1 Institute of Developmental Genetics, GSF-National Research Center for
Environment and Health, Ingolstädter Landstrasse 1, 85764 Neuherberg,
Germany
2 Institute of Pathology, GSF-National Research Center for Environment and
Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
3 Department of Developmental Medicine (Pediatrics), Osaka University Graduate
School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
4 Gene Center and Institute of Biochemistry, Ludwig Maximilians University,
Feodor Lynenstrasse 25, 81377 Munich, Germany
Author for correspondence (e-mail:
imai{at}gsf.de)
Accepted 17 August 2004
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SUMMARY |
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Key words: Lrp6, Wnt signaling, Somitogenesis, Osteoporosis, Mouse
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Introduction |
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The Wnt pathway has recently been implicated in the control of
somitogenesis (Aulehla et al.,
2003; Hamblet et al.,
2002
) and of bone mass in adults in humans and mice
(Boyden et al., 2002
;
Gong et al., 2001
;
Kato et al., 2002
;
Little et al., 2002
). In the
context of somite development, Wnt signaling mediated by Wnt3a has been
implicated in the specification and propagation of progenitor cells of the
paraxial mesoderm in the primitive streak or in the tail bud
(Takada et al., 1994
;
Yoshikawa et al., 1997
), and
this Wnt signaling is transduced through the canonical ß-catenin
signaling pathway (Galceran et al.,
1999
; Galceran et al.,
2001
). As late functions, Wnt signaling is also known to play
essential roles in the dorsoventral patterning of formed somites, which is
required for proper development of the dermomyotome and the myotome
(Capdevila et al., 1998
;
Fan et al., 1997
;
Münsterberg et al., 1995
;
Wagner et al., 2000
). However,
whether Wnt signaling plays any significant role in the periodic morphogenetic
movement of somitogenesis that takes place in the presomitic mesoderm (PSM)
had not been clear until recently. Mouse dishevelled 2 (Dvl2),
together with its paralog dishevelled 1 (Dvl1), has recently been
shown to be required for somite segmentation, through the analysis of
Dvl2-single and Dvl1;Dvl2-double knockout mice
(Hamblet et al., 2002
).
Furthermore, it has recently been demonstrated that a paralog of Axin,
Axin2 (also called conductin) exhibits a dynamic and cyclic expression
profile in the PSM. This finding, together with the detailed analysis of the
notch-delta signaling activity in Wnt3a mutants, has provided clear
evidence for the involvement of Wnt signaling in the process of somitogenesis
in the PSM, functioning upstream of notch-delta signaling
(Aulehla et al., 2003
). On the
other hand, recent studies have also elucidated another, unexpected functional
aspect of Wnt signaling in the postnatal life, with the identification of Lrp5
as one of the key genetic factors that control bone mass. Positional cloning
of the gene responsible for osteoporosis-pseudoglioma syndrome (OPPG), an
autosomal recessive disorder in humans, revealed that loss-of-function
mutations in LRP5 lead to a low bone mass phenotype (osteoporosis)
(Gong et al., 2001
).
Despite the availability of Lrp6-null mouse mutants
(Pinson et al., 2000), whether
Lrp6 plays any roles in somitogenesis during development and in the control of
bone mass during adult life has not been known, because of strong pleiotropic
effects of Lrp6 deficiency that leads to neonatal lethality
(Pinson et al., 2000
). In the
present study, we demonstrate that Lrp6 is required for somitogenesis and
osteogenesis, through the analysis of a novel spontaneous mouse mutation
ringelschwanz (rs), identified in this study as a viable
hypomorphic allele of Lrp6.
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Materials and methods |
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Genotyping by PCR
Lrp6 genotyping was performed by PCR-based RFLP analysis as
follows. A 456-bp including exon 12 of Lrp6 was amplified by PCR with
primers: 5'-TTTCCCAAAATAGGACTCAACCG-3' (forward) and
5'-CCCCAGTTTCAACCTTTGGATTATAC-3' (reverse), under the following
condition: initial denaturing at 94°C for 4 minutes, followed by 40 cycles
of 94°C/30 seconds, 60°C/45 seconds, 72°C/45 seconds, and final
elongation at 72°C for 8 minutes. A single-nucleotide difference between
rs-mutant and wild-type alleles was detected by digestion of the PCR
products with HpaII.
Skeletal preparations
Skeletons of E14 and newborn specimens were prepared by a double staining
procedure with Alcian blue 8 GX (Sigma) and Alizarin red S (Sigma), according
to the procedure described previously
(Kessel et al., 1990).
Scanning electron microscopy and semi-thin histology
Embryos (E11.5) for semi-thin histology and for scanning electron
microscopy (SEM) were fixed in 4% paraformaldehyde (PFA) and post-fixed in 2%
OsO4. For semi-thin histology, specimens were dehydrated and
embedded in EPON 812 (Merck). Sections (1 µm) were made with a Reichert
Ultracut E (Leica) and stained with 1% Toluidin Blue (Merck). For SEM, fixed
samples were critical-point-dried with CO2 and sputter-coated with
platinum. Coated specimens were examined in a JSM-6300F (JEOL).
Whole-mount RNA in situ hybridization
Whole mount in situ hybridization was performed as described
(Kokubu et al., 2003) using
digoxigenin-labeled riboprobes. The following cDNA probes were used in this
study: Sox10 (Kuhlbrodt et al.,
1998
), Mesp2 (Saga et
al., 1997
), Uncx4.1
(Mansouri et al., 1997
),
Tbx18 (Kraus et al.,
2001
), Dll1
(Bettenhausen et al., 1995
),
paraxis (Burgess et al., 1995
)
and Lfng (Evrard et al.,
1998
).
Assay for the Wnt-ß-catenin pathway in cultured fibroblasts
Primary fibroblasts were prepared from minced dorsal skin of newborn
animals. Fibroblasts were grown at 37°C in Dulbecco's modified Eagle's
medium containing 15% fetal calf serum. Transient transfection was performed
with lipofectamine (Invitrogen) according to the manufacturer's protocol. A
LEF-luciferase (LUC) reporter plasmid, containing seven multimerized
LEF1-binding sites linked to fos promoter-LUC gene, was transfected alone or
in combination with expression plasmids for LEF1, ß-catenin or Wnt1 as
described (Hsu et al., 1998).
A Rous sarcoma virus-ß-galactosidase control plasmid was included in each
transfection experiment to control for the efficiency of transfection.
Luciferase and ß-galactosidase assays were performed as described
(Hsu et al., 1998
).
X-ray radiography and bone histology
For X-ray radiography and bone histology of adult mice
(Fig. 10), radiographs of the
cadavers were taken in a cabinet X-ray system (Hewlett-Packard). Subsequently,
the specimens were fixed in 4% PFA, decalcified in EDTA, and embedded in
paraffin. Sections (5 µm thickness) were stained with hematoxylin and
eosin. For histology of non-decalcified bones
(Fig. 9), the left limbs were
separated and fixed in 4% PFA. The right limbs were used for skeletal
preparation. PFA-fixed limbs were dehydrated and embedded in
methylmethacrylate and sectioned (5 µm) on a motorized Minot microtome
(Jung). Serial sections was stained either with Alcian blue at pH 1.0 or with
Alizarin red followed by hematoxylin.
|
|
Peripheral quantitative computed tomography (pQCT)
Computed tomography was performed with the XCT Research SA+ and its
associated software version 5.40 (Stratec Medizintechnik). Metaphyseal pQCT
scans of tibiae were performed to determine the cortical and trabecular
volumetric BMD and cortical thickness. The scan was positioned in the
metaphysis at a distance of 1.7 mm from the proximal end of the epiphysis. The
trabecular bone region was defined by peel mode 2, using a threshold at 395
mg/cm3. Student's t-test was used for statistical
evaluations.
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Results |
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Dysfunction of the somite segmentation clock in rs
The notch-delta signaling pathway is known to play an essential role in
somitogenesis as part of the segmentation clock machinery that drives periodic
formation of somites (Pourquié,
2001). Thus, we examined expression of some key players in the
notch-delta pathway, including Dll1, Mesp2 and Lfng, in
rs embryos. Dll1 was normally expressed very strongly in the
PSM, except in prospective anterior-half compartments in the rostral part of
the PSM (Bettenhausen et al.,
1995
). In rs mutants, expression of Dll1
appeared indistinguishable from that in wild-type controls until early E9
(Fig. 7A,B). Remarkably, while
strong expression of Dll1 in the caudal two-thirds of the PSM was
well maintained, expression of Dll1 in a striped pattern in the
rostral PSM was disturbed in rs embryos at mid to late E9
(Fig. 7C-F). At early E10,
Dll1 expression in the caudal PSM of rs mutants was
significantly reduced (Fig. 7H,
compared with G), and by mid E10, Dll1 expression in the PSM was
totally abolished (Fig. 7I).
Cyclic expression of Lfng in the PSM reflects the activity of the
segmentation clock (Evrard et
al.,1998
). Consistent with the progressive downregulation of
Dll1 in the PSM of rs embryos, Lfng expression in
the PSM was also strongly downregulated by mid E10
(Fig. 7O, compared with N).
Interestingly, Mesp2 expression in the rostral part of the PSM
appears to be maintained for a longer time, although at a reduced level, even
at mid to late E10 in rs embryos (n=20)
(Fig. 7J-M).
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|
Osteoporosis in adult rs mice
We next examined the integrity of adult bones in rs mutants. The
status of lumbo-sacral vertebrae in 9-month-old rs/rs and its control
animal was examined by X-ray radiography
(Fig. 10A,B). Aside from the
strong vertebral malformations in rs/rs, vertebrae were more
translucent in rs. Consistently, on histological sections the
reductions in the number of trabecules and in the thickness of the cortical
bone were remarkable in rs (Fig.
10C,D). Interestingly, a clump of cells of chondrocytic morphology
(arrowhead in Fig. 10F) was
frequently seen in rs, suggesting the presence of foci undergoing the
recovery process from multiple microfractures. In order to quantitatively
assess bone density and cortical bone thickness, we further performed a
peripheral quantitative computed tomography (pQCT) analysis on the proximal
part of the tibia from 14 month-old female animals, and the summary of pQCT
data from the metaphysis region of the tibia is shown in graphs
(Fig. 10G-I). In
rs/rs, the bone density was significantly reduced to 84% of wild-type
controls in the whole metaphysis (P<0.05)
(Fig. 10G) and to 94% in
cortical bones (P<0.01) (Fig.
10H). In the metaphysis, the cortical bone thickness was
significantly reduced to 71% of wild-type controls (P<0.01)
(Fig. 10I). rs/+
samples exhibited intermediate values between wild-type and rs/rs,
suggesting the semidominant nature of the rs mutation with respect to
these traits. Thus, we demonstrated the presence of a low bone mass phenotype
in rs mutants, which was similar to that of Lrp5 mutants.
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Discussion |
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Wnt/Lrp6 pathway in somitogenesis
The morphogenetic movement to form somites is regarded as the intrinsic
property of the PSM. However, some extrinsic signal(s) from the overlaying
surface ectoderm is required to complete somite segmentation
(Borycki et al., 2000;
Correia and Conlon, 2000
;
Sosic et al., 1997
). The bHLH
transcription factor paraxis is thought to mediate this extrinsic signal
(Correia and Conlon, 2000
;
Sosic et al., 1997
), and
paraxis deficiency leads to disturbances in the epithelialization and AP
polarity determination of somites (Burgess
et al., 1996
; Johnson et al.,
2001
). Thus we assumed that defects in somitogenesis in
rs might be in part due to paraxis deficiency. However, we found that
paraxis expression is unexpectedly well maintained in rs mutants,
suggesting the presence of additional player(s) that is/are controlled by Wnt
signaling.
Notch-delta signaling is required for the upregulation of Mesp2 in
the rostral PSM, and the induced Mesp2 in turn represses Dll1
(Takahashi et al., 2000). In
the prospective somite posterior halves at the somite stage I level
(Pourquié and Tam,
2001
), where Mesp2 expression has been downregulated,
Dll1 is re-upregulated via Psen1-dependent notch-delta signaling (the
notch-delta-Mesp2 regulatory loop)
(Takahashi et al., 2000
;
Saga and Takeda, 2001
). Thus,
our observation that this Dll1 re-upregulation is disturbed in
rs mutants suggests that this process of Dll1
re-upregulation is also dependent on Wnt signaling. This finding points to the
possibility of the regulatory interactions between the Wnt and notch-delta
signaling pathways in the control of somitogenesis at the rostral part of the
PSM. The present study does not define when and how Wnt signaling is required
for the maintenance of the notch-delta-Mesp2 regulatory loop. Further study is
needed to address these issues.
In the PSM of rs/rs, we could not detect significant change in the
status of cell proliferation and programmed cell death
(Fig. 8). This suggests that
the apparent reduction in the number of cells in the PSM corresponding to the
sacral and tail region of rs/rs mutants is mainly due to the
reduction in the rate of production of paraxial mesoderm cells in the tail
bud. Recently, Wnt3a signaling has been implicated in the proliferation of PSM
cells in the chick (Galli et al.,
2004), but our observation appears inconsistent with this notion.
The proliferative role of Wnt3a in the chick is proposed based on observations
from overexpression studies. Thus, our result from a loss-of-function study in
rs may not necessarily be contradictory. With noting the hypomorphic
nature of the rs mutation, we do not rule out the possibility that
Lrp6-mediated Wnt signaling is indeed required for cell proliferation in the
PSM.
Genetic factors for the pathogenesis of vertebral segmentation defects
In humans, a number of hereditary disorders with vertebral segmentation
defects have been reported. However, the molecular pathogenesis remains
unknown in most cases. Spondylocostal dysostosis is one form of vertebral
segmentation defect, and involves characteristic rib malformations with
proximal fusions, called crab-like chest. Vertebral segmentation defects in
rs mice, due to the disturbances in somitogenesis, are frequently
associated by rib fusions at the proximal part
(Fig. 1C-E). Thus, Lrp6 may be
one of genetic factors for the pathogenesis of spondylocostal dysostosis in
humans.
In the mouse, a group of classical mutations, collectively referred to as
`Wirbel-Rippen-Syndrom (vertebra-rib syndrome)'
(Theiler, 1968;
Theiler, 1988
), including
Crooked tail (Morgan,
1954
), Malformed vertebrae
(Theiler et al., 1975
),
pudgy (Grüneberg,
1961
), Rachiterata
(Theiler et al., 1974
),
Rib fusions (Theiler and Stevens,
1960
), Rib-vertebrae
(Theiler and Varnum, 1985
) and
Fused (Theiler and
Glücksohn-Wälsch, 1956
), affect vertebral segmentation
with rib malformations. Their characteristic dysmorphologies in the vertebrae
and ribs strikingly resemble those seen in individuals suffering from
spondylocostal dysostosis. Indeed, delta-like 3 (Dll3),
encoding a ligand for the notch receptor, has been shown to be mutated in the
pudgy mutation in the mouse
(Kusumi et al., 1998
).
Accordingly, the human counterpart DLL3 is mutated in the
Jarcho-Levin syndrome, an autosomal recessive hereditary disorder,
representative of spondylocostal dysostosis in humans
(Bulman et al., 2000
). On the
other hand, Axin, encoding a negative regulator of the
Wnt/ß-catenin signaling pathway, was identified as a gene disrupted in an
allelic series of Fused (Zeng et
al., 1997
). Furthermore, vertebral column malformations of
knockout mutants in lunatic fringe (Lfng)
(Evrard et al., 1998
;
Zhang and Gridley, 1998
) and
Hes7 (Bessho et al.,
2001
) also phenocopy spondylocostal dysostosis symptoms, thus they
can also be regarded as mouse models for spondylocostal dysostosis. It should
be noted that these genes discussed here are components of the notch-delta or
Wnt pathways in somitogenesis. This notion is consistent with the emerging
view from the recent studies (Aulehla et
al., 2003
; Hamblet et al.,
2002
) and our present work that somitogenesis is controlled by a
concerted interaction between the notch-delta and Wnt signaling pathways. It
is thus conceivable that various components of the notch-delta and Wnt
pathways comprise genetic factors for the pathogenesis of vertebral
segmentation defects including spondylocostal dysostosis. As discussed below,
in rs mutants, delayed ossification and osteoporosis associate with
vertebral segmentation defects. Since the notch-delta pathway has not been
implicated in osteogenesis, this association might be of diagnostic importance
in sorting out the potential molecular etiology of individuals with vertebral
segmentation defects.
Lrp6 as a novel genetic factor for osteoporosis
Our analyses of rs animals at postnatal stages revealed two types
of bone defects. First, a delay in ossification in rs mutants was
confirmed in phalangeal bones in fingers and toes, which is very similar to
that reported for Lrp5-null mice (Kato et
al., 2002). Our histological analysis suggests that this defect is
probably secondary to preceding disturbances in chondrocyte differentiation in
the context of endochondral ossification. Indeed, our preliminary analysis of
embryos between E14 and E18 suggests that delayed ossification is a general
problem in rs mutants, because it is also present in other bones
including the zeugopod and stylopod of the limbs (data not shown). Second,
bone density and mass are reduced, which are also similar to those in
Lrp5-deficient mice. Lrp5 and Lrp6 are highly similar in the primary
structure and in the function as coreceptors in Wnt signaling. Furthermore,
both Lrp5 and Lrp6 are induced by bmp2 treatment in
osteoblastic ST2 cells (Gong et al.,
2001
). Thus, it is very likely that there is a functional
redundancy between Lrp5 and Lrp6 in bone formation. Indeed, a genetic
interaction between Lrp5 and Lrp6 has recently been demonstrated during
osteogenesis and during gestation (Kelly
et al., 2004
). We confirmed co-expression of Lrp5 and Lrp6 in
embryonic fibroblasts by RT-PCR (see Fig. S2 in the supplementary material).
Nevertheless, our in vitro assay system to assess the function of the mutated
Lrp6 could detect significantly reduced Wnt signal transduction in
rs/rs (Fig. 5). These
observations together suggest that the contribution made by either Lrp5 or
Lrp6 in their cooperation may significantly vary, presumably in a
tissue-specific manner. This idea is consistent with the notion that Lrp6
functions more significantly than Lrp5 at least during gestation
(Kelly et al., 2004
).
The strong malformations in the axial skeleton in rs mutants are, surprisingly, not associated with disturbances in nerve functions that affect locomotion. Therefore, at least, the observed osteoporosis phenotype in rs mutants is unlikely to be a secondary consequence of the vertebral malformations. Whether Lrp6, like Lrp5, positively regulates osteoblast proliferation and function is currently under investigation. Further study is required to define how the functional roles of Lrp5 and Lrp6 are shared in the control of bone development and homeostasis. If Lrp5 and Lrp6 function redundantly also in adult bones, pharmacological activation of Lrp6-mediated signaling can be a therapeutic means even in LRP5-deficient individuals suffering from osteoporosis.
Our observation that rs/+ animals show a slight decrease in bone
mass suggests that the function of Lrp6 in bones is haploinsufficient. A
similar dosage effect has been observed for Lrp5 in humans and mice:
Lrp5-null heterozygotes exhibit reduced bone mass
(Gong et al., 2001;
Kato et al., 2002
). Together
with the notion that activating mutations in Lrp5 increases bone mass in a
dominant manner (Boyden et al.,
2002
; Little et al.,
2002
), these observations suggest that the activity of the Wnt
canonical pathway may have to be tightly regulated within a certain range with
the intact two copies of each of the Lrp5 and Lrp6
genes.
Recent studies on Lrp5 have also shed light on its unexpected roles in
cholesterol metabolism and in glucose-induced insulin secretion, thereby its
potential involvement in atherosclerosis and in diabetes has been indicated
(Magoori et al., 2003;
Fujino et al., 2003
). It is
currently not known whether Lrp6 exerts similar functions in these biological
processes. However, these issues can certainly be addressed in the rs
mutant mouse line by taking advantage of the hypomorphic nature of the
Lrp6 mutation. Thus, it is conceivable that future studies with
rs mutant mice will bring further so-far uncovered insights into Lrp6
functions in both the pre- and postnatal stages.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/21/5469/DC1
* These authors contributed equally to this work
Present address: Department of Social and Environmental Medicine, Graduate
School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
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