We have developed a mouse in which the Cre recombinase gene has been targeted to exon 1 of the matrilin-1 gene (Matn1) to investigate the origins of articular chondrocytes and the development of the knee joint. Analysis of joints from offspring of Matn1-Cre/R26R crosses demonstrated that articular chondrocytes are derived from cells that have never expressed matrilin-1 whereas the remainder of the chondrocytes in the cartilage anlagen expresses matrilin-1. A band of chondrocytes adjacent to the developing interzone in the E13.5 day knee joint became apparent because these chondrocytes did not turn on expression of matrilin-1 in contrast to the other chondrocytes of the anlagen. The chondrocytes of the presumptive articular surface therefore appear to arise directly from a subpopulation of early chondrocytes that do not activate matrilin-1 expression rather than by redifferentiation from the flattened cells of the interzone. In addition, lineage tracing using both Matn1-Cre/R26R and Col2a1-Cre/R26R lines indicated that non-cartilaginous structures in the knee such as cruciate ligament, synovium and some blood vessels are formed by cells derived from the early chondrocytes of the anlagen.

The bone morphogenetic protein-2 (BMP-2) is a potent secreted factor that promotes osteoblast differentiation during development. Exposure to BMP-2 is sufficient to cause a lasting change in cell fate presumably by activating specific target genes. To identify genes downstream of BMP-2 we treated the murine pluripotent embryonic cell line, C3H10T1/2 that can be induced to form an osteoblastic phenotype, with 100 ng/ml BMP-2 for 24 h. Using suppression subtractive hybridisation we found the novel zinc finger transcription factor, ZNF450 was upregulated. The single-copy ZNF450 gene spans 15.6 kb on chromosome 10B1 and consists of seven exons, the first of which is untranslated. The open reading frame encodes a 710 reside protein. Analysis of the protein sequence reveals a highly conserved amino-terminal BTB/POZ dimerisation domain, an AT-hook motif, and eight C2H2 zinc fingers. Library screening identified a second mRNA isoform encoding a short protein isoform with one zinc finger. Using reverse transcriptase-real time PCR to measure mRNA expression we found that ZNF450, Runx2/Cbfa-1, and Sp7/osterix were induced by BMP-2 after 4 h in C2C12 myoblast cells. Treatment of C2C12 cells with BMP-2 causes a shift from a myoblastic to osteoblastic phenotype. ZNF450 was upregulated three to fivefold after 24 h in C3H10T1/2 cells and required 100 ng/ml BMP-2. Expression of the 3 kb major transcript was highest in liver, testis, and kidney. However, ZNF450 mRNA was found also in a wide range of adult tissues. The consistent induction of ZNF450 by BMP-2 after 4 h in three murine pluripotent cell lines suggests that ZNF450 may play a role in the BMP-2 signalling pathway.

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