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First published online 12 April 2006
doi: 10.1242/dev.02363
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Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
* Author for correspondence (e-mail: jkramer{at}northwestern.edu)
Accepted 15 March 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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B mutants, suggesting that
DGN-1 serves as a receptor for laminin. dgn-1(0)/+ animals are
fertile but show gonad migration defects in addition to the defects seen in
homozygotes, indicating that DGN-1 function is dosage sensitive. Phenotypic
analyses show that DGN-1 and dystrophin-associated protein complex (DAPC)
components have distinct and independent functions, in contrast to the
situation in vertebrate muscle. The DAPC-independent functions of DGN-1 in
epithelia and neurons suggest that vertebrate DG may also act independently of
dystrophin/utrophin in non-muscle tissues.
Key words: Dystroglycan, Laminin, Dystrophin, Basement membrane, Extracellular matrix, Epithelia, Neurons
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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subunit and a
transmembrane ß subunit produced by proteolytic processing of a single
precursor (Ibraghimov-Beskrovnaya et al.,
1992
). Laminin G-like domains in extracellular matrix (ECM)
proteins, such as laminin, agrin and perlecan, and the neural cell surface
neurexins bind O-linked glycans on -DG
(Hohenester et al., 1999;
Michele et al., 2002).
ß-DG binds to -DG (Sciandra et
al., 2001) and mediates its transmembrane linkage to intracellular
cytoskeletal and signaling proteins
(Winder, 2001). DG is a
component of the skeletal muscle dystrophin-glycoprotein complex (DGC) that
includes the transmembrane sarcoglycan complex and the cytoplasmic
dystrophin-associated protein complex (DAPC), which contains the actin-binding
protein dystrophin, dystrobrevin and syntrophin
(Cohn and Campbell, 2000;
Winder, 2001).
Loss-of-function mutations in DG and other DGC components result in
sarcolemmal damage and muscular dystrophy
(Cote et al., 1999;
Cohn and Campbell, 2000;
Cohn et al., 2002;
Michele and Campbell, 2003;
Parsons et al., 2002).
DG also functions as a BM receptor in non-muscle tissues. Unconditional
knockout of DG in mice results in early embryonic lethality due to failure of
extraembryonic BM formation (Williamson et
al., 1997). DG functions as an important laminin receptor in
developing kidney, lung and salivary epithelia
(Durbeej et al., 1995;
Durbeej et al., 2001). The
loss of DG in epithelial-derived breast tumor cells leads to a failure of
ECM-induced cell polarization and enhanced invasiveness
(Muschler et al., 2002).
Brain-specific knockout of DG results in pial BM discontinuities and cortical
neuron migration defects (Michele and
Campbell, 2003; Moore et al.,
2002). Knockout of DG in Schwann cells produces defects in
myelination and nodal architecture (Saito
et al., 2003). The importance of DGC components to DG function
outside of muscle is unclear, as mice lacking both dystrophin and utrophin, or
the sarcoglycan complex, do not display the nervous system defects or
embryonic lethality seen in the brain-specific and unconditional DG knockouts,
respectively (Rafael et al.,
1999; Imamura et al.,
2000).
Homologs of DG and other DGC components have been identified in
Drosophila melanogaster and Caenorhabditis elegans
(Dekkers et al., 2004;
Deng et al., 2003;
Grisoni et al., 2002).
Drosophila DG is expressed in follicle and imaginal disc epithelia
and the oocyte, and its loss disrupts epithelial and oocyte polarity
(Deng et al., 2003).
Drosophila DG and other DGC components are also expressed in the
nervous system and some muscle (Dekkers et
al., 2004). In C. elegans, DAPC complex homologs function
in muscle with the acetylcholine transporter SNF-6 to regulate cholinergic
stimulation (Bessou et al.,
1998; Gieseler et al.,
2001; Grisoni et al.,
2003; Kim et al.,
2004), but the roles of other DGC components have not been
extensively characterized.
We report the characterization of DGN-1, the C. elegans ortholog of vertebrate DG. DGN-1 is expressed in epithelia in the gonad and other tissues, and in neurons. DGN-1 is not found in muscle and does not function with the conserved DAPC complex in C. elegans muscle. DGN-1 plays an important role in gonad epithelial development, where it is likely to mediate the function of laminin. DGN-1 also affects guidance and the extension of cell processes along BM surfaces. These findings suggest a conserved role for DG in mediating epithelial and neural cell responses to the ECM.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) at 20°C. The following strains were used: wild-type N2
var. Bristol; NJ52 epi-1(rh27); NJ244 epi-1(rh92); NJ590
epi-1(rh199); LS292 dys-1(cx18); LS505 dyb-1(cx36);
LS721 stn-1(ok292). The following GFP markers were used: DA/DB
motoneurons, evIs82 [unc-129::GFP]; early somatic gonad
cells/distal tip cell, qIs19 or qIs56 [lag-2::GFP];
spermatheca, jcIs1 [AJM-1::GFP]; gonad sheath, tnIs6
[lim-7::GFP]; anchor cell and body wall muscle, syIs49
[zmp-1::GFP]; mIs11 [myo-2::GFP,
pes-10::GFP, gut promoter::GFP], chromosome III integrant;
qIs54 [myo-2::GFP, pes-10::GFP, gut promoter::GFP],
X chromosome integrant; oxIs12 [unc-47::GFP], X chromosome
integrant. dgn-1(cg121) was maintained as a heterozygote balanced by
visible X chromosome markers. Alleles of epi-1 were balanced by
mIs11. Extrachromosomal arrays of dgn-1 promoter reporter or
GFP fusion protein constructs were created by germline injection
(Mello et al., 1991). The
laminin-ß GFP fusion, urEx131 [LAM-1::GFP], and EPI-1 antibody
were generous gifts of Bill Wadsworth (Robert Wood Johnson Medical
School).
dgn-1 genomic and cDNA constructs
The C. elegans DG homolog (T21B6.2) was identified by BLAST
searches and designated dgn-1. Sequencing of the yk671e7 cDNA
extended the predicted 5' end of exon 1 to nucleotide 10315 of cosmid
T21B6 (GenBank Z68011), and the 3'UTR to nucleotide 1837. A genomic
dgn-1 clone (pJK600) was constructed by inserting 1181-12990 of T21B6
into the XbaI site of BlueScribe M13 Plus (Stratagene). This region
includes 2680 bp upstream of exon 1 through the entire 3'UTR of
dgn-1. pJK600-containing transgenes rescue dgn-1(cg121)
phenotypes, with 
90% of transgenic animals having restored fertility.
dgn-1::GFP
Plasmid pJK602 contains 2680 bp upstream of exon 1 through the middle of
exon 2 (4679-12990 of T21B6) inserted between the MluI and
HindIII sites of pJJ471. The GFP fusion product contains the first 54
amino acids of DGN-1, a synthetic transmembrane region and GFP. An identical
expression pattern was observed with a similar reporter containing only 213 bp
upstream of exon 1, suggesting that the major dgn-1 expression
controls are in intron 1.
DGN-1::GFP
Plasmid pJJ516 was made by inserting GFP from pPD114.38
(www.ciwemb.edu/pages/resources.html)
into the HindIII site near the end of the dgn-1 coding
sequence. The product contains GFP inserted after residue 575 of DGN-1, with
the final seven DGN-1 residues at the C terminus. Expression of DGN-1::GFP is
identical to that of the dgn-1::GFP promoter reporter, and DGN-1::GFP
rescues the sterility of dgn-1(cg121).
Bacterially expressed DGN-1 fusions
DGN-1 regions corresponding to vertebrate -DG (amino acids
20-379;TRVF...NSFT) and ß-DG (amino acids 392-584;VAFS...FIPP) were PCR
amplified from yk671e7 and cloned into pGEX-4T1 (Pharmacia) and pMal-c2 (New
England Biolabs) to produce glutathione S-transferase (GST) and maltose
binding protein (MBP) fusions. Primers used were:
forward, 5'-CCGCTCGAGACCCGTGTGTTTATTGG-3'; reverse, 5'-GGCCTCGAGCTAAGTGAAACTGTTGACTGG-3';
Deletion mutagenesis
Deletion mutagenesis was performed essentially as described
(Barstead, 1999). Progeny of
trimethylpsoralen/UV mutagenized animals were screened by PCR for deletions in
dgn-1. The cg121 deletion removes nucleotides 2045-4439 of
cosmid T21B6. The cg121 mutant strain was backcrossed to wild type at
least six times before further analyses.
Western blot analysis
Embryos and mixed larval stage animals were pulverized in liquid nitrogen
and extracted in PBS, 1% NP40 containing protease inhibitor cocktail (Roche
Biochemical). Extracts were centrifuged (50,000 g, 20 minutes,
4°C) to remove insoluble material. Alternatively, 25 adults were boiled
for 15 minutes in 2% SDS containing protease inhibitors and centrifuged
(14,000 g, 10 minutes, 4°C). Extracts were subjected to
SDS-PAGE and transferred to nitrocellulose. Filters were blocked in PBS
containing 5% non-fat dried milk, incubated with affinity-purified anti-DGN-1
antibody followed by horseradish peroxidase-conjugated secondary antibody
(Vector Laboratories), and developed for ECL chemiluminescent detection
(Amersham Pharmacia Biotechnology).
For deglycosylation experiments, extracts were adjusted to 1% SDS and boiled for 10 minutes. After cooling, nine volumes of 0.5% NP40, 50 mM Tris-HCl (pH 8.0) containing protease inhibitors was added and samples were digested for 16 hours at 37°C with 1 unit of protein-N-glycosidase F (PNGaseF; New England Biolabs).
Microscopy
Animals were mounted on thin pads of 2% agarose in a drop of M9 buffer
(Wood, 1988) containing
levamisole (0.1 mM) or sodium azide (10 mM) to immobilize them.
Immunohistochemistry was performed as described
(Kang and Kramer, 2000).
Monoclonal anti-MHC-A myosin, polyclonal anti-LET-2, polyclonal anti-NID-1,
and polyclonal anti-EPI-1 antibodies were used as described
(Kang and Kramer, 2000;
Huang et al., 2003).
Polyclonal anti-DGN-1 was used at a 1:100 dilution. Images were collected on a
Zeiss Axiophot microscope equipped with a CCD camera. Some fluorescent images
were deconvolved (VayTek MicroTome) to remove out-of-focus signals.
Expression of differentiated gonad cell markers in dgn-1(0)
Assessment of L4 or adult stage dgn-1(0) animals using integrated
GFP markers and/or morphology revealed that: 98% have one to two distal tip
cells (strong qIs56[lag-2::GFP]); 99% have one to two
clusters of presumptive spermathecal cells (strong
jcIs1[ajm-1::GFP]); 90% form one to two anchor cells
(syIs50[zmp-1::GFP]); 89% have presumptive gonad sheath
cells (tnIs6[lim-7::GFP]); and 63% show a peri-vulval lumen
in mid/late L4 stage, indicative of uterine tissue (n=95-100 animals
scored for each marker).
Behavioral assays
For activity on plates, individual L4 stage animals were transferred to NGM
plates without bacteria and allowed to recover for 1 minute before counting
the number of body bends in a 2-minute interval. For thrashing in liquid, L4
animals were transferred to a drop of M9 medium on an unseeded NGM plate,
allowed to recover for 1 minute, then filmed for 2 minutes. The number of body
bends, defined as a reversal in direction of head movement, was counted from
recordings. For defecation assays, the timing of successive posterior body
contraction and expulsion steps (Avery and
Thomas, 1997) on seeded NGM plates was recorded for 10 defecation
cycles. Assays were performed at room temperature (approximately
22°C).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-DG (Bozic et al.,
2004), which is missing from Drosophila DG
(Fig. 1B, N-terminal). A
threonine-rich, mucin-like region is present in all DGs, although it is much
shorter in DGN-1 (Fig. 1B,
N-terminal). dgn-1 orthologs in the nematodes C. briggsae
(CBG17551) and C. remanei (ORF on Contig1797.2) show strong
conservation of sequence (80-90% identity) and domain organization with
DGN-1.
Specific functional amino acid residues are conserved in all DGs, including
two cysteine pairs that form disulfide bonds in vertebrate DG
(Brancaccio et al., 1998;
Deyst et al., 1995) and
several predicted N-linked glycosylation sites
(Fig. 1B). Two regions of
sequence divergence are noteworthy. First, neither invertebrate DG shows
strong sequence conservation in the vertebrate /ß proteolytic
cleavage region (Fig. 1B,
core). Second, crucial residues for binding WW and SH3 domain-containing
proteins such as dystrophin (Huang et al.,
2000) are not conserved in DGN-1
(Fig. 1B, cytoplasmic). These
differences are also true for the C. briggsae and C. remanei
orthologs (data not shown).
DGN-2 and DGN-3 share only the DG core with vertebrate DG, corresponding to
the -DG C terminus and the ß-DG N terminus
(Fig. 1B). In addition to the
dgn-1 ortholog, C. briggsae (CBG16240) and C.
remanei also have other predicted gene products containing a DG core.
This region therefore defines a family of DG-like proteins.
Drosophila DG contains two adjacent, divergent copies of this core
region (Sciandra et al.,
2001). Outside of the core, DGN-2, DGN-3 and CBG16240 show no
similarity to each other or to other DG family members. Thus DGN-1 is the
unique nematode ortholog of vertebrate DG, and additional species-specific
DG-like proteins with variable N-terminal and cytoplasmic domains have arisen
in nematodes.
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and ß subunits- or
ß-DG were used to detect DGN-1 in lysates of embryos and larvae. Both
antibodies detected a band of 85 kDa, higher than the 68 kDa predicted
mass (Fig. 2A). No bands
corresponding to separate DGN-1 or ß species were detected,
indicating that DGN-1 is not processed into separate and ß
subunits. Embryonic DGN-1 migrates as a compact 85 kDa band and a minor 95 kDa
species (Fig. 2C, lane 1), and
larval DGN-1 as a heterogeneous band centered around 85 kDa
(Fig. 2C, lane 3). PNGase F
digestion of embryo extracts shifts the 85 kDa species to a 75 kDa band
(Fig. 2C, lane 2). The 95 kDa
band shifts slightly to 90 kDa, suggesting differential N-glycosylation
of this species. PNGase F digestion of larval extracts results in widening of
the DGN-1 band and a 10-15 kDa decrease in median apparent weight
(Fig. 2C, lane 4). Thus, larval
DGN-1 is also N-glycosylated, but its heterogeneity is largely determined by
other modifications, possibly including O-glycosylation as occurs with
vertebrate -DG.
DGN-1 is expressed in epithelial and neural tissues, but not in muscle
Sites of dgn-1 expression were determined using reporters driven
by dgn-1 upstream sequences. In early (pre-morphological) embryos,
dgn-1::GFP expression is evident in many epithelial and neural
precursors comprising the outer layer of cells
(Fig. 3A). As elongation begins
at comma stage, expression becomes most prominent in several specialized
epithelial cells, including pharyngeal e2 and marginal cells, excretory cells,
the somatic gonad precursors (SGPs) Z1 and Z4, and rectal epithelial cells
(Fig. 3B). Weaker expression is
apparent in hypodermal precursors and neuroblasts along the ventral midline.
Pharyngeal expression persists through the L3 larval stage, whereas excretory
and rectal cell expression persists throughout development. SGP expression
(Fig. 3C,E) persists in SGP
descendants, such as the distal tip cells (DTCs;
Fig. 3I), and increases
throughout the gonad during the L4 stage
(Fig. 3J). Variable, generally
weak expression is seen throughout larval development in several neurons,
although PVP neurons show strong expression throughout development
(Fig. 3C,F). Transient
increased expression occurs in new P cell-derived neurons in the ventral nerve
cord in late L1/early L2 stage animals
(Fig. 3H). Variable weak
expression is seen in hypodermal cells, principally hyp5 in the head
(Fig. 3D). Preceding the
L4/adult molt, expression increases in the vulval epithelium
(Fig. 3K).
The expression pattern was confirmed and subcellular localization determined using anti-DGN-1 antibody staining and analysis of a rescuing DGN-1::GFP fusion protein. Both approaches yielded similar results, which are described for antibody staining. In pre-morphological embryos, DGN-1 is diffuse around the surface of outer ectodermal cells before BMs form (Fig. 4A), but begins to polarize towards basal surfaces as BMs assemble between germ layers (Fig. 4B). Throughout subsequent development, DGN-1 localizes to the basal surfaces of pharyngeal (Fig. 4C), gonadal (Fig. 4C-G), rectal (Fig. 4H), vulval (Fig. 4E,F) and excretory cell (Fig. 4I,K) epithelia, in close apposition to the underlying BM. DGN-1 accumulation is more variable in the hypodermis (Fig. 4K,L) and neurons (Fig. 4J).
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Sterility results from an early disruption of gonad morphogenesis. The
wild-type gonad primordium contains two central primordial germ cells (PGCs)
flanked at anterior and posterior poles by the two SGPs. The primordium is
compact, with a sharp DIC image boundary, indicating a robust surrounding BM
(Fig. 5C). In newly hatched
cg121 homozygotes, the primordium is usually compact but often
displays bulging of PGCs and a weak DIC boundary
(Fig. 5D). SGPs are frequently
displaced from the poles, sometimes interposing between PGCs
(Fig. 5D,H). In some
cg121 larvae, no DIC boundary is detectable and gonadal cells spread
dorsally along the body wall (Fig.
5E). Older dgn-1 mutants often show swelling in the
mid-body region as a result of body wall muscle cell fusion with, or
engulfment of, loose germ cells (data not shown), as is also seen in
epi-1 laminin mutants (Huang et
al., 2003).
The aberrant gonad morphology of dgn-1 mutants suggests defects in the gonad BM. Antibodies to collagen IV and nidogen, and a LAM-1(laminin-ß)::GFP fusion were used to examine the distribution of BM components. In early cg121 L1 larvae with compact gonad primordia, localization of BM components around the primordium appears normal (Fig. 6). In larvae with disrupted primordia, localization of BM components is still seen, but staining is weak and diffusely distributed over the surface of the gonad tissue (not shown). Thus, a BM organizes around the dgn-1(0) gonad but is not maintained. No gross alteration in other BMs is seen in dgn-1 mutants.
Failure of gonad primordium BM function in dgn-1 mutants may result from an inability of SGPs to form a stable epithelial layer around the PGCs. Mispositioned SGPs, showing strong association of laminin-ß::GFP, can be observed covering one PGC while excluding the other (Fig. 6F,G). Thus, the extrusion of germ cells probably reflects a failure of the somatic gonad epithelium and its associated BM to provide a stable barrier.
Organization of gonad tissue in dgn-1(0) animals was examined following the L1/L2 molt, using lag-2::GFP expression and nuclear morphology to discriminate somatic and germ cells. Early L2 dgn-1(0) animals contained 8.8±1.0 somatic gonad cells (n=25), which is close to the wild-type number of 8. The somatic gonad cells of dgn-1(0) animals cluster together in a central clump, or in a contiguous line (sometimes separated into two clusters) along the ventral surface (Fig. 5J,K). In either organization the somatic cells remain in contact, suggesting somatic cell-cell adhesion is retained. By contrast, germ cells are either not in contact or only in peripheral contact with somatic cells (Fig. 5J). These results further indicate the inability of somatic cells to associate with or ensheath germ cells in dgn-1(0) animals. Monitoring of dgn-1(0) gonads throughout the L2 stage did not reveal any overt re-organization of dgn-1(0) somatic gonad cells, indicating a failure of somatic primordium formation in dgn-1(0) animals. By contrast, wild-type gonads showed reorganization of somatic cells into a central somatic primordium and distal tip cells at the ends of the two emerging gonad arms.
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Heterozygous cg121/+ animals form a grossly normal gonad, but
produce 15% fewer progeny than do wild type (wild type, 309±41;
cg121/+, 263±32; n=18). Twenty-three percent of
cg121/+ heterozygotes have defects in gonad arm migration
(Fig. 7). The arms migrate on
the underlying BM led by DTCs, which express DGN-1. Although a range of
migration defects is seen in cg121/+ animals, the occurrence of
oblique turns and abnormal midline crossings indicates a failure of DTC
responses to BM guidance cues (Su et al.,
2000).
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dgn-1 mutants do not have strong movement defects, indicating a severe perturbation of muscle or neural function. However, in 31% of dgn-1 mutants, at least one DA/DB type neuron commissure extends on the wrong side of the body (Fig. 9A). Individual animals also show additional axonal defects, such as defasciculation or abnormal branching (Fig. 9B-D). Heterozygous cg121/+ animals show similar DA/DB guidance defects but at a lower penetrance.
The vulval epidermis shows prominent dgn-1 expression starting when the invaginated epidermis everts and tightens into a slit-shaped opening. Forty-five percent of dgn-1 adults have a protruding vulva (Pvl) phenotype, suggesting a detachment of the vulval epithelium from the underlying tissue (Fig. 5, Table 1). Frequently, rupture at the protruding vulva leads to the herniation of internal organs, indicating that dgn-1 function is important in the anchorage of the vulval epidermis.
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). Using the
AC marker zmp-1::GFP (Inoue et
al., 2002), we found 14% of dgn-1 mutants generate two
ACs, while 10% fail to generate an AC (n=95). All wild-type animals
produced one AC (n=100). This AC specification defect is likely to
result primarily from a disruption of gonad structure in dgn-1
mutants. dgn-1 mutants do not show phenotypes associated with defects in muscle function, such as Pat (paralyzed at two-fold) or Unc (uncoordinated movement). No gross disorganization of body wall muscle was apparent by DIC microscopy. The muscles associated with the alimentary system function normally, while function of uterine and vulval muscles cannot be assessed because of the failure of gonad formation in dgn-1 mutants.
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B gene epi-1
chains and
two chains, LAM-3/laminin-A and EPI-1/laminin-B
(Huang et al., 2003). Both
laminin- chains are distributed broadly in BMs, but EPI-1 uniquely
localizes to the gonad BM. Gonad epithelialization fails in epi-1
mutants (Huang et al., 2003),
resulting in gonad rupture like that seen in dgn-1(0)
(Fig. 5F). The penetrance of
the gonad defect varies between epi-1 alleles, but is 100% in the
putative null allele rh199 (Table
1). SGP mispositioning is seen in newly hatched rh199
animals, although at a higher penetrance than in dgn-1 mutants
(Fig. 5H). The similarity of
gonad phenotypes suggests that DGN-1 may be a crucial receptor for EPI-1 in
the gonad primordium. epi-1 mutants show other morphological defects reminiscent of dgn-1 phenotypes, although they often are more severe (Table 1). On average, three to four excretory cell arms are missing in rh199 animals, compared with one to two in cg121, and remaining arms are short, and exhibit aberrant morphology and guidance. epi-1 mutants also show Pvl defects comparable to dgn-1 mutants. These results are consistent with DGN-1 mediating some EPI-1 function in the development of the excretory cell and vulval epithelium.
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). We examined
C. elegans DAPC mutants of dys-1 dystrophin, dyb-1
dystrobrevin and stn-1 syntrophin for morphological phenotypes
similar to those of dgn-1. None of these putative functional null
mutants show the morphological defects prevalent in dgn-1(0)
(Fig. 5,
Table 1), indicating that the
morphological functions of DGN-1 are largely independent of DAPC function.
Distinct behavioral phenotypes of dgn-1 and dystrophin complex mutants
C. elegans DAPC mutants display a head muscle hypercontraction
phenotype (Fig. 10A) due to
excessive acetylcholine neurotransmission
(Kim et al., 2004).
Hypercontraction is apparent in 100% of dys-1, dyb-1 or
stn-1 mutants and occurs in at least 97% of movement cycles in each
mutant (n=20 L4 animals, each observed for 50 movement cycles), but
is not seen in dgn-1 mutants. Double mutants of dgn-1 with
dys-1, dyb-1 or stn-1 show hypercontraction as in the DAPC
single mutants (Fig. 10B),
indicating that dgn-1(cg121) does not epistatically suppress this
phenotype.
DAPC mutants also display hyperactivity on agar plates
(Bessou et al., 1998;
Gieseler et al., 2001;
Grisoni et al., 2003),
generating 37%-57% more body bends per minute than wild type
(Fig. 10C). The
dgn-1(cg121) mutant shows comparable hyperactivity in plate
locomotion (Fig. 10C). Both
DAPC and dgn-1 mutants also show a 9%-29% increase in thrashing rate
when suspended in liquid (Fig.
10C). Double mutants show activity levels comparable to or
slightly lower than wild type (Fig.
10C). Cross-suppression of hyperactivity in double mutants is
inconsistent with DGN-1 and DAPC acting in a common functional pathway, and
indicates that hyperactivity of dgn-1 and DAPC mutants involves
genetically distinct mechanisms.
dgn-1 homozygotes (Fig.
10D) show a 27% increase in defecation cycle time
(Avery and Thomas, 1997) when
compared with wild type (61±6 seconds versus 48±3 seconds).
dys-1 mutants display essentially normal periodicity (51±4
seconds), and dys-1;dgn-1 double mutants have periodicity comparable
with dgn-1 single mutants (66±4 seconds). Together, these
phenotypic comparisons indicate that DGN-1 and the DAPC complex of C.
elegans function in different processes and act independently of one
another.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-DG and the extracellular region of ß-DG. The
vertebrate DG core contains the site of post-translational /ß
cleavage and sites within and ß subunits mediating their
association (Sciandra et al.,
2001). DGN-1 and Drosophila DG
(Deng et al., 2003) are not
processed into separate and ß subunits, although interaction of
the corresponding regions may occur. Interestingly, a mutant DG in which
/ß cleavage is disrupted dominantly produces altered DG
glycosylation and muscular dystrophy in mice
(Jayasinha et al., 2003),
suggesting a regulatory role for vertebrate /ß cleavage. The core
region also contains conserved potential N-linked glycosylation sites
important in intracellular trafficking of DG
(Holt et al., 2000). The core
domain may mediate conserved interactions with as yet unidentified
extracellular or transmembrane factors.
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;
Parsons et al., 2002;
Winder, 2001) (this work).
Multiple Caenorhabditis species contain additional species-specific,
more divergent, DG-like genes whose roles remain to be elucidated. C.
elegans dgn-2 and dgn-3 are expressed in a few neural and
epithelial cells, and deletion mutations cause no overt phenotypes (J.M.K.,
unpublished). It is possible that the N-terminal and C-terminal domains of
these divergent DG-like proteins mediate distinct transmembrane linkages
between specific extracellular ligands and intracellular effectors.
DGN-1 is not generally required for BM assembly but is a likely mediator of laminin function in early gonad epithelium
BMs form and most are maintained in dgn-1(0) mutants, although the
gonad BM is not maintained in the absence of DGN-1. Similarly, vertebrate DG
is not generally essential for BM assembly
(Li et al., 2002) but may play
an important role in some contexts (Henry
and Campbell, 1998). Mouse embryos lacking DG do not form
Reichert's membrane, but form the epiblast BM in the embryo proper
(Williamson et al., 1997). In
a brain-specific DG knockout, the pial BM forms but contains focal
discontinuities (Moore et al.,
2002). Thus, in both nematodes and vertebrates the DG ortholog has
roles in the maintenance of specific BMs but is not required for all BM
assembly.
The similar gonad primordium defects in epi-1 and dgn-1
mutants suggest that DGN-1 is a likely mediator of EPI-1 function in the early
gonad. The single C. elegans ß integrin PAT-3 is another
potential laminin receptor, but dominant interference of PAT-3 function does
not appear to produce the same early gonad phenotype
(Lee et al., 2001). DGN-1 is
not required for the initial localization of laminin to the surface of the
primordium, indicating that other factors mediate laminin recruitment. EPI-1
functioning through DGN-1 appears to be essential for promoting the epithelial
function of the SGPs, although the nature of this nascent epithelium is
unclear. Somatic cells of the early gonad do not display junctional complexes
indicative of mature polarized epithelia and do not express identified
junctional components (Miskowski et al.,
2001). A BM signal through DGN-1 may play a role in the apicobasal
polarization of somatic gonad cells in the early gonad, before a mature
epithelium has differentiated.
Early gonad disruption in dgn-1 and epi-1 mutants results in the escape of germ cell precursors into the body cavity. The gonad BM may be important structurally to maintain ensheathment by the somatic gonad cells, but it is also possible that an unidentified DGN-1-mediated signal from the gonad BM promotes germ-soma interaction. DGN-1 function is not required for the adhesion of somatic gonad cells with one another. Somatic gonad cells cluster together in dgn-1(0) L2 stage animals, and differentiated spermathecal and uterine cells displaying AJM-1-containing cell junctions and lumen formation are found in L4 stage animals.
A role for DGN-1 in the migration of cells and specialized cell processes
Migrations of axons and the tubular arms of the excretory cell involve the
extension of cell processes between the hypodermis and its BM
(Wood, 1988), and several cell
adhesion and cytoskeletal factors, including laminin, are involved in their
guidance (Buechner, 2002). The
excretory cell and axonal guidance phenotypes of dgn-1 and
epi-1 mutants suggest that DGN-1 partially mediates laminin function
in guiding cell processes along the hypodermal BM.
Heterozygous dgn-1(0)/+ animals show defects in excretory cell and axon migration, as well as in DTC guidance along BMs. DGN-1 may have an essential role in DTC migration that cannot be assessed directly because of the gonad disruption in dgn-1(0) homozygotes. The defects in excretory cell, DTC and axon migration in cg121/+ heterozygotes must represent haploinsufficiency, in which the reduced DGN-1 levels in heterozygotes are insufficient for normal function. DGN-1 overexpression from extrachromosomal arrays can also cause dgn-1(0)-like defects in wild-type animals, particularly in excretory cell morphology (R.P.J., unpublished). The appearance of similar phenotypes from increased or decreased expression suggests that DGN-1 activity may be required dynamically and/or in precise stoichiometry relative to other components for normal function.
Conserved epithelial and neural roles for DG
DGN-1 functions in a variety of epithelia and neurons but is not expressed
in muscle. Vertebrate DG also functions in the nervous system
(Moore et al., 2002;
Saito et al., 2003) and in at
least some types of epithelia (Durbeej et
al., 1995; Durbeej et al.,
2001), as well as in muscle. In Drosophila, DG is
important in the polarization of follicular epithelia and oocytes
(Deng et al., 2003), although
it is expressed and may have additional roles in muscle and neural tissue
(Dekkers et al., 2004). These
findings suggest that either the DGN-1/DG subfamily originated as an ECM
receptor in epithelial/neural tissue and that muscle function was acquired
later, or that an original muscle role for DG was not retained in nematodes.
Notably, each sarcomere in nematode muscle is anchored to the underlying BM
via integrin-containing dense bodies
(Moerman and Fire, 1997), and
these numerous attachments may preclude the need for further sarcolemmal
stabilization by DGN-1.
Divergence of DGN-1 and DAPC complex functions in C. elegans
DGN-1 functions in epithelia and neurons do not depend on the conserved
DAPC complex, which is consistent with the poor conservation of the
dystrophin-binding site in the DGN-1 cytoplasmic domain. The major site of
DAPC function is in muscle, where it regulates contraction intensity via the
acetylcholine transporter SNF-6 (Bessou et
al., 1998; Gieseler et al.,
2001; Grisoni et al.,
2003; Kim et al.,
2004). It is unclear whether the hyperactivity of DAPC mutants
(Bessou et al., 1998;
Gieseler et al., 2001;
Grisoni et al., 2003) is also
due to altered cholinergic stimulation. dgn-1 mutants show similar
hyperactivity that is genetically distinct from that of the DAPC mutants and
may reflect neuronal DGN-1 function.
The divergence of DGN-1 and DAPC complex function in C. elegans
has intriguing implications for the normal and pathological roles of their
vertebrate homologs. The progressive muscle degeneration in C.
elegans DAPC and snf-6 mutants is reminiscent of vertebrate
muscular dystrophy (Bessou et al.,
1998; Gieseler et al.,
2001; Grisoni et al.,
2003; Kim et al.,
2004; Cohn and Campbell,
2000) and may reflect a conserved muscle function of the DAPC that
is separable from its interaction with DG. Conversely, evidence from
vertebrate systems suggests that non-muscle DG may have roles not requiring
DAPC function. Several studies indicate that vertebrate DG has important
functions in the early embryo and in non-muscle tissues that do not appear to
depend on DAPC or sarcoglycan complex function, but do depend on the ECM
ligand-binding activity of -DG
(Durbeej et al., 1995;
Durbeej et al., 2001;
Michele et al., 2002;
Moore et al., 2002;
Rafael et al., 1999;
Imamura et al., 2000;
Saito et al., 2003;
Williamson et al., 1997).
Thus, non-muscle DG probably mediates important ECM interactions that are
wholly or partly independent of DAPC function, suggesting that other
intracellular factors transduce DG function in these contexts. The
DAPC-independent roles of DGN-1 in C. elegans may thus help to
elucidate conserved non-muscle roles of DG as a BM receptor and to identify
novel downstream partners of DG.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) in N-terminal and core domains,
conserved predicted N-glycosylation sites (•) in the core
domain, and the 
) 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
