Elucidating the Transcriptional Regulation of col2a1a in Zebrafish

Cartilage is an important tissue in vertebrates beginning in the early embryo where it lays down the scaffolding for the skeleton, and continuing through adulthood where it makes up joints and intervertebral disks. The major component of cartilage is a filamentous protein known as Collagen type II, alpha 1 (Col2a1). Mutations in col2a1 in humans can lead to multiple congenital disorders and the early onset of joint and retinal deterioration. Our laboratory is interested in understanding the conserved transcriptional regulation of this critical structural gene utilizing the zebrafish, Danio rerio. Previously, we identified a 310 bp regulatory region (R2) 1.7 kb upstream of the transcriptional start site that is critical for cartilage, ear, and notochord expression of the col2a1a gene in zebrafish. Utilizing transgenic zebrafish EGFP reporter analysis, we have now narrowed R2 down to 120 bp that can still reproduce the full col2a1a mRNA expression seen by in situ hybridization. Further characterization allowed us to identify a 60 bp sequence that specifies cartilage and ear but eliminates notochord expression. By employing comparative genomic analysis we have identified three highly conserved putative transcription factor binding sites for the transcription factor families of Ets, Runx, and Sox. Generation of targeted deletions of these transcription factor binding sites in our transgenic reporter results in loss of EGFP reporter activity, suggesting their necessity for col2a1a R2 activity