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First published online 12 April 2006
doi: 10.1242/dev.02363

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C. elegans dystroglycan DGN-1 functions in epithelia and neurons, but not muscle, and independently of dystrophin

Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

View larger version (43K): [in a new window]   Fig. 1. Structure of the dgn-1 gene and product. (A) Coding (gray) and non-coding (white) exons of dgn-1; regions corresponding to - and ß-DG, the site of /ß cleavage in vertebrates (arrowhead), and the extent of the cg121 deletion are indicated. (B) Three regions of similarity to vertebrate (mouse) and Drosophila DGs were identified, corresponding to the N-terminal domain of -DG, the C-terminal region of vertebrate -DG plus extracellular region of ß-DG (core domain), and the ß-DG cytoplasmic domain. A transmembrane (TM) region follows the core. Amino acids retained in a majority of the sequences are shown in color (N-terminal, purple; core and transmembrane, green; cytoplasmic, red), similar residues in gray. Two additional predicted DG-like proteins in C. elegans, DGN-2 and DGN-3, contain a core domain but lack similarity to DG outside of the core (not shown). Conserved cysteine pairs () in N-terminal and core domains, conserved predicted N-glycosylation sites (•) in the core domain, and the /ß cleavage sitein vertebrate DG () are indicated. A 20-residue threonine-rich region in the DGN-1 N-terminal domain may function as a mucin-like region. Residues mediating interaction of the ß-DG cytoplasmic tail with WW/SH3 proteins such as dystrophin are indicated (asterisks). (C) Pairwise sequence similarity (% identity/% similarity) between conserved regions. Regions of similarity were identified by BLAST (Altschul et al., 1990) and sequence alignments constructed using Clustal W (Thompson et al., 1994).

View larger version (32K): [in a new window]   Fig. 2. DGN-1 is glycosylated but not cleaved into /ß subunits. (A) Antibodies to DGN-1 regions corresponding to -DG and ß-DG detect a single major band at 85 kDa (arrow) on western blots of embryo or mixed larval extracts. The anti-DGN-1ß antibody cross-reacts with a bacterial antigen in the larval extract (asterisk). (B) DGN-1 is absent from dgn-1(cg121) lysates probed with anti-DGN-1, demonstrating that cg121 is a molecular null allele. The extract from 25 adult animals was run in each lane. (C) Extracts digested with PNGaseF (protein N-glycosidase F) to remove N-linked glycans (lanes 2,4) were western blotted with anti-DGN-1 antibody. The compact 85 kDa species in embryos shifts to 75 kDa after digestion, near to the predicted polypeptide size. In larval extracts, the broad DGN-1 band decreases in molecular weight after digestion but remains heterogeneous, indicating the presence of N-glycosylation and some additional modification(s), possibly O-glycosylation.

View larger version (125K): [in a new window]   Fig. 3. dgn-1 is expressed in epithelia and neurons. (A) In early embryos, dgn-1::GFP is expressed in epithelial and neural precursors in the outer layer of cells. (B) As elongation begins, expression becomes prominent in pharyngeal epithelia (pe), the excretory cell (exc), the two somatic gonad precursors in the gonad primordium (gp), and in rectal epithelia (rect). Weaker expression is seen in a number of hypodermal and neural precursors. (C) In early larvae, expression persists in the e2 and marginal (mc) cells of the pharyngeal epithelium, the excretory cell, the gonad primordium and the rectal epithelium. Strong expression is seen in the PVP neurons. (D-G) Enlarged images showing expression in: (D) the pharyngeal epithelium and hypodermal cell hyp5 (hyp); (E) somatic gonad precursors, which project processes (arrowheads) around the primordial germ cells (asterisks); (F) PVP neurons; (G) rectal epithelial cells, which project processes (arrowhead) around the rectal sphincter. (H) Transient expression during late L1 stage in P-cell-derived ventral cord neurons (arrowheads). (I) A L3 stage animal showing expression in the distal tip cell and gonad epithelium (arrowheads). (J) Strong expression in L4/adult stage gonad, including the uterus (ut), spermatheca (st) and sheath (sh). (K) Expression in late L4 stage vulval epithelium. Scale bar: 10 µm.

View larger version (79K): [in a new window]   Fig. 4. Localization of DGN-1 in epithelia and neurons, but not muscle. (A) In early embryos (pre-lima stage) before BM formation, DGN-1 (red) is distributed around the surface of many epithelial and neural precursors. (B) As BMs assemble (lima to comma stages), identified by staining for NID-1/nidogen (green), DGN-1 (red) redistributes to the basal surface (arrowheads). (C) In late embryos (pretzel stage), DGN-1 (red) is prominent in marginal cells (mc) and the gonad primordium (gp), and localizes to basal surfaces marked by NID-1 (green). Hypodermal DGN-1 colocalizes with NID-1 at the edges of the body wall muscles (hme). (D) DGN-1 (red) and EPI-1/laminin-B (green) colocalize in the gonad primordium of late embryos (pretzel stage). (E) DGN-1 (green) is prominent in spermatheca (st), gonad sheath (sh) and distal tip cells (dtc) of the gonad and the vulval epithelium (vul). (F,G) In the gonad and vulva, DGN-1 (red) localizes to the BM surface (NID-1, green). (H,I) DGN-1 (green) is at the basal surface of the rectal epithelium (H, rect) and the excretory cell (I, exc). (J) DGN-1 (green) in the PVP neurons. (K) DGN-1 (green) localization in the hypodermis (hyp) and excretory cell, but not in body wall or pharyngeal muscle, visualized by staining of MHC-A myosin in muscle M-lines (red). (L) DGN-1 (green) is seen weakly throughout the hypodermis covering muscle and is slightly concentrated adjacent to the body wall muscle (arrowheads), but not in the underlying muscle (red). Scale bar: 10 µm.

View larger version (98K): [in a new window]   Fig. 5. A dgn-1 null mutant is viable but sterile due to early gonad disruption. (A,B). Wild-type (A) and homozygous dgn-1(cg121) null mutant (B) adults. Gonad tissue in cg121 forms a disorganized mass (white outline) and the vulva protrudes (arrowheads). (C) Wild-type four-cell gonad primordium with two central PGCs (asterisk) and two SGPs surrounded by a BM creating a sharp DIC boundary. (D) Newly-hatched cg121 homozygotes retain a compact primordium but have mispositioned SGPs, a weak DIC boundary and bulging PGCs. (E) Ruptured gonad primordium of a cg121 L1 larva; gonadal cells spread along the body wall (white outline). (F) Early epi-1(rh199) laminin B L1 larva showing similar rupture of the primordium (white outline). (G) Normal gonad primordium in dys-1(cx18) dystrophin mutant L1 larvae. (H) Percentage of mispositioned SGPs in newly-hatched dgn-1(cg121) (n=104) and epi-1(rh199) (n=58) larvae. (I) Overlay of DIC and lag-2::GFP images of wild-type gonad (black outline) at early L2 stage. Somatic gonad cells expressing lag-2::GFP (green) are in close association with germ cells (asterisks). (J,K) DIC/lag-2::GFP overlays of gonads in early L2 stage dgn-1(0) animals. (J) In 60% (n=25) of animals, somatic gonad cells (white outline) form a central cluster separated from, or in only peripheral contact with, germ cells (asterisks). (K) In 40% of animals, somatic gonad cells adopt a contiguous linear arrangement along the ventral surface, sometimes split into two clusters. In I-K, additional lag-2::GFP signal from non-gonadal cells is visible along the ventral midline. Scale bar: 10 µm.

View larger version (89K): [in a new window]   Fig. 6. Localization of BM components to the dgn-1(0) gonad. (A,B) Wild-type (A) and cg121 homozygote (B) L1 larvae stained with antibodies to collagen 2(IV) LET-2 (green) and to DGN-1 (red). (C,D) Wild-type (C) and cg121 homozygote (D) L1 larvae stained with antibodies to nidogen NID-1 (green) and to DGN-1 (red). Early cg121 larvae retaining a compact gonad primordium show wild-type staining of the gonad primordium with LET-2 and NID-1 antibodies (arrows). (E) Early cg121 homozygote expressing LAM-1::GFP (green); association with the compact gonad primordium is apparent (enlarged with DIC overlay in inset). (F) Early cg121 homozygote with a mispositioned SGP; both SGPs with an associated strong LAM-1::GFP signal (green) ensheath only one (arrowheads) of the two primordial germ cells (asterisks). The unsheathed PGC is only weakly associated with laminin (arrow). (G) DIC image of F. Scale bar: 10 µm.

View larger version (18K): [in a new window]   Fig. 7. Gonad migration defects in cg121 heterozygotes. The percentages of cg121/+ animals showing the indicated type of DTC migration defects is shown.

View larger version (46K): [in a new window]   Fig. 8. Defects in excretory cell processes in dgn-1 mutants. (A) H-shaped excretory cell of a wild-type animal. (B) Homozygous cg121 null mutants frequently have short (arrow) or missing (arrowhead) excretory cell arms. (C) Null mutants also show arm duplications (arrowheads) but at a lower frequency (ra/la, right/left anterior; lp, left posterior arms). (D) Percentage of wild type, cg121 homozygotes or cg121/+ heterozygotes with the indicated defect in at least one excretory cell arm. Multiple defects can be present in a single animal, so values do not add to 100%.

View larger version (74K): [in a new window]   Fig. 9. Neural guidance defects in dgn-1 mutants. Homozygous and heterozygous dgn-1 mutants have axon guidance, branching and fasciculation defects in the DA/DB motoneurons (marker: evIs82[unc-129::GFP]). (A) Approximately 31% of dgn-1(cg121) homozygotes have side-switching defects in commissural DA/DB axons, which migrate from the ventral nerve cord (vnc) to the dorsal nerve cord (dnc). Heterozygotes show side-switching at a lower frequency. In this cg121/+ heterozygote, the DA6 commissure is abnormally extending on the right side, while the neighboring DB6 commissure is correctly extending on the right. (B-D) A variety of other, low penetrance defects are also seen in dgn-1 mutants. (B) Extra process arising from the neural cell body (arrowheads). (C) Abnormal axon branching (arrow) and aberrant anterior/posterior migration of processes (arrowheads). (D) Defasciculation (arrowhead) of axons in the dorsal nerve cord. Anterior is down in each image. Scale bar: 10 µm.

View larger version (29K): [in a new window]   Fig. 10. C. elegans DAPC and dgn-1 mutants have distinct behavioral phenotypes. (A) Hypercontraction of the head musculature (arrowhead, arrow) in DAPC component mutants dys-1(cx18) and dyb-1(cx36), but not in dgn-1(cg121), during movement on plates. (B) Double mutants of dgn-1 and DAPC components show hypercontraction. (C) Activity (body bends per minute) during movement on agar plates or during thrashing in liquid. Measurements were normalized to wild-type (wt) values (plate movement, 100%=30.1 bends/minute; thrashing, 100%=167.9 bends/minute). Both dgn-1(cg121) and DAPC mutants are hyperactive, but double mutants show wild-type or lower rates. (D) Prolongation of the Pboc-to-Pboc defecation cycle time in the dgn-1(cg121) and in dgn-1;dys-1 double mutants, but not in the dys-1(cx18) mutant itself. In C and D, the mean and standard deviation (error bars) of 8-10 animals is reported.

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