline functions (see Discussion). The NEXTDBexpression patterns and RNAi phenotypes both supportour detection of these genes as germline-expressed.To gain a general picture of the functions of the 157 geneswith the highest expression in the germ line, as well as thefunctions of all 4699 genes with SAGE tags in the germlinelibrary, genes were annotated with KOG (eukaryoticorthologous groups) descriptions and classified into KOGcategories and sub-categories (Additional file 2; Figure 3a)[28,29]. For all germline-expressed genes, we determinedthe number of genes in each KOG category, as well as thenumber of tags in each category (Figure 3a). Of all of thegenes identified in the germline SAGE library, ~75% haveassociated KOG terms. However, ~90% of tags identifiedcorrespond to genes with associated KOG terms; there-fore, many of the genes lacking a KOG term have relativelylow expression levels. The KOG category with by far themost germline-expressed genes is Information storage andprocessing, particularly its sub-category Translation, ribos-omal structure and biogenesis [compared to soma SAGElibrary (see below) there is a significant enrichment of thissub-category in the germ line, p < 0.001] (Figure 3); 66 ofthe 157 most highly expressed genes fall within this sub-category (Additional file 2). While most of the genes inthis sub-category encode ribosomal proteins, alsoincluded are some genes encoding translational regula-tors; puf-3, puf-5 and puf-11 each encode proteins homol-ogous to the Drosophila Pumilio. The Puf (Pumilio andfbf) family of proteins consists of similar RNA bindingproteins that have been shown to control many cellularand developmental processes in many tissues, includingthe C. elegans germ line [30-36].Among the 157 genes with the highest tag counts in theWhile proteasome mediated degradation of proteins wasonce thought to be primarily involved in the degradationof misfolded or damaged proteins, increasing evidencehas shown that proteasome mediated degradation is animportant regulatory mechanism in the development andfunction of cells and tissues [37]. In the C. elegans germline, the proteasome has been implicated in the regula-tion of germline sex determination and the proliferationvs. meiotic entry decision in the germline stem cells [38-40].Many of the 157 genes most highly expressed in the germline are often referred to as 'house-keeping' genes, or genesthat are necessary for the general function of virtually anycell, including genes involved in mitochondrial function,ribosome structure and other cellular processes. To deter-mine if the number of genes and their expression levelsobserved in the germ line are typical of the soma, we firstdetermined the number of genes and number of SAGEtags for each KOG category and sub-category for the entiregermline SAGE library (Figure 3a). We then compared thenumber of genes and number of tags in each KOG sub-cat-egory in the germline SAGE library with the number ofgenes and tags in each sub-category from a soma SAGElibrary (Figure 3b). A soma C. elegans SAGE library hasalready been published, which was generated from wormslacking a germ line due to the glp-4(bn2ts) temperaturesensitive mutation [24]. Germ cells in glp-4(bn2ts) worms,grown at the restrictive temperature, arrest early in mitosisallowing only ~12 germline nuclei to be formed pergonad arm; however, the soma in glp-4(bn2ts) animalsappears wild type [41]. Therefore, the soma library wasgenerated from virtually all tissues not used in the gener-ation of our germline SAGE library; however, some cells ofthe somatic gonad are common to both libraries. In thegermline SAGE library there is a modest increase in thenumber of expressed genes in the Translation, ribosomalstructure and biogenesis KOG sub-category as compared tothe soma (Figure 3b). However, when gene expressionlevels are taken into account, based on tag number, thereis a dramatic increase in the Translation, ribosomal structureand biogenesis sub-category in the germline SAGE library ascompared to the soma library (p < 0.001; Figure 3b). Thisincrease in tag numbers in the germline SAGE library isprimarily due to the large number of tags associated withribosomal genes. Among the 87 ribosomal protein encod-ing genes in the C. elegans genome [42], 53 are identifiedby SAGE to be highly expressed (tag count >60) in thegerm line (23 ribosomal small subunits and 30 large sub-units; Additional file 2). The expression levels for theribosomal genes are strikingly high, as they produce ~27%of all tags in the germline SAGE library (Figure 3a). Thehigh level of expression of these ribosomal genes suggestsPage 5 of 19(page number not for citation purposes)germ line are a number of genes of the ubiquitin/proteas-ome protein degradation pathway (Additional file 2).that ribosome biogenesis is an essential component tomaintain normal germline function and developmentBMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213Page 6 of 19(page number not for citation purposes)KOG classification of the germline SAGE libraryFigure 3KOG classification of the germline SAGE library. (A) KOG distribution of the germline library based on either the number of genes or the number of tags. A total of 3555 genes can be assigned a KOG classification in the germline SAGE library. "Information Storage and Processing" is over-represented in distribution by the number of tags, and this over-represen-tation is mainly contributed by the subcategory "Translation, Ribosomal Structure and Biogenesis". The over-representation of "Information Storage and Processing" is not observed in the soma library (data not shown). Overall, the KOG distribution by the number of genes is highly similar between the germline library and the soma library; however, some categories or sub-cat-egories do show some enrichment in the germ line or soma, the most striking of which are described in the text (data not shown). (B) Shown are the percentages of the KOG sub-categories with respect to the total number of genes (upper panel) or the total number of tags (lower panel) assigned with KOGs.BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213(see Discussion). Supporting this idea, nearly all of theribosomal genes show RNAi phenotypes consistent with agermline function, such as embryonic lethality/develop-mental defect and sterility [26,27] (Additional file 2).Other KOG sub-categories that have a higher percentageof tag counts (p < 0.001), suggesting higher expressionlevels, in the germ line than the soma are; (1) Cell cyclecontrol, chromosome partitioning, (2) Replication, recombina-tion and repair, (3) Transcription, and (4) RNA processingand modification. It is not surprising that genes in the firsttwo sub-categories are expressed at a higher level in thegerm line. Since both the soma and germline librarieswere generated from adult tissue, and since the germ lineis the only tissue with dividing cells in the adult, it is log-ical that these two sub-categories, which deal with thedivision of cells, have a higher level of expression in thegerm line than the soma. The third and fourth sub-catego-ries listed above have to do with the transcription ofmRNA and its modification. It seems reasonable that ifmore translation occurs in the germ line than other tis-sues, based on the high level of expression of ribosomalgenes, more mRNA would need to be made and proc-essed.All other KOG sub-categories have either similar expres-sion levels in the soma and germ line, or have higherexpression levels in the soma. Perhaps the two most strik-ing examples of KOG sub-categories with higher expres-sion levels in the soma than germ line are Lipid transportand metabolism and Signal transduction mechanisms (p <0.001). The intestine is likely the tissue in which mostenergy production occurs, and many genes involved inlipid metabolism are enriched in the intestine [24]; there-fore, it is logical that expression levels are higher in thesoma than germ line. The greater than two-fold increase inexpression in genes involved in Signal transduction mecha-nisms suggests that the soma relies more heavily on sign-aling for proper function.Genes encoding RNA binding proteins are expressed at a higher level in the germ lineMany RNA binding proteins have essential functions inthe germ line of C. elegans, with RNA metabolism being apredominant mechanism for the control of gene expres-sion in this tissue [43,44]. It has recently been suggestedthat the expression of proteins in the C. elegans germ linerelies primarily upon the control of translation or mRNAstability regulated through the 3'UTR [45]. In order todetermine if RNA regulators are expressed at a higher levelin the germ line than the soma, we identified a list of 319RNA binding proteins (See Methods; Additional file 3).We found that 190 of these genes are expressed in theare expressed in the soma. Taking into account thenumber of tags found in each library, we found that tran-scripts of these RNA binding proteins are expressed >4fold in the germ line than the soma; 3267 tags were iden-tified in the germline SAGE library, whereas only 775 wereidentified in the soma library (Fisher's exact p-value <0.001). The higher expression level in the germ line is notdue to one or few genes being expressed at much higherlevels in the germ line; rather, most genes are expressed ata higher level in the germ line as compared to the soma(Additional file 4). The higher level of expression of genesencoding RNA binding proteins supports the model of theexpression of genes in the germ line relying more heavilyupon RNA metabolism for control.Identification of germline-enriched and germline-specific genesThe germline SAGE library allows us to identify genestranscribed in the germ line, as well as their relativeexpression levels, irrespective of their expression in thesoma. Previous studies of germline expression usingmicroarrays have produced valuable lists of genes whosetranscripts are enriched in the germ line [11,12]. Genesthat are expressed at a higher level in the germ line thanthe soma may have roles that are more specific to germ-line function. As mentioned above, a SAGE library hasbeen generated from somatic tissues of adult hermaphro-dites [24]. By comparing the expression levels (tag counts)of genes in our germline SAGE library with the expressionlevels of the same genes in the soma, as determined by thesoma SAGE library, we can identify genes whose transcrip-tion is enriched in the germ line. This comparison is anal-ogous to the microarray analyses that identified germline-enriched genes, in which animals with a germ line werecompared to animals lacking a germ line (Figure 1b)[11,12]. All technological platforms, including SAGE andmicroarrays, have inherent strengths and weaknesses [22];therefore, identifying germline-enriched genes using a dif-ferent technological platform (SAGE instead of microar-ray), may allow for the identification of additionalgermline-enriched genes. Additionally, SAGE will help tovalidate many of the genes identified by microarray asbeing germline-enriched. Finally, an analysis of the lists ofgermline-enriched genes obtained by microarray andSAGE will help to uncover some of the inherent strengthsand weaknesses of each technological platform.Before we describe the comparison between the germlineand soma SAGE libraries, it is important to emphasizethat these two libraries were not made at the same timeand differ somewhat in their construction, which compli-cates their comparison and the interpretation of results(see Methods). The primary difference is that the somaPage 7 of 19(page number not for citation purposes)germ line, based on the presence of one or more tags inthe germline SAGE library, while only 131 of these geneslibrary was made using short SAGE, while the germlinelibrary was made using long SAGE. 'Short' and 'long' refersBMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213to the size of the SAGE tag produced upon restrictiondigestion [46]. Therefore, the soma and germline SAGElibraries differ somewhat in the genes that have associatedunique SAGE tags. Genes that do not have SAGE tags inone or both of the libraries were not included in the com-parison (see Methods). Other differences between thegermline and soma SAGE libraries include the genotypesof the animals; the germline library was constructed fromwild-type worms (N2), while the soma library was con-structed from glp-4(bn2) worms [24], which caused themto lack germline tissue. However, these were the same gen-otypes that were used in the microarray analysis [11]. As afinal difference, the wild-type worms for the germlineSAGE library were grown at 20°, while the glp-4(bn2ts)worms were shifted from 15° to 25°, causing the animalsto have severely reduced germ cell number [41]. Whilethese differences in library construction may have aneffect on the expression of some genes, independent ofthe inherent differences in germline and soma expressionlevels, the microarray data [11] and large scale in situexpression data (NEXTDB) [25] provide excellent tools tohelp determine the extent the differences in library con-struction may have on interpretation.To obtain a list of germline-enriched genes, we comparedthe number of tags in the germline SAGE library with thenumber of tags in the soma SAGE library for each gene(Figure 4) [47]. 87% of genes that were identified in thegermline SAGE library show a germline/soma tag ratio ≥1, and 75% show a germline/soma ratio ≥ 2 (Figure 4a).Since differences in tag counts for genes with low numbersof tags are less likely to be statistically significant, we onlyanalyzed genes with a tag count ≥ 9 in the germline SAGElibrary (Figure 4b). By using this cut-off, we remove themajority of genes that fail to reach the p < 0.01 confidencelevel (Figure 4b). This cut-off was also used in a previouslypublished comparison of SAGE libraries [24]. We classi-fied genes with a two-fold or greater increase in thenumber of SAGE tags in the germ line compared to somaas "germline-enriched", while those with ≥ 9 tags in thegermline library and no tags in the soma library as "germ-line-specific" (see Methods). Using these criteria, we iden-tified 733 genes that are germline-enriched and 330 genesthat are germline-specific, for a total of 1063 genes that areeither specifically expressed or enriched in the germ line(Additional file 5). The proportion of germline-expressedgenes that are specific/enriched in the germ line is higherfor genes with higher expression levels, based on tagcounts, than for genes with lower expression levels (Figure2b). Since germline-specific/enriched genes are expressedat a higher level in the germ line than soma, they mayhave functions that are more specific to germline func-tion. The functions of many of these genes, based on KOGrapid formation of gametes (Additional file 5; see Discus-sion).Comparison of SAGE and Microarray dataWe have identified 1063 genes whose transcripts areeither enriched or specific in the germ line. We comparedthis list of genes to the list of germline-enriched genes inthe adult obtained by microarray [11,12] to generate amore complete list of germline-enriched genes. Of the1063 genes that we identified as being either germline-specific or enriched, 43.3% were also identified in thegermline microarray analysis as being enriched in thegerm line (This overlap is significant as compared to theoverlap between the entire germline SAGE library and themicroarray data; Fisher's exact p-value < 0.0001; Figure5a) [11]. Therefore, we identified 603 genes as beinggermline-specific/enriched that were not identified bymicroarray. To determine how accurately SAGE identifiedgermline-enriched genes, we analyzed the mRNA in situexpression patterns of these 603 genes using the NEXTDBdatabase. 344 of the 603 genes have expression patterns inthe NEXTDB database. Of these 344, 86% showed germ-line-specific/enriched expression and 11% show similarexpression levels in the germ line and soma (Additionalfile 6). Only 10 genes (2.9%) showed soma-enrichedexpression (Additional file 6). Therefore, the large major-ity of the 603 genes that we identified as germline/specificenriched, but which were not identified as such by micro-array, have mRNA in situ staining patterns consistent withour classification. We suspect that some of the genes notshowing germline-enriched expression by in situ mayactually be germline-enriched, but that we were unable tosee a two-fold difference with the images provided in theNEXTDB database. Other genes may have been misclassi-fied as germline-enriched by SAGE, although overall theSAGE classification correlates very well with the mRNA insitu expression patterns (Additional file 6).The microarray analysis identified far more germline-enriched genes than SAGE in the adult hermaphroditegerm line (2304); only 20.0% germline-enriched genesidentified in the microarray analysis were also identifiedas being germline-specific/enriched by SAGE (Figure 5a).To increase our understanding of why genes were identi-fied in the microarray analysis but not SAGE, we looked atthe 1844 germline-enriched genes in the microarray anal-ysis that were not identified by SAGE. The majority(59.9%) of these genes were not identified by SAGEbecause they were expressed at too low of a level (Figure5b); either no SAGE tags were detected (24.5%), or thegermline SAGE tag count was below our threshold (≤ 9)and not counted in our analysis (35.4%). An additional23.9% of genes could not be identified by SAGE becausePage 8 of 19(page number not for citation purposes)classification, are in keeping with the tremendous amountof cell division and tissue generation that occurs in thethe genes do not have an associated SAGE tag in the germline library (long SAGE) or soma library (short SAGE)BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213Page 9 of 19(page number not for citation purposes)Distribution of transcript levels between the germline and the somaFigure 4Distribution of transcript levels between the germline and the soma. (A) Histogram showing the distribution of the Germline/Soma tag count ratio for those common genes between the germline library and the soma library. The data corre-spond to 981 genes with germline tag counts ≥ 9 and soma tag counts ≥ 1. ~75% of these appear to have the Germline/Soma tag count ratio ≥ 2. (B) Scatter plot showing the distribution of tag counts for those common genes between the germline library and the soma library. The data represent 1552 genes with tag counts ≥ 2 in both the germline library and the soma library. Data points for genes that are expressed at the same level in the germ line and soma should fall along the mid-diagonal with a G/S tag ratio of 1, while genes that have enriched germline expression should be to the right of the diagonal with a G/S ≥ 2. The dashed blue line provides a measurement of sampling confidence based on the statistical analysis of [47]. Given a par-ticular number of tags generated for a gene in the soma library, there is a 99% chance that the number of tags generated in the germline library for the same gene will lie to the left of the dashed blue line. In the set of germline-enriched genes that were selected, there are 41 genes below this 99% confident line; however, 19 out of the 23 genes that have a discrete in situ expres-sion pattern in the NEXTDB appear to be germline-enriched.BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213(15.9%), or the tag was ambiguous (8.0%; tags that corre-spond to more than one gene; see Methods). 5.9% of the1844 genes did have more tags in the germline SAGElibrary than the soma SAGE library, thus were germline-enriched, but were below our two-fold threshold, so werenot included with the list of germline-enriched genes. Ofthe 1844 germline-enriched genes identified in the micro-array analysis, 188 (10.2%) were identified to have highertranscripts level in soma than the germ line by SAGE.However, most of these had soma tag counts less thannine or had less than two-fold increase in tag counts;therefore, these genes do not meet our criteria to be confi-dently classified as tissue enriched. Only 25 genes (1.4%)were soma specific/enriched by SAGE (two fold moresoma than germline tags with soma tag ≥ 9; Figure 5b).Therefore, there is very little discrepancy between theSAGE and microarray data; the vast majority of genes notidentified in the SAGE (but germline-enriched by micro-array), were not identified due to low levels of expressionor the lack of unique SAGE tags associated with the genes.200 genes were also randomly selected from the 1844germline-enriched genes that were only identified bymicroarray and their in situ expression patterns we ana-lyzed in NEXTDB. 85 of these 200 genes are included inthe NEXTDB database and have discernable expressionpatterns. 77/85 have germline-specific/enriched expres-sion patterns, suggesting that their identification as germ-line enriched in the microarray analysis is accurate.Germline-specific/enriched genes identified only by SAGESAGE identified 1063 germline-specific/enriched genes,but only 460 of these were also identified as such by themicroarray analysis (Figure 5a) [11]. We further analyzedthe 603 genes that were not identified by microarray todetermine the potential reasons for only identifying themby SAGE (Additional file 6). Of the 603 genes, 64.0% werenot identified in the microarray analysis either because nodata was available for the gene on the microarray (34.0%;includes genes lacking a probe on the microarray), or thedata obtained from the microarray was below the confi-dence level (30.0%) (Figure 5c). The remaining 217 genes(36.0%) gave ratios below two in the microarray analysis,suggesting that they are either enriched in the soma, orexpressed exclusively in the soma (ratio of one). As anindependent means of determining if these 217 genes areexpressed at a higher level in germ line or soma, we ana-lyzed their mRNA in situ expression patterns in the NEX-TDB database [25]. 126 of the 217 genes are included inthe NEXTDB database and have discernable expressionpatterns. Of these 126, 87% have an expression patternthat is either germline-specific or enriched, 10% showexpression at similar levels in the germ line and soma,while only 2.3% show a higher level of expression in thespecific/enriched, but which were identified as notenriched in the microarray analysis, do indeed showgermline-specific/enriched expression patterns consistentwith the SAGE classification.To further investigate the cause of identifying genes asbeing germline-specific or enriched by one analysis (SAGEor microarray) and not the other, we determined if therewas a correlation between gene expression level and thelikelihood of being identified by both technological plat-forms. For this analysis, we compared the percentage ofgenes identified by SAGE that were also identified bymicroarray, relative to the number of tags identified forthe genes. We divided the genes in this comparison intotwo groups; germline-specific genes (Figure 5d) and germ-line-enriched genes (Figure 5e), as determined by SAGE.Overall, genes that we labeled as germline-specific weremore likely to be identified in the microarray data(65.2%) than genes that we labeled as germline-enriched(33.6%). Therefore, genes with little or no soma expres-sion were more likely to be identified by both SAGE andmicroarray. Additionally, for germline-specific genes thepercentage of genes identified by both platformsremained high, or even increased, as the expression level,based on tag count, increased. Surprisingly, this trend didnot hold true for germline-enriched genes (Figure 5e). Forgermline-enriched genes (as determined by SAGE), alower percentage of genes were identified as germline-enriched by microarray as the expression level (tag count)increased. Even though only 13% of the 60 SAGE identi-fied germline-enriched genes with tag counts over 128were also identified by microarray, 77% of those includedin the NEXTDB in situ database (40/52) show germline-specific/enriched expression patterns; the other 23% showsimilar expression levels in germ line and soma. Noneappear to be enriched in the soma. Therefore, the SAGEclassification of germline-enriched is consistent with theNEXTDB expression patterns for the majority of thesehighly expressed genes, even though few of these geneswere identified by microarray as being germline-enriched.Chromosomal distribution of germline-expressed genesThe microarray analyses of germline-enriched genes,described above, demonstrated a chromosomal bias forgenes that are enriched for expression in the germ line[11,12,48]. It was found that genes with enriched germ-line expression are under-represented on chromosomes Vand X, and over-represented on chromosomes I and III,based on an expected random distribution of expressedgenes [11,12]. To determine if this chromosomal bias isonly for germline-enriched genes, or if it also applies togermline-expressed genes that have similar or higherexpression in the soma, as well as to determine if the levelPage 10 of 19(page number not for citation purposes)soma than the germ line (Additional file 7). Therefore, themajority of genes identified by SAGE as being germline-of expression is also chromosomally biased, we used ourgermline SAGE data to study the chromosomal distribu-BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213Page 11 of 19(page number not for citation purposes)Comparison of the germline-specific/enriched genes identified by SAGE with the germline-enriched genes identified by the microarrayFigure 5Comparison of the germline-specific/enriched genes identified by SAGE with the germline-enriched genes identified by the microarray. (A) Overview of the comparison between SAGE and microarray. Germline-specific/enriched SAGE data were generated by selecting genes that are identified only by the germline library (germline-specific) and genes that have a 2-fold enrichment of the tag counts in the germline library over the soma library (germline-enriched). Genes were fur-ther selected to have a germline tag count ≥ 9. Data correspond to 330 germline-specific genes and 733 germline-enriched genes. Microarray data used were the adult data, with wt/glp-4 F.I. ≥ 2 (99% confidence >0). (B) SAGE data for the germline-enriched genes identified only by microarray. 'Ambiguous genes' are genes that were not identified as germline-specific due to having an ambiguous tag in the soma (Short-SAGE) library, or that have an ambiguous Long-SAGE tag (Germline SAGE library). 'No SAGE tags' do not have an assigned Long and/or Short-SAGE tag. 'SAGE tags available but not identified' are genes that do have an associated Long-SAGE tag and Short-SAGE tag, and the tags are not ambiguous, but no tags were identified in either library. 'Germline tag < 9' are genes identified in the germline SAGE library, and either were not identified in the soma SAGE library or have a tag count ratio ≥ 2, but have a germline tag count < 9. '1 ≤ germline/soma < 2' refers to genes that were iden-tified in both the germline and the soma SAGE libraries and have a germline/soma tag ratio ≥ 1 and < 2. The expanded category are genes that are only identified in the soma SAGE library, or genes that are identified in both the germline and the soma SAGE libraries with a soma/germline tag ratio > 1. (C) The corresponding microarray data for the germline-specific/enriched genes identified only by the SAGE. 'Microarray F.I.' refers to the adult/glp-4 fold induction value. 'Below confidence level' refers to genes with a 99% confidence level < 0. (D) Germline-specific or (E) germline-enriched genes identified by SAGE compared to germline-enriched genes identified by microarray plotted against the distribution of transcript levels based on tag counts.BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213tion of germline-expressed genes. We first determined thechromosomal distribution of all 4699 germline-expressedgenes, irrespective of their soma expression (Figure 6a).The chromosomal distribution of these genes is similar tothe distribution determined by microarray analysis ongermline-enriched genes [11,12]; germline-expressedgenes are over-represented on chromosomes I and III, andunder-represented on chromosomes V and X, assuming arandom distribution of germline-expressed genes in thegenome (Figure 6a). For comparison, we analyzed thechromosomal distribution of soma-expressed genes usingthe soma SAGE library data and found that the number ofexpressed genes for all chromosomes was similar to theexpected value, except chromosomes III and V; somaexpressed genes are over-represented on chromosome IIIand under-represented on chromosome V, although theobserved numbers are closer to the expected values for thesoma than the germ line. To determine if the chromo-somal bias for soma and germline-expressed genes per-tains only to the number of genes expressed on eachchromosome, or if it also corresponds to expression levelsof the expressed genes, we analyzed the number of SAGEtags for the genes expressed on each of the chromosomes.It was previously found that the average expression levelof 258 oocyte-expressed genes was lower on the X chro-mosome than other chromosomes, based on microarrayspot intensities [48]. By analyzing the number of SAGEtags for all 4699 germline-expressed genes, we found thatgenes on the X chromosome have a dramatic decrease inexpression levels as compared to the other chromosomes(Figure 6a). In other words, not only are fewer germline-expressed genes found on the X chromosome thanexpected, but also the germline-expressed genes that areon the X chromosome are expressed at a lower level thanexpected. This differs from soma-expressed genes; boththe number of genes and the expression levels of thosegenes are close to expected levels for the X chromosome.To determine if the chromosomal bias of germline-expressed genes is limited to genes that are enriched in thegerm line, or if it applies to all germline-expressed genes,even if they are also expressed at a similar or higher levelin the soma, we split the germline-expressed genes intotwo groups; germline-expressed genes that are germline-specific/enriched, and germline-expressed genes that areexpressed at a similar or higher level in the soma than thegerm line (Figure 6b). We found that all chromosomesshowed the same general trend for the number of genesexpressed in the germ line on a given chromosome,whether they were germline-enriched or not, except forthe X chromosome. Genes that are expressed at a higherlevel in the germ line than in the soma are less likely to befound on the X chromosome, while genes that arevalues, based on a random distribution (Figure 6b). How-ever, both sets of germline-expressed genes, whethergermline-enriched or not, are expressed at a lower levelthan expected (Figure 6b). It is interesting that the numberof X chromosome genes expressed in the germ line isbelow the expected value for germline-enriched genes, butnot for germline-expressed genes that are expressed in thesoma at a similar or higher level (Figure 6b, top panel);both sets of genes are expressed at a lower level thanexpected (Figure 6b, lower panel). The lower level of X-chromosome genes expressed in the germ line is due tohistone modifications, which leads to global silencing[48-50]. Germline-enriched genes presumably have func-tions that are more critical to the germ line; therefore, theymay require higher expression that cannot be achieved onthe X chromosome. Genes that are expressed in the somaand germ line may be more likely to be genes needed forembryogenesis. Since the X chromosome becomes reacti-vated as germ cells progress through oogenesis [48], germ-line-expressed genes that are not germline-enriched maybe expressed in more mature germ cells as the X chromo-some is becoming reactivated; therefore, this class ofgenes may not need to be excluded from the X chromo-some.DiscussionWe have identified 4699 genes that are expressed in the C.elegans adult hermaphrodite gonad (Additional file 1),which corresponds to roughly 1/5th of all annotated C. ele-gans genes. We further show that 1063 of these germline-expressed genes have either enriched or specific expres-sion in the germ line (Additional file 5). While 460 ofthese genes were previously identified as being germline-enriched [11], 603 are new to this classification (Addi-tional file 6). These lists are an important resource forunderstanding the genes that are necessary for gamete for-mation and early embryogenesis. We also compared thedata obtained from the microarray and SAGE platforms,identifying strengths and limitations of each. Finally, weadded to our understanding of the chromosomal distribu-tion of germline-expressed genes by including an analysisof expression levels, as well as including genes that areexpressed in the germ line, but which are not enriched inthe germ line. We found that, unlike genes that are germ-line-enriched, non-germline-enriched (but germline-expressed) genes are not under-represented on the X chro-mosome.High level of ribosomal gene expressionThe class of genes with the highest representation in thegermline SAGE library contains genes necessary for ribos-omal formation and function (Additional file 2; Figure 3).These genes are expressed at a higher level in the germ linePage 12 of 19(page number not for citation purposes)expressed at similar or higher levels in the soma than germline are found on the X chromosome near the expectedthan soma (Figure 3b). Their high level of expressionlikely has to do with the amount of tissue generated in theBMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213adult germ line. In the adult soma, all tissues are fullyformed and no further cell division occurs [9]. However,in the germ line, cell division continues throughout muchof the life of the adult. In the distal end of the gonad,~200–250 mitotic cells divide to replenish the cells thatenter into meiotic prophase [3,51]. The cells that entermeiotic prophase progress through meiosis as they moveproximally, finally culminating in surviving cells formingoocytes, which ovulate every ~23 minutes per gonad arm[4]. Along with a large amount of cell division occurringin the germ line, a significant amount of cell growthoccurs. Ovulating oocytes have a final volume of ~20,000um3, while cells in the distal end of the gonad are only afraction of that volume [4]. Therefore, in the adult her-maphrodite germ line, a tremendous amount of cell divi-Tissue generation requires protein, and protein produc-tion requires ribosomes; therefore, it is logical that ribos-omal genes are actively transcribed in a dividing tissue.Indeed, the correlation between cell growth and ribosomenumber was first observed in E. coli, in which it was foundthat the growth rate is proportional to the number ofribosomes [52]. The same correlation between growthrate and ribosome number has also been shown in anumber of eukaryotic species [53,54].Comparison of SAGE and microarray technological platformsBy comparing our SAGE derived list of germline-enrichedgenes with the list generated by microarray [11], we haveidentified additional genes that are germline-enriched,Gene expression shows a biased chromosomal distributionFigur  6Gene expression shows a biased chromosomal distribution. Distribution was generated based on either the number of genes (A & B, upper panel) or the number of tags (A & B, lower panel). (A) The fraction of observed germline (black) and soma (gray) expressed genes compared to the expected value per chromosome is plotted (upper panel). The expected value is based on a random distribution of expressed genes throughout the genome as described in Methods. To compare expression levels of expressed genes on each chromosome (measured by the number of SAGE tags; lower panel), the average number of tags per gene in each gene set was first calculated then was multiplied by the number of genes observed in the same gene set for each chromosome to obtain the expected total number of tags (expression level) per chromosome. (B) Two sets of germ-line-expressed genes are compared; germline-expressed genes that are enriched or specifically expressed in the germ line (black), and germline-expressed genes that are expressed at a similar or higher level in the soma, as compared to the germ line (gray). The fraction of expressed genes compared to the expected value (upper panel), and the level of expression (lower panel), were determined as in (A). Statistical significance is marked by an asterisk (P < 0.001; hypergeometric probability test for the number of genes; re-sampling method for the number of tags, see Methods for details).Page 13 of 19(page number not for citation purposes)sion and cell growth is occurring. Indeed, the entirevolume of the gonad is turned over every ~6.5 hours [2].adding confidence to the genes that were identified inboth platforms, and have analyzed some of the strengthsBMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213and weaknesses of each platform. Generally speaking, thedegree of overlap between the SAGE generated list ofenriched genes and the microarray list is relatively high.For the most part, the differences in the lists reflect thelack of data for certain genes in one of the analyses, prima-rily due to limitations of the platform, rather than con-flicting data between the two analyses. For SAGE, far fewergermline-enriched genes were identified as compared tomicroarray; 1063 germline-enriched genes were identifiedby SAGE, while 2304 genes were identified by microarray(Figure 5a). For those genes that were identified in themicroarray analysis, but not the SAGE, over 60% of thegenes had few or no associated tags identified in the germ-line SAGE library, suggesting that expression levels werevery low. Therefore, the cause of not identifying thesegenes may be that the sequencing depth was not sufficientfor detection, and that greater sequencing depth may over-come this limitation. Newer sequencing technologies,which greatly increase the amount of sequence obtained,and at a much lower cost, will likely overcome this limita-tion. Indeed, recently prepared SAGE libraries are cur-rently being sequenced at ~25× the depth of those in thisstudy (millions of tags rather than hundreds of thou-sands; [55]; libraries of four million tags are now standard(DGM, unpublished results). Another limitation of SAGE,accounting for ~1/4 of the genes identified by microarraybut not by SAGE, is that not all genes have unique SAGEtags; some genes lack the restriction site needed to gener-ate a SAGE tag, while others generate SAGE tags that arenot unique to a gene, and thus are ambiguous. The lack ofa unique SAGE tag associated with some genes is an inher-ent limitation of SAGE and is difficult to overcome. How-ever, similar problems exist for other technologies; forexample, genes within a gene family may have similarsequence causing probes corresponding to more than onegene hybridizing to a single spot on a microarray. The useof Long SAGE rather than Short SAGE increases the prob-ability that a given tag will be unique to single gene [46].For those genes that were identified as being germline-enriched by SAGE, but not by microarray, over half werenot detected by microarray because corresponding probeswere not on the array, or because the data obtained for agiven probe on the chip was deemed unreliable. UnlikeSAGE, microarrays are a closed system that requires priorknowledge of genes; therefore, the microarrays are limitedby the quality of gene annotation. The quality of the C.elegans annotation is very high; therefore, a similar analy-sis in a system with less extensive annotation would likelymiss a higher percentage of genes.The remaining ~48% of genes that were only identified bySAGE as being germline-enriched were detected by micro-soma. We are confident that many of these genes, if notthe majority, are germline-enriched because ~87% ofthese genes that are included in the NEXTDB databasehave staining patterns showing germline enrichment(Additional file 6). We are unsure as to why the microar-ray analysis did not identify these genes as being germ-line-enriched; however, we find it intriguing that thedegree of overlap between the SAGE and microarray datafor germline-enriched genes decreases as the expressionlevel increases. Perhaps highly expressed genes, whichwould presumably have a significant level of labeledcDNA and a very bright hybridization signal on the micro-array for both channels, could saturate the signal and pre-vent the difference in levels of expression from beingdetected [56].This work has highlighted the benefit of using multipletechnological platforms to generate a more complete listof the transcripts present in a given tissue. Although theamount of conflicting data between the two technologieswas very low (Figure 5), the number of genes identified byonly one of the technologies was large; ~56% of SAGEdetected germline-enriched genes were not identified bymicroarray, and ~80% of microarray detected germline-enriched genes were not identified by SAGE.Other forms of regulationOur germline SAGE library, and the microarray analyses[11,12], identified genes that are transcribed in the germline. These analyses give a general idea as to the genes thatare involved in gamete formation and early embryogene-sis. However, it should be emphasized that transcriptionis only one level of regulation; the presence and relativeabundance of mRNA transcripts may not always accu-rately reflect the function of the gene in the germ line. Forexample, post-translational control through phosphoryla-tion regulates many aspects of germline development andfunction. Indeed, MAP kinase signaling, which presuma-bly culminates in the phosphorylation of many proteintargets, regulates at least eight distinct processes in C. ele-gans hermaphrodite germline development and function[57]. Translational control has also been implicated inmultiple aspects of C. elegans germline development. Forexample, the proliferation vs. meiotic entry decision uti-lizes numerous proteins that regulate the translation and/or stability of target mRNAs [58,59]. The 3'UTR has beenidentified as the primary means by which the expressionof germline-expressed genes is controlled [45]. We havefound that mRNAs encoding RNA binding proteins areexpressed at ~4× the level in the germ line than the soma.Therefore, the presence of an mRNA transcript does notfully predict a germline function. Indeed, some genestranscribed in the germ line may not function in the germPage 14 of 19(page number not for citation purposes)array analysis, but were determined to be expressed at anequal or lower level in the germ line as compared to theline at all, but rather are needed for embryogenesis. Simul-taneous disruption of the activities of two translationalBMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213regulators, GLD-1 and MEX-3, results in the formation ofsomatic tissues, such as muscles and neurons, in the germline [60]. Presumably, genes necessary for the generationof these tissues are transcribed in the germ line; however,repression by GLD-1 and MEX-3 prevents the translationof these target mRNAs until the proteins are needed forembryogenesis. Therefore, the identification of genes tran-scribed in the germ line will need to be combined withprotein expression and modification data to obtain amore complete picture of the factors necessary for propergermline function.ConclusionUsing SAGE we found that 4699 genes (~21% of allgenes) are expressed in the C. elegans hermaphrodite germline. A majority of the highest expressed genes areinvolved in translation, ribosome structure and biogen-esis, and this general class of genes is expressed at a higherlevel in the germ line than the soma. Additionally, RNAbinding proteins are expressed at a higher level in thegerm line than the soma, suggesting that the control ofgene expression through RNA metabolism is more pre-dominant in the germ line than the soma. A comparisonof germline-enriched genes identified through SAGE witha previously published list of germline-enriched genesidentified by microarray found overlap with 460 genes,corroborating their classification as germ line enriched.Analysis of the genes identified by only one of the techno-logical platforms identified potential strengths and weak-nesses of each platform, as well as emphasized theimportance of using more than one technological plat-form to obtain a more complete picture of global geneexpression. Finally, the number of germline-enrichedgenes on the X chromosome is lower than that predictedassuming a normal distribution. However, the number ofgenes on the X chromosome that are expressed at a loweror equal level in the germ line than the soma is near whatis expected.MethodsProduction and analysis of the germline SAGE libraryYoung adult wild type (N2) hermaphrodites grown at 20°were dissected ~18 hours after the fourth larval stage toisolate the gonad arms. Animals were dissected in 2 mL ofPBS-EDTA-ATA (125 mM NaCl, 16.6 mM Na2HPO4, 8.4mM NaH2PO4, 0.1 mM EDTA, 1 mM auxin tricarboxylicacid) with 0.2 mM Levamisole. Animals were dissectedwith two 25-gauge needles at the pharynx, allowing forthe gonads and intestine to extrude from the body. Thegonads were dissected away from the body by cutting at ornear the spermatheca. ~150 gonad arms were placed inTRIZOL (Invitrogen, Carlsbad California) and the RNAwas isolated following the manufacturers instructions.and we used the established LongSAGE technique, whichuses the enzyme MmeI to generate 21-bp tags [63]. Theraw data for the germline SAGE library is deposited athttp://tock.bcgsc.ca/cgi-bin/sage160, along with the datafor other C. elegans SAGE libraries. SAGE tags weremapped to C. elegans genes using Wormbase WS160.Gene identification criteria used were: removal of dupli-cated ditags; resolve to lowest tag position; hide ambigu-ous tags; hide antisense tags; sequence quality > 99%;only coding RNA. Using these criteria we identified92,007 tags in the germline SAGE library. We used thesame criteria to analyze the soma SAGE library, which hasbeen previously published [24]. From the soma library91,888 tags were identified. For subsequent analysis, thetotal number of tags for each library was normalized to100,000 tags, and only genes with a tag position 1 wereused. If different unique tags were assigned to the samegene, both at position 1, the tag counts were combined toprovide the total tag count for that gene. With these cor-rections, a total of 4699 and 5900 genes with unique tagswere identified in the germline library and the somalibrary, respectively. There are 33 more genes identified inthe soma library than previously reported [24]; thisincrease in the number of genes is likely due to the use ofa more recent Wormbase freeze to map the tags to C. ele-gans genes (we used WS160 while the soma tags were orig-inally mapped using WS140 [24]).Classification of RNA binding proteinsTo identify proteins with putative RNA binding activity, weidentified genes that encode proteins with a predicted RNAbinding domain. Proteins were identified that had one ormore of the RNA binding domains described previously[43]. We then filtered from this list, genes that encode pro-teins that are unlikely to have RNA binding function, such asthose that are predicted to bind DNA, and genes encodingribosomal proteins, using KOG classifications and otherdescriptions found in Wormbase freeze 190. Using these cri-teria we identified 319 putative RNA binding proteins (Addi-tional file 3). 190 of these genes are expressed in the germline, and 130 are expressed in the soma, based on the pres-ence of one or more tags in the respective SAGE library. Forthis analysis, we wanted to obtain lists of RNA binding pro-teins, which could potentially be expressed in the germ lineand in the soma, that are as complete as possible; therefore,in these lists we included genes that have one or more tags.Some of the RNA binding proteins that are associated withlow tag counts may be background; for the entire germlineSAGE library, ~25% of the genes are represented by just onetag.Identification of germline-specific/enriched genesThe germline and soma SAGE libraries were generatedPage 15 of 19(page number not for citation purposes)The germline SAGE library was prepared by standardmethods as described in detail elsewhere [24,61,62].Starting material was 143 ng of purified germline RNAusing the same anchoring enzyme, NlaIII, but the taggingenzyme for each was different. The soma library was gen-erated using the tagging enzyme BsmFI, a 14 bp cutter,BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213while in the construction of the germline library we usedthe tagging enzyme MmeI, a 21 bp cutter. This means thetwo libraries have tags of different lengths, which some-what complicates a comparison between the libraries[46]. Since different tags are generated in long and shortSAGE, genes may have a unique tag in one library but notthe other. Therefore, in order to compare the germlineand soma libraries to identify germline-specific genes, weexcluded genes that have ambiguous tags with either shortor long SAGE. We also removed genes that did not haveassigned short SAGE tags. We defined "germline-enriched" genes as those that have associated SAGE tags inboth the germline and the soma SAGE libraries, the germ-line tag count was ≥ 9 and there was at least a two-foldincrease in the number of SAGE tags in the germline SAGElibrary as compared to the soma SAGE library. We defined"germline-specific" genes as those that have associatedSAGE tags in the germline SAGE library, but not the somaSAGE library, and the number of tags in the germlinelibrary was ≥ 9. By using the ≥ 9 cut-off, we removed themajority of genes that failed to reach the p < 0.01 confi-dence level, which increases our confidence in the properidentification of germline-specific/enriched genes (Figure4b). We chose a two-fold increase in SAGE tags based ona comparison with previously published microarray anal-ysis of germline-expressed genes [11]. We compared genesthat are found in both the microarray data and the SAGEdata based on their fold increase. We compared the foldincrease of genes that had at least two tags in both thesoma and germline SAGE libraries with the fold increaseof the same genes in the microarray data, in which a foldincrease ≥ 2 was deemed to be germline-enriched [11]. Wefound that genes with 2 to 2.49 folds increase by SAGEhad an average fold increase of 2.3 by microarray, andgenes with 2.5 to 2.99 fold increase by SAGE had an aver-age fold increase of 2.7 by microarray. Therefore, choos-ing a two-fold increase as the threshold for germline-enriched genes was roughly consistent with the microar-ray data. The distinction between germline-specific andgermline-enriched is also likely dependent on the depthof sequencing of the SAGE libraries. It is likely that at leastsome genes with 0 tags in the soma SAGE library wouldhave 1 or more tags if sequencing depth were increased.Classification of NEXTDB expressionAvailable expression patterns in NEXTDB were classifiedas follows: Class "I" = germ line is the only (obvious) siteof expression in the adult worm; Class "II" = germ line isthe major site of expression (with, say, > 70% of theexpression intensity detected in the germ line – the 70%expression intensity level was determined by measuringthe in situ staining intensity in the germ line, as comparedto other tissues, of five randomly chosen Class II genesbetween germ line and soma is roughly the same); Class"IV" = gene is not expressed in the adult germ line; Class"?" = there is no staining, or expression pattern cannot bedetermined, usually because of weak signals; Class "-" =gene is not available in the NEXTDB database, or the insitu hybridization pattern is not available for the L4 –adult stage. Cross-referencing to the NEXTDB databasewas performed manually. Genes were searched in theNEXTDB database one at a time and assigned the corre-sponding in situ hybridization pattern according to theabove classifications. Genes were searched in the NEX-TDB, and the classifications assigned, by one person inorder to minimize potential variation. The same individ-ual blindly analyzed (not knowing previous classifica-tion) all in situ patterns a second time and determined aclassification. <3% of classifications differed between thetwo replicates. Genes with different classifications wereanalyzed a third time to assign a final classification.In addition to the genes identified in the germline SAGElibrary that we analyzed using the NEXTDB, we also ran-domly selected 1089 genes from the entire genome andclassified accordingly using NEXTDB. 342 of these geneswere in NEXTDB and showed a discernable in situ hybrid-ization pattern. 54% of the 342 genes showed Class I orClass II expression patterns, while 46% showed Class IIIand Class IV expression pattern. Therefore, it is highly sig-nificant that 81% of the 124 genes (with discernable insitu pattern) that are highly represented in the germlineSAGE library showed Class I or Class II expression pat-terns, as well as 86% of the 344 genes (with discernable insitu pattern) that are germline-specific/enriched onlyidentified by SAGE showed Class I or Class II expressionpatterns (Fisher's exact p-value < 0.001).Chromosomal distribution of germline-expressed genesTo determine the expected number of expressed genes ona given chromosome, "SAGE tags available protein codinggenes" were obtained from Wormbase http://www.wormbase.org from the WS160 data freeze. For the germlineSAGE library, only genes with associated Long-SAGE tagswere used, while for the soma SAGE library, only geneswith associated Short-SAGE tags were used. The fraction ofgenes from each chromosome with associated SAGE tags,as compared to the total number of genes in the entiregenome with associated SAGE tags, was determined. Foreach chromosome, this fraction was multiplied by thetotal number of genes expressed in the germline or somaSAGE libraries; this provided the expected number ofgermline or soma expressed genes for each chromosomebased on a random distribution of expressed genes. Theobserved number of genes expressed in the germline orsoma SAGE libraries for each chromosome was thenPage 16 of 19(page number not for citation purposes)using Photoshop CS3 (Adobe); average 77.4% ± 5.4%; n= 5); Class "III" = germ line is one expression site amongothers in the adult worms (the expression intensitydivided by the expected number of expressed genes perchromosome to obtain the observed/expected ratio that isplotted in the upper panel of figure 6a.BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213For comparing the observed versus expected expressionlevels for each chromosome, based on the number of tags,the expected value was determined by first calculating theaverage tags per expressed gene for the respective library(13.7 tags/gene for the germline SAGE library, 10.5 tags/gene for soma SAGE library). This average tags/gene valuewas then multiplied by the number of genes expressed oneach chromosome from the corresponding library. Thisprovided the expected number of SAGE tags expressedfrom each chromosome. The observed number of tagsexpressed was then divided by the expected number oftags expressed to obtain the observed/expected ratio thatwas plotted in the lower panel of figure 6a. Similar analy-ses were used on the germline-specific/enriched and thenon-germline-specific/enriched datasets to obtain theobserved/expected ratios that were plotted in figure 6b.Statistical significance of the differences between theobserved and the expected values was determined with ap-value < 0.001. For the comparisons based on thenumber of genes, hypergeometric probability tests wereused [64]. For the comparisons based on the number oftags, a re-sampling approach was used. For example, inthe case of chromosome I of the germline SAGE library,the corresponding number of tags of the 4694 germlineSAGE genes was considered as the population, and thenumber of genes identified on chromosome I (1008), wasconsidered as the sample size. Re-sampling was per-formed such that 1008 values were randomly picked fromthe population and then summed. The summed valuecorresponded to the total number of tags that was ran-domly determined. This procedure was performed100,000 times to generate a probability distribution,which was used to obtain the p-value.SAGE and microarray comparisonWe compared our SAGE generated list of germline-spe-cific/enriched genes with the published list of microarrayidentified germline-enriched genes expressed in the adult[11]; we removed the 585 genes from the microarray datathat only showed germline-enriched expression in the lar-vae because the SAGE data was obtained using adults. Weanalyzed the microarray generated germline-expresseddata with WS160 Wormbase freeze genome annotationsand updated gene IDs, removed non-protein codinggenes, removed pseudogenes, removed retired genes andremoved duplicated data entries. This resulted in anupdated list of 2304 genes that were determined to begermline-enriched in the adult hermaphrodite by micro-array. All 2304 genes have wt/glp-4 two-fold differencewith a t-value over 99% confidence (p < 0.01). For geneswith duplicated data entries in the microarray data, theaverage fold induction value was used. For the 603 germ-Authors' contributionsDH isolated the gonadal tissue, contributed to the experi-mental design of the work and contributed to the dataanalysis. He also coordinated the study and drafted themanuscript. XW contributed to the experimental design ofthe work and performed the data analysis classifyinggenes into their KOG terms, classifying expression pat-terns from NEXTDB, classifying genes according to expres-sion levels, comparing the microarray and SAGE data,determining the distribution of genes and tags in thegenome and assisted in drafting the manuscript. YZ con-tributed to the construction and sequencing of the germ-line SAGE library. KW contributed to the initialbioinformatics analysis of the SAGE tags, posting andmaintaining the tag library at Multisage. PE performed thestatistical analysis of the chromosomal distribution oftags. DGM, SJJ, MAM, and RAH coordinated the construc-tion and sequencing of the germline SAGE library. SJJ alsocritiqued the manuscript. DGM contributed to the experi-mental design of the work, and critiqued the manuscript.YK provided the in situ data in NEXTDB.Additional materialAdditional File 1All of the genes identified by germline SAGE library. A list of genes that were identified by the germline SAGE library used for analysis.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-213-S1.xls]Additional File 2Set of 157 highly expressed genes identified in the germline library. A list of genes, organized according to KOG classification, that have a tag count >60 in the germline SAGE library.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-213-S2.xls]Additional File 3Proteins with putative RNA binding activity. A list of genes that were identified to encode proteins with putative RNA binding activity.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-213-S3.xls]Additional File 4Tag distribution of the RNA binding proteins identified in the germ-line and soma SAGE libraries. Tag distribution of the RNA binding pro-teins identified in the germline and soma SAGE libraries. Proteins with potential RNA binding activity were identified as described in Methods. In total, 319 proteins were identified (Additional file 3), with 190 genes present in the germline SAGE library and 131 genes present in the soma SAGE library. Plotted is the total number of genes, of the 190 genes in the germ line and 131 genes in the soma, that have a given tag distribution. The number of genes in each tag range was determined and plotted against the tag distribution.Page 17 of 19(page number not for citation purposes)line-specific/enriched genes that were only identified bySAGE, the fold induction microarray data was kindly pro-vided by Valerie Reinke (Yale University).Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2164-10-213-S4.pdf]BMC Genomics 2009, 10:213 http://www.biomedcentral.com/1471-2164/10/213AcknowledgementsWe thank Jim McGhee and members of the Hansen lab for helpful discus-sions and for critical reading of the manuscript, and Paul Cliften for provid-ing programming support. We also thank Valerie Reinke for providing microarray raw data. This work was supported by funds from the Canadian Institutes of Health Research (CIHR) to D.H. and funds to D.G.M., D.L. Baillie, M.A.M. and S.J.J. from Genome Canada and Genome British Colum-bia. M.A.M. and S.J.J. are scholars of the Michael Smith Research Foundation for Health Research. Y.K. was supported by KAKENHI (Grant-in-Aid for Scientific Research) from the Ministry of Education, Culture, Sports, Sci-ence and Technology of Japan. D.H. is a scholar of the Alberta Heritage Foundation for Medical Research.References1. Hubbard EJ, Greenstein D: The Caenorhabditis elegans gonad:a test tube for cell and developmental biology.  Dev Dyn 2000,218(1):2-22.2. Hirsh D, Oppenheim D, Klass M: Development of the reproduc-tive system of Caenorhabditis elegans.  Dev Biol 1976,49(1):200-219.3. Killian DJ, Hubbard EJ: Caenorhabditis elegans germline pat-terning requires coordinated development of the somaticgonadal sheath and the germ line.  Dev Biol 2005,279(2):322-335.4. 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