1 Department of Animal Biology and Centre for Environmental Biology, Faculty of
Sciences, University of Lisbon, 1749-016 Lisbon, Portugal
2 Gulbenkian Institute of Science, Oeiras, Portugal
3 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht,
The Netherlands
4 Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, The
Netherlands
5 Institute of Molecular Embryology and Genetics, Kumamoto University School of
Medicine, Kumamoto, Japan
Present address: Faculty of Medicine, INSERM U385, Nice, France
Author for correspondence (e-mail:
solveig{at}fc.ul.pt)
Accepted 9 January 2003
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SUMMARY |
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Key words: ß1 integrins, Knock-in, Myogenesis, Muscle mass, Cell migration, Placentation, Mouse
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INTRODUCTION |
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During mouse development, ß1A and ß1D expression is mainly
non-overlapping: ß1A is expressed ubiquitously, while ß1D is
detectable in the heart from E11.0, increasing sharply around birth, and in
skeletal muscle from E17.5 (van der Flier
et al., 1997; Brancaccio et
al., 1998
). In both cases ß1D completely displaces ß1A
perinatally, suggesting distinct roles for each in striated muscle in
vivo.
In vertebrates, myogenesis occurs sequentially, starting when proliferating
myoblasts induce myogenic regulatory factors, such as Myf5, MyoD and myogenin,
followed by an irreversible exit from the cell cycle, phenotypic
differentiation and fusion of myoblasts to form multinucleated elongated
myotubes (Buckingham, 2001).
During mouse development, there are two waves of myogenesis involving three
distinct populations of myoblasts. Between E11.5 and E15.5 the formation of
primary myotubes results from the fusion of primary (embryonic) myoblasts,
while secondary (foetal) myoblasts remain proliferative. From E15.5, secondary
myoblasts progressively enter the muscle differentiation programme. Finally, a
third population of myoblasts (adult) develops and contributes to secondary
myogenesis until well after birth and also gives rise to the quiescent
satellite cells of adult muscle (Wigmore
and Dunglison, 1998
).
Antibody perturbation experiments have suggested a role for ß1
integrins during several steps of myogenesis, including migration,
differentiation and fusion (McDonald et
al., 1995; Gullberg et al.,
1998
). However, in wild type/ß1-null chimeric mice,
ß1-deficient myoblasts migrate, differentiate and fuse with wild-type
myoblasts/myotubes (Fässler and
Meyer, 1995
). Thus the role of ß1 integrins in myogenesis in
vivo is presently not clear. In vitro studies show that exogenous ß1D
inhibits cell cycle progression in cultured C2C12 cells
(Belkin and Retta, 1998
),
while exogenous ß1A maintains proliferation in quail myoblasts when
paired with a permissive
chain
(Sastry et al., 1999
).
Together, these in vitro results suggest not only that integrin ß1 may
play a role in myogenesis, but that ß1A and ß1D might have different
functions.
We studied in detail the role of ß1A and ß1D during myogenesis in vivo in the ß1D knock-in mice, which were crossed into a less penetrant genetic background than used previously. This revealed a second period of lethality. Our results show that the replacement of ß1A with ß1D supports normal cell migration during development, but placentation is abnormal and the most likely cause of midgestational death. Furthermore, we show for the first time that ß1 plays an important role during myogenesis in vivo, ß1D being incapable of replacing ß1A functionally during primary myogenesis.
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MATERIALS AND METHODS |
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Primordial germ cells
E10.5 embryos were fixed in 4% paraformaldehyde (PFA) for 2.5 hours at
4°C, embedded in Paraplast and stained for alkaline phosphatase (AP)
activity (Lawson et al.,
1999). Primordial germ cells (PGCs) in each embryo (n=8
for wild-type and n=6 for ß1D1D ki/ki embryos) section
were counted on the basis of the strong cytoplasmic AP activity and morphology
(Chiquoine, 1954
).
PGCs were isolated by flow cytometry as described by Abe et al.
(Abe et al., 1996) and cDNA
isolated by standard procedures. RT-PCR was performed on 2 ng cDNA and PCR
conditions were 94°C for 2 minutes, 35 cycles of 94°C for 30 seconds,
54°C for 30 seconds and 72°C for 30 seconds. Primers for integrin
ß1A (282 bp) and ß1D (363 bp) were
5'-GGCAACAATGAAGCTATCGT-3' and
5'-CCCTCATACTTCGGATTGAC-3'; for Oct4,
5'-GGAGAGGTGAAACCGTCCCTAGG-3' and
5'-AGAGGAGGTTCCCTCTGAGTTGC-3'
(Anderson et al., 1999
); and
for HPRT, 5'-GCTGGTGAAAAGGACCTCT-3' and
5'-CACAGGACTAGAACACCTGC-3'
(Johansson and Wiles,
1995
).
In situ hybridisation
Digoxigenin-labelled RNA probes were prepared from linearised plasmids
(Sambrook et al., 1989).
Whole-mount in situ hybridisation was performed as described by Henrique et
al. (1995
). mRNA was
visualised using BM Purple (Roche). The expression of Snail
(n=8 for ß1D1D ki/ki and wild type or heterozygous) and
pax3 (n=2 for ß1D1D ki/ki and heterozygous)
were analysed in toto; some embryos were embedded in Technovit
8100TM and sectioned.
Histology
E18.5 embryos were fixed for 10 days in 4% PFA, washed for 10 days in PBS,
5 days in 0.83% NaCl, 10 days in 1:1 mix of 0.83% NaCl and 100% ethanol and
stored in 70% ethanol, all at 4oC. After embedding in paraffin
(Histowax), 7 µm sections were processed for Haematoxylin and Eosin
staining.
Immunohistochemistry
E18.5 cleidomastoideus muscle was fixed in 2% PFA, dissected out and
embedded in Tissue-Tek OCT compound. E14.5 placenta, E18.5 thoracic body wall,
and E14.5, E16.5, E17.5 and E18.5 lower hindlimb muscles were embedded without
fixation, but immediately frozen in liquid nitrogen-chilled isopentane.
Transverse cryosections (5-10 µm) were fixed in 2% PFA and processed as
described by Venters et al. (Venters et
al., 1999). Some E14.5 placentae were fixed in 4% PFA and embedded
in paraffin. Paraffin sections (7 µm) were processed as described
previously (Zwijsen et al.,
1999
) and some were processed for Haematoxylin and Eosin
staining.
Primary antibodies used were rabbit anti-EHS laminin (Sigma), rat
anti-mouse endothelial glycoprotein (MECA32, DSHB), mouse anti-slow myosin
heavy chain (slow MHC, NOQ7.1.1A) (Draeger
et al., 1987), mouse anti-neonatal/fast MHC (MY-32, Sigma), rabbit
anti-protein gene product 9.5 (PGP9.5)
(Thompson et al., 1983
), rat
anti-muscle acetylcholine nicotinic receptor (mAb35; DSHB) and rabbit
anti-phospho-histone H3 (UBI). Secondary antibodies were Alexa 488 (Molecular
Probes) or TRITC-conjugated (Sigma) goat anti-rabbit IgG, FITC- or
TRITC-conjugated goat anti-rat IgG (Sigma) and HRP-conjugated goat anti-mouse
IgG (Southern Biotech). For the latter, diaminobenzidine was used as a
substrate.
Myotube quantification
Primary and secondary myotubes were quantified by counting slow
MHC-positive (primary) and slow MHC-negative (secondary) myotubes
(Harris et al., 1989;
Sheard and Duxson, 1996
).
Consecutive sections were labelled with the MY32 antibody, which stains all
myotubes at E18.5.
In E18.5 body wall muscles, primary and secondary myotubes were counted per unit area of intercostal and serratus dorsalis muscles. The number of myotubes from the sternum to the vertebra in one section per individual (n=2 for wild type and n=3 for ß1D1D ki/ki) was counted and the number of myotubes/mm2 calculated.
In E18.5 cleidomastoideus and lower hindlimb muscles, primary and secondary myotubes were counted in 3-6 serial sections, from each individual (n=3 or 4 per genotype). The widest part of the hindlimbs and the red area of cleidomastoideus were used. Primary myotubes were counted in E14.5 lower hindlimb and the myotube diameter measured in extensor digitorum longus (EDL) muscle in at least 15 myotubes from each individual (n=3 per genotype). Primary and secondary myotubes were also counted in E16.5 (n=1 per genotype) and E17.5 (n=2 per genotype) EDL muscle.
Differences in the numbers of myotubes were tested either by analysis of variance (body wall, E16.5 hindlimb) or nested analysis of variance. Differences in myotube diameter were tested by nested analysis of variance.
TUNEL assay
Apoptosis was analysed on cryosections of E14.5 placentae (n=2 or
3 per genotype), and E14.5, E16.5 and E17.5 (n=2, 1, 2 per genotype,
respectively) hindlimb muscles using the Cell Death Detection Kit (Roche).
TUNEL-positive nuclei were counted in the tibialis anterior, (TA), EDL and
peroneus group (P) muscles using at least 5 serial sections per
individual.
C2C12 differentiation
The mouse myoblast cell line C2C12 (ATTC CRL 1772) was grown in DMEM
supplemented with 20% (v/v) foetal calf serum and high glucose (5 g/l). The
C2C12/ß1D and C2C12/ß1A cells were generated by retroviral
transduction with cDNA constructs for human ß1A and ß1D, as
described previously (Gimond et al.,
1999), but using 2x104 C2C12 cells and infection
for 6 hours. Myogenic differentiation was induced as described by van der
Flier et al. (van der Flier et al.,
2002
).
For immunofluorescence, cells were processed as described by van der Flier
et al. (van der Flier et al.,
2002), using mouse anti-sarcomeric MHC (MF20; DSHB) and
FITC-conjugated goat anti-mouse IgG (Sigma). To-Pro 3 (Molecular Probes) was
added to the last PBS wash. The number of MF20-positive cells
(MHC+, total myoblasts and myotubes) and respective nuclei were
counted in three microscopic fields at 10x magnification and three to
seven microscopic fields at 16x magnification. The fusion index (%) was
calculated as the ratio of nuclei in myotubes (>3 nuclei) to the total
number of nuclei in MHC+ cells. Student's t-test was used
to analyse the differences between wild-type and infected cells.
Western blotting was performed as in van der Flier et al.
(van der Flier et al., 1997).
Primary antibodies were mouse anti-p21 (UBI), rabbit anti-pRB (Santa Cruz),
MF20, rabbit anti-connexin43 (Sigma), rabbit anti-ß1Acyto (kind gift from
U. Mayer) and mouse anti-ß1Dcyto (2B1)
(van der Flier et al., 1997
).
Secondary antibodies were HRP-conjugated sheep anti-mouse and donkey
anti-rabbit (Amersham Pharmacia).
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RESULTS |
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Migratory cells behave normally in ß1D ki/ki embryos
One of the most prominent defects in E11.5 ß1D1D ki/ki
embryos on a mixed background was a reduction in the size of the first
branchial arch (Baudoin et al.,
1998), raising the possibility that the migration of
ß1D1D ki/ki cranial neural crest cells into the branchial arches
could be impaired. Although on a mainly FVB background, only 7.4% of the
ß1D1D ki/ki embryos had a phenotype suggesting problems with
cell migration, we determined whether a general failure in cell migration
contributed to the first period of lethality. The behaviour of three migratory
cell populations (cranial neural crest cells, limb muscle precursor cells and
primordial germ cells; PGCs) was analysed in ß1D1D ki/ki embryos
and their littermates.
In E9.5 embryos there were no differences between ß1D1D ki/ki
embryos and their littermates in the expression of snail
(Fig. 1A,D), which encodes a
transcription factor in cranial neural crest cells
(Nieto et al., 1992) and
pax3 (Fig. 1B,E), a
marker for muscle precursor cells
(Goulding et al., 1994
).
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Although we cannot exclude defective cell migration in the few ß1D1D ki/ki embryos showing external defects, our data show that three cell populations that undergo long-range cell migration behave normally in the ß1D1D ki/ki embryos analysed. These results suggest that on a predominantly FVB genetic background, ß1D is able to support cell migration in vivo as efficiently as ß1A.
Abnormal vascularisation and increased apoptosis in the placental
labyrinth causes early death of ß1D ki/ki embryos
The ß1D1D ki/ki embryos that ceased development between
E13.5-E14.5 were exceptionally pale (not shown), raising the possibility of
placental malfunction. We examined the distribution of laminin, a marker for
foetal blood vessels (Harbers et al.,
1996), in placentae of E14.5 embryos. The results showed that
embryonic blood vessels present in the labyrinth of both heterozygous and
morphologically normal, living ß1D1D ki/ki embryos were uniform
in diameter and regularly spaced (Fig.
2A,B,D,E,G,H). In contrast, the labyrinth of the pale
ß1D1D ki/ki embryos contained a reduced number of large calibre
blood vessels, the lumen of the vessels was irregular and many were
obstructed. Furthermore, there was less branching, although strong laminin
immunoreactivity was maintained (Fig.
2C,F,I). TUNEL assay and immunostaining with MECA32 antibody
(recognising endothelial cells) revealed increased apoptosis in labyrinthine
endothelial cells in two of three pale ß1D1D ki/ki placentae,
compared to wild-type or morphologically normal ß1D1D ki/ki
placentae (Fig. 2J-L). However,
phospho-histone H3 immunostaining showed no difference in the number of
proliferating cells (not shown). Interestingly, the one ß1D1D
ki/ki placenta that did not show increased apoptosis, did show a marked
increase in proliferation (not shown), suggesting placental recovery
(Plum et al., 2001
).
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Reduction in muscle mass in late gestation ß1D ki/ki
embryos
The ß1D ki/ki embryos recovered alive between E14.0 and E18.5
were as advanced in development as their wild-type littermates. However, a
large proportion was thinner and a few were also shorter than heterozygous and
wild-type embryos (Fig. 3A). This was particularly evident between E17.0-E18.5 where 13 of 16
ß1D1D ki/ki embryos exhibited this phenotype.
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Histological analysis of E18.5 ß1D1D ki/ki embryos did not
reveal obvious gross morphological abnormalities, and all major organs, e.g.
lung (Fig. 3C,D), heart
(Fig. 3E,F) and kidneys (not
shown), appeared normal. However, there was a marked reduction in skeletal
muscle mass (Fig. 3G-L),
probably causing the overall size reduction. Furthermore, while wild-type and
heterozygous embryos/neonates had a straight spine and upright head, the
posture of ß1D1D ki/ki embryos was characterised by pronounced
spinal curvature and downward facing head
(Fig. 3A). Interestingly,
myogenin-deficient mice (Hasty et al.,
1993; Nabeshima et al.,
1993
) displayed a similar posture and gross phenotype, and also
died perinatally due to a severe reduction in skeletal muscle mass.
Reduced primary and secondary myotube numbers in E18.5 ß1D ki/ki
embryos
The next question to be considered was whether the reduction in muscle mass
resulted from selective loss of either primary or secondary myotubes, or
whether these were affected equally. This is important since maternal
malnutrition has been shown to affect the development of secondary, but not
primary myotubes (Wilson et al.,
1988). Thus, a selective reduction in secondary myotubes could
indicate an indirect effect on skeletal muscle development due to placental
insufficiency, rather than a direct effect of the ß1D1D ki/ki
genotype in skeletal muscle cells.
The number of primary and secondary myotubes were compared in three different muscle groups in E18.5 wild-type and ß1D1D ki/ki embryos, namely cleidomastoideus (neck), intercostal/serratus dorsalis (trunk) and lower hindlimb muscles. A comparable reduction in the number of both primary (27%) and secondary (30%) myotubes per mm2 in crosssections of trunk muscles of ß1D1D ki/ki embryos was observed but this was not statistically significant (Fig. 4A-C).
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Furthermore, ß1D1D ki/ki lower hindlimb muscles showed a
significant reduction (37%) in primary myotubes as did the individual muscles
assayed (tibialis anterior, TA; extensor digitorum longus, EDL; and the
peroneus group, P) (Fig. 4G-I).
Secondary myotubes were counted only in EDL muscles
(Fig. 4J-L); the results showed
a significant decrease (44%) in ß1D1D ki/ki embryos. The ratio
of secondary/primary myotubes in EDL muscles was similar in wild-type (1.71)
and ß1D1D ki/ki (1.67) embryos at this developmental stage.
These results demonstrate that the reduction in muscle mass observed in E18.5
ß1D1D ki/ki embryos is due to a reduction in the number of both
primary and secondary myotubes. However, since primary myotubes serve as a
scaffold for the formation of secondary myotubes
(Duxson et al., 1989), a
reduction in primary myotubes will cause a proportional reduction in secondary
myotubes, evident from E18.5 onwards (Ashby
et al., 1993
; Kegley et al.,
2001
). Together these results suggested that the reduction in
muscle mass of the ß1D1D ki/ki embryos was not due to placental
insufficiency, but was rather a direct effect of defective primary
myogenesis.
Primary myotube formation and survival are affected in ß1D ki/ki
embryos
The reduction in the number of primary myotubes in E18.5 ß1D1D
ki/ki embryos may result from the formation of fewer primary myoblasts,
their reduced proliferation or subsequent loss by, for example, apoptosis.
Primary myogenesis starts around E12.0 in the mouse, with the number of
primary myotubes reaching a maximum at around E14.5 in hindlimb muscles,
without secondary myotubes being present
(Ashby et al., 1993;
Kegley et al., 2001
).
Serial sections of the lower hindlimb of E14.5 ß1D1D ki/ki and wild-type embryos were stained with anti-slow MHC antibody. ß1D1D ki/ki hindlimb muscles were smaller than wild type at all levels (Fig. 5A-F), and showed a significant average reduction in the number of primary myotubes (16%; Fig. 5I). Fewer serial sections of each muscle were obtained (not shown), suggesting that these muscles were also shorter. In addition, myotubes of ß1D1D ki/ki embryos were smaller (Fig. 5G,H), EDL myotube diameter being 81% of those of wild type (Fig. 5J). This suggested that the exclusive presence of ß1D resulted in a reduced capacity to form primary myotubes, which was reflected both in the lower number formed and their smaller size.
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At E14.5, apoptosis is a normal feature of developing muscle
(Ashby et al., 1993). TUNEL
assay carried out on sections of E14.5 hindlimbs revealed no differences in
apoptosis between wild-type and ß1D1D ki/ki embryos
(Fig. 5M,N), suggesting that
the presence of ß1D does not lead to increased cell death in primary
myoblasts or early myotubes.
It is, however, clear from the results above (see
Fig. 4I and
Fig. 5I) that the reduction in
the number of primary myotubes in ß1D1D ki/ki hindlimbs is much
greater at E18.5 (37%) than at E14.5 (16%). Thus about 21% of ß1D1D
ki/ki primary myotubes are apparently lost during this period. To
pinpoint the exact period of myotube loss, we exposed alternate cryosections
of wild-type and ß1D1D ki/ki E16.5 and E17.5 hindlimbs to
anti-slow MHC antibody and TUNEL assay. Quantification of primary myotubes in
E16.5 EDL showed a similar difference between wild type and ß1D1D
ki/ki as observed in E14.5 (not shown), suggesting that myotube loss had
not yet started. However, at E17.5, primary myotube numbers in ß1D1D
ki/ki EDL (Fig. 5O,P) were
reduced by 18% as compared to the wild type
(Fig. 5S), an intermediate
value between the reduction observed at E14.5 (11%) and E18.5 (40%)
(Fig. 4I and
Fig. 5I). The number of
secondary myotubes was, however, identical both at E16.5 (not shown) and E17.5
(Fig. 5S) between genotypes,
confirming that reduced number of secondary myotubes as a result of defective
primary myogenesis becomes evident only by E18.5
(Ashby et al., 1993). The TUNEL
assay revealed a twofold increase in apoptosis in ß1D1D ki/ki
hindlimb muscles compared with those of wild type both at E16.5 (not shown)
and E17.5 (Fig. 5Q,R,T).
Together these data show that there is an increase in apoptosis in
ß1D1D ki/ki muscles at E16.5, but myotube loss is only evident
by E17.5.
Denervation has been shown to result in reduction of primary myotube
numbers, although the myotube loss was observed before E15.5
(Condon et al., 1990;
Ashby et al., 1993
). Neurites
differentiate and migrate normally on a permissive substrate in ß1D1D
ki/ki embryoid bodies (Gimond et al.,
2000
). No evidence for abnormal innervation was observed in E14.5
ß1D1D ki/ki hindlimb sections stained for PGP9.5, a nerve marker
(not shown). Clusters of acetylcholine receptors were also present in normal
numbers in hindlimb muscles of E18.5 ß1D1D ki/ki embryos (not
shown), suggesting normal development of neuromuscular junctions.
Thus, these results show a significant impairment in primary myogenesis in E14.5 ß1D1D ki/ki embryos, not caused by reduced myoblast proliferation, increased apoptosis or absence of nerves. In addition, our data indicate that long-term primary myotube survival is affected when ß1A is replaced by ß1D.
Differential effects of ß1A and ß1D overexpression during
myogenic differentiation of C2C12 cells
To gain insight in the putative roles of ß1A and ß1D during
myogenic differentiation, C2C12 cells were stably infected with either
ß1D or ß1A constructs and differentiation and fusion parameters
analysed. ß1A is expressed by proliferating C2C12 cells and is
downregulated during myogenic differentiation, while ß1D becomes
upregulated (Belkin et al.,
1996). Infection of C2C12 cells with either splice variant caused
constant and high expression of that splice variant
(Fig. 6A,B) and infection with
ß1D totally inhibited the expression of ß1A
(Fig. 6B).
|
MHC, a marker for myoblast differentiation, was upregulated 2 days after
induction of differentiation in C2C12 cells, as expected, and the levels of
expression steadily increased until day 7
(Andrés and Walsh, 1996;
Dedieu et al., 2002
). Although
both C2C12/ß1A and C2C12/ß1D cells upregulated MHC at a similar rate
(not shown), immunostaining for MHC showed clear differences between infected
and control cultures (Fig.
6F-H,J-L).
After 3 days of differentiation, both C2C12/ß1A and C2C12/ß1D cells showed a similar reduction in number of multinucleated MHC+ myotubes/MHC+ cells (30% reduction compared to C2C12 control) and in fusion index (Fig. 6L,M). After 5 days of differentiation, the number of multinucleated MHC+ myotubes/MHC+ cells formed by C2C12/ß1A cells was similar to the C2C12 control, but the number of nuclei present per myotube was significantly lower. In contrast, both the number of multinucleated MHC+ cells and the number of nuclei per myotube were reduced in C2C12/ß1D cells (Fig. 6L,M).
During differentiation, C2C12/ß1D myotubes elongated normally, but the
elongation of C2C12/ß1A myotubes was blocked and myotubes were rounded
and had little cytoplasm (Fig.
6H,L). Finally, we analysed the expression of connexin43, a gap
junction protein downregulated in C2C12 myotubes
(Reinecke et al., 2000).
C2C12/ß1A cells did not downregulate this protein, in contrast to
C2C12/ß1D cells (Fig. 6E), despite a similar fusion index. Thus, we propose that the failure to
downregulate connexin43 correlates with the failure in myotube elongation
rather than fusion as such.
These data thus show that overexpression of ß1A and ß1D in C2C12 cells differentially affects myogenic differentiation.
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DISCUSSION |
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We demonstrated that the great majority of ß1D1D ki/ki embryos lost during midgestation, died not because of abnormal cell migration or haemorrhage as previously, but from defective placentation. Furthermore, the remaining ß1D1D ki/ki embryos were lost at birth, most likely as a consequence of reduced muscle mass, which affected, among other muscles, the diaphragm and therefore their ability to breath.
By studying the phenotype of the ß1D knock-in mice in a predominantly FVB background, we revealed the importance of ß1 during placental labyrinth development and also demonstrated that precocious and exclusive expression of ß1D in skeletal muscle leads to a reduction in muscle mass.
Replacement of ß1A with ß1D supported cell migration in
vivo
Cell migration on a predominantly FVB background was not perturbed by the
substitution of ß1A by ß1D in any of the migratory cells analysed in
vivo (neural crest cells, limb muscle precursor cells and PGCs), meaning that
their migratory behaviour was unaffected by the exclusive presence of
ß1D. Independent experiments have shown that myoblasts and neural crest
cells derived from mES cells deficient in ß1 were able to migrate
normally in vivo in wild-type/ß1-null chimeric embryos
(Fässler and Meyer, 1995;
Hirsch et al., 1998
). This
suggested that migration in general was not dependent on ß1 integrins. An
alternative explanation, however, could be the low percentage of chimerism
used in those experiments. Thus ß1-null cells could have adhered to
wild-type cells and been passively transported to their destination. However,
PGC colonisation of the gonads has been shown to be dependent on ß1
integrins, by analysis of wild-type/ß1-null chimeric mice
(Anderson et al., 1999
) and
therefore ß1 is necessary for normal PGC migration even in the presence
of a large number of wild-type cells. Our results clearly show that
replacement of ß1A with ß1D had no effect on either the total number
of PGCs or their migration towards the gonads. This strongly suggests that
ß1D is able to support normal cell migration in vivo on a predominantly
FVB background.
Placental labyrinth defects are responsible for early lethality in
ß1D ki/ki embryos
The placental labyrinth starts forming immediately after chorioallantoic
fusion, when foetal blood vessels growing from the allantois contact the
chorionic plate, the precursor of the labyrinthine trophoblast. The chorionic
trophoblast cells then proliferate, differentiate and fuse into multinucleated
syncytiotrophoblast, a prerequisite for labyrinthine trophoblast branching
morphogenesis and subsequent embryonic vascular invasion (Anson-Cartwright et
al., 2000; Rossant and Cross,
2001).
In the mouse, ß1 integrin is strongly expressed in both the
labyrinthine trophoblast and foetal blood vessels throughout their development
(Bowen and Hunt, 1999).
4ß1 in the chorionic plate plays an important role in
chorioallantoic fusion by binding to VCAM1 expressed by the allantois
(Yang et al., 1995
;
Kwee et al., 1995
;
Gurtner et al., 1995
). This
early event does not appear to be affected in ß1D1D ki/ki
embryos. However, labyrinthine branching defects were observed in the minority
of VCAM1-null embryos that underwent chorioallantoic fusion
(Gurtner et al., 1995
),
suggesting that VCAM1-
4ß1 interactions are also important during
later stages.
vß1 and
vß3 heterodimers are expressed
in the labyrinth (Bowen and Hunt,
1999
), which is poorly developed in 80% of
v-null embryos
(Bader et al., 1998
).
Furthermore,
7ß1 is strongly expressed in the chorionic plate, but
is downregulated during labyrinthine branching
(Klaffky et al., 2001
).
Although placental defects have not been described in
7-null embryos,
about half of these embryos are lost at midgestation
(Mayer et al., 1997
), raising
the question of whether
7 is important in trophoblast development as
suggested by in vitro assays (Klaffky et
al., 2001
). Although
6ß1 is present in labyrinthine
blood vessels,
6-null embryos develop to term
(Georges-Labouesse et al.,
1996
), indicating normal placental development. Laminin receptors
are likely to play a role in labyrinthine development since inactivation of
the laminin
5 chain (present in laminin 10/11) causes a reduction in
labyrinthine branching and leads to embryonic lethality between E13.5 and
E16.5 (Miner et al.,
1998
).
ß1A is the only ß1 splice variant present in the early placenta
and trophoblast cell lines (Klaffky et
al., 2001). Based on the phenotype observed in two out of three
ß1D1D ki/ki embryos, we hypothesise that the replacement of
ß1A by ß1D might impair chorionic trophoblast morphogenesis, leading
to abnormal vascularisation of the labyrinth
(Rossant and Cross, 2001
).
Alternatively, ß1D expression on invading embryonic blood vessels might
impair their morphogenesis directly.
The placenta was not examined in ß1D1D ki/ki embryos on a
mixed background (Baudoin et al.,
1998). We cannot rule out placentation defects, at least in part,
causing the observed embryonic lethality, since 5 of the 13 ß1D1D
ki/ki embryos analysed at E12.5 were severely anaemic. It was suggested
that the anaemia was the result of the extravasation of red blood cells
through weak-walled vessels throughout the bodies of ß1D1D ki/ki
embryos. However, in the predominantly FVB background described here,
extravascular blood cells were not observed and we believe that the anaemia
resulted largely or entirely from abnormal placentation.
Precocious expression of ß1D inhibits primary myogenesis
From its expression pattern it is clear that ß1D is not involved in
primary myogenesis. ß1D is detected on the surface of myotubes from E17.5
and then becomes enriched in myotendinous junctions and at costameres, where
it might be important for the formation of strong adhesion sites
(van der Flier et al.,
1997).
We show here that precocious and exclusive expression of ß1D in skeletal muscle affects primary myogenesis. Not only were there fewer primary myotubes at E14.5, but they were also smaller in diameter and shorter.
Fewer primary myotubes could be the result of: (1) a reduction in the number of primary myoblast precursors, (2) a reduction in the proliferation rate of primary myoblasts, (3) their increased apoptosis and (4) an inhibition in myoblast differentiation and subsequent fusion.
Our results suggest that myogenic precursor cells delaminate and migrate
normally towards the limb buds in ß1D1D ki/ki embryos and that
proliferation and apoptosis is not altered at E14.5. However, we cannot
exclude an earlier reduction in the numbers of myoblast precursors. The fact
that primary myotubes are smaller in ß1D1D ki/ki embryos does,
however, strongly suggest a defect in myoblast differentiation and/or fusion.
A significant reduction in primary myotubes was also found in embryos lacking
the transcription factor NFATC3 (Kegley et
al., 2001). However, there, the E15.0 EDL primary myotubes were
normal in size and morphology, leading the authors to exclude a defect in
differentiation and/or fusion and favour a reduction in the pool of primary
myoblasts.
Several studies have suggested a role for ß1 integrins in the
differentiation and fusion of myoblasts in vitro
(Sastry et al., 1996;
Gullberg et al., 1998
), but
because ß1-null cells participate in the formation of skeletal muscle in
ß1-null/wild type chimeras
(Fässler and Meyer, 1995
)
the role of ß1 in this process in vivo has been questioned. However,
Hirsch et al. (Hirsch et al.,
1998
) have shown that while ß1-null myoblasts isolated from
E16.0 embryos (i.e. secondary myoblasts) fuse and form normal myotubes,
myoblasts derived from ß1-null mES cells (i.e. predominantly primary
myoblasts) show a significant inhibition in myotube formation. This study
together with our data strongly suggest that ß1A plays an important role
in the formation of primary myotubes and that substitution of ß1A by
ß1D significantly impedes this process.
Long-term myotube survival is clearly affected in ß1D1D ki/ki
embryos. This could be due to inhibition of myotube growth caused by a
progressive impairment in the addition of myoblast nuclei to the early
myotubes (Zhang and McLennan,
1995). Alternatively, the precocious presence of ß1D might
interfere with the establishment of myotube connections to the surrounding
basement membrane (Vachon et al.,
1996
) or the myotendinous junction
(Miosge et al., 1999
),
compromising myotube survival by forming strong adhesion sites too early.
Overexpression of ß1D and ß1A produces different effects on
C2C12 differentiation
C2C12 cells overexpressing ß1A or ß1D exhibit inhibition of
myotube formation, suggesting that excessive amounts of either ß1 splice
variant interferes with this process. However, the more long-term effect of
ß1D overexpression shows that this splice variant causes a stronger
inhibition. Strikingly, myotube morphology was severely affected in
C2C12/ß1A cells and downregulation of connexin43 did not occur,
suggesting that ß1A inhibits myotube maturation. This is probably due to
overexpression since inhibition in myotube maturation is not observed in
ß1D knock-out mice, where ß1A expression persists in mature muscle
fibres (Baudoin et al., 1998).
Overall, these results suggest that C2C12 myotube formation and maturation is
dependent on a quantitative balance of different integrins, an idea supported
by studies on the effect of integrin
-subunit ratios on ß1A
signalling during myoblast differentiation
(Sastry et al., 1996
;
Sastry et al., 1999
).
Secondary myogenesis is unaffected in ß1D ki/ki embryos,
suggesting different roles for ß1 integrins in primary versus secondary
myogenesis
At E16.5 and E17.5, the number of secondary myotubes was similar between
ß1D1D ki/ki and wild-type embryos, while at E18.5, it was
significantly lower in ß1D1D ki/ki embryos. The ratio of
secondary to primary myotubes, however, was similar in the muscles studied at
E18.5. Thus the number of secondary myotubes is only reduced when the reduced
number of primary myotubes in ß1D1D ki/ki embryos becomes a
limiting factor for the formation of more secondary myotubes
(Ashby et al., 1993). These
results suggest that secondary myogenesis is unaffected in ß1D1D
ki/ki embryos. It is becoming evident that different regulatory pathways
control these two waves of myogenesis in vivo. For example, in myogenin null
embryos secondary myogenesis is much more affected than primary myogenesis
(Venuti et al., 1995
), while
inactivation of NFATC3 causes a selective reduction in primary myotubes
(Kegley et al., 2001
). Our
data show that primary myogenesis is more sensitive to the precocious presence
of ß1D than secondary myogenesis. This not only demonstrates different
roles for ß1A and ß1D, but also strongly suggests that ß1
integrins play distinct roles in primary versus secondary myogenesis in
vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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