Northwestern University Medical School, Department of Cell and Molecular Biology, Chicago, Illinois 60611
Type IV collagen in Caenorhabditis elegans
is produced by two essential genes, emb-9 and let-2,
which encode 1- and
2-like chains, respectively. The
distribution of EMB-9 and LET-2 chains has been characterized using chain-specific antisera. The chains colocalize, suggesting that they may function in a single heterotrimeric collagen molecule. Type IV collagen is
detected in all basement membranes except those on
the pseudocoelomic face of body wall muscle and on
the regions of the hypodermis between body wall muscle quadrants, indicating that there are major structural
differences between some basement membranes in C. elegans. Using lacZ/green fluorescent protein (GFP) reporter constructs, both type IV collagen genes were
shown to be expressed in the same cells, primarily body
wall muscles, and some somatic cells of the gonad. Although the pharynx and intestine are covered with
basement membranes that contain type IV collagen,
these tissues do not express either type IV collagen
gene. Using an epitope-tagged emb-9 construct, we
show that type IV collagen made in body wall muscle
cells can assemble into the pharyngeal, intestinal, and
gonadal basement membranes. Additionally, we show
that expression of functional type IV collagen only in
body wall muscle cells is sufficient for C. elegans to
complete development and be partially fertile. Since
type IV collagen secreted from muscle cells only assembles into some of the basement membranes that it has
access to, there must be a mechanism regulating its assembly. We propose that interaction with a cell surface-associated molecule(s) is required to facilitate type IV collagen assembly.
Basement membranes are thin sheets of specialized
extracellular matrix that underlie or surround
groups of cells, separating them from other cells
and/or adjacent connective tissue. The major constituents
of basement membranes are type IV collagen, laminin, nidogen (entactin), and heparan sulfate proteoglycan (perlecan) (59, 72). The functions and interactions of these molecules have primarily been studied in vertebrates using in
vitro systems. These proteins, and presumably their functions, have been conserved throughout metazoan evolution. We are analyzing type IV collagen in vivo in the nematode Caenorhabditis elegans (26, 27) to further understand
the assembly and functions of this basement membrane
molecule.
The structure of type IV collagen has been conserved
from nematodes to mammals (27, 29). The majority of the
molecule is the central Gly-X-Y repeat domain, which folds
into a triple-helical structure. There are ~20 sites at which
the triple-helical structure is interrupted by amino acids in
which glycine is not at every third position. At the amino
terminus, there is a short non-(Gly-X-Y) domain containing four conserved cysteines, and at the carboxyl terminus,
there is the globular NC1 domain that contains 12 conserved cysteines. In basement membranes, type IV collagen molecules form a complex polygonal network stabilized by intermolecular disulfide bonding (59). NC1 domains
associate to form dimers, while amino terminal (7S) domains associate into tetramers. Lateral associations between type IV molecules are also involved in forming the
network seen in basement membranes (70, 71).
Six type IV collagen genes have been identified in mammals, The diversity of type IV collagen is increased in C. elegans by alternative splicing of the The predominant form of type IV collagen in mammals,
( Mutations have been characterized in the C. elegans Here we show that the EMB-9 and LET-2 type IV collagen chains are colocalized and are found in most, but not
all, basement membranes in C. elegans. We show that the
emb-9 and let-2 genes are expressed in mesoderm, primarily body wall muscle and somatic gonadal cells. We demonstrate that EMB-9 produced in body wall muscles can
assemble into the basement membranes that surround other tissues and that body wall muscle expression is sufficient for C. elegans development. Our results indicate that
a mechanism must exist to control the sites of assembly of
type IV collagen in basement membranes.
C. elegans Strains
Animals were maintained as described by Brenner (6). The C. elegans variety Bristol isolate N2 is designated as wild-type. The mutant strain used
in this study, unc-36(e251) emb-9(g23cg45)/qC1[unc-36(+)emb-9(+)], was
generated by reverting the temperature-sensitive dominant lethality of
emb-9(g23) (20a). The cg45 mutation converts Gln1388 of EMB-9 to a termination codon.
Preparation of Type IV Collagen Antisera
cDNA fragments encoding the entire NC1 domains of EMB-9 and LET-2
were generated by reverse transcriptase-PCR amplification and cloned
into pGEX fusion protein vectors (Pharmacia LKB Biotechnology, Inc.,
Piscataway, NJ). Fusion proteins were isolated in inclusion bodies after
overexpression in Escherichia coli. The inclusion bodies were solubilized
in SDS/ Rabbit antisera against fusion protein and peptide antigens were generated commercially (Hazelton Research Products, Inc., Denver, PA). Sera
against synthetic peptides were affinity purified using Actigel (Serogene
Bioseparations, Arcadia, CA) columns coupled with the same peptide.
Fusion protein antisera were first affinity purified on columns coupled
with the fusion protein to which they were generated. Cross-reacting antibodies were then removed by passing the affinity-purified anti-EMB-9
sera over a column containing the LET-2 fusion protein, and vice versa.
Purified antisera were stored at Western Blots
C. elegans extracts were prepared by washing mixed-stage populations extensively with large volumes of distilled water (dH2O).1 After pelleting,
they were mixed immediately in an equal volume of 2× Laemmli sample
buffer (30), containing 5% freshly added Western blots were performed by standard procedures (21). C. elegans
protein extracts were electrophoresed on either 7.5 or 4% polyacrylamide
gels and transferred to nitrocellulose using 25 mM Tris, 192 mM glycine,
0.1% SDS, 10% methanol. Nitrocellulose filters were stained with Ponceau S to determine the position of molecular mass markers and then
blocked for at least 1 h in BB (BB: 10% dry milk, 0.3% Tween 20 in PBS).
Filters were incubated with anti-type IV collagen antibodies diluted
1:500-1:1,000 in BB for 1 h at room temperature and washed with 0.3%
Tween 20 in PBS. They were then incubated with alkaline phosphatase-
conjugated goat anti-rabbit antibodies (Sigma Chemical Co., St. Louis,
MO) diluted 1:1,000 in BB, washed, and developed with BCIP/NPT (21).
For competition experiments, anti-type IV collagen antibodies were
preincubated for 1 h at room temperature with 200-400 µg/ml of the fusion protein or 100 µg/ml of the peptide used as the original antigen. The
antibody/antigen mixture was then used to stain blots as described above,
or to stain animals as described below.
Antibody Staining of C. elegans
Animals were prepared for immunofluorescence analysis by one of three
methods. In the first, eggs were collected from alkaline hypochlorite-
treated animals (55), rinsed twice with dH2O, fixed with 3% paraformaldehyde in PBS for 15 min at room temperature, rinsed twice with dH2O,
and stored at After fixation, animals were rehydrated and either blocked immediately for 1 h in PBS containing 0.1% Triton X-100 (PBS-T) and 10% normal donkey serum (NDS), or treated with 6 M urea, 0.1 M glycine, pH 3.5, for 15 min (69) and rinsed 3× with PBS before blocking. Samples were
incubated with antibodies against type IV collagen (EMB-9 or LET-2),
myosin heavy chain B (UNC-54), or mouse anti-hemagglutinin (HA)
monoclonal antibody (Boehringer-Mannheim Corp., Indianapolis, IN) in
PBS-T-NDS overnight at 4°C for 1 h at 37°C, washed with PBS-T, incubated 1 h at 37°C with FITC donkey anti-rabbit and LRSC donkey anti-
mouse secondary antibodies (Jackson ImmunoResearch, West Grove,
PA) in PBS-T-NDS, washed with PBS-T containing 1 µg/ml diamidinophenolindole, followed by PBS alone.
To prepare frozen sections, animals were fixed with 3% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.0, for 20 min at room temperature. After washing in water, they were resuspended in 30% sucrose
in PBS at 4°C. After the animals sank to the bottom of the tube, the overlying sucrose solution was removed and an equal volume of OCT compound (Miles, Inc., Elkhart, IN) was added. Frozen sections were collected on polylysine-coated slides and dried for 30 min before being
blocked and stained as described above.
Construction and Analysis of Type IV Collagen
Expression Reporters
Reporter constructs for emb-9 were generated from the plasmid pJJ359,
which contains the complete emb-9 gene flanked by 4.3 kb of 5
Another emb-9:lacZ reporter construct, pJJ318, was made by inserting
a HindIII-PvuI ( Two let-2 reporter constructs were generated from the plasmid pJJ353,
which contains the complete let-2 gene with 2.2 kb of 5 DNAs were injected into hermaphrodite gonads as described (36) using pRF4[rol-6(su1006)] (28) as a transformation marker. The emb-9 reporter construct pJJ318 was integrated into the genome by Construction and Analysis of Epitope-tagged Type
IV Collagen
The pJJ400 construct was made by cloning the 3.6-kb HindIII-XbaI fragment from pJJ359 (Fig. 1 A) into the pCRII vector (InVitrogen, San Diego, CA), which is useful because it has no AatII sites. PCR was performed on first strand cDNA derived from mixed-stage C. elegans RNA
using primers emb9ectA ( Transgenic Rescue of emb-9(null) by Expression in
Muscle Cells
Plasmid pJJ427 was constructed by ligating the 3.5-kb ApaI-KpnI fragment from pPD30.38 with the 7.8-kb PvuI-PstI fragment from pJJ359 after
blunt ending. This construct has the wild-type emb-9 gene ( Characterization of Anti-type IV Collagen Antisera
We have used four antisera to examine the distribution of
type IV collagen chains in C. elegans. Two antisera were
generated against fusion proteins containing the NC1 domains of EMB-9 (NW155) or LET-2 (NW153), one against
a peptide from the NC1 domain of EMB-9 (NW1910), and
one against a peptide derived from an interruption of the
Gly-X-Y domain of LET-2 (NW68). Each of the sera was
affinity purified using the original antigen. In addition, the
fusion protein sera were cross adsorbed on columns carrying the fusion protein from the other type IV collagen chain.
The purified anti-EMB-9 antisera do not cross-react with
the LET-2 fusion protein, and the purified anti-LET-2 antisera do not cross-react with the EMB-9 fusion protein
(data not shown).
On Western blots of total C. elegans extracts, both
EMB-9 antisera react with a major band of ~240 kD, and
both LET-2 antisera react with a major band of ~210 kD
(Fig. 2). The predicted molecular masses of EMB-9 and
LET-2, after cleavage of signal peptides, are 168 and 165 kD,
respectively. Their higher apparent molecular masses are
at least partly due to the abnormally slow migration of collagens relative to globular protein molecular mass standards on SDS-polyacrylamide gels (17). The apparent molecular masses of the reacting bands differ depending on
the percentage acrylamide in the gels, another characteristic of collagens. The fact that the EMB-9 and LET-2 antisera specifically react with proteins of different mobilities
indicates that they are specific for the chains against which
they were made. Activity on Western blots is eliminated
by competition with the original antigen, but not by competition with antigen from the other type IV collagen chain (Fig. 2 B). Similar results were obtained in competition experiments on embryos prepared for immunofluorescence.
The specificity of the antisera is further supported by the
fact that the anti-EMB-9 antisera do not react with embryos homozygous for either a stop codon or an internal
deletion of emb-9 (20a).
Distribution of Type IV Collagen
Ultrastructural analyses of C. elegans (1, 64) have identified basement membranes on the pseudocoelomic face of
the hypodermis and surrounding the body wall muscles,
pharynx, intestine, and gonad (Fig. 3). We have determined the distributions of the
At 20°C, C. elegans embryogenesis takes ~800 min from
first division to hatching (55). Most cell divisions are completed during the first 350 min, after which the embryo begins elongation from an ovoid ball into a tube approximately four times the length of the egg. We refer to the
stages of morphogenesis, from the start of elongation to
hatching, by the length of the embryo relative to the egg
shell, progressively as lima, comma, 1 1/2-fold, twofold, threefold, and fourfold.
Type IV collagen is first detectable in body wall muscle
cells at the onset of morphogenesis, just preceding the lima
stage (data not shown). At this early stage, the type IV collagen stain appears to be completely intracellular. By the
1 1/2-fold stage, strong type IV collagen stain is seen in
body wall muscle cells and at the interface between them
and the hypodermis (Fig. 4, A and B). Weak stain is seen
surrounding the pharyngeal and intestinal primordia beginning at the comma stage, and this stain becomes intense
by the 1 3/4-fold stage (Fig. 4, C and D). Intracellular stain
is also seen in the head mesodermal cell and its lineal homologue MS.pppaaa by this stage (Fig. 4, E and F).
From about the comma stage on, stain for type IV collagen accumulates in four stripes at the interfaces between
the hypodermis and the four body wall muscle quadrants
(Fig. 4, G-J). There is no stain detectable in the hypodermal basement membrane that is located between the muscle quadrants. There is also no detectable stain on the
pseudocoelomic face of body wall muscle cells. This same
staining pattern is seen with all four type IV antisera and is
not altered after treatment with acid urea to unmask potentially hidden epitopes (69).
After the twofold stage, six cells in the nerve ring frequently exhibit stain for type IV collagen (Fig. 4 K). Based
on their positions, these cells appear to be the GLR cells,
which are considered to have glial-like characteristics (66).
The GLR cells have broad thin processes that separate the
muscle arms that project under the nerve ring from the
pharynx. At the time of hatching, strong type IV collagen
stain is seen at the interface between the body wall muscle
cells and the hypodermis and surrounding the pharynx, intestine, and primordial gonad (Fig. 4 L).
In larval and adult animals, the distribution of type IV
collagen generally remains the same as that seen in late
embryos; underlying the body wall muscles and surrounding the pharynx, intestine, and gonad (Fig. 5 C). As in embryos, type IV collagen is not detectable in the hypodermal
basement membranes between body wall muscle quadrants or on the pseudocoelomic face of body wall muscles
(Fig. 5, A and B). In larvae and adults, coelomocytes can
be seen to stain with the type IV antisera (Fig. 5 D). The basement membrane surrounding the gonad stains for
type IV collagen throughout larval development and in
adults. However, in many L4 and adult animals the gonadal stain is not uniform, being more intense around the
spermatheca (Fig. 5 E) and around the distal ends of the
gonad arms. Although the basement membranes surrounding the pharynx and intestine stain with the type IV
antisera, intracellular stain in these tissues was never seen
in embryos, larvae, or adults. A schematic summary of the
observed type IV collagen staining pattern is shown in Fig. 3.
Type IV Collagen Gene Expression
The sites of emb-9 and let-2 expression have been determined using lacZ and GFP reporter constructs in transgenic animals. For both genes, the reporter constructs
were derived from clones that are capable of rescuing mutant animals when present in transgenic arrays. For emb-9,
constructs in which lacZ or GFP are inserted into exon 10 and that have 4.3 kb of 5 For let-2, 3.2 kb of 5 Weak
The emb-9 and let-2 reporters showed activity in the
same cells that stained with the anti-EMB-9 and -LET-2
antisera, with the exception of some accessory muscles.
Although the intestinal, anal, vulval, and uterine muscles
showed activity with type IV expression reporters, intracellular staining of these cells with anti-type IV collagen
antisera was not apparent. It is possible that antibody
staining of these cells was weak and/or obscured by the intense stain of adjacent intestinal, gonadal, and body wall
muscle basement membranes.
It is notable that no emb-9 or let-2 reporter activity was
seen in pharyngeal, intestinal, or hypodermal cells of embryos, larvae, or adults. This result is consistent with the
fact that anti-type IV collagen antisera did not show intracellular stain in these tissues. However, it is surprising because the pharynx and intestine are covered by basement
membranes that do stain with the anti-type IV collagen
antisera. Although the pharyngeal and intestinal basement
membranes contain EMB-9 and LET-2, the genes encoding them are not expressed in these tissues.
Type IV Collagen Expressed in Muscle Cells Can
Assemble on Other Tissues
The strong expression of emb-9 and let-2 in body wall muscle cells suggests that they could be the source of the type
IV collagen found in the pharyngeal and intestinal basement membranes. To determine if type IV collagen produced in body wall muscle cells can assemble on other tissues, we constructed an epitope-tagged emb-9 gene that is
transcriptionally controlled by the body wall muscle-specific unc-54 promoter and enhancer, pJJ414 (Fig. 1 A). The
unc-54 gene encodes the body wall muscle myosin heavy
chain MHC-B (33), and the specificity of its promoter has
been well documented (44). The epitope tag consists of two
tandem copies of the nine-amino acid HA peptide (67).
Transgenic lines carrying the construct were generated in
the wild-type N2 background and stained with anti-HA
and -type IV collagen antibodies.
In lima through 1 1/2-fold embryos, the anti-HA stain is
seen in body wall muscle cells (Fig. 7 A), coincident with
stain for the LET-2 chain (Fig. 7 D). In later embryos,
when wild-type type IV collagen is localized largely in
basement membranes (Fig. 7 E), the HA-tagged molecules are mostly still intracellular (Fig. 7 B). This intracellular retention appears similar to that seen in animals with
mutations in either of the type IV collagen genes (20a). In
very late embryos that are close to hatching, weak HA
staining is detectable on the surface of the pharynx and intestine (Fig. 7, C and F). Thus, the epitope-tagged EMB-9
chain is expressed in body wall muscle as expected; however, the presence of the HA epitope tag at the carboxyl
end of EMB-9 appears to interfere with its proper assembly and/or secretion.
The inhibition of HA-tagged EMB-9 secretion is not
complete, however, since in larvae and adults carrying
pJJ414, strong staining with anti-HA antibodies is seen
covering the pharynx and intestine (Fig. 7, G and H). This
staining is coincident with that seen with anti-type IV collagen antisera (Fig. 7, I and J). Staining with HA antibodies beneath body wall muscles and over the gonad can also
be detected. Animals in the same preparation that do not
carry the transgenic array show no staining, demonstrating the specificity of the HA antibody (Fig. 7, G-J). These results show that type IV collagen produced in body wall
muscle cells can assemble into the basement membranes
covering other tissue.
Expression of EMB-9 in Body Wall Muscle Can Rescue
emb-9 Mutant Animals
As a test of how much type IV collagen function can be
provided by expression in body wall muscle cells, we asked
how far animals could develop if they produced functional
type IV collagen only in body wall muscle cells. The body
wall muscle-specific unc-54 promoter and enhancer were
placed immediately upstream of the initiation codon of the
complete wild-type emb-9 gene to create pJJ414 (Fig. 1 A).
This construct was used to generate transgenic arrays in animals carrying an emb-9 nonsense mutation linked to an Unc
marker maintained over a balancer chromosome, unc-36
(e251) emb-9(g23cg45)/qC1. Normally, this stain segregates no Unc progeny because of the strict embryonic lethality
of the emb-9 nonsense mutation.
We expected that, at best, sterile adults could develop
from emb-9(g23cg45) animals carrying pJJ427, since the normal gonadal type IV collagen expression would be absent.
To our surprise, viable and fertile transgenic Unc animals
were generated by injecting the unc-54:emb-9 construct.
We have established several lines that are homozygous
unc-36(e251) emb-9(g23cg45) and are rescued by arrays
carrying the pJJ427 construct. The rescued animals grow slowly; many arrest during larval development, and those
that reach adulthood have small brood sizes. However, the
strains can be maintained. These results indicate that expression of functional type IV collagen only in body wall
muscles is sufficient for C. elegans to develop to adulthood
and to reproduce.
It is possible that the presence of the complete emb-9
coding region in the pJJ427 construct could alter the expression pattern of the unc-54 promoter and enhancer. To
test this possibility, GFP was inserted into exon 10 of the
construct to create pJJ429 (Fig. 1 A). Transgenic animals
carrying pJJ429 show strong GFP activity only in body
wall muscle cells (Fig. 8 A). No activity was seen in the
pharynx, intestine, or gonad. Therefore, rescue of emb-9
(g23cg45) mutant animals is possible by expression of functional emb-9 solely in body wall muscle cells.
Embryos homozygous for emb-9(g23cg45) do not stain
with anti-EMB-9 antisera and show only intracellular
accumulation, no basement membrane stain, with anti-
LET-2 antisera (20a). We stained pJJ427-rescued emb-9
(g23cg45) embryos with anti-EMB-9 antisera and found
that EMB-9 is present in pharyngeal, intestinal, and gonadal basement membranes and under body wall muscles
(Fig. 8, B-D). This result further demonstrates that type
IV collagen produced in body wall muscle cells can assemble into basement membranes covering other tissues, and
that it can do so in the absence of any other source of type
IV. It also shows that type IV collagen derived from body wall muscles is sufficient for development of C. elegans to
the adult stage and for at least partial fertility.
We have used specific antisera to localize the Antisera against EMB-9 and LET-2 give the same spatial and temporal staining patterns in wild-type animals,
indicating that these chains are colocalized. Additionally,
in animals with missense mutations of either chain, the distributions of the chains are altered but still colocalized, and
in animals with nonsense mutations in emb-9, the LET-2
chain is retained intracellularly and not secreted (20a).
The colocalization of EMB-9 and LET-2 and the inability
of LET-2 to be secreted in the absence of EMB-9 indicates that the two chains do not function independently. Since
EMB-9 and LET-2 are Basement membranes in two regions of C. elegans did
not stain with anti-type IV collagen antisera at any stage,
those on the hypodermis between the body wall muscle
quadrants and those on the pseudocoelomic face of body
wall muscles (Fig. 3). The basement membranes in these
regions appear ultrastructurally similar to those that cover
the intestine and gonad (66) and do stain for type IV collagen. There are three possible explanations for the failure
to detect type IV collagen in these regions of basement membrane.
First, it is possible that EMB-9 and LET-2 are present
but their epitopes are masked in these regions. Acid-urea
extraction, which can uncover masked epitopes in vertebrate basement membranes (69), of whole animals or frozen sections of C. elegans had no effect on the type IV collagen staining pattern. All four type IV antisera fail to
stain these regions in animals that are fixed with paraformaldehyde or methanol/acetone and in frozen sections. These results make the possibility of epitope masking unlikely and support the idea that EMB-9 and LET-2 are absent from these regions.
Second, it is possible that there is type IV collagen
present that is produced by an unidentified gene(s). Southern blot analyses indicated that emb-9 and let-2 are likely
to be the only type IV collagen gene in C. elegans (19). At
this time, with approximately one-half of the C. elegans genome sequence complete (61), no other type IV collagen
genes have been identified. Multiple cDNA clones derived
from emb-9 and let-2 have been identified by C. elegans
EST database projects (62; Kohara, Y., personal communication), but no other type IV collagen transcripts have
been seen. Although it is not yet possible to exclude the
existence of another type IV collagen gene(s) in C. elegans,
the probability is small.
Finally, it is possible that there is no type IV collagen in
these regions of basement membrane. Although basement
membranes can vary in the collagen isoforms they contain
(37, 48), they all appear to contain some type IV collagen.
There is evidence that during angiogenesis, newly forming
basement membranes are laminin rich and lack type IV
collagen (15), and there are cell lines that secrete matrix lacking type IV collagen (5, 16). However, there are no reports
of fully formed in vivo basement membranes that do not
contain type IV collagen. The regions in C. elegans on the
surface of the hypodermis between body wall muscle quadrants and on the pseudocoelomic face of body wall muscle cells may be basement membrane-like structures that lack
type IV collagen.
Whichever of the above possibilities is true, it is clear
that the composition and/or modifications of the basement
membranes between body wall muscle quadrants and on
the pseudocoelomic face of muscles must be different
from the other basement membranes in C. elegans. Another basement membrane component, the UNC-52 perlecan homologue of C. elegans, is also not detected in these same regions of basement membrane (42).
We have identified the cells that express the emb-9 and
let-2 type IV collagen genes using reporter constructs in
transgenic animals. With minor exception, the type IV collagen-expressing cells in C. elegans are mesodermal. The
mesoderm of C. elegans is derived from four founder cells,
MS, D, Cap, and Cpp (54, 55). The D, Cap, and Cpp
founders generate only body wall muscle cells, all of which
express type IV collagen. The MS cell generates body wall
muscles and several other type IV collagen-expressing cell
types (Fig. 9). Notably, all of the embryonic MS-derived cells express type IV collagen, except the pharyngeal cells.
Type IV collagen expression is also confined to mesodermal cells in Drosophila (25, 32, 39) and sea urchin (9, 63).
In vertebrates, type IV collagen expression has been reported in both mesenchymal and epithelial cells of several
tissues (34, 49, 57, 58), although it is restricted to the mesenchyme in mouse intestine (52).
The combination of antibody localization and gene reporter results indicated that the pharynx and intestine do
not express the These results raise the question of how the sites of type
IV collagen assembly into basement membranes are determined. Presumably, body wall muscle cells secrete type IV
collagen from both their hypodermal and pseudocoelomic
faces. Body wall muscle cells are closely apposed to the hypodermis, and collagen secreted into the small intervening
space could assemble into the stripes seen beneath the
body wall muscle quadrants. The type IV collagen that assembles into the pharyngeal, intestinal, and gonadal
basement membranes is likely to be secreted from the
pseudocoelomic face of the muscle cells. Notably, the basement membranes that cover the pseudocoelomic face of
body wall muscle cells do not contain detectable type IV
collagen. Since the body wall muscle quadrants extend
from the head to the tail of the animal, secretion into and
diffusion within the pseudocoelomic cavity could provide
the necessary type IV collagen for all other tissues.
The remaining question is why all basement membranes
that are exposed to the pseudocoelomic cavity do not contain type IV collagen? As noted above, type IV collagen is
not detectable in the basement membranes on the pseudocoelomic face of muscle cells or on the hypodermis between body and wall muscle quadrants. Either something
is present in these regions that blocks type IV collagen assembly, or something that is required to promote assembly
is absent from these regions. We favor the latter possibility, since assembly could be promoted by simply increasing
the local concentration of type IV collagen, while no inhibitors of assembly are known.
Type IV collagen can assemble into a polygonal lattice
in vitro in a concentration-dependent manner (70). However, the in vivo concentration of type IV collagen may be
too low to allow such unassisted assembly. Higher local
concentrations of type IV collagen could be achieved if it
bound to cell surface receptors or other cell-associated
basement membrane components. Likely candidates for
this function are integrins and/or nidogen, both of which
have been identified in C. elegans (18; Kramer, J., unpublished results). Binding to these molecules could produce
local concentrations of type IV collagen high enough for
assembly to occur. Thus, the localization of type IV collagen may be determined by the localization of cell-associated molecules to which it binds.
The sources and final locations of type IV collagen have
been determined in some vertebrate systems. In cocultures
of mouse epithelium with chick endothelium, the developing kidney glomerular basement membrane was shown to
have type IV collagen contributed by both species (49). In
cocultures of fetal intestinal chick mesenchyme with rat endoderm, the resulting subepithelial basement membrane
was found to have type IV collagen derived only from the
mesenchyme (52, 53). In cocultures of bovine keratinocytes and human fibroblasts, both cell types contributed
type IV collagen to the dermal-epidermal basement membrane (34), while nidogen was derived only from fibroblasts (14). So, the type IV collagen in a basement membrane can be derived from cells on either or both sides of
it. In these cases, the two groups of cells contributing to
basement membrane formation are generally present
along a continuous interface. In C. elegans, the situation is
different because the body wall muscle cells that produce
type IV collagen are not immediately adjacent to all sites
at which it assembles (Fig. 3). However, the localized assembly of type IV collagen in both the vertebrate and C. elegans studies suggest that it must interact with other molecules to direct its assembly.
The assembly properties of the perlecan homologue of
C. elegans, UNC-52, are different from those of type IV
collagen. UNC-52 is deposited in the basement membrane
close to its sites of synthesis (42). The tissues that have
UNC-52 in their basement membranes all express the unc-52
gene. Laser ablation of body wall muscle cells results in a
hole in the UNC-52 antibody staining pattern where the
cells are missing, indicating that UNC-52 does not move far from its site of synthesis. In C. elegans, two basement
membrane components assemble in different ways; perlecan is deposited locally, while type IV collagen can assemble at sites distant from its site of synthesis.
1-
6 (22, 29). The predominant form of type IV
collagen in mammals is a heterotrimer of two
1 and one
2(IV) chains. There are two genes that encode type IV
collagen chains in C. elegans, emb-9 and let-2 (19, 20, 50).
EMB-9 is an
1(IV)-like chain and LET-2 is an
2(IV)-
like chain. The six mammalian genes are organized as
closely spaced pairs of one
1-like and one
2-like gene in
head-to-head orientation (22, 29). In contrast, the C. elegans type IV collagen genes are located on different chromosomes. Two type IV collagen genes have been identified in sea urchin (9, 10), and one each in Drosophila (3,
4), and the parasitic nematodes Brugia (7) and Ascaris
(46). There is no evidence for more than two type IV collagen genes in any invertebrate.
2(IV) chain gene let-2
(50). One splice variant is predominant in embryos, while
the other is predominant in larvae and adults. The alternative splicing does not change the length of the molecule
but changes a small number of amino acids in the Gly-X-Y
repeat domain. The same alternative splicing occurs in the
2(IV) chain of Ascaris (47). The importance of these splice
variants is unknown, but they may increase the complexity
of type IV collagen functions.
1)2
2, is abundant in most basement membranes (22, 24,
48). The four additional mammalian chains,
3-
6(IV),
have more restricted tissue distributions, being most abundant in the kidney (37, 43, 45, 68). Mutations in the human
3,
4, and
5(IV) collagen genes can cause Alport syndrome, a progressive glomerulonephritis variably associated with hearing loss and ocular lesions (2, 31, 41, 60). A
similar nephrotic syndrome is seen in dogs with mutations
in the
5(IV) collagen gene (73) and in mice with a knockout of the
3(IV) collagen gene (8, 38). The primarily renal focus seen in Alport syndrome is consistent with the
localization of the
3-
5(IV) chains primarily in kidney. Mutations have not been identified in the mammalian
1
or
2(IV) collagen genes.
1
and
2(IV) collagen genes, emb-9 and let-2 (20, 20a, 51).
Mutations in either of the two genes cause similar defects.
Most of the mutations are substitutions for glycines in the
Gly-X-Y repeat domain and cause temperature-sensitive
phenotypes. At nonpermissive temperature (25°C), most
of these mutations cause arrest at the twofold stage of embryonic development. At intermediate temperature (20°C),
they generally cause larval arrest or adult sterility. At the
permissive temperature (15°C), most of the mutations allow animals to develop and reproduce. Thus, normal EMB-9
and LET-2 are required for both viability and fertility. The
evidence that emb-9 and let-2 are the only type IV collagen
genes in C. elegans and their embryonic lethal phenotypes
suggest that their products may be widely distributed,
analogously to the mammalian
1 and
2 chains.
Materials and Methods
-mercaptoethanol sample buffer (30) and electrophoresed on
SDS-polyacrylamide gels, and the fusion protein bands were excised from
the gels and used to immunize rabbits. Synthetic peptides derived from
the NC1 domain of EMB-9 (CVDQDKQFRKPMSQ) or from exon 9 of
LET-2 (CREFTGSGSIVGPR) were coupled to BSA using m-maleinidobenzoyl-N-hydroxysuccinimide ester (21).
20°C in PBS with 50% glycerol, 1% BSA.
-mercaptoethanol, and boiled
for 10 min. Extracts were then incubated for 12-24 h at room temperature,
boiled 10 min, and stored at
20°C.
80°C in 100% methanol for 30 min to 7 d. Alternatively,
animals in dH2O were pipetted onto slides subbed with 0.2% gelatin,
0.1% polylysine, 0.01% chrome alum, covered with a coverslip, and frozen
on dry ice. The coverslip was cracked off with a razor blade, and the slide
was immersed in
20°C methanol for 5 min, followed by
20°C acetone
for 3 min, and air dried. Lastly, larvae and adults were prepared as described by Finney and Ruvkun (11).
upstream
and 0.5 kb of 3
sequence (Fig. 1 A). pJJ359 is capable of transgenic rescue
of emb-9 mutant animals. To generate the emb-9:lacZ reporter construct
pJJ444, the 10-kb XmaI-SaII fragment from pJJ359 was inserted into
XmaI-XhoI cut pPD34.110. PPD34.110 contains the E. coli lacZ gene with a
membrane stop transfer sequence and 3
untranslated sequences from
unc-54 (13.). An emb-9:green fluorescent protein (GFP) reporter, pJJ446,
was made by removing the lacZ region of pJJ444 by KpnI-SpeI digestion and inserting the KpnI-SpeI GFP-containing fragment from pPD95.75 (provided by A. Fire, S. Xu, J. Ahnn, and G. Seydoux).
Fig. 1.
Diagrams of emb-9 and let-2 expression constructs. Constructs involving emb-9
were derived from the genomic clone pJJ359
(A). Constructs involving let-2 were derived
from the genomic clone pJJ353, or cosmid
C12F7 (B). Exons are indicated as open
boxes, introns as thick lines, and flanking sequences as thin lines. The unc-54 promoter/
enhancer is stippled, and the HA epitope tag
and reporter sequences are solid. The emb-9
and let-2 genomic sequences are drawn to
scale, but exogenous sequences are not.
[View Larger Version of this Image (28K GIF file)]
1672 to
18 relative to emb-9 start codon) fragment
from the lambda phage clone CH#2 into HindIII-SmaI-digested pPD21.28.
CH#2 contains the complete emb-9 gene and is capable of transgenic rescue of emb-9 mutant animals (20). pPD21.28 contains the E. coli lacZ
gene with a nuclear localization signal and 3
untranslated sequences from
unc-54 (13).
upstream and 430 bp
of 3
untranslated sequence (Fig. 1 B). pJJ353 is capable of transgenic rescue of let-2 mutant animals (50). pJJ360 was made by ligating the 6-kb SphIEcoRV fragment from pJJ353 into SphI-SmaI-digested pPD34.110. pJJ361
was made by ligating the 4-kb SmaI-ApaI fragment from pPD34.110 with
the 11-kb EcoRV-ApaI fragment from pJJ353. pPD34.110 contains the E. coli lacZ gene with a membrane stop transfer sequence and 3
untranslated sequences from unc-54 (13). An additional let-2 reporter, pJJ325,
was generated by cloning the 3.2-kb AvaI fragment from cosmid C12F7
(
3.2 to +20 kb relative to let-2 initiation codon) into SmaI-digested
pPD22.04 (13).
-irradiation
and selection of a line that segregates 100% roller progeny.
-galactosidase activity was visualized by staining as described (12), after fixation for
15 min with 3% paraformaldehyde in PBS and washing with PBS.
44 to
9, relative to translation start) and
emb9ectB2 (+3268 to +3250). Emb9ectA has a single nucleotide change,
T-38G, relative to wild-type to create a HindIII site. Emb9ectB2 has a
synthetic HindIII site appended. PCR products were HindIII digested and
cloned into HindIII-digested pJJ400 to generate pJJ401. The 1.6-kb AatII
fragment was removed from pJJ401 and replaced with the 1.6-kb AatII
fragment from pJJ359 to generate pJJ402. PCR was performed on first
strand cDNA derived from mixed-stage C. elegans RNA using primers
emb9ectD (+5725 to +5750) and embtwoHA, which is the complement of
+7217 to +7231 with two HA epitope tags (67), a termination codon, and
an Xba site appended. PCR products were SaII-XbaI digested and cloned into SaII-XbaI-digested pJJ402 to generate pJJ404. The insert in pJJ404
was isolated after NsiI-ApaI digestion and ligated into NheI-KpnI-digested
pPD30.38, after both fragments had been blunt ended with T4 DNA polymerase. pPD30.38 contains the unc-54 body wall muscle myosin promoter
and enhancer (35). The resulting clone, pJJ414 (Fig. 1 A), has the unc-54
promoter and enhancer, the emb-9 gene with introns 1-6, 10, and 11 removed and two copies of HA inserted immediately preceding the termination codon, and 3
untranslated sequences from pPD30.38.
15 to +7750)
under control of the unc-54 promoter and enhancer. pJJ427 was used to
generate transgenic strains in unc-36(e251) emb-9(g23cg45)/qC1 animals.
The constructs pJJ445 and pJJ429 were generated by ligating the emb-9
gene and reporter containing NsiI-SpeI fragments from pJJ444 or pJJ446
with the unc-54 promoter containing NsiI-XbaI fragment from pJJ427.
Results
Fig. 2.
Antibodies against the 1 and
2(IV) collagen chains,
EMB-9 and LET-2, react specifically to their respective antigens.
Western blots of C. elegans extract were probed with type IV collagen antisera. (A) Strips cut from a blot of C. elegans extract
were reacted with the anti-EMB-9 peptide antiserum 1910 (lane
1) or the anti-LET-2 peptide antiserum 68 (lane 2). (B) C. elegans extract was reacted with fusion protein-generated antiserum
NW153 against LET-2 (lanes 1-3) or antiserum NW155 against
EMB-9 (lanes 4-6). Before reacting with the blot, the antibodies
were incubated with no competitor (lanes 1 and 4), LET-2 fusion
protein (lanes 2 and 5), or EMB-9 fusion protein (lanes 3 and 6).
Antibodies incubated with the antigen to which they were made
no longer react to the worm extract (lanes 2 and 6), while those
incubated with the other fusion protein still react to the appropriate size band (lanes 3 and 5).
[View Larger Version of this Image (54K GIF file)]
1 and
2(IV) collagen chains
EMB-9 and LET-2 in whole mount animals using specific
anti-type IV collagen antisera. All four antisera generate
essentially identical immunofluorescence patterns, so below we refer to type IV collagen localization rather than
to the separate chains. The colocalization of EMB-9 and
LET-2 chains further supports the specificity of the antisera and suggests that EMB-9 and LET-2 may assemble
into the same heterotrimeric collagen molecule.
Fig. 3.
Schematic cross
sections of C. elegans
through the head (left) and
midbody (right) showing the
locations of basement membranes. Basement membranes that stain with anti-
EMB-9 and anti-LET-2 antisera are shown as thick black
lines; those that do not stain are shown as thick stippled
lines. Tissues that express the
emb-9 and let-2 genes are indicated with white text on a
black background; those that
do not are shown as black
text on a white background. Note that only a subset of somatic gonadal cells express
emb-9 and let-2.
[View Larger Version of this Image (48K GIF file)]
Fig. 4.
Type IV collagen distribution in wild-type C. elegans embryos. Embryos were stained with anti-LET-2 antiserum 68 (A, E, G, and K) or anti-EMB-9 antiserum 1910 (C, I, and L). The same staining patterns are seen with all anti-EMB-9 and anti-LET-2 antisera. The same embryos were stained with anti-UNC-54 myosin monoclonal antibody to identify body wall muscle cells (B, D, F, H, and J). (A) A 1 1/2-fold embryo shows type IV collagen accumulation in body wall muscle cells. (B) Anti-UNC-54 staining of the same embryo in A. (C) A 1 3/4-fold embryo shows type IV collagen in body wall muscle cells and covering the pharynx primordium (p). (D) Anti- UNC-54 staining of the same embryo in C. (E) A 1 1/2-fold embryo showing type IV collagen in body wall muscle cells, and the head mesodermal cell (hmc) and its lineal homologue. (F) Anti-UNC-54 staining of the same embryo in E. Note that the hmc and its homologue do not stain for UNC-54. (G) A twofold embryo showing type IV collagen staining under the body wall muscle quadrants (two of the four are indicated with arrowheads), but not between them. Stain around the pharynx (p) appears as a ring in the head in this optical
cross-section. Stain around the intestine (i) is seen in the posterior of the embryo. (H) Anti-UNC-54 staining of the same embryo in G. (I) Surface focal plane of a threefold embryo shows type IV collagen as a stripe overlying a body wall muscle quadrant. (J) Anti-UNC54 staining of the same embryo in I. (K) Type IV collagen staining of two ring ganglion cells (rg), believed to be GLR cells, in a fourfold
embryo. The posterior bulb of the pharynx (p) is indicated. (L) An embryo at hatching showing type IV collagen stain of basement
membranes covering the pharynx (p), intestine (i), and gonad primordium (g).
[View Larger Version of this Image (88K GIF file)]
Fig. 5.
Type IV collagen distribution in wild-type C. elegans larvae and adults. Anti-LET-2 (A) and anti-UNC-54 myosin (B) stain of a frozen section through the head of an adult animal. The Type IV stain is associated with the pharynx (p) and four body wall muscle
quadrants. There is not type IV collagen stain between muscle quadrants. Type IV collagen stain is localized near the myosin stain, which
is closely apposed to the hypodermal face of body wall muscle cells. (C) Anti-EMB-9 stain of an L2 larva showing localization to basement membranes surrounding the pharynx (p), intestine (i), and gonad (g). (D) Anti-EMB-9 stain of an L4 larva showing reaction with
the gonad basement membrane (g) and with two coelomocytes (cc). (E) Anti-LET-2 stain of an adult showing intense stain surrounding
the spermatheca (sp).
[View Larger Version of this Image (92K GIF file)]
upstream sequences, pJJ444/446 (Fig. 1 A), show strong activity in the cells that exhibit intracellular stain with anti-EMB-9 antisera. A construct
with 1.7 kb of 5
upstream sequences in a transcriptional
fusion with lacZ, pJJ318, has also been analyzed, both as
an extrachromosomal array and integrated into the genome. This construct has an expression pattern very similar to pJJ444 but often shows activity in two to four of the
most posterior intestinal cells in early larvae and has lower
activity in body wall muscles of late larvae and adults.
upstream sequences, which is more
than is required for transgenic rescue of mutant animals,
shows very little activity when used in a transcriptional fusion with lacZ, pJJ325 (Fig. 1 B). However, insertion of
lacZ into exon 13 of let-2 generates a translational fusion,
pJJ360, that shows strong activity in the same cells that stain
intracellularly with type IV collagen antisera. The presence of further 3
exons and flanking sequences, pJJ361,
have no effect on the observed expression pattern. The let-2
expression reporters, pJJ360/361, show the same spatial
and temporal expression patterns as the emb-9 pJJ444/446 reporter constructs.
-galactosidase and GFP activity can first be detected in body wall muscle cells of lima stage embryos (data
not shown). In embryos of all later stages,
-galactosidase
and GFP activity is strong in body wall muscle cells (Fig.
6, A and B), and no activity is seen in pharyngeal, intestinal, or hypodermal cells. In situ hybridization of C. elegans
embryos with a probe derived from emb-9 also shows localization in the body wall muscles (56). In L1 larvae (Fig.
6 D), body wall muscle cells continue to show activity, and
activity is also seen in the ring ganglion (presumptive GLR
cells), head mesodermal cell, coelomocytes, and intestinal and anal muscles. Beginning at the L2 larval stage, the distal tip cells of the developing gonad show strong activity
(Fig. 6 C). In L3-L4 animals, the level of activity in the distal tip cells often appears to be greater than that seen in
the body wall muscles (Fig. 6 E). In L4 animals, spermathecal cells begin to show
-galactosidase activity, and this activity can still be seen in adults (Fig. 6 F). In adult animals,
type IV collagen reporter activity is seen in vulval and
uterine muscles (Fig. 6 F), body wall muscles (Fig. 6 G), as
well as distal tip cells (Fig. 6 H).
Fig. 6.
Expression of type IV collagen genes in C. elegans. Expression reporters for emb-9 and let-2 show activity in the same cells and
at the same times. (A) A 1 1/2-fold embryo showing GFP activity from the emb-9 reporter pJJ446 in body wall muscle cells. (B) A twofold embryo showing -galactosidase activity from pJJ318 in body wall muscle cells. (C) An L2 larva showing
-galactosidase activity from
pJJ318 in the distal tip cells (dtc) of the gonad. (D) An L1 larva showing
-galactosidase activity from pJJ444 in body wall muscle cells
and ring ganglion cells (rg). (E) A late L3 larva showing
-galactosidase activity from pJJ444 in the distal tip cells (dtc) of the gonad. (F)
A young adult animal showing
-galactosidase activity from pJJ318 in spermathecal cells (sp) and vulval muscles (vul). (G) A young
adult animal showing GFP activity from pJJ446 in body wall muscle cells. (H) An adult animal showing GFP activity from pJJ446 in a
distal tip cell (dtc) of the gonad.
[View Larger Version of this Image (111K GIF file)]
Fig. 7.
Distribution of HA-tagged EMB-9 expressed from the unc-54 body wall muscle myosin promoter construct pJJ414. Animals
were stained with anti-HA monoclonal antibody (A-C, G, and H), and with anti-LET-2 antiserum (D-F, I, and J). (A) In a 1 1/2-fold embryo, anti-HA staining colocalizes in body wall muscle cells with LET-2 (D). (B) In a threefold embryo, the HA-tagged EMB-9 is retained within the body wall muscle cells, while LET-2 is seen in basement membranes (E). (C) In a fourfold embryo, a small amount of
HA-tagged EMB-9 can be seen in the pharyngeal basement membrane, while LET-2 staining of basement membranes is intense (F). In
L2-L3 larvae, HA-tagged EMB-9 is seen in pharyngeal (p) and intestinal (i) basement membranes (G and H) and can also be detected
under body wall muscle quadrants and around the gonad. Animals that do not carry the transgenic array, and do not stain with anti-HA
antibody, are indicated with arrowheads. Staining of the same animals with anti-LET-2 (I and J) shows that the HA-tagged EMB-9 colocalizes with LET-2.
[View Larger Version of this Image (55K GIF file)]
Fig. 8.
Transgenic rescue
of emb-9(g23cg45) embryos
with an unc-54 promoter:
emb-9 construct. (A) An L2
larva carrying the unc-54: emb-9:GFP reporter pJJ429.
The GFP activity is seen only
in body wall muscle cells.
Muscle arm projections to
the nerve ring are indicated
(ma). (B-D) Homozygous
emb-9(g23cg45) embryos
carrying the unc-54 promoter:emb-9 construct pJJ427
show anti-EMB-9 staining of
basement membranes covering the pharynx (p), intestine
(i), and gonad and under
body wall muscle quadrants
(bwm). In the absence of the
rescuing construct, embryos
that are homozygous for the putative null mutation emb9(g23cg45) show no staining
with anti-EMB-9 antisera.
[View Larger Version of this Image (82K GIF file)]
Discussion
1 and
2(IV) collagen chains of C. elegans, EMB-9 and LET-2.
Type IV collagen was first detectable within body wall muscle cells of C. elegans embryos just beginning morphogenesis. Consistent with this result, the temperature-sensitive
periods for both type IV collagen genes, emb-9 and let-2,
also begin at this time (23,40; Kramer, J., unpublished results). Soon after, at the comma stage, secreted type IV
collagen begins to form stripes under the body wall muscle
quadrants. At approximately the same time, type IV collagen first appears around the primordial pharynx and intestine. By the end of embryogenesis, type IV collagen underlies the body wall muscle cells and surrounds the
pharynx, intestine, and gonad primordium. The same general pattern of type IV collagen localization continues in
larval and adult animals.
1 and
2-like type IV collagen
chains, respectively (19), they may assemble into a single
heterotrimeric collagen molecule as do the vertebrate
1
and
2 chains. These results are consistent with the notion
that EMB-9 and LET-2 function in a single heterotrimeric
type IV collagen molecule.
Fig. 9.
Diagram of the embryonic MS blastomere lineage showing type IV collagen-expressing cells. Cells
that express type IV collagen are shown as filled circles. Cells that divide during
larval development (M, Z1,
Z4) are circled at the bottom
of the figure, and their progeny that express type IV collagen are indicated. Cell fates are indicated as: bm, body
wall muscle; cc, coelomocyte;
dtc, distal tip cell; hm, head
mesodermal cell; im, intestinal muscle; ph, pharynx; rg,
ring ganglion; sp, spermatheca; um, uterine muscle; vm,
vulval muscle. Several body
wall muscle cells derived from the C and D blastomeres and
one body wall, one intestinal,
and two anal muscles derived
from the AB blastomere also
express type IV collagen. The
lineage was modified from
Sulston et al. (55).
[View Larger Version of this Image (29K GIF file)]
1 or
2(IV) genes, emb-9 or let-2, even
though these tissues are covered by basement membranes
that contain their products. Also, type IV collagen antisera
stain the entire surface of the gonadal basement membrane, even though only a few cells located at the distal
and proximal ends of the gonad express the type IV collagen genes. We demonstrated that
1(IV) chains synthesized in body wall muscle can assemble into basement
membranes on these other tissues. Additionally, we found
that expression of functional type IV collagen only in body
wall muscles is sufficient for C. elegans to complete development and be at least partially fertile.
Received for publication 17 February 1997 and in revised form 22 March 1997.
1. Abbreviations used in this paper: dH2O, distilled water; GFP, green fluorescent protein; HA, hemagglutinin; NDS, normal donkey serum.We wish to thank David Miller (Vanderbilt University, Nashville, TN) for providing antibodies and Andrew Fire (Carnegie Institute, Baltimore, MD) for providing expression vectors.
This work was supported by National Institutes of Health grant HD22027 (J.M. Kramer) and NRSA GM15268 (P.L. Graham).
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