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First published online 13 June 2007
doi: 10.1242/dev.000877

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Defective osteoblast function in ICAP-1-deficient mice

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.

View larger version (43K): [in this window] [in a new window]   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).

View larger version (63K): [in this window] [in a new window]   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.

View larger version (73K): [in this window] [in a new window]   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.

View larger version (66K): [in this window] [in a new window]   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.

View larger version (48K): [in this window] [in a new window]   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.

View larger version (102K): [in this window] [in a new window]   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.

View larger version (61K): [in this window] [in a new window]   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).

View larger version (36K): [in this window] [in a new window]   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.

View larger version (73K): [in this window] [in a new window]   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.

View larger version (20K): [in this window] [in a new window]   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.