Regulation of regulators: Tbx5 impacts cardiac expression through miR-218 modulation in zebrafish

Background: Tbx5, a member of the T-box gene family, is one of the key transcription factor of vertebrate heart development Minor alterations in its dosage cause Holt-Oram syndrome (HOS) an autosomal dominant disease characterized upper limb malformation and congenital heart defects of variable severity. In mouse model of HOS gene expression profiling revealed that TBX5 regulate a complextranscriptional network probably acting directely on gene expression or indirectely by " regulating regulators" such as TF and miRNAs. The integration of miRNAs and TFs into the genetic cardiac circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Looking for TBX5/miRNA regulatory circuit we identified miR-218 which, together with its host gene, Slit2, has been already shown to be involved in heart development. Purpose: the goal of our project is to demonstrate the existence of a regulatory circuit involving Tbx5/slit2/miR-218 and its importance in cardiac development. Methods: For in vitro studies P19CL6, a murine cell line able to differentiate cardiomyocites were utilized. For in vivo functional studies, gain and loss of function experiments of both, Tbx5 and Results: in cardiomyocyte differentiation of mouse P19CL6 cell culture, we confirmed a correlation between Tbx5 and miR-218 expression. Using zebrafish model, we showed that alterations of miR-218 expression have a deep impact on heart development. Interestingly, down-regulation of miR-218 was able to almost rescue defects generated by Tbx5 over-expression, confirming a functional relation between these two regulators. Conclusions: these data demonstrate that TBX5 directly and indirectly controls TF/miRNA regulatory circuitries and support the importance of miRNA regulationin HOS.