Chondrocyte maturation bridges two cross-inhibitory subnetworks of the skeletal cell gene regulatory network

The mechanisms by which crucial transcription factors of a gene regulatory network (GRN) interact continue to be revealed. In the vertebrate skeleton, SOX9 and RUNX2 combine to specify three different cell types. Sox9 drives immature chondrocyte differentiation, Runx2 regulates osteoblast differentiation, and both Sox9 and Runx2 are somehow required for mature chondrocyte formation. To elucidate mechanisms of GRN regulation in mature chondrocytes, transcriptomic data were examined from all three skeletal cell types isolated by laser capture microdissection of embryonic mouse. Multiple bioinformatic analyses supported the hypothesis that SOX9 and RUNX2 operate two cross-inhibitory subnetworks of the skeletal cell GRN during immature chondrocyte and osteoblast formation, but mature chondrocyte differentiation involves cooperation between these subnetworks. Several mature chondrocyte gene clusters had expression levels that represented an averaging of SOX9 and RUNX2 subnetworks, while one cluster, containing the hallmark mature chondrocyte genes collagen type 10a1 and Indian hedgehog, suggested a synergistic interaction between subnetworks. Generally, this in vivo LCM-RNA-seq approach enabled new understanding of interactions between distinct GRN subnetworks during cell differentiation and can similarly reveal regulatory control of any developmental process.