Bone Abstracts

The c-Myb transcription factor is associated with proliferation of undifferentiated cells in number of tissues, but recent data suggests its role also in differentiation. c-Myb is important in formation of the cartilage, bone and apparently also in hard tissue mineralization (Matalova et al. 2011).

Embryonic micromasses were established from mouse front limbs at the embryonic day E12. Micromass cultures represent an effective tool for experimental biology and they are routinely used in molecular studies of embryogenesis. Moreover, the micromass technology approach enables investigators to follow tissue formation from a single cell to organized spheres in a controlled environment (Meyer et al. 2007).

Techniques of electroporation and lipofection were applied in the gain-in-function experiments, siRNA approach for loss-of-function investigation. Sox9 was investigated as a marker of chondrogenesis, gene expression was followed using qPCR.

Transient transfection by constructs carrying Sox9 overexpressing vectors markedly decreased c-Myb expression in cultured micromasses, whereas Sox9 level was enhanced. Transient transfection using constructs carrying c-Myb overexpressing vectors enhanced markedly both, c-Myb and Sox9 expression. Along with overexpression, siRNA c-Myb and siRNA Sox9, respectively, were transfected, representing downregulation impact. siRNA. c-Myb treated cultures expressed significantly lower level of Sox9, whereas siRNA Sox9 treated cells showed considerably increased level of c-Myb expression. These findings suggest a possible signalling connection between these two proteins. Furthermore, the results indicate a negative feedback loop during chondrogenesis.

Further experiments will apply explant mouse mandibles and limbs to investigate impact of c-Myb overexpression and downregulation, respectively, on tooth and bone phenotype in the intact organs and to compare endochondral- and intramembranous-types of ossification.

The research was supported by the GACR project (P302/12/J059), international collaboration runs under M200451201 and the Brno lab under RVO 67985904.