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Background: Fetal alcohol spectrum disorder (FASD) describes a range of birth defects including various congenital heart defects (CHDs). Mechanisms of FASD-associated CHDs are not understood. Whether alcohol interferes with a single critical event or with multiple events in heart formation is not known. Results: Our zebrafish embryo experiments showed that ethanol interrupts different cardiac regulatory networks and perturbs multiple steps of cardiogenesis (specification, myocardial migration, looping, chamber morphogenesis, and endocardial cushion formation). Ethanol exposure during gastrulation until cardiac specification or during myocardial midline migration did not produce severe or persistent heart development defects. However, exposure comprising gastrulation until myocardial precursor midline fusion or during heart patterning stages produced aberrant heart looping and defective endocardial cushions. Continuous exposure during entire cardiogenesis produced complex cardiac defects leading to severely defective myocardium, endocardium, and endocardial cushions. Supplementation of retinoic acid with ethanol partially rescued early heart developmental defects, but the endocardial cushions did not form correctly. In contrast, supplementation of folic acid rescued normal heart development, including the endocardial cushions. Conclusions: Our results indicate that ethanol exposure interrupted divergent cardiac morphogenetic events causing heart defects. Folic acid supplementation was effective in preventing a wide spectrum of ethanol-induced heart developmental defects. Developmental Dynamics 242:1184–1201, 2013. © 2013 Wiley Periodicals, Inc.

The anteroposterior (A-P) patterning of the developing heart underlies atrial and ventricular lineage specification and heart chamber morphogenesis. The posteriorization of cardiomyogenic phenotype with retinoic acid (RA) treatment of primitive streak stage chicken embryos is suggestive of a role for the clustered homeobox (Hox) genes in early heart patterning (Yutzey et al. [1994] Development 120:871-873; [1995] Dev. Biol. 170:531-541). A screen for Hox genes expressed in chick heart primordia and primitive heart led to the isolation of anterior genes of the Hox clusters expressed during cardiogenesis. Specific hoxd-3, hoxa-4, and hoxd-4 transcripts were detected at the early stages of heart formation and full-length cDNA clones were isolated. Expression of hoxd-3 was detected in the heart forming region of embryos prior to heart tube formation. Expression of hoxa-4, hoxd-3, and hoxb-5 was increased in cardiogenic tissue treated with RA in culture conditions that also produced changes in positionally restricted cardiomyogenic phenotypes. Hox genes expressed in cardiac explants exhibited distinct sensitivities to RA and ouabain treatment when compared to genes, such as nkx-2.5, that are involved in cardiac commitment and differentiation. These studies support a role for Hox genes in early heart patterning and suggest that positional information in the cardiogenic region is established by regulatory mechanisms distinct from early heart lineage specification.

By utilizing myosin immunostaining, we were able to identify early rat myocardium as a thin epithelial sheet and realized that its cohesive movement toward the midline leads to the straight heart tube formation. Localization study of fibronectin mRNA and protein was, therefore, carried out to investigate its tissue origin and possible roles in facilitating mesoderm migration and heart formation. Fibronectin mRNAs were first detected throughout the mesoderm during the early primitive streak stage, suggesting that the mesoderm is the source of fibronectin. By pre-head fold (pre-somite) and head fold (early somite) stages, the mesoderm became largely down-regulated for fibronectin mRNAs, while it was also at these stages when myosin-positive myocardium formed itself into the epithelium and was subsequently folding toward the midline. Thus, there appears to be little fibronectin synthesis during and directly relevant to early heart tube formation. Later, during the early straight heart tube stage (5 somite and older), endocardium became highly positive for fibronectin mRNAs, suggesting that the endocardium is the major source of fibronectin for the cardiac jelly. Based on the results, we present a map for the early mammalian heart in which the heart is a single crescentic band lying in front of the prechordal plate. We also suggest a process for heart tube formation based on the cohesive movement of the myocardial epithelium. During heart tube formation, fibronectin protein had been deposited previously by the mesoderm and was found uniformly in the ECM and not newly produced by any adjacent tissue. The data contradict the endodermal guidance of heart migration by fibronectin gradient and suggest, instead, a permissive role for the fibronectin substrate. © 1995 wiley-Liss, Inc.

Pericardial cavity formation and epithelialization of the cardiac precursor cell population constitute a critical developmental period that precedes stable cardiac cell commitment and differentiation. These events delineate the myocardial and endocardial precursor population in the embryo. Restriction of Na/K-ATPase (the sodium pump) expression to the pre-cardiomyocyte lateral membranes coincides with these events. Na/K-ATPase has been implicated developmentally in cavitation and in maintaining membrane potential. Experiments were undertaken to determine if the effects of perturbing sodium pump activity will affect pericardial cavity formation and, in turn, whether heart formation and/or cardiac cell commitment will be affected. We incubated whole chick embryos in vitro between stages 5 to 8 in the presence of the highly specific Na/K-ATPase inhibitor ouabain. Exposure of whole embryos to 10 μM ouabain (10−5 M) demonstrated that heart development and precardiomyocyte differentiation are inhibited principally between stage 5 through stage 7. In each stage the degree of inhibition follows a rostrocaudal gradient as development proceeds along the anterior to posterior axis. After stage 8 ouabain no longer affects heart development or cardiomyogenesis. The inhibition is concentration- and developmental stage-dependent. The inhibition is reversible by elevating the outside potassium ion concentration [Ko] in the culture medium or by transferring the embryos into normal medium minus ouabain even after 20 hr of ouabain exposure. The results also suggest that the regulation of the formation of the three-dimensional organ is independent from regulation of myogenesis. ©1995 Wiley-Liss, Inc.