1 Center for Biochemistry, Medical Faculty, University of Cologne, D-50931
Cologne, Germany
2 Institute for Neurophysiology, Medical Faculty, University of Cologne, D-50931
Cologne, Germany
* Author for correspondence (e-mail: neil.smyth{at}uni-koeln.de)
Accepted 26 November 2002
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Summary |
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Key words: Laminin-nidogen complex, Basement membrane permeability, Epithelial differentiation
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Introduction |
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Nidogen-1, a sulphated 150 kDa glycoprotein, co-purifies with laminin-1
upon EDTA extraction (Paulsson et al.,
1987). The nidogen-binding site on laminin-1 has been localized to
laminin EGF-like (LE) module 4, which has subsequently been determined at
atomic resolution (Stetefeld et al.,
1996
; Baumgartner et al.,
1996
). It was shown that this interaction might be significant for
proper basement membrane formation in organ cultures, as the presence of
antibodies raised against the nidogen-binding site on laminin-1 disrupted the
basement membrane and reduced branching epithelial morphogenesis in a number
of different organs (Ekblom et al.,
1994
; Kadoya et al.,
1997
). The results suggested that the formation of the
laminin/nidogen-1 complex would be a key event during basement membrane
deposition and epithelial differentiation. Detailed analysis of the sites of
laminin-1 and nidogen-1 gene expression by in situ hybridization
(Dong and Chung, 1991
;
Thomas and Dziadek, 1993
;
Ekblom et al., 1994
;
Fleischmajer et al., 1995
)
have revealed that laminin-1 is predominantly produced by epithelial cells,
whereas nidogen-1 is secreted by mesenchymal cells. The binding of mesenchymal
nidogen-1 to epithelial laminin-1 is believed to occur at the interface
between epithelial and mesenchymal tissues and possibly dictate the site of
basement membrane formation (Dziadek et al., 1995).
Surprisingly, the loss of nidogen-1 in the mouse by homologous
recombination does not result in any gross changes in development, and the
basement membranes in these animals appear to be structurally normal. However,
it could be shown that the related protein (nidogen-2) was redistributed
and/or upregulated in certain tissues in the nidogen-1-null animals. Nidogen-2
was initially isolated from an osteoblast-like cell line and shown to have
27.4% identity to nidogen-1 at the amino acid level
(Kimura et al., 1998).
Recombinant human nidogen-2 has a similar structure to nidogen-1 but binds to
the nidogen-binding site
1III4 of the murine laminin
1 chain
with a 100- to 1000-fold lower affinity than murine nidogen-1
(Kohfeldt et al., 1998
). Like
nidogen-1, nidogen-2 also binds collagens I and IV, and perlecan in vitro
(Kohfeldt et al., 1998
). These
results suggested that nidogen-2 could take over certain of the roles of
mammalian nidogen-1 in its absence, but they did not explain why there
appeared to be no basement membrane defect in Caenorhabditis elegans
upon the loss of the single nidogen family member in this species
(Kim and Wadsworth, 2000
). The
possibility cannot be ruled out that, in the original perturbation
experiments, the binding of antibodies prevented other interactions of the
laminin molecule by steric hindrance caused by the large immunoglobulin
molecule.
To study the importance of laminin-nidogen binding in an alternative and less artefactual manner, we attempted to block the interaction by the introduction of an excess of recombinantly expressed nidogen-binding sites during basement membrane formation in F9 derived embryoid bodies. F9 cells have a very limited differentiation ability and so offer a simpler model of basement membrane development than that seen in vivo or in organ culture systems, presumably with a lesser ability to compensate for molecular defects. Here, we show that differentiation of such cells in the presence of the exogenously added or endogenously recombinantly expressed nidogen-binding site led to defects in the basement membrane, increased permeability and abnormal differentiation suggesting that the laminin-nidogen interaction might be highly significant in epithelial development.
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Materials and Methods |
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Cell culture
Mouse teratocarcinoma F9 (CRL 1720; American Type Culture Collection; DSM
ACC 112) cells were maintained in DMEM containing 200 U ml-1
penicillin, 200 µg ml-1 streptomycin, 2 mM L-glutamine and 10%
foetal calf serum (Gibco BRL) and grown at 37°C in a humidified incubator
with a 5% CO2 atmosphere. For embryoid body cultures, F9 cells were
maintained in normal growth medium supplied with 5x10-8 M
all-trans-retinoic acid (R-2625; Sigma-Aldrich) for 12 days using the
cell spin system (Integra Bioscience).
1x106 F9 cells were electroporated with 5 µg circular plasmid DNA. Selection was carried out with 1 mg ml-1 G418 (Gibco BRL) and G418 resistant clones were picked and grown in 48-well plates. These were screened for expression by immunoblotting with the Bio M2 monoclonal antibody directed against the FLAG epitope (F9291; Sigma-Aldrich).
Immunofluorescence staining
Rabbit polyclonal antisera to laminin-1 (M.P.), perlecan and nidogen-2 (R.
Timpl, Max Planck Institute for Biochemistry, Munich), a rat monoclonal
anti-nidogen G2 domain antibody (MAB 1884; Chemicon) and a rat monoclonal
TROMA-1 antibody (Kemler et al.,
1981) were used as primary antibodies. As secondary antibodies,
fluorescein-conjugated goat anti-rabbit IgG, Cy3TM-conjugated goat
anti-rabbit IgG or Cy3TM-conjugated goat anti-rat IgG (all from Jackson
ImmunoResearch Laboratories) were used. All stainings were performed in PBS,
pH 7.4. The embryoid bodies were either fixed with 1% paraformaldehyde for 30
minutes at room temperature and embedded in Tissue tek (Sakura) for subsequent
cryosectioning or fixed in ice-cold methanol:acetone (7:3) for 1 hour at
-20°C, washed with 0.1% Triton-X 100, stored in PBS at 4°C and used
for wholemount immunostaining.
7 µm cryosections were briefly fixed with 0.5% paraformaldehyde, blocked with 5% normal goat serum (ICN Biomedicals) and 0.2% Tween for 30 minutes, then the primary antibody was applied for 1 hour followed by three washes with the blocking solution. The sections were incubated with the secondary antibody for 45 minutes, washed, mounted in fluorescent mounting medium (DAKO) and examined using a Axiophot microscope (Carl Zeiss) equipped with a fluorescent light source. Whole embryoid bodies were blocked in 10% milk powder for 1 hour, incubated with the primary antibody for 1.5 hours on a rocking device, washed with 0.01% Triton-X100, incubated with the second antibody for 1 hour, washed again and analysed using a laser scanning confocal microscope (LSM 410; Carl Zeiss) with a Plan-Neofluar 25x/0.8NA objective and 4.5x zoom.
Immunoblot analysis
To determine the expression level of 1-FLAG fusions by transfected
F9 cells, culture supernatants were TCA precipitated and separated by SDS-PAGE
on a 15% polyacrylamide gel. Proteins were transferred onto nitrocellulose
membranes and probed with 10 µg ml-1 BioM2 monoclonal antibody
against the FLAG epitope (F9291; Sigma-Aldrich) and a rabbit polyclonal
antiserum against mouse BM40 (Nischt et
al., 1991
). For determination of the endogenous level of laminin-1
and nidogen-1, embryoid bodies were lysed in SDS sample buffer
(Laemmli, 1970
), submitted to
SDS-PAGE on a 3-10% polyacrylamide gel in the presence of 5%
ß-mercaptoethanol. Proteins were transferred to nitrocellulose and
incubated with a rabbit polyclonal antiserum against laminin-1, a rat
monoclonal antibody against the nidogen (entactin) G2 domain (MAB 1884;
Chemicon) and a mouse monoclonal antibody against human actin (sc-8432; Santa
Cruz Biotechnology). As secondary reagents, either streptavidin-biotinylated
horseradish peroxidase (HRP) complexes (RPN1051; Amersham Life Science) or HRP
conjugated immunoglobulins from swine anti-rabbit (P0399; DAKO), rabbit
anti-rat (P0450; DAKO) or rabbit anti-mouse (P0260; DAKO) immunoglobulin G
antisera were used. Immunoreactive proteins were detected using the enhanced
chemiluminescent detection system.
Diffusion assay
At day 8 of culture, embryoid bodies were rinsed in E1 solution (135 mM
NaCl, 5.4 mM KCl, 1.8 mM CaCl2.2H2O, 1 mM
MgCl2.6H2O, 10 mM glucose, 10 mM HEPES pH 7.5),
transferred into 10 µM rhodamine dextran solution of either 10 kDa (neutral
D-1824; Molecular Probes) or 70 kDa (neutral D-1841; Molecular Probes) with a
hydrodynamic radius comparable to those of 40 kDa and 280 kDa globular
proteins. After incubation for 5 minutes, the embryoid bodies were briefly
washed twice in E1 solution to reduce background staining and analysed with a
laser scanning microscope (LSM 410; Carl Zeiss) by means of the optical probe
technique (Wartenberg et al.,
1998a). The diffusion coefficient D was calculated based
on the Einstein and Smoluchowski equation D=x2
÷ 2t, where x describes the distance of diffusion in
a distinct time period t
(Wartenberg et al.,
1998b
).
Treatment of wild-type F9 embryoid bodies with affinity-purified FLAG
fusion protein 1III3-5
The FLAG fusion protein 1III3-5 was expressed in human embryonic
kidney 293-EBNA cells using the CMV promoter
(Smyth et al., 2000
), loaded
onto a anti-FLAG M2 affinity gel (A1205; Sigma-Aldrich) column and eluted with
100 µg ml-1 FLAG® peptide (F3290; Sigma-Aldrich) following
the manufacturer's instructions. SDS-PAGE analysis of the purified protein
showed a single band after Coomassie staining (data not shown).
Untransfected F9 cells were grown to confluence, trypsinized and
transferred into the Cell Spin system (Integra Bioscience), where
all-trans-retinoic acid (R-2625; Sigma-Aldrich) was added to a
concentration of 5x10-8 M to induce differentiation. At day
two of culture, embryoid bodies were transferred into single wells of a
96-well plate filled with 100 µl normal cell culture medium supplemented
with affinity purified FLAG fusion protein 1III3-5 at a concentration
of 10 µg ml-1. Every day, the medium was changed and new protein
was added. At day 8 of culture, embryoid bodies were fixed and stained for
laminin-1.
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Results |
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For control purposes, F9 cells were either transfected with the empty
expression vector, a similar but inactive set of three LE domains also present
in laminin 1 (
1V1-3) or a mutant-binding-site-expressing
construct (
1III3-5)N802S. The LE domains
1V1-3 have no known
nidogen-binding activity but this fragment has a similar structure and size to
1III3-5 (Fig. 1).
However, biochemical studies have shown that Asn residue 802 is crucial for
nidogen binding, with the N802S change (present in the homologous region of
the laminin
2 chain) reducing the interaction between nidogen and its
binding domain upon laminin by
5000 times
(Poschl et al., 1996
).
|
F9 cells electroporated with the empty expression vector and the FLAG
fusion constructs were selected for resistance to G418 and surviving clones
isolated. Expressing clones were identified by immunoblotting with a
monoclonal antibody against the FLAG epitope (Bio M2, Sigma-Aldrich;
Fig. 2). For comparison of
protein expression levels in these cells, loading of the medium was
standardized by the detection of BM40, a calcium-binding extracellular matrix
protein (Nischt et al., 1991)
that is also secreted by F9 cells
(Nishiguchi et al., 1996
). For
further studies, a representative clone containing the empty expression vector
was chosen, along with two independent clones (
1III3-5/A and
1III3-5/B) that express the nidogen-binding site, one expressing the
three LE domains
1V1-3, and a fifth producing the mutated binding site
1III3-5mut, all at similar levels.
|
Analysis of the deposition of basement-membrane proteins in the F9
embryoid bodies
The embryoid body system offers a well-studied model of basement membrane
deposition (Prehm et al.,
1982; Cooper et al.,
1983
). Pluripotent F9 cells
(Alonso et al., 1991
) form
parietal or visceral endoderm only upon treatment with, for example, retinoic
acid (Strickland and Mahdavi,
1978
) or cAMP (Hogan et al.,
1983
) accompanied by a 5-20-fold increase in the synthesis of
basement membrane proteins. These are secreted to the extracellular surface
and deposited between the outer endodermal cell layer and the inner core of
the differentiating embryoid body. These rudimentary basement membrane
structures contain laminin-1, nidogen-1, perlecan and collagen IV
(Carlin et al., 1983
;
Durkin et al., 1986
;
Kleinman et al., 1987
;
Chakravarti et al., 1993
).
The cell clones were expanded and used to produce embryoid bodies in suspension culture. The embryoid bodies from each of the clones grew at a comparable rate and showed a similar gross morphology. After 12 days in culture, these were harvested, cryo-embedded, sectioned and stained with a rabbit polyclonal antibody raised against laminin-1. Sections from two sets of independent experiments and at least 20 embryoid bodies from each culture were examined for the presence of a subepithelial basement membrane. A near-continuous signal for laminin-1 at the periphery of the embryoid body underlying the outer layer of differentiated endodermal cells was considered to be a correctly formed basement membrane, whereas a disrupted, intermittent staining (covering less than 50% of the periphery) was taken as a sign of perturbation.
By these criteria, a formed basement membrane was produced in the empty
vector control, 1V1-3 and
1III3-5mut expressing embryoid bodies
(Fig. 3B). In the
1III3-5/A and
1III3-5/B expressing embryoid bodies the laminin-1
signal had a disrupted appearance, with little continuous staining and
immunoreactive material was often deposited irregularly within the more
central regions of the aggregates. This punctate laminin-1 staining pattern
suggests a disruption of the basement membrane and/or a defect in cellular
differentiation. To analyse the former further, we then stained for another
basement membrane component, perlecan. This showed a similar change in its
staining pattern in the
1III3-5 expressing embryoid bodies as described
for laminin-1 (Fig. 3A).
|
A double-staining, using a rabbit polyclonal antibody for laminin-1
(laminin subunits 1, ß1,
1 and nidogen-1) and a
nidogen-1-specific monoclonal antibody reacting with the G2 domain of mouse
nidogen-1, was used to provide evidence of a successful competition for
nidogen-1 binding (Fig. 3A).
This revealed colocalization of both proteins in each of the control forms of
embryoid bodies, whereas the
1III3-5 expressing embryoid bodies showed
imperfect colocalization indicating the presence of laminin molecules not
bound with nidogen. Human nidogen-2 has a similar binding repertoire to
nidogen-1 with the exception of a markedly weaker interaction with the murine
laminin
1III4 module. To see whether F9-derived embryoid bodies
produced and incorporated nidogen-2 into a basement membrane, they were double
labelled using a rabbit polyclonal specific for nidogen-2 and the
nidogen-1-specific monoclonal antibody. These showed that nidogen-2 was
expressed and was seen present with a similar staining pattern to nidogen-1,
being incorporated only into a basement membrane in the three control clones
(Fig. 3C).
To ensure that the disrupted appearance of the basement membrane proteins
in 1III3-5 expressing embryoid bodies is caused by efficient blocking
of the laminin/nidogen-1 complex and not by changes in nidogen-1 production,
mRNA and protein blots were performed. Total RNA was isolated from embryoid
bodies and hybridized with cDNA probes for mouse nidogen-1 and human GAPDH.
This revealed an similar levels of nidogen-1 mRNA expression in all
sets of embryoid bodies (results not shown). When the embryoid bodies were
lysed in SDS sample buffer (Laemmli,
1970
), no major variation in laminin-1 or nidogen-1 could be
observed between control,
1V1-3 and
1III3-5mut or
1III3-5
expressing embryoid bodies (Fig.
4).
|
Altered permeability in embryoid bodies expressing the nidogen
binding site
An important biological role of the basement membrane is its function as a
permeability barrier. To test whether the disruption of the laminin/nidogen-1
interaction resulted in an altered permeability of the embryoid bodies, a
diffusion assay was established. Embryoid bodies were incubated with
rhodamine-labelled 10 kDa and 70 kDa dextrans, which correspond in their
hydrodynamic radius to globular proteins of 40 kDa and
280 kDa,
respectively. After 5 minutes the distance of polymer diffusion into the
embryoid bodies was determined by confocal microscopy
(Fig. 5A) and diffusion
coefficients were calculated to compare the permeability properties of the
different clones (Fig. 5B).
These diffusion coefficients of
5x10-8 cm2
sec-1 for control or
1V1-3 expressing embryoid bodies, and
1x10-7 cm2 sec-1 for the
1III3-5
expressing clones
1III3-5/A and
1III3-5/B, fit to inhibited and
facilitated diffusion, respectively, as previously measured for
embryonic-stem-cell-derived embryoid bodies
(Wartenberg et al., 1998b
).
This shows that the control and
1V1-3 expressing embryoid bodies have
effective diffusion barriers for both 10 kDa and 70 kDa dextrans, whereas the
expression of the nidogen-binding site results in drastically increased
diffusion rates (Fig. 5). This
suggests that the disruption of the basement membrane upon expression of the
nidogen-binding site correlates with an increased permeability of the embryoid
body.
|
Expression of the nidogen-1-binding site results in altered
differentiation
The differentiation of F9 cell aggregates in the presence of retinoic acid
differentiation induces the formation of an external polarized endoderm. To
discover whether this is seen in the transfected F9 cells, TROMA-1
(cytokeratin 8) expression was analysed. This protein is normally synthesized
in mature endoderm and would be expected to be seen in the outermost cell
layer (Oshima, 1982). The
control forms of embryoid bodies, including that expressing the mutated
binding domain, showed the expected strong staining in the flattened surface
cell layer. The embryoid body was covered with TROMA-1-positive cells but they
were only occasionally present internally. However, where the nidogen-binding
site was expressed, TROMA-1 signals occurred widely and were not merely
restricted to the outer cells (Fig.
6).
|
Exogenous addition of the nidogen-binding site alters laminin
deposition in wild-type embryoid bodies
Recombinant expression, genetic manipulation and cell cloning might lead to
artefacts independent of the effect of the induced protein. Although two
independent clones gave the same result, changes not seen with our three
different types of control, we attempted to further exclude the possibility of
such artefacts by the exogenous addition of the fusion protein to
wild-type-derived embryoid bodies. The laminin FLAG fusion protein
1III3-5 was recombinantly expressed in 293-EBNA cells, purified and
added to the medium of wild-type embryoid bodies at a concentration of 10
µg ml-1. Non-supplemented controls were also cultured. Both
supplemented and non-supplemented embryoid bodies grew in a similar manner to
those described earlier. Whole-mount staining for laminin-1 was analysed by
confocal microscopy and revealed in wild-type embryoid bodies a network-like
laminin-1 pattern (Fig. 7B)
similarly organized to that observed on the cell surface of embryonic stem
cells (Henry et al., 2001
),
whereas embryoid bodies treated with exogenous
1III3-5 FLAG fusion
protein showed a highly disrupted punctate laminin-1 signal
(Fig. 7A).
|
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Discussion |
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We chose to use a method of continuous agitation for growth of the F9
aggregates, rather than the more conventional hanging drop method, after
preliminary experiments showed that the control embryoid bodies in such a
system were far more uniform in size and growth characteristics. Cell
differentiation of these embryonic carcinoma cells is limited, in embryoid
bodies this results in an ordered outer single endodermal layer sitting upon a
basement membrane. The disorganization in the basement membrane and the
reduced colocalization between laminin and nidogen-1 in embryoid bodies
expressing the binding site suggest the successful competition by the
recombinant nidogen-binding-site fragment for the binding to nidogen-1.
Indeed, where laminin is completely absent, nidogen appears to be partially
lost from the tissue (De Arcangelis et
al., 1996; Smyth et al.,
1999
). In our competition experiments, redistribution of nidogen-1
was more marked than actual loss from the embryoid body (Figs
3,
4). This might indicate that
nidogen-1 released from laminin finds other binding partners, with which it
interacts using an independent site. The possibility cannot be excluded that
the severity of the defects described here were exacerbated by the culture
system used, and that the embryoid bodies were placed under sheer stress
during spinner culture. This could result in the changes seen here occurring
owing to a less stable rather than unformed basement membrane, and indeed no
alteration was described in embryonic stem cells carrying genetic defects in
the nidogen-binding site (Willem et al.,
2002
) when grown in hanging drops. However, in initial experiments
with the F9 derived cells cultured in hanging drops, we produced changes
similar to those described from the spinner culture.
Disruption or weakness of the basement membrane has been a consistent
effect seen upon the loss of other basement membrane components and receptors,
although only the lack of laminin 1 or ß1 integrin have led to a
complete absence of basement membrane formation. That receptors for individual
basement membrane components are significant in the organization of the
basement membrane has been shown by basement membrane defects in
-dystroglycan and integrin deletion mice. Laminin
1 chain
expression, which is required for laminin secretion, is regulated by ß1
integrin (Aumailley et al.,
2000
; Li et al.,
2002
). Hence, basement membrane defects in the absence of ß1
integrin might be due to the lack of laminin rather than to abnormalities in
basement membrane organization per se. Our results, together with those
derived from the antibody perturbation experiments, suggest strongly that the
nidogen-1-binding region of laminin plays a highly significant role in the
molecular organization of the basement membrane.
Basement membranes with varying ultrastructure have been identified in vivo
(Ogawa et al., 1999;
Eyden, 1999
), and
developmental stages have been shown in basement membrane formation. For
example, in the six-day-old mouse embryo, although a classical basement
membrane has formed under the ectoderm in the extraembryonic egg cylinder, the
basement membrane is unstructured within the embryo itself even though
laminin-1 is deposited in a linear pattern
(Miosge et al., 1993
). In
fact, only upon the full ultrastructural development of the basement membrane
is nidogen-1 found by immunogold histochemistry, suggesting that in the early
embryo nidogens might have a role in the maturation of basement membranes
(Miosge et al., 2000
), albeit
one that is not crucial for embryonic survival or that might be compensated
for by other molecules.
F9 cells also produce and incorporate nidogen-2 into their basement
membrane, but this did not prevent basement membrane disorganisation in the
presence of the nidogen-binding site. Our results indicate that nidogen-2
indeed has an affinity for this site despite that fact previous results
suggested the use of an alternative binding surface upon the laminin trimer
(Kohfeldt et al., 1998).
In glomerular capillaries, the basement membrane has been identified as the
primary filtration barrier to graded dextrans
(Caulfield and Farquhar,
1974). Many studies have been carried out characterizing the role
of basement membrane components, such as proteoglycans and collagen IV in the
permselectivity process (Morello et al.,
2001
; Tryggvason and
Wartiovaara, 2001
). The function of the basement membrane as a
barrier to molecules diffusing into the embryoid body was tested with 10 kDa
and 70 kDa dextran molecules. Both sizes of dextran were excluded from the
control embryoid bodies with diffusion coefficients clearly indicating higher
permeability for embryoid bodies expressing the nidogen-binding site than for
controls. This might either be caused directly by the disruption of the
basement membrane proteins or be produced by secondary changes in the
development of the outer epithelial layers.
To study the consequences of basement membrane disruption on cellular
differentiation, the embryoid bodies were stained for TROMA-1, which is
synthesized by mature endoderm (Oshima,
1982). All embryoid body types had TROMA-1 positive cells, but
there was a marked alteration in their localization. Although control embryoid
bodies revealed the expected strong peripheral staining,
1III3-5
producing clones show patchy staining distributed over the embryoid bodies and
a reduction in staining of the marginal cells. Ectopic (stromal) TROMA-1
production in the
1III3-5 expressing embryoid bodies indicates a
separation of expression of this usually epithelium-specific gene from
epithelial formation. In F9 embryoid bodies, changes in the expression pattern
of integrins (Morini et al.,
1999
), Indian hedgehog (Becker
et al., 1997
) and extracellular matrix components such as
laminin-1 and collagen IV(
1)
(Rogers et al., 1990
)
characteristically occur concurrently with the formation and subsequent
organization of the epithelium. In the absence of laminin, parietal endoderm
differentiation occurs in embryonic-stem-cell-derived embryoid bodies with the
peripheral cells showing the morphological markers and high matrix production
usual for such cells. However, parietal endodermal cells are found in greater
numbers and as here in a disorganized manner through the embryoid body
(Murray and Edgar, 2001
).
Hence not only is laminin itself a prerequisite for ordered parietal
endodermal differentiation but its regulated deposition is also needed.
Cytokeratin 8 (the TROMA-1 antigen) is expressed by embryonic epithelia but,
in its absence, differentiation in these tissues is able to occur
(Baribault et al., 1994
;
Brock et al., 1996
). It is
possible that the endoderm formed upon the surface of the
nidogen-binding-site-expressing embryoid bodies could in other respects be
normal.
A mouse line in which the nidogen-binding site (LE module 1III4) has
been deleted by homologous recombination has been very recently described
(Willem et al., 2002
). These
animals show far more severe phenotypes than that seen in the nidogen-1
knockout. Even so, basement membrane defects often appeared to be discrete or
confined to certain tissues, suggesting that the requirement for the
laminin-nidogen interaction is not needed for the formation of all basement
membranes. The early lethality of
40% of mutant animals lacking the
nidogen-binding site could be a defect, similar to that described here, which
is partially overridden by a compensatory mechanism in a proportion of
embryos. Furthermore, the lack of basement membrane alterations in C.
elegans mutants lacking nidogen suggests that requirements of a basement
membrane alter between tissues, through development and during evolution.
Together, the present results, the binding-site deletion
(Willem et al., 2002) and the
nidogen-1 gene inactivation
(Murshed et al., 2000
) suggest
that the roles of nidogen-1 can be compensated for. This is possibly through
the action of nidogen-2 because both proteins are generally found in the same
basement membranes (Kohfeldt et al.,
1998
; Miosge et al.,
2000
) and, in the absence of nidogen-1, there is obvious
redistribution of this protein in particular within the basement membranes of
striated muscles (Murshed et al.,
2000
). Although initial in vitro studies indicated that the
binding of the murine
1III3-5 region to human nidogen-2 to be at least
100 times weaker than that occurring with nidogen-1
(Kohfeldt et al., 1998
), it is
conceivable that this interaction, in vivo, is sufficient to provide this
compensation. Indeed, recent in vitro results suggest that the murine
nidogen-2 can bind recombinant and proteolytic fragments of laminin in a
manner comparable to murine nidogen-1, suggesting a species variation in
binding activity (Salmivirta et al.,
2002
). Alternatively other proteins might interact with this
region and affect the molecular structure of the basement membrane. One
further, less likely, possibility is that free nidogen is itself able to
direct aberrant differentiation, an effect not seen in the total absence of
this protein. The discrepancy between the in vitro results and the in vivo
deletion experiments might be a result of altering requirements for a stable
basement membranes in different differentiation systems. A resolution of the
apparent discrepancies between these experiments is likely to come from our
ongoing studies of the mice lacking both nidogen-1 and
nidogen-2 genes. However, it is evident that different tissues and
different basement membranes show varying susceptibility to the loss of the
laminin-nidogen interaction.
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Acknowledgments |
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References |
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Alonso, A., Breuer, B., Steuer, B. and Fischer, J. (1991). The F9-EC cell line as a model for the analysis of differentiation. Int. J. Dev. Biol. 35,389 -397.[Medline]
Aumailley, M., Wiedemann, H., Mann, K. and Timpl, R. (1989). Binding of nidogen and the laminin-nidogen complex to basement membrane collagen type IV. Eur. J. Biochem. 184,241 -248.[Abstract]
Aumailley, M., Battaglia, C., Mayer, U., Reinhardt, D., Nischt, R., Timpl, R. and Fox, J. W. (1993). Nidogen mediates the formation of ternary complexes of basement membrane components. Kidney Int. 43,7 -12.[Medline]
Aumailley, M., Pesch, M., Tunggal, L., Gaill, F. and Faessler, R. (2000). Altered synthesis of laminin 1 and absence of basement membrane component deposition in ß1 integrin-deficient embryoid bodies. J. Cell. Sci. 2,259 -268.
Baribault, H., Penner, J., Iozzo, R. V. and Wilson-Heiner, M. (1994). Colorectal hyperplasia and inflammation in keratin 8-deficient FVB/N mice. Genes Dev. 8,2964 -2973.[Abstract]
Baumgartner, R., Czisch, M., Mayer, U., Poschl, E., Huber, R.,
Timpl, R. and Holak, T. A. (1996). Structure of the nidogen
binding LE module of the laminin 1 chain in solution. J.
Mol. Biol. 257,658
-668.[CrossRef][Medline]
Becker, S., Wang, Z. J., Massey, H., Arauz, A., Labosky, P., Hammerschmidt, M., St-Jacques, B., Bumcrot, D., McMahon, A. and Grabel, L. (1997). A role for Indian hedgehog in extraembryonic endoderm differentiation in F9 cells and the early mouse embryo. Dev. Biol. 187,298 -310.[CrossRef][Medline]
Brock, J., McCluskey, J., Baribault, H. and Martin, P. (1996). Perfect wound healing in the keratin 8 deficient mouse embryo. Cell. Motil. Cytoskeleton 35,358 -366.[CrossRef][Medline]
Carlin, B. E., Durkin, M. E., Bender, B., Jaffe, R. and Chung,
A. E. (1983). Synthesis of laminin and entactin by F9 cells
induced with retinoic acid and dibutyryl cyclic AMP. J. Biol.
Chem. 258,7729
-7737.
Caulfield, J. P. and Farquhar, M. G. (1974).
The permeability of glomerular capillaries to graded dextrans. Identification
of the basement membrane as the primary filtration barrier. J. Cell
Biol. 63,883
-903.
Chakravarti, S., Hassell, J. R. and Phillips, S. L. (1993). Perlecan gene expression precedes laminin gene expression during differentiation of F9 embryonal carcinoma cells. Dev. Dyn. 197,107 -114.[Medline]
Cooper, A. R., Taylor, A. and Hogan, B. L. (1983). Changes in the rate of laminin and entactin synthesis in F9 embryonal carcinoma cells treated with retinoic acid and cyclic AMP. Dev. Biol. 99,510 -516.[Medline]
De Arcangelis, A., Neuville, P., Boukamel, R., Lefebvre, O., Kedinger, M. and Simon-Assmann, P. (1996). Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation. J. Cell Biol. 133,417 -430.[Abstract]
Dong, L. J. and Chung, A. E. (1991). The expression of the genes for entactin, laminin A, laminin B1 and laminin B2 in murine lens morphogenesis and eye development. Differentiation 48,157 -172.[Medline]
Durkin, M. E., Phillips, S. L. and Chung, A. E. (1986). Control of laminin synthesis during differentiation of F9 embryonal carcinoma cells. A study using cDNA clones complementary to the mRNA species for the A, B1 and B2 subunits. Differentiation 32,260 -266.[Medline]
Dziadek, M. (1995). Role of laminin-nidogen complexes in basement membrane formation during embryonic development. Experientia 51,901 -913.[Medline]
Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H. Y.,
Kadoya, Y., Chu, M. L., Mayer, U. and Timpl, R. (1994). Role
of mesenchymal nidogen for epithelial morphogenesis in vitro.
Development 120,2003
-2014.
Eyden, B. (1999). Perivascular amorphous matrices containing laminin and type IV collagen not organized as a conventional basal lamina: identification by electron microscopy and implications for the control of cell biological processes. Ultrastruct. Pathol. 23,355 -357.[CrossRef][Medline]
Fleischmajer, R., Schechter, A., Bruns, M., Perlish, J. S., Macdonald, E. D., Pan, T. C., Timpl, R. and Chu, M. L. (1995). Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro. J. Invest. Dermatol. 105,597 -601.[Abstract]
Fox, J. W., Mayer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H., Mann, K., Timpl, R., Krieg, T. and Engel, J. (1991). Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV. EMBO J. 10,3137 -3146.[Abstract]
Henry, M. D., Satz, J. S., Brakebusch, C., Costell, M.,
Gustafsson, E., Fassler, R. and Campbell, K. P. (2001).
Distinct roles for dystroglycan, ß1 integrin and perlecan in cell surface
laminin organization. J. Cell Sci.
114,1137
-1144.
Hogan, B. L., Barlow, D. P. and Tilly, R. (1983). F9 teratocarcinomas as a model for the differentiation of parietal and visceral endoderm in the mouse embryo. Cancer Surv. 2,115 -140.
Hopp, T. P., Prickett, K. S., Price, V. L., Libby, R. T., March, C. J., Cerretti, D. P., Urdal, D. L. and Conlon, P. J. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology 6,1204 -1210.
Kadoya, Y., Salmivirta, K., Talts, J. F., Kadoya, K., Mayer, U.,
Timpl, R. and Ekblom, P. (1997). Importance of nidogen
binding to laminin 1 for branching epithelial morphogenesis of the
submandibular gland. Development
124,683
-691.
Kang, S. H. and Kramer, J. M. (1990). Nidogen
is nonessential and not required for normal type IV collagen localization in
Caenorhabditis elegans. Mol. Biol. Cell
11,3911
-3923.
Kemler, R., Brulet, P., Schnebelen, M. T., Gaillard, J. and Jacob, F. (1981). Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development. J. Embryol. Exp. Morphol. 64,45 -60.[Medline]
Kim, S. and Wadsworth, W. G. (2000).
Positioning of longitudinal nerves in C. elegans by nidogen.
Science 288,150
-154.
Kimura, N., Toyoshima, T., Kojima, T. and Shimane, M. (1998). Entactin-2: a new member of basement membrane protein with high homology to entactin/nidogen. Exp. Cell Res. 241, 36-45.[CrossRef][Medline]
Kleinman, H. K., Ebihara, I., Killen, P. D., Sasaki, M., Cannon, F. B., Yamada, Y. and Martin, G. R. (1987). Genes for basement membrane proteins are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues. Dev. Biol. 122,373 -378.[Medline]
Kohfeldt, E., Maurer, P., Vannahme, C. and Timpl, R. (1997). Properties of the extracellular calcium binding module of the proteoglycan testican. FEBS Lett. 414,557 -561.[CrossRef][Medline]
Kohfeldt, E., Sasaki, T., Gohring, W. and Timpl, R. (1998). Nidogen-2: a new basement membrane protein with diverse binding properties. J. Mol. Biol. 282,99 -109.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Li, S., Harrison, D., Carbonetto, S., Fassler, R., Smyth, N.,
Edgar, D. and Yurchenco, P. D. (2002). Matrix assembly,
regulation, and survival functions of laminin and its receptors in embryonic
stem cell differentiation. J. Cell Biol.
157,1279
-1290.
Mann, K., Deutzmann, R. and Timpl, R. (1988). Characterization of proteolytic fragments of the laminin-nidogen complex and their activity in ligand-binding assays. Eur. J. Biochem. 178,71 -80.[Abstract]
Mayer, U., Nischt, R., Poschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y. and Timpl, R. (1993). A single EGF-like motif of laminin is responsible for high affinity nidogen binding. EMBO J. 12,1879 -1885.[Abstract]
Miosge, N., Gunther, E., Becker-Rabbenstein, V. and Herken, R. (1993). Ultrastructural localization of laminin subunits during the onset of mesoderm formation in the mouse embryo. Anat. Embryol. 187,601 -605.[CrossRef][Medline]
Miosge, N., Kother, F., Heinemann, S., Kohfeldt, E., Herken, R. and Timpl, R. (2000). Ultrastructural colocalization of nidogen-1 and nidogen-2 with laminin-1 in murine kidney basement membranes. Histochem. Cell Biol. 113,115 -124.[CrossRef][Medline]
Mizushima, S. and Nagata, S. (1990). pEf-Bos, a powerful mammalian expression vector. Nucleic Acids Res. 18,5322 .[Medline]
Morello, R., Zhou, G., Dreyer, S. D., Harvey, S. J., Ninomiya, Y., Thorner, P. S., Miner, J. H., Cole, W., Winterpacht, A., Zabel, B. et al. (2001). Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome. Nat. Genet. 27,205 -208.[CrossRef][Medline]
Morini, M., Piccini, D., de Santanna, A., Levi, G., Barbieri, O. and Astigiano, S. (1999). Localization and expression of integrin subunits in the embryoid bodies of F9 teratocarcinoma cells. Exp. Cell Res. 247,114 -122.[CrossRef][Medline]
Murray, P. and Edgar, D. (2000). Regulation of
programmed cell death by basement membranes in embryonic development.
J. Cell Biol. 150,1215
-1221.
Murray, P. and Edgar, D. (2001). Regulation of laminin and COUP-TF expression in extraembryonic endodermal cells. Dev. Mech. 101,213 -215.[CrossRef]
Murshed, M., Smyth, N., Miosge, N., Karolat, J., Krieg, T.,
Paulsson, M. and Nischt, R. (2000). The absence of nidogen 1
does not affect murine basement membrane formation. Mol. Cell.
Biol. 20,7007
-7012.
Niimi, T. and Kitagawa, Y. (1997). Distinct
roles of mouse laminin ß1 long arm domains for
1ß1
1 trimer formation.
FEBS Lett. 400,71
-74.[CrossRef][Medline]
Nischt, R., Pottgiesser, J., Krieg, T., Mayer, U., Aumailley, M. and Timpl, R. (1991). Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40. Eur. J. Biochem. 200,529 -536.[Abstract]
Nishiguchi, S., Sakuma, R., Nomura, M., Zou, Z., Jearanaisilavong, J., Joh, T., Yasunaga, T. and Shimada, K. (1996). A catalogue of genes in mouse embryonal carcinoma F9 cells identified with expressed sequence tags. J. Biochem. Tokyo 119,749 -767.[Abstract]
Ogawa, S., Ota, Z., Shikata, K., Hironaka, K., Hayashi, Y., Ota, K., Kushiro, M., Miyatake, N., Kishimoto, N. and Makino, H. (1999). High-resolution ultrastructural comparison of renal glomerular and tubular basement membranes. Am. J. Nephrol. 19,686 -693.[CrossRef][Medline]
Oshima, R. G. (1982). Developmental expression
of murine extra-embryonic endodermal cytoskeletal proteins. J.
Biol. Chem. 257,3414
-3421.
Paulsson, M., Aumailley, M., Deutzmann, R., Timpl, R., Beck, K. and Engel, J. (1987). Laminin-nidogen complex. Extraction with chelating agents and structural characterization. Eur. J. Biochem. 166,11 -19.[Abstract]
Poschl, E., Mayer, U., Stetefeld, J., Baumgartner, R., Holak, T.
A., Huber, R. and Timpl, R. (1996). Site-directed mutagenesis
and structural interpretation of the nidogen binding site of the laminin
1 chain. EMBO J.
15,5154
-5159.[Abstract]
Prehm, P., Dessau, W. and Timpl, R. (1982). Rates of synthesis of basement membrane proteins by differentiating teratocarcinoma stem cells and their modulation by hormones. Connect. Tissue Res. 10,275 -285.[Medline]
Rogers, M. B., Watkins, S. C. and Gudas, L. J. (1990). Gene expression in visceral endoderm: a comparison of mutant and wild-type F9 embryonal carcinoma cell differentiation. J. Cell Biol. 110,1767 -1777.[Abstract]
Salmivirta, K., Talts, J. F., Olsson, M., Sasaki, T., Timpl, R. and Ekblom, P. (2002). Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens. Exp. Cell Res. 279,188 -201.[CrossRef][Medline]
Schymeinsky, J., Nedbal, S., Miosge, N., Poschl, E., Rao, C.,
Beier, D. R., Skarnes, W. C., Timpl, R. and Bader, B. L.
(2002) Gene structure and functional analysis of the mouse
nidogen-2 gene: nidogen-2 is not essential for basement
membrane formation in mice. Mol. Cell. Biol.
22,6820
-6830.
Smyth, N., Vatansever, H. S., Murray, P., Meyer, M., Frie, C.,
Paulsson, M. and Edgar, D. (1999). Absence of basement
membranes after targeting the LAMC1 gene results in embryonic
lethality due to failure of endoderm differentiation. J. Cell
Biol. 144,151
-160.
Smyth, N., Odenthal, U., Merkl, B. and Paulsson, M. (2000). Eukaryotic expression and purification of recombinant extracellular matrix proteins carrying the Strep II tag. Methods Mol. Biol. 139,49 -57.[Medline]
Stetefeld, J., Mayer, U., Timpl, R. and Huber, R.
(1996). Crystal structure of three consecutive laminin-type
epidermal growth factor-like (LE) modules of laminin 1 chain harboring
the nidogen binding site. J. Mol. Biol.
257,644
-657.[CrossRef][Medline]
Strickland, S. and Mahdavi, V. (1978). The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15,393 -403.[Medline]
Thomas, T. and Dziadek, M. (1993). Genes coding for basement membrane glycoproteins laminin, nidogen, and collagen IV are differentially expressed in the nervous system and by epithelial, endothelial, and mesenchymal cells of the mouse embryo. Exp. Cell. Res. 208,54 -67.[CrossRef][Medline]
Tryggvason, K. and Wartiovaara, J. (2001). Molecular basis of glomerular permselectivity. Curr. Opin. Nephrol. Hypertens. 10,543 -549.[CrossRef][Medline]
Wartenberg, M., Hescheler, J., Acker, H., Diedershagen, H. and Sauer, H. (1998a). Doxorubicin distribution in multicellular prostate cancer spheroids evaluated by confocal laser scanning microscopy and the `optical probe technique'. Cytometry 31,137 -145.[CrossRef][Medline]
Wartenberg, M., Gunther, J., Hescheler, J. and Sauer, H. (1998b). The embryoid body as a novel in vitro assay system for antiangiogenic agents. Lab. Invest. 78,1301 -1314.[Medline]
Willem, M., Miosge, N., Halfter, W., Smyth, N., Jannetti, I.,
Burghart, E., Timpl, R. and Mayer, U. (2002). Specific
ablation of the nidogen-binding site in the laminin 1 chain interferes
with kidney and lung development. Development
129,2711
-2722.
Yurchenco, P. D. and Schittny, J. C. (1990).
Molecular architecture of basement membranes. FASEB J.
4,1577
-1590.
Yurchenco, P. D., Quan, Y., Colognato, H., Mathus, T., Harrison,
D., Yamada, Y. and O'Rear, J. J. (1997). The chain of
laminin-1 is independently secreted and drives secretion of its ß- and
-chain partners. Proc. Natl. Acad. Sci. USA
94,10189
-10194.