Loss of muscleblind-like 1 promotes invasive mesenchyme formation in endocardial cushions by stimulating autocrine TGF[beta]3

Valvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor [beta] (TGF[beta]) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGF[beta]-dependent endocardial cell invasion. In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGF[beta]3 after it is no longer required to induce basal levels of invasion. TGF[beta]3, but not TGF[beta]2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGF[beta]3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGF[beta]3 production in the endocardium. More TGF[beta]3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGF[beta]3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGF[beta]3 secretion, and early exposure to excess TGF[beta]3 induces precocious invasion. MBNL1 expression precedes TGF[beta]3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGF[beta]3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGF[beta] is necessary and sufficient to mediate this effect. Taken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGF[beta]3 production.