Tesi di dottorato di ricerca
Identification and study of microRNAs regulated by Tbx5during cardiovascular development and differentiation in zebrafish (Danio rerio)
Settore scientifico disciplinare
Corso di studi
SCIENZE BIOLOGICHE E MOLECOLARI
tutor Pitto, Letizia
Data inizio appello
Cardiac malformations are the most prevalent birth defects and the main cause of infant mortality, moreover the heart diseases remain the primary cause of mortality also in the adult life. The T-box gene Tbx5 is a key transcription factor of vertebrate heart development. Tbx5 function in heart is gene dosage sensitive, as either haploinsufficiency or gene duplication give rise to Holt−Oram syndrome (HOS), an highly penetrant autosomal dominant disease characterized by congenital malformations of heart and upper limb. Nonetheless, molecular mechanisms accounting for gene dosage sensitiveness are not known. Several genes regulated by mouse Tbx5 are transcription factors (TFs), or are involved in transcriptional regulation, thus suggesting that HOS might be in part the results of the “regulation of regulators”. MicroRNAs (miRNAs) are small 22 nucleotide RNA molecules that are emerging as important regulators of gene expression. Indeed, recent work has begun to elucidate the key role of miRNAs in cardiac development and disease. There is increasing evidence that TFs and miRNAs can work cooperatively regulating each other. Up to date over 500 miRNAs have been cloned and sequenced, and for many, their targets are unknown. Thanks to recent advances in miRNAs sequencing by Next Generation Sequencing (NGS) platforms, the characterization and quantification of miRNAs at massive scale is now a new powerful instrument for miRNA profiling to be exploited for investigate the complexity of HOS miRNA-mRNA interaction network. However the power of NGS technology requires animal models which allow a massive-scale screening, such as Danio rerio (zebrafish). By coupling the high-throughput DNA sequencing with the advantages offered by zebrafish as model system we investigated the role of Tbx5 related miRNAs during heart development. Among the identified miRNAs we demonstrated that miR-218a have a crucial role in mediating the effects of Tbx5 dosage on heart development, so we focus our attention in the construction of two specific transgenic lines for the temporal and tissue specific regulation of miR-218a. We also identified a second Tbx5 regulated miRNA: miR-19a, establishing a complete novel role for miR-19a as heart and proepicardial (PE) development regulator. Moreover our data shows that miR-19a is capable to partially rescue the HOS-like phenotype by exerting its action through its target S1Pr1. As result we propose a model where mir-19a, acting as functional effector of Tbx5, controls different aspects of heart morphogenesis also by repressing S1Pr1.