We used MAPCeL 14 to assess mRNA expression in embryonic muscle cells. This technique involves the dissociation of blastomeres from embryos expressing a cell-type restricted GFP reporter gene, thus allowing FACS-enrichment of specific cell types (Figure 1). To mark embryonic muscle cells, we used an integrated myo-3::GFP transgene 15. myo-3::GFP expression begins early, in the 'pre-comma' stage embryo that is readily dissociated into individual blastomeres. This reporter is expressed in all 81 embryonic body wall muscle cells (Figure 2), the anal depressor, and sphincter muscles. We used MAPCeL to profile muscle cells from two cell populations (Figure 2) 16: myo-3::GFP labeled blastomeres sorted directly from freshly dissociated embryos; and myo-3::GFP expressing muscle cells from dissociated embryos cultured for 24 hours before sorting.

The microarray profile of freshly dissociated muscle cells is labeled 'M0' to denote direct isolation from embryos at '0' hours (normalized intensity values are listed in Additional data file 1). The M0 profile is expected to include transcripts that are highly expressed in nascent muscle cells. The embryonic myo-3::GFP positive body wall muscle cells comprise about 15% of the total cell population (81/550 total cells), which is consistent with the frequency at which myo-3::GFP expression is detected in dissociated embryonic cells (Figure 2b) 17.

myo-3::GFP expression persists in fully differentiated muscle cells after the comma stage, when embryos become resistant to dissociation. We have previously shown that C. elegans neurons and muscle cells can differentiate in vitro from early embryonic blastomeres 1617. Therefore, to obtain a profile of mature embryonic muscle cells, dissociated myo-3::GFP embryos were cultured for 24 hours before sorting; the microarray dataset from these myo-3::GFP cells is labeled 'M24' (Additional data file 1). mRNAs in the M24 profile are expected to represent transcripts expressed in differentiated body wall muscle cells. Although myo-3::GFP is also expressed in post-embryonically derived muscle cells (for instance, vulval), larval cells apparently do not differentiate under these culture conditions and therefore should not be directly profiled in these experiments 17.

The coefficient of determination (R²) was calculated for each set of microarray replicates. An average R² of 0.94 (n = 3) was obtained for the reference dataset (R0) obtained from freshly dissociated embryonic cells. The reproducibility of these data is illustrated graphically in the representative scatter plot shown in Figure 3. A similar high value of R² (0.96; n = 4) was previously determined for the reference data (R24) obtained from all embryonic cells after 24 hr in culture 14. R² values for pair-wise combinations of the M0 (average R² = 0.92) and M24 (average R² = 0.87) datasets are shown in Figure 3.

We initially identified all transcripts that are reliably detected in the muscle datasets. These lists of 'present' genes for the experimental M0 and M24 datasets were adjusted to remove transcripts that could easily be attributed to contamination by non-GFP cells (about 10%) in FACS-derived myo-3::GFP cell populations (see Materials and methods, below) 14. The resultant list of 'expressed genes' includes 7,070 unique mRNAs from the M0 and M24 populations of C. elegans body wall muscle cells (Figure 4a and Additional data file 2). A total of 10,455 unique expressed genes are included in the sum (R0 + R24) of the reference datasets; overall, 10,939 transcripts were detected in these experiments. A substantial number of expressed genes (6,586) are expressed in both muscle cells and in the reference dataset (Figure 4b and Additional data file 3). These transcripts are likely to include 'housekeeping' genes that play universal roles in cell differentiation and homeostasis; for example, transcripts for 75 ribosomal proteins are included in this group (Additional data file 3). Expressed genes that are selectively detected in the M0 and M24 profiles are likely to provide functions that are largely restricted to muscle cells (Figure 4b and Additional data file 3). These 'muscle-specific' genes, as well as transcripts showing 'enriched' expression in muscle cells relative to other embryonic cells, are described in detail below.

A scatter plot comparing the M0 muscle dataset with the R0 reference reveals significant differences in gene expression levels (Figure 3b). Enrichment for known muscle genes is evident, because transcripts for the abundant muscle structural proteins MYO-3 (myosin heavy chain), UNC-54 (myosin heavy chain), and UNC-15 (paramyosin) 181920 are highly elevated (red) relative to reference data obtained from all embryonic cells. Other transcripts, including those encoding SNAP-25 (a synaptic vesicle protein expressed in neurons) 21, are depleted (green; Figure 3b). A similar scatter plot was obtained for a comparison of the M24 muscle and R24 reference profiles (data not shown).

Transcripts that are differentially expressed in the M0 and M24 muscle datasets were identified by a statistical comparison of the paired experimental and reference datasets (for instance, M0 versus R0 and M24 versus R24; see Materials and methods, below). This treatment identified a total of 770 genes that are significantly enriched in the M0 muscle dataset and 937 transcripts with elevated expression relative to reference in the M24 profile. A comparison of these data identified 1,312 unique transcripts that are enriched in at least one of these datasets (Figure 5 and Additional data file 4). Conversely, 2,542 genes are depleted in embryonic body wall muscles in comparison with all cells (Additional data file 5).

A survey of the literature and a comprehensive search of WormBase 22 (see Materials and methods, below) identified 1,003 genes with known expression in myo-3::GFP-positive embryonic muscle cells (body wall muscle and defecation muscles; Additional data files 6 and 7; also see Materials and methods, below). A majority of these genes (773/1,003 [77%]) are detected as expressed genes in myo-3::GFP muscle cells (Additional data file 2). In contrast, only 28% (1,003/3,544) of all genes with expression patterns listed in WormBase are annotated as expressed in muscle (Additional data file 5).

Consistent with the low false discovery rate calculated for these datasets, we detected limited overlap with microarray profiles generated from other cell types. For example, only 100 out of 1,685 intestine or germline-enriched transcripts 23 are also listed in our enriched muscle dataset (Additional data file 8). These intestine and germline genes are thus under-represented in the embryonic muscle profile (representation factor = 0.8, P < 0.036). (Hypergeometric calculations were performed as described by Von Stetina and coworkers 24.) In contrast, a similar comparison of the embryonic muscle enriched genes detected significant overlap with transcripts that are also elevated in a MAPCeL dataset obtained from embryonic A-class motor neurons 14. In this case 159 of the approximately 1,000 embryonic A class motor neuron enriched transcripts are detected in the muscle profile (representation factor = 2.3, P < 1.5 × e^-24; Additional data file 9). The significantly higher fraction of shared transcripts between neurons and muscles could be indicative of the common functions of excitable cells. For example, transcripts for the acetylcholine receptors (unc-38 and unc-63), ryanodine calcium receptor (unc-68), and innexin gap junction protein (unc-9) are detected in both the muscle and A-class motor neuron datasets. This view is consistent with the finding that the embryonic muscle dataset also shows significant overlap with a MAPCeL profile of the C. elegans embryonic nervous system (representation factor = 2.0, P < 2.5 × e^-24; Additional data file 7) 24.

Two previous studies, using different methodologies, have also reported body wall muscle gene expression, and these can serve as validation tests for our methods. Fukushige and coworkers 7 used the same microarray platform (Affymetrix) to examine body wall muscle-like gene expression resulting from nearly uniform myogenic conversion of early C. elegans blastomeres by the transcription factor HLH-1 (CeMyoD). Of the 1,312 transcripts that are enriched in at least one of the embryonic MAPCeL muscle datasets, 592 (about 45%) are upregulated in body wall muscle-like cells at 6 hours post-induction of HLH-1 (representation factor = 3.6, P < 6.5 × e^-205; Figure 6 and Additional data file 8). This finding is clearly indicative of highly similar muscle profiles. In contrast, the MAPCeL list of embryonic muscle enriched genes shows less overlap with a microarray profile of larval body muscle cells obtained by the mRNA-tagging method 25, although the 249 transcripts shared by both datasets are indicative of significant similarity (representation factor = 2.8, P < 1.8 × e^-54; Additional data file 6). It is unclear whether this disparity is due to the different profiling strategies used to generate these data or to developmentally regulated differences in gene expression between embryonic and larval muscles.

The finding that a majority of known muscle genes is detected in our microarray profiles, and that these datasets exhibit substantial overlap with an independent profile of embryonic myogenesis 7 suggested that other uncharacterized transcripts in these datasets are also likely to be expressed in body wall muscle cells. To test this idea, we generated promoter-GFP reporter genes for representative transcripts in the M0 and M24 datasets and scored expression in embryonic and post-embryonic muscle cell types. A 'promoter' was defined as the region upstream of the ATG start codon for a distance of 4 kilobases or the distance to the end/beginning of the 5' flanking gene, whichever was less. In some cases, the promoter region tested was quite small (as little as 450 base pairs) and therefore may not have included necessary regulatory elements for expression of the transgene (Additional data file 11).

We found that about 70% (36/52) of transgenic lines generated from these reporter genes exhibited GFP-positive muscle cells in vivo (Additional data file 11). This finding is comparable to the finding that 61% (238/393) of genes in the total muscle enriched dataset for which expression patterns are listed in WormBase are annotated as expressed in muscle. In contrast, only 28% (1,003/3,544) of all genes with expression patterns in WormBase are identified as muscle expressed (Additional data files 6 and 7). The majority of muscle positive promoters (20/36) drove expression in both embryonic and post-embryonic muscle, although 16 had no detectable embryonic expression. We saw no correlation between the rank order of transcripts identified by MAPCeL and the likelihood of muscle expression of the corresponding GFP reporters, suggesting that these microarray datasets are robust (Additional data file 11).

Figure 7a depicts expression of representative GFP reporters in three myo-3::GFP positive muscle cell types (body wall, vulval, and defecation) and pharyngeal muscle as scored in late larvae and adults. Given that body wall cells are the predominant muscle cell type, it is not surprising that most (35/36) of the muscle positive reporters showed expression in this tissue. The one exception, zig-6::GFP, is detected in embryonic anal muscles, a finding that underscores the sensitivity of our methods to transcripts that may be selectively expressed in a subset of embryonically generated muscles. Twenty-two GFP reporters were also expressed in the vulval muscles, although these post-embryonically derived cells are likely to be absent from primary cultures 17 and therefore were not directly profiled by our methods (Figures 7b and 8). This finding must reflect underlying similarities between vulval and body wall muscle cells. Interestingly, six reporters show expression in all four muscle types and may be indicative of genes required for general muscle function (Figure 8). It is noteworthy that a majority of the corresponding endogenous genes for the body wall muscle positive GFP reporters (31/36 [86%]) were also strongly upregulated (≥1.7 fold) during HLH-1 induced embryonic myogenesis (Figure 6). In comparison, only 19% of muscle negative GFP reporters (3/16) exhibit similar upregulation (Additional data file 11). The analysis of GFP reporters constructed from the muscle enriched datasets confirms muscle expression in vivo and also potentially reveals interesting examples of genes with roles common to all four major muscle types as well as other transcripts with functions that may be selectively required in specific subsets of body muscle cells.

The experiments performed in this study profile muscle cells that presumptively differ in developmental age. The M0 dataset is comprised of early pre-morphogenesis embryonic cells whereas the M24 dataset includes muscle cells that have differentiated in culture for 24 hours. A comparison of transcripts enriched in both datasets reveals 401 common genes (Figure 5). Interestingly, of 38 transcripts encoding muscle structural proteins, 74% (28/38) are common to both datasets (Additional data file 12). This finding indicates that other genes in this list of 395 transcripts may also fulfill key roles in both nascent and fully differentiated muscle cells, and may therefore constitute a class of fundamental muscle function genes.

In addition to transcripts that are elevated in both datasets, we also detected genes that are selectively enriched in either the M0 or M24 profiles. Overall, 375 genes show elevated expression in the M0 dataset only whereas a separate group of 542 transcripts are exclusively enriched relative to all other cells in the M24 dataset (Figure 5). Of genes that are differentially detected in these datasets, we note that pat-3 and pat-6, which are required for initial muscle assembly 112627, are selectively enriched in the M0 profile. Conversely, unc-70 is detected as an expressed gene in the M0 dataset but it is exclusively elevated in the M24 profile, a result that is consistent with the finding that UNC-70 (β-spectrin) is expressed in all embryonic cells early in development but is localized to muscles and neurons at hatching 28. It is also possible that some of these differences could be induced by differences in the cellular environments of the M0 (intact embryo) and M24 (in vitro culture) muscle cells. For example, 24 genes encoding proteosome subunits show elevated expression in the M24 dataset whereas none of these transcripts are enriched in the M0 profile. This finding could be indicative of the general lack of innervation of muscle cells in culture because the removal of motor neuron activity in vivo results in increased muscle protein degradation via a proteosome dependent mechanism 29. Despite this caveat, these MAPCeL data appear to reveal differences in gene expression that correlate with the developmental 'age' of the M0 and M24 muscle cell populations, suggesting that this technique may be generally useful for detecting temporal changes in gene expression during development.

Genetic studies in C. elegans have identified a large number of genes that are required for muscle structure, development, and function 23. To assess the potential utility of our microarray data for expanding this catalog of muscle genes, we organized transcripts in these profiles according to functional categories. A sampling of these findings is presented below. Genes exhibiting enriched transcript levels are highlighted in bold when they are first identified in the text. All genes discussed in this section are listed in Table 1.

The overall organization of C. elegans body wall muscle cells is similar to that of vertebrate skeletal muscle. The primary functional component is the sarcomere, a structure composed of myosin-containing thick filaments (A-band) that interdigitate with actin-containing thin filaments (I-band). The nematode sarcomere resembles vertebrate striated muscle, although it is obliquely striated with myosin and actin-containing filaments oriented at an angle of 6° with respect to sarcomere end plates 1230. The sarcomere maintains functional alignment through attachment of thin filaments to dense bodies, which link thin filaments to the basement membrane of the cell. Thick filaments are stabilized within the sarcomere by the M-line, a specialized region in the A-band that links adjacent thick filaments. The dense bodies and the M-line are the primary mediators of tension generated during muscle contraction 10. Hemidesmosomes that connect each muscle cell to the overlying cuticle transmit this force to deform the exoskeleton and thereby propel locomotion 3031 (Figure 9).

Thick filaments are largely comprised of two myosin heavy chain (MHC) proteins, MHC A and MHC B, encoded by the myo-3 and unc-54 genes, respectively 1820. Interestingly, myo-3 is enriched in both the M0 and M24 datasets whereas unc-54 is selectively elevated in the M24 profile but detected as an expressed gene in M0 muscle cells. The elevation of myo-3 transcript levels before unc-54 mRNA during body wall muscle development is consistent with the observation that MHC A protein is also more abundant than UNC-54 in early embryonic muscle cells 32. The apparent sequential expression of myo-3 and unc-54 parallels their distinct roles in thick filament assembly; MHC A establishes a bipolar nucleation complex to which UNC-54 is added as the filament elongates 83334. Differential roles in muscle development are also underscored by the findings that myo-3 null mutants are nonviable as embryos whereas genetic ablation of unc-54 disrupts muscle structure and impairs movement but does not result in lethality 31335. Two additional transcripts, F45G2.2 and Y11D7A.14, with sequence similarity to the myosin heavy chain genes, are elevated in the M24 dataset. On the basis of strong similarity to the amino-terminal actin-binding and ATPase domain, F45G2.2 is a member of the myosin II class of striated muscle MHCs that includes myo-3 and unc-54. However, the carboxyl-terminal sequence of F45G2.2 is unusually short, with only about 100 amino acids, as opposed to the extended α-helical domain of about 1,000 amino acids in the MYO-3 and UNC-54 proteins. Because this so-called 'rod' domain drives thick filament assembly, it will be interesting to determine whether the foreshortened carboxyl-terminal region of F45G2.2 contributes to this structure. Y11D7A.14 encodes an unconventional myosin that is more distantly related to other structural myosins expressed in muscle. Potential functions for these additional myosin molecules in muscle can now be explored by genetic or RNA interference methods.

The myosin light chain proteins regulate the ATPase activity of the MHCs. Three myosin light chain genes (mlc-1, mlc-2, and mlc-3) are enriched in both datasets. Genetic data indicate that mlc-3 is an essential muscle component, whereas mlc-1 and mlc-2 appear to have redundant functions 3. Paramyosin (UNC-15), a core component of thick filaments that interacts with MHC A (MYO-3) and MHC B (UNC-54) 3236, shows elevated transcript levels in both M0 and M24 profiles. UNC-45, a highly conserved myosin binding protein and chaperone that directs assembly of these components, is enriched (Figure 9) 37.

Muscle structure and function also depend on a family of very large cytoplasmic proteins, which contain multiple fibronectin and immunoglobulin domains 3839. The founding member of this gene family, unc-22, encodes 'twitchin', which when mutated causes constant twitching movements 310. unc-22 is enriched in both datasets. A second member of this family, titin, adopts an elongated structure that spans half of the mammalian muscle sarcomere (from Z line to M line) and functions in myofibril assembly and elasticity 40. C. elegans titin is somewhat smaller and largely localized to the thin filament or I band region of the body wall muscle sarcomere. Previous work identified a 90 kilobase gene that encodes three distinct titin isoforms in C. elegans 38. We find that one of the identified titin transcripts (ttn-1) 39 is enriched in both M0 and M24 datasets (Table 1). Additionally, we identify ketn-1, an invertebrate-specific titin-like protein 41.

Thin filaments are primarily composed of actin, troponin, and tropomyosin (Figure 9). Although actin transcripts are not enriched in the muscle datasets because of high expression in nonmuscle cells, actin genes (act-2, act-3, act-4, and act-5) are detected as expressed genes (Additional data file 2). We do see elevated expression of several actin-binding and regulatory proteins. Among these are members of the profilin (for example, pfn-3) and gelsolin (for example, K06A4.3) families, which are proposed to regulate thin filament assembly (Table 1) 13. Sarcomeres are initially assembled during embryonic development. Muscle cells add new sarcomere repeats and expand in size as the animal grows 42. The continuous growth of the contractile apparatus during development could account for the expression of key structural components (for example, tni-1 and troponin) in both of the datasets. On the other hand, as noted above, genes identified in the M0 dataset may play important roles in the initial formation or organization of the sarcomere, whereas transcripts that are uniquely enriched in the M24 profile are required for sarcomere maintenance during expansion of the body wall muscle cells.

Troponin and tropomyosin form a complex that regulates actin-myosin interactions in response to calcium 13. In C. elegans, tropomyosin is encoded by lev-11, which is enriched in both datasets. Troponin is comprised of three subunits, TnI, TnT, and TnC. There are four TnI genes in the nematode, of which three (tni-1, unc-27, and tni-3) are expressed in body wall muscle 43 and each is enriched in our datasets. Similarly, all three body wall muscle expressed TnT transcripts, mup-2, tnt-2, and tnt-3, and the TnC transcript pat-10 are enriched in both datasets (Table 1 and Figure 8).

The sarcomere is attached to the cell membrane by both dense bodies and M-lines. Many components of the dense bodies are known, including the enriched genes encoding vinculin (deb-1), talin (Y71G12B.11a,b), afadin (W03F11.6a) and β-1 integrin (pat-3). Previous studies have also identified unc-112 and dim-1 as components required for dense body assembly and maintenance, respectively 4445. Other enriched actin-binding proteins with potential roles in body muscle assembly include tag-138, a Huntingtin-interacting protein, and cor-1, which is a homolog of coronin (Table 1).

Muscle contraction is triggered by the release of intracellular calcium stores in response to neurotransmitters secreted from adjacent motor neurons at the neuromuscular junction. Cytoplasmic calcium initiates myofibril contraction and is rapidly pumped back into the sarcoplasmic reticulum via an ATP-dependent calcium channel 46. As expected, calcium channel and calcium ion binding proteins are detected as enriched transcripts in our datasets (Table 1). For example, the ryanodine receptor, UNC-68, mediates the release stored calcium 4748 and sca-1 encodes the nematode sarco-endoplasmic reticulum calcium ATPase (SERCA), which returns cytoplasmic calcium to the sarcoplasmic reticulum 46. We also find three genes that are predicted to function as calcium sensors (C54E10.2, F21A10.1, and ncs-3), as well as other components of calcium signaling including calsequestrin and calmodulin (Table 1).

In humans, Duchenne and Becker muscular dystrophies arise from mutations in a single gene encoding the large membrane-associated protein, dystrophin; these diseases are characterized by severe muscle weakening and degeneration 49. Dystrophin is localized beneath the sarcolemma and is attached to actin filaments as well as to the dystrophin glycoprotein complex (DGC) 50. This protein complex stabilizes the sarcolemma and prevents damage to muscle fibers induced by long-term contraction. Mutations in the C. elegans homolog of dystrophin (dys-1) lead to hyperactivity, but muscle degeneration has not been observed 5152. However, in C. elegans, as in mouse, mild MyoD (hlh-1) mutations in conjunction with dystrophin deficiencies act synergistically to induce muscle disassembly 5053; this finding suggests that C. elegans may be a useful model for studying these degenerative diseases. Most of the major DGC components 52, (dys-1 [dystrophin], dyc-1 [CAPON], stn-1 [syntrophin], and sgn-1 [sarcoglycan]) are enriched in our C. elegans body wall muscle profiles; the one exception is dyb-1 (dystrobrevin), which is not detected. Other enriched transcripts with potentially related functions include the sarcoglycan-like genes sgcb-1 and H22K11.4. A GFP reporter for H22K11.4 confirms expression in body wall, anal, and vulval muscles (Figure 7b). Additionally, the M0 profile detects stn-2, a second syntrophin-like gene. Given the conservation between the nematode and vertebrate homologs of the DGC components, it is probable that our datasets include transcripts for additional proteins that may be required to maintain muscle integrity.

Coordinated locomotion is mediated by signaling between motor neurons and postsynaptic muscle partners. Neurotransmitters released by motor neurons activate ion channels, thereby altering the local membrane potential to regulate muscle cell activity. These transmitter-gated ionotropic receptors are clustered at the synapse and open transiently in response to neurotransmitter binding. Consistent with the importance of these molecules in facilitating synaptic signals, we find a large number of ligand-gated ion channels to be enriched in body wall muscle cells. All of the subunits of the levamisole-sensitive nicotinic acetylcholine receptor (nAChR; namely lev-1, lev-8, unc-29, unc-38, and unc-63) are enriched 545556. Two additional uncharacterized nAChR genes, acr-8 and acr-16, were also detected. We recently confirmed that acr-16 is an essential component of the levamisole-insensitive nAChR, thereby substantiating the utility of these data for identifying new muscle genes 1657.

The neurotransmitter γ-aminobutyric acid (GABA) inhibits muscle activation and thus works in opposition to acetylcholine receptors. The GABA receptor gene unc-49 that mediates this response is enriched in our datasets 55. In addition to the expected acetylcholine and GABA receptors, we also detected ionotropic receptors for other classes of neurotransmitters that have not previously been shown to regulate muscle activity directly in C. elegans. Prominent among these is mod-1, which encodes a serotonin-gated chloride channel required for 5-hydroxytryptamine dependent inhibition of C. elegans locomotion 58. Additional amine responsive ionotropic receptors include T24D8.1 and Y113G7A.5, which are activated by 5-hydroxytryptamine and tyramine, respectively, when they are expressed in Xenopus ooctyes (Abe N, Ringstad N, Horvitz B, personal communication). Other candidate ionotropic anion channel receptor genes include glc-4 (glutamate-gated chlorine channel) and T27A1.4 (similar to GABA-A receptor). Excitatory responses to glutamate could be mediated by glr-8 (glutamate-gated kainite-type ion channels).

Mammalian myogenesis is initiated by a family of HLH transcription factors that includes MyoD, myogenin, MRF-4/herculin/Myf-6, and Myf-5 6. In C. elegans, hlh-1 encodes CeMyoD and is detected in all embryogenic lineages (MS, C, D, and AB) that give rise to the body wall muscle cells 5. Consistent with the important role of HLH-1 in early myogenesis, the hlh-1 transcript is enriched in both muscle datasets. Also enriched is the MADS transcription factor UNC-120. unc-120 is expressed early in embryonic development in muscle precursor cells and appears to cooperate with HLH-1 to drive muscle gene expression 59. Additionally, we detect ceh-13, a member of the HOX family of transcription factors that regulate cellular differentiation in specific body regions. ceh-13 expression in the embryo is limited to the anterior body wall muscles and other cell types in the head region 60.

In addition to these characterized transcription factors, we detect eight putative nuclear hormone receptors (NHRs; Table 1). In C. elegans, the NHR family consists of about 280 receptors that are presumptively regulated by lipophilic hormones to control a variety of processes from sex determination to lifespan 61. Functions for the NHR transcripts showing enriched expres