1 Department of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ
08854, USA
2 Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New
York, NY 104661, USA
3 Department of Biology, Johns Hopkins University, Baltimore, MD 21218,
USA
4 Medical Biotechnology Center, University of Maryland Biotechnology Institute,
725 West Lombard Street, Baltimore, MD 21201, USA
5 Max-Planck-Institut Für Medizinische Forchung, Heidelberg, 69120
Germany
6 Department of Biology, Sinsheimer Laboratories, University of California,
Santa Cruz, CA 95064, USA
* These authors contributed equally to the paper
Author for correspondence (e-mail:
william.wadsworth{at}umdnj.edu)
Accepted 14 April 2003
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SUMMARY |
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Key words: Laminin, Basement membranes, Extracellular matrix, C. elegans, Cell adhesion, Cell polarity, Cell migration, Differentiation, Cell-cell signaling
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INTRODUCTION |
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Although genetic studies have established the diversity and complexity of
laminin functions in vivo, the manner by which laminins mechanistically
regulate development is not well understood. Traditionally, laminin and
basement membranes have been viewed at substrates that support cell adhesion
and migration. However, the idea that the supramolecular organization of
laminin itself has an instructive role has gained support
(Colognato and Yurchenco,
2000). On the surface of cells, laminins are known to bind several
receptors and receptor-like molecules, including integrins,
/ß-dystroglycan, and syndecans. One model predicts that laminin
receptors anchor laminin and drive laminin polymerization on cell surfaces by
causing the critical concentration for laminin self-assembly to be locally
exceeded (Colognato et al.,
1999
). The formation of a laminin polymer appears to be necessary
before other components are able to assemble into a basement membrane
(Aurelio et al., 2002
;
Smyth et al., 1999
).
Polymerization further triggers the reorganization of the receptors within the
plasma membrane and facilitates the reorganization of cytoskeletal components.
It has been observed that on the surface of cultured myotubes, this
reorganization drives laminin, laminin receptors and cytoskeletal components
into a polygonal network (Colognato et
al., 1999
).
The receptor-facilitated laminin self-assembly model predicts that in vivo
secreted laminin associates with receptors on exposed cell surfaces. Loss of
laminin function is predicted to cause defective basement membrane assembly
and spatial organization of receptor complexes and cytoskeletal components.
The detailed description of the anatomy and cell lineages of C.
elegans makes it a particularly attractive genetic system to examine
these predictions. In particular, serial section electron microscopy has
allowed every cell and cell contact to be described in the wild-type animal,
allowing the genetic analyses of cellular development to be studied in
remarkable detail (see
www.wormatlas.org).
Four members of the laminin family have been predicted in C. elegans:
there are two , one ß and one
, which are encoded by
epi-1, lam-3, lam-1 and lam-2, respectively
(Hutter et al., 2000
). We
report that both laminin
subunits are secreted between the primary
tissue layers and become localized in different patterns to exposed cell
surfaces, consistent with a receptor-facilitated process. Mutations within
each laminin
subunit gene cause abnormal cell-cell adhesions at
regions associated with the localization of the subunit. Some cells fail to
make the proper connections to adjacent tissues, while other cells
inappropriately adhere to and invade neighboring tissues. Affected cells may
fail to properly differentiate or migrate, suggesting widespread disruption of
inductive interactions between adjacent tissues. Using electron microscopy, we
observe missing or abnormal extracellular matrix, mispositioned adhesion
complexes and disoriented cytoskeletal elements. For example, we observe on
the surface of body wall muscle cells laminin organizes into a polygonal array
and in mutants muscle cells may fail to properly adhere to the overlying
epidermis. Muscle adhesion complexes and myofibrillar components are
improperly positioned and in the epidermis the cytoskeleton is defective
adjacent to where the muscle cells attach. Taken together, our results are
consistent with the idea that laminin plays a crucial role in organizing a
supramolecular architecture comprising extracellular matrix, receptors and
cytoskeletal components, and that this architecture is important for
regulating adhesion and signals between adjacent tissues.
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MATERIALS AND METHODS |
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Chromosome I: MT6550, lam-3(n2651)/dpy-5(e61)unc-75(e950); PD9753, ccIs9753; PD4251, ccIs4251.
Chromosome II: SP756, unc-4(e120) mnDf90/mnC1.
Chromosome IV: GG23, emb-9(g23); RW3600, pat-3(st564)/qC1; NG2324, ina-1(gm86)/qC1; NJ52, epi-1(rh27); NJ244, epi-1(rh92); NJ497, epi-1(rh152); NJ569, epi-1(rh165); NJ572, epi-1(rh191); NJ590, epi-1(rh199); NJ594, epi-1(rh200); IM131, epi-1(rh233); PD4251, ccIs4251; PD9753, ccIs9753; IM19, urIs13.
Chromosome V: IM336, nid-1(ur41)rhIs4(glr-1:GFP).
The epi-1 (rh152) allele was isolated by screening F2 progeny of
mutagenized N2 animals for the presence of defective epithelial conversion of
the gonad. This allele was three factor mapped to lie between dpy-20
and unc-5 on LGIV. To isolate additional alleles of epi-1,
F2 progeny of mutagenized dpy-13/mec-3 animals were screened for
embryonic larval lethals or adult steriles linked to dpy-13. These
mutants were examined for phenotypes that were similar but more severe than
those of rh152. Each selected allele was tested and shown to fail to
complement rh152. epi-1(rh199) and epi-1(rh200) were
maintained from heterozygous mothers because the homozygotes are early lethal
or sterile (Zhu et al.,
2000).
Mutations in lam-3 were isolated in a genome-wide screen for EMS-induced larval lethal mutations causing morphological defects (A.D.C., unpublished). Four mutations (n2488, n2493, n2561 and n2563) confer similar defects in integrity of the pharyngeal basement membrane, as judged using Nomarski microscopy, and result in a fully penetrant lethal phenotype. All four mutations display linkage to chromosome one and fail to complement the reference allele n2488. n2563 was mapped in the lin-11 unc-75 interval: from heterozygotes of genotype n2563/n566 e950, 36 Lin non-Uncs were picked of which three segregated Lam (Pha/Let) worms; 2/2 Unc non-Lin recombinants segregated Pha/Let worms. Map data for other alleles were less extensive but consistent with this position.
Electron microscopy
Animals were immersion fixed using buffered aldehydes and then osmium
tetroxide as described previously (Hall,
1995). Three or four animals were aligned within an agar block,
then embedded and sectioned together. Serial thin sections were collected on
slot grids and post-stained with uranyl acetate and lead citrate, and examined
with a Philips CM10 electron microscope. To fix young L1s from RNAi
experiments, animals were exposed to microwave irradiation during the primary
fixation in buffered aldehydes, using a model 3450 oven (Ted Pella) at half
power (Paupard et al., 2001
).
Subsequent fixation steps follow our normal protocols
(Hall, 1995
).
Molecular biology
RNA-mediated interference (RNAi) was performed as described previously
(Guo and Kemphues, 1995;
Rocheleau et al., 1997
). RNA
was prepared by in vitro transcription (Promega kit) using both T3 and T7 RNA
polymerase, and the products were pooled. As cDNA templates, a cloned 0.4 kb
PstI fragment of lam-3, which encodes for G1, and a cloned
1.0 kb BamHI fragment of epi-1, which encodes for G1 and
part of G2, were used. N2 hermaphrodites were placed on separate plates 12-24
hours after injection, and allowed to lay eggs. These plates were examined
every 24 hours for 3 days to determine the numbers of eggs that hatched and to
which larval stage the animals would develop. To score lam-3 and
epi-1 genetic null mutants, transheterozygous lam3/dpy-5;
unc-75 and epi-1/mec-3 animals were placed on separate plates to
lay eggs. Each parent was transferred to a fresh plate after 5, 10 and 15
hours. Development was scored as above. From the lam-3 and
epi-1 heterozygous parents, one quarter of the progeny (227/863 and
324/1260, respectively) arrest as embryos or larvae. We inferred that these
were homozygous for the laminin mutation as dpy-5; unc-75 and
mec-3 homozygotes develop to the adult stage.
For sequencing of epi-1 alleles, four sets of primers were
designed to create PCR fragments that would span the entire epi-1
genomic region (12,371 bp) with 200 to 300 bp overlaps between fragments.
Primer sets were designed to produce NotI and SpeI sites at
either end of a fragment. For templates, genomic DNA from five to seven mutant
hermaphrodites was prepared as described
(Williams et al., 1992).
Expand High Fidelity PCR enzyme (Roche) was used to generate the PCR fragments
in order to minimize PCR based errors. The PCR fragments were gel purified,
digested with NotI and SpeI, and ligated into
NotI/SpeI-digested pBluescript SK(+) vector (Stratagene).
Each product was completely sequenced. In all cases, at least two cloned
fragments from two independent PCR reactions were sequenced.
Laminin A was deduced from the analysis RT-PCR products in
combination with sequence analysis of cDNA clones obtained from Y. Kohara
(Gene Library Laboratory, National Institute of Genetics, Japan). PCR of
reverse transcribed C. elegans RNA was performed using primers
selected based on the genomic sequence of cosmids T22A3 and H10E24 (C.
elegans genome sequence project). The lam-3 mRNA sequence was
deposited under Accession Number AF074902.
The promoter sequence for the lam-3 reporter construct was PCR
amplified from cosmid T22A3.8. The sequence starts at 2.6 kb 5' of the
predicted ATG start codon. The promoter sequence for the epi-1
reporter construct was PCR amplified from cosmid K08C7.3 and starts at 2.8 kb
5' of the predicted ATG start codon. Both fragments were cloned into the
GFP bearing vector, pPD96.62 [provided by A. Fire (Carnegie Institution of
Washington, Baltimore, MD)]. Transgenic strains expressing the GFP reporters
driven by each promoter were generated by standard methods
(Mello and Fire, 1995;
Mello et al., 1991
). A plasmid
containing wild type dpy-20 sequence was co-injected with the
reporter construct into dpy-20(e1282ts) animals as an injection
marker.
To detect lam-3 and epi-1 RNA, in situ hybridization was
performed as described previously for detection of RNA in whole-mount C.
elegans embryos (Seydoux and Fire,
1995). AP-anti-DIG antibody (Boehringer Mannheim, IN) was used for
alkaline phosphatase (AP)-mediated detection. DAPI (1 mg/ml) was included in
the staining solution to allow nuclei to be identified by epifluorescence
microscopy.
Preparation of antisera and morphological analysis
To generate antisera against laminin A, a plasmid construct was made
by subcloning into the vector pQE (Qiagen, CA) an 800 bp cDNA fragment that
contains the sequence encoding the G3 domain. To generate antisera against
laminin
B, plasmid constructs were made by subcloning cDNA fragments
encoding the G2 domain. The fusion proteins produced contain 6xHIS-tags
and were purified according to the instructions of Qiagen and were used to
immunize rabbits. Antisera against each fusion protein were also raised in
chickens by Pocono Rabbit Farm & Laboratory (Canadensis, PA). Immune serum
was affinity purified on columns coupled to the fusion protein to which they
were generated. Immunostaining was performed as described for embryos
(Wadsworth et al., 1996
) and
for larvae and adults (Finney and Ruvkun,
1990
). Anti-rabbit and anti-chicken fluorescein- and
rhodamine-conjugated secondary antibodies were used. The epi-1(rh199)
mutant embryos and epi-1(RNAi) embryos lack detectable laminin
B antiserum staining and lam-3(RNAi) and lam-3(n2561)
larvae lack detectable laminin
A antiserum staining (see Fig. S2 at
http://dev.biologists.org/supplemental/).
The following antibodies were also used to visualize tissues: MH4, a
monoclonal antibody that recognizes an intermediate filament subunit
(Francis and Waterston, 1991
;
Hresko et al., 1994
); MH27, a
monoclonal antibody that recognizes the adherens junction protein JAM-1
(Francis and Waterston, 1991
;
Leung et al., 1999
;
Mohler et al., 1998
);
anti-myotactin, formerly named MH46
(Hresko et al., 1999
);
anti-UNC-54, a monoclonal antibody that recognizes myosin heavy chain B
(Miller et al., 1983
); and
MH25, a monoclonal antibody that recognizes PAT-3ß integrin
(Gettner et al., 1995
). Images
were obtained using a Zeiss LSM 410 Inverted Laser Scan microscope.
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RESULTS |
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Expression of laminin subunit genes
Using antisense cDNA probes and the expression of laminin promoter-GFP
transgenes, we examined the expression of the subunit genes during
embryogenesis and larval development (Table
1). In situ hybridization was used to examine early epi-1
gene expression. Expression of epi-1, the gene encoding laminin
B, is first detected in the nucleus of cells entering the gastrula
(Fig. 7A). As the cells arrange
into the endodermal and mesodermal layers, epi-1 mRNA is detected
within cytoplasm (Fig. 7B).
Expression continues as the intestinal cells and the precursors of the pharynx
form a central cylinder with the myoblasts filling in between this cylinder
and the outer layer of cells. Strong expression by the myoblasts is detected
(Fig. 7C). Besides the cell
movements, this period of development is characterized by rapid cell divisions
as the number of cells increase from 28 to
350. These results indicate
that the regulation of epi-1 expression is coordinated with the
events of gastrulation. In larvae, epi-1 is expressed in the body
wall, vulva and anal depressor muscles, as well as intestinal cells and in
somatic cells of the gonad (not shown).
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To determine whether the localization of each laminin isoform depends on
the other isoform, the distribution of each laminin subunit in the
loss-of-function mutant of the other laminin
subunit was examined. The
results showed that the localization of laminin
A does not depend on
laminin
B, nor does the localization of laminin
B depend on
laminin
A (see Fig. S1 at
http://dev.biologists.org/supplemental/).
Collagen type IV is not required to localize the laminin
subunits
Basement membranes comprise networks of laminin and collagen type IV. These
networks interact with other extracellular matrix proteins and cell-surface
receptors, such as integrins. Collagen type IV is first detected
intracellularly as the embryo begins to elongate and is detected
extracellularly after the animals have elongated by 1.5-fold
(Graham et al., 1997). This
expression is later than the expression of laminin, suggesting that laminin is
localized to cells before collagen type IV and that laminin does not require
collagen type IV to associate with cell surfaces. To test this experimentally,
the distribution of the laminin
subunits in collagen type IV mutant
animals was observed. We used the mutation emb-9 (g23), which is
semidominant and causes both the collagen type IV chains to accumulate
intracellularly, apparently by blocking assembly or secretion of the type IV
collagen heterotrimers (Graham et al.,
1997
). Both laminin
A and laminin
B show normal
distribution patterns until after early elongation, when collagen type IV is
secreted (see Fig. S3 at
http://dev.biologists.org/supplemental/).
These results indicate that the correct distribution of the laminin
subunits does not initially require collagen type IV and is consistent with
the notion that laminin is localized to cell surfaces before a prototypical
basement membrane assembles.
epi-1 and lam-3 loss-of-function mutations cause
lethality
Mutations in the epi-1 gene were isolated in a screen devised to
isolate mutants defective in gonad conversion from mesenchyme to epithelium.
The female somatic gonad of C. elegans is a cylindrical myoepithelium
that surrounds the germ cells and sustains their maturation
(Buechner et al., 1999;
Hirsh et al., 1976
;
Kimble and Hirsh, 1979
). Like
epidermis, pharynx and intestine, the gonad will epithelialize during its
morphogenesis. However, unlike other tissues in which the cell polarization
occurs during gastrulation, the gonad polarizes late in larval development and
is larger. As a result, its morphogenesis is more easily observed. Mutants
were isolated at the L3 and L4 stage, in which the uterine precursors failed
to exclude germ cells from the center of the gonad (the uterus), spermathecae
failed to form a closed lumen, or the ovarian sheath cells failed to spread
over the adjacent germ cells. One of the genes identified in this screen, was
designated epi-1 (epithelialization).
Mutations in the lam-3 gene were isolated after the possible
phenotypes of lam-3 mutants were identified by examining animals made
deficient for laminin A using RNA-mediated interference, or RNAi
(Fire et al., 1998
). The
lam-3(RNAi) animals arrest during early elongation or at the L1
larval stage. The L1 animals have abnormal pharyngeal development and have
shorter bodies (posterior to the pharynx) at the time of arrest. From a screen
for mutations that cause pharyngeal defects, mutants with phenotypes similar
to the lam-3(RNAi) phenotype were isolated. Four non-complementing
alleles, n2488, n2493, n2561 and n2563, were mapped to the
region of linkage group I where the physical sequence data suggested the
lam-3 gene should reside. A 20 kb laminin
A DNA sequence was
amplified by PCR and was found to rescue the mutant phenotypes when expressed
in n2561 animals.
The strongest alleles of epi-1 and lam-3 and
RNAi animals cause embryonic and larval lethality
(Table 2). The
epi-1(rh199) mutant embryos and epi-1(RNAi) embryos lack
detectable Laminin B antiserum staining and lam-3(n2561) and
lam-3(RNAi) larvae lack detectable Laminin
A antiserum
staining. Animals deficient for both
subunits were made by double RNAi
as the genetic construction of epi-1 and lam-3 double
mutants was problematic. Compared with single epi-1- or
lam-3-null mutants or RNAi animals made deficient for a single
subunit, the double RNAi animals were more likely to arrest development during
embryogenesis. This suggests that each laminin has separate functions required
for viability during embryogenesis. Although a pharyngeal development is not
required for embryonic viability, the observation that only lam-3
larvae always arrest at the L1 stage with abnormal pharynxes is also
consistent with the idea of distinct developmental roles for the laminins
during embryogenesis.
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Body wall muscles appear to support different basement membranes on each
face, with a thickened membrane facing the outer epidermis and cuticle
(featuring laminin B expression; see below), and a very thin membrane
facing lateral and inward-facing surfaces (pseudocoelom). In many places the
basement membranes of the muscle and neighboring epidermis are together;
however, in various places where the tissues are further separated, the muscle
basement membrane appears separately very thick, whereas the neighboring
epidermal membrane is thin, just as in other parts of the anatomy. This
suggests that the basement membrane associated with the muscle mostly
contributes to the thick basement membrane between the cells. A single layer
of basement membrane similarly covers the gonadal cells, both over the sheath
cells of the proximal arm and over the bare germ cells of the distal arm
(Hall et al., 1999
). A rather
thick basement membrane lies over the distal tip cell (DTC) of the gonad that
merges at the trailing edge of the DTC with the thin layer covering the bare
germ cells. These descriptions of the basement membrane come from TEM of
immersion-fixed specimens. Preliminary studies of fast-frozen worms suggest
the basement membranes are more lacey or flocculent and seem to extend farther
into the space between tissues (D.H.H., unpublished).
One striking feature revealed is the degree of asymmetry of basement membranes associated with some cells (see Fig. S5 at http://dev.biologists.org/supplemental/). Besides the bodywall muscles, this is a feature of many classes of alimentary and sex muscles, often to a more dramatic extent. Thus, the muscles of the alimentary tract [intestinal, sphincter and anal depressor (DA)] show very thick basement membranes in limited regions where they are anchored to the bodywall or rectal cuticle via thin epidermal interfaces (an example for the muDA anchorage is shown on SW-Worm Tiler, Slice 726, at wormatlas.org/SW/SW.htm/WormTiler.htm). Similarly the sex muscles of the hermaphrodite (vulval and uterine muscles) and of the male tail (spicule, gubernacular and bursal muscles) show very robust basement membranes at their anchorages to various specialized cuticle regions, but thin membranes elsewhere. The uterine epithelium also shows this asymmetry, with a very thick basement membrane where it anchors to the seam and alar cuticle, and a thin basement membrane elsewhere (shown in SEAM FIG5 at wormatlas.org/handbook/hypodermis/hypsupportseam.htm).
Laminin subunits are required for intact basement
membranes
We examined the laminin subunit requirements for basement membrane
structures by comparing basement membranes in wild-type and laminin
subunit mutants (Table 3).
Basement membranes where laminin
B is primarily localized are disrupted
in epi-1 mutants. For example, in severe alleles broken pieces of
thick membrane are occasionally found in the body cavity at midbody, perhaps
derived from the spermatheca. The final TEM appearance of these broken
membranes, as described below, are subject to secondary tissue displacement
after initial weakness of the basement membranes. Thus, when a tissue breaks
through its covering, the membrane pieces may snap back, fold, clump or become
distorted as viewed by EM. The thinner basement membranes surrounding the
epidermis, intestine and gonad show a wide variety of defects in all
epi-1 alleles. Multiple layers, large whorls and clumping of material
are very common in adult epi-1 animals
(Fig. 8). More rarely, the
membrane material forms a diffuse granular lumpy substance filling the
pseudocoelom. In epi-1 mutants, the thicker basement membrane
covering the pharynx does not show many disruptions. There are only rare
ruptures of pharyngeal basement membranes in embryos and none in adults,
although the entire pharynx is sometimes grossly twisted in adults. This
latter phenotype could be a secondary consequence of the disruption of
basement membranes elsewhere.
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Using electron microscopy we observe that embryonic lethality caused by
mutations in laminin subunit genes is primarily caused by improper
separation of tissues and/or detachment of cells
(Fig. 10). In addition, we
observe in larvae and adult epi-1 mutants areas where basement
membrane is ruptured and adjoining tissues are adherent, indicating places
where tissues can not slide past each other. In adults, failure of the sheath
cells to cover the developing gonad leads to germ cells breaking through the
lamina and invading neighboring tissues
(Fig. 11B), and producing germ
cells free in the pseudocoelom (Fig.
8). In lam-3 mutants, cell bodies of both muscle cells
and marginal cells from the pharynx are sometimes displaced into the
surrounding tissue. Despite this dramatic displacement of cell bodies, all the
pharyngeal cells remain connected to the lumen of the pharynx. On their
lumenal surface, as in wild type, apical adherens junctions are found
connecting adjacent cells (Fig.
9C).
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Germ cell invasion of neighboring tissues is a characteristic phenotype in mutants that manage to develop to later stages (Fig. 11B). These cells inappropriately remain in mitosis and proliferate within the neighboring tissues. This proliferation defect causes a gross enlargement of the midbody in adult animals. This is a predominant phenotype observed in the rh165 strain.
In the mutants, striking defects in cell and axon outgrowth are observed,
apparently owing to misguidance along broken or misassembled basement
membrane. This phenotype is easily observed in the more active and more
fertile mutants, particularly with the temperature sensitive allele
rh191. All longitudinal nerves show occasional defects in final
positions, and the ventral nerve cord often wanders from its normal position
at the ventral midline (Fig.
11D). This phenotype was also observed by Forrester and Garriga
(Forrester and Garriga, 1997).
Interestingly, individual axons can become surrounded by separate sheets of
basement membrane and leave the fascicle. Axons may also defasciculate in
regions where the basement membrane forms clumps rather than sheets. Such
errors probably cause defects in synaptic connectivity, although we have not
tried to reconstruct the nerve circuits. The rh191 allele frequently
retains cuticle on the tail, perhaps owing to difficulties in molting.
All of the weak or moderate alleles may show `strong phenotypes' at low
frequencies. Alleles such as rh92 and rh152 are almost
normal in fertility, but still show many tissue defects, including some
disorganization of the gonad, extracellular accumulation of yolk granules and
whorls of material (presumed to be basement membranes), milder muscle defects
and occasional guidance errors by touch dendrites and excretory canals.
Although the gonad sheath cells usually cover the gonad successfully in these
alleles, their processes show irregular folds where they oppose the basement
membrane, and sheath cell somata fail to flatten normally and may contain
whorls of membranous material (data not shown). Excess yolk accumulates in the
pseudocoelom, possibly owing to poor development of the oocytes, which
normally take up yolk only at maturity, or possibly owing to failure to form
sheath pores that admit yolk from the pseudocoelom to the oocytes
(Hall et al., 1999;
Grant and Hirsh, 1999
). Weak
epi-1 alleles such as rh233 and rh27 show milder
versions of these same defects.
Finally, the allele rh233 is unique in that it has specific
effects on the migrations of the canals of the excretory cell. The excretory
cell is the largest mononucleate cell in the animal
(Buechner et al., 1999;
Nelson et al., 1983
). Its cell
body is positioned ventrally near the terminal bulb of the pharynx. Two arms
of the cell, the canals, extend laterally along the length of the animal.
Frequently, in the mutants, these canals are ventrally mispositioned.
Sometimes the canals are shortened or both canals travel along the same side.
Similar excretory guidance defects are observed in other alleles, but less
frequently.
The reconstructions of lam-3 mutants also demonstrate the importance of laminin in regulating cell polarity. Although wild-type pharynx cells show radial organization, with myofilaments or intermediate filaments oriented between apical and basal membranes, in lam-3 animals these filaments become disordered, with some running to the lateral membranes in the pharyngeal muscle cells and marginal cells, respectively (Fig. 9C). We also observe adherens junctions between pharyngeal cells at abnormal locations (Fig. 9D) and greatly increased basal cell membrane surrounded by extracellular matrix, resulting in very little lateral membrane and little cell-cell contact (Fig. 9E). These results suggest that in lam-3 mutants the apicobasal polarity of the pharyngeal cells is compromised, as well as the ability to maintain or establish lateral identity.
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DISCUSSION |
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The C. elegans subunits are members of
phylogenetically conserved protein families
The C. elegans Sequencing Consortium has revealed only two
subunits and a ß and a
subunit predicting that only
Aß
and
Bß
laminin heterotrimers are
present. Alternatively spliced forms of laminin ß or
subunit have
not been detected by RT-PCR (C.-c.H., D.H.H., E.M.H., G.K., V.K., B.E.V.,
H.H., A.D.C., P.D.Y. and W.G.W., unpublished). Overall, the size and primary
structure of laminin
subunits are conserved between phyla. We find
that the C. elegans laminin
A chain is typical with two
exceptions: there are only four LG domains instead of the usual five and there
are an additional two LE modules (10 instead of eight). In
Drosophila, two
subunits and a ß and
subunit
have been described (Chi and Hui,
1988
; Chi and Hui,
1989
; Chi et al.,
1991
; Garcia-Alonso et al.,
1996
; Haag et al.,
1999
; Henchcliffe et al.,
1993
; Kusche-Gullberg et al.,
1992
; Martin et al.,
1999
; Montell and Goodman,
1988
; Montell and Goodman,
1989
; Yarnitzky and Volk,
1995
). One of the Drosophila
subunits is similar
to the C. elegans
A subunit and the vertebrate
1 and
2 subunits, whereas the other is similar to the C. elegans
B subunit and to the vertebrate
3, and
5 subunits
(Martin et al., 1999
). In
general, the
B-like laminins are the most widely expressed of the
laminin
subunits, whereas the
A-like laminins appear to have
more restricted expression patterns
(Martin et al., 1999
;
Miner et al., 1995
;
Miner et al., 1997
). The
laminin
subunits of C. elegans and Drosophila appear
during gastrulation, suggesting a common requirement for having different
laminin
subunits during early development.
Each laminin subunit is segregated to different cell
surfaces
Our study reveals early events that lead to the assembly of basement
membranes in vivo. Both laminin subunit genes are apparently expressed
under the control of signals that initiate and regulate gastrulation. Gene
expression is first detected in the nuclei of cells that are ingressing
through a furrow along the ventral midline and, as the tissue layers begin to
be organized, cytoplasmic RNA is detected. At this time, the gene encoding
laminin
A, lam-3, is expressed in pharyngeal and epidermal
cells, and weakly in intestinal cells, whereas the gene encoding laminin
B, epi-1, is expressed in intestinal, pharyngeal and myoblast
cells. Both laminin
subunit proteins are then deposited between the
tissue layers. Near the end of embryogenesis, laminin
subunit gene
expression changes, the laminin
A gene being expressed most notably in
the pharynx and the laminin
B gene in the muscle cells.
The distribution of the different laminin subunits is probably a
cell-surface receptor-mediated process. Although both laminin proteins are
secreted between tissue layers during gastrulation, they do not
indiscriminately assemble. Rather, each subunit is distributed in a different
pattern to cell surfaces and, furthermore, they are not necessarily associated
with the cells that express the subunit
(Table 1). The staining pattern
of laminin A along the nerve tracts is revealing because the basement
membrane associated with the nerve tracts is not morphologically distinguished
from other regions of the epidermal basement membrane. We hypothesize that
laminin
A is concentrated at neuronal cell surfaces by specific
cell-surface receptor(s) and that the laminin
A containing trimer mixes
with the
B trimer in the basement membrane at these locations. The
association of laminin
A even when the axons are mispositioned supports
this conclusion. Also revealing is the finding that even when the two laminins
might appear to be able to intermingle, such as where the pharynx and body
wall basement membranes are juxtaposed, they in fact remain separated,
indicating that each is anchored to a particular architecture.
The laminin subunits associate with cell surfaces before the
reported expression of other basement membrane components and they are
required to assemble stable basement membranes. Evidence from other systems
also suggests that early laminin expression is essential for further basement
membrane assembly. For example, antisense experiments that disrupt laminin
subunit expression in Caco2 epithelial cells blocks laminin secretion
and prevents the subepithelial accumulation of entactin/nidogen and type IV
collagen, and the formation of a basement membrane
(De Arcangelis et al., 1996
).
In addition, the laminin
1 knockout arrests at peri-implantation and
neither the embryos nor derived embryoid bodies form basement membrane
(Aurelio et al., 2002
;
Smyth et al., 1999
). In both
these cases other basement membrane proteins are detected but only as
disorganized extracellular deposits.
Like the laminin subunits, other extracellular matrix proteins in
C. elegans also localize to different basement membranes and are not
necessarily associated with the cells that expressed the protein
(Graham et al., 1997
;
Kang and Kramer, 2000
;
Kim and Wadsworth, 2000
). This
suggests that cell surface-associated molecules are required for the assembly
of the extracellular matrix proteins into basement membranes
(Graham et al., 1997
).
Collagen IV localizes to all basement membranes except those between the
pseudocoelomic cavity and the body wall muscles or the epidermis
(Graham et al., 1997
). Nidogen
(entactin), which can bind both collagen IV and laminin with high affinity
(Fox et al., 1991
), is
associated with muscle cells as the embryo begins to elongate and subsequently
is detected at the pharynx, intestine and gonad primordia
(Kang and Kramer, 2000
;
Kim and Wadsworth, 2000
). In
larvae and adults, nidogen is detected in most basement membranes, but is most
strongly detected around the nerve ring and developing gonad. It becomes
concentrated at the edges of muscle quadrants and on the sublateral nerves,
which run longitudinally along the center margin of the muscle quadrants. This
staining pattern is different from either laminin
subunit, although
there are striking similarities to the laminin
A pattern in regards to
nervous system staining. Nidogen is found at all locations where collagen IV
localizes. The C. elegans homolog of mammalian perlecan, a heparan
sulfate proteoglycan that binds nidogen/entactin and laminin, plays an
essential role in muscle development has been shown by antibody staining to
localize to basement membranes at the bodywall and anal muscles, and at the
pharynx and gonad (Rogalski et al.,
1993
). Perlecan may be produced by epidermal cells and recruited
to the body wall muscles cells (Spike et
al., 2002
).
Our results suggest that although laminin may associate with many different surfaces of cells, it is normally excluded from doing so. In lam-3 mutants, an inappropriate matrix can accumulate between the pharyngeal cells, presumably because defective adhesions between the pharyngeal cells allow laminin inappropriate access to lateral surfaces. Where body wall basement membrane is defective in epi-1 mutants, growth cones, which normally migrate between the body wall basement membrane and the basal surface of the epidermis, may become inadvertently exposed to secreted non-polymerized laminin. As a result, laminin is inappropriately assembled at exposed surfaces all around the axons.
The laminin subunits are required to control cell-cell
adhesions between tissues
Basement membranes are observed to separate different tissues. The basement
membrane appears to distinguish discrete (basal) borders between specialized
tissues, but is generally excluded from any spaces formed by the lateral
surfaces of adjacent cells within a tissue, perhaps permitting these cells to
stick tightly to their neighbors. In a few locations, two neighboring tissues
may make close approaches and need to stick together. In these locales, their
basement membranes appear to be able to fuse end-to-end (as at the joining of
the pharynx to its valve or at the joining of the DTC to the germline) or to
fuse face-to-face (as at neuromuscular junctions of the nerve ring and the
longitudinal nerve cords, where the parallel membranes of the nerves and of
the somatic muscles seem to become one layer at the point of neuromuscular
contacts). In all other locales, it is notable that each tissue is bounded by
its own basal lamina, perhaps to permit easy sliding of one tissue past
another, as during the flexion of the nematode body. Preventing cell-cell
adhesions between tissues is critical. For example, where body wall muscles
face the cuticle, the basement membranes form an integral link from muscle to
epidermis and to cuticle. During normal body flexions when the nematode moves
through the environment, most other internal adhesions must be minimized or,
as in the case of epi-1 mutants, the animal will be paralyzed.
Laminin forms a network between the body wall muscle cell surface and the
basal surface of the epidermis. Our results suggest that this is important for
forming the apparatus that physically link the muscle, basement membrane and
the overlying epidermis and cuticle. This apparatus is necessary for
transferring contractile force. In the muscle, the myofilament lattice is just
below the cell surface and is anchored to the muscle cell membrane and
adjacent basement membrane by integrin-containing dense bodies and M-line
complexes. In turn, the complexes are linked through the basement membrane to
the intermediate filament arrays that extend across the compacted epidermis
and attach to the cuticle (Francis and
Waterston, 1991; Hresko et
al., 1999
; Moerman and Fire,
1997
).
In epi-1 mutants, dense bodies are missing and can ectopically
assemble, the myofilament lattice is disrupted, and the overlying epidermis
may not compact. These observations suggest that laminin B is required
for properly organizing the receptor complexes and filament arrays of muscle
and epidermis. During early body wall muscle development, myofibrillar
components accumulate at membranes adjacent to the epidermis and other muscle
cells, whereas in the epidermis hemidesmosome components become restricted to
regions adjacent to the muscle cells
(Hresko et al., 1994
). After
this polarization, integrin-containing cell-matrix adhesion complexes appear,
followed by the assembly of the highly ordered dense body and M-line
structures. Thick filaments associate with the M-line and thin filaments with
the dense bodies (Barstead and Waterston,
1991
; Gettner et al.,
1995
; Hresko et al.,
1994
; Rogalski et al.,
1993
; Waterston,
1989
). The myofilaments run longitudinally in the animal, while
the intermediate filaments in the epidermis become organized in
circumferentially oriented bands. The assembly of the dense body and M-line
components requires the extracellular matrix protein UNC-52/perlecan and
UNC-112, an intracellular protein that colocalizes with integrin at the
cell-matrix adhesion complexes (Francis
and Waterston, 1991
; Rogalski
et al., 2000
; Rogalski et al.,
1993
).
The ectopic assembly of dense bodies in epi-1 mutants suggest that
laminin is required during the early step of muscle cell polarization. This is
consistent with observations that laminin B is present before the
adhesion complexes are assembled and before UNC-52/perlecan and the muscle
integrins are functionally required for muscle development. Defective
polarization may not preclude assembly of adhesion complexes if the signals
responsible for driving the assembly are still present.
The lam-3 phenotypes suggest laminin is required to polarize cells
and to organize adhesion complexes. In the mutants, adherens junctions are
positioned at inappropriate cell surfaces of the pharyngeal cells. Current
models predict that epithelial cell polarity is initiated by contact between
cells or between a cell and the extracellular matrix
(Yeaman et al., 1999). These
contacts trigger the assembly of cytoskeletal and signaling networks at the
contact sites and the establishment of an apical membrane domain at the
non-contacting surface. In a process that is not well understood, global
changes in the organization of the cytoskeleton are induced. Microtubules are
redistributed into long bundles along the apicobasal axis of the cell. The
mechanism regulating the orientation of the microtubule assembly is not known,
although linkages to the cytoskeletal networks at the lateral and basal
membranes or to the actin cytoskeleton assembly at the apical membrane domain
have been suggested. There is evidence that suggests cell adhesion to the
extracellular matrix is important for organizing the apicobasal axis. In
Madin-Darby canine kidney (MDCK) culture, tight junctions become localized to
the apicolateral membrane boundary only after extracellular matrix accumulates
on one side of the cells (Wang et al.,
1990
). The tight junctions and the axis of polarity can be
reoriented by exposing a different surface to extracellular matrix
(Wang et al., 1990
). In the
lam-3 mutant, all pharyngeal cells form mature apical adherens
junctions and remain attached to the lumen, indicating that apical membrane
domains are established and maintained. However, the adherens junctions
forming at abnormal locations suggests that the loss of contact sites because
of the absence of laminin
A might establish other `non-contacting
surfaces' and, in turn, induced the formation of apical-like membrane domains.
As a result, some cytoplasmic filaments orient relative to these domains,
instead of perpendicular to the lumenal surface.
We observe other cases as well where epithelial cells fail to form the
specialized structures of the apical and basolateral domains. For example, the
spermatheca will not form a closed lumen. Furthermore, the accumulation of
yolk and the molting defects of some epi-1 mutants are associated
with the improper uptake or secretion of proteins from mature epithelial
cells. In the most severe events, cells may fail to assemble into tissues at
all. In mutant embryos with a predicted laminin null allele, some
cells were seen to lose contact with all neighbors and round up into isolated
spherical units. The inability of cells to polarize and correctly adhere to
their neighbors probably accounts for the embryonic lethality of
epi-1 null animals.
The laminin subunits are required for inductive
signaling
A dramatic case where cellular development is altered occurs when the gonad
sheath cells fail to enclose the germline. The normal proliferation and
differentiation of the germ cells requires cell-cell signaling from elements
of the somatic gonad (Austin and Kimble,
1987; Buechner et al.,
1999
). Once the developing germline is unsheathed, the germ cells
can remain in mitosis and create a vigorous pathological phenotype, in which
immature germ cells invade and multiply within other tissues. In strong
epi-1 alleles, dividing germ cells are observed in all regions of the
body from head to tail.
Cell and axon migrations also require laminin. In C. elegans,
axons migrate between the basement membranes and the epidermis, whereas
mesodermal cells migrate on the opposite side of the basement membrane
(Hedgecock et al., 1990;
Hedgecock et al., 1987
).
Extracellular cues, such as the laminin-related guidance molecule, netrin
UNC-6 are thought to be associated with the basement membrane
(Wadsworth et al., 1996
). In
the epi-1 mutants, both cell and axon migrations are disrupted (see
also Forrester and Garriga,
1997
). In most cases this may be the result of the physical
disruption of the basement membrane; however, it is also possible that some
alleles of epi-1 interfere with the ability of guidance cues or
guidance receptor molecules to interact with basement membrane components.
This may be the case for epi-1(rh233), which specifically affects the
migrations of the excretory cell canal processes.
A model for laminin function
It is proposed that receptor-facilitated laminin self-assembly sets up a
supramolecular architecture that is necessary for organizing adhesion and cell
signaling between adjacent tissues
(Colognato et al., 1999;
Colognato and Yurchenco,
2000
). This model predicts dynamic reorganization involving cell
receptors, intracellular proteins and the extracellular matrix. We show that
laminin associates with cell surfaces in a process that is probably receptor
mediated and occurs before the assembly of a prototypical basement membrane.
These observations, coupled with the mutant phenotypes that show disorganized
basement membranes, receptor complexes and cytoskeletal components, provide in
vivo evidence for this model. A particularly attractive system for further
study is the role of laminin in the development of the body wall muscle
attachments. We are struck by the similarity between the polygonal arrays
observed on the body wall muscles and myotube surfaces in culture. In C.
elegans, laminin polymerization between the surfaces of the epidermis and
muscle cells might be a key event for the organization of muscle
integrin-containing receptor complexes, extracellular matrix components and
epidermal hemidesmosomes into a supramolecular configuration that spans the
tissues.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Aberdam, D., Galliano, M. F., Vailly, J., Pulkkinen, L., Bonifas, J., Christiano, A. M., Tryggvason, K., Uitto, J., Epstein, E. H., Jr, Ortonne, J. P. et al. (1994). Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5). Nat. Genet. 6, 299-304.[Medline]
Albertson, D. G. and Thomson, J. N. (1976). The pharynx of Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 275,299 -325.[Medline]
Aurelio, O., Hall, D. H. and Hobert, O. (2002).
Immunoglobulin-domain proteins required for maintenance of ventral nerve cord
organization. Science
295,686
-690.
Austin, J. and Kimble, J. (1987). glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans. Cell 51,589 -599.[Medline]
Barstead, R. J. and Waterston, R. H. (1991). Vinculin is essential for muscle function in the nematode. J. Cell Biol. 114,715 -724.[Abstract]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Buechner, M., Hall, D. H., Bhatt, H. and Hedgecock, E. M. (1999). Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell. Dev. Biol. 214,227 -241.[CrossRef][Medline]
Burgeson, R. E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinman, H., Martin, G. R., Meneguzzi, G., Paulsson, M., Sanes, J. et al. (1994). A new nomenclature for the laminins. Matrix Biol. 14,209 -211.[CrossRef][Medline]
Chi, H. C. and Hui, C. F. (1988). cDNA and amino acid sequences of Drosophila laminin B2 chain. Nucleic Acids Res. 16,7205 -7206.[Medline]
Chi, H. C. and Hui, C. F. (1989). Primary
structure of the Drosophila laminin B2 chain and comparison with human, mouse,
and Drosophila laminin B1 and B2 chains. J. Biol.
Chem. 264,1543
-1550.
Chi, H. C., Juminaga, D., Wang, S. Y. and Hui, C. F. (1991). Structure of the Drosophila gene for the laminin B2 chain. DNA Cell Biol. 10,451 -466.[Medline]
Colognato, H., Winkelmann, D. A. and Yurchenco, P. D.
(1999). Laminin polymerization induces a receptor-cytoskeleton
network. J. Cell Biol.
145,619
-631.
Colognato, H. and Yurchenco, P. D. (2000). Form and function: the laminin family of heterotrimers. Dev. Dyn. 218,213 -234.[CrossRef][Medline]
De Arcangelis, A., Neuville, P., Boukamel, R., Lefebvre, O., Kedinger, M. and Simon-Assmann, P. (1996). Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation. J. Cell Biol. 133,417 -430.[Abstract]
Deng, W. M. and Ruohola-Baker, H. (2000). Laminin A is required for follicle cell-oocyte signaling that leads to establishment of the anterior-posterior axis in Drosophila. Curr. Biol. 10,683 -686.[CrossRef][Medline]
Finney, M. and Ruvkun, G. (1990). The unc-86 gene product couples cell lineage and cell identity in C. elegans. Cell 63,895 -905.[Medline]
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391,806 -811.[CrossRef][Medline]
Forrester, W. C. and Garriga, G. (1997). Genes
necessary for C. elegans cell and growth cone migrations.
Development 124,1831
-1843.
Fox, J. W., Mayer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H., Mann, K., Timpl, R., Krieg, T., Engel, J. et al. (1991). Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV. EMBO J. 10,3137 -3146.[Abstract]
Francis, R. and Waterston, R. H. (1991). Muscle cell attachment in Caenorhabditis elegans. J. Cell Biol. 114,465 -479.[Abstract]
Garcia-Alonso, L., Fetter, R. D. and Goodman, C. S.
(1996). Genetic analysis of Laminin A in Drosophila:
extracellular matrix containing laminin A is required for ocellar axon
pathfinding. Development
122,2611
-2621.
Gettner, S. N., Kenyon, C. and Reichardt, L. F. (1995). Characterization of beta pat-3 heterodimers, a family of essential integrin receptors in C. elegans. J. Cell Biol. 129,1127 -1141.[Abstract]
Graham, P. L., Johnson, J. J., Wang, S., Sibley, M. H., Gupta,
M. C. and Kramer, J. M. (1997). Type IV collagen is
detectable in most, but not all, basement membranes of Caenorhabditis elegans
and assembles on tissues that do not express it. J. Cell
Biol. 137,1171
-1183.
Grant, B. and Hirsh, D. (1999).
Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte.
Mol. Biol. Cell 10,4311
-4326.
Guo, S. and Kemphues, K. J. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81,611 -620.[Medline]
Haag, T. A., Haag, N. P., Lekven, A. C. and Hartenstein, V. (1999). The role of cell adhesion molecules in Drosophila heart morphogenesis: faint sausage, shotgun/DE-cadherin, and laminin A are required for discrete stages in heart development. Dev. Biol. 208, 56-69.[CrossRef][Medline]
Hall, D. H. (1995). Electron microscopy and three-dimensional image reconstruction. Methods Cell Biol. 48,395 -436.[Medline]
Hall, D. H., Winfrey, V. P., Blaeuer, G., Hoffman, L. H., Furuta, T., Rose, K. L., Hobert, O. and Greenstein, D. (1999). Ultrastructural features of the adult hermaphrodite gonad of Caenorhabditis elegans: relations between the germline and the soma. Dev. Biol. 212,101 -123.[CrossRef][Medline]
Hedgecock, E. M., Culotti, J. G., Hall, D. H. and Stern, B. D. (1987). Genetics of cell and axon migrations in Caenorhabditis elegans. Development 100,365 -382.[Abstract]
Hedgecock, E. M., Culotti, J. G. and Hall, D. H. (1990). The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 4,61 -85.[Medline]
Helbling-Leclerc, A., Zhang, X., Topaloglu, H., Cruaud, C., Tesson, F., Weissenbach, J., Tome, F. M., Schwartz, K., Fardeau, M., Tryggvason, K. et al. (1995). Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy. Nat. Genet. 11,216 -218.[Medline]
Henchcliffe, C., Garcia-Alonso, L., Tang, J. and Goodman, C.
S. (1993). Genetic analysis of laminin A reveals diverse
functions during morphogenesis in Drosophila.
Development 118,325
-337.
Hirsh, D., Oppenheim, D. and Klass, M. (1976). Development of the reproductive system of Caenorhabditis elegans. Dev. Biol. 49,200 -219.[Medline]
Hresko, M. C., Williams, B. D. and Waterston, R. H. (1994). Assembly of body wall muscle and muscle cell attachment structures in Caenorhabditis elegans. J. Cell Biol. 124,491 -506.[Abstract]
Hresko, M. C., Schriefer, L. A., Shrimankar, P. and Waterston,
R. H. (1999). Myotactin, a novel hypodermal protein involved
in muscle-cell adhesion in Caenorhabditis elegans. J. Cell
Biol. 146,659
-672.
Hutter, H., Vogel, B. E., Plenefisch, J. D., Norris, C. R.,
Proenca, R. B., Spieth, J., Guo, C., Mastwal, S., Zhu, X., Scheel, J. et
al. (2000). Conservation and novelty in the evolution of cell
adhesion and extracellular matrix genes. Science
287,989
-994.
Iivanainen, A., Sainio, K., Sariola, H. and Tryggvason, K. (1995). Primary structure and expression of a novel human laminin alpha 4 chain. FEBS Lett. 365,183 -188.[CrossRef][Medline]
Kang, S. H. and Kramer, J. M. (2000). Nidogen
is nonessential and not required for normal type IV collagen localization in
Caenorhabditis elegans. Mol. Biol. Cell
11,3911
-3923.
Kim, S. and Wadsworth, W. G. (2000).
Positioning of longitudinal nerves in C. elegans by nidogen.
Science 288,150
-154.
Kimble, J. and Hirsh, D. (1979). The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Dev. Biol. 70,396 -417.[Medline]
Kusche-Gullberg, M., Garrison, K., MacKrell, A. J., Fessler, L. I. and Fessler, J. H. (1992). Laminin A chain: expression during Drosophila development and genomic sequence. EMBO J. 11,4519 -4527.[Abstract]
Kuster, J. E., Guarnieri, M. H., Ault, J. G., Flaherty, L. and Swiatek, P. J. (1997). IAP insertion in the murine LamB3 gene results in junctional epidermolysis bullosa. Mamm. Genome 8,673 -681.[CrossRef][Medline]
Leung, B., Hermann, G. J. and Priess, J. R. (1999). Organogenesis of the Caenorhabditis elegans intestine. Dev. Biol. 216,114 -134.[CrossRef][Medline]
Martin, D., Zusman, S., Li, X., Williams, E. L., Khare, N.,
DaRocha, S., Chiquet-Ehrismann, R. and Baumgartner, S.
(1999). wing blister, a new Drosophila laminin alpha
chain required for cell adhesion and migration during embryonic and imaginal
development. J. Cell Biol.
145,191
-201.
McGrath, J. A., Kivirikko, S., Ciatti, S., Moss, C., Dunnill, G. S., Eady, R. A., Rodeck, C. H., Christiano, A. M. and Uitto, J. (1995). A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in Herlitz junctional epidermolysis bullosa: prenatal exclusion in a fetus at risk. Genomics 29,282 -284.[CrossRef][Medline]
Mello, C. and Fire, A. (1995). DNA transformation. Methods Cell Biol. 48,451 -482.[Medline]
Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10,3959 -3970.[Abstract]
Miller, D. M., 3rd, Ortiz, I., Berliner, G. C. and Epstein, H. F. (1983). Differential localization of two myosins within nematode thick filaments. Cell 34,477 -490.[Medline]
Miner, J. H., Lewis, R. M. and Sanes, J. R.
(1995). Molecular cloning of a novel laminin chain, alpha 5, and
widespread expression in adult mouse tissues. J. Biol.
Chem. 270,28523
-28526.
Miner, J. H., Patton, B. L., Lentz, S. I., Gilbert, D. J.,
Snider, W. D., Jenkins, N. A., Copeland, N. G. and Sanes, J. R.
(1997). The laminin alpha chains: expression, developmental
transitions, and chromosomal locations of alpha1-5, identification of
heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.
J. Cell Biol. 137,685
-701.
Miner, J. H., Cunningham, J. and Sanes, J. R.
(1998). Roles for laminin in embryogenesis: exencephaly,
syndactyly, and placentopathy in mice lacking the laminin alpha5 chain.
J. Cell Biol. 143,1713
-1723.
Moerman, D. and Fire, A. (1997). Muscle: structure, function, and development. In C. elegans II (ed. D. Riddle T. Blumenthal B. Meyer and J. Priess), pp.417 -470. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Mohler, W. A., Simske, J. S., Williams-Masson, E. M., Hardin, J. D. and White, J. G. (1998). Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis. Curr. Biol. 8,1087 -1090.[Medline]
Montell, D. J. and Goodman, C. S. (1988). Drosophila substrate adhesion molecule: sequence of laminin B1 chain reveals domains of homology with mouse. Cell 53,463 -473.[Medline]
Montell, D. J. and Goodman, C. S. (1989). Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis. J. Cell Biol. 109,2441 -2453.[Abstract]
Nelson, F. K., Albert, P. S. and Riddle, D. L. (1983). Fine structure of the Caenorhabditis elegans secretory-excretory system. J. Ultrastruct. Res. 82,156 -171.[Medline]
Noakes, P. G., Gautam, M., Mudd, J., Sanes, J. R. and Merlie, J. P. (1995). Aberrant differentiation of neuromuscular junctions in mice lacking s-laminin/laminin beta 2. Nature 374,258 -262.[CrossRef][Medline]
Parsons, M. J., Campos, I., Hirst, E. M. and Stemple, D. L. (2002). Removal of dystroglycan causes severe muscular dystrophy in zebrafish embryos. Development 129,3505 -3512.[Medline]
Paupard, M. C., Miller, A., Grant, B., Hirsh, D. and Hall, D.
H. (2001). Immuno-EM localization of GFP-tagged yolk proteins
in C. elegans using microwave fixation. J. Histochem.
Cytochem. 49,949
-956.
Rocheleau, C. E., Downs, W. D., Lin, R., Wittmann, C., Bei, Y., Cha, Y. H., Ali, M., Priess, J. R. and Mello, C. C. (1997). Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell 90,707 -716.[CrossRef][Medline]
Rogalski, T. M., Williams, B. D., Mullen, G. P. and Moerman, D. G. (1993). Products of the unc-52 gene in Caenorhabditis elegans are homologous to the core protein of the mammalian basement membrane heparan sulfate proteoglycan. Genes Dev. 7,1471 -1484.[Abstract]
Rogalski, T. M., Mullen, G. P., Gilbert, M. M., Williams, B. D.
and Moerman, D. G. (2000). The UNC-112 gene in Caenorhabditis
elegans encodes a novel component of cell-matrix adhesion structures required
for integrin localization in the muscle cell membrane. J. Cell
Biol. 150,253
-264.
Seydoux, G. and Fire, A. (1995). Whole-mount in situ hybridization for the detection of RNA in Caenorhabditis elegans embryos. Methods Cell Biol. 48,323 -337.[Medline]
Smyth, N., Vatansever, H. S., Murray, P., Meyer, M., Frie, C.,
Paulsson, M. and Edgar, D. (1999). Absence of basement
membranes after targeting the LAMC1 gene results in embryonic lethality due to
failure of endoderm differentiation. J. Cell Biol.
144,151
-160.
Spike, C. A., Davies, A. G., Shaw, J. E. and Herman, R. K.
(2002). MEC-8 regulates alternative splicing of unc-52
transcripts in C. elegans hypodermal cells.
Development 129,4999
-5008.
Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100,64 -119.[Medline]
Sunada, Y., Bernier, S. M., Kozak, C. A., Yamada, Y. and
Campbell, K. P. (1994). Deficiency of merosin in dystrophic
dy mice and genetic linkage of laminin M chain gene to dy locus. J.
Biol. Chem. 269,13729
-13732.
Wadsworth, W. G., Bhatt, H. and Hedgecock, E. M. (1996). Neuroglia and pioneer neurons express UNC-6 to provide global and local netrin cues for guiding migrations in C. elegans. Neuron 16,35 -46.[Medline]
Wang, A. Z., Ojakian, G. K. and Nelson, W. J. (1990). Steps in the morphogenesis of a polarized epithelium. II. Disassembly and assembly of plasma membrane domains during reversal of epithelial cell polarity in multicellular epithelial (MDCK) cysts. J. Cell Sci. 95,153 -165.[Abstract]
Waterston, R. H. (1989). The minor myosin heavy chain, mhcA, of Caenorhabditis elegans is necessary for the initiation of thick filament assembly. EMBO J. 8,3429 -3436.[Abstract]
White, J. G., Southgate, E., Thompson, J. N. and Brenner, S. (1976). The structure of the ventral nerve cord of Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 275,327 -348.[Medline]
White, J., Southgate, E., Thompson, J. and Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 314,1 -340.
Williams, B. D., Schrank, B., Huynh, C., Shownkeen, R. and
Waterston, R. H. (1992). A genetic mapping system in
Caenorhabditis elegans based on polymorphic sequence-tagged sites.
Genetics 131,609
-624.
Xu, H., Wu, X. R., Wewer, U. M. and Engvall, E. (1994). Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene. Nat. Genet. 8, 297-302.[Medline]
Yarnitzky, T. and Volk, T. (1995). Laminin is required for heart, somatic muscles, and gut development in the Drosophila embryo. Dev. Biol. 169,609 -618.[CrossRef][Medline]
Yeaman, C., Grindstaff, K. K. and Nelson, W. J.
(1999). New perspectives on mechanisms involved in generating
epithelial cell polarity. Physiol. Rev.
79, 73-98.
Zhu, X., Joh, K., Hedgecock, E. and Hori, K.
(2000). Identification of Epi-1 locus as a laminin chain
gene in the nematode Caenorhabditis elegans and characterization of Epi-1
mutant alleles. DNA Seq.
10, 1-11.