2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada, and 3Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
Received on June 7, 2001; revised on July 12, 2001; accepted on July 13, 2001.
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Abstract |
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Key words: Caenorhabditis elegans/core 2 N-acetylglucosaminyltransferase/O-glycans
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Introduction |
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Mammalian genes that encode these enzyme specificities are subject to developmental regulation (Bierhuizen and Fukuda, 1992; Bierhuizen et al., 1993
; Yeh et al., 1999
; Schwientek et al., 2000
). Human erythrocytes switch the fetal (i) antigen for the adult (I) antigen as a result of stage-specific expression of I-GlcNAc-T during late embryonic development (Bierhuizen et al., 1993
). In mice, core 2 GlcNAc-T(L) is widely expressed at embryonic day (E) 7, becoming progressively more restricted to a subset of mucin- and cartilage-producing tissues between E11.5 and E17.5 (Granovsky et al., 1995
). Activation of both human and mouse T cells results in up-regulation of core 2 GlcNAc-T (Barran et al., 1997
; Piller et al., 1988
). Transgenic mice that ectopically express core 2 GlcNAc-T prematurely during the elaboration of the T cell lineage have reduced delayed-type hypersensitivity and proliferation after stimulation as well as poorer substratum adherence (Tsuboi and Fukuda, 1997
). Mice lacking core 2 GlcNAc-T(L) exhibit a restricted phenotype with neutrophilia and a partial deficiency of selectin ligands but without affecting lymphocyte homing (Ellies et al., 1998
). PSGL-1 is the glycoprotein ligand for P- and E-selectin, and the O-glycan formed by core 2 GlcNAc-T is essential for its high affinity binding to these receptors (Kumar et al., 1996
).
In mammals, more than one gene product can add a particular monosaccharide in a given linkage to maturing glycoproteins. Multiple homologues of several glycosyltransferases have been characterized, each representing a subtly different enzyme specificity or expression pattern. For example, humans have at least three gene products producing core 2 O-glycans, core 2 GlcNAc-T(L), core 2 GlcNAc-T(M), and core 2 GlcNAc-T3 (Bierhuizen and Fukuda, 1992; Yeh et al., 1999
; Schwientek et al., 2000
). The Caenorhabditis elegans genome sequencing project revealed numerous sequences homologous to glycosyltransferase or nucleotidesugar transporter activities, several of which have been demonstrated functionally authentic (C. elegans Sequencing Consortium, 1998; Hagen and Nehrke, 1998
; DeBose-Boyd et al., 1998
; Chen et al., 1999
; Bulik et al., 2000
; Berninsone et al., 2001
; Warren et al., unpublished data). In fact, with the exception of sialylation, the C. elegans genome encodes a repertoire of genes that have the potential to elaborate the same glycosylation machinery as mammals (Dennis et al., 1999
). There are eight "squashed-vulva" genes defined by mutation in C. elegans of which sqv-3, sqv-7, and sqv-8, homologues of galactosyltransferase, glucuronyltransferase, and a nucleotidesugar transporter, respectively, have been confirmed to encode functional components of the proteoglycan glycosylation pathway (Herman and Horvitz, 1999
; Bulik et al., 2000
; Berninsone et al., 2001
). These gene products direct invagination of epithelia during vulval morphogenesis as well as support oocyte maturation and early embryogenesis (Herman et al., 1999
). The "surface" mutations cause ectopic wheat germ agglutinin binding to the cuticle. srf-4, srf-8, and srf-9 have pleiotropic defects caused by improper migrations of the distal tip cell and axon morphology (Link et al., 1992
). At least one C. elegans mucin homologue, let-653, is essentialhomozygous mutant animals arrest early in larval development with abnormal, cystic excretory canals (Jones and Baillie, 1995
).
A characteristic of metazoan radiation is the duplication of genomic blocks resulting in the appearance of multiple diverged homologues of ancestral precursors (Rubin et al., 2000). The genome of C. elegans is typical, containing many gene families, including glycosyltransferase gene families (C. elegans Sequencing Consortium, 1998). There are 18 predicted genes at least somewhat homologous to core 2 GlcNAc-T in C. elegans, the 167th largest gene family in this organism (Apweiler et al., 2001
). A subset of six that are more closely related to the mammalian core 2/I GlcNAc-T sequences are the best candidates to encode nematode O-glycan ß6-glucosaminyltransferases. We report here the initial characterization of this family of core 2 N-acetylglucosaminyltransferase genes from C. elegans named gly-1 and gly-15 to gly-19. We show that the spatial and temporal patterns of expression of this group are virtually nonoverlapping. We also demonstrate that for three of the family, deletion mutagenesis does not disrupt development under laboratory conditions.
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Results |
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gly-1: an internal cistron within an operon
Both, 91, isolated by library hybridization, and the EST yk244g8 contained the full coding region of gly-1 as well as portions of the predicted upstream gene sra-12, a 7TM receptor (Troemel et al., 1995
). Comparison with genomic sequence indicates a 451-bp intron between the two genes, with splice donor and acceptor sequences immediately after the end of the ultimate sra-12 exon and preceding the first gly-1 exon, respectively. The separation between cistrons within C. elegans operons is bimodal, typically
100 bp or 300400 bp apart. The closely spaced class is trans-spliced to splice leader (SL) type 2 sequences, whereas genes of the less dense class are always trans-spliced to both SL1 and SL2 (Blumenthal and Steward, 1997
). Attempts to derive SL2 trans-spliced gly-1 cDNA by 5' rapid amplification of cDNA ends (RACE) or directly by PCR using SL2 primers have been unsuccessful, but an SL1 trans-spliced product corresponding to the first splice acceptor sequence upstream of the ATG was isolated. These data are consistent with gly-1 being an internal cistron within an operon. Although the genomic interval between sra-12 and gly-1 is sufficient to contain an additional internal promoter, the main promoter likely resides in an upstream distal location.
gly-15 to gly-19 are expressed from their own promoters
gly-16 and gly-17 as well as gly-18 and gly-19 occur as tandem pairs in the genome, separated by 601 and 578 bp, respectively. The proximal upstream gene to gly-16 is 775 bp away, encoded by the opposite strand. For gly-18, the nearest upstream neighbor is 3514 bp away on the same strand. The nontandem gene gly-15 is 2156 bp downstream of its nearest neighbor, but there is an orphan exon situated 280 bp upstream that may be a part of gly-15. These spacings suggest that the promoters for these genes are proximal upstream, and they are not members of operons. SL2 trans-spliced transcripts, a feature of internal cistrons, were not detectable (Blumenthal and Steward, 1997). These inferences are also supported by the expression patterns of glyp::GFP reporter constructs (see Family members are expressed in nonoverlapping patterns).
The 5' RACE fragments isolated from gly-16 and gly-19 terminated exactly at the initiator methionine. This characteristic is typical of an "outron," where a genomic 5' terminal splice acceptor is trans-spliced to SL1 (Blumenthal and Steward, 1997). The genomic sequence immediately upstream of the tandem gene pairs, gly-16 to gly-19, has canonical splice acceptor sequences abutted exactly prior to the predicted or observed start codons in all cases suggesting that these are the 5' ends of the mature mRNA and are probably trans-spliced to SL1. Splice leader sequences were not recovered in the RACE amplimers from gly-16, but the gly-19 5' RACE fragment and the EST yk740h11 included an SL1 trans-spliced leader sequence abutted exactly prior to the ATG. No amplimers in which sequences from the upstream gene or an SL2 leader indicative of gly-19 being an internal cistron within an operon initiated by gly-18 were isolated.
The deduced primary sequences are characteristic of glycosyltransferases
Core 2 GlcNAc-T homologues from C. elegans conform to the secondary structure characteristic of glycosyltransferases (Munro, 1995; Unligil and Rini, 2000
). The optimized alignment of amino acid sequences is shown in Figure 2. The mammalian enzymes have between 5 and 16 residues before a 1419-residue predicted transmembrane domain, the remaining 375424 amino acids are believed to be lumenal. The six C. elegans homologues are similar consisting of 516 cytoplasmic amino acids prior to 1020 predicted transmembrane residues, whereas the C-terminal 366446 amino acids are likely the stem and catalytic domains.
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Family members are expressed in nonoverlapping patterns
Representative expression patterns in hermaphrodites from transcriptional fusions of a GFP reporter to presumptive promoter regions of gly-1 and gly-15 to gly-19 are shown in Figure 5. The distribution of signal was unique for each reporter construct and expression patterns were highly restricted with respect to tissue and/or stage of development. With the exception of minor aspects of gly-18, the expression pattern of any family member did not overlap temporally or spatially with that of another. Expression did not correlate with the elaboration of particular cell sublineages, nor was it restricted by tissue type. A composite cartoon mapping the expression of all six gene products is shown in panel r.
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The GFP fluorescence produced by gly-15 (Figure 5eg) was exquisitely specific; observed solely in a cellular process, probably the secretory network, of the G2 gland cells (Figure 5g). Signal appeared in the embryo as soon as pharyngeal structures could be discerned and remained through adulthood. It is possible that this construct is a translational fusion including the coding region for the transmembrane domain of GLY-15. The 5' end of gly-15 could not be isolated experimentally, so the initiator methionine predicted by GENEFINDER was used. However, the genomic sequence contains an orphan exon slightly 5' to the predicted gene that could encode an initiator methionine and potential transmembrane domain. Plausibly this is the authentic N-terminal of GLY-15, in which case the reporter construct would direct expression of a message containing this exon fused to the GFP cassette resulting in a polypeptide that contained residues sufficient for localization to the Golgi apparatus.
gly-16p directed expression of GFP (Figure 5h, i) was complementary to that of gly-1. Fluorescence was observed only in the late embryonic seam cells, fading exponentially during early larval development and completely absent from L2 onward.
Patterns of gly-17 expression are not shown because fluorescence was rarely observed in any samples. The few cases that did display a GFP signal were all malformed embryos. gly-16 and gly-17 are tandem in the genomic sequence, separated by only 601 bp, and thus could be polycistronic. The gly-17p::GFP expression construct therefore contained the region comprising the entire gly-16 gene as well as the intervening sequence upstream of the gly-17 initiator codon. The expression patterns of the gly-16p::GFP and gly-17p::GFP constructs are distinct and specific, indicating that unique promoters drive these genes.
Expression of GFP from gly-18p::GFP arrays (Figure 5jo) was most complex of the family. Fluorescence was detected in the ALA, GLR, and other neurons. Muscle expression was observed in the VM1, intestinal muscle cells, certain anterior body wall muscles and probably the anal sphincter. GFP was also present in the seam, distal tip cells, celomocytes, a socket cell associated with a nonamphid neuron, as well as other as yet unidentified cells. gly-18 expression occurs in a set of cells that execute diverse functions and are not related by lineage, tissue type, or anatomy. The pattern of gly-18 expression overlaps with two other genes in the family. Seam cell expression seems contemporaneous with that of gly-1, and gly-18 and gly-19 may be coexpressed in the anal sphincter muscle.
gly-18 and gly-19 are also tandem in the genome, the intergenic distance is only 578 bp. The gly-19p::GFP expression construct therefore included genomic DNA that encompassed gly-18 as well. gly-19p::GFP was well expressed (Figure 5p, q) in intestine and anal sphincter as soon as these structures appeared in the developing embryo and continued to be expressed until and including adulthood, a pattern quite distinct to that of gly-18. We infer that the 578 bp of genomic DNA interposed between the genes contains a fully qualified promoter for gly-19.
gly-1, gly-16, and gly-18 are nonessential
ev686 contains a 2-bp insertion in the third exon of gly-1, a footprint generated by "precise" Tc1 excision from ev588 (TableI and Figure 1). The frameshift prematurely truncates the authentic polypeptide at a third of its length. qa702 is an imprecise Tc1 excision allele of ev588 that replaced almost the entire conserved coding region with 30 random nucleotides during the religation-repair of the double-strand break caused by Tc1 excision. Both ev686 and qa702 are probably null alleles. gly-1 mutant strains homozygous for either ev686 or qa702 had no defect observable under a dissecting microscope. The expression of gly-1 in the adult seam suggested a function in the development of the male tail, because the seam cells are known to participate in the formation of this structure. Loss and/or mispositioning of ray 1 in the adult male tail (25% at 20°C) was observed in NW1287 gly-1(ev686) but could be complemented by gly-1(qa702), indicating a linked but unrelated extragenic mutation.
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Introduction of double stranded cRNA into the hermaphrodite gonad leads to F1 embryos in which the endogenous gene is silenced, typically copying the null phenotype (Fire et al., 1998). RNA-mediated interference was attempted for four of the homologues (except gly-1 for which a mutant allele was already available, and gly-17 since expression in normal circumstances was not observed) using the transcript targets shown in Figure 1. In our hands, both positive and negative control experiments were consistently successful but none of the core 2 GlcNAc-T genes assayed caused visible embryonic or adult defects. Experiments combining all the homologues were not attempted because ablation efficiency generally drops greatly when more than two cRNAs are combined and the effect is concentration dependent (Fire et al., 1998
).
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Discussion |
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Only gly-1 appears to be expressed as part of an operon downstream of sra-12, a 7TM receptor, raising the possibility of a functional relationship. All other family members, even the downstream members of the tandem pairs, gly-17 and gly-19, have independent promoters. All six homologues have specific and distinct patterns of expression but even collectively are not expressed in all tissues or stages of the animal. The GFP reporter transgenes indicate that gly-1, gly-15, gly-18, and gly-19 are expressed in generally nonoverlapping spatial domains during adult life. The exceptions are gly-1 and gly-18, which are both expressed in the seam, and gly-18 and gly-19, which are present in the anal sphincter muscle. gly-16 is also expressed in the seam, but only during embryonic stages. gly-15 and gly-19 are expressed in a gland cell and gut, respectively, possibly involved with the production of lubricant mucins. The potential roles of gly-1, gly-16, and gly-18 in their hypodermal, neuronal, or muscular domains or gly-17 in defective embryos are harder to envisage. Deletion, presumably null, alleles were derived for gly-1, gly-16, and gly-18, but homozygous mutant animals manifested no visible defects. Likewise, RNA-mediated interference of gly-15 or gly-19 did not lead to aberrant phenotypes. The expression pattern data is suggestive of essentially independent domains of function for each gene, and the emergence and adaptation of the six core 2 GlcNAc-T homologues should be associated with selective pressure to contribute to fitness. However, under laboratory conditions ablation of gene activities did not affect development, behavior, or propagation at growth temperatures from 15°C to 25°C. Because GFP reporter transgenes are multicopy mosaics, both ectopic and absent expression are possible. It remains a possibility that expression of some of the homologues, and perhaps the other 12 distantly related genes, overlap to create functional redundancy. Experiments are underway to mutate gly-15, gly-17, and gly-19 so that strains lacking all six members of the family can be constructed.
The sequence variation among the C. elegans homologues is at least as great as that between mammalian members of the core 2 GlcNAc-T family (Figure 4). From sequence comparison it is not possible to discern whether the nematode genes encode core 2M, core 2L, I GlcNAc-T enzymes, or a novel but related specificity. Using nuclear magnetic resonance and mass spectrometry, a series of O-glycans were identified from C. elegans, the majority based on a core 1 structure. In several of these oligosaccharides the polypeptide linked GalNAc was ß1-6 branched, but the substitutent monosaccharide was glucose or galactose rather than GlcNAc (Guerardel et al., 2001). We are investigating whether the gene products described here are the enzymes responsible for this linkage.
Curiously, no relative could be found in the Drosophila melanogaster genome. Molecular data currently suggests that the fly is a closer relative of humans than worms but alternative models where nematodes are closer relatives of vertebrates than arthropods cannot be robustly excluded (Fitch and Thomas, 1997). With the exception of gly-15, all comprise 8 coding exons spread over
2kb of genomic sequence. The tandem homologues are the most related to each other and then to the other tandem pair, gly-15 is the most different. This pattern is consistent with the polypeptide phylogeny where gly-1 and gly-15 arose by duplication from a common ancestor. Primordial gly-1 or gly-15 then duplicated forming primitive gly-19 which in turn generated a tandem copy that was duplicated en bloc most recently giving rise to the progenitors of gly-16 and gly-17. All the homologues, with the exception of gly-1, which occurs on linkage group (LG) II, are found on LGI. gly-15, gly-16, and gly-17 are near each other in the genome embedded within a region that is rich in sequences related to glycosylation; multiple glycosyltransferase homologues, C-type and S-type lectin domains, as well as nucleotidesugar synthases (Jungmann and Munro, 1998
; Apweiler et al., 2001
). Natural selection on the nematode has fixed multiple duplications of the primordial coding potential and specific promoters have evolved for each. The most parsimonious interpretation of this divergence is that a similar core 2/I GlcNAc-T like enzyme specificity is conserved by all homologues but expressed in distinct temporal-spatial boundaries.
Published studies on glycan biosynthesis in C. elegans lend support to this view. There are nine genes homologous to polypeptide GalNAc-T that give rise to 13 transcripts. Of these, five isoforms from three of the genes were shown to be catalytically active (Hagen and Nehrke, 1998). GlcNAc-TI has three homologues, all expressed in the gut but distinctly elsewhere in the animal, and enzyme activity has been demonstrated for two of them (Chen et al., 1999
). There is biochemical evidence for at least 2
1,3 fucosyltransferases (DeBose-Boyd et al., 1998
). A GlcNAc-TV orthologue, gly-2, can rescue Lec4 mutation of Chinese hamster ovary cells (Warren et al., unpublished data). The components of the proteoglycan pathway encoded by sqv-3, sqv-7, and sqv-8 all possess the biochemical activity expected from their homologies (Herman and Horvitz, 1999
; Bulik et al., 2000
; Berninsone et al., 2001
). Sequence analysis of the genomes of C. elegans and Drosophila melanogaster has revealed that it is the nematode which has more genes, despite it being the simpler organism (Rubin et al., 2000
). Our data are consistent with the view that C. elegans tends to regulate processes through the replication and adaptation of unique genes for specific tasks, rather than by modifying a single gene for multiple tasks requiring complex promoters or alternative splicing. For example, there are two alternative splicing products of murine I-branching enzyme (Figure 4, mIGnT-A and -B) that are as diverged as the tandem pairs of nematode core 2 GlcNAc-T homologues. C. elegans has fixed larger families of diverged homologues, also with generally non-overlapping patterns of expression (Troemel et al., 1995
; Aspock et al., 1999
).
The experimental tractability of C. elegans may complement studies in mammals. In particular, screening for extragenic enhancers of the mutations reported here could reveal a set of genes that interact genetically with the core 2 N-acetylglucosaminyltransferase homologues, candidate effector glycoproteins whose function is dependent on being correctly O-glycosylated, and so discover the contribution to fitness made by ß6-GlcNAc branched O-glycans. Constructing strains where all six are deleted might reveal the survival value of the core 2 GlcNAc-T genes. We are also testing whether core 2 GlcNAc-T genes in C. elegans function in environments to which laboratory animals are not ordinarily subjected and performing genetic "precomplementation" screens.
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Materials and methods |
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Mutagenesis
The gly-1 alleles, ev686 and qa702 were generated by Tc1-mediated mutagenesis with minor modifications (Zwaal et al., 1993). Detection of ev686, which is a 2-bp insertion (TableI), was possible because it destroys an NdeI site present in the wild-type genome so that amplimers encompassing the mutated site failed to digest with this enzyme. Alleles of gly-16(qa701) and gly-18(qa704) were isolated directly from an ethyl methanesulphonateinduced deletion library (Liu et al., 1999
). In both cases, PCR-directed sib-selection was used to identify and recover animals bearing deletions that were outcrossed then backcrossed at least nine times with N2 (the exception being qa702, which was outcrossed to DR466 then DR435 and finally to N2).
RNA-mediated interference
RNA was transcribed from linearised pBluescript (Stratagene) containing PCR-amplified portions of cDNA using T7 and T3 RNA polymerases (Promega). Complementary strands were annealed and microinjected into the gonads of young adult N2 animals (Fire et al., 1998). F1 progeny derived from eggs laid on the following day were examined under a dissecting microscope as well as by differential interference contrast microscopy using a Leica DMR photomicroscope. The NcoI-BamHI cassette of pCITE4a.GLD-1(84-457) was subcloned into pBluescript. Double stranded gld-1 cRNA (Francis et al., 1995
) and media alone were used as injection controls. gld-1 injected animals showed a progressive conversion of the hermaphrodite germ line over the following days to the tumorous state concomitant with loss of fertility. Injection of media only caused no defect.
RT-PCR and cDNA isolation
Unless otherwise noted, standard molecular biology techniques were employed (Ausubel et al., 1992). Poly-A+ RNA was isolated using a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech). Poly-A+ RNA was made from unstarved, mixed-stage, wild-type (N2) hermaphrodites, as well as from populations enriched for dauers by starvation, for males by mating, and from embryos. To enrich for embryo-specific transcripts, gravid hermaphrodites were treated with alkaline hypochlorite (Wood et al., 1988
). Lysates were washed seven times with M9, once with M9 containing 1% Tween 20, three more times with M9 alone, then snap frozen in ethanol/dry ice before poly-A+ RNA extraction. Reverse transcription was performed using either random or gene specific primers and cDNA fragments were amplified by PCR. Smart-race (Clontech) and 5' RACE System v2 (Life Technologies) kits were used for isolation of 5' and 3' cDNA ends. The general strategy was to design gene-specific primers to a conserved portion of the predicted gene. Using this approach fragments were amplified, subcloned, and sequenced from gly-1, gly-15, gly-16, gly-17, and gly-18. Using the 579-bp amplimer from gly-1, cDNA,
91, was isolated by plaque hybridization.
91 contained the full coding region of gly-1 as well as the last 1.5 exons of the predicted upstream gene, sra-12 (Troemel et al., 1995
). Consistent with this, a gly-1 EST, yk244g8, was also identified, sequenced, and found to contain a contiguous cDNA encoding full-length gly-1 and sra-12. gly-19 was represented by two ESTs in the genome databases, yk150d10 and yk171f8, both of which were sequenced and found to contain 1250 and 740 nucleotides, respectively, with the same 3' termini, representing most of the coding sequence. Since then three more gly-19 ESTs have been added to the database. yk740h11 includes an SL1 sequence at its 5' end that corresponds exactly to a 5' RACE product isolated for gly-19. yk707a7 and yk726b10 are very similar and contain an untranslated exon separated from the start codon by a 43-bp intron. All three terminate at the 3' end within the coding region. Thus partial cDNAs representing transcripts from all six genes were obtained. Using these sequences, primers were designed for 5' and 3' RACE. Additional fragments of gly-16, gly-17, and gly-18 were obtained by 3' RACE. 5' RACE fragments were obtained for gly-1, gly-16, gly-18, and gly-19. Composite sequences and cDNA fusions were then constructed by standard methods.
GFP reporter transgenes
Precise transcriptional fusions of promoter regions to GFP were constructed using at least 4 kb of cosmid DNA, upstream of and including the initiator codon, which was ligated into pPD95.77 or pPD95.79. CB1282 hermaphrodites were transformed by gonad injection (Mello et al., 1991) of a mixture of reporter construct and pMH6, a plasmid containing a region of C. elegans genomic DNA capable of rescuing the dpy-20(e1282) mutation. Multiple independent non-Dpy F1 progeny were selected, transgenic lines established from them, and epifluorescence microscopy performed using a Leica DMR photomicroscope. The inheritance of extrachromosomal arrays is mosaic, and the fine structure of the array in each strain is different. Consequently, several individuals from each line were examined to compile consensus expression patterns. Animals bearing the reporter construct for gly-15 were also examined using an Olympus IX70 deconvolution microscope. Cell identification was accomplished by comparison to morphological features visualized by DIC microscopy, dye filling of amphid neurons (Starich et al., 1995
) or phalloidin staining of muscle (Priess and Hirsh, 1986
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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