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Study of developmental heart defects requires the visualiza- tion of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xe- nopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence mi- croscopy with subcellular 1t o 2m resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed up to 900 frames per second that enables real-time 4-D high resolution imaging in vivo. These im- aging modalities can provide a comprehensive picture of the morpho- logic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investi- gation of the teratogenic effects of ethanol. Microstructural abnormali- ties associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecu- lar mass. © 2007 Society of Photo-Optical Instrumentation Engineers.

Caffeine is a naturally occurring product that acts as a mild central nervous system stimulant. In humans the major sources of caffeine are coffee, tea, and soft drinks, as well as cocoa, chocolate, and certain medications. Caffeine is metabolized more slowly in pregnant women and due to the hydrophobic properties of caffeine it can cross the placenta and the brain-blood barrier. Studies in human and animal models have shown that caffeine exposure during pregnancy affects the perinatal cardiovascular system as well as central nervous system and can result in intrauterine growth retardation and stillbirth. Recent studies show that caffeine intake increases risk of first-trimester spontaneous abortion in human. However, the extent and the mechanism by which maternal caffeine intake influences embryonic cardiovascular function during early morphogenesis is not known. We hypothesized that caffeine ingestion during early pregnancy impairs embryonic cardiac function by delaying the onset of heart beat and alters the normal increase in heart rate (HR) resulting in growth delay and first-trimester spontaneous abortion. Eight to 12 week-old pregnant CD-1 mice and 81embryos were studied under an approved IACUC protocol. Caffeine was dissolve in distilled water and administered daily by gavage at a dose of 120mg/kg from gestational days 0.5 to 10.5. We monitored embryonic heart rate (HR) from gestational days 8.5 to 10.5 at 24 hour intervals using a 40MHz ultrasound biomicroscope. At gestational day 10.5, embryos were fixed and somite number and external morphology was assessed. This period of gestational includes the onset of heart beat of the primitive heart tube through the completion of heart looping. Onset of heart beat was significantly delayed in caffeine group at gestational day 8.5 (heart beat was detected in 41% of caffeine treated embryos versus 79% of sham treated embryos). HR increase was higher in caffeine group at gestational days 9.5 (127±4 in caffeine vs. 112±5 in sham, respectively, p

During the first 20 days of development, the human embryo has no cardiovascular structure. Over the next month, the heart and great vessels complete their development and look very much like they will at full gestation. This amazing process transforms isolated angiogenic cell islets into a complex, four-chambered structure. During this transformation, the single heart tube begins to beat at 23 days of development and by 30 days blood circulates through the embryo.

Measurements of local area changes of the embryonic myocardial surface indicate that the prospective right side expands during looping. Observations from sectioned specimens showing that the right side of the myocardium also becomes thinner during looping suggest that the increase in area may result from myocardial cell flattening. Light microscopic examination of silver impregnated hearts suggests that individual cells increase in apical surface area as the right side becomes convex. Computer-assisted analyses of scanning electron micrographs show that cells of the prospective convex side increase significantly in apical surface area during looping, although the relative alignment of their major surface axes remains unchanged and random. Myocardial cells of the concave side of the heart have a smaller apical surface area and their major surface axes are aligned circumferentially in relation to the heart. We propose that these regional changes in cell shape and alignment mediate heart looping.

Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

In human development, it is postulated based on histological sections, that the cardiogenic mesoderm rotates 180° with the pericardial cavity. This is also thought to be the case in mouse development where gene expression data suggests that the progenitors of the right ventricle and outflow tract invert their position with respect to the progenitors of the atria and left ventricle. However, the inversion in both cases is inferred and has never been shown directly. We have used 3D reconstructions and cell tracing in chick embryos to show that the cardiogenic mesoderm is organized such that the lateralmost cells are incorporated into the cardiac inflow (atria and left ventricle) while medially placed cells are incorporated into the cardiac outflow (right ventricle and outflow tract). This happens because the cardiogenic mesoderm is inverted. The inversion is concomitant with movement of the anterior intestinal portal which rolls caudally to form the foregut pocket. The bilateral cranial cardiogenic fields fold medially and ventrally and fuse. After heart looping the seam made by ventral fusion will become the greater curvature of the heart loop. The caudal border of the cardiogenic mesoderm which ends up dorsally coincides with the inner curvature. Physical ablation of selected areas of the cardiogenic mesoderm based on this new fate map confirmed these results and, in addition, showed that the right and left atria arise from the right and left heart fields. The inversion and the new fate map account for several unexplained observations and provide a unified concept of heart fields and heart tube formation for avians and mammals.

Our understanding of the means by which the left–right axis is patterned is not fully understood, although a number of key intermediaries have been recently described. We report here that retinoic acid (RA) excess affects heart situs concomitant with alterations in the expression of genes implicated in the establishment of the left–right axis. Specifically, RA exposure during a specific developmental window evoked bilateral expression of lefty-1, lefty-2, nodal, and pitx-2 in the lateral plate mesoderm. Time course experiments, together with analysis of midline markers, suggest that nascent mesoderm constitutes a predominant RA target involved in this process. These events are likely to underlie the perturbations of heart looping provoked by excess RA and suggest a means by which retinoids influence the early steps in establishment of the left–right embryonic axis.

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensen’s node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left–right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.

The left–right body axis is coordinately aligned with the orthogonal dorsoventral and anterioposterior body axes. The developmental mechanisms that regulate axis coordination are unknown. Here it is shown that the cardiac left–right orientation in zebrafish (Danio rerio) is randomized in notochord-defectiveno tailandfloating headmutants.no tail(Brachyury) andfloating head(Xnot) encode putative transcription factors that are expressed in the organizer and notochord, structures which regulate dorsoventral and anterioposterior development in vertebrate embryos. Results from dorsal tissue extirpation and cardiac primordia explantation indicate that cardiac left–right orientation is dependent on dorsoanterior structures including the notochord and is specified during neural fold stages inXenopus laevis.Thus, the notochord coordinates the development of all three body axes in the vertebrate body plan.

Vertebrate organisms are characterized by dorsal–ventral and left–right asymmetry. The process that establishes left–right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left–right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left–right pattern formation during embryogenesis in the mouse.

The mechanism by which asymmetric signals induce left-right-specific morphogenesis has been elusive. Pitx2 encodes a transcription factor expressed throughout the left lateral plate mesoderm and subsequently on the left side of asymmetric organs such as the heart and gut during organogenesis in the chick embryo. Pitx2 is induced by the asymmetric signals encoded by Nodal and Sonic hedgehog, and its expression is blocked by prior treatment with an antibody against Sonic hedgehog. Misexpression of Pitx2 on the right side of the embryo is sufficient to produce reversed heart looping and heart isomerisms, reversed body rotation, and reversed gut situs.

The early embryo is initially bilaterally symmetrical. One of the first distinct indications of asymmetry in the embryo occurs during heart looping. The midline tubular heart begins to bend to the right to form a C-shaped structure around 30 hr of development in the avian model. A molecular basis for heart asymmetry and direction of looping is not known, although factors inherent to the myocardium are believed to underlie looping. A left–right asymmetric localization of a specific molecule in the bilateral heart forming regions has not been reported previously. One molecule that we are calling flectin (flectere,in L., to bend or to loop) shows a bilateral asymmetric localization early in the heart forming mesoderm and continues to be expressed asymmetrically in a highly organized manner in the cardiac jelly during heart looping. This large extracellular matrix molecule has been identified using a monoclonal antibody F-22 (Mieziewskaet al.,1994a,b). Flectin shows a discrete spatiotemporal pattern of extracellular matrix expression during avian heart development. An asymmetric expression of flectin is observed during heart development at stage 7+/8− (approximately at 24 hr of development around the 3-somite stage). It is predominantly expressed in the left precardiac mesoderm at this developmental period. Between stages 12 and 14, flectin continues to be asymmetrically expressed in the myocardium and is localized at high levels on the basal side of the myocardium and within the cardiac jelly extending to the endocardial cell surfaces. In the same plane of the looping part of the heart it is differentially organized within the cardiac jelly on the convex side and in the outer loop areas. A reduced expression is apparent anteriorly and posteriorly along the tubular heart. The initial asymmetry of localization is maintained throughout the tubular heart. At stage 22 (Embryonic Day 3.5), intensity of immunolocalization of flectin is significantly decreased, with left–right asymmetry becoming less discernible or absent. It again is expressed in Day 10 embryonic hearts. Flectin expression appears to be modulated by retinoids. In vitamin A-deficient quail embryonic hearts that do not loop (Dersch and Zile, 1993; Twalet al.,1995), flectin protein expression is decreased and disorganized, as are other extracellular matrix components comprising the cardiac jelly.

We assessed the arrangement of actin bundles in the looping chick heart. Actin filaments were stained with rhodamine-labeled phalloidin, and their total arrangement was observed in whole mount specimens. Before the straight heart tube was formed, actin bundles were in a net-like arrangement as if to indicate the cell borders. With progress of the heart tube formation, actin bundles were gradually arranged in a circumferential direction. In the looped heart, regional differences in actin arrangements were observed. In the truncus arteriosus, actin bundles ran in a net-like arrangement. In the bulbus cordis, actin bundles ran in random directions. In the ventricle, actin bundles were roughly arranged in a circumferential direction. Between these three regions, actin bundles ran in a circumferential direction especially on the concave side. Near the right contour on the ventral face, some actin bundles ran in a longitudinal direction along the axis of the tubular heart. In the bulbus cordis and the ventricle at the looped stage, there was another group of actin bundles in the inner layer of the myocardium which ran in a circumferential direction. We presume that the arrangement of actin bundles is related to heart looping.

The transformation of the straight embryonic heart tube into a helically wound loop is named cardiac looping. Such looping is regarded as an essential process in cardiac morphogenesis since it brings the building blocks of the developing heart into an approximation of their definitive topographical relationships. During the past two decades, a large number of genes have been identified which play important roles in cardiac looping. However, how genetic information is physically translated into the dynamic form changes of the looping heart is still poorly understood. The oldest hypothesis of cardiac looping mechanics attributes the form changes of the heart loop (ventral bending → simple helical coiling → complex helical coiling) to compressive loads resulting from growth differences between the heart and the pericardial cavity. In the present study, we have tested the physical plausibility of this hypothesis, which we call the growth-induced buckling hypothesis, for the first time. Using a physical simulation model, we show that growth-induced buckling of a straight elastic rod within the confined space of a hemispherical cavity can generate the same sequence of form changes as observed in the looping embryonic heart. Our simulation experiments have furthermore shown that, under bilaterally symmetric conditions, growth-induced buckling generates left- and right-handed helices (D-/L-loops) in a 1:1 ratio, while even subtle left- or rightward displacements of the caudal end of the elastic rod at the pre-buckling state are sufficient to direct the buckling process towards the generation of only D-loops or L-loops, respectively. Our data are discussed with respect to observations made in biological ‘models’. We conclude that compressive loads resulting from unequal growth of the heart and pericardial cavity play important roles in cardiac looping. Asymmetric positioning of the venous heart pole may direct these forces towards a biased generation of D- or L-loops.

lefty-1 , lefty-2 , and nodal are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) determination. The role of lefty-1 was examined by analyzing mutant mice lacking this gene. The lefty-1 –deficient mice showed a variety of L-R positional defects in visceral organs. Unexpectedly, however, the most common feature of lefty-1 −/− mice was thoracic left isomerism (rather than right isomerism). The lack of lefty-1 resulted in bilateral expression of nodal , lefty-2 , and Pitx2 (a homeobox gene normally expressed on the left side). These observations suggest that the role of lefty-1 is to restrict the expression of lefty-2 and nodal to the left side, and that lefty-2 or nodal encodes a signal for "leftness."

Microtubule-dependent motor, murine KIF3B, was disrupted by gene targeting. The null mutants did not survive beyond midgestation, exhibiting growth retardation, pericardial sac ballooning, and neural tube disorganization. Prominently, the left–right asymmetry was randomized in the heart loop and the direction of embryonic turning. lefty-2 expression was either bilateral or absent. Furthermore, the node lacked monocilia while the basal bodies were present. Immunocytochemistry revealed KIF3B localization in wild-type nodal cilia. Video microscopy showed that these cilia were motile and generated a leftward flow. These data suggest that KIF3B is essential for the left–right determination through intraciliary transportation of materials for ciliogenesis of motile primary cilia that could produce a gradient of putative morphogen along the left–right axis in the node.