Fig. 6

ESRRa directly binds ESRRE consensus elements located upstream of sox9b.

(A) Consensus DNA sequences to where ESRRa is known to bind (ESRRE consensus) are identified upstream of both sox9a and sox9b. In addition, esrra also contains an ESRRE consensus in the proximal promoter region (esrra 70 b). (B) Embryos at 1-cell stage were injected with hESRRa mRNA, raised and collected at 36 hpf for chromatin immunoprecipitation using anti-ESRRa antibody. Significant binding of hESRRa is detected at the ESRRE consensus sites located upstream of sox9b (8.8~8.5 kb denoted as sox9b-1and 3.6 kb as sox9b-2), but not detected at the site found upstream of sox9a (6.5 kb). hESRRa also binds to the proximal promoter of esrra itself. Statistical analysis of pair-wise samples was performed using IBM SPSS Statistics 22 software. Values with p < 0.05 were considered to be statistically significant (indicated by asterisks). (C–F) Embryos were injected with MOesrra together with sox9a or sox9b mRNA, and subject to alcian blue staining at 4 dpf. White arrows indicate correctly-oriented ceratohyal cartilage, and white asterisks point to ceratobranchial arches. Note that overexpression of sox9b efficiently rescues defective cartilage induced by esrra knockdown, while overexpression of sox9a only partially rescues proper formation of ceratohyal cartilage.