Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva

Alicia N. Minniti*, Mariana Labarca*, Claudia Hurtado and Enrique Brandan{ddagger}

Centro de Regulación Celular y Patología, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, MIFAB, P. Universidad Católica de Chile, Casilla 114-D, Santiago, Chile

{ddagger} Author for correspondence (e-mail: ebrandan{at}bio.puc.cl)

Accepted 5 July 2004


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In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. In the present study, we show that C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in western blot analysis. In the sdn-1 (ok449) deletion mutant allele we observed the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 (ok449) deletion mutants lack two glycosylation sites. Hence, the missing protein in the western blot analysis probably corresponds to SDN-1. In addition, we show that SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 (ok449) deletion mutants. These mutants show a defective egg-laying phenotype. Our results show for the first time, the identification, localization and some functional aspects of syndecan in the nematode C. elegans.

Key words: C. elegans, Proteoglycans, Syndecan, Nervous system, Extracellular matrix, Egg-laying


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nematode Caenorhabditis elegans and other genetically accessible organisms such as Drosophila melanogaster have been crucial in elucidating the biological functions of diverse gene products during development. In C. elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special relevance in recent years (Berninsone and Hirschberg, 2002Go). The genes that encode the GAG biosynthetic enzymes were found through traditional genetic screens (Herman et al., 1999Go) that uncovered their essential role during development as well as their high degree of conservation in eukaryotes. These `squashed vulva' genes, sqv-1 to sqv-8, encode enzymes such as galactosyltransferases (sqv-2 and sqv-3) (Bulik et al., 2000Go; Hwang et al., 2003aGo), chondroitin synthase (sqv-5) (Hwang et al., 2003bGo) and xylosyltransferase (sqv-6) (Hwang et al., 2003aGo). Mutations in all of these genes were shown to cause defects in vulva development, specifically in the invagination of the vulval epithelium. Loss of function of these genes also led to decreased fertility as well as embryonic developmental arrest (Herman et al., 1999Go). In addition, a gene homologue of the EXT protein family has been found in C. elegans (rib-2), which is probably involved in the initiation and elongation of heparan sulfate synthesis (Kitagawa et al., 2001Go).

Certain heparan sulfate modifying enzymes have also been analyzed recently. Mutants for three such genes (hst-2, hse-5 and hst-6) show axonal and cellular guidance defects in specific neuronal classes (Bulow and Hobert, 2004Go). Moreover, two heparan sulfate modifying enzymes (hse-5 and hst-6) were seen to regulate the activity of KAL-1, the C. elegans homologue of the human KAL-1 secreted protein that is linked to a human genetic neurological disorder, the Kallman syndrome (Berninsone and Hirschberg, 2002Go; Bulow et al., 2002Go).

Although most of the enzymes involved in the biosynthesis and modification of GAGs have been extensively characterized in C. elegans, little is known regarding the core proteins to which these GAGs are attached in proteoglycans. The only mammalian proteoglycan homologue that has been widely studied is unc-52, the C. elegans perlecan homologue. Lack of UNC-52 blocks the assembly of the myofilament lattice during embryogenesis, resulting in embryonic developmental arrest. The sequencing of the C. elegans genome revealed the presence of at least three other proteoglycan core proteins, homologues to the vertebrate syndecans, glypicans and agrin (Berninsone and Hirschberg, 2002Go; The C. elegans Sequencing Consortium, 1998Go).

Syndecans are a particular family of heparan sulfate proteoglycans with highly conserved cytoplasmic and transmembrane domains and more divergent extracellular domains (Bellin et al., 2002Go; Bernfield et al., 1999Go; Carey, 1997Go). The extracellular domain contains specific attachment sequences where heparan sulfate GAGs are covalently linked. The cytoplasmic domain of some syndecans (e.g. mammalian syndecan-4) can be phosphorylated at a specific serine residue (Horowitz and Simons, 1998Go). Both, the GAG chains in the extracellular domain, as well as the phosphorylation site of the cytoplasmic domain of some syndecan family members, have been implicated in numerous biological functions such as cell differentiation (Larrain et al., 1998Go), synapse development (Yamaguchi, 2001Go), growth factor signaling (Simons and Horowitz, 2001Go), regulation of cytoskeletal organization (Yoneda and Couchman, 2003Go) and embryonic patterning (Kramer et al., 2002Go). There are four syndecan family members in mammals, whereas only one, involved in neuronal axon guidance, has been found in Drosophila (Johnson et al., 2004Go; Steigemann et al., 2004Go). As mentioned above, a single syndecan homologue (sdn-1) was identified in the C. elegans genome by sequence analysis. However, sdn-1 expression as well as the glycosylation, localization and biological function of its protein product have yet to be explored.

In this work, we show for the first time, that C. elegans synthesizes sulfated proteoglycans, seen as three distinct species by western blot analysis. In the sdn-1 (ok449) deletion mutant allele we observed the lack of one species that corresponds to a 50 kDa product after heparitinase treatment. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 (ok449) deletion mutants lack two glycosylation sites. Hence, the missing protein in the western blot analysis corresponds to SDN-1. In addition, SDN-1 was localized to the C. elegans nervous system and vulva. Interestingly, SDN-1 was found specifically phosphorylated in nerve ring neurons and to a lesser extent in the vulva in both wild-type and sdn-1 mutant worms. The deletion mutants showed an egg-laying phenotype that could be related to sqv phenotypes observed in mutants lacking specific GAG biosynthetic enzymes and/or to defective migration of hermaphrodite-specific neurons.


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Worm strains and handling
Worms were cultured on 100, 60 or 30 mm NGM (nematode growth medium) agar plates seeded with Escherichia coli strain OP50, as described elsewhere (Brenner, 1974Go; Stiernagle, 1999Go). All strains were stored at 20°C and experiments conducted at 20-22°C. Strains used were N2 (var. Bristol) as the wild type; RB690, sdn-1 (ok449) X; NW1229, dpy-20 (e1362) IV; evIs 111 (pan-neural GFP reporter strain); VC233, gpn-1 (ok377) X.

Radiolabeling and concentration of proteoglycans
For biochemical analyses, six 100 mm plates of multistage worms were synchronized using the NaOH-hypoclorite method (Brenner, 1974Go). Eggs were placed on NGM plates seeded with bacteria and cultured at 20°C until they reached the L4 young adult stage. Worms were collected, washed and resuspended for radiolabeling on S Medium without sulfate (Stiernagle, 1999Go) and 100 µCi/ml of [35S]H2SO4 (NEN, USA). The worms were then incubated in this medium for 18 hours with gentle shaking. Excess radioactive isotope was eliminated by two washes in S Medium. Radiolabeled worms were homogenized in 4 M Guanidine-HCl sodium acetate buffer (50 mM NaAc pH 5.8, 0.2 M NaCl, 0.5% Triton X-100 supplemented with a cocktail of protease inhibitors: 10 µg/ml leupeptin, 50 µg/ml aprotinin, 100 µg/ml benzamidine chlorure, 10 µg/ml pepstatin, 100 µg/ml soy trypsin inhibitor and 1 mM PMSF) through six passages on an Ultraturrax homogenizer for 10 seconds each, and then incubated overnight at 4°C with gentle shaking. Homogenates were finally pelleted to remove worm debris and guanidine-soluble extracts dialyzed twice using the same buffer containing first 4 M and then 8 M urea. Incorporated radioactivity was measured by scintillation counting. Samples were incubated with 1 ml anion exchange chromatography DEAE-Sephacel (BioRad, USA), washed extensively, packed on columns and eluted using a linear 0.2-1.0 M NaCl gradient. The conductivity and radioactivity of each collected fraction was then determined. Radioactive fractions were pooled, concentrated and dialyzed against heparitinase buffer (20 mM Tris-HCl pH 7.4, 50 mM NaCl) to remove excess salt and analyzed by gel filtration chromatography on either Sepharose CL-6B or Sephadex G-50 columns, after being subjected to the various chemical or enzymatic treatments described below.

Biochemical analysis of proteoglycans and GAGs
Pooled intact radiolabeled proteoglycans were subjected to alkali extraction in the presence of 1.0 M NaBH4, as described previously (Brandan and Inestrosa, 1987Go) to release intact GAG chains. Core proteins were degraded by proteinase K (Sigma) treatment: 50 µg/ml of enzyme at 65°C for 1 hour, in buffer containing 50 mM Tris-HCl pH 7.4, 0.1 M NaCl and 5 mM CaCl2 (Veiga et al., 1997Go). To determine GAG identities, samples were either treated with nitrous acid to specifically hydrolyze heparan sulfate chains, as described by (Brandan and Inestrosa, 1987Go), or incubated with 50 mU chondroitinase ABC (Seikagaku, Japan) for 18 hours at 37°C to hydrolyze chondroitin/dermatan sulfate chains (Riquelme et al., 2001Go).

Electrophoresis and western blotting
Proteoglycan extracts were obtained from wild-type and sdn-1 (ok449) mutant worms as described above. After incubation on DEAE-Sephacel, samples were eluted with 1 M NaCl, dialyzed against heparitinase buffer, treated with 0.5 mU heparitinase obtained from Flavobacterium heparinum (Seikagaku, Japan) and separated by SDS-PAGE on a 4-15% gradient gel. Proteins were transferred onto nitrocellulose and subjected to western blot analysis with anti-{Delta}-heparan sulfate monoclonal antibody (3G10 epitope, US Biologicals, USA), named anti-{Delta}-heparan sulfate (Steinfeld et al., 1996Go; Casar et al., 2004aGo). Alternatively, one 100 mm plate of synchronized adult worms was used to obtain worm extracts for western blot analyses. Worms were collected, washed twice in either phosphate-buffered saline (PBS) or M9 buffer, resuspended in 300 µl of PBS supplemented with the cocktail of protease inhibitors and sonicated three times for 10 seconds, with a 2 minute break between each sonication round. Samples were cleared by centrifugation at 10,000 g for 10 minutes, and soluble fractions collected, treated with heparitinase and analyzed by western blotting, as described above.

Molecular biology techniques
An sdn-1 gene fragment was amplified by PCR from single wild-type and sdn-1 (ok449) mutant worms, as described (Williams, 1995Go). The primers used, originally designed by The Gene Knockout Consortium, were as follows: forward primer, 5'-GAAAGTGGTCCCTTCGTCAA-3'; reverse primer, 5'-CTGTAATCACCGCAACGAGA-3' (Keystone Labs, USA). These primers allowed the amplification of both a 2688 bp fragment of wild-type genomic sdn-1 and wild-type cDNA fragment of 632 bp using a Platinum® Taq DNA polymerase high fidelity (Invitrogen, USA). These primers also amplify a cDNA fragment of 425 bp from sdn-1 (ok449) mutants.

RNA was extracted from four 100 mm plates of synchronized adult worms as described (Johnstone, 1999Go). Reverse transcription (RT) reactions were performed with 4 µg total RNA, random hexamers and the MMLV Reverse Transcriptase Kit (GibcoBRL, USA). The mixture was incubated at 37°C for 40 minutes and then heated at 95°C for 5 minutes. 1 µl cDNA was used as a template for standard PCR procedures. Northern blotting was carried out as described (Casar et al., 2004bGo) using the 632 bp cDNA fragment as a radiolabeled probe. Automated sequencing was used to corroborate all cDNAs identities (Molecular Biology Facility, Catholic University of Chile, Chile).

Immunofluorescence
For immunofluorescence of heparan sulfate proteoglycans, worms were treated as described (Finney and Ruvkun, 1990Go), with an additional treatment that involved the inclusion of the enzyme heparitinase (0.5 mU) after the worms had been permeabilized. The enzyme was diluted in heparitinase buffer, added to the permeabilized worms and incubated at 37°C for 3-4 hours (Casar et al., 2004aGo). The worms were then blocked in PBS pH 7.4, 1% BSA, 0.5% Triton X-100, 1 mM EDTA plus 10% normal goat serum for 1 hour at room temperature, pelleted and incubated overnight at 4°C with anti-{Delta}-heparan sulfate antibody (1:800 dilution). Goat anti-mouse antibody conjugated with rhodamine (Pierce, USA), previously pre-absorbed with fixed worms (1:200 dilution) was used as a secondary antibody (Casar et al., 2004aGo).

Immunofluorescence of phosphorylated SDN-1 was performed as above with the exception that heparitinase treatment was omitted. Permeabilized worms were incubated with anti-syndecan-4 [pS179] phospho-specific polyclonal antibody (anti-P-syndecan-4) (BioSource, USA) at a concentration of 4 µg/ml overnight at 4°C. As a specific control, the anti-P-syndecan-4 antibody was pre-incubated with the immunogenic peptide in its phosphorylated and nonphosphorylated form at 100-fold molar excess. Goat anti-rabbit IgG, conjugated with rhodamine from Pierce (1:200 dilution), was used as a secondary antibody. Double labeling using the anti-P-syndecan-4 and anti-{Delta}-heparan sulfate antibodies was performed in fixed, permeabilized worms treated with heparitinase, as described above. Donkey anti-mouse IgG-Cy5 and donkey anti-rabbit IgG-RRX (1:100 dilutions) were used as secondary antibodies. The worms were mounted using the DAKO Fluorescent Mounting Medium (DAKO Co., USA), examined by fluorescence microscopy and images were obtained with a Zeiss LSM 2000 MT confocal microscope. Some of the images shown are 3D reconstructions of image stacks.

Egg retention quantification
Mid-L4 stage hermaphrodites were picked onto a plate, left for 41-42 hours and then individually dissected through the body midline, using a tuberculine needle to allow for the release of eggs stored in the uterus, as described (Herman et al., 1999Go). The embryos were then counted and classified according to their developmental stage. Pictures of reproductive adults were taken using Differential Interference Contrast microscopy (DIC) optics and a digital camera.


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C. elegans synthesizes heparan sulfate proteoglycans
Wild-type C. elegans were radiolabeled in liquid culture containing radioactive sulfate in the absence of bacteria and after 18 hours, extracted with 4 M guanidine-HCl. The extracts were dialyzed against an 8 M urea buffer and then chromatographed on DEAE-Sephacel columns. The resulting elution pattern is shown in Fig. 1A. Approximately 80% of the radioactive material bound to the DEAE-Sephacel column. The profile shows a major peak eluting at 0.3-0.5 M NaCl. In order to determine whether this bound radioactive material corresponded to proteoglycans, the bound material was pooled, concentrated and incubated with either proteinase K or treated with alkaline NaBH4 in order to release the GAGs present in the proteoglycan fractions. The samples were then fractionated on a Sepharose CL-6B column. Fig. 1B shows that both treatments released radioactive material that eluted as species with a distribution coefficient, Kav of 0.7-0.8, in comparison with untreated material that eluted with a polydisperse Kav of 0.2 to 0.8, strongly suggesting the proteoglycan nature of the radioactive material isolated from C. elegans. Finally, to determine the nature of these proteoglycans, the concentrated material obtained from the DEAE-Sephacel was incubated with nitrous acid or chondroitinase ABC, which degrade heparan sulfate and chondroitin/dermatan sulfate chains, respectively, and then separated on a Sephadex G-50 column. The resulting elution profile is shown in Fig. 1C. Untreated samples as well as chondroitinase ABC-treated samples eluted near the V0 of the column, whereas nitrous acid treatment revealed small fragments that eluted close to the total volume of the column. Together, these results strongly suggest that C. elegans synthesizes heparan sulfate proteoglycans.



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Fig. 1. Caenorhabditis elegans synthesize heparan sulfate proteoglycans. (A) Wild-type worms were incubated with radioactive sulfate and after solubilization in 4 M guanidine-HCl followed by dialysis against 8 M urea, were loaded onto a DEAE-Sephacel column of 1.0 ml bed volume, pre-equilibrated with the urea buffer. The column was washed and eluted using a linear gradient from 0.2 to 1.0 M NaCl. Aliquots of each fraction were counted (closed circles) and conductivity was determined (open circles). (B) Proteoglycans eluted from the DEAE indicated by the bar, were pooled, concentrated on a small DEAE column and fractionated on a Sepharose CL-6B column. The chromatographic profiles of control samples (closed circles), samples pre-incubated with proteinase K (open circles) and samples treated with alkaline borohydride to release the GAG chains (open triangles) are shown. (C) Proteoglycans eluted from the DEAE and concentrated as in B, were fractionated on a Sephadex G-50 column. The profiles shown correspond to untreated samples (closed diamonds) and samples previously incubated with either chondroitinase ABC (open triangles) or nitrous acid (open diamonds).

 

C. elegans synthesizes at least three species of heparan sulfate proteoglycans
To identify the different heparan sulfate proteoglycans present in adult wild-type C. elegans, samples were obtained as above. The core protein of any proteoglycan that comprises heparan sulfate GAGs can be detected by western blot analyses of the heparitinase-digested proteoglycan, using the monoclonal antibody anti-{Delta}-heparan sulfate (3G10 epitope). This antibody recognizes a neo-epitope generated in the proteoglycan core protein after GAG digestion with the heparitinase enzyme (Steinfeld et al., 1996Go). Fig. 2A shows the presence of three bands of ~80, 50 and 30 kDa in C. elegans, different to those presented in Fig. 2B, which correspond to several myoblast heparan sulfate proteoglycan core proteins previously characterized in our laboratory for other animal species (Casar et al., 2004aGo; Fuentealba et al., 1999Go; Henriquez et al., 2002Go; Olguin and Brandan, 2001Go). The intensity of the bands corresponds to the number of neo-epitopes generated by the heparitinase treatment and to the amount of core proteins containing it. These results indicate that wild-type C. elegans synthesize at least three different heparan sulfate proteoglycans, as revealed by their different core proteins.



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Fig. 2. C. elegans synthesize at least three species of heparan sulfate proteoglycans. (A) Western blot analysis was carried out using post-DEAE samples obtained from wild-type worms, dialyzed against heparitinase buffer and incubated with heparitinase. Samples were separated by SDS-PAGE on a 4-15% gradient gel, transferred onto nitrocellulose, incubated with anti-{Delta}-heparan sulfate antibody (3G10 epitope) and subjected to chemiluminescence detection. Three different core proteins migrating with apparent molecular masses of 80, 50 and 30 kDa were detected. (B) The electrophoretic migration pattern of the core proteins of heparan sulfate proteoglycans, obtained from C2C12 myoblast extracts incubated with the same anti-{Delta}-heparan sulfate antibody, is shown as a comparison. The pattern shows the following previously characterized murine heparan sulfate core proteins and their corresponding molecular weights: syn-3, syndecan-3; syn-1, syndecan-1; gly, glypican; syn-2, syndecan-2 and syn-4, syndecan-4.

 

C. elegans heparan sulfate proteoglycans localize to the nervous system and the vulva
The localization of the C. elegans heparan sulfate proteoglycans was first studied by immunofluorescence, using the monoclonal antibody anti-{Delta}-heparan sulfate. Immunoreactivity in heparitinase-treated adult nematodes showed a pattern restricted to neuronal cells (Fig. 3), and more specifically, to a set of neuronal processes in the nerve ring and along the ventral and dorsal nerve cords (Fig. 3a). Immunoreactivity was also seen associated with the vulva of adult worms (Fig. 3b) and the nervous system of L2-L3 larvae (Fig. 3c). The lower panels of Fig. 3 show the immunostaining patterns in a pan-neural green fluorescent protein (GFP) reporter strain of C. elegans. A close localization of heparan sulfate proteoglycans (Fig. 3e) to the nervous system (Fig. 3d) is shown in the merged image (Fig. 3f). The inset shows a higher magnification indicating a close relationship between the neuronal bodies and the heparan sulfate proteoglycans along the ventral nerve cord, probably present in the extracellular matrix. These results suggest that heparan sulfate proteoglycans localize to the nerve ring and close to the nerve cords that run ventrally and dorsally in C. elegans, and also to the vulva.



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Fig. 3. Heparan sulfate proteoglycans in C. elegans localize to the nerve ring, nerve cords and vulva. Whole worms were permeabilized, incubated with heparitinase and stained with anti-{Delta}-heparan sulfate antibody. (a) Staining shows that heparan sulfate proteoglycans are located at the nerve ring (arrowheads), ventral nerve cord (white arrows) and dorsal nerve cord (gray arrows). (b) Heparan sulfate proteoglycans are also located in the vulva as seen from a ventral view (gray arrowheads). The white arrows indicate the ventral nerve cord and the gray arrows the dorsal nerve cord. (c) The staining of L2-L3 larva showed the location of heparan sulfate proteoglycans at the nerve ring (white arrowhead) and at the ventral and dorsal nerve cords (white and gray arrows, respectively). (d) The nervous system of a transgenic strain expressing the pan-neural GFP was visualized. (e) Heparan sulfate proteoglycans as seen in panel a were also immunodetected in this reporter strain. The white arrows indicate ventral nerve cord. (f) Merged images of panels d and e. The insert shows an enlarged area, with details of the neural bodies (green) and their close localization to the heparan sulfate proteoglycans (red). Controls without heparitinase treatment and without primary antibody are shown in panels g and h respectively. All images are 3D reconstructions of image stacks, except for d, e and f, which correspond to a single optic slice. Bar, 20 µm.

 

The 50 kDa core protein bearing heparan sulfate GAGs corresponds to SDN-1
The genome of C. elegans contains a single homologue of the mammalian proteoglycan syndecan, thereby providing an excellent model for studying syndecan function in vivo. In addition, the C. elegans mutant sdn-1 (ok449) carries a deletion in the syndecan homologue gene sdn-1 (The C. elegans Sequencing Consortium, 1998Go). The expression of sdn-1 was analyzed by RT-PCR analysis followed by sequencing of the wild-type and sdn-1 mutant cDNA. The RT-PCR analysis shown in Fig. 4A indicates that, with the primers used (arrows shown in Fig. 5A), wild-type C. elegans and sdn-1 (ok449) mutants expressed the expected mRNA fragments of 632 bp and 425 bp, respectively. Fig. 4B shows the northern blot analysis of mRNA isolated from the wild type and sdn-1 (ok449) mutants, where the latter expressed a smaller mRNA product than the wild type, as predicted by RT-PCR analysis (Fig. 4A). Furthermore, western blot analysis failed to detect the 50 kDa protein in sdn-1 (ok449) mutants, unlike the other remaining bands recognized by the monoclonal antibody that were still present (Fig. 4C).



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Fig. 4. The 50 kDa core protein bearing heparan sulfate GAGs corresponds to the single C. elegans orthologue of vertebrate syndecans. (A) mRNA was isolated from wild-type and sdn-1 (ok449) mutant strains and RT-PCR was performed using primers in the positions indicated in Fig. 5A. The migration of the RTPCR products, by agarose gel electrophoresis, is shown. wt, wild-type worms; sdn-1, sdn-1 (ok449) mutant worms. (B) Northern blot analysis carried out using mRNA (30 µg samples) obtained as described from wild-type and sdn-1 (ok449) mutant worms. Blots were probed with specific 32P-labeled 632 bp hybridization probes. The migration of 18S and 28S ribosomal sub-units is shown as a standard. (C) Western blot analysis of wild-type and sdn-1 (ok449) mutant worms. Samples were obtained by sonication in PBS and protease inhibitors, treated directly with heparitinase, separated on a 10% polyacrylamide slab gel and visualized with anti-{Delta}-heparan sulfate antibody. Total absence of the 50 kDa core protein was observed in the sdn-1 mutants (low and high exposure), in contrast to its prominence in wild-type samples, whereas the other two core proteins (80 and 30 kDa) remained almost unaffected (high exposure).

 


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Fig. 5. Schematic representation of C. elegans SDN-1 and sequence comparison of its transmembrane and cytoplasmic domains with those of other species. (A) Model of SDN-1 showing its type I transmembrane protein nature. The locations of the GAG attachment sites in the ectodomain (white arrowheads), including a putative site closer to the transmembrane region, are shown. The postulated phosphorylated serine residue of the cytoplasmic domain (SerP) and the location of primer used to analyze wild-type and sdn-1 (ok449) worm strains (black arrows). The lower model shows the most likely form of the deleted SDN-1 protein, as deduced from sdn-1 (ok449) cDNA sequencing. (B) SDN-1 sequence for the conserved transmembrane and cytoplasmic domains is shown aligned to other syndecan sequences. A consensus sequence arose when the SDN-1 sequence was compared with syndecan-1, -2, -3 and -4 from different species.

 

Amino acid sequence analysis together with Worm Base (http://www.wormbase.org/) information revealed that SDN-1 has the four structural domains, identified previously in other organisms and found to be characteristic of syndecan proteoglycans: a signal peptide (26 amino acids), an ectodomain containing glycosylation sequences (205 amino acids), a hydrophobic transmembrane domain (21 amino acids), and a cytoplasmic domain (36 amino acids) (Fig. 5A). The mature core protein of SDN-1 is predicted to have a molecular mass of 31.0 kDa. Interestingly, the sdn-1 (ok449) mutation resulted in a deletion of 69 amino acids in the ectodomain of SDN-1, as inferred from cDNA sequencing, where two putative GAG attachment sites are located (Fig. 5A). However, this deletion did not produce a shift in the reading frame of the mutant sdn-1 (ok449) messenger as determined by cDNA sequencing. Fig. 5B shows a sequence alignment of the transmembrane and cytoplasmic domains of SDN-1 with other syndecan family members. These two domains present a high degree of conservation with other syndecans. Although a consensus sequence is indicated in Fig. 5B, no homology could be found in the extracellular domain of SDN-1 consistent with that described for each syndecan subgroup. Together, these results strongly suggest that the syndecan expressed in C. elegans has a core protein with an apparent molecular weight of 50 kDa, as visualized on denaturating polyacrylamide gels, that is not recognized in sdn-1 (ok449) mutants by the anti-{Delta}-heparan sulfate antibody, owing to the absence of two glycosylation sites in the core protein synthesized by the mutants.

SDN-1 immunoreactivity is lost along the nerve cords and vulva in sdn-1 (ok449) mutant worms
The immunolocalization of heparan sulfate proteoglycans was evaluated in the sdn-1 mutant strain. In wild-type C. elegans, SDN-1 was present in the nerve ring and along the nerve cords (Fig. 6a,b), mainly in the ventral region and the vulva (Fig. 3a,b). In contrast, in sdn-1 (ok449) mutants, all immunostaining along the nerve cords and vulva was lost (Fig. 6c,d), with only diminished staining noted at the nerve ring (Fig. 6c). These results show that other heparan sulfate proteoglycans (probably those species detected as 80 and 30 kDa bands in the western blots of Fig. 4B) are probably expressed specifically in some neurons of the nerve ring. These results indicate that the monoclonal antibody cannot recognize SDN-1 along the nerve cords, vulva and perhaps in certain neurons of the nerve ring, owing to the absence of heparan sulfate chains in the mutant form of SDN-1 expressed in sdn-1 (ok449).



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Fig. 6. C. elegans sdn-1 (ok449) mutants show loss of heparan sulfate proteoglycan immunoreactivity mainly in the nerve cords and vulva. Immunolocalization of heparan sulfate proteoglycans was carried out in whole worms as described for Fig. 3, for both wild-type (panels a and b) and sdn-1 (ok449) mutants (panels c and d). In sdn-1 (ok449) mutants, some immunoreactivity remained in the nerve ring shown in c (white arrowheads) but no staining was observed along the neural cords and the vulva (d). White arrows indicate the ventral nerve cord, gray arrows the dorsal nerve cord. All images are 3D reconstructions of image stacks. Bar, 20 µm.

 

Phosphorylated SDN-1 is found in a specific group of nerve ring neurons in sdn-1 (ok449) mutants and the wild type
The cytoplasmic domain of mammalian syndecan-4 is known to undergo in vivo phosphorylation on a serine residue in its membrane-proximal region (Horowitz and Simons, 1998Go). Fig. 7A shows that SDN-1 has a high degree of sequence homology to mammalian syndecan-4 in the cytoplasmic domain, especially along the peptide sequence used to generate the specific anti-P-syndecan-4 antibody (75% identity at the amino acid level and exact position when both sequences are compared; Fig. 7A, boxed region). The anti-P-syndecan-4 peptide sequence used as immunogen is only found in SDN-1 of C. elegans genome. Antibodies raised against this peptide region (anti-P-syndecan-4) were tested for their ability to recognize SDN-1 in wild-type C. elegans. Fig. 7B panel a shows a wild-type worm immunostained with the anti-{Delta}-heparan sulfate antibody, which recognized the structures described in Fig. 3a, whereas panel b shows immunostaining with the anti-P-syndecan-4 antibody. This antibody recognized the phosphorylated form of SDN-1 in the nerve ring and some projections to the anterior part of the head. Occasionally some animals showed a weak reaction with the anti-P-syndecan-4 antibody at the vulva (data not shown). The inserts in part b show control images of wild-type worms immunostained with the anti-P-syndecan-4 antibody in the presence of the phosphorylated peptide, which completely eliminates the immunostaining at the nerve ring (upper insert) and the non-phosphorylated peptide that has no effect on the immunostaining observed at the nerve ring and at the vulva (lower insert). Panel c of Fig. 7B shows the merged image of Fig. 7A and B and indicates some degree of colocalization between heparan sulfate proteoglycans and SDN-1 phosphorylated at the nerve ring and to the tip of the head. The insert in panel c shows the immunolocalization of phosphorylated SDN-1 performed in a pan-neural GFP reporter strain. The antibody recognizes a group of neurons at the nerve ring and some projections to the anterior cephalic area. Panel d shows immunostained neurons at the nerve ring at increased magnification.



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Fig. 7. SDN-1 located at the C. elegans nerve ring is specifically phosphorylated at its cytoplasmic domain. (A) Amino acid sequences of the cytoplasmic domain of SDN-1 from C. elegans (C.e) and syndecan-4 from M. musculus (M.m). The synthetic peptide used to generate the specific anti-phosphoserine syndecan-4 antibody (anti-P-syndecan-4) is boxed and the phosphorylated serine indicated. (B) (a) Anti-{Delta}-heparan sulfate immunoreactivity as shown in Fig. 3a. (b) Anti-P-syndecan 4 immunoreactivity in wild-type worms. The inserts show immunostaining with preincubated antibody with phosphorylated (upper insert) and non-phosphorylated peptide (lower insert). (c) Merged image of a and b. The insert shows anti-P-syndecan-4 immunoreactivity in the pan-neural GFP transgenic strain. The anti-P-syndecan-4 antibody stained specific neurons at the nerve ring and some anterior structures, but no immunoreactivity was detected in either the nerve cords or the vulva. (d) An enlarged area of the neurons stained in the nerve ring. In all images, white arrowheads indicate the nerve ring. All images are 3D reconstructions of image stacks. Bar, 20 µm.

 

Immunolocalization of the phosphorylated form of SDN-1 in sdn-1 (ok499) mutants is shown in Fig. 8. The anti-P-syndecan-4 antibody recognized the same specific group of neurons at the nerve ring as described in Fig. 7 (Fig. 8a). The sdn-1 (ok499) mutants immunostained with the anti-{Delta}-heparan sulfate antibody and anti-P-syndecan-4 (Fig. 8b,c, respectively). The anti-{Delta}-heparan sulfate antibody recognized heparan sulfate proteoglycans other than SDN-1 in the nerve ring, as described above, whereas the anti-P-syndecan-4 antibody recognized the phosphorylated form of SDN-1 in the nerve ring and some projections of the anterior cephalic region but not along the dorsal or ventral cords. The merged image is shown in Fig. 8d and reveals no colocalization of the other heparan sulfate proteoglycans and the phosphorylated form of SDN-1 in the sdn-1 (ok499) mutants. The insert shows a higher magnification of the nerve ring area. Together, these results suggest that in the wild type and in mutant worms, SDN-1 localizes to the nerve ring and to the anterior cephalic area and is likely to be phosphorylated at its cytoplasmic domain.



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Fig. 8. C. elegans sdn-1 (ok449) mutants express a protein recognized by antibodies against the phosphorylated syndecan-4 epitope, with immunoreactivity localizing to the nerve ring. (a) Anti-P-syndecan-4 immunoreactivity in sdn-1 (ok449) mutants. (b,c) Double immunostaining of anti-{Delta}-heparan sulfate and anti-P-syndecan-4 immunoreactivity in sdn-1 (ok449) mutants respectively. (d) Merged images of b and c. The insert shows an enlarged area, with detail of the heparan sulfate proteoglycans (green) and their close localization to the phosphorylated SDN-1 (red). White arrowheads indicate the immunoreaction at the nerve ring. The image in panel a corresponds to a 3D reconstruction of an image stack, whereas the rest represent two optic slices. Bar, 20 µm.

 

sdn-1 (ok449) mutants accumulate embryos in the uterus compared to the wild type
Mutations in many enzymes involved in the synthesis of GAG chains (sqv mutants) have been described to generate defective phenotypes related to vulva formation. Therefore, the egg-laying ability of sdn-1 mutants was evaluated compared to that of wild-type worms. Table 1 shows that sdn-1 (ok449) mutants accumulated almost twice the number of eggs in the uterus compared to that in the wild-type strain, although in most cases all embryos are ultimately expelled, suggesting that the absence of heparan sulfate chains in SDN-1 may be affecting vulval function. An extreme example of such accumulation of late stage embryos in sdn-1 (ok449) mutants (Fig. 9B) compared to wild-type worms (Fig. 9A) is shown. One evident phenotypic characteristic of worms carrying mutations in GAG biosynthetic enzymes is the partial collapse of vulval cells during invagination (Herman et al., 1999Go). The possibility that sdn-1 mutants also showed this kind of defect that could account for the heightened accumulation of eggs was therefore tested in sdn-1 (ok449) mid-L4 hermaphrodites. In many cases, the space between the invaginating vulval cells and the cuticle seemed to be slightly smaller than in wild-type worms (Fig. 9C,D). These results suggest that the absence of heparan sulfate chains in SDN-1 causes accumulation of embryos in the uterus.


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Table 1. sdn-1 (ok449) hermaphrodites retain eggs

 


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Fig. 9. C. elegans sdn-1 mutants show accumulation of embryos in the uterus and weak morphological defects of the invaginating vulva. (A,B) DIC images of in uterus embryos in wild-type worms and sdn-1 (ok449) mutants respectively. (C,D) DIC micrographs of the vulvae of mid-L4 larvae from both strains. In sdn-1 (ok449), the space between the vulva cells and the cuticle is slightly smaller than in wild-type samples. Bar, 10 µm.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study shows, for the first time, the molecular identity of the proteoglycan syndecan in C. elegans, its cellular localization and some phenotypic defects of sdn-1 (ok449), a syndecan deletion mutant strain. These results are particularly relevant in light of the important phenotypic alterations reported for strains carrying mutations in the enzymes required for heparan sulfate chain synthesis (Berninsone and Hirschberg, 2002Go; Bulik and Robbins, 2002Go; Selleck, 2001Go). In contrast to the comprehensive knowledge accumulated for these biosynthetic enzymes, there is almost no information regarding the characteristics and specific functions of the proteoglycans expressed in C. elegans.

Through biochemical analyses, C. elegans was shown to incorporate radioactive sulfate into macromolecules with an affinity for DEAE-Sephacel, indicating the anionic nature of these molecules. Moreover, these molecules eluted at a salt concentration consistent with the elution of proteoglycans in other systems (Brandan et al., 1996Go; Brandan and Inestrosa, 1987Go). The proteoglycan nature of this eluted material (a protein core with covalently attached GAG chains) was demonstrated by the release of GAGs after protease treatment or ß-elimination in the presence of NaBH4. Under both experimental conditions, radioactive material eluting at a Kav of 0.8 was observed, consistent with the presence of short GAG chains in the proteoglycan population. Interestingly, most (if not all) of this material was sensitive to nitrous acid degradation (Brandan et al., 1985Go) and resistant to chondroitinase ABC treatment, suggesting that the labeled material corresponded mainly to heparan sulfate proteoglycans. It has also been shown by structural analysis that treatment of GAGs with heparin lyases in C. elegans releases disaccharides bearing N-, 2-O- and 6-O-sulfations also found in vertebrates, consistent with the presence of N-deacetylase/N-sulfotransferase, C5 glucuronyl epimerase and heparan-sulfate sulfotransferase enzymes (Berninsone and Hirschberg, 2002Go; Bulik et al., 2000Go). Selleck's group described the presence of chondroitin GAGs in C. elegans that were not sulfated, in contrast to the chondroitin sulfate found in many tissues of a wide range of animals (Toyoda et al., 2000Go). These findings are consistent with our current observations that the radioactive material isolated from C. elegans was resistant to chondroitinase ABC treatment, indicating that in adult worms, the radioactive sulfate was mostly incorporated into heparan sulfate proteoglycans. However, other studies provide evidence consistent with the existence of chondroitin/dermatan sulfate in C. elegans (Beeber and Kieras, 2002Go; Schimpf et al., 1999Go). The reason for these differences is unclear and may be related to variations in the preparation and/or methods used.

The identification of SDN-1 in C. elegans was elucidated by the absence of the 50 kDa core protein in the sdn-1 (ok499) deletion mutants, observed in western blots using the anti-{Delta}-heparan sulfate antibody (Steinfeld et al., 1996Go) compared to wild-type worms. Analyses of the SDN-1 amino acid sequence revealed the presence of three putative GAG attachment consensus sequences also present in other syndecan family members. In sdn-1 (ok449) mutants, SDN-1 is probably expressed as a protein with a deletion of 69 amino acids (exons 2 and 3), encompassing two of the three putative GAG attachment sites. This is confirmed by western blot analyses, followed by RT-PCR and sequencing of the products obtained from wild-type and sdn-1 (ok449) strains. These analyses indicated that there is no frame shift in the mutant gene product. Therefore, a protein without heparan sulfate chains is probably expressed. Through GAG lyase treatment and western blotting, we confirmed that the glycine-serine sequence near the transmembrane domain is not substituted with either chondroitin or dermatan GAGs (data not shown). At present, we are developing specific antibodies against SDN-1 to confirm that a protein product is still expressed in sdn-1 (ok449) mutants. However, our current results showing the phosphorylation and localization of SDN-1 at the nerve ring, in the wild type and sdn-1 mutants, strongly suggest that the protein is expressed in the mutant strain. Sequence analysis of the C. elegans genome failed to reveal other proteins bearing a reasonable degree of homology to the cytoplasmic domain of syndecan. In this respect, the syndecan family was thus first grouped because its members had high sequence similarities in their transmembrane and cytoplasmic domains, with little resemblance in the extracellular regions (Bernfield et al., 1999Go; Carey, 1997Go).

The localization of SDN-1 in C. elegans is very specific. It was found associated to the nervous system (nerve ring, dorsal and ventral nerve cords) and the vulva. The localization of SDN-1 along the nerve cords was lost in sdn-1 (ok449) mutants. The elegant work of Bulow and Hobert, shows that C. elegans mutants lacking three heparan sulfate-modifying enzymes exhibit distinct and sometimes overlapping axonal guidance defects in specific neuronal classes (Bulow and Hobert, 2004Go). Based on these observations and the results presented here, it is possible to speculate that SDN-1 may be participating in axonal and cellular guidance, probably through its heparan sulfate chains. However, the specific neuronal events concerning SDN-1, as well as more detailed phenotypic analysis of the neurons involved, require further study. In this work, SDN-1 was also observed at the nerve ring by immunofluorescence analyses. Interestingly, sdn-1 (ok449) mutants exhibited reduced heparan sulfate proteoglycan immunostaining compared to wild-type worms. This could indicate the presence, only at the nerve ring, of other heparan sulfate proteoglycans that could be acting on the establishment of the nerve ring architecture or operating in concert with SDN-1. This protein was also found associated with the vulva. Several mutant phenotypes for vulval development, which display embryonic arrest and decreased fertility, have been linked to defects in the genes involved in heparan sulfate synthesis (Berninsone and Hirschberg, 2002Go). The absence of heparan sulfate chains in SDN-1 might therefore be responsible for the heightened accumulation of eggs observed in the uterus of sdn-1 (ok449) mutants (see below).

Particularly interesting was the finding that in both the wild type and sdn-1 (ok449) mutants, antibodies against a chemically synthesized phosphopeptide derived from a region of syndecan-4 highly conserved in the cytoplasmic domain of SDN-1, showed immunoreactivity in specific neuronal processes localized at the nerve ring and some reaction to the most anterior part of the animal. Occasionally a weaker immunoreaction was also observed at the vulva. The same results were obtained when the fixation and permeabilization processes were carried out in the presence of phosphatase inhibitors (data not shown). The specificity of this reaction was confirmed by using the phosphorylated and the nonphosphorylated peptide. In addition, when fixed and permeabilized worms were treated with highly purified acid phosphatase, which removed all phosphate groups from serine residues, no staining was observed (data not shown). This highly specific localization indicates that the phosphorylation of SDN-1, at the cellular structures mentioned, is independent of the presence of heparan sulfate chains. It has been shown that phosphorylation of syndecan-4 has the ability to bind and activate protein kinase C-{alpha} by reducing its affinity to PIP2 and inhibiting the oligomerization of its cytoplasmic domains (Couchman et al., 2002Go; Horowitz and Simons, 1998Go). The mechanisms that regulate the phosphorylation of SDN-1 and the biological effects of this post-translational modification remain unknown, but we are currently in the process of evaluating how different genetic backgrounds for neural defects determine the extent of SDN-1 phosphorylation in the cytoplasmic domain.

Through western blot analysis using the anti-{Delta}-heparan sulfate antibody, three different protein bands were detected after heparitinase treatment, suggesting the presence of three different heparan sulfate proteoglycans at the nerve ring. The 50 kDa protein has been shown to correspond to SDN-1, but further studies are required to determine the identity of the other two bands detected at 80 and 30 kDa. Although sequence analysis predicts a molecular mass of 31 kDa for the mature core protein of SDN-1, discrepancies have been described previously for other proteoglycans between molecular weight and the observed polyacrylamide gel migration (Bernfield et al., 1999Go; Carey, 1997Go). Using the same experimental approach described for SDN-1, analyses were attempted with the C. elegans gpn-1 (ok377) mutant strain for the glypican homologue, also isolated by the Gene Knockout Consortium. However, gpn-1 mutants displayed the same migration for the three bands as wild-type worms (data not shown). The deletion in gpn-1 (ok377) mutants is known to affect the second and third introns of the gene, with a small exon inside (174 bp). This genomic deletion, but not the resulting mRNA sequence, was confirmed by our laboratory. These results could therefore not resolve whether GPN-1 was one of the other bands detected by western blot analysis in sdn-1 (ok449) mutants. A gene, unc-52, encoding a protein related to perlecan has been studied in detail in C. elegans and has been shown to affect muscle attachment and sarcomere organization (Moerman et al., 1996Go; Mullen et al., 1999Go; Rogalski et al., 1993Go). Perlecan/UNC-52 is found in basement membranes between the body-wall muscle and the hypodermis and is concentrated in muscle dense bodies and M-lines (Francis and Waterston, 1991Go; Rogalski et al., 1993Go). Using the anti-{Delta}-heparan sulfate antibody, no immunoreactivity was detected in the muscle of C. elegans in the present study. UNC-52 as well as its mammalian homologue contains several domains (I to V), yet the three consensus sequences for GAG attachment are not present in domain I of UNC-52. Although this domain contains one serine-glycine motif (Mullen et al., 1999Go), a careful analysis with experimental confirmation of the amino acid context is needed to determine whether this sequence corresponds to an O-glycosylation site on the core protein. This suggests that UNC-52 may be present in C. elegans in its unglycosylated form.

Variations in the expression of heparan sulfate proteoglycans during development and differentiation have been observed in several systems (Bandtlow and Zimmermann, 2000Go; Fuentealba et al., 1999Go; Olguin and Brandan, 2001Go; Rapraeger, 2001Go). Analysis of expression of the three proteoglycan species by western blotting shows that they are expressed through all developmental stages (data not shown). The anti-{Delta}-heparan sulfate antibody recognized the same anatomical structures of the nervous system, in both adult and larval stages (Fig. 3a-c).

In this study, sdn-1 (ok449) mutants presented a clear phenotype: the significant accumulation of embryos in the uterus. Immunostaining of SDN-1 was observed in the vulva of wild-type worms. This result was not surprising, given that sqv genes have been shown to be required zygotically for the invagination of vulval epithelial cells and that sqv mutations manifest abnormal embryo retention. It is important to mention that the more severe phenotypic alterations observed in organisms presenting mutations in sqv genes ultimately affect all proteoglycan types (Berninsone and Hirschberg, 2002Go; Hwang et al., 2003bGo; Mizuguchi et al., 2003Go). The number of embryos found in sdn-1 mutants was similar to those counted for the majority of sqv phenotypes (Herman et al., 1999Go), although the overall phenotype observed in sdn-1 mutants is weaker. As genetic evidence indicates that vulva morphogenesis depends on GAG expression, it is highly likely that the proteoglycan SDN-1 is implicated in this process. The egg-laying defect and the slight collapse during vulval invagination in sdn-1 (ok449) are probably due to the lack of heparan sulfate GAGs in these mutants. Other studies have demonstrated that unsulfated chondroitin proteoglycans are essential for vulval morphogenesis (Hwang et al., 2003bGo). However, the results of this work show that sdn-1 heparan sulfate chains may also be contributing to this process.

In summary, this study shows for the first time that heparan sulfate proteoglycans localizing mainly at the nerve ring, nerve cords and vulva, are synthesized in C. elegans. The main heparan sulfate proteoglycan species identified through immunofluorescence was SDN-1, although other heparan sulfate proteoglycans, such as glypican and/or agrin homologues, may also be present, as some immunostaining at the nerve ring was also seen in sdn-1 (ok449) mutants. The fact that the cytoplasmic domain of SDN-1 is phosphorylated in both wild-type and sdn-1 (ok449) strains opens the possibility that other signaling pathways not involving heparan sulfate chains may occur. It has been shown that worms defective in particular heparan sulfate modifying enzymes exhibit axonal and cellular guidance defects in specific neuronal classes (Bulow and Hobert, 2004Go). Hence, we speculate that SDN-1 may be one of the proteoglycans involved in this process. Therefore, one could hypothesize that the egg-laying defect observed in sdn-1 (ok449) mutants may be related to flawed neuronal wiring resulting in abnormal vulval function. When sdn-1 null alleles become available, the full range of biological processes involving this proteoglycan may finally be unveiled, and functional distinctions could be made between the core protein, its post-transductional modifications and heparan sulfate components.


    Acknowledgments
 
Our special thanks to Juan Larraín for careful reading of the manuscript and useful suggestions, to Mark Edgley for his gift of primers and to Francisca Bronfman for some secondary antibodies. We are indebted to Yasna Sanhueza and Alejandro Roth for confocal microscopy assistance and to Paula Grez for help with DIC microscopy. This work was partly supported by grants from FONDAP-Biomedicine N° 13980001. Strains used in this work were kindly provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources; and by the International C. elegans Gene Knockout Consortium (C. elegans Gene Knockout Facility at the Oklahoma Medical Research Foundation, which is funded by the NIH; and the C. elegans Reverse Genetics Core Facility at the University of British Columbia, which is funded by the Canadian Institute for Health Research, Genome Canada, Genome BC, the Michael Smith Foundation and the NIH). The research of E.B. was supported in part by an International Research Scholar grant from the Howard Hughes Medical Institute. The Millennium Institute for Fundamental and Applied Biology (MIFAB) receives funding from the Ministerio de Planificación y Cooperación, Government of Chile.


    Footnotes
 
* These authors contributed equally to this work Back


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 Introduction
 Materials and Methods
 Results
 Discussion
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