Abstract
tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt−Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations.
Highlights
The formation of the mature vertebrate heart with separated chambers and valves involves a complex orchestration of gene expression
We focused our attention on miR-218 since: i) it is conserved from human to zebrafish; ii) the miR-218-1 host gene, slit2, is highly sensitive to Tbx5 mis-expression [8]; iii) the secreted Slit ligands, together with their Robo receptors, contribute to the control of oriented cell tissue growth during chamber morphogenesis of the mammalian heart [18]; iv) Slit/ miR-218/Robo are part of a regulatory loop required during heart tube formation in zebrafish [16]
P19CL6 increased the expression of cardiac differentiation markers such as GATA4, a-MHC, CX40 and decreased the expression of the marker of pluripotency Oct4 after a few days of culture in differentiation medium (DM), compared to cells maintained in growth medium (GM) (Fig. 1A)
Summary
The formation of the mature vertebrate heart with separated chambers and valves involves a complex orchestration of gene expression. Tbx function in the heart is gene dosage sensitive, as both haploinsufficiency and gene duplication give rise to Holt2Oram syndrome (HOS). Comparable defects are seen in the zebrafish Tbx mutant heartstrings, suggesting that Tbx expression and function have been conserved throughout vertebrate evolution [10,11]. In the murine model of HOS, gene expression profiling of a tbx allelic series demonstrated that Tbx could regulate hundreds of downstream genes [8]. Examination of the expression dynamics of mouse genes regulated by Tbx indicates that Tbx can act via an array of independent mechanisms, some of which include direct DNA binding as has been shown for Gja5 [9], and others via indirect mechanisms that may involve complex regulatory networks
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