C. elegans Agrin Is Expressed in Pharynx, IL1 Neurons and Distal Tip Cells and Does Not Genetically Interact with Genes Involved in Synaptogenesis or Muscle Function

Agrin is a basement membrane protein crucial for development and maintenance of the neuromuscular junction in vertebrates. The C. elegans genome harbors a putative agrin gene agr-1. We have cloned the corresponding cDNA to determine the primary structure of the protein and expressed its recombinant fragments to raise specific antibodies. The domain organization of AGR-1 is very similar to the vertebrate orthologues. C. elegans agrin contains a signal sequence for secretion, seven follistatin domains, three EGF-like repeats and two laminin G domains. AGR-1 loss of function mutants did not exhibit any overt phenotypes and did not acquire resistance to the acetylcholine receptor agonist levamisole. Furthermore, crossing them with various mutants for components of the dystrophin-glycoprotein complex with impaired muscle function did not lead to an aggravation of the phenotypes. Promoter-GFP translational fusion as well as immunostaining of worms revealed expression of agrin in buccal epithelium and the protein deposition in the basal lamina of the pharynx. Furthermore, dorsal and ventral IL1 head neurons and distal tip cells of the gonad arms are sources of agrin production, but no expression was detectable in body muscles or in the motoneurons innervating them. Recombinant worm AGR-1 fragment is able to cluster vertebrate dystroglycan in cultured cells, implying a conservation of this interaction, but since neither of these proteins is expressed in muscle of C. elegans, this interaction may be required in different tissues. The connections between muscle cells and the basement membrane, as well as neuromuscular junctions, are structurally distinct between vertebrates and nematodes

Identification and characterization of agrin in "Caenorhabditis elegans"

Agrin is a large basement membrane (BM) proteoglycan expressed in many tissues in vertebrates, with particularly important function at the neuromuscular junction (NMJ) where it clusters acetylcholine receptors (AChRs) and maintains structural stability of postsynaptic specializations. In order to cluster the receptors it has to activate muscle-specific kinase (MuSK) through an indirect interaction via an unidentified myotube-associated specificity component (MASC). Agrin has also been implicated in providing structural integrity to different tissues by connecting the extracellular matrix (ECM) to α-dystroglycan (α-DG) which is part of a large supramolecular dystrophyn-associated glycoprotein complex (DGC) spanning the cell membrane and binding the actin cytoskeleton. Since an agrin orthologue was identified in the C. elegans genome, we decided to experimentally confirm its expression and characterize the protein. Based on the predicted sequences, we cloned the agr-1 cDNA and assembled the ORF of 4422 bp from overlapping fragments. The putative protein domain architecture shared high similarity to the vertebrate agrin, except for missing one laminin G (lamG) domain, serine/threonine-rich regions and the SEA module. Since in vertebrates agrin exists in two main isoforms varying at the amino (N)-terminal side, it was surprising to identify only one isoform in C. elegans. Likewise, additional alternative splicing that occurs at conserved sites in the vertebrate agrin orthologues having strong impact on the AChRs clustering activity, was not identified in AGR-1. Reporter constructs revealed agr-1 expression in the buccal epithelium of the pharynx, in four IL1 sensory neurons in the head, and the distal tip cell (DTC) of the gonad, but surprisingly no expression was found in the muscles or the motoneurons innervating them. The specific anti-AGR-1 antibodies detected the protein in the basement membrane of the pharynx. We analyzed several agr-1 mutant strains and performed many different assays with the goal to identify its function. No defects related to the NMJ could be found and some indications suggested that it might be implicated in the gonad migrations through genetic interaction with other factors. Based on the expression pattern in the head neurons and pharynx, we expected a sensory or feeding-related function but did not see clear defects. AGR-1 probably acts in parallel with several other proteins in a redundant fashion. This is the first characterization of an invertebrate agrin orthologue which sets a substantial basis for further research

<i> Agr-1</i> expression in IL1d and IL1v neurons.

<p>A–C, DiI staining in <i>hdEx25</i> trangenic worms; no co-staining is observed between <i>agr-1::YFP</i> (A) and DiI (B). In D–F, no costaining is observed between <i>agr-1::GFP</i> (D) and DiI+CaAcetate (E) in <i>kdIs66</i> transgenic animals. In G–I, costaining is observed in <i>eat-4::GFP</i> (G) and <i>agr-1::dsRED</i> (H) in <i>adIs1240; kdEx71</i> transgenic worms. Figures C, F, I show merged channels. In all panels dashed arrows point out dendrites; arrows point to neuronal cell bodies; arrowheads mark buccal epithelial cells and asterisks indicate the nerve ring.</p

Detection of endogenous <i>C. elegans</i> agrin by western blot and immunofluorescence.

<p>Lysates of wild type (N₂) and agrin mutant worms (<i>eg1770</i>, <i>eg153</i>, <i>tm2051</i>) were analysed on western blots (A). Two prominent bands of about 160 kDa and 75 kDa were present exclusively in the wild type (Wt) worms and not the mutants. The larger band corresponds to the calculated size of the full length AGR-1 protein and the smaller band may represent an agrin degradation product. Asterisks denote two additional background bands present in all the strains. B, Worms were immunostained with the monoclonal antibody pool against <i>C. elegans</i> agrin (green) and Rim, a synaptic marker prominent in nerve ring (red). Agrin was detected in the basal lamina around the pharynx procorpus (arrow) and anterior bulb (asterisk). Posterior bulb staining was weaker possibly due to poor antibody penetration (dashed arrow). (C–H) Polyclonal antiserum staining resulted in the same pattern in the pharynx of wild type worms (C and D, asterisk for anterior bulb) whereas it was clearly absent in agrin mutants (F–H). Prominent background staining of the gut was present in all strains (C–H, arrowhead). Preimmune serum of the same rabbit was used as negative control on wild type worms (E) where both pharyngeal and gut staining was clearly missing.</p

Genomic organization of the <i>agr-1</i> gene and mutant alleles.

<p>The assembled transcript consists of 29 exons which span over almost 14.5 kb on chromosome 2. Black arrows indicate the three locations where a nucleotide is missing in the database genomic sequence (exons 14, 15 and 20; cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000731#pone-0000731-g001" target="_blank">Fig. 1</a>). Three mutations in the agrin gene were isolated. In the <i>eg1770</i> mutant strain (black arrowhead) <i>Mos1</i> transposon was inserted into the seventh exon which results in an out-of-frame transcript, therefore causing a putative strong loss of function mutation. The <i>eg153</i> strain (asterisk) was created by imprecise excision of the <i>Mos1</i> transposon leaving 5 bp at the insertion site and resulting in a +2 frameshift mutation. Mutant <i>tm2051</i> (dotted line) carries a deletion of 423 bp including exons 26 and 27 resulting in an in-frame loss of 42 amino acids.</p

<i> Agr-1::reporter</i> expression in transgenic animals.

<p>A, Reporter genes were fused to different portions of agrin non-coding and coding sequences as shown in the schematic representation of the genomic region containing the <i>agr-1</i> promoter and <i>agr-1</i> 5′-end. The lengths of the promoter or gene sequences and the names of the the <i>pagr-1</i>::<i>reporter</i> plasmids and DNA arrays are indicated. Since all of these constructs resulted in the same expression patterns, representative micrographs of the <i>kdIs66</i> transgenic worms are shown in B–J. B Expression starts in 2 cells in the anterior part of the embryo at around the 64 AB cell stage. C, Towards the end of gastrulation expression is seen in about 40 cells throughout the embryo including neuronal precursors, several ventral hypodermal cells and pharyngeal precursor cells (ventral view). D At the 1 1/2 to 2 fold stage expression is seen in IL1 neurons (identity determined postembryonically), embryonic motoneurons and a number of additional cells in the head, most likely arcade cells and epithelial buccal cells in the pharynx, and in few apoptotic cells (marked by +). E, In the 3fold stage embryos expression is seen in the IL1 neurons (6 neurons), most of the arcade cells (3 anterior arcade cells and the DL and DR posterior arcade cells) and the buccal epithelial cells in the pharynx. The 2 lateral IL1 neurons express GFP only weakly and only in early larval stages, wheras the remaining 4 IL1 neurons express GFP strongly throughout all larval stages. F (dorsal view) and I In L1 larvae expression is observed, in the buccal epithelial cells (dashed arrow), in 3 anterior arcade cells and the DL and DR posterior arcade cells (arrowheads), and in IL1v and IL1d neurons (arrows) and posterior gut cells (asterisk). In F, the worm was co-stained with DiI. G and H Head of a young adult worm; expression is visible in the buccal epithelial cells (dashed arrows) and in the IL1v and IL1d neurons (arrows); open arrowheads point at the IL1 processes in the nerve ring. J, L2 larva; expression in the migrating distal tip cells (arrows) and posterior gut (asterisk). Bars are 10μm.</p

<i> In vitro</i> interaction between <i>C. elegans</i> agrin and vertebrate α-dystroglycan.

<p>Purified chicken α-DG (lanes 1–3 and 5) or crude COS cell extract (lane 4) was transferred to the membrane after separation by SDS-PAGE and membrane strips were incubated with different samples of agrin: lane 1, chicken muscle agrin isoform; lane 2, chicken neuronal isoform; lanes 3 and 4, <i>C. elegans</i> agrin in conditioned medium of transfected COS cells; lane 5, conditioned medium of non-transfected COS cells. Binding of the respective agrins was detected by anti-chick agrin antibody (lanes 1 and 2) or with the Tn60 antibody recognizing the short tenascin C fragment which was fused to the <i>C. elegans</i> agrin fragment (lanes 3, 4 and 5). Binding of <i>C. elegans</i> agrin to α-DG was detected in lane 3, but not in the negative controls (lanes 4 and 5).</p

A, Reporter genes were fused to different portions of agrin non-coding and coding sequences as shown in the schematic representation of the genomic region containing the promoter and 5′-end

plasmids and DNA arrays are indicated. Since all of these constructs resulted in the same expression patterns, representative micrographs of the transgenic worms are shown in B–J. B Expression starts in 2 cells in the anterior part of the embryo at around the 64 AB cell stage. C, Towards the end of gastrulation expression is seen in about 40 cells throughout the embryo including neuronal precursors, several ventral hypodermal cells and pharyngeal precursor cells (ventral view). D At the 1 1/2 to 2 fold stage expression is seen in IL1 neurons (identity determined postembryonically), embryonic motoneurons and a number of additional cells in the head, most likely arcade cells and epithelial buccal cells in the pharynx, and in few apoptotic cells (marked by +). E, In the 3fold stage embryos expression is seen in the IL1 neurons (6 neurons), most of the arcade cells (3 anterior arcade cells and the DL and DR posterior arcade cells) and the buccal epithelial cells in the pharynx. The 2 lateral IL1 neurons express GFP only weakly and only in early larval stages, wheras the remaining 4 IL1 neurons express GFP strongly throughout all larval stages. F (dorsal view) and I In L1 larvae expression is observed, in the buccal epithelial cells (dashed arrow), in 3 anterior arcade cells and the DL and DR posterior arcade cells (arrowheads), and in IL1v and IL1d neurons (arrows) and posterior gut cells (asterisk). In F, the worm was co-stained with DiI. G and H Head of a young adult worm; expression is visible in the buccal epithelial cells (dashed arrows) and in the IL1v and IL1d neurons (arrows); open arrowheads point at the IL1 processes in the nerve ring. J, L2 larva; expression in the migrating distal tip cells (arrows) and posterior gut (asterisk). Bars are 10μm