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

Abstract

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.

Figure 1. C. elegans agrin DNA and protein sequence with predicted domain architecture.

The C. elegans agrin coding sequence was assembled from overlapping cDNA fragments, amplified by RT-PCR. The positions of the primers are shown by black arrows, where the corresponding pairs are depicted with the same line pattern (full line, dotted line, “dash-dot-dash” line). The three nucleotides missing in the genomic sequence of the database entry are framed with red rectangles. Based on the nucleotide numbering in cosmid F41G3, their positions are: C after 30028, A after 29776 and C after position 28351. The coding region of the gene is 4422 bp long with 5′ and 3′ untranslated regions of 212 and 160 bp, respectively (dark gray boxes; EMBL/GeneBank Accession AM773423). The predicted protein sequence is 1473 amino acids long and the domain architecture is shown in different colors. A putative signal sequence (purple box) is followed by seven follistatin domains (blue), two epidermal growth factor domains of the laminin-type (light gray), a follistatin domain (blue), an EGF-like domain (orange) and two laminin G domains (yellow).

Figure 2. Genomic organization of the agr-1 gene and mutant alleles.

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. Fig. 1). Three mutations in the agrin gene were isolated. In the eg1770 mutant strain (black arrowhead) Mos1 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 eg153 strain (asterisk) was created by imprecise excision of the Mos1 transposon leaving 5 bp at the insertion site and resulting in a +2 frameshift mutation. Mutant tm2051 (dotted line) carries a deletion of 423 bp including exons 26 and 27 resulting in an in-frame loss of 42 amino acids.

Figure 3. Domain architecture of the C. elegans agrin protein in comparison to the vertebrate orthologues.

C. elegans agrin starts with a signal sequence (SS; purple), followed by seven follistatin-like domains (F; blue), two epidermal growth factor (EGF) domains of the laminin-type (LE; gray), one follistatin-like domain (F; blue), an EGF-like domain (EG; orange), and two laminin G domains at the C terminus (LamG; yellow). The color scheme follows the same pattern as presented in the Fig. 1. Predicted N-glycosylation sites are shown with blue circles. Vertebrate agrins have two alternative N-termini: a secreted form, with a signal sequence (SS; dark purple) and a laminin-binding N-terminal agrin domain (NtA; light purple). These are followed by follistatin domains, including one more than in C. elegans (F; blue), a sea urchin sperm protein, enterokinase and agrin domain (SEA; light blue), two serine/threonine rich regions (S/T; light orange), and three laminin G domains (LamG; yellow). O-linked heparan sulphate and chondroitin sulphate chains are schematically shown as branches and several N-linked glycoslation sites as blue circles. Alternative splicing at the last two LamG domains of vertebrate agrin (A/y and B/z) gives rise to several agrin isoforms with different functions, but no alternative splicing was found in C. elegans agrin. Three separate segments of C. elegans agrin marked by dashed lines were used in a Blast search. The resulting degrees of identity/similarity to the corresponding parts of chicken agrin (Swissprot entry P31696-2) are indicated. Recombinant fragments 1 and 2 of C. elegans agrin indicated above the LamG domains were used as antigens for raising monoclonal and polyclonal antibodies, respectively.

Figure 4. Alignment of the C. elegans lamG domains to the corresponding domains of other proteins.

A, The first LamG domain of the C. elegans protein (Agrin_LamG1_C.elegans) was used as a query for Swissprot, Trembl and Refseq databases. After the analysis of an extensive alignment, the best hits were selected for this representation and include: the predicted agrin orthologue of C. briggsae (Agrin_LamG1_C.briggsae), the agrin LamG2 domains of the human, electric ray and chicken proteins, the LamG2 and LamG1 of human perlecan and the LamG4 of a laminin-like protein 2 (LAML2) identified in C. elegans. The similarities between each of the sequences compared to the C. elegans lamG1 are expressed as % identity/% similarity. B, The second LamG domain of the C. elegans protein (Agrin_LamG2_C.elegans) was used as a query for Swissprot, Trembl and Refseq databases. After the analysis of a more extensive alignment, the best hits were selected for this representation and include: the predicted agrin orthologue of C. briggsae (Agrin_LamG2_C. briggsae), the LamG4 of human lamininA4, the LamG3 of human perlecan and the agrin LamG3 domains of the human, electric ray and chicken proteins. The similarities between each of the sequences compared to the C. elegans lamG1 are expressed as % identity/% similarity. C, The C. elegans agrin sequence aligns best with the B₀/z₀ isoforms of chick and rat agrin, respectively. The conserved alternatively spliced agrin exons, encoding 8 aa, 11 aa or 19 aa inserts at this site, do not exist in C. elegans.

Figure 5. Agr-1::reporter expression in transgenic animals.

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 agr-1 promoter and agr-1 5′-end. The lengths of the promoter or gene sequences and the names of the the pagr-1::reporter plasmids and DNA arrays are indicated. Since all of these constructs resulted in the same expression patterns, representative micrographs of the kdIs66 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.

Figure 6. Agr-1 expression in IL1d and IL1v neurons.

A–C, DiI staining in hdEx25 trangenic worms; no co-staining is observed between agr-1::YFP (A) and DiI (B). In D–F, no costaining is observed between agr-1::GFP (D) and DiI+CaAcetate (E) in kdIs66 transgenic animals. In G–I, costaining is observed in eat-4::GFP (G) and agr-1::dsRED (H) in adIs1240; kdEx71 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.

Figure 7. Antibodies against C. elegans agrin.

A, Schematic representation of the recombinant fragments used as antigen to raise monoclonal and polyclonal antibodies. For eukaryotic expression the C-terminal lamG domains were fused to a short fragment of chicken tenascin C (Tn-C), including a secretion signal and the epitope of the anti-Tn60 antibody. The specificity of both polyclonal and monoclonal antibodies was tested by western blotting on conditioned medium of COS cells transfected with the construct encoding the two LamG domains with the Tn-C-tag (B). Lanes 1 and 2 were incubated with polyclonal antisera from two different rabbits, lane 3 with the monoclonal antibody pool raised against the bacterially expressed fragment, and lane 4 with pre-immune serum. All antibodies detected a band of about 80 kDa, which corresponds to the size of the recombinant protein. Additional smaller bands (asterisk) most likely correspond to degradation products which are not recognized by the monoclonal antibodies. C–J, Immunofluorescence staining of transfected COS cells was performed with the anti-agrin monoclonal antibody pool (C–F) and compared to the anti-Tn60 control (G–J). In transfected cells (C and D; G and H) the secreted agrin fragment was detected on cell surfaces of non-permeabilized cells (C and G) or in the endoplasmatic reticulum/Golgi apparatus of permeabilized cells (D and H). Non-transfected cells were used as a negative control (E and F; I and J).

Figure 8. Detection of endogenous C. elegans agrin by western blot and immunofluorescence.

Lysates of wild type (N₂) and agrin mutant worms (eg1770, eg153, tm2051) 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 C. elegans 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.

Figure 9. In vitro interaction between C. elegans agrin and vertebrate α-dystroglycan.

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, C. elegans 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 C. elegans agrin fragment (lanes 3, 4 and 5). Binding of C. elegans agrin to α-DG was detected in lane 3, but not in the negative controls (lanes 4 and 5).

Figure 10. Recombinant C. elegans agrin clusters endogenous dystroglycan in COS cells.

COS cells were transfected with the recombinant fragment of C. elegans agrin and immunostained for agrin and endogenous β-DG. A–D, agrin staining; E–F, anti-β-DG staining; I–L, overlay including nuclear staining. A–C, In transfected cells, secreted agrin bound to the cell surface in a patchy pattern to cells producing large quantities of agrin (A) as well as to cells expressing little or no agrin themselves (B, C). The cells were co-immunostained for endogenous β-DG (E–H) which, in transfected cell cultures, colocalized with agrin on cell surfaces. In non-transfected cells (D–L), no agrin staining was present (D) and β-DG showed diffuse staining (H and L).