Sox9b Negatively Regulates Kcnj2a During Zebrafish Heart Development

Sox9 plays regulatory roles as a transcription factor in cell differentiation and development in numerous organs/tissues including skeleton, gonads, glia and heart. Zebrafish has two co-orthologs of mammalian Sox9 gene, sox9 and sox9 b. Previous studies showed sox9 b expression in myocardial cells in zebrafish. However, the mechanisms underlying how Sox9 b contributes to heart development and function are still unknown. Here, we generated two zebrafish sox9 b mutant lines using CRISPR-Cas9 technology. Homozygous mutant fish can survive to adults but exhibit low survival rate and reduced body size. Approximately 85.7 % of the homozygous adults display cardiac malformations and die from congestive heart-failure. Transcriptome sequencing analysis of sox9 b mutants revealed some up-regulatd genes including kcnj2a(potassium inwardly-rectifying channel, subfamily J, member 2a) whose mutation led to human Andersen-Tawil syndrome(ATS). Luciferase reporter and Ch IP assays showed that Sox9 b represses kcnj2 a expression through binding to its promoter. Overexpression of kcnj2 a showed similar heart function defects as sox9b-/-. Knockdown of kcnj2 a also showd phenotypes including heart malformatioin and deformed trunk. Taking together, our studies indicate that Sox9 b functions in heart development and also in the inwardly rectifying of potassium in heart through negative regulation on kcnj2 a. The sox9 b mutants we generated can provide a disease model for studying Andersen-Tawil syndrome(ATS).