Targeted mutation of NOV/CCN3 in mice disrupts joint homeostasis and causes osteoarthritis-like disease.

OBJECTIVE: The matricellular protein NOV/CCN3, is implicated in osteoarthritis (OA) and targeted mutation of NOV in mice (Nov(del3)) leads to joint abnormalities. This investigation tested whether NOV is required for joint homeostasis and if its disruption causes joint degeneration. METHOD: NOV expression in the adult mouse joint was characterized by immunohistochemistry. A detailed comparison of the joints of Nov(del3)-/- and Nov(del3)+/+ (wild-type) males and females at 2, 6 and 12 months of age was determined by X-ray, histology and immunohistochemistry. RESULTS: NOV protein was found in specific cells in articular cartilage, meniscus, synovium and ligament attachment sites in adult knees. Nov(del3)-/- males exhibited severe OA-like pathology at 12 months (OARSI score 5.0 ± 0.5, P < 0.001), affecting all tissues of the joint: erosion of the articular cartilage, meniscal enlargement, osteophytic outgrowths, ligament degeneration and expansion of fibrocartilage. Subchondral sclerosis and changes in extracellular matrix composition consistent with OA, were also seen. The density of articular cartilage cells in Nov(del3)+/+ knee joints is maintained at a constant level from 2 to 12 months of age whereas this is not the case in Nov(del3)-/- mice. Compared with age and sex-matched Nov(del3)+/+ mice, a significant increase in articular cartilage density was seen in Nov(del3)-/- males at 2 months, whereas a significant decrease was seen at 6 and 12 months in both Nov(del3)-/- males and females. CONCLUSION: NOV is required for the maintenance of articular cartilage and for joint homeostasis, with disruption of NOV in ageing Nov(del3)-/- male mice causing OA-like disease.

Mouse Nov gene is expressed in hypaxial musculature and cranial structures derived from neural crest cells and placodes.

NOV is a member of an emerging family of proteins, the CCN family, implicated in the control of cell growth and differentiation. During mouse development Nov is expressed predominantly in the skeletal and visceral muscles and in the nervous system. Transcripts are first detected in muscle precursor cells from 10.0 dpc and later in the hypaxial muscles of the trunk and shoulder/hip, as well as in the muscles of the head and in the smooth muscle of major vessels. In the nervous system, Nov is observed in the somatic motor neurons of the spinal cord from 12.5 dpc and in cranial structures derived either from neural crest cells or placodes, including V, VII, VIII, and IX ganglia and olfactory neuroepithelia.

Characterisation, chromosomal localisation and expression of the mouse Kid3 gene.

Kid1 encodes a transcriptional repressor implicated in the differentiation of renal epithelial cells. Here we report the characterisation of Kid3, a novel mouse gene related to Kid1. Kid3 encodes a C2H2 zinc finger protein with an N-terminal KRAB transcriptional repression domain. It maps to chromosome 11, adjacent to Kid1 and another related gene Kid2. Northern analysis shows that Kid3 is highly expressed in embryonic and adult brain, with lower levels in adult and embryonic (E16.5) kidney, gut, lung and heart. Expression of Kid3 in the kidney is developmentally regulated and suggests a role for Kid3 in the early stages of nephrogenesis.

Characterization of the mouse Kid1 gene and identification of a highly related gene, Kid2.

Kid1 encodes a zinc finger protein that has been implicated in renal cell differentiation. Levels of Kid1 mRNA correlate with maturation of kidney tubule epithelia in rat post-natal kidney development and during kidney regeneration following injury. KID1 is a putative transcriptional repressor, containing a KRAB domain at its amino terminus that mediates transcriptional repression in transient cell transfection assays when fused to a heterologous DNA-binding domain. In this paper, we describe the isolation and characterization of the mouse homologue of Kid1 and the identification of a novel highly related mouse gene, Kid2, Kid1 and Kid2 are tightly linked on mouse chromosome 11 and show conservation across mammals. Both genes are expressed predominantly in the mouse adult kidney and brain, but transcripts are also detected in embryonic brain, kidney, gut and lung, suggesting an additional role for these genes during mouse development.

A PCR-based method for isolation of genomic DNA flanking a known DNA sequence.

We describe a simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence. The method is based on the directional cloning of digested genomic DNA into the multiple cloning site of a pUC-based plasmid to generate a limited genomic library. The library is plated onto a number of selective LA plates which are incubated overnight, and recombinant plasmid DNA is then isolated from resistant colonies pooled from each plate. PCR amplification is performed on the pooled recombinant plasmid DNAs using primers specific for the pUC vector and the known genomic sequence. The combination of efficient directional cloning and bacterial transformation gives relative enrichment for the genomic sequence of interest and generates a simple DNA template, enabling easy amplification by PCR.

Genomic structure and chromosomal mapping of the mouse nov gene.

The nov gene encodes a cysteine-rich protein that is overexpressed in avian nephroblastomas. It is a member of the CCN family of proteins, all of which are involved in cell growth. Genomic and cDNA clones encompassing the mouse nov gene have been isolated and characterized. The mouse nov gene is highly conserved with the human and chick nov genes at the level of nucleotide sequence and genomic organization. The exon structure reflects the modular organization of the NOV protein in a number of structural domains. These are highly conserved with other members of the CCN family, as is the distribution of 38 of its 40 cysteine residues. The nov gene maps to chromosome 15, between D15 Mit 153 and D15 Mit 183, in a region of conserved synteny with human chromosome 8.

Multiple cloning sites carrying loxP and FRT recognition sites for the Cre and Flp site-specific recombinases.

Plasmids were constructed carrying the loxP and FRT recognition sites for the Cre and Flp site-specific recombinases, respectively, within multiple cloning sites. Vectors carrying single and tandemly repeated targets are available with various flanking restriction enzyme sites. In addition, a series of plasmids carrying both loxP and FRT sites is available. These vectors facilitate construction of target molecules for these site-specific recombinases which are becoming increasingly important tools for the in vivo manipulation of DNA.

Isolation of genomic sequences flanking a retroviral insertion site using a novel PCR-based method.

A novel PCR approach for the isolation of genomic sequences that flank retroviral insertion sites uses two standard primers and defined PCR conditions. The method is based on the directional cloning of digested genomic DNA into the multiple cloning site of pUC-based plasmids, isolation of recombinant plasmid DNA from pooled colonies and PCR amplification using primers specific for the pUC vector and retroviral LTR sequences. The method is simple and can be adapted to enable rapid isolation of any genomic sequences tagged by insertion of foreign DNA.

Expression of v-src induces aberrant development and twinning in chimaeric mice.

The role of the proto-oncogene c-src in mouse development has been investigated by studying the consequences of expressing its viral homologue, v-src. Embryonic stem (ES) cell lines with differing levels of v-src tyrosine kinase activity have been used to generate chimaeric mice. Whereas a low level of v-src expression is compatible with embryogenesis, chimaeras derived from ES clones with high levels of v-src activity develop abnormally and die on the 8th-9th day of gestation. These abnormalities are characterized by the formation of twin or multiple embryos within the same Reichert's membrane, and by the arrest of embryonic development at the late egg cylinder stage, accompanied by relative expansion of the visceral yolk sac (VYS) and hyperplasia of the VYS endoderm. These results demonstrate for the first time that deregulated expression of the src protooncogene product can induce developmental abnormalities during early embryogenesis.

Expression of v-src arrests murine glial cell differentiation.

A replication-defective retroviral vector carrying the v-src oncogene and the gene for neomycin resistance was used to infect neurone- and fibroblast-depleted embryonic mouse brain cells in vitro. Cells resistant to the antibiotic G418 were obtained and continually passaged. Several cell lines were isolated which express high levels of v-src mRNA and v-src tyrosine kinase activity. The antigenic marker profile of either the pooled cells from an individual infection or sublines isolated from individual foci showed the cells to be immature glia: most cells expressed vimentin, A2B5 antigen and/or J1/tenascin glycoproteins, but not fibronectin. Sublines expressed different antigen profiles suggesting that the immortalised cells were derived from glial cells of different phenotypes. The cell lines expressed the 120 and 140 but not the 180 kd components of N-CAM as well as voltage-activated potassium channels, typical for glial cells. 01 antigen-positive oligodendrocytes were never observed in the lines or sublines after long term passage (over 1 year), but some cells expressed glial fibrillary acidic protein, a marker for mature astrocytes. Thus, expression of v-src in murine glial cells appears to arrest their development and prevent their differentiation.

Tumour induction in fetal brain transplants exposed to the viral oncogenes polyoma middle T and v-src.

Using a neural transplantation model, we have studied the effect of polyoma virus middle T antigen and of the viral src oncogene on the developing rat brain. For this purpose, single-cell suspensions of fetal rat brain were prepared on day 14 of gestation (E14), infected with a replication-defective retroviral vector which carries the middle T oncogene driven by an internal thymidine kinase promoter, and stereotaxically injected into the caudoputamen of adult host rats. With mock-infected donor cells, the transplants developed into an organotypically differentiated population of neurons, astrocytes, oligodendrocytes and other central nervous system cell types and expressed marker proteins of mature neuroectodermal cells, including neuron-specific enolase, synaptophysin, neurofilament protein, glial fibrillary acidic protein and S-100 protein. A high percentage of rats carrying transplants infected with the middle T-encoding vector died within two to six weeks from massive haemorrhage into the transplant. Upon microscopic examination, gross abnormalities of the microvasculature were seen, with formation of large blood-filled spaces lined by a thin layer of proliferating endothelial cells. This effect of middle T was apparently cell type-specific, since the differentiation of neuroectodermal cells within the graft proceeded in a regular fashion. In order to assess further the cell-specificity of the oncogene, analogous experiments were carried out with a retroviral construct in which the middle T cDNA had been replaced by the viral src gene (v-src). Transplants infected with the v-src vector developed astrocytomas, but no vascular abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of c-src and c-abl in embryonal carcinoma cells and adult mouse tissues.

We have studied the expression of c-src and c-abl proto-oncogenes in early mouse development using embryonal carcinoma (EC) cells as a model system, and compared this to the expression pattern in adult tissues. In all three EC lines tested (F9, PC13, and P19), c-src and c-abl mRNA can be detected. When F9 and PC13 are induced to differentiate they form endodermal cells characteristic of the early embryo, and we found no change in c-src or c-abl expression. In contrast, P19 cells showed increased levels of both mRNAs both mRNAs when induced to differentiate along the neural pathway by retinoic acid, whereas differentiation along the muscle pathway by dimethyl sulfoxide resulted in decreased levels of c-abl expression. These results are consistent with the idea that c-src and c-abl have important functions in the differentiation of the cell types of the later embryo, but not in those of the early embryo.

The effects of v-src expression on the differentiation of embryonal carcinoma cells.

We have used replication-defective selectable retroviruses to express inducibly or constitutively the v-src gene in the embryonal carcinoma (EC) cell lines F9, PC13 and P19. High v-src expression in F9 and PC13 cells does not induce or disrupt their differentiation. In contrast, P19 cells expressing high levels of v-src have a 'differentiated' morphology and are unable to respond to signals that normally induce differentiation along the neural or muscle pathways. Regulation of v-src transcription from the inducible human metallothionein (MT) promoter has shown that v-src expression above a threshold level induces differentiation of these cells, as defined by loss of the stem cell marker ECMA-7. These results suggest that v-src expression may be compatible with differentiation events occurring early in embryogenesis, represented by F9 and PC13 cell differentiation, but might disrupt the differentiation of cell types present at later developmental stages.