Abnormal skeletal and cardiac development, cardiomyopathy, muscle atrophy and cataracts in mice with a targeted disruption of the Nov (Ccn3) gene - PubMed (original) (raw)

Abnormal skeletal and cardiac development, cardiomyopathy, muscle atrophy and cataracts in mice with a targeted disruption of the Nov (Ccn3) gene

Emma Heath et al. BMC Dev Biol. 2008.

Abstract

Background: Signals from the extracellular environment control many aspects of cell behaviour including proliferation, survival, differentiation, adhesion and migration. It is increasingly evident that these signals can be modulated by a group of matricellular proteins called the CCN family. CCN proteins have multiple domains through which they regulate the activities of a variety of signalling molecules including TGFbeta, BMPs and integrins, thereby influencing a wide range of processes in development and disease. Whilst the developmental roles of CCN1 and CCN2 have been elucidated, very little is known about the function of CCN3 (NOV). To investigate this, we have generated mice carrying a targeted mutation in the Nov gene (Novdel3) which reveal for the first time its diverse functions in embryos and adults.

Results: By replacing Nov exon 3 with a TKneomycin cassette, we have generated Novdel3-/- mice which produce no full length NOV protein and express at a barely detectable level a mutant NOV protein that lacks the VWC domain. In Novdel3-/- embryos, and to a lesser extent in Novdel3+/- embryos, development of the appendicular and axial skeleton was affected with enlarged vertebrae, elongated long bones and digits, delayed ossification, increased bone mineralization and severe joint malformations. Primary embryo fibroblasts from Novdel3-/- mutant embryos showed enhanced chondrogenesis and osteogenesis. Cardiac development was also influenced leading to enlargement and abnormal modelling of the endocardial cushions, associated with septal defects and delayed fusion. In adults, cardiomyopathy was apparent, with hypertrophy and calcification of the septum and left ventricle dilation. Muscle atrophy was seen by 5 months of age, associated with transdifferentiation to fat. Premature tissue degeneration was also seen in the lens, with cataracts present from 6 months.

Conclusion: We have generated the first mice with a mutation in the Nov gene (Novdel3). Our data demonstrate that NOV is a regulator of skeletal and cardiac development, and implicates NOV in various disease processes including cardiomyopathy, muscle atrophy and cataract formation. Novdel3 mutants represent a valuable resource for studying NOV's role in the modulation and co-ordination of multiple signalling pathways that underpin organogenesis and tissue homeostasis.

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Figures

Figure 1

Figure 1

Gene targeting at the Nov locus. A. Schematic diagram showing wild type and targeted Nov alleles, the targeting construct, and the positions of the 5' and 3' external probes used to confirm targeting by Southern blotting. The TkneopolyA cassette replaces exon 3 in the targeted allele while the HSV TK cassette allows negative selection with Gancyclovir. B. Southern analysis of DNA from wild type, heterozygous and homozygous embryos using 5' and 3' external probes to confirm targeting. Digestion with _Bam_HI gave a 16 kb wild type band with the 5'external probe and 14 kb targeted allele with the 5' probe, while digestion with _Eco_RI gave a 14 kb wild type band and 8.5 kb targeted band with the 3' probe. C. Western blotting of whole cell lysates and conditioned media from Nov del3 homozygous and wild type E13.5 PEFs, using an anti-NOV antibody (59.3). Although NOV protein is readily detectable in wild type PEF whole cell lysates, no NOV protein can be detected in Nov del3 homozygous PEFs. Conditioned medium from wild type PEFs contains high levels of secreted NOV, whereas that from Nov _del3_-/- PEFs contains trace amounts of mutant NOV protein lacking the VWD domain.

Figure 2

Figure 2

Skeletal staining of wild type and Nov _del3_-/- E19.5 embryos and adults with Alcian blue (cartilage) and Alizarin red (bone). A-F: Nov _del3_-/- E19.5 embryos. A. Staining of rib cages showing barrel chest in Nov del3 -/- with overgrowth of ribs. B. Hind foot showing fusion of the tarsal cartilage elements (white arrow) and elongation of the digits in Nov del3 -/-. Note increased intensity of Alizarin red staining and thickening of the tibia in Nov _del3_-/- compared to wild type. C. Knee abnormalities in Nov _del3_-/- compared with wild type, including flattening of the patella. D. Malformation of the wrist elements in Nov del3 -/- compared to wild type. E. Dislocation of hip (arrow) in Nov _del3_-/- compared to wild type. F. Kinking of tail with compression of the vertebral body (arrow) in Nov del3 -/- embryo. G-J: Adult skeletons. Overgrowth of the appendicular skeleton in Nov del3 -/-, and to a lesser extent in Nov del3 +/-, compared to wild type (+/+) littermate. H. Overgrowth of the axial skeleton in Nov _del3_-/- compared to wild type, with increased length of individual vertebral bodies. I. Frontal view of right (upper panel) and left (lower panel) knee joints from Nov _del3_-/- and wild type (+/+) littermates showing abnormal patella (P) and grossly enlarged medial meniscus (arrow head) in mutant compared with wild type. J. Lateral view of knee showing flattening of patella in Nov _del3_-/-. F: femur; T: tibia and P: patella.

Figure 3

Figure 3

Nov expression and phenotypes of E16.5 skeletons. A and B. RNA in situ hybridization showing Nov expression in the hind foot at E16.5. A. Strong expression in the myotendenous junctions (arrow) and in the mesenchyme of the joints adjacent to the cartilage elements, but no expression in condensing cartilage. B. High expression in the mesenchyme overlying the cartilage elements (arrow) and in the digital tendons (arrow head). C-G. Skeletal staining with Alizarin red and Alcian blue of E16.5 Nov del3+/-, Nov _del3_-/- and wild type (+/+) embryos. C. Wild type forefoot showing ossification of digits (arrow). D. No ossification in Nov _del3_-/- digits (arrow), but intense staining with Alizarin red of the radius and ulna indicating increased bone mineralization. E-G Vertebrae and rib cages of wild type (E), Nov _del3_-/- (F) and Nov del3+/- (G) showing delay in ossification of the vertebrae in the mutant embryos. No ossification is present in the Nov _del3_-/- embryo, and whilst ossification is taking place in the Nov del3+/- embryo (G), it is not occurring in the ordered, sequential manner observed in the wild type. Thoracic vertebrae numbered 1–13. H-Q. Histological sections of E16.5 wild type and Nov _del3_-/- embryos. H, J, L, N, P: wild type; I, K, M, O, Q: Nov _del3_-/- littermate. H, I: Haematoxylin and Eosin stained sections showing expanded perichondrium and periosteum in Nov _del3_-/- and a thicker bone collar (arrow head) compared to the wild type. o: ossified bone; h: hypertrophic cartilage; ph: pre-hypertrophic cartilage; c: columnar chondroctyes. J, K: Haematoxylin and Eosin stained sections at higher magnification at the junction of pre-hypertrophic/hypertrophic chondrocytes, adjacent to the border of the perichondrium/periosteum, showing abnormal morphology of chondrocytes and matrix in the mutant (K). L, M: Alcian blue staining of cartilage, showing expansion of the cartilage element and blurring of its borders in the Nov _del3_-/- embryo (M) compared to wild type (L). N, O: PCNA staining showing a sharp demarcation (black dashed line) between proliferating chondrocytes and pre-hypertrophic chondroctes in the wild type (N), but not in the Nov _del3_-/- mutant (black dashed line) (O). P, Q: Von Kossa staining for mineralised bone showing a shorter and thicker bone collar (indicated by the black bar) in the pre-hypertrophic/hypertrophoic zone of the Nov _del3_-/- mutant (Q) compared to wild type (P). Scale bars in A B = 20 μm; H,I,N,O,P,Q = 10 μm; J-M = 5 μm.

Figure 4

Figure 4

Enhanced chondrogenesis and osteogenesis in Nov _del3_-/- PEFs. A-H: Staining of micromass cultures of PEFs derived from wild type (A-D) and Nov _del3_-/- (E-H) embryos. Alcian blue staining after 5 days (A, E) and 9 days (B, F) in culture showing enhanced cartilage differentiation in Nov _del3_-/- cells, with the dashed line in B and F marking the extent of the micromass. Alkaline phosphatase staining after 5 days (C, G) and 9 days (D, H) in culture showing enhanced osteoblast differentiation in Nov _del3_-/- cells. I-L: Morphology of monolayer PEF cultures derived from wild type (I, J) and Nov _del3_-/- (K,L) embryos with (I, K) and without (J, L) phase contrast. Alkaline phosphatase staining after 10 days in culture (I, J, K, L) showing osteoblast differentiation in the Nov _del3_-/- PEFs but not in the wild type PEFs. Scale bar in I-N = 10 μm. M: Semi quantitative RT-PCR of mRNA from wild type (+/+) and Nov _del3_-/- PEFs using primers for alkaline phosphatase, collagen I and osteocalcin. Two fold serial dilutions of cDNA were used and normalised to gapdh.

Figure 5

Figure 5

Nov expression and histology of embryonic and adult hearts. A, B: RNA in situ hybridization showing Nov expression in the endothelial and smooth muscle cells of the aortic outflow tract, pulmonary trunk and in a subset of cells near the origins of the great vessels at E16.5. C-F: Haematoxylin and Eosin stained sections of wild type (C,D) and Nov _del3_-/- (E,F) E13.5 embryonic hearts showing abnormal expansion of the endocardial cushions (EC) and delay in fusion of the septum (S) in the mutant mouse (arrow). G-M: Haematoxylin and Eosin stained sections of wild type (G, J) and Nov _del3_-/- (H, I, K) adult hearts, showing accumulation of blood in the sub-endothelial space between the right ventricle and septum (H) and hypertrophy of the septum near the origins of the great vessels, (H, I, K). Areas of calcification on the septal wall of the right ventricle (K, L), stain with Von Kossa (M). There is no associated fibrosis. Scale bars in A,D,F = 20 μm; B,L = 10 μm; C,E = 50 μm; G,H,I = 1 mm; J,K = 50 μm; M = 5 μm.

Figure 6

Figure 6

Nov expression and phenotype of subcutaneous and hypaxial muscles. A: RNA in situ hybridization showing Nov expression in subcutaneous (arrow head), body wall (filled arrow) and intervertebral muscles (arrow) in E16.5 embryos. B, C: photographs of wild type (B) and Nov _del3_-/- (C) body walls, showing thinning and transparency in the mutant. D-I: Haematoxylin and Eosin stained sections of adult skin from wild type (D, G), Nov del3+/- (E, H) and Nov _del3_-/- (F, I) mice showing atrophy of subcutaneous muscles in Nov del3+/- and Nov _del3_-/- mice, with arrow heads marking the residual muscle layer. Histology of the subcutaneous adipose tissue in wild type (J) and Nov _del3_-/- (K) showing large mature fat cells in the wild type in contrast to the mixture of large (mature) and small (immature) fat cells in the mutant. L, M: Haematoxylin and Eosin stained section of adult intercostal muscles in wild type (L) and Nov _del3_-/- (M) mice showing muscle atrophy and transdifferentiation to fat in the mutant. Scale bars in A = 20 μm; D-F = 50 μm; G-M = 10 μm.

Figure 7

Figure 7

Phenotype of adult lens. A, B: Photograph of eyes of wild type (A) and Nov _del3_-/- (B) adult mice of six months of age, showing cataracts in both eyes in the mutant. The small white colouration in the wild type eye is an artefact due to reflected light. C-F: Haematoxylin and Eosin stained sections of wild type (C,E) and Nov _del3_-/- (D,F) adult eyes showing degeneration of the mutant lens, with vacuolation and loss of surface epithelium. Scale bars in E,F = 5 μm; C,D = 15 μm.

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