Chick embryos exposed to trichloroethylene in an ex ovo culture model show selective defects in early endocardial cushion tissue formation

BACKGROUND: Formation of the primitive heart is a critical step for establishing a competent circulatory system necessary for continued morphogenesis, and as such has significant potential as a target for environmental insult. The goal of this study was to identify the initial cellular events that precede more superficially observable abnormalities resulting from exposing early chick embryos to trichloroethylene (TCE). METHODS: A whole embryo culture method was used to assess the susceptibility of endocardial epithelial-mesenchymal transformation in the early chick heart to TCE. This method has the benefits of maintaining the anatomical relationships of developing tissues and organs, instantaneously exposing precisely staged embryos to quantifiable levels of TCE in a protein-free medium, and the ability to directly monitor developmental morphology. RESULTS: A minority of embryos (Hamburger and Hamilton [HH] stage 13-14) exposed to TCE (10-80 ppm) were not viable after 24 hr in culture and exhibited a variety of gross malformations in a dose-dependent fashion. However, the majority of treated embryos remained viable and developed into HH stage 17 embryos that were superficially indistinguishable from vehicle-treated controls. Further analysis of the hearts of these superficially normal embryos by whole-mount confocal microscopy revealed selective reduction in the number of atrioventricular canal mesenchymal cells. Additionally, those mesenchymal cells that did develop migrated abnormally as long thin cords of adherent cells. CONCLUSIONS: The regional selectivity of these effects in the chick heart suggests a critical window of susceptibility to TCE in the epithelial-mesenchymal transformation of atrioventricular canal endocardium.     

Origins and patterning of avian outflow tract endocardium

Outflow tract endocardium links the atrioventricular lining, which develops from cardiogenic plate mesoderm, with aortic arches, whose lining forms collectively from splanchnopleuric endothelial channels, local endothelial vesicles, and invasive angioblasts. At two discrete sites, outflow tract endocardial cells participate in morphogenetic events not within the repertoire of neighboring endocardium: they form mesenchymal precursors of endocardial cushions. The objectives of this research were to document the history of outflow tract endocardium in the avian embryo immediately prior to development of the heart, and to ascertain which, if any, aspects of this history are necessary to acquire cushion-forming potential. Paraxial and lateral mesodermal tissues from between somitomere 3 (midbrain level) and somite 5 were grafted from quail into chick embryos at 3-10 somite stages and, after 2-5 days incubation, survivors were fixed and sectioned. Tissues were stained with the Feulgen reaction to visualize the quail nuclear marker or with antibodies (monoclonal QH1 or polyclonals) that recognize quail but not chick cells. Many quail endothelial cells lose the characteristic nuclear heterochromatin marker, but they retain the species-specific epitope recognized by these antibodies. Precursors of outflow tract but not atrioventricular endocardium are present in cephalic paraxial and lateral mesoderm, with their greatest concentration at the level of the otic placode. Furthermore, the ventral movement of individual angiogenic cells is a normal antecedent to outflow tract formation. Cardiac myocytes were never derived from grafted head mesoderm. Thus, unlike the atrioventricular regions of the heart, outflow tract endocardial and myocardial precursors do not share a congruent embryonic history. The results of heterotopic transplantation, in which trunk paraxial or lateral mesoderm was grafted into the head, were identical, including the formation of cushion mesenchyme. This means that cushion positioning and inductive influences must operate locally within the developing heart tubes.     

The antidysrhythmic effect of metoprolol in the epinephrine treated chick embryo

It was confirmed through electrocardiography that within two hours after epinephrine treatment, four day chick embryos either maintained normal rhythm or developed a severe cardiac dysrhythmia (22/93, 24% dysrhythmic). The ECG dysrhythmia in epinephrine treated embryos were characterized by periods of bradycardia, asystole, and various supraventricular or ventricular dysrhythmias. Within four hours after treatment, dysrhythmic embryos either reestablished normal rhythmicity or died. Electrocardiographic data also demonstrated that metoprolol pretreatment will block epinephrine induced dysrhythmias (0/46, 0% dysrhythmic). We conclude that metoprolol possesses antidysrhythmic properties in the epinephrine treated chick embryo.     

Myocardial cell relationships during morphogenesis in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum

Sarcomere formation has been shown to be deficient in the myocardium of axolotl embryos homozygous for the recessive cardiac lethal gene c. We examined the developing hearts of normal and cardiac mutant embryos from tailbud stage 33 to posthatching stage 43 by scanning electron microscopy in order to determine whether that deficiency has any effect on heart morphogenesis. Specifically, we investigated the relationships of myocardial cells during the formation of the heart tube (stage 33), the initiation of dextral looping (stages 34-36), and the subsequent flexure of the elongating heart (stages 38-43). In addition, we compared the morphogenetic events in the axolotl to the published accounts of comparable stages in the chick embryo. In the axolotl (stage 33), changes in cell shape and orientation accompany the closure of the myocardial trough to form the tubular heart. The ventral mesocardium persists longer in the axolotl embryo than in the chick and appears to contribute to the asymmetry of dextral looping (stages 34-36) in two ways. First, as a persisting structure it places constraints on the simple elongation of the heart tube and the ability of the heart to bend. Second, after it is resorbed, the ventral myocardial cells that contributed to it are identifiable by their orientation, which is orthogonal to adjacent cells: a potential source of shearing effects. Cardiac lethal mutant embryos behave identically during these events, indicating that functional sarcomeres are not necessary to these processes. The absence of dynamic apical myocardial membrane changes, characteristic of the chick embryo (Hamburger and Hamilton stages 9-11), suggests that sudden hydration of the cardiac jelly is less likely to be a major factor in axolotl cardiac morphogenesis. Subsequent flexure (stages 38-43) of the axolotl heart is the same in normal and cardiac lethal mutant embryos as the myocardial tube lengthens within the confines of a pericardial cavity of fixed length. However, the cardiac mutant begins to exhibit abnormalities at this time. The lack of trabeculation (normally beginning at stage 37) in the mutant ventricle is evident at the same time as an increase in myocardial surface area, manifest in extra bends of the heart tube at stage 39. Nonbeating mutant hearts (stage 41) have an abnormally large diameter in the atrioventricular region, possibly the result of the accumulation of ascites fluid. In addition, mutant myocardial cells have a larger apical surface area compared to normals.     

Response of the quiescent heart tube to mechanical stretch in the intact chick embryo

Quiescent heart tubes in intact eight-somite chick embryos were mechanically stretched by injecting excess fluid into the heart lumen. The stretch stimuli sometimes caused precocious focal twitches in the ventricular portion of the primitive heart tube. The contractile response to mechanical stretch was confirmed by recording electrograms before, during, and after the pressure pulse injections. The results prompt us to suggest that the precise timing of the initial heartbeats in the intact embryo may involve an increase in the intraluminal fluid volume and pressure stretching the heart tube wall.     

Development of aortic and mitral valve continuity in the human embryonic heart

The anatomic relationship of the aortic and mitral valves is a useful landmark in assessing congenital heart malformations. The atrioventricular and semilunar valve regions originate in widely separated parts of the early embryonic heart tube, and the process by which the normal fibrous continuity between the aortic and mitral valves is acquired has not been clearly defined. The development of the aortic and mitral valve relationship was studied in normal human embryos in the Carnegie Embryological Collection, and specimens of Carnegie stages 13, 15, 17, 19, and 23, prepared as serial histologic sections cut in the sagittal plane, were selected for reconstruction. In stage 13, the atrioventricular valve area is separated from the semilunar valve area by the large bend between the atrioventricular and outflow-tract components of the single lumen heart tube created by the left interventricular sulcus. In stages 15 and 17, the aortic valve rotates into a position near the atrioventricular valves with development of four chambers and a double circulation. In stage 19, there is fusion of aortic and mitral endocardial cushion material along the endocardial surface of the interventricular flange, and this relationship is maintained in subsequent stages. Determination of three-dimensional Cartesian coordinates of the midpoints of valve positions shows that, while there is growth of intervalvular distances up to stage 17, the aortic to mitral distance is essentially unchanged thereafter. During the period studied, the left ventricle increases in length over threefold. The relative lack of growth in the saddle-shaped fold between the atrioventricular and outflow tract components of the heart, contrasting with the rapid growth of the outwardly convex components of most of the atrial and ventricular walls, may be attributed to the different mechanical properties of the two configurations. It is postulated that the pathogenesis of congenital heart malformations, which characteristically have failure of development of aortic and mitral valve continuity, may involve abnormalities of rotation of the aortic region or malpositioning of the fold in the heart tube.     

Myocardialization of the cardiac outflow tract.

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricular myocardium did not produce factors capable of supporting myocardialization.     

A New Oxidative Stress Model, 2,2-Azobis(2-Amidinopropane) Dihydrochloride Induces Cardiovascular Damages in Chicken Embryo

It is now well established that the developing embryo is very sensitive to oxidative stress, which is a contributing factor to pregnancy-related disorders. However, little is known about the effects of reactive oxygen species (ROS) on the embryonic cardiovascular system due to a lack of appropriate ROS control method in the placenta. In this study, a small molecule called 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), a free radicals generator, was used to study the effects of oxidative stress on the cardiovascular system during chick embryo development. When nine-day-old (stage HH 35) chick embryos were treated with different concentrations of AAPH inside the air chamber, it was established that the LD₅₀ value for AAPH was 10 µmol/egg. At this concentration, AAPH was found to significantly reduce the density of blood vessel plexus that was developed in the chorioallantoic membrane (CAM) of HH 35 chick embryos. Impacts of AAPH on younger embryos were also examined and discovered that it inhibited the development of vascular plexus on yolk sac in HH 18 embryos. AAPH also dramatically repressed the development of blood islands in HH 3+ embryos. These results implied that AAPH-induced oxidative stress could impair the whole developmental processes associated with vasculogenesis and angiogenesis. Furthermore, we observed heart enlargement in the HH 40 embryo following AAPH treatment, where the left ventricle and interventricular septum were found to be thickened in a dose-dependent manner due to myocardiac cell hypertrophy. In conclusion, oxidative stress, induced by AAPH, could lead to damage of the cardiovascular system in the developing chick embryo. The current study also provided a new developmental model, as an alternative for animal and cell models, for testing small molecules and drugs that have anti-oxidative activities.     

The development of the conduction system in the mouse embryo heart: IV. Differentiation of the atrioventricular conduction system

The development of the atrioventricular conduction system in the mouse heart has been studied by light and electron microscopy from the time of the completion of ventricular septation to fetal stage II, 13–16 days postcoitum. At the beginning of this period the already established atrioventricular node (AVN) enlarges rapidly into the dorsal AV cushion from the primitive AV tract, reaching almost its full fetal size when septation is complete. The development of the atrionodal interconnections is a slow and complex process. The dorsal atrial myocardium develops on both sides of the node, establishing a muscular overlay over its proximal aspect, and also incorporating the former AV tract. At this time also, the developing muscular interatrial septum grows downward to establish contact with the node, the sinus venosus, and the myocardium of the right and left atrial walls. The distally proceeding differentiation of the ab initio continuous conduction pathway along the AVN, His bundle, and bundle branches demonstrates a progressive and sequential development of high cellular glycogen content. Progressive isolation of the atrioventricular conduction system leading to (still incomplete) insulation by connective tissue, has been observed.     

Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail

In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left-right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty,Pitx2 or cSnR expression were observed. In addition, Flectin expression remained higher in the left dorsal mesocardial folds of embryos with leftwardly looped hearts resulting from Claudin-1 overexpression on the right side of the embryo. We demonstrated that Claudin-1's C-terminal cytoplasmic tail is essential for this effect: mutation of a PKC phosphorylation site in the Claudin-1 C-terminal cytoplasmic domain at threonine-206 eliminates Claudin-1's ability to randomize the direction of heart looping. Taken together, our data provide evidence that appropriate expression of the tight junction protein Claudin-1 is required for normal heart looping and suggest that phosphorylation of its cytoplasmic tail is responsible for mediating this function.     

Hemodynamic effects of acetylcholine in the chick embryo and differences from those in the rat embryo

It has been reported that acetylcholine induces cardiac anomalies in the chick embryo. Thus, we studied hemodynamic effects of this drug in the chick embryo and also compared them with those in the rat embryo since we found that the effect of caffeine was different between the chick and rat embryos. Acetylcholine was given at doses of 5, 0.5, and 0.05 micrograms into the vitelline vein in chick embryos at Hamburger-Hamilton stage 21 and at a dose of 0.5 micrograms into the placenta in rat embryos at gestational day 12. In the chick embryo, heart rate was reduced to 91, 88, and 87% of control at the end of injection of 0.05, 0.5, and 5 micrograms, respectively, then returned to the baseline level. Vitelline arterial blood pressure was 110% of control with 0.05 micrograms, 134% with 0.5 micrograms, and 142% with 5 micrograms at 1 min after injection. The dorsal aortic blood flow decreased with time after injection, but it was increased only by a 5 micrograms dose at the end of injection. The vascular resistance increased in a dose-dependent manner. In the rat embryo, the change of heart rate was qualitatively similar to that of the chick embryo. The blood pressure did not change significantly. The blood flow velocity at the outflow tract decreased at the end of injection, which indicated the decrease in cardiac output, along with slowing of heart rate, then returned to the control level thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibroblast growth factor (FGF)-4 can induce proliferation of cardiac cushion mesenchymal cells during early valve leaflet formation

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the growth and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by growth factors are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized fibroblast growth factor (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage 20-25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24-25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5'-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial beta-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.     

Chronic ethanol exposure in the embryonic chick heart: effect on myocardial function and structure

To investigate the effect of chronic ethanol exposure on the embryonic chick heart, chick embryos were exposed daily to one of seven graded doses of ethanol or to saline only (shams) from 0 to 96 hr of incubation. One hour before and after exposure at 72 hr, and 1 hr before and after exposure at 96 hr, embryos were analyzed for changes in heart function, embryo tissue ethanol content, occurrence of anomalies, and embryo weights. At both 71 and 73 hr of incubation (during cardiogenesis), when compared to shams, heart rate (HR) in embryos receiving ethanol doses greater than 0.0375 ml increased significantly (P less than .05) with commensurate increases in injected ethanol. Additionally, at 73 hr, depressed cardiac contractility, measured as shortening fraction, was noted at doses greater than or equal to .0375 when compared to shams. While slight increases in shortening fraction (SF) across dose were noted at 95 and 97 hr, only random doses were statistically significant from shams, with no specific trend in either HR or SF at this postcardiogenesis stage. Within each time group, gas chromatography analysis of embryo tissue ethanol content demonstrated a linear relationship between dose injected and tissue ethanol content retrieved. With increasing dose and stage, viability decreased. Weights of ethanol-injected embryos were not significantly different from shams within each time group. Our studies of the response of the embryonic chick heart to ethanol indicate both dose and stage susceptibility, with greater susceptibility to ethanol injury during active cardiogenesis.     

Right and left ventricular wall deformation patterns in normal and left heart hypoplasia chick embryos

The vertebrate embryonic ventricle transforms from a smooth-walled single tube to trabeculated right ventricular (RV) and left ventricular (LV) chambers during cardiovascular morphogenesis. We hypothesized that ventricular contraction patterns change from globally isotropic to chamber-specific anisotropic patterns during normal morphogenesis and that these deformation patterns are influenced by experimentally altered mechanical load produced by chronic left atrial ligation (LAL). We measured epicardial RV and LV wall strains during normal development and left heart hypoplasia produced by LAL in Hamburger-Hamilton stage 21, 24, 27, and 31 chick embryos. Normal RV contracted isotropically until stage 24 and then contracted preferentially in the circumferential direction. Normal LV contracted isotropically at stage 21, preferentially in the longitudinal direction at stages 24 and 27, and then in the circumferential direction at stage 31. LAL altered both RV and LV strain patterns, accelerated the onset of preferential RV circumferential strain patterns, and abolished preferential LV longitudinal strain (P < 0.05 vs. normal). Mature patterns of anisotropic RV and LV deformation develop coincidentally with morphogenesis, and changes in these deformation patterns reflect altered cardiovascular function and/or morphogenesis.     

Versican/PG-M is essential for ventricular septal formation subsequent to cardiac atrioventricular cushion development

Versican (Vcan)/proteoglycan (PG)-M is a large chondroitin sulfate proteoglycan which forms a proteoglycan/hyaluronan (HA) aggregate in the extracellular matrix (ECM). We tried to generate the Vcan knockout mice by a conventional method, which resulted in mutant mice Vcan(Δ3/Δ3) whose Vcan lacks the A subdomain of the G1 domain. The Vcan knockout embryos died during the early development stage due to heart defects, but some Vcan(Δ3/Δ3) embryos survived through to the neonatal period. The hearts in Vcan(Δ3/Δ3) newborn mice showed normal cardiac looping, but had ventricular septal defects. Their atrioventricular canal (AVC) cushion was much smaller than those of wild-type (WT) embryos, and the extracellular space for cardiac jelly was narrow. The Vcan deposition in the Vcan(Δ3/Δ3) AVC cushion had decreased, whereas the HA deposition was maintained and condensed. In the tip of ventricular septa, both Vcan and HA had decreased. The cell proliferation based on the number of Ki67-positive cells had remarkably increased in both the AVC cushion and ventricular septa, compared with that of WT embryos. Vcan(Δ3/Δ3) seemed to have endocardial and mesenchymal mixed characteristics. When the ex vivo explant culture of these regions was performed on the collagen gel, hardly any migration to make sufficient space for the ECM construction was apparent. Our results suggest that the proteoglycan aggregates are necessary in both the AVC cushion and ventricular septa to fuse interventricular septa, and the Vcan A subdomain plays an essential role for the interventricular septal formation by constituting the proteoglycan aggregates.     

Uptake and blood pressure studies in the chick embryo following treatment with a teratogenic dose of dopamine

We report on the ventricular pressor response and regional (head, heart, and body) uptake of dopamine hydrochloride (DA) following topical application of a teratogenic dose to the stage 24 chick embryo. Embryos treated with DA exhibited a significant increase in mean ventricular blood pressure (MVBP) at 0.5 and 1 hour after treatment when compared to saline-treated control embryos. The time of the elevated MVBP correlated with the time of the peak uptake of DA in the embryonic heart. Even though the level of DA in the heart remained high for 9 hours, there was no measurable increase in blood pressure beyond 1.5 hours, suggesting unresponsiveness to DA or perhaps conversion of DA to its metabolites. These experiments have correlated a physiologic response of a known cardiovascular teratogen to its uptake in the embryo.     

Atrioventricular valves development in human heart: the Paris embryological collection revisited

29 human embryos staging from stage 15 to stage 23 (post-somitic period, collection of the UER Biomedicale des Saints-Péres, Université René Descartes Paris V) have been studied. The most important morphological events of the atrioventricular valves development have been reinvestigated and photographed. This is a complementary information about cardiac development analysing this french collection of human embryos (Mandarim-de-Lacerda, in press). At stage 15, we can observe the gelatinous reticulum well organized when cardiac valves will become established; progressively the fused endocardial cushions and right and left lateral cushions encircle the atrioventricular channels indicating the site of the tricuspid valves. These cushions, however, have a temporary influence being replaced gradually by atrial and ventricular myocardium. At stage 23, the heart presents a complete atrioventricular valvular structure.     

Migratory behavior of cardiac cushion tissue cells in a collagen-lattice culture system

A culture system was devised for the study of factors which influence the migration of cardiac cushion tissue cells. Explants of isolated chick atrioventricular canal cushions were placed on hydrated collagen lattices. Cells grew out of the explants from the endocardium and across the surface of the collagen lattices. During further incubation, mesenchyme-type cells seeded from the surface population into the underlying collagen matrix. These cells were morphologically similar to the mesenchymal cushion tissue cells which are derived from the endocardium and which migrate into and through the cardiac jelly matrix in the embryonic heart. The events observed in the culture system mimicked those occurring in the developing chick atrioventricular cushion.     

Contribution of the aortopulmonary septum to the muscular outlet septum in the human heart

The development of the outlet septum has been studied microscopically in 14 human embryos, ranging from 9 to 28 mm crown-rump length. Three tissue components are involved in the septation process: condensed mesenchyme of extracardiac origin, myocardium and endocardial cushion tissue. At the stage of 9 1/2 mm the condensed mesenchyme, which is embedded in the endocardial cushion tissue, is in contact with the myocardium at two sites. Graphic reconstructions of a 16 mm embryo show that at these sites of contact the myocardium is "drawn inwards" to form two bulges interconnected by the condensed mesenchyme. With further development the two myocardial bulges become the main mass of what then can be called the outlet septum. Thus the sites of contact between condensed mesenchyme and myocardium can be considered to represent the sites of attachment of the muscular outlet septum. This has important implications for the elucidation of the development of outlet malformations.