A predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart looping dynamics

How left-right patterning drives asymmetric morphogenesis is unclear. Here, we have quantified shape changes during mouse heart looping, from 3D reconstructions by HREM. In combination with cell labelling and computer simulations, we propose a novel model of heart looping. Buckling, when the cardiac tube grows between fixed poles, is modulated by the progressive breakdown of the dorsal mesocardium. We have identified sequential left-right asymmetries at the poles, which bias the buckling in opposite directions, thus leading to a helical shape. Our predictive model is useful to explore the parameter space generating shape variations. The role of the dorsal mesocardium was validated in Shh-/- mutants, which recapitulate heart shape changes expected from a persistent dorsal mesocardium. Our computer and quantitative tools provide novel insight into the mechanism of heart looping and the contribution of different factors, beyond the simple description of looping direction. This is relevant to congenital heart defects.

Role of Mechanical Feedback in Restoration of Normal Cardiac C-Looping Following Perturbed Loading

Cardiac c-looping is an important developmental phase, as the initially straight heart tube (HT) is transformed into a c-shaped tube. Looping consists of two distinct processes: ventral bending, which is likely driven by actin polymerization, and dextral torsion, which is likely due to external forces. These forces are applied by a membrane enveloping the ventral side of the heart, the splanchnopleure (SPL, Fig. 2A) and a pair of atria that flank the caudal end of the heart tube (HT, Fig 1A). In particular, the atria provide the initial push, biasing the HT towards the right while the SPL applies a ventrally directed force, which causes the HT to rotate using the dorsal mesocardium (DM, Fig. 2A) as a pivot (the DM attaches the dorsal length of the heart to the body of the embryo).

Development of the human pulmonary vein and its incorporation in the morphologically left atrium

Objective: Using a newly acquired archive of previously prepared material, we sought to re-examine the origin of the pulmonary vein in the human heart, aiming to determine whether it originates from the systemic venous sinus (“sinus venosus”), or appears as a new structure draining to the left atrium. In addition, we examined the temporal sequence of incorporation of the initially solitary pulmonary vein to the stage at which four venous orifices opened to the left atrium. Methods: We studied 26 normal human embryos, ranging from 3.8 mm to 112 mm crown-rump length, and representing the period from the 12th Carnegie stage to 15 weeks of gestation. Results: The pulmonary vein canalised as a solitary vessel within the mediastinal tissues so as to connect the intraparenchymal pulmonary venous networks to the heart, using the regressing dorsal mesocardium as its portal of cardiac entry. The vein was always distinct from the tributaries of the embryonic systemic venous sinus. The orifice of the solitary vein became committed to the left atrium by growth of the vestibular spine. During development, a marked disparity was seen between the temporal and morphological patterns of incorporation of the left-sided and right-sided veins into the left atrium. The pattern of the primary bifurcation was asymmetrical, a much longer tributary being formed on the left than on the right. Contact between the atrial wall and the venous tributary on the left initially produced a shelf, which became effaced with incorporation of the two left-sided veins into the atrium. Conclusions: The initial process of formation of the human pulmonary vein is very similar to that seen in animal models. The walls of the initially solitary vein in humans become incorporated by a morphologically asymmetric process so that four pulmonary veins eventually drain independently into the left atrium. Failure of incorporation on the left side may provide the substrate for congenital division of the left atrium.

Conotruncal myocardium arises from a secondary heart field

The primary heart tube is an endocardial tube, ensheathed by myocardial cells, that develops from bilateral primary heart fields located in the lateral plate mesoderm. Earlier mapping studies of the heart fields performed in whole embryo cultures indicate that all of the myocardium of the developed heart originates from the primary heart fields. In contrast, marking experiments in ovo suggest that the atrioventricular canal, atria and conotruncus are added secondarily to the straight heart tube during looping. The results we present resolve this issue by showing that the heart tube elongates during looping, concomitant with accretion of new myocardium. The atria are added progressively from the caudal primary heart fields bilaterally, while the myocardium of the conotruncus is elongated from a midline secondary heart field of splanchnic mesoderm beneath the floor of the foregut. Cells in the secondary heart field express Nkx2.5 and Gata-4, as do the cells of the primary heart fields. Induction of myocardium appears to be unnecessary at the inflow pole, while it occurs at the outflow pole of the heart. Accretion of myocardium at the junction of the inflow myocardium with dorsal mesocardium is completed at stage 12 and later (stage 18) from the secondary heart field just caudal to the outflow tract. Induction of myocardium appears to move in a caudal direction as the outflow tract translocates caudally relative to the pharyngeal arches. As the cells in the secondary heart field begin to move into the outflow or inflow myocardium,they express HNK-1 initially and then MF-20, a marker for myosin heavy chain. FGF-8 and BMP-2 are present in the ventral pharynx and secondary heart field/outflow myocardium, respectively, and appear to effect induction of the cells in a manner that mimics induction of the primary myocardium from the primary heart fields. Neither FGF-8 nor BMP-2 is present as inflow myocardium is added from the primary heart fields. The addition of a secondary myocardium to the primary heart tube provides a new framework for understanding several null mutations in mice that cause defective heart development.

An experimental study of the relation of cardiac jelly to the shape of the early chick embryonic heart

The structural roles of cardiac jelly components were examined in the early developing chick embryonic heart. Cardiac jelly matrix components were enzymically removed in situ by injecting nanogram quantities of enzymes directly into the cardiac jelly. Injection of ovine testicular hyaluronidase caused shrinkage and the heart became flaccid, but overall heart shape did not change. These responses were the result of enzymatic removal of glycosaminoglycan sugar moieties and were not due to lumenal collapse. Although purified collagenase did not cause any noticeable change, enzymes with non-specific proteolytic activity induced marked cardiac shape changes. In such hearts the dorsal mesocardium opened completely, and the myocardium as well as splanchnic mesoderm of foregut detached from their substrata and the entire heart region swelled. Consequently the shape of the heart was altered completely. These results suggested that in the normal condition the myocardial envelope was under an internal pressure due to the presence of glycosaminoglycans in the cardiac jelly space, and that some matrical non-collagenous protein components were essential to control the internal pressure. Therefore it is suggested that the internal pressure of cardiac jelly may be the direct driving force for the looping process and protein components of cardiac jelly may be important in directing the force for the morphogenetic process.

Compositional and structural heterogenicity of the cardiac jelly of the chick embryo tubular heart: a TEM, SEM and histochemical study

The hearts of chick embryos of stages 9–13 were subjected to SEM, TEM and histochemical studies to ascertain possible regional differences in the structure and composition of the cardiac jelly. Two distinct regions, the cardiac jelly filling the space located between the myocardium and the endocardium (MECJ) and the cardiac jelly filling the dorsal mesocardium (EECJ), were distinguished by their structural and histochemical properties. MECJ is formed by amorphous and fibrillar material arranged between the endocardial and myocardial layer. The amount of its components increases when cetylpyridinium chlorideis introduced into the fixative, and it appears intensely stained by ruthenium red and alcian blue at low concentrations of MgCl2. The amount and arrangement of its componentsincrease during the beginning of the looping process of the heart tube. The EECJ is very rich in ruthenium-red-positive basal-lamina-like material and the addition of cetylpyridinium chloride to the fixative does not modify its appearance. It also appears poorly stained by alcian blue at low concentrations of MgCl2 and its arrangement undergoes modifications closely associated with the events of endocardial fusion. The possible significance of these results in the early morphogenesis of the heart is discussed.

The heart of the salamander ( salamandra salamandra l. ), with special reference to the conducting (connecting) system and its bearing on the phylogeny of the conducting systems of mammalian and avian hearts

Nine salamander hearts have been studied histologically by means of serial sections, cut in each of three planes (transverse, frontal and sagittal), and stained with haemalum and eosin, van Gieson's acid fuchsin and iron-haematoxylin, and by the protargol method of Bodian. I his study has demonstrated muscular continuity between the several cardiac chambers, and the entile absence of any specialized muscle or ‘nodal tissue’ at the junctional sites or in any other part of the heart. The heart muscle forms a continuum. The cardiac muscle fibres are characterized by their large size (i.e. breadth); they have the same general histological characters in all parts of the heart. Measurements are given for the fibres from various parts of the hearts of the salamander and frog. The muscular connexions between the various cardiac chambers have been studied in detail. In each of the chambers the musculature is arranged in a basket-work fashion, but at each of the junctional sites the muscle suddenly changes to a regular circular arrangement. The sinus, at its junction with the right atrium, contains muscle only in its ventral wall, and it is this wall only of the sinus which thus establishes muscular continuity with the ring of muscle (S-A ring) around the sinu-atrial opening. The musculature of both atria is continuous with that of the ventricle in two ways. From the ring of muscle (A-V ring) surrounding the common opening of the atria into the ventricle, the atrio-ventricular funnel dips down into the ventricle, and the caudal border of this funnel is continuous ( a ) with an invaginated part of the base of the ventricle, and ( b ) more extensively with ventricular papillary muscles, which, in their turn, are continued into the inner ventricular trabeculae about the middle level of the ventricle. The A-V funnel is homogeneous in structure; no one part of its circumference differs from another. The ventricular muscle is directly continued into that of the bulbus cordis, in which latter chamber the muscle is entirely circular. The course which the wave of contraction takes during its transmission from the sinus throughout the heart has been deduced from the study of the details of the continuity of the musculature of the various cardiac chambers. To a large extent this deduction has been confirmed by superimposing tracings of the outline of the pulsating heart, made from the slow-motion cinephotographic records. This latter study has revealed many of the details of the phases of the cardiac cycle. The delay in the transmission of the wave of contraction from one cardiac chamber to the next is accounted for by the relatively long path which the impulse has to traverse at the junctional sites, where the muscle is arranged in a circular fashion, without postulating the existence of specialized ‘block fibres’ at these sites. The branching of the muscle fibres has the effect of converting the morphological circular arrangement of the fibres at these junctions into a physiological spiral. The glycogen content of the various parts of the frog’s heart, as revealed by staining with carmine, is found to increase in the order sinus, atria, ventricle and bulbus cordis. This is correlated with a similar increasing order of density of musculature and work done, the glycogen being a reserve potency for the energy of muscular contraction. The fact that the intrinsic rhythmic rates of the several chambers decrease in the same order as the glycogen content increases may or may not be coincidental. Cutting and ligature experiments, with cinephotographic and kymographic records, reveal the intrinsic rhythmic rates of the various cardiac chambers of the salamander heart. No satisfactory reason has yet been adduced to account for the different intrinsic rhythmic rates of the several parts of the heart when these are isolated from each other. The dorsal mesocardium has been traced in its entirety. The sinu-ventricular fold is a part of the continuous dorsal mesocardium and does not constitute a direct muscular sinuventricular connexion. The distribution of the intracardiac nerve cells has been noted and the probable pathway of migration of these nerve cells in the embryo has been suggested. The significance of the results of this investigation in relation to the phylogeny of the specialized conducting system of the hearts of homoiothermal vertebrates (mammals and birds) is discussed. The view is expressed that the cardiac conducting systems of homoiothermal vertebrates constitute a neomorphic development, correlated with functional requirements, and are not remnants of more extensive tissues of similar structure in the lower vertebrate heart. Variations in this newly evolved formation probably account for the different descriptions of such elements in various mammalian and avian hearts.