Abstract
Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih−/−) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk−/−) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk−/− embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfks11 variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.
Highlights
The genetic programs and developmental processes that lead to organ formation are still poorly understood
The specific absence of endocardial cushion (EC) formation in these embryos clearly demonstrates that myocardial function is required for EC formation
We have shown that the expression of bmp-4 is not affected by myocardial function defects, it is possible that myocardial function is somehow required for another aspect of this signaling event
Summary
The genetic programs and developmental processes that lead to organ formation are still poorly understood. To determine the expression pattern of cfk, we performed in situ hybridization with a probe corresponding to its 39 untranslated region (UTR), which is distinct from the 39 UTR sequences of zebrafish a-cardiac and a-skeletal actins These data showed that cfk is expressed in the myocardial but not endocardial cells of zebrafish embryos from 24 to 48 hpf (Figure 3C and 3D), suggesting a nonautonomous role in EC formation. Because most cfkÿ/ÿ embryos have a dilated heart and lack blood flow and because subsequently approximately 50% of these embryos fail to form ECs, we wondered whether these two phenotypes were causally related or whether lack of EC formation could occur independently of poor early myocardial function To address this question, 379 embryos from multiple cfk clutches were assayed for cardiac dilation and lack of blood flow at 36 hpf and for lack of endocardial ring formation at 48 hpf. The results from the 2,3-BDM and tricaine treatments suggest that it is poor myocardial function, and not lack of blood flow, which is primarily responsible for the loss of EC formation in cfkÿ/ÿ and sihÿ/ÿ embryos
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