SOX9 Is an Activator and Repressor in Chondrocytes Figure 4. Mutation of SOX9 binding consensus results in derepression of Col10a1 transgene expression in vivo. The expression pattern of mutant Flag-tagged Col10a1 mini-genes, in which the SOX9 site (Col10Flag-ED1) or the TG-rich motif (Col10Flag-ED2) within the element A was mutated, was examined by in-situ hybridization of proximal humeri in E15.5 expressing fetuses. In a major (5/7) portion of Col10Flag-ED1 fetuses (a–d), the expression in the proliferating zone (*) was relatively higher than in the hypertrophic zone. In a minor (2/7) portion (e–h), the expression was predominant in nearly all immature chondrocytes (*). For Col10Flag-ED2, a minor (2/6) portion of fetuses (m–p) showed up-regulated expression in the non-hypertrophic zone (*), while a major (4/6) portion showed relatively slight mis-expression (i–l). The prehypertrophic and hypertrophic zones are encircled. PC, proliferating chondrocytes; PHC, prehypertrophic chondrocytes; HC, hypertrophic chondrocytes. doi:10.1371/journal.pgen.1002356.g004 PLoS Genetics | www.plosgenetics.org 7 November 2011 | Volume 7 | Issue 11 | e1002356