First published online 13 June 2007
doi: 10.1242/dev.000877
Development 134, 2615-2625 (2007)
Published by The Company of Biologists 2007
Defective osteoblast function in ICAP-1-deficient mice
Daniel Bouvard1,2,3,*,
Attila Aszodi3,
Günter Kostka3,
Marc R. Block1,2,
Corinne Albigès-Rizo1,2 and
Reinhard Fässler3
1 Université Joseph Fourier, CNRS, UMR 5538, LEDAC, Institut Albert
Bonniot, La Tronche Cedex, F-38706, France.
2 INSERM, U823, Equipe DySAD, Institut Albert Bonniot, F-38042, France.
3 Max Planck Institut für Biochemie, Department of Molecular Medicine, Am
Klopferspitz 18a, 82152 Martinsried, Germany.
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Fig. 1. Disruption of the mouse Icap-1 gene. (A) Partial
structure of the mouse Icap-1 gene and the targeted allele after
homologous recombination. Black boxes represent exons (E2 to E7). The
initiation codon (ATG) is located in exon 2. The expected fragment sizes for
wildtype and recombinant alleles are 20 and 10 kb, respectively, following
digestion with BamHI and hybridization with the indicated external
probe (ext pb). (B) Southern blot analysis of tail DNA isolated from
wild-type, heterozygous mutant and homozygous mutant mice. (C) Northern
blot analysis of total RNA derived from adult kidney of wild-type,
heterozygous and homozygous mutant mice. The filter was hybridized with probes
specific for Icap-1 and Gapdh, respectively. (D)
Western blot analysis of wild-type, heterozygous and homozygous mutant brain
extracts. (E,F) Whole-mount lacZ staining of heterozygous
mutant embryos at E8.5 (E) and E14.5 (F).
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Fig. 2. Growth delay, craniofacial malformation and delayed bone mineralization
in Icap-1-deficient mice. (A) Gross appearance of
Icap-1+/+ and Icap-1-/- P0
littermates. Note that Icap-1-/- mice are smaller in size
than their control littermates. A subset of the Icap-1-/-
offspring has an empty stomach (arrow). (B) Growth curves of control
(Icap-1+/+ or Icap-1+/-) versus
Icap-1-/- (male and female) offspring of two pooled
representative littermates. Mice were weighed every other day over a period of
34 days. Each point represents the mean±s.d. (C) Lateral view
(upper panel) or top view (lower panel) of 30-day-old wild-type and
Icap-1-deficient mice. Note the abnormal shape (short nose and
bulged-head) of the Icap-1-deficient skull compared with the wild
type. (D) X-ray analysis of Icap-1+/+ and
Icap-1-/- 5.5-month-old mice. Note that in the
Icap-1-null mouse the skull shape is severely affected, long bones
are shorter and vertebrae are only poorly ossified (arrows and insets for
higher magnification). (E) Alizarin Red/Alcian Blue staining of E16.5
skeletons showing reduced Alizarin Red staining intensity of the maxilla
(arrowheads), the radius and ulna of the forelimb (arrows) in
Icap-1-null tissues. (F) Alizarin red/Alcian Blue skeletal
staining at newborn stage. No obvious difference in staining intensity or
patterning could be seen between the two genotypes.
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Fig. 3. Defect of calvarial ossification in Icap-1 mutant mice.
(A,B) Whole-mount Alizarin Red staining of the skulls of
Icap-1+/+ and Icap-1-/- newborn mice.
(A) Mineralization of the skull base is comparable in wild-type and mutant
animals. (B) Mineralization of the skull vault. The mineralized areas of the
interparietal (ip), parietal (p) and frontal bones (f) are smaller and
fontanelles are open in Icap-1-/- mice. (C-H)
Whole-mount Alizarin Red/Alcian Blue staining of wild-type and
Icap1-null 2-month-old calvariae. The area of the metopic suture is
boxed in C-E, and displayed at high magnification in F-H. In
Icap1-null mice (D,E), the sagittal suture is shorter, the coronal
sutures are V-shaped and irregular as compared with wild-type littermates (C).
At this age, the metopic suture (arrow) is closed in wild type (F), whereas in
Icap-1-deficient mice the posterior part of the metopic suture is not
ossified and is stained with Alcian Blue (G,H). Some
Icap-1-/- mice display non-mineralized, Alcian
Blue-positive areas in the frontal bones (arrowhead in H). (I,J)
Whole-mount skeletal staining of the axis (I) and the pelvic region (J) of
21-day-old wild-type and Icap-1-/- (mt) mice. Arrows point
to the fusion defect observed in the Icap-1-deficient mice, whereas
fusion is completed in control animals. Scale bars: 2 mm in A,B; 5 mm in C-E;
2 mm in F-H.
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Fig. 4. Defective formation of the osteogenic front in
Icap-1-/- calvaria. (A) Whole-mount
lacZ staining of newborn calvariae of Icap-1+/+
and Icap-1-/- mice. Strong ß-gal activity visualizes
Icap-1 expression in the sutural regions and the edges of the bony
plates of the Icap-1-deficient calvaria. (B,C) Hematoxylin and
Eosin-stained frontal section of the parietal region. The distance between the
ossified ends of the parietal bones (arrows) is wider in
Icap-1-/- (C) compared with wild-type mice (B).
(D-G) Frozen frontal sections through the sagittal suture of wild-type
(D,F) and Icap-1-/- (E,G) animals stained with Hematoxylin
and Eosin (D,E) or for ß-gal activity (F,G). Note that the wild-type
suture presents a typical condensed cell population corresponding to the
osteogenic front (arrowheads) that is severely reduced in the
Icap-1-/- mice. lacZ staining indicates extensive
expression of ICAP-1 in this region. B, bone; i.m, intersutural mesenchyme;
SO, supraoccipital bone; P, parietal bone; F, frontal bone; S, sagittal
suture; L, lambdoid suture; C, coronal suture; IF, interfrontal suture. Scale
bars: 2 mm in A; 50 µm in B-G.
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Fig. 5. Reduced proliferation of calvarial osteogenic cells in
Icap-1-null mice. (A) Immunodetection of the proliferation
marker Ki67 in the sagittal sutural region of newborn
Icap-1+/+ and Icap-1-/- calvaria. The
osteogenic front of the Icap-1-/- mice displays a reduced
number of Ki67-positive cells. Boxes indicate regions used for KI-67 and BrdU
quantification in B. Scale bar: 25 µm. (B) Quantification of Ki67-
and BrdU-positive cells in the osteogenic front of control and mutant animals.
Error bars represent s.d.; asterisks indicate a statistically significant
difference between Icap-1+/+ and
Icap-1-/- (**, P<0.0001).
(C) Immortalized calvarial osteoblasts. ICAP-1-deficient cells
(SV2.1-Icap-1-/-) show significantly reduced BrdU-labeling
index compared with wildtype cells (SV6.5-Icap-1+/+).
Retroviral transfection of the Icap-1 cDNA into the
Icap-1-/- cells rescues the proliferation defect
(SV2.1-Icap-1resc) (**, P<0.0001).
(D) SV2.1 and SV2.1-Icap-1resc cells were cultured
for 24 hours in 1% FCS before replating them onto 10 µg/ml FN. After 5
hours of spreading, cells were washed with PBS and directly lysed onto Petri
dishes with RIPA buffer. Protein (30 µg per lane) was gel separated and
then transferred onto PVDF membrane before processing for western blotting
with anti-cyclin D1 antibody. The same gel was blotted with anti-actin
polyclonal antibodies for normalizing protein loading.
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Fig. 6. Osteogenic differentiation is abnormal in Icap-1-/-
calvaria. Frontal sections through the parietal bones and the sagittal
suture of Icap-1+/+ and Icap-1-/-
newborn mice stained for (A) AP, (B,C) osteonectin, (D,E)
COL1, (F) FGFR1 and (G) FGFR3. C and E are 400x
magnification views of the osteogenic front region in B and D, respectively.
Dashed lines represent bone borders and osteogenic front area. B, bone; i.m.,
intersutural mesenchyme. Note the reduced expression of the osteogenic and
differentiation markers in Icap-1-/- tissue. (H,I)
Whole-mount in situ hybridization on calvaria of E17.5 embryo was performed to
detect either Runx2 (Cbfa1) (H) or Bsp (I)
transcripts in Icap-1+/+ and Icap-1-/-
embryos. The upper panel of each pair is an overview of the full calvaria; the
lower panel is a closer view of the osteogenic front (boxed in the overview).
Scale bars: 2 mm.
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Fig. 7. Bone nodule formation by the calvarial osteoblast is defective in
Icap-1-/- mice. (A) Primary
Icap-1+/+ and Icap-1-deficient osteoblasts were
cultured for 4 weeks in inductive medium and stained with Alizarin Red for
monitoring bone nodule formation. Icap-1-/- cultures show
fewer and smaller mineralized nodules (arrows) compared with wild-type
cultures (Icap-1+/+). The result is representative of at
least three independent experiments from three different animals. Scale bar: 1
mm. (B) Immortalized osteoblasts SV2.1 Icap-1-/-,
SV6.5 Icap-1+/+ or SV2.1-Icap-1resc
were cultured in inductive medium for 3 weeks and mineralized nodules were
identified by Alizarin Red staining. Icap-1-/- osteoblasts
show a significantly reduced nodule formation compared with wild-type or
Icap-1resc osteoblasts. The means and s.d. were calculated
from three independent experiments (*, P<0.05;
**, P<0.0001).
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Fig. 8. Increases in ß1 integrin activity in
Icap-1-/- mouse cells. (A) Sagittal sections
were stained with the 9EG7 monoclonal antibody (red) that recognizes
ligand-bound ß1 integrins. ß1 integrins are
highly expressed and strongly activated in wild-type cells
(Icap-1+/+) at the osteogenic front (arrowheads) and at
the surface of the bony plates (arrows). In Icap-1-/-
tissues, the cells at the osteogenic front show a moderate staining for
activated ß1 integrin. Scale bar: 50 µm. (B) FACS
analyses demonstrate a slight reduction in the surface expression of ß1
integrins (assayed by the MB1.2 monoclonal antibody) on
Icap-1-/- primary osteoblasts (red) compared with
wild-type osteoblasts (blue). (C) Adhesion assays. The adhesion of
Icap-1-/- primary osteoblasts to FN and COL1 is moderately
increased compared with that of Icap-1+/+ cells. Adhesion
is expressed as a percentage of the maximal adhesion and measured in duplicate
in two independent experiments from two different animals
(P<0.05). (D) FACS analysis demonstrates increased binding
of FITC-Fn7-10 fragment to Icap-1-/- osteoblasts (green).
(E) The activation index (AI) of the ß1 integrin is
increased in Icap-1-/- osteoblats. The maximum AI obtained
is used to normalize both genotype groups and designated as 100.
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Fig. 9. Defects in bone nodule formation and spheroid compaction with
Icap-1-/- osteoblasts. (A) Immortalized SV6.5
(wild-type) or SV2.1 (Icap-1-/-) preosteoblasts were
induced to differentiate in vitro for 15 days, then bone nodule formation and
organization were visualized by phase contrast microscopy. The inset is a
higher magnification view of the boxed region. Note that SV2.1 cells are less
cohesive than control cells. (B) Spheroids were formed from SV2.1, from
SV2.1 rescued with Icap-1 cDNA or from SV6.5 preosteoblasts and
analyzed after 16 hours incubation at 37°C using a standard protocol for
the hanging drop assay. We used 25,000 cells per drop in each experimental
condition. For antibody treatment, 9EG7 or control antibodies were added at a
final concentration of 10 µg/ml during the condensation process in a medium
supplemented with an FN-depleted serum. Note that cellular compaction is
delayed in both Icap-1-/- and 9EG7-treated wild-type
osteoblast compared with untreated wild-type cells.
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Fig. 10. Schematic representation of bone formation in calvaria. Bone
formation is a multistep process that requires an initial condensation stage.
In this step, which enables the cells to start the differentiation process,
early markers such as RUNX2 and osterix (also known as SP7) are expressed. At
later stages, other osteogenic markers such as BSP, osteocalcin (also known as
BGLAP) and osteonectin are produced. Lack of ICAP-1 expression slows down
proliferation and the ability of mesenchymal cells to compact. Since the
compaction is a very early event in the osteoblast differentiation pathway,
the expression of more-distal markers is consequently reduced in
Icap-1-/- mice.
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