1 Renal Division, Washington University School of Medicine, St Louis, MO 63110,
USA
2 Department of Cell Biology and Physiology, Washington University School of
Medicine, St Louis, MO 63110, USA
3 Department of Cell Biology, University of Virginia, Charlottesville, VA 22908,
USA
* Author for correspondence (e-mail: minerj{at}wustl.edu)
Accepted 29 January 2004
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SUMMARY |
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Key words: Laminin, Basement membrane, Embryogenesis, Mouse
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Introduction |
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Both naturally occurring and targeted mutations in laminin genes have
demonstrated the importance of laminin and its isoform composition to basement
membrane structure and function. For example, mutations in Lama2
(2) cause congenital muscular dystrophy
(Helbling-Leclerc et al.,
1995
; Xu et al.,
1994
); mutations in laminin 5 (
3ß3
2) component
genes cause severe skin blistering (reviewed by
Pulkkinen and Uitto, 1999
);
mutation of Lama4 causes a mild muscular dystrophy and a mild
bleeding disorder (Patton et al.,
2001
; Thyboll et al.,
2002
); mutation of Lama5 causes defects in neural tube
closure, digit septation, placentation, kidney formation and vascularization,
lung lobe septation, hair morphogenesis, and intestinal smooth muscle
differentiation (Bolcato-Bellemin et al.,
2003
; Li et al.,
2003a
; Miner et al.,
1998
; Miner and Li,
2000
; Nguyen et al.,
2002
); and mutation in Lamb2 causes severe defects in
neuromuscular junction differentiation and kidney filtration
(Noakes et al., 1995a
;
Noakes et al., 1995b
).
Finally, null mutation in Lamc1 results in very early embryonic
lethality (embryonic day [E] 5) because of the absence of basement membranes,
resulting in failure of endoderm differentiation
(Smyth et al., 1999
).
Similarly, in vitro laminin
1 is required for proper organization and
differentiation of embryoid bodies (reviewed by
Li et al., 2003b
;
Li et al., 2002
;
Murray and Edgar, 2000
;
Murray and Edgar, 2001
).
That mutation in Lamc1 causes such early lethality probably stems
from the fact that laminin 1 is found in 10 out of the 15 known laminin
trimers. Thus, removing
1 effectively prevents assembly of almost all
basement membrane-associated laminins, though only two laminins, laminin 1
(
1ß1
1) and laminin 10 (
5ß1
1), are
detectable at significant levels in peri-implantation embryos
(Klaffky et al., 2001
). We now
report the generation and characterization of mice with mutations in
Lama1 and Lamb1, which encode the remaining two laminin 1
subunits,
1 and ß1. These mice were derived from embryonic stem
(ES) cell clones isolated in a gene trap screen designed to capture genes
encoding secreted and cell surface proteins
(Leighton et al., 2001
;
Mitchell et al., 2001
;
Skarnes et al., 1995
). The
gene trap vector contained a splice acceptor, a transmembrane (TM) domain, and
ßgeo, a reporter (lacZ)/selectable marker
(neo) fusion, but it lacked a promoter to drive expression. Upon
electroporation into ES cells, this vector integrated into introns of many
genes (Mitchell et al., 2001
),
resulting in production of membrane-tethered fusion proteins that contained
the N terminus of the protein encoded by the trapped gene, a TM domain, and a
cytoplasmic ßgeo. By forcing production of such fusion proteins, the gene
trap vector not only prevents expression of the full-length endogenous
proteins (i.e. it is mutagenic), but it also results in expression of the
lacZ reporter under the control of the endogenous regulatory elements
of the trapped genes. Thus, the gene trap insertions in Lama1 and
Lamb1 have allowed us to: (1) generate knockout mice that lack
laminin
1 or ß1, and (2) characterize the expression pattern of
the two genes using X-gal staining. In addition, we have used a widely
expressed laminin
5 transgene to show that
5 can compensate
somewhat for the absent
1 in the embryonic basement membrane but not in
Reichert's membrane (RM). This compensation is highly significant in that it
allows for initiation of gastrulation even in the absence of RM.
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Materials and methods |
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Mice carrying a laminin 5 transgene (Mr5) have been previously
described (Kikkawa et al.,
2002
; Kikkawa et al.,
2003
; Moulson et al.,
2001
). The full-length mouse laminin
5 cDNA-coding region
was placed under the control of the widely active regulatory element miw,
which contains the Rous Sarcoma long terminal repeat inserted into the chicken
ß-actin promoter (Suemori et al.,
1990
). Of five transgenic founders, two produced offspring that
expressed the transgene widely throughout embryogenesis. One of these lines
was used in the experiments described here.
X-gal staining
For wholemount staining, embryos or dissected tissues were fixed in 2%
paraformaldehyde in PBS for 1 to 6 hours at room temperature. They were then
incubated at 37°C for 2-12 hours in staining solution containing 4 mM
potassium ferricyanide, 4 mM potassium ferrocyanide, 2 mM MgCl2,
0.1% NP-40, 0.2% sodium deoxycholate and 0.8 mg/ml
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) in PBS.
For staining of sections, tissues were first immersed in OCT and frozen in dry ice/ethanol-cooled 2-methylbutane. Sections were cut in a cryostat (10 µm), air-dried at room temperature, and then fixed and stained as described above. After staining, sections were rinsed in PBS, counterstained with 0.1% nuclear Fast Red (Fluka/Sigma-Aldrich, St. Louis, MO) in 5% aluminum sulfate, and mounted in 90% glycerol.
Antibodies and immunofluorescence
The antibodies to mouse laminin chains used were: rat anti-1 clone
8B3 and rat anti-ß1 clone 5A2
(Abrahamson et al., 1989
;
St John et al., 2001
); rabbit
anti-
5 serum 8948 (Miner et al.,
1997
); rabbit anti-ß1 and anti-ß2
(Sasaki et al., 2002
); rat
anti-
1, MAB1914 (Chemicon, Temecula, CA); and rabbit anti-laminin-1
(
1ß1
1) serum R23 (Sanes
et al., 1990
). Other antibodies used were: goat anti-collagen
1,
2(IV) (Southern Biotechnology Associates, Birmingham, AL); rat
anti-nidogen/entactin, MAB1946 (Chemicon); and rat anti-cytokeratin 8 clone
TROMA-1 (Developmental Studies Hybridoma Bank, developed under the auspices of
the NICHD and maintained by the University of Iowa, Department of Biological
Sciences, Iowa City, IA).
For cryosectioning of peri- and post-implantation embryos (E5 to E7.5), uterine horns were removed from timed mated females, immersed in OCT, and frozen in dry ice-ethanol cooled 2-methylbutane. In some cases, decidua were first removed from the uterus and then frozen in OCT. Sections (7-10 µm) were cut in a cryostat and either fixed in 2% paraformaldehyde in PBS for 10 minutes or stained unfixed. After rinsing in PBS, antibodies diluted in 1% BSA in PBS were applied for 1 hour. Sections were then rinsed in PBS, and FITC- or Cy3-conjugated secondary antibodies (Chemicon), along with 1 µg/ml Hoechst 33258 (Molecular Probes, Eugene, OR) to label nuclei, were applied for 1 hour. After rinsing, sections were mounted in 1 mg/ml p-phenylenediamine/90% glycerol/0.1x PBS and viewed under epifluorescence with a Nikon Eclipse 800 microscope. Images were captured with a Spot2 cooled color digital camera (Diagnostic Instruments, Sterling Heights, MI).
In situ hybridization
E7.5 embryos were frozen and sectioned as described above. In situ
hybridizations were carried out essentially as described
(Schaeren-Wiemers and Gerfin-Moser,
1993) using a T (brachyury) riboprobe labeled with a
digoxigenin-UTP labeling kit (Roche Molecular Biochemicals, Indianapolis, IN).
The probe was made from an 800 bp EcoRI fragment of a brachyury
expressed sequence tag (GenBank accession number AA163572) subcloned into
Bluescript II SK+ (Stratagene, La Jolla, CA).
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Results |
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We obtained the Lama1 and Lamb1 gene trap ES cell lines
and produced mice carrying the insertions
(Yin et al., 2003). Based on
5' RACE analysis and comparisons with the Celera Genomics Mouse Genome
Assembly, we determined that in the Lama1 trap line the gene trap
vector (pGT0TMp) inserted in the 20.4 kb third intron of Lama1 to
produce a fusion protein containing the first 120 amino acids of laminin
1 (part of domain VI), the vector-encoded TM domain and ßgeo
(Fig. 1A)
(Yin et al., 2003
). In the
Lamb1 trap line, the vector (pGT1TMpfs) inserted into the 10.2 kb
23rd intron to produce a fusion protein containing the first 1178 amino acids
of laminin ß1 (domains III through VI), the TM domain and ßgeo
(Fig. 1B) (Yin et al., 2003
).
|
Lama1
In general, Lama1 is considered to have a restricted pattern of
expression (Virtanen et al.,
2000), and our results are consistent with this notion. The major
site of expression from E9 to E13 was the central nervous system (CNS).
Intense X-gal staining was observed in several regions of the brain, as well
as in the ependymal layer of the spinal cord, which lines the central canal,
and in meningeal cells (Fig. 2
and J.H.M., unpublished). This is consistent with in situ hybridization
analyses (Lentz et al., 1997
;
Thomas and Dziadek, 1993
). The
major basement membrane associated with the CNS that contains laminin
1
is the pial or meningeal basement membrane, which covers the entire CNS outer
surface. This basement membrane is synthesized by meningeal cells, which
explains their staining with X-gal. However, it is not clear why ependymal
cells express Lama1 so robustly. Staining could represent the radial
glia, which extend from the ependyma to the pia and may deposit laminin 1
(
1ß1
1) in the pial basement membrane. Expression was also
observed in the lens of the eye (Fig.
2E), as we showed previously by in situ hybridization
(Lentz et al., 1997
). Embryos
lacking the Lama1 gene trap insertion did not stain significantly
with X-gal (Fig. 2B).
|
Lamb1
Lamb1 is considered to be deposited almost ubiquitously in
basement membranes. Consistent with this, X-gal stained whole
Lamb1+/- gene trap embryos completely blue
(Fig. 2G,H). Tissues dissected
out of older embryos were also stained completely blue; however, X-gal
staining of frozen sections, albeit a less sensitive assay, revealed that not
all cells were stained (J.H.M., unpublished). Interestingly, many cells not
adjacent to basement membranes were stained blue. This indicates that there
may be promiscuous expression of Lamb1 in cells that do not normally
secrete laminin. Indeed, we found significant expression of Lamb1 by
a large population of CNS neurons, but very little laminin ß1 protein
associated with most of them (Yin et al.,
2003).
Both laminin 1 (1ß1
1) and laminin 10 (
5ß1
1) are present in early postimplantation embryos
Owing to the peri-implantation lethality caused by targeted mutation of
Lamc1, we predicted that Lama1-/- and
Lamb1-/- embryos would show phenotypes at similarly early
gestational ages. We therefore examined expression of laminin and
ß chains in early postimplantation embryos to gain a better understanding
of where
1 and ß1 were expressed and whether compensation by other
chains might be expected.
From E5.5 to E7.5, we could only detect laminins 1,
5,
ß1 and
1 (Fig. 3
and J.H.M., unpublished), which indicates the presence of laminin 1 and
laminin 10. But these isoforms did not accumulate equally in basement
membranes. Laminin 1 was the prominent laminin in RM at all stages
(Fig. 3, arrows), though
laminin 10 was also present at E6.5 in the region of RM nearest the
ectoplacental cone (Fig. 3B,
asterisks), as previously shown (Klaffky
et al., 2001
). The other major basement membrane at these early
stages is the embryonic basement membrane, which lies between the embryonic
ectoderm and the visceral endoderm (Fig.
3, arrowheads). Laminin 10 appeared most prominent in the
embryonic basement membrane (Fig.
3B,C,F,G), though laminin 1 was also clearly detectable
(Fig. 3A,E). Given the nature
of immunofluorescence, it is not possible to make a quantitative assessment of
the level of laminin 1 versus that of laminin 10 in this basement
membrane.
|
Identification of Lama1-/- and Lamb1-/- embryos
Lama1+/- and Lamb1+/- mice were
intercrossed to generate potential Lama1-/- and
Lamb1-/- embryos at an expected Mendelian frequency of
25%. The uterine horns of timed pregnant females were removed, frozen in OCT
and sectioned on a cryostat. Embryo implantation sites (E5.5 to E7.5) were
localized using the trophoblast marker antibody TROMA 1 (anti-cytokeratin 8),
which labeled the invading trophoblast cells as well as endoderm cells in the
embryo. We then used monoclonal antibodies to laminin 1 (8B3) and
ß1 (5A2) to identify control Lama1+/- and
Lamb1+/- embryos, respectively. Embryos that were not
stained with these antibodies in a basement membrane-like pattern were judged
to be Lama1-/- and Lamb1-/-,
respectively (see below).
In many cases, several sections were stained with X-gal to take advantage
of the expression of ßgeo to determine which embryos contained at least
one mutated (gene trapped) allele. In Lama1+/- and
Lama1-/- embryos, only cells of the parietal endoderm
(PE), which secrete the components of RM, stained blue
(Fig. 3H-J); the lack of
staining in the visceral endoderm, which contributes to the embryonic basement
membrane, indicates that Lama1 expression is weak in those cells.
However, while the blue parietal endodermal cells were localized at the
periphery of laminin 1 antibody-positive embryos
(Fig. 3H,I), blue cells were
clustered in those embryos that were laminin
1-negative
(Fig. 3J), all of which were
small, suggesting that the PE was unable to migrate on the inner surface of
the blastocoele, a phenotype reported for Lamc1-/- embryos
(Smyth et al., 1999
). Absence
of X-gal staining was used to identify wild-type Lama1+/+
embryos. Of 55 scored embryos from Lama1+/- intercrosses,
25% were +/+, 51% were +/-, 20% were -/- and 4% could not be unambiguously
genotyped. Of 77 scored embryos from Lamb1+/-
intercrosses, 13% were +/+, 57% were +/-, 22% were -/- and 8% could not be
genotyped.
Phenotypes of Lama1-/- and Lamb1-/- embryos
Lama1
At E7.5, after gastrulation has normally ensued and embryos should be
relatively large (Fig. 3C),
Lama1-/- embryos had already died, as little embryonic
tissue remained at the center of the deciduum (J.H.M., unpublished). However,
at E6.5, Lama1-/- embryos still exhibited some degree of
organization and had cavitated, though they were significantly smaller than
wild-type and heterozygous littermates
(Fig. 4C-F). RM was not present
in the Lama1-/- embryos, as judged by staining with
antibodies to type IV collagen, nidogen and laminin 1
(Fig. 4F and J.H.M.,
unpublished). At E5.5, Lama1-/- embryos were already
smaller than control embryos (Fig.
4A,B). RM was not present at this earlier stage either, indicating
that it did not form and then break down because of the absence of laminin 1.
Staining with antibodies to laminin
5, ß1 and
1 revealed
that there were significant levels of laminin 10 (
5ß1
1) in
both control and Lama1-/- embryonic basement membranes
(Fig. 4A-D and J.H.M.,
unpublished). It is therefore likely that compensation by laminin 10 for the
missing laminin 1 allows for maintenance of the embryonic basement membrane
and for successful polarization and cavitation of the embryo proper.
|
|
Overexpression of laminin 5 promotes increased compensation in Lama1 mutants
If our hypothesis that endogenous laminin 5 compensates for the
missing
1 to promote formation and maintenance of the embryonic
basement membrane is correct, then increasing the level of
5 expression
in the embryo should improve embryonic development in the mutant and perhaps
even rescue RM formation. To test this possibility, we crossed a widely
expressed laminin
5 transgene (Mr5) onto the
Lama1+/- background and intercrossed the resulting
offspring to generate Lama1+/-; Mr5/Mr5 mice (homozygous
for the Mr5 transgene). Then, in order to generate
Lama1-/- embryos that carry the Mr5 transgene,
Lama1+/-; Mr5/Mr5 males were mated to
Lama1+/- females to produce Lama1-/-;
Mr5 embryos at an expected frequency of 25%. The resulting anti-laminin
1-negative, obligate hemizygous Mr5 embryos always appeared larger and
more developed than Lama1-/- embryos at E6.5 (compare
Fig. 4 with
Fig. 6A,B). At E7.5, when
Lama1-/- embryos were already dead,
Lama1-/-; Mr5 embryos were alive, although they were
significantly smaller than controls (Fig.
6C-F). The embryonic region was particularly small, while the
extra-embryonic ectoderm and ectoplacental cone regions were relatively
larger. The embryonic ectoderm had cavitated and exhibited strong staining for
laminin in the embryonic basement membrane. Significantly, there were very few
trophoblast giant cells at the periphery of the embryo, and the blood sinuses
of the yolk sac placenta, which were quite evident in controls, were absent in
Lama1-/-; Mr5 embryos
(Fig. 6E,F).
|
|
Because gastrulation begins on the seventh day of gestation, we asked
whether the Lama1-/-; Mr5 embryos were capable of
initiating gastrulation, despite their abnormalities. To investigate this, we
performed in situ hybridization using a probe for brachyury (T), a
mesodermal marker that is expressed only in embryos that have begun to
gastrulate. E7.5 Lama1-/-; Mr5 embryos did express
brachyury (Fig. 8A-D),
suggesting that they initiated gastrulation even in the absence of laminin 1
and RM. Lama1-/- embryos without the transgene were never
found alive at E7.5. Conversely, Lama5-/- embryos initiate
gastrulation (Fig. 8E,F) and
carry out a somewhat normal developmental program
(Miner et al., 1998). Taken
together, these data suggest that either laminin 1 or laminin 10 alone in the
embryonic basement membrane can support initiation of gastrulation, but
supernormal levels of laminin-10 are required in the absence of laminin 1.
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Discussion |
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The first requirement involves the embryonic basement membrane that
separates the epiblast/embryonic ectoderm from the visceral endoderm. This
basement membrane has been shown to be necessary in vitro for survival,
differentiation, polarization, and cavitation of the epiblast
(Coucouvanis and Martin, 1995;
Li et al., 2002
;
Li et al., 2001
;
Murray and Edgar, 2000
;
Murray and Edgar, 2001
). The
embryonic basement membrane normally contains both laminin 1
(
1ß1
1) and laminin 10 (
5ß1
1)
(Fig. 3)
(Klaffky et al., 2001
),
neither of which is able to form in the Lamb1 and Lamc1
nulls because ß1 and
1 are components of both trimers. However,
laminin 10 is present in the Lama1 null and appears capable - at
least to some extent - of promoting embryonic basement membrane formation and
cavitation of the embryonic ectoderm (Fig.
4). Thus, Lamb1 and Lamc1 mutants have no
detectable laminin trimers or basement membranes, but Lama1 mutants
have laminin 10 and an embryonic basement membrane; this makes them somewhat
healthier, but they are still small and abnormal at E6.5. Dag1
mutants also have an embryonic basement membrane, suggesting that dystroglycan
is not required for its formation. Interestingly, Dag1 mutants are
found as late as E10, though they are severely disrupted and do not gastrulate
(Williamson et al., 1997
). In
comparison with the Lama1, Lamb1 and Lamc1 nulls, the
integrity of the embryonic basement membrane is greater in the
Dag1-/- and Lama1-/-; Mr5 embryos. Why
the latter but not the former embryos are able to initiate gastrulation is
unknown, but it probably indicates a requirement for dystroglycan in this
crucial developmental process.
The inability of laminin 10 to fully compensate for a lack of laminin 1 in the embryonic basement membrane is an important question to be answered with future studies. It is unlikely that the structure of the basement membrane is obligatorily disrupted by the absence of laminin 1, for there are many basement membranes at various stages of development that contain only laminin 10. One possibility is that laminin 1 interacts with an important molecule - perhaps another basement membrane component; perhaps a cellular receptor, such as dystroglycan; or perhaps a growth factor that needs to be concentrated in the basement membrane - for which laminin 10 has less affinity. Presumably, by increasing the amount of laminin 10 in the embryonic basement membrane through expression of the Mr5 transgene, we have increased the binding of such a molecule to the basement membrane and enhanced its function.
The second requirement for laminin involves RM. The exact function of RM is
not known, but it appears to serve as a barrier between the maternal blood in
the yolk sac placenta and the developing embryo, and as a facilitator of
materno-embryonic exchange of nutrients and gases
(Williamson et al., 1997).
Mutation in any of the laminin 1 subunit genes, or in Dag1, results
in the absence of RM. Consideration of the different phenotypes in the
Dag1-/- mice and the Lama1-/- mice
illustrate two major functions of laminin 1 in RM. The first is structural:
laminin polymerization into a basement membrane provides for the barrier
function of RM. The importance of the structural attributes is evident in the
Dag1-/- mutants, where the PE cells do migrate, but are
unable to assemble the secreted laminin 1 into an organized RM
(Williamson et al., 1997
). In
these embryos, the resulting RM has its normal cellular components (PE and
trophoblast giant cells) and architecture, but loses its barrier capacity. The
second function of laminin 1 in RM is informational: interaction of both PE
and trophoblast with laminin 1 influences their differentiation and behavior.
When there is a complete lack of laminin, as demonstrated in the Lama1,
Lamb1 and Lamc1 mutants, both the cellular and molecular
components of RM are lacking. The PE cells fail to differentiate and fail to
migrate properly to the periphery of the embryo
(Smyth et al., 1999
), while
the trophoblast cells do not differentiate to giant cells and do not form the
blood sinuses. Dystroglycan is, thus, the crucial receptor for assembly of RM,
but not for transmitting specific signals from laminin 1 to the overlying
trophoblast cells, or for promoting PE migration. It will be interesting to
determine the mechanism of RM assembly in Myd (Large - Mouse
Genome Informatics) mutant mice, which have defects in glycosylation and
produce a dystroglycan molecule unable to interact with laminin in adults
(Michele et al., 2002
).
It has been suggested that the absence of RM contributes to death of the
embryo due to exposure to maternal blood
(Williamson et al., 1997).
This may be true for the Dag1-/- phenotype, but in
Lama1-/- embryos the lack of RM does not lead to exposure
to maternal blood. Rather, in the absence of laminin 1 the trophoblast cells
do not undergo their normal morphogenesis to form the blood sinuses of the
yolk sac placenta. The lack of these spaces leads to a severe deficit in
nutrients and oxygen for the embryonic region, and this is likely the fatal
deficit in these embryos.
The compositional requirement for laminin 1 in RM is somewhat surprising,
because laminin 10 is present in part of RM in normal embryos
(Fig. 3). However, laminin 10
is neither necessary (Fig. 6D) (Miner et al., 1998) nor
sufficient (Figs 4,
6) for formation of RM. In
addition, despite the fact that the Mr5 transgene can direct deposition of
laminin 10 into RM (Fig. 7A),
the presence of the transgene in Lama1-/-; Mr5 embryos
does not restore RM, and does not rescue the formation of the yolk sac
placenta (Fig. 6F). These
observations suggest that there is a unique and crucial function of laminin 1
in RM for initiating morphogenesis of the yolk sac placenta. Based on the
phenotype of the Lama1-/-; Mr5 embryos, we hypothesize
that the crucial step may be between the onset of invasive behavior and the
final differentiation of the trophoblast cells to the giant cell phenotype
(Sutherland, 2003
). The
trophoblast cells of the Lama1-/-; Mr5 mutant embryos are
able to initiate invasion and displace the uterine epithelium, but they do not
have significant numbers of giant cells and do not undergo appropriate
morphogenesis.
Finally, the ßgeo insertion in Lama1 and Lamb1
allowed us to determine their expression patterns. Of note, we identified a
previously unreported site of robust Lama1 expression, the presomitic
mesoderm (Fig. 2). Currently,
we are unable to determine the importance of this expression, but it could be
required for the mesenchyme to epithelium transition that these cells undergo.
If true, this would be analogous to the situation in developing kidney. There,
loose mesenchyme condenses to form the epithelial renal vesicle in the first
step of nephrogenesis (Saxen,
1987), and this is accompanied by intense laminin
1
expression (Ekblom et al.,
1990
) (Fig. 2).
Inhibition of laminin polymerization or its recognition by cellular receptors
with monoclonal antibodies to
1 prevents polarization of these cells in
organ culture, demonstrating the importance of laminin
1 in this
process (Falk et al., 1996
;
Klein et al., 1988
;
Sorokin et al., 1992
).
The ßgeo insertion in Lamb1 revealed very widespread
expression, even in cells that are not adjacent to basement membrane and would
not be expected to secrete laminin. We recently reported similar findings in
the brains of these mice, which exhibit widespread neuronal expression of
Lamb1 but little accumulation of immunoreactive protein
(Yin et al., 2003). A
widespread pattern of gene expression was not found in studies aimed at
defining the Lamb1 regulatory region, suggesting that the transgenic
constructs used in those studies contained some but not all regulatory
elements that normally regulate Lamb1 expression
(Sharif et al., 2001
).
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Abrahamson, D. R., Irwin, M. H., St. John, P. L., Perry, E. W., Accavitti, M. A., Heck, L. W. and Couchman, J. R. (1989). Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains. J. Cell Biol. 109,3477 -3491.[Abstract]
Bolcato-Bellemin, A. L., Lefebvre, O., Arnold, C., Sorokin, L., Miner, J. H., Kedinger, M. and Simon-Assmann, P. (2003). Laminin alpha5 chain is required for intestinal smooth muscle development. Dev. Biol. 260,376 -390.[CrossRef][Medline]
Burgeson, R. E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinman, H., Martin, G. R., Ortonne, J.-P., Paulsson, M., Sanes, J. et al. (1994). A new nomenclature for laminins. Matrix Biol. 14,209 -211.[CrossRef][Medline]
Cheng, Y.-S., Champliaud, M.-F., Burgeson, R. E., Marinkovich,
M. P. and Yurchenco, P. D. (1997). Self-assembly of laminin
isoforms. J. Biol. Chem.
272,31525
-31532.
Colognato, H., Winkelmann, D. A. and Yurchenco, P. D.
(1999). Laminin polymerization induces a receptor-cytoskeleton
network. J. Cell Biol.
145,619
-631.
Colognato, H. and Yurchenco, P. D. (2000). Form and function: the laminin family of heterotrimers. Dev. Dyn. 218,213 -234.[CrossRef][Medline]
Coucouvanis, E. and Martin, G. R. (1995). Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83,279 -287.[Medline]
Ekblom, M., Klein, G., Mugrauer, G., Fecker, L., Deutzmann, R., Timpl, R. and Ekblom, P. (1990). Transient and locally restricted expression of laminin A chain mRNA by developing epithelial cells during kidney organogenesis. Cell 60,337 -346.[Medline]
Falk, M., Salmivirta, K., Durbeej, M., Larsson, E., Ekblom, M.,
Vestweber, D. and Ekblom, P. (1996). Integrin alpha 6B beta 1
is involved in kidney tubulogenesis in vitro. J. Cell
Sci. 109,2801
-2810.
Helbling-Leclerc, A., Zhang, X., Topaloglu, H., Cruaud, C., Tesson, F., Weissenbach, J., Tome, F. M., Schwartz, K., Fardeau, M., Tryggvason, K. et al. (1995). Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy. Nat. Genet. 11,216 -218.[Medline]
Kikkawa, Y., Moulson, C. L., Virtanen, I. and Miner, J. H.
(2002). Identification of the binding site for the Lutheran blood
group glycoprotein on laminin 5 through expression of chimeric laminin
chains in vivo. J. Biol. Chem.
277,44864
-44869.
Kikkawa, Y., Virtanen, I. and Miner, J. H.
(2003). Mesangial cells organize the glomerular capillaries by
adhering to the G domain of laminin alpha5 in the glomerular basement
membrane. J. Cell Biol.
161,187
-196.
Klaffky, E., Williams, R., Yao, C. C., Ziober, B., Kramer, R. and Sutherland, A. (2001). Trophoblast-specific expression and function of the integrin alpha 7 subunit in the peri-implantation mouse embryo. Dev. Biol. 239,161 -175.[CrossRef][Medline]
Klein, G., Langegger, M., Timpl, R. and Ekblom, P. (1988). Role of laminin A chain in the development of epithelial cell polarity. Cell 55,331 -341.[Medline]
Koch, M., Olson, P. F., Albus, A., Jin, W., Hunter, D. D.,
Brunken, W. J., Burgeson, R. E. and Champliaud, M. F. (1999).
Characterization and Expression of the Laminin gamma3 Chain: A Novel,
Non-Basement Membrane-associated, Laminin Chain. J. Cell
Biol. 145,605
-618.
Leighton, P. A., Mitchell, K. J., Goodrich, L. V., Lu, X., Pinson, K., Scherz, P., Skarnes, W. C. and Tessier-Lavigne, M. (2001). Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 410,174 -179.[CrossRef][Medline]
Lentz, S. I., Miner, J. H., Sanes, J. R. and Snider, W. D. (1997). Distribution of the ten known laminin chains in the pathways and targets of developing sensory axons. J. Comp. Neurol. 378,547 -561.[CrossRef][Medline]
Li, J., Tzu, J., Chen, Y., Zhang, Y. P., Nguyen, N. T., Gao, J.,
Bradley, M., Keene, D. R., Oro, A. E., Miner, J. H. et al.
(2003a). Laminin-10 is crucial for hair morphogenesis.
EMBO J. 22,2400
-2410.
Li, S., Edgar, D., Fassler, R., Wadsworth, W. and Yurchenco, P. D. (2003b). The role of laminin in embryonic cell polarization and tissue organization. Dev. Cell 4, 613-624.[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.
Li, X., Chen, Y., Scheele, S., Arman, E., Haffner-Krausz, R.,
Ekblom, P. and Lonai, P. (2001). Fibroblast growth factor
signaling and basement membrane assembly are connected during epithelial
morphogenesis of the embryoid body. J. Cell Biol.
153,811
-822.
Libby, R. T., Champliaud, M. F., Claudepierre, T., Xu, Y.,
Gibbons, E. P., Koch, M., Burgeson, R. E., Hunter, D. D. and Brunken, W.
J. (2000). Laminin expression in adult and developing
retinae: evidence of two novel CNS laminins. J.
Neurosci. 20,6517
-6528.
Michele, D. E., Barresi, R., Kanagawa, M., Saito, F., Cohn, R. D., Satz, J. S., Dollar, J., Nishino, I., Kelley, R. I., Somer, H. et al. (2002). Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies. Nature 418,417 -422.[CrossRef][Medline]
Miner, J. H. and Li, C. (2000). Defective
glomerulogenesis in the absence of laminin 5 demonstrates a
developmental role for the kidney glomerular basement membrane.
Dev. Biol. 217,278
-289.[CrossRef][Medline]
Miner, J. H., Patton, B. L., Lentz, S. I., Gilbert, D. J.,
Snider, W. D., Jenkins, N. A., Copeland, N. G. and Sanes, J. R.
(1997). The laminin alpha chains: expression, developmental
transitions, and chromosomal locations of alpha1-5, identification of
heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.
J. Cell Biol. 137,685
-701.
Miner, J. H., Cunningham, J. and Sanes, J. R.
(1998). Roles for laminin in embryogenesis: Exencephaly,
syndactyly, and placentopathy in mice lacking the laminin 5 chain.
J. Cell Biol. 143,1713
-1723.
Mitchell, K. J., Pinson, K. I., Kelly, O. G., Brennan, J., Zupicich, J., Scherz, P., Leighton, P. A., Goodrich, L. V., Lu, X., Avery, B. J. et al. (2001). Functional analysis of secreted and transmembrane proteins critical to mouse development. Nat. Genet. 28,241 -249.[CrossRef][Medline]
Moulson, C. L., Li, C. and Miner, J. H. (2001). Localization of Lutheran, a novel laminin receptor, in normal, knockout, and transgenic mice suggests an interaction with laminin alpha5 in vivo. Dev. Dyn. 222,101 -114.[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
the differentiation and behaviour of extra-embryonic endodermal cells by
basement membranes. J. Cell Sci.
114,931
-939.
Nguyen, N. M., Miner, J. H., Pierce, R. A. and Senior, R. M. (2002). Laminin alpha5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung. Dev. Biol. 246,231 -244.[CrossRef][Medline]
Noakes, P. G., Gautam, M., Mudd, J., Sanes, J. R. and Merlie, J. P. (1995a). Aberrant differentiation of neuromuscular junctions in mice lacking slaminin/laminin ß2. Nature 374,258 -262.[CrossRef][Medline]
Noakes, P. G., Miner, J. H., Gautam, M., Cunningham, J. M., Sanes, J. R. and Merlie, J. P. (1995b). The renal glomerulus of mice lacking slaminin/laminin ß2: nephrosis despite molecular compensation by laminin ß1. Nat. Genet. 10,400 -406.[Medline]
Parsons, M. J., Pollard, S. M., Saude, L., Feldman, B.,
Coutinho, P., Hirst, E. M. A. and Stemple, D. L. (2002).
Zebrafish mutants identify an essential role for laminins in notochord
formation. Development
129,3137
-3146.
Patton, B. L., Cunningham, J. M., Thyboll, J., Kortesmaa, J., Westerblad, H., Edstrom, L., Tryggvason, K. and Sanes, J. R. (2001). Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4. Nat. Neurosci. 4,597 -604.[CrossRef][Medline]
Pulkkinen, L. and Uitto, J. (1999). Mutation analysis and molecular genetics of epidermolysis bullosa. Matrix Biol. 18,29 -42.[CrossRef][Medline]
Sanes, J. R., Engvall, E., Butkowski, R. and Hunter, D. D. (1990). Molecular heterogeneity of basal laminae: Isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J. Cell Biol. 111,1685 -1699.[Abstract]
Sasaki, T., Mann, K., Miner, J. H., Miosge, N. and Timpl, R.
(2002). Domain IV of mouse laminin ß1 and ß2 chains:
structure, glycosaminoglycan modification and immunochemical analysis of
tissue contents. Eur. J. Biochem.
269,431
-442.
Saxen, L. (1987). Organogenesis of the Kidney. Cambridge, UK: Cambridge University Press.
Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100,431 -440.[Medline]
Sharif, K. A., Li, C. and Gudas, L. J. (2001). cis-acting DNA regulatory elements, including the retinoic acid response element, are required for tissue specific laminin B1 promoter/lacZ expression in transgenic mice. Mech. Dev. 103, 13-25.[CrossRef][Medline]
Skarnes, W. C., Moss, J. E., Hurtley, S. M. and Beddington, R. S. P. (1995). Capturing genes encoding membrane and secreted proteins important for mouse development. Proc. Natl. Acad. Sci. USA 92,6592 -6596.[Abstract]
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.
Sorokin, L. M., Conzelmann, S., Ekblom, P., Battaglia, C., Aumailley, M. and Timpl, R. (1992). Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development. Exp. Cell Res. 201,137 -144.[Medline]
St John, P. L., Wang, R., Yin, Y., Miner, J. H., Robert, B. and
Abrahamson, D. R. (2001). Glomerular laminin isoform
transitions: errors in metanephric culture are corrected by grafting.
Am. J. Physiol. Renal Physiol.
280,F695
-F705.
Suemori, H., Kadodawa, Y., Goto, K., Araki, I., Kondoh, H. and Nakatsuji, N. (1990). A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous beta-galactosidase expression. Cell Differ. Dev. 29,181 -186.[CrossRef][Medline]
Sutherland, A. (2003). Mechanisms of implantation in the mouse: differentiation and functional importance of trophoblast giant cell behavior. Dev. Biol. 258,241 -251.[CrossRef][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]
Thyboll, J., Kortesmaa, J., Cao, R., Soininen, R., Wang, L.,
Iivanainen, A., Sorokin, L., Risling, M., Cao, Y. and Tryggvason, K.
(2002). Deletion of the laminin alpha4 chain leads to impaired
microvessel maturation. Mol. Cell. Biol.
22,1194
-1202.
Virtanen, I., Gullberg, D., Rissanen, J., Kivilaakso, E., Kiviluoto, T., Laitinen, L. A., Lehto, V. P. and Ekblom, P. (2000). Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues. Exp. Cell Res. 257,298 -309.[CrossRef][Medline]
Williamson, R. A., Henry, M. D., Daniels, K. J., Hrstka, R. F.,
Lee, J. C., Sunada, Y., Ibraghimov-Beskrovnaya, O. and Campbell, K. P.
(1997). Dystroglycan is essential for early embryonic
development: disruption of Reichert's membrane in Dag1-null mice.
Hum. Mol. Genet. 6,831
-841.
Xu, H., Wu, X.-R., Wewer, U. M. and Engvall, E.
(1994). Murine muscular dystrophy caused by a mutation in the
laminin 2 (Lama2) gene. Nat. Genet.
8, 297-302.[Medline]
Yin, Y., Kikkawa, Y., Mudd, J. L., Skarnes, W. C., Sanes, J. R. and Miner, J. H. (2003). Expression of laminin chains by central neurons: analysis with gene and protein trapping techniques. Genesis 36,114 -127.[CrossRef][Medline]