MicroRNA-23 Restricts Cardiac Valve Formation by Inhibiting Has2 and Extracellular Hyaluronic Acid Production

Abstract

Objective: To study the role of microRNAs in cardiac valve formation. Methods and Results:We show that zebrafish dicer mutant embryos, lacking mature miRNAs, form excessive endocardial cushions. By screening miRNAs expressed in the heart, we found that miR-23 is both necessary and sufficient for restricting the number of endocardial cells that differentiate into endocardial cushion cells. In addition, in mouse endothelial cells, miR-23 inhibited a transforming growth factor-␤-induced endothelial-to-mesenchymal transition. By in silico screening of expression data with predicted miR-23 target sites combined with in vivo testing, we identified hyaluronic acid synthase 2 (Has2), Icat, and Tmem2 as novel direct targets of miR-23. Finally, we demonstrate that the upregulation of Has2, an extracellular remodeling enzyme required for endocardial cushion and valve formation, is responsible for the excessive endocardial cushion cell differentiation in dicer mutants. T he development of valve structures occurs in distinguishable phases, which are highly conserved. 1 The first event during valve development is the induction of endocardial cushions (ECs) within the atrioventricular (AV) canal (AVC) and outflow tract of the primitive heart tube. It has been well established that bone morphogenetic protein (BMP), a member of the transforming growth factor (TGF)-␤ superfamily, is the major myocardial signal that initiates EC formation from human to zebrafish. [2][3][4][5] Compromised BMP signaling results in downregulation of multiple pathways including TGF-␤, Has2 (hyaluronic acid synthase 2), and Notch1, as well as the transcription factors Snail1 and Twist1. 2,4 The importance of regulating Has2 expression in the endocardium has been exemplified by the observation that in Has2 deficient mice the cardiac jelly does not expand and ECs fail to form. 6 Has2 is responsible for the production of hyaluronic acid (HA), one of the ECM components of cardiac jelly. 6 HA production leads to expansion of the extracellular space because it binds salt and water and induces PI3K and ErbB signaling (reviewed by B. Toole 7 ). Conclusions: MiR-23 in the embryonic heart is required to restrict endocardial cushion formation by inhibitingDespite differences in the cellular processes that precede valve formation the molecular signals regulating valve formation (eg, Notch, NFAT, ErbB, and TGF-␤ signaling) have been conserved between amniotes and zebrafish. 8 -11 MiRNAs are a class of 21-to 25-nucleotide single-stranded noncoding RNAs transcribed from DNA but not translated into protein. Instead, miRNAs interact with messenger RNAs in the cytosol and regulate their final output at the protein level. 12 Dicer, an RNase III endonuclease, processes hairpinlike pre-miRNAs into double-stranded miRNA molecules. MiRNA function during organogenesis has been studied mainly by generating conditional Dicer knockout mouse models because conventional Dicer mutant mice are embryonic lethal. 13 Conditional depletion of Dicer from the endocardium has been pre...

Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish

“…This regulation of Tbx5 is critical to restrict the expression of has2, one of the critical genes for the formation of AVC, in the specific region of the heart tube. The ectopic expression of has2 causes heart malfornations 8,22 , that are very similar to the phenotype of kctd10 mutant. We confirmed that the heart defect in kctd10 mutant is due to the ectopic expression of has2.…”

“…Has2 catalyses the synthesis of hyaluronic acid, the major component of cardiac jelly, thus the cardiac jelly swells at the AVC. Has2 also plays a pivotal role in the epithelium-to-mesenchyme transformation of the endocardium at the AV boundary, which gives rise to the endocardial cushions and valves 22,23,27 . In the forming heart of the kctd10 mutant, Tbx5 is hyperactive due to the absence of functional Kctd10.…”

“…It also restricts the expression of Tbx2 (tbx2b in zebrafish) and has2 (hyaluronan synthase 2) at the AVC to facilitate the formation of the endocardial cushion and valve 21 . Deregulation of has2 abrogates the normal cardiac morphogenesis 22,23 .…”

“…Has2 is essential for AVC formation [21][22][23] . Its expression was detected at the AVC region in the wild-type embryos (Fig.…”

“…Has2 protein is the synthetase of hyaluronic acid, an extracellular glycosaminoglycan that is the major component of the cardiac jelly. Ectopic expression of Has2 is reported to impair cardiac looping, results in lumen narrowing and increases the space between the endocardium and myocardium in the heart chambers 22 . This phenotype was also observed in the histological sections of kctd10 mutants at 48 hpf (Fig.…”