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doi: 10.1242/10.1242/dev.00481
subunits and their role in C. elegans development
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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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß/
) glycoproteins that
form a major polymer within basement membranes. Different , ß and
subunits can assemble into various laminin isoforms that have
different, but often overlapping, distributions and functions. In this study,
we examine the contributions of the laminin subunits to the
development of C. elegans. There are two , one ß and one
laminin subunit, suggesting two laminin isoforms that differ by their
subunit assemble in C. elegans. We find that near the end of
gastrulation and before other basement membrane components are detected, the
subunits are secreted between primary tissue layers and become
distributed in different patterns to the surfaces of cells. Mutations in
either subunit gene cause missing or disrupted extracellular matrix
where the protein normally localizes. Cell-cell adhesions are abnormal: in
some cases essential cell-cell adhesions are lacking, while in other cases,
cells inappropriately adhere to and invade neighboring tissues. Using electron
microscopy, we observe adhesion complexes at improper cell surfaces and
disoriented cytoskeletal filaments. Cells throughout the animal show defective
differentiation, proliferation or migration, suggesting a general disruption
of cell-cell signaling. The results suggest a receptor-mediated process
localizes each secreted laminin to exposed cell surfaces and that laminin is
crucial for organizing extracellular matrix, receptor and intracellular
proteins at those surfaces. We propose this supramolecular architecture
regulates adhesions and signaling between adjacent tissues.
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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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, three
laminin ß and three laminin subunits are known in vertebrates and
over 12 heterotrimeric laminin isoforms are thought to be assembled
(Burgeson et al., 1994
;
Iivanainen et al., 1995;
Miner et al., 1995). In
vertebrates and invertebrates, laminin isoforms are widely distributed and a
variety of phenotypes have been associated with the disruption of different
laminins. For example, laminin 2 mutations are found in some congenital
muscular dystrophies (Helbling-Leclerc et
al., 1995; Sunada et al.,
1994; Xu et al.,
1994); laminin 3, ß3 or 2 mutations are found
in junctional epidermolysis bullosa, a skin blistering disease
(Aberdam et al., 1994;
Kuster et al., 1997;
McGrath et al., 1995); and
mutations of the laminin ß2 chain gene disrupts neuromuscular and renal
function (Noakes et al.,
1995). Multiple defects are observed in mice that lack the laminin
5 chain and the animals arrest late in embryogenesis
(Miner et al., 1998).
Mutations in zebrafish indicate that laminin ß and subunits are
important for notochord development
(Parsons et al., 2002). In
Drosophila, the two laminin subunits are required for
embryonic viability; mutants have defects in the morphogenesis of heart,
somatic muscle and trachea (Henchcliffe et
al., 1993; Martin et al.,
1999; Yarnitzky and Volk,
1995). Furthermore, there is evidence that one of the
Drosophila laminin subunits is required for the follicle
cell/oocyte signaling that establishes the anteroposterior axis of the
organism (Deng and Ruohola-Baker,
2000).
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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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Bristol strain
N2 was used as wild type. The strains used in this study are as follows. 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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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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genes, one
ß and one subunits are identified
(Zhu et al., 2000). Therefore,
two laminin isoforms each containing a different subunit are predicted
to form in C. elegans. The C. elegans laminin genes
lam-3 and epi-1 encode for laminin A and laminin
B, respectively (Fig.
1). A description of the isolation and sequencing of the
epi-1 gene and a sequence comparison of some alleles have been
presented (Hutter et al.,
2000). The laminin B protein has a predicted structure that
is similar to other reported laminin subunits. We analyzed
lam-3 mRNA and found that it is derived from 13 exons and is 9450
nucleotides long plus a SL1 trans-spliced leader and a poly A tail. We predict
that the lam-3 mRNA sequence encodes a protein, laminin A,
that is similar to the laminin 1 and laminin 2 subunits found in
other species, with the exception that that are only four LG domains instead
of the usual five, and that there are 10 LE modules instead of the usual
eight.
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subunits have different distribution
patterns subunits, chicken and
rabbit polyclonal antibodies were raised against laminin A and laminin
B fusion proteins. The antisera indicate that early expression of the
subunits occurs during gastrulation and that the subunits have
distinct distributions as early organogenesis proceeds
(Table 1). In C.
elegans, gastrulation begins at the 28-cell stage as intestinal precursor
cells move from the ventral surface into the interior
(Sulston et al., 1983). As
gastrulation proceeds, cell divisions give rise to a central cylinder of
intestinal and pharyngeal precursor cells surrounded by body myoblasts and an
outer cell layer. At this point cell proliferation largely ceases and the
embryo, a spheroid of about 550 cells, begins to elongate. During this stage
of morphogenesis, the organs develop into their mature form and the embryo
elongates about fourfold along its longitudinal axis.
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A is first detected between tissue layers near the end of
gastrulation (Fig. 2A) and then
becomes localized along the muscle cells as the embryo begins to elongate
(Fig. 2B). By the threefold
stage of elongation, staining along the muscle quadrants is weaker and becomes
restricted to a band at the center of each quadrant, which colocalizes with
the dorsal and ventral sublateral nerve tracts in the adult. Staining is
intense around the pharynx, pharyngeal-intestinal valve, and intestine during
morphogenesis (Fig. 2C). In
larvae and adults, the antiserum stains the spermatheca strongly
(Fig. 2D) and only weakly
stains the pharynx, the intestine and the excretory canal
(Fig. 2F). Laminin A is
also associated with the nervous system. During elongation and throughout the
rest of development, A is localized at the nerve ring, a bundle of
100 axons that encircles the outside of the pharynx, at the right
fascicle of the ventral nerve cord and at the sublateral nerves
(Fig. 2C,E).
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A association with the nervous system is interesting because
nerves are sandwiched between the epidermis and the overlying basement
membrane. Using electron microscopy, the membrane overlying the nerves is
indistinguishable from the adjacent membrane. To examine whether laminin
A is localized by interactions that involve neurons or whether it
localizes to the nerve pathways regardless of whether the axons are present,
we examined the distribution of laminin A in nid-1(ur41)
animals (Fig. 3). This mutation
causes the dorsal sublateral axons to migrate along the dorsal midline and
axons of the right ventral nerve cord fascicle to migrate in the left fascicle
(Kim and Wadsworth, 2000). We
observe that Laminin A is localized with the mispositioned axons rather
than along the normal nerve pathways (Fig.
3D). This suggests that laminin A is localized to neuronal
cell surfaces by specific laminin A receptors.
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B antiserum shows that, like laminin
A, the B subunit accumulates between the primary tissue layers
near the completion of gastrulation (250 minutes)
(Fig. 4A). Laminin B
antiserum stains all the major basement membranes during the remainder of
embryogenesis and throughout larval development and in the adult
(Fig. 4B-E). Although laminin
B staining is weak in the basement membranes surrounding pharynx,
intestine, body wall muscle and epidermis, the staining is strong in the
basement membranes surrounding gonad, distal tip cell, vulval muscle,
intestinal muscle, anal depressor muscle and coelomocytes
(Fig. 4F-H). These basement
membranes are notable in that they are thick and appear to have mainly the
B-containing laminin isoform. Although the staining is diffuse within
most basement membranes, at the muscle/epidermis membrane a distinct pattern
is observed (Fig. 4B). On the
muscle surface, a grid-like network that comprises regularly spaced bands
running circumferentially and longitudinally is observed
(Fig. 5). The longitudinal
bands correspond to the thin-filament-containing (I-band) region of the muscle
myofilament lattice. In this region, thin filaments are anchored by dense body
structures that are in turn linked by transmembrane complexes to the basement
membrane (Moerman and Fire,
1997). Laminin B is also strongly detected at muscle-muscle
cell boundaries.
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subunits, we co-stained for both subunits using species-specific secondary
antibody conjugates. As the germ layers develop, both subunits are
deposited between the layers. However, the staining for laminin A is
most intense around the pharyngeal and intestinal precursor cells, whereas
staining for laminin B is most intense around the myoblast cells and
along epidermal cells (Fig.
6A). By the onset of elongation, distinct layers of laminin
A and laminin B staining can be distinguished, particularly
anteriorly between the developing pharynx and the body wall
(Fig. 6B). This indicates that
the segregation of the laminin isoforms begins early, before or as
organogenesis proceeds.
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subunits retain an
association with different basement membranes as elongation proceeds, we used
mutants homozygous for the deficiency mnDf90. In these animals, the
pharynx differentiates, but it does not attach to the buccal cavity, causing
the pharynx to become displaced from the anterior body wall (M. Portereiko and
S. E. Mango, personal communication). This allows the two basement membranes,
which in mature wild-type animals are juxtaposed, to be visualized separately.
We observe that the two subunits remain differentially localized after
elongation. Laminin A is localized to the pharyngeal basement membrane,
whereas laminin B is associated mainly with the body wall basement
membranes and only weakly with the pharyngeal basement membrane
(Fig. 6C). These results
indicate that each laminin subunit is segregated in the embryo to
different adjacent basement membranes and that each membrane retains its
unique subunit composition.
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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A, was not successful. However, promoter-GFP transgenes indicated that
lam-3 is expressed during gastrulation and through embryogenesis in
pharyngeal, intestinal and epidermal cells
(Fig. 7D). In the larvae,
lam-3 gene expression is maintained in the spermatheca
(Fig. 7E) and in the pharyngeal
m3-m8 and mc cells (Fig. 7F).
These results show that the laminin subunit genes have different
expression patterns and, together with the protein localization studies,
indicate that each subunit can localize to basement membranes not
associated with the cells that express the gene.
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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; White et al.,
1986; White et al.,
1976); however, a methodical comparison of membranes has not been
carried out. We compare here, using transmission electron microscopy (TEM) of
thinly sectioned wild-type adults, the distinctive basement membrane that
separate each major tissue (see Figs S4, S5 at
http://dev.biologists.org/supplemental/).
For example, the bodywall consists of two distinct parts, the somatic
musculature and the epidermal tissue. All muscle cells within a given quadrant
are surrounded by a single basement membrane, which covers their surfaces
without entering the spaces between neighboring cells. Similarly, the cells of
the epidermis, including all hypodermal cells, neurons, glial cells and the
excretory and gland cells are covered by a single layer of basement membrane,
which covers the entire epidermis without invading between cells. It is very
often possible to see a periodic series of dark dots, or `lollypops'
(terminology of J. White), which lie on the outer surface of the epidermal
basement membrane in transverse sections (facing away from the tissue of
origin), that seem to comprise a second layer to this very thin membrane. In
favorable oblique sections, these dots comprise thin parallel rods (about 2 nm
diameter) encircling the basement membrane (not shown). Their spacing is often
20 nm or 40 nm between dots or rods. Endodermal tissues are similarly covered
by a single basement membrane. Thus, the pharynx displays a very thick
basement membrane that covers basal surfaces of muscles, support cells, glands
and neurons without separating any of them. This thicker basement membrane
seems to consist of a single layer. Where the pharynx meets the intestine, the
thick basement membrane stretches to cover the pharyngeal/intestinal valve
cells on their basal surfaces, before merging into the thinner basement
membrane that covers the basal side of the intestine. Only a few other cells
display similar thick basement membranes, including the mature oocyte before
fertilization (Hall et al.,
1999), the uterine epithelium, and many specialized alimentary and
sex muscles where they oppose the cuticle (see below). The extracellular
material surrounding the oocytes in some other organisms is referred to as a
glycocalyx; this distinction might also be appropriate in C. elegans
once molecular compositions are known.
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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B mutants, the body wall
structure and most other tissues in the lam-3 laminin A
mutants appear normal by electron microscopy. However, in lam-3
mutants, the thick pharyngeal basement membrane has distinct gaps that permit
pharyngeal cells to adhere to surrounding tissues
(Fig. 9B). These results are in
agreement with the unique distribution patterns of each subunits, the
morphological distinctions between membranes, and the idea that each laminin
isoform contributes specific properties.
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subunits are required to prevent cells from adhering
to and invading neighboring tissues
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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subunits are required throughout development for cell
polarity, differentiation, proliferation and migrationGerm 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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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits play in the
development of C. elegans. Each subunit is distributed to
specific cell surfaces that are exposed between tissue layers near the end of
gastrulation. Based on epi-1 and lam-3 phenotypes, laminin
is necessary to assemble a stable basement membrane and for organizing
receptor complexes and cytoskeletal components to the proper cell surfaces. We
consider that these results are consistent with an idea that laminin can
organize extracellular matrix, receptor and cytoskeletal elements into a
supramolecular configuration that is crucial for regulating interactions
between adjacent tissues.
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 o
