While Pax3 acts as a transcriptional activator to promote myogenesis. It will be of interest to determine whether Pax3 inhibits chondrogenesis by acting as a transcriptional repressor or activator in the satellite cells. It will be also of interest to investigate whether other myogenic factors play inhibitory roles in Atropine sulfate chondrogenic differentiation. We also discovered a novel function for Sox9 in this study. Sox9 is the master regulator of chondrogenesis, as no cartilage formation takes place in the absence of Sox9. Sox9 acts as a transcriptional activator in chondrogenic precursor cells by binding to the Tulathromycin B promoters of cartilage-specific matrix genes collagen II and aggrecan. We found that Sox9 strongly induced collagen II and aggrecan expression, as well as glycosaminoglycan level in the muscle satellite cells, which normally are non-chondrogenic precursors, consistent with its activity in the somite. In the meantime, Sox9 also significantly, although weakly, inhibited the expression of early muscle lineage marker Pax3 and Pax7, as well as myosin heavy chain. It has been reported that Sox9 is expressed in the satellite cells, and has the ability to inhibit a-sarcoglycan expression in the C2C12 myoblast cell line and the myogenin promoter in 10T1/2 cells. Our data are consistent with these reports. While Sox9 may be expressed in satellite cells, it is apparent from our work and others that Sox9 is much more strongly expressed in chondrocytes, and that ectopic expression of Sox9 leads to chondrogenic differentiation and maintenance of the chondrocyte phenotype. Our data suggest that Nkx3.2 plays a central role in the chondrogenic differentiation of satellite cells, and that its activity is required for Sox9 to promote chondrogenesis and inhibit myogenesis. Like Sox9, Nkx3.2 is expressed in the cartilage precursors in the embryo, and promotes cartilage cell fate in the somites. Nkx3.2 null mice exhibit reduced cartilage formation including a downregulation of Sox9 expression. 
Inactivating mutations of Nkx3.2 in human lead to spondylo-megaepiphyseal-metaphyseal dysplasia, a disease that causes abnormalities of the vertebral bodies, limbs and joints. Here we show that Nkx3.2 is activated in the muscle satellite cells during chondrogenic differentiation in vitro as well as in the adult fracture healing process in vivo, suggesting that Nkx3.2 may also be involved in a cell fate determination process at a stage later than early embryogenesis. Furthermore, we show that Nkx3.2 acts as a transcriptional repressor to inhibit Pax3 promoter activity. While there are consensus Nkx3.2 binding sites on the Pax3 promoter, we have not determined whether Nkx3.2 binds to the Pax3 promoter. Interestingly, Nkx3.2 has also been shown to act as a repressor to inhibit osteogenic determining factor Runx2, suggesting that Nkx3.2 may be used to inhibit other noncartilage cell fates. We have also uncovered a pivotal role for Nkx3.2 in the induction of chondrogenic genes. We found that without the repressing activity of Nkx3.2, Sox9, despite its ability to bind to collagen II and aggrecan promoters, was unable to activate those genes or inhibit myogenesis. Additionally, Nkx3.2 potentiates the ability of Sox9 to induce aggrecan expression, which may be due to its repression of chondrogenic inhibitor Pax3. This data is consistent with the time course experiment, which indicated that the high level expression of collagen II and aggrecan is clearly correlated with the induction of Nkx3.2, as Sox9 expression is reduced at later stages of chondrogenesis.