* Department of Medicine, Renal Division and Department of Anatomy and Neurobiology, Washington University School of
Medicine, St. Louis, Missouri 63110
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Abstract |
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Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are /
/
heterotrimers assembled from 10 known chains, and
they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and
therefore provide little insight into laminin's roles in
embryonic development. Here, we show that the laminin
5 chain is required during embryogenesis. The
5
chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as
development proceeds, including those of the surface
ectoderm and placental vasculature. BLs that lose
5
retain or acquire other
chains. Embryos lacking laminin
5 die late in embryogenesis. They exhibit multiple
developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to
defects in BLs that are
5 positive in controls and that
appear ultrastructurally abnormal in its absence. Other
laminin
chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.
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Introduction |
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CELLS in most tissues of vertebrates and invertebrates bear coats of basal laminae (BLs),1 thin
layers of extracellular matrix whose main components are laminin, collagen IV, entactin/nidogen, and
sulfated proteoglycans (Timpl, 1996; Timpl and Brown,
1996
). Laminins are glycoproteins that self-assemble to
form the major noncollagenous network of all BLs (Chung
et al., 1979
; Timpl et al., 1979
; Yurchenco and O'Rear,
1994
). All laminins studied to date are heterotrimers composed of one
, one
, and one
chain (Timpl, 1996
). Five
, three
, and two
chains have now been identified,
which can associate to form at least 11 heterotrimers. Different BLs contain distinct complements of laminin trimers,
allowing BLs to subserve distinct functions while maintaining a uniform structure.
In adults, BLs provide structural support for tissues,
serve as scaffolds to organize regeneration after tissue
damage, and underlie physiological functions such as renal glomerular filtration (Timpl, 1989; Yurchenco and
O'Rear, 1994
). The importance of laminin to the proper
function of BLs is underscored by the phenotypes of
known mutations in laminin chain genes: the laminin
2
gene is mutated in some congenital muscular dystrophies
(Sunada et al., 1994
; Xu et al., 1994
; Helbling-Leclerc et al.,
1995
); mutations in any one of the
3,
3, or
2 chain
genes causes junctional epidermolysis bullosa, a skin blistering disease (Aberdam et al., 1994
; Pulkkinen et al.,
1994
; McGrath et al., 1995
; Kuster et al., 1997
); and targeted mutagenesis of the laminin
2 chain gene impairs
both neuromuscular and renal function (Noakes et al., 1995a
,b). BLs and the laminins they contain are also likely
to play critical roles in embryos by providing signals for
cell proliferation, migration, and differentiation and by
serving as cell attachment sites (Timpl, 1989
; Yurchenco
and O'Rear, 1994
). However, no hitherto described mutations in laminin genes lead to major embryonic defects. Thus, either other laminin chains play predominant roles
during development, or laminins might be dispensable for
embryogenesis.
One good candidate for a laminin chain that could be involved in developmental processes is the laminin 5 chain
(Miner et al., 1995
). This chain associates with the
1 chain
and either
1 or
2 to form laminins 10 and 11, respectively (Miner et al., 1997
). The laminin
5 gene is expressed in many fetal and adult tissues, including kidney,
lung, skeletal muscle, skin, and intestine, and laminin
5
protein is present in specific BLs within these tissues (Durbeej et al., 1996
; Lentz et al., 1997
; Miner et al., 1997
; Patton et al., 1997
; Sorokin et al., 1997a
,b). Recently, purified
laminin 11 has been shown to induce responses from cultured neurons and Schwann cells that are qualitatively different from those induced by laminins 1, 2, or 4 (
1/
1/
1,
2/
1/
1, and
2/
2/
1, respectively) (Patton et al., 1997
,
1998
). These results suggested that laminin
5 might have
unique developmental roles.
To explore roles of laminin 5 in embryos, we have documented its localization and generated mice lacking this
chain. We show that laminin
5 is present in most embryonic and extraembryonic BLs at early stages, that it becomes restricted to a distinct subset of BLs as development proceeds, and that its loss leads to fetal lethality.
Numerous defects were apparent in mutant homozygotes, including failure of neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis
of the placenta. In each case, we consider mechanisms that
may explain how loss of laminin
5 disrupts normal development.
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Materials and Methods |
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Histology
Immunofluorescence microscopy was performed on cryostat sections as
described previously (Miner et al., 1997). Antisera to mouse laminins
4
and
5 were described previously (Miner et al., 1997
). The recombinant
fragment used to generate anti-laminin
5 antiserum comprised amino
acids 1415-1642 (Miner et al., 1995
), which are COOH-terminal to the region deleted in Lama5
/
embryos. Several other antibodies were provided by generous colleagues: anti-laminin
2 from Peter Yurchenco
(Robert Wood Johnson Medical School, Piscataway, NJ; Cheng et al.,
1997
); anti-laminin-5 (
3
3
2) from Robert Burgeson (CBRC, Harvard
Medical School, Charlestown, MA; Marinkovich et al., 1992
); anti-keratin
14 from Elaine Fuchs (University of Chicago, Chicago, IL; Stoler et al.,
1988
); and anti-laminin
1 (5A2) and anti-
1 (8B3) from Dale Abrahamson (University of Alabama at Birmingham; Abrahamson et al., 1989
).
We showed previously that 5A2 specifically recognizes the
1 chain (Martin et al., 1995
) and have now used similar methods to show that 8B3 recognizes mouse laminin
1 (data not shown). A commercial antibody to
mouse laminin
1 (MAB1914) was from Chemicon International (Temecula, CA). Cy3- and FITC-conjugated secondary antibodies were from
ICN/Cappel (Costa Mesa, CA).
For semithin and thin sectioning, embryos were fixed in 4% paraformaldehyde, 4% glutaraldehyde in 0.1 M cacodylate buffer and processed
as described (Noakes et al., 1995b). 2-µm sections were cut with glass
knives and stained with toluidine blue for light microscopy. Thin sections
were cut with a diamond knife and stained with lead citrate plus uranyl acetate for electron microscopy.
To label and detect dividing cells in embryos, we used the 5-Bromo-2'-deoxy-uridine (BrdU) Labeling and Detection Kit II (Boehringer Mannheim Corp., Indianapolis, IN). Pregnant females were injected intraperitoneally with 0.15 ml of 10 mM BrdU per 10 g of body weight. After 1 h, the mice were killed, and the embryos were removed, frozen, and sectioned at 7 µm on a cryostat. The label was detected in nuclei according to the manufacturer's instructions.
To stain cartilage in embryos, we used Alcian blue 8GX (Sigma Chemical Co., St. Louis, MO). Fixation, staining, and clearing were performed
as described (Jegalian and De Robertis, 1992).
Generation and Genotyping of Mutant Mice
A clone containing exons encoding parts of domains VI, V, and IVb of
laminin
5 was obtained by screening a 129sv mouse genomic library
(Stratagene, La Jolla, CA) with the 5' 900 bp of the cDNA previously described by Miner et al. (1995)
. To construct a targeting vector, two consecutive XbaI fragments totaling 3.5 kb and encoding 113 amino acids (129-
241 in Miner et al., 1995
) were replaced with an in frame lacZ cDNA and a
PGK neo cassette. The neo cassette was derived from the vector pPNT,
which also supplied PGK HSV-tk for negative selection (Tybulewicz et al., 1991
; see Fig. 2 A). The mutated chromosomal segment was transferred to
R1 ES cells by electroporation, and transfectants were selected with G418
(400 µg/ml) and FIAU (0.2 µM). Approximately 450 clones were
screened by PCR, and a single homologous recombinant clone was obtained. Cells from this clone were injected into C57BL/6J blastocysts by
standard methods. Three chimeras transmitted the mutation to their offspring. Homozygotes derived from each of these founders exhibited the
same phenotypes, and all phenotypes segregated with the mutation, even
after five generations of crossing to C57BL/6J mice. We never detected
lacZ activity in heterozygotes or homozygotes, presumably because the
signal sequence of the laminin
5 protein forces the catalytic domain of
the lacZ protein into the endoplasmic reticulum, where it is inactive.
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Mice were genotyped either by Southern blot or by PCR. For Southern analysis, genomic DNA was prepared from embryos, digested with NcoI, fractionated on an agarose gel, transferred to nitrocellulose, and probed with a fragment just outside the short arm of homology (Fig. 2 A). For PCR, lacZ-specific primers were used to identify the mutated allele, and Lama5 primers from within the deleted region were used to identify the wild-type allele.
Ra/+ mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
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Results |
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Widespread Expression of Laminin 5 in Embryos
We began this study by determining the distribution of the
laminin 5 chain in embryonic tissues. Sections were double-labeled with a previously characterized rabbit antiserum to
5 (Miner et al., 1997
) and a monoclonal antibody
to the
1 chain.
1 is present in all BLs described to date
and therefore serves as a general marker for them.
Laminin 5 was present in virtually all
1-positive BLs
at embryonic day (E) 8.5 (Fig. 1, A and B). These included
the BL underlying the neural folds and the surface ectoderm, as well as BLs associated with gut epithelium. Thus,
laminin
5 is not only a major
chain in adult BLs (Miner
et al., 1997
) but also a prominent component of BLs at
early somite stages. At later stages, however, the distribution of
5 became restricted to a subset of BLs. In the spinal cord, for example,
5 was found throughout the pial
BL at E9.5 (Fig. 1 E), but levels then decreased in dorsal regions, and by E13.5, this chain was confined to the floorplate at the ventral midline (Fig. 1 H). On the other hand,
5 remained abundant in the surface ectodermal BL
throughout embryogenesis (Fig. 1 and data not shown).
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The presence of BLs rich in 1 but poor in
5 at later
stages suggested that other
chains were being expressed.
We tested this idea by staining sections with antibodies
specific for the
1-
4 chains. Levels of
2-
4 were low at
E8.5-9.5, but the laminin
1 chain was codistributed with
5 in most BLs at this stage (Fig. 1, C and F, and data not
shown). Later, as
5 became restricted to a subset of BLs,
1 became restricted to a largely complementary subset,
so the extent of their colocalization decreased. For example, the ectodermal and neuroectodermal BLs were rich in
both
1 and
5 at the earliest stages examined, but each
chain became restricted to distinct regions as development
proceeded. Whereas
5 remained abundant in ectodermal
BL but became restricted to the floor plate of the neural
tube,
1 remained abundant in the neural tube (Fig. 1, H
and I) but was gradually lost from the ectoderm. Though
3 was not detected at E8.5-10.5, it became a major component of ectodermal BL by E12.5, as shown previously
(Aberdam et al., 1994
) and discussed below.
In some BLs, levels of both 1 and
5 were low. In
heart, for example,
1 and
5 were both detected in atria
at E9.5 and later ages, but ventricular expression of
1 and
5 was low and regionally restricted throughout the embryonic period (Fig. 1, J-L). Likewise, small
1-positive
blood vessels that arose within the brain, spinal cord, and
mesenchymal regions lacked both
1 and
5 (Fig. 1, G-I; see also Klein et al., 1990
). At least one of the other
known
chains,
2-
4, was found in these regions. For
example, BLs of ventricular muscle were rich in
2, and
blood vessels were rich in
4 (data not shown).
We also examined the distribution of the laminin 1 and
5 chains in extraembryonic tissues. At all stages examined, Reichert's membrane was rich in laminin
1 but contained comparatively little
5, whereas the yolk sac and
amnion contained both
1 and
5 at E10.5 and later ages
(data not shown). The few blood vessel BLs found inside
the nascent placental labyrinth at E9.5 contained
1 and
5 but not
1, whereas all three chains were found in vessel BLs near their origins in the ectoplacental plate (Fig. 1,
M-O). Finally, at E13.5, the BLs of fetal vessels in the
more mature placental labyrinth were rich in
1 and both
5 and
1 (Fig. 1, P-R). Thus,
5 is a prominent component of many extraembryonic as well as embryonic BLs.
Production of Laminin 5-deficient Mice
To determine the function of laminin 5, we mutated the
laminin
5 gene (Lama5) using a targeting vector that deleted exons encoding portions of the NH2-terminal domains VI and V (Fig. 2 A). With this strategy, only 211 of
3,718 amino acids were upstream of the deletion; transcription originating from the Lama5 promoter should be
terminated by SV-40 sequences in the vector; and translation of any resulting mRNAs should be terminated by
multiple stop codons. The targeting vector was transferred
to R1 ES cells (Nagy et al., 1993
) by electroporation, and a
single homologous recombinant clone was obtained from
450 screened. Cells from this clone were injected into
C57BL/6J blastocysts to produce three germline chimeras. Heterozygotes, which displayed no obvious abnormalities,
were back-crossed to wild-type C57BL/6J mice for at least
three generations to obtain a more defined genetic background. Initial and back-crossed heterozygotes were bred,
but no live homozygotes were detected among ~40 offspring, indicating that mice require laminin
5 to complete
development.
To determine when homozygotes were dying, we killed
timed pregnant females at E8.5-17.5. PCR (not shown),
and Southern blot analyses (Fig. 2 B) showed that homozygotes were alive at E13.5. Immunostaining of tissues
from confirmed homozygotes showed a complete absence
of laminin 5 protein (Fig. 2, C and D). Of 267 E8.5-16.5
embryos scored, 61 (23%) were Lama5
/
, suggesting that most homozygotes survived well past the implantation stage. However, defects became apparent after E9,
and some homozygotes were dead in litters taken between
E13.5 and 16.5. No homozygotes lived past E17. Consistent with the broad distribution of laminin
5, defects
were visible in many tissues, including the limb, neural
tube, and placenta. In the following sections, we describe these defects and consider mechanisms that could account
for them. Defects in some internal organs, including lung,
heart, intestine, and kidney, were also observed; these will
be described elsewhere (Miner, J.H., manuscript in preparation).
Syndactyly
In normal mice, distal extremities of limbs are initially
paddle-shaped, then septation occurs to form digits. The
distal limbs of Lama5 /
embryos were also initially
paddle-shaped, but then became club-like and failed to
form separate digits, a phenomenon known as syndactyly.
This defect was visible at E12.5, as soon as septation began
in controls. It became more obvious at later ages and was
apparent in all Lama5
/
mice examined (Fig. 3, A and
B). The forelimbs were more severely affected than the
hindlimbs; little if any septation occurred in mutant forelimbs, but digits 1 and 5 exhibited partial separation in
hindlimbs by E14.5.
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Syndactyly could result either from a failure of pattern
formation within the distal limb, or from failure to form
septa between digits that were otherwise well formed. To
distinguish between these alternatives, we stained limbs
from mutant and control embryos with Alcian blue, which
selectively stains cartilage. Digit pattern was established
normally in Lama5 /
embryos at E13.5 (Fig. 3, C and
D), indicating that
5 was not required for the complex developmental processes that initially pattern the distal
limb. Later, however, the phalanges of digits 2 and 3 fused
(Fig. 3, E and F). This sequence suggests that the defective
cartilage pattern is secondary to, rather than a cause of,
the syndactyly in Lama5 mutants.
Genesis of the Limb Defect
We used light and electron microscopy to learn how absence of laminin 5 results in syndactyly. The following
description refers to forelimbs, but similar results were obtained in hindlimbs. In normal development, the forelimb
bud is visible at E9. It is covered by a continuous ectodermal epithelium, which overlies the mesenchymal cells of
the dermis. The basal surface of the epithelial cells is
coated by a laminin
5-rich BL, which separates ectoderm
from dermis (Fig. 2 C). The ectodermal and dermal layers appeared similar in mutants and controls during early embryogenesis (not shown).
As the limb bud elongates, cells are added to the ectoderm so that it completely covers the limb at all times. In
mutants, however, ~200-µm gaps appeared in the distal
epidermis slightly ventral to the tip of each digit condensation (Fig. 4, A and B). Mesenchymal cells migrated or
were extruded through these gaps. Some of these cells may
have been sloughed into the amniotic fluid, but many remained associated with the external (peridermal) surface of the epithelium and migrated from the hole to form a
continuous, multilayered coat over each digit tip (Fig. 4, B
and C). As a result, the surface ectoderm thickened and
became covered on both sides by a dermis at the limb tip
(Fig. 4 C), and a second BL formed at the ectopic ectodermal/dermal interface (Fig. 4 D). Apparently, either the
surface ectoderm "doubled back" on itself to maintain a
basal relationship with the displaced mesenchymal cells,
or, alternatively, the presence of displaced mesenchyme on the apical side of the epithelial cells induced a repolarization and/or a reorganization of some of these cells,
producing a second epithelial/mesenchymal interface. In
either case, the additional cells in the epithelium were
clearly of epidermal lineage, as shown by immunostaining
with an antibody to the keratinocyte-specific antigen keratin 14 (Stoler et al., 1988). This antibody stained all surface
ectodermal cells as well as the full width of the thickened
mutant epithelium at this stage (data not shown). Because the interdigital mesenchyme is intimately involved in digit
septation (see Jiang et al., 1998
and references therein), its
depletion from the interior might be expected to impede
this process. Depleted mesenchyme could also lead to the
observed digit fusion, and the doubled ectoderm could
physically impede proper septation. Together, these abnormalities appear sufficient to account for the syndactyly observed in Lama5
/
embryos.
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How does loss of laminin 5 lead to the discontinuities
and a thickened epithelium at the limb tip? Ultrastructural
analyses showed that the BL associated with the mutant
ectoderm in the limb tip was patchy and discontinuous
compared with that seen in controls (Fig. 4, E and F). In
contrast, the ectodermal BL in the proximal part of the
limb was similar in mutants and controls (data not shown).
We surmise that the discontinuities observed in the distal
BL rendered it unable to maintain either its own integrity
or that of the surface ectoderm, leading to rupture of the
ectoderm under the stress of distal limb outgrowth. As a
result, mesenchymal cells from within the limb passed
through the hole and migrated along the outer surface of
the limb, thereby both depleting the interior of mesenchyme and capping the limb tip.
We also considered the possibility that syndactyly resulted from defects in the apical ectodermal ridge (AER).
The AER is a morphologically distinct epithelium that
runs from anterior to posterior at the distal margin of the
limb bud. It forms in the ectoderm at E10.5 and is necessary for limb outgrowth (Martin, 1998). That limb outgrowth occurs in the Lama5 mutants indicates that AER
function is not severely disrupted by the absence of
5
from the ectodermal BL. On the other hand, we did observe that the most distal phalanges of mutant forelimb
digits 2, 3, and 4 were missing or severely shortened (Fig. 3
E). Thus, the discontinuities at the distal tips of the limbs
may lead to subtle defects in the AER that contribute to
defects in both outgrowth and patterning (see also Jiang et
al., 1998
). Such subtle defects in the AER could combine
with the mesenchymal and ectodermal abnormalities discussed above to account for both syndactyly and the missing distal phalanges.
Exencephaly
In ~60% of Lama5 /
fetuses examined, the brain was
enlarged and misshapen, and it was not covered by skin or
skull (Fig. 5, A and B); the remaining mutants appeared to
have grossly normal heads (data not shown). This partially
penetrant defect is termed exencephaly, a condition in
which the cranial vault fails to develop, and the tissues of
the brain are exposed (Wallace and Knights, 1978
). We do
not know why this defect is partially penetrant, but we do
note that partial penetrance has been observed in other mutant strains that exhibit exencephaly (Wallace and
Knights, 1978
; Macdonald et al., 1989
; Vogelweid et al.,
1993
; Harris and Juriloff, 1997
).
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Exencephaly is caused by failure of the anterior neural tube to close. As a result, the left and right sheets of nonneural surface ectoderm do not fuse at the dorsal midline or detach from the neural tube, but remain contiguous with the neuroepithelium throughout development. Histological analyses of embryos at multiple gestational ages confirmed that this aspect of exencephaly was evident in the affected embryos (data not shown).
A major consequence of exencephaly is that the neuroepithelium is topologically "inside out." In normal mice,
neurons are born in a ventricular zone of proliferating
cells that abuts an interior sealed ventricle, and then they
migrate toward the outer pial surface to form the cortical
plate. The topology of exencephalic brains predicts that
the proliferating cells should lie on the outer surface or
abut a "pseudoventricle" that is actually continuous with
the extraembryonic space. To assess the polarity of the
neuroepithelium in Lama5 /
embryos, we labeled dividing cells in E13.5 embryos with BrdU for 1 h and detected incorporation immunohistochemically in frozen
sections. Control brains and brains of Lama5
/
mice that were not exencephalic showed characteristic labeling
near the ventricles, where proliferating cells normally reside at this stage (Fig. 5 C and data not shown). In exencephalic brains, labeling was observed in cells on the outer
surface (Fig. 5 D) as well as near the pseudoventricles (not
shown). These patterns confirm that the cranial defect observed in the absence of laminin
5 is exencephaly.
Genesis of the Neural Defect
To determine how a lack of laminin 5 leads to exencephaly, we examined the ultrastructure of the cranial neural
tube and its neighboring ectoderm in controls and mutants
at E8.7, the age just before the anterior neuropore closes
in normal embryos (Kaufman, 1992
). Low-magnification
views of sections representative of those that were used
for electron microscopy demonstrated that the anterior
neuropore was nearly but not completely closed in both
control and mutant embryos (Fig. 5, E and F). If the absence of laminin
5 led to significant ultrastructural defects, we expected them to be detectable at this stage.
In control embryos, the basal surfaces of the ectoderm
and neuroectoderm were coated by a continuous BL, but
discontinuities were apparent at the junction of ectoderm
and neuroectoderm, the area through which neural crest
cells later migrate from the dorsal neural tube to the periphery (Fig. 6, B-E). The mutant BL was similar to that of
controls in being continuous beneath the neuroectoderm and ectoderm but discontinuous at the ectodermal-neuroectodermal junction (Fig. 6, F, H, and I). Lama5 /
ectodermal BL differed from that of controls, however, along a
strip bordering the neural folds: the ectodermal BL of controls was continuous, whereas that of mutants was thin and
patchy (Fig. 6 G). This difference was seen in several
mutant/control littermate pairs. Interestingly, Schoenwolf
and colleagues have suggested that this strip of ectoderm is involved in generating forces that are necessary to close
the neural tube (Schoenwolf and Smith, 1990
; Hackett
et al., 1997
). We speculate that weakness of the BL in this
region decreases the amount of lateral force the ectoderm
can generate on the neural folds, thereby leading to sporadic failure of cranial neural tube closure.
Molecular Compensation
In some laminin mutants, loss of one chain results in compensatory upregulation of other chains. For example,
laminin 1 is found ectopically in the mature glomerular
basement membrane in kidneys of mice with a targeted
mutation in the laminin
2 chain gene. This compensatory
response produces a structurally intact BL that nevertheless does not function properly as a filter (Noakes et al.,
1995b
). Likewise, loss of laminin
2 in dy/dy mice results in upregulation of
4, which is, however, insufficient to
prevent muscular dystrophy (Patton et al., 1997
). In view
of these precedents, we assessed the consequences of laminin
5 deficiency on the composition of ectodermal BL. In
control embryos,
3,
5, and
1 were present in ectodermal BL at E13.5, but
1,
2, and
4 were undetectable (Fig. 7, A-E). In mutants, the entire surface ectoderm was
coated by a laminin
1-positive BL, consistent with electron microscopic observations that the discontinuities in
this structure were small and sparse (data not shown), except at the particular sites discussed above. The
5 chain
was absent, as expected, but the
3 chain was retained at
apparently normal levels. In addition, the
1,
2, and
4
chains were present in many but not all segments of mutant ectodermal BL (Fig. 7, F-J, and data not shown).
Thus, absence of laminin
5 led to retention and/or ectopic accumulation of the
1,
2, and
4 chains. The preservation of the mutant BL may result from the retention
of
3-containing laminins or the ectopic accumulation of
1-,
2-, and
4-containing laminins. Conversely, the localized defects in mutant BL that lead to exencephaly and
syndactyly may reflect either regional differences in the extent of compensation or uniform weakness that renders
the BL vulnerable in regions subjected to greatest stress.
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Placental Dysmorphogenesis
Syndactyly and exencephaly seemed unlikely to account
for the death of homozygous mutants at E14-17. Moreover, even mutants without exencephaly died by E17. Cardiac abnormalities were also observed, but these were
relatively minor and only seen in some of the Lama5 /
embryos (Miner, J.H., manuscript in preparation). We
therefore examined extraembryonic tissues from mutant
and control embryos. Reichert's membrane and the yolk
sac of Lama5
/
embryos were similar to those in controls, but the placental labyrinth was clearly malformed.
The labyrinth is the part of the placenta in which the fetal
and maternal circulations come into close proximity to exchange gases, nutrients, and wastes. In mice, the placental labyrinth is composed primarily of trophoblasts and endothelial cells, both derived from the embryo. The endothelial cells form the vessels through which the embryonic
blood circulates. Three layers of trophoblasts surround
these vessels and line the adjacent spaces through which
the maternal blood flows (Cross et al., 1994
; Hogan et al.,
1994
). The endothelium and the innermost trophoblast layer are separated from each other by a BL that, as shown
in Fig. 1, is normally rich in laminin
5.
The placental labyrinth was significantly smaller in mutants than controls by E13.5 and remained smaller at later
ages (data not shown). Immunostaining with antibodies to
laminin 1 (to localize BLs) and to PECAM (to localize
endothelial cells) revealed that the network of fetal blood
vessels was present and associated with BLs in the mutants
at E13.5. However, the complexity of vessel branching was
markedly reduced in mutant homozygotes, and the diameter of the vessels was significantly increased (Fig. 8, A and
B). This defect was still apparent at E16.5, indicating that branching of the blood vessels was not merely developmentally delayed. The resulting simplification of the labyrinth reduces the surface area available for exchange of
molecules between the fetal and maternal bloodstreams,
and could thereby lead to placental insufficiency in mutants. Consistent with this possibility, mutant embryos
were almost always smaller than their normal littermates after E14.
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High-resolution light microscopic and ultrastructural
studies of plastic embedded tissue revealed an additional
defect in mutant labyrinths. In controls, the fetal endothelium was separated from the trophoblasts only by a BL, to
which both cell layers were tightly adherent (Fig. 8, C and
E). In mutants, in contrast, the two cell layers were frequently separated from each other by a cell-free space. In
these regions of separation, the BL was associated exclusively with the endothelial cell, leaving the basal surface of
the trophoblast uncoated (Fig. 8, D and F). Ultrastructurally, the mutant BL was heterogeneous in width and exhibited splits and discontinuities (Fig. 8, G and H, and data
not shown). Thus, the trophoblasts failed to form stable
adhesions with the laminin 5-deficient BL, suggesting
that
5-containing laminins are necessary to maintain trophoblast adhesion to the BL. The abnormal separation of
the fetal blood vessels from the trophoblasts would have
serious consequences for placental function and, together
with the branching defects discussed above, provide a
plausible explanation for death of the mutant embryos.
Phenotype of Lama5/ragged Trans-heterozygotes
The naturally occurring dominant mutation ragged (Ra)
leads to sparse fur in heterozygotes (Ra/+). Ragged homozygotes (Ra/Ra) are almost completely hairless and
usually die before weaning (Carter and Phillips, 1954). We
(Miner et al., 1997
) and Durkin et al. (1997)
previously
noted that Lama5 maps to a region of mouse chromosome
2 very close to the ragged gene and raised the possibility
that Ra is, in fact, a neomorphic allele of the Lama5 gene.
If this were the case, Ra/Lama5 mice might resemble Ra/ Ra mice because only mutant protein would be produced
in both cases. To test this possibility, we mated Ra/+ mice
with Lama5 +/
mice. We identified 11 mice out of 36 offspring from 4 litters that were genotypically Lama5 +/
(as determined by PCR) and phenotypically Ra/+ (by coat
abnormality). This frequency was not significantly different from that expected (9/36). None of these 11 mice had the severe abnormalities observed in Ra/Ra homozygotes,
and none died before weaning. Moreover, these mice had
apparently normal amounts of laminin
5 protein in their
kidney BLs, as assessed immunohistochemically (data not
shown). It remains formally possible that Ra is a dominant-negative mutation in Lama5 that is only lethal or severe when present in two copies. However, we suspect that
the ragged defect does not result from a mutation in the Lama5 gene.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutations have been generated or discovered for 5 of the
10 laminin genes identified to date (see introduction). In
all of these cases, embryogenesis proceeds essentially normally, and defects only become severe at or after birth. In
contrast, the absence of laminin 5 leads to embryonic lethality. Lama5
/
homozygotes appear normal until
about E9, but development is then progressively aberrant,
and no embryos live beyond E16.5. Multiple defects were
observed, consistent with the wide expression pattern of
Lama5. Here, we focused on the most severe defects:
those observed in limb, head, and placenta. In all three
cases, ultrastructural analysis suggests that the phenotypes
result fairly directly from defects in BLs that normally
contain laminin
5.
Despite the multiple defects observed in Lama5 /
embryos, we are unsure of why they die by E17. Exencephaly is an unlikely cause, since mice routinely live
to birth in other models of exencephaly (Wallace and
Knights, 1978
; Macdonald et al., 1989
; Vogelweid et al.,
1993
), and the Lama5
/
fetuses with normal neural
tube closure also die by E17. Similarly, syndactyly is unlikely to be lethal. The observed placental defects may be
at least partly responsible for mortality. First, the percentage of the placenta that contained the labyrinth of fetal
blood vessels and maternal blood spaces was reduced in
mutants. Second, the complexity of the fetal capillary network was significantly reduced compared with controls;
the presence of
5 but not
1 in the BLs at the distal tips
of blood vessels in the nascent placental labyrinth (Fig. 1)
is consistent with it having a role in elaboration of the fetal
vessel network. Third, portions of many fetal vessels were
no longer juxtaposed to the trophoblasts by E16.5. Fourth, the vessel BLs themselves were aberrant, varying in width
and exhibiting splits and discontinuities. Thus, laminin
5
may be important in placental endothelial cell migration
and blood vessel branching, in trophoblast adhesion to
BL, and in formation of a proper BL. Together, these defects would be expected to impair exchange of gases, nutrients, and wastes between the fetal and maternal circulation, and thus contribute to death of the fetuses by E17.
We plan to test whether these placental defects are wholly responsible for fetal death by aggregation of Lama5
/
ES cells with tetraploid wild-type embryos, which can contribute to formation of the placenta but not to the embryo
itself (Nagy et al., 1993
).
In addition to exencephaly, syndactyly, and placentopathy, several defects were seen in internal organs of Lama5
/
embryos, including small or absent kidneys, small or
absent eyes, defects in lung lobe septation and bronchiolar
branching, and reduced size of the left ventricle of the
heart (Miner, J.H., manuscript in preparation). Some of
these defects were variable in severity and penetrance, but
all can be associated with BLs that normally contain laminin
5. Though none of these defects alone appears sufficient to be responsible for fetal death, together with exencephaly and syndactyly, they may compromise the health
of the fetus enough that an otherwise tolerable level of
placental malfunction becomes lethal.
The limb and cranial defects in Lama5 mutants may
have a common pathogenesis. Both are associated with localized structural deficiencies in regions of BLs that are
likely to be subjected to greater mechanical stress than are
neighboring regions. In the limb, robust outgrowth occurs
in the distal portion of the limb bud under the direction of
the AER. In Lama5 mutants, this outgrowth may stress
the laminin 5-deficient BL in this region, leading in turn
to rupture of the overlying ectoderm and extrusion of mesenchymal cells. More proximal segments of the ectoderm
and its underlying BL did not exhibit obvious defects, and
the proximal limb was normal. Likewise, neural tube closure was defective in cranial regions, but not in more caudal sections of the neural tube, consistent with the notion
that elevation and closure of the large cranial neural folds
that give rise to the brain generates more stress than elevation and closure of the much smaller caudal neural folds
that give rise to the spinal cord. This stress may be maximal in the strip of ectoderm just lateral to the neural folds,
which Schoenwolf and colleagues have shown to play a
critical role in neural tube closure (Hackett et al., 1997
). In
their experiments, removal of this portion of the ectoderm
in chick embryos impaired elevation of the neural plate
and prevented its bending towards the dorsal midline. It is
in this region that the BL is discontinuous in Lama5
/
mice but not in controls. We favor the idea that impaired
interactions with the defective BL decrease the amount of
lateral force the overlying ectoderm can generate. Thus, in both limb and head, mechanical stresses on weakened,
5-deficient BLs could result in rupture of the BL, which in
turn would impair function of the overlying ectoderm.
This explanation is essentially mechanical. Another explanation, not mutually exclusive, is that laminin 5 has
unique signaling functions necessary for development. Indeed, laminins are known to be signaling molecules that
interact with numerous signaling receptors required for
cellular integrity and motility, the best studied of which
are the integrins (Mercurio, 1995
). Thus, some defects observed in the mutants could reflect lack of activation of
laminin
5 receptors, or lack of some other ligand that is absent because the BL is disrupted. For example, the neuroepithelial BL is rich in laminin
5 at early stages, and
this laminin might be required for the neuroepithelium to
generate intrinsic forces necessary, along with ectoderm-derived forces, for neural tube closure (for review see
Schoenwolf and Smith, 1990
). Likewise, matrix-bound factors such as FGFs and Wnts have been implicated in patterning of the vertebrate limb (Martin, 1998
); their accumulation or localization might be perturbed in laminin
5-deficient BLs. In any event, our results show that BLs
and the laminins they contain are required for neurulation
and for proper digit septation.
The finding that laminin 5 deficiency leads to localized
structural defects in the BL is noteworthy, in that BLs
lacking some other major components do not exhibit obvious structural defects. For example, laminin
2 is the only
chain so far found in renal glomerular or neuromuscular
synaptic BLs, but these BLs appear structurally normal in
mice lacking laminin
2, apparently because laminin
1
substitutes for the absent
2 chain (Noakes et al., 1995a
,b;
Patton et al., 1997
). These BLs are functionally defective,
however, so synaptic and glomerular functions are impaired. Thus, molecular compensation permits formation
of a structurally intact but functionally inadequate BL.
Likewise, in humans and mice lacking the collagen
3-
5(IV) chains, which are normally the major collagen
chains of the glomerular BL, the
1 and
2(IV) chains
substitute to form an ultrastructurally normal BL. Eventually, however, this BL becomes severely damaged, and the
kidney ceases to function properly (Kashtan and Kim,
1992
; Cosgrove et al., 1996
; Miner and Sanes, 1996
; Kalluri
et al., 1997
). In Lama5
/
mice, in contrast, at least some
BLs are ultrastructurally defective despite ectopic deposition of other
chains. It remains to be determined
whether the severity of these defects reflects quantitatively inadequate compensation by other
chains, or
whether
5 is uniquely qualified to maintain the structural
integrity of BLs.
![]() |
Footnotes |
---|
Address correspondence to Jeffrey H. Miner, Renal Division, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: (314) 362-8235. Fax: (314) 362-8237. E-mail: minerj{at}thalamus.wustl.edu
Received for publication 4 September 1998 and in revised form 27 October 1998.
We thank D. Abrahamson, P. Yurchenco, R. Burgeson, and E. Fuchs for antibodies; A. Nagy for ES cells; M. Nichol for ES cell culture and blastocyst injections; C. Kenoyer, J. Mudd, E. Ryan, and R. Lewis for technical assistance; C. Borgmeyer, J. Gross, and S. Weng for care of mice; T. Tolley (supported by National Institutes of Health PO1HL29594) for help with histology; and R.M. Grady, Y. Sadovsky, D.M. Nelson, Z. Werb, and R. Kopan for helpful discussions.
This work was supported by grants from the National Institutes of Health to J. Miner and to J.R. Sanes.
![]() |
Abbreviations used in this paper |
---|
AER, apical ectodermal ridge; BL, basal lamina; BrdU, 5-bromo-2'-deoxy-uridine; E, embryonic day.
![]() |
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