MicroRNA 218 Mediates the Effects of Tbx5a Over-Expression on Zebrafish Heart Development

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.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

“…We also targeted the lateral plate mesoderm-expressed transcription factor genes tbx5a and hand2, which when mutated display compound heart and pectoral fin defects (Garrity et al, 2002;Yelon et al, 2000). Analogous to the tbx5a mutant heartstrings (hst) and morpholinos (Ahn et al, 2002;Chiavacci et al, 2012;Garrity et al, 2002), RNP-mediated targeting of tbx5a caused a spectrum of phenotype expressivity (n=232), including bilateral loss of pectoral fins and elongated heart tubes in efficiently injected embryos ( Fig. 3J-L,P).…”

Interactions between microRNAs and long non-coding RNAs in cardiac development and repair

“…Additionally, miR-218 family belongs to the regulatory circuit through which tbx5 transcription factor controls cardiac morphogenesis. Both the overexpression of tbx5 [32] and the downregulation of miR-218 family caused heart-looping defects with chamber abnormalities, while the upregulation of miR-218-1 is able to rescue tbx5 overexpression-related cardiac defects [33]. Moreover, during cardiogenesis the involvement of miR-99a/let-7c in embryonic development has been described.…”

MicroRNAs: pleiotropic players in congenital heart disease and regeneration

“…Based on mutual interactions between transcription factors and miRs (59), an in silico approach was used to identify miRs within introns of genes that are regulated by TBX5 with the help of integrated comparisons of several gene databases with miR databases. This approach identified four candidate miRs: mir-218-1, miR-678, miR-719 and miR-335 (23). Of these, miR-218-1 was highly promising due to its high cross-species conservation, its location within a TBX5-regulated gene, and the documented role of its host gene (SLIT2) in heart development.…”

“…Of these, miR-218-1 was highly promising due to its high cross-species conservation, its location within a TBX5-regulated gene, and the documented role of its host gene (SLIT2) in heart development. Indeed, the expression of Slit2 and miR-218-1 was clearly modulated by Tbx5 during the differentiation of mouse embryonic carcinoma cells (P19CL6) into beating cardiomyocytes (23). A functional role for miR-218-1 during heart morphogenesis is strongly supported by the fact that miR-218-1 overexpression dosedependently induced cardiac defects in zebrafish embryos, possibly by affecting the migratory properties of cardiac precursors (23).…”