Development of cranial flexure and Rathke's pouch in the chick embryo

Experiments were done to investigate the cause of the cranial (mesencephalic) flexure of the chick brain during stages 10 to 14. Measurements of the length and thickness of the roof and floor of the mesencephalon gave values similar to the values obtained previously by others. The labeling index was determined in the roof and floor of the prosencephalon, mesencephalon, and rhombencephalon as a preliminary measure of cell division. The labeling index was about the same in all regions, and was high enough to suggest that most of the cells were dividing. The labeling indices did not suggest that differential growth was caused by differential rates of cell division in the roof and floor of the mesencephalon. It was found through time lapse photography that the foregut and heart remained stationary along the rostrocaudal axis, whereas the prosencephalon moved rostrally and the mesencephalon underwent flexure. Measurements suggested that the neural tube cranial to the otic primordium grew in volume exponentially at a rate consistent with the labeling index. The rostral tip of the neural tube was observed to be linked to the rostral tip of the foregut by the ectoderm that formed Rathke's pouch at the neural tube and the pharyngeal membrane (prospective stomodeum) at the foregut. As the neural tube grew in length, the link between the neural tube and the foregut did not. We suggest that because of this link, the growing neural tube had to bend around the foregut, forming the cranial flexure, and the ectoderm folded where it attached to the prosencephalon, forming Rathke's pouch. © 1994 Wiley-Liss, Inc.     

Correlation between the embryonic head flexures and cardiac development

The aim of the present study is to examine whether the formation of the cranial and cervical flexures is involved in the process of cardiac looping, and whether looping anomalies are causally involved in the development of cardiac malformations. For this purpose, the formation of the cranial and cervical flexures was experimentally suppressed in chick embryos by introducing a straight human hair into the neural tube. In the experimental embryos, the absence of the cervical flexure, alone or in combination with a reduced cranial flexure, was always associated with anomalies in the looping of the tubular heart. The convergence of the primary distant venous and arterial ends of the heart, as well as the normal movement of the ventricular region from its original position, cranial and ventral from the cardiac inflow, to its final position caudal to the presumptive atria, was suppressed to an extent related to the degree to which the formation of the flexures was prevented. Positional immaturity of the heart loop (increased distance between its inflow and outflow, and cranio-ventral position of the ventricular region) was associated with incomplete deformations (reduced angulations) of the cardiac wall at the atrioventricular or conoventricular junctional areas. Reduced angulations were associated with the hypoplasia of the anlagen of the cardiac septa at the level of the angulation (av-cushions, conal ridges). Hypoplasia of these anlagen was followed by incomplete or absent fusion of their opposite free edges, which finally resulted in atrioventricular or ventricular septal defects. These results show that the convergence of the venous and arterial ends of the tubular heart and the caudo-dorsal movement of its ventricular region are related to the formation of the cervical flexure, and that the mesenchymal septa of the heart seem to develop in response to deformations of the embryonic heart, which are generated by the process of cardiac looping. Therefore, the positional and morphological changes of the looping heart are regarded as playing a key role in the process of normal and abnormal morphogenesis of the heart.     

The mechanism of cervical flexure formation in the chick

Summary Chick embryos, during stages 14 to 25, undergo an arching of the hindbrain and cervical neural tube that is termed cervical flexure. We have found that if the truncus arteriosus is severed during stage 12–13, the embryos survive for more than 24 h and do not show cervical flexure. The embryos have a beating heart, the expected number of somites, and often have discernible wing and leg buds. Light and electron micrographs reveal no histological abnormalities. The percentage of cells that become labeled with tritiated thymidine is close to normal, indicating that most of the cells are healthy. These results suggest that cervical flexure is related to normal morphogenesis of the heart. At stage 10, the heart is almost straight, with the prospective ventricle cranial to the prospective sinus venosus. The heart tube loops between stage 10 and stage 23, first to the right and then caudad, so that the ventricle becomes caudal to the sinus venosus. The heart undergoes these morphogenetic movements autonomously. The truncus arteriosus does not increase in length during caudal movement of the ventricle, so the cervical region is pulled into an arch. Bending of the cervical region into an arch can be prevented in intact embryos by injecting agar into the foregut, so that the foregut cannot bend. However, after about 24 h of further growth, if the axis cannot bend, the truncus begins to leak blood and the embryo dies. We conclude that cervical flexure is a response of the embryonic axis to the morphogenesis of the heart.     

Cardiac looping in the chick embryo: the role of the posterior precardiac mesoderm

Summary Grafts of mesoderm taken from the precardiac region of quail embryos of stages 5–7 were inserted into the precardiac mesoderm of chick embryos of stages 5–7. The experiments were of four types and were codenamed to indicate the origin and the destination of the graft. QACP: tissue from the anterior end of the quail precardiac area was inserted into the posterior end of the chick precardiac mesoderm; QPCA: tissue from the posterior end of the quail precardiac area was inserted into the anterior end of the chick precardiac mesoderm; QACA: tissue from the anterior end of the quail precardiac area was inserted into the anterior end of the chick precardiac mesoderm; QPCP: tissue from the posterior end of the quail precardiac area was inserted into the posterior end of the chick precardiac mesoderm. In no case was precardiac tissue removed from the host. Three main types of anomaly were obtained: inverted hearts, in which looping took place to the left rather than to the right; compact hearts, in which no looping occurred, and hearts in which extra tissues or regions were apparent. The incidence of compact hearts was significantly greater with QPCA than with any other category of experiment. When older donors were used (stages 8–9), the incidence of compact hearts fell. No variations in the origin of the graft, nor in its ultimate destination in the host, were found to affect the frequency of any of the anomalies. Sections showed that quail hearts tended to have thicker walls than chick hearts; although quail tissues were often incorporated into the host chick hearts, they retained the histological characteristics of the donors. The fact that no compact hearts resulted from the experiment QACA, or from the mock operations, leads us to conclude that failure to loop in the compact hearts was not due to mechanical trauma caused by the operation, but to some specific difference between grafts taken from the anterior and posterior precardiac mesoderm. The fact that compact hearts were obtained when chick donors were used instead of quails, shows that the effect is not species-specific. We propose that a morphogen is secreted by the posterior end of the precardiac mesoderm and this plays a role in controlling the cessation of looping.     

Association of the cardiac neural crest with development of the coronary arteries in the chick embryo

Background: Chick coronary arteries orginate as penetrating channels from a subepithelial peritruncal ring into the wall of all three aortic coronary sinuses. Two of these capillaries develop a muscular wall and become the definitive coronary arteries. Since cardiac neural crest (CNC) contributes ectomesenchyme to the tunica media (TM) of the aortic arch vessels, we wished to learn if the CNC also contributes to the media of the coronary arteries and if CNC plays an inductive role in determining the site of aortic penetrations and influences which channels persist to hatching. Methods: Quail-to-chick chimeras were made by bilaterally removing the chick CNC and replacing it with quail CNC. The chimeras and unoperated controls were collected on embryonic days (ED) 7–18, fixed in Carnoy's fixative, serially sectioned, stained with Feulgen-Rossenbeck stain, and analyzed. Several ED 18 controls and chimeras were also stained with Gomori's trichrome stain, or labeled with antineurofilament or antivascular smooth muscle alpha actin. Results: The TM of the coronary arteries and the aortic coronary sinuses did not consist of CNC cells. The media of the surviving coronary arteries was disrupted by clusters of CNC cells scattered in the wall of the base of the coronary artery on ED 14 and 18. Persisting coronary arteries were always associated with large neural crest-derived parasympathetic ganglia near their origin. Branches from parasympathetic nerves entered the base of the coronary arteries where the clusters of neural crest cells were located. Quail cells were also associated with tiny vessels exiting the ostia of the coronary arteries and traveling in the outer aortic wall. Labeling with antibodies confirmed a disruption of the TM at the base of the coronary arteries, and showed innervated clusters of quail cells in the disrupted part of the TM. Conclusion: Although the TM of the coronary arteries and the aortic coronary sinuses contained no CNC cells, clusters of innervated quail cells disrupted the TM at the base of the coronary arteries. CNC does not appear to induce capillary penetration direclty; however, the exclusive association of CNC-derived parasympathetic ganglia and nerves with persisting coronary arteries suggests that the presence of parasympathetic ganglia is essential to the survival of the definitive coronary arteries. CNC cells may also be associated with the development of the aortic vasa vasorum. © 1994 Wiley-Liss, Inc.     

3-D observation of actin filaments during cardiac myofibrinogenesis in chick embryo using a confocal laser scanning microscope

Summary Using a confocal laser scanning microscope (CLSM), we observed subcellular three-dimensional (3-D) arrangements of actin filaments stained with fluorescein-labeled phalloidin during myofibrinogenesis of chick embryonic heart (7- to 13-somite stages). Serial optical tomograms were obtained from whole-mounted heart tubes and reconstructed into stereoscopic images. Development of myofibrils in myocardial differentiation considerably differed in inner and outer myocardial cell layers. In the outer layer, initial myofibrils appeared along cell membranes at the 8-somite stage. They increased rapidly and constituted network structures with spatial extension over cell-cell junctions. In the inner layer, myofibrils appeared at the bottom, facing the cardiac jelly, at the 10-somite stage, and, when the straight heart tube began to bend, they were already aligned circumferentially in the direction of the heart tube. Double staining of fluorescein-phalloidin and DiI [1,1-dioctadecyl-3, 3,3,3-tetramethylindo-carbocyanine perchlorate; DiI-C₁₈-(3)] of the looped heart revealed that while myocytes in the outer layer were round, those of the inner layer were spindle-shaped, and their long axes coincided with the circumferential direction. These results suggest that the circumferentially arranged myofibrils at the bottom of the inner layer may play an important role in the looping of the heart tube.     

Structural arrangement of the extracellular matrix network during myocardial development in the chick embryo heart

Summary We analyzed the extracellular matrix and the connective tissue of the developing chick myocardium (myocardial interstitium). The importance of this myocardial element for heart function has been well documented both for the normal and pathologic adult hearts. However, little information is available on the organization of the embryonic myocardial interstitium and its modifications during development and increasing intracardiac pressure. In the present study we used light and scanning electron microscopic techniques, and lectin probes to study the interstitium of the ventricular myocardium of chick embryos from stage 29 (day 6 of development) until hatching. Our observations trace the progressive appearance and organization of the elements of the extracellular matrix, comprising the epimysium, perimysium and endomysium, which form a well-defined architectural network. Finally, we discuss the role of these elements of the extracellular matrix and their possible relation with the biomechanical properties of developing heart.     

The development of cholinergic ganglia in the chick embryo heart

Summary Cholinesterase activity was investigated in the heart of the developing chick from the 6th to 20th day of incubation. The earliest cholinesterase-positive nerve cells and fibers could be demonstrated between the 7th and 9th day. On the 13th day the nervous structure attained full development comparable with that seen in the hatched chicken.The number of ganglia increases up to the 15th day, and remains constant thereafter. The right ventricle is associated with the largest number of ganglia.     

The development of pericardial villi in the chick embryo

Summary The development of pericardial villi and their relation to the development of the cardiac surface was studied in chick embryos from the 3rd to 10th day of incubation by scanning electron microscopy. During the 3rd day of incubation (stage 14–17 HH) the coelomic epithelium covering the ventral wall of the sinus venosus forms villous protrusions. By the end of the 3rd day (stage 17 HH) these protrusions contact the dorsal wall of the heart, so that a secondary dorsal mesocardium is formed. This bridges the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles. This sinu-ventricular mesocardium exists only temporarily, as on the 8th day of incubation it becomes a part of the cardiac wall due to fusion with the epicardium of the coronary sulcus. During the 4th and 5th day of incubation (stage 17 – 25 HH), the formation of the epicardium proceeds from the point of attachment of the sinu-ventricular mesocardium. Although these findings suggest that the epithelium of the villous protrusions spreads over the surface of the embryonic heart, one cannot exclude other hypotheses on epicardial origin. The impression of a spreading epicardium could also be created if epicardial cells were to delaminate from a local epithelium in a temporally and spatially organized pattern.     

Scanning and light microscope studies of the development of the chick embryo semilunar heart valves

Summary The development of the semilunar valves of the great arteries was studied by light and scanning electron microscopy in the chick embryo. The results show that three distinct developmental periods can be distinguished. The formation of the anlage of the valves takes place in the first period (stages 26–29). These early anlage consist of three pyramidal shaped cusps formed by a core of loosely packed mesenchymal cells covered by a flattened endothelium. In the second period (stages 30–35) the cusps undergo excavation on their distal face. Morphological evidence is reported suggesting that this excavation process is produced by an initial solid ingrowth of the endothelium of the arterial face of the cusps which is immediately luminated by detachment of cells towards the bloodstream and by cell death. The histogenesis of the valves takes place in the third period (from stage 36 until hatching). It was observed that during this period some myocardial cells of the outflow tracts of the ventricles invade the valvular tissue and that in the upper part of the cusps a prominent fibrous layer is formed.     

The development of the circulating blood volume of the chick embryo

Summary The circulating blood volume of the chick embryo was determined from the 4th up to the 18th day of hatching. In contrast to former studies, there was employed a radioisotope dilution method with albumin-bound I¹³¹. The findings are in close correspondence to those of the earlier studies. The blood volume does not display an entirely perfect curve of exponential growth, i.e., the doubling time increases steadily. The blood volume attains a peak value between the 16th and 18th day and decreases somewhat toward the end of the hatching period. There has been postulated a reduction of total red cell volume and hemoglobin caused by the involution of the extraembryonic circulatory system. The destruction of the erythrocytes seems to take place in the endodermal epithelium of the proximal yolk sac, where an accumulation of iron could be demonstrated on the 19th and 20th days.Head of Department: Prof. G. TönduryDissertation under the direction of Prof. J. Rickenbacher.     

Expression of α-tropomyosin during cardiac development in the chick embryo

A new monoclonal antibody (mAb) that recognizes α-tropomyosin in cardiac muscle cells was used in a qualitative (polyacrylamide gel electrophoresis and indirect immunofluorescence) and quantitative (fluorescence-activated cell sorting) study of the expression of this protein during heart development. α-Tropomyosin expression was weak in early stages of chick embryo development (Hamburger and Hamilton stage 18), and increased steadily until Hamburger Hamilton stage 40. In early stages, the protein was found mainly in cytoplasm, whereas by the final stages, it was more abundant in the cytoskeletal compartment. The mAb cross-reacted with α-tropomyosin in smooth and striated muscle cells from chickens, mice, and humans, but did not cross-react with nonmuscle tropomyosin.© Willey-Liss, Inc.     

Changes in the arrangement of actin bundles during heart looping in the chick embryo

Summary 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.     

Experimental study of the development of the truncus arteriosus of the chick embryo heart. I. Time of appearance

The time of appearance of the truncus arteriosus was studied in the chick embryo using an in ovo labeling technique. Three hundred embryos at stages 13–18 of Hamburger and Hamilton were selectively labeled at the distal end of the heart tube, using gelatine-india ink label; 122 of these embryos were reincubated and 111 of them reached stages 25–28. In these stages the final location of the label was determined. Only 95 of these embryos showed both a normal heart and a label located in it. The remaining embryos were discarded due to abnormal cardiac morphology or because the label was not found. Embryos labeled at stages 13–14 had label in the conus in 42.8% of the cases and in the boundary between the conus and the truncus arteriosus in 57.1% of the cases. Label placed at stages 15–16 was located in the conus in 6.1% of the cases, in the boundary between the conus and the truncus arteriosus in 44.8% of the cases, and in the truncus arteriosus in 48.9% of the cases. Finally, label placed at stages 17–18 was located in the boundary between the conus and the truncus arteriosus in 18.7% of the cases and in the truncus arteriosus in 81.2% of the cases. Our results permit us to conclude that the truncus arteriosus appears in the chick embryo as early as stages 15–16 of Hamburger and Hamilton (50–56 hours of incubation). © 1993 Wiley-Liss, Inc.     

An ultrastructual topographical study on myofibrillogenesis in the heart of the chick embryo during pulsation onset period

Summary Ultrathin sections of the chick embryonic heart at the 8-, 9- and 10-somite stage were cut serially at an interval of 20 m and mounted for transmission electron microscopic examination on a copper grid with a sufficiently large hole to survey the entire section area. The grid was supported by a formvar film. Thick filaments were first found to assemble into well-defined bundles in several cells composing the caudal region of the newly formed heart just before onset of the pulsation at the 8-somite stage. Then, at the 9-somite stage when pulsation commences, the cells possessing nascent myofibril(s) increase in number, slightly more in the right side of ventricular region. At the 10-somite stage, the rhythmical contraction is established and striated myofibrils become distinctly discernible. Right side dominance is more conspicuous at this stage than previously. Then, myofibrillogenesis gradually progresses toward the cranial or bulbar region.     

Perivascular astrocytes and endothelium in the development of the blood-brain barrier in the optic tectum of the chick embryo

The role played by perivascular astrocytes in neural vessel maturation was investigated in microvessels of the chick embryo optic tectum. Three-dimensional reconstructions and quantitative analyses were made, and permeability was studied. On embryonic days 14–16, 12.5% of the microvessel wall is surrounded by astrocyte endfeet which, in most cases (82%), are located under endothelium junctions; the latter, at this stage, partly prevent the extravascular escape of the marker horseradish peroxidase. On days 18–21, the astrocyte processes form a nearly complete perivascular sheath enveloping 96% of the microvessel perimeter; the junctions of the endothelial cells are much wider and impermeable owing to extensive fusion of the endothelial plasma membranes. This investigation suggests a close relationship between the perivascular arrangement of glia and differentiation of the endothelium tight junctions and indicates that the morphofunctional maturation of the latter takes place progressively during the prenatal organogenesis of the chick central nervous system.     

Actin bundles on the right side in the caudal part of the heart tube play a role in dextro-looping in the embryonic chick heart

Summary The role of actin bundles on the heart looping of chick embryos was examined by using cytochalasin B, which binds to the barbed end of actin filaments and inhibits association of the subunits. It was applied to embryos cultured according to New's method. Looping did not occur when cytochalasin B was applied diffusely in the medium. Further, we disorganized actin bundles in a limited part of the heart tube to examine the role of actin bundles in each part in asymmetry formation. A small crystal of cytochalasin B was applied to the caudal part of the heart tube on either the left or right side. The disorganization of actin bundles on the left side resulted in the right-bending of the heart, an initial sign of dextro-looping (normal pattern), and right side disorganization resulted in left-bending. We suggest that actin bundles on the right side of the caudal part of a heart tube generate tension and cause dextro-looping. Embryos whose hearts bent to the right rotated their heads to the right, and embryos with left-bent-hearts rotated their heads to the left. The rotation of the heart tube may therefore decide in which direction the body axis rotates.     

Migration and distribution of circumpharyngeal crest cells in the chick embryo

The cardiac neural crest is located in a transitional area on the neuraxis between trunk and cephalic regions and gives rise to both the dorsolateral and ventrolateral crest cell populations. Around stage 18 of chick development, a mass of E/C8⁺ cells surrounds the postotic pharyngeal arches and forms a crescent-shaped arch, termed the circumpharyngeal ridge. Using immunohistochemistry and quail-chick chimeras, it was determined that the E/C8⁺ cell mass located in the circumpharyngeal ridge derives from the dorsolateral component of the cardiac neural crest. The ventrolateral cell population of the cardiac crest is located more medially and shows long-persistent HNK-1 immunoreactivity dorsolateral to the foregut. The crest cells that populate the gut arise from the caudal portion of the circumpharyngeal crest and are always located caudal to the caudalmost pharyngeal ectomesenchyme. Circumpharyngeal crest cells continuously populate the pharyngeal arch ectomesenchyme and enteric nervous system on the lateral side of the foregut wall, as well as the hypoglossal pathway which develops within the ventral portion of the circumpharyngeal ridge. E/C8 and HNK-1 immunoreactivity are associated with the cells migrating via the dorsolateral (circumpharyngeal) and ventrolateral pathways, respectively, with one exception: there is a population of putative crest cells along the proximal course of the vagal intestinal branch that shows both immunoreactivities around stage 20. Dil labeling of the cells in the circumpharyngeal ridge suggests that the cells are contributed from the circumpharyngeal ridge to this population. Thus, the distribution of the circumpharyngeal crest cells and their derivatives coincides with the peripheral branch distribution of the cranial nerves IX, X, and XII, whose development is selectively affected in the absence of the cardiac neural crest, the source of the circumpharyngeal crest.© Willey-Liss, Inc.     

Nervous system development in normal and atresic chick embryo intestine: an immunohistochemical study

Intestinal motility disorders are a common complication after surgery for neonatal intestinal atresia. Although intestinal atresia causes alterations in the enteric nervous system, especially in its inner structures (nervous fibers in the mucosa, submucous and deep muscular plexuses), how these alterations develop is unclear. The chick model is a useful research tool for investigating the ontogenesis of the enteric nervous system and the pathogenesis of congenital bowel diseases. More information is needed on the overlap between the developing enteric nervous system and intestinal atresia. Because vasoactive intestinal polypeptide and substance P are typical intestinal neuropeptides, and vasoactive intestinal polypeptide acts as a modulator in neurodevelopment and an inhibitor of smooth muscle cell proliferation, our aim in this study was to investigate the distribution of their immunoreactivity in the developing enteric nervous system of normal and experimental chick models. We studied gut specimens excised from normal chick embryos (aged 12–20 days) and experimental chick embryos (aged 15–20 days) that underwent surgical intervention on day 12 to induce intestinal atresia (atresic embryos) or simply to grasp the bowel loop (sham-operated embryos). In normal chick embryos we showed vasoactive intestinal polypeptide and substance P immunoreactivity from day 12 in the submucous and myenteric plexuses. The distribution of peptide immunoreactivity differed markedly in atresic and normal or sham-operated gut embryos. These differences especially affected the inner structures of the enteric nervous system of specimens proximal to atresia and were related to the severity of dilation. Because nerve structures in the gut wall mucosa and submucous and deep muscular plexuses play a role in motility control and stretch sensation in the intestinal wall, our findings in the chick embryo may help to explain how gut motility disorders develop after surgery for neonatal intestinal atresia.