Spectrum of looping disturbances in stage 34 chicken hearts after retinoic acid treatment

Background: In a recently developed chick model the teratogen retinoic acid has appeared to induce a spectrum of double outlet right ventricle, which needs further detailed evaluation. It is known that retionic acid is able to induce cardiac malformations. Although the exact mechanism is not known, an interaction with neural crest cell function is thought to exist. Methods: After treatment with 1 μg all-trans retinoic acid at Hamburger and Hamilton stage 15 and reincubation until stage 34 of development 41 chicken embryos were evaluated macroscopically and microscopically, supported by graphic reconstructions. These retinoic acid treated embryos were compared with a control group (n = 8). Results: The retinoic acid treated embryos could be divided in three groups. Group 1 (23/41) had an intact septum, group 2 (11/41) had an isolated ventricular septal defect (VSD), and group 3 (7/41) had a double outlet right ventricle (DORV). Besides, in the group with an intact septum 11 hearts showed an abnormal course of the subaortic outflow tract. In the group with DORV a straddling tricuspid orifice (7/8) and a double inlet left ventricle (1/8) could be distinguished. Considering the external contour, the hearts in the DORV group all showed a dextroposed arterial pole. Malformed pharyngeal arch arteries were found in all three groups (11/41) and with a great diversity. Conclusions: The present cardiac malformations in the chicken as a result of retinoic acid treatment are part of a continuous spectrum, varying from hearts with an intact ventricular septum and a normal course of the subaortic outflow tract to a double outlet right ventricle with a straddling tricuspid orifice or even a double inlet left ventricle. A remarkable observation in this spectrum concerns the correlation of malformations of the inflow and outflow tracts, which is explained as a cardiac looping disturbance. The disturbance of the looping process seems to lead to malalignment of septal components, although, in the chick, retinoic acid does not in general interfere with the formation of these septal components themselves. © 1995 Wiley-Liss, Inc.     

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.     

Gap junction proteins exhibit early and specific expression during intramembranous bone formation in the developing chick mandible

The spatial and temporal expression of three closely related members of the connexin family of gap junction proteins (connexin42, Cx42; connexin43, Cx43; and connexin45, Cx45) was evaluated during bone formation in the mandibular process of the chick embryo. Mandibles of chick embryos from Hamburger and Hamilton stage 25 (approximately 5 days) through 19 days of development were dissected, serially sectioned and processed for immunocytochemical localization, employing site-specific anti-connexin antibodies. Our data revealed that (1) Cx43 was present throughout mandibular bone formation; (2) although it appeared to be associated with all bone cell types, Cx43 was concentrated in mesenchymal cells during the earliest stages in the osteogenic lineage; (3) most importantly, the localization of Cx43 at sites of bone formation appeared to precede the overt expression of the osteogenic phenotype; (4) by contrast, Cx45 was more restricted, spatially and temporally, in its distribution; (5) Cx42 expression was not detected in osteogenic tissue during mandibular bone formation. From all of the data obtained, Cx45 appeared to be associated with stages of bone formation characterized by the elaboration of matrix and the progressive expression of the differentiated osteogenic phenotype. Cx43 appeared to be associated with condensation of mesenchyme and the earliest stages of osteogenesis. Because of these associations, we propose that connexin expression may be necessary for the initiation of bone formation and the full expression of the osteogenic phenotype.     

Ventrally emigrating neural tube cells migrate into the developing vestibulocochlear nerve and otic vesicle

Virtually all cell types in the inner ear develop from the cells of the otic vesicle. The otic vesicle is formed by the invagination of non-neural ectodermal cells known as the otic placode. We investigated whether a recently described cell population, originating from the ventral part of the hindbrain neural tube known as the ventrally emigrating neural tube (VENT) cells, also contributes cells to the otic vesicle. The ventral hindbrain neural tube cells were labeled with the fluorescent vital dye DiI or replication-deficient retroviruses containing the LacZ gene in chick embryos on embryonic day 2, after the emigration of neural crest from this region. One day later, the labeled cells were detected only in the hindbrain neural tube. Shortly thereafter, the labeled cells began to appear in the eighth (vestibulocochlear) cranial nerve and otic vesicle. From embryonic day 3.5-5, the labeled cells were detected in the major derivatives of the otic vesicle, i.e. the endolymphatic duct, semicircular canals, utricle, saccule, cochlea, and vestibulocochlear ganglion. That the emigrated cells originated from the ventral part of the hindbrain neural tube was confirmed by focal application of DiI impregnated filter paper and with quail chimeras. It is concluded that, in addition to the otic placode cells, the otic vesicle also contains the ventrally emigrating neural tube cells, and that both cell populations contribute to the structures and cell types in the inner ear. It is well known that inductive signals from the hindbrain are required for the morphogenesis of the inner ear. The migration of the hindbrain neural tube cells into the otic vesicle raises the possibility that the inductive effect of the hindbrain might be mediated, at least in part, by the ventrally emigrating neural tube cells and that, therefore, a mechanism exists that involves cells rather than diffusible molecules only.     

Effects of opiates on the growth of neuron-enriched cultures from chick embryonic brain

Neuron-enriched cultures derived from 6-day-old chick embryo cerebral hemispheres were treated with morphine or methadone, 10(-5) M or 10(-6) M, on days 4-6 or 6-8 in culture and were evaluated morphologically and biochemically at day 9 using phase contrast microscopy and choline acetyltransferase activity (ChAT) as a cholinergic marker. The treatment of the cultures with morphine markedly affected their growth pattern; specifically, we observed an increased number of flat cells presumptively glia, and aggregates sided by flat cells and devoid of thick bundles of neuritic processes that normally characterize neuron-enriched cultures. These morphologic changes were reflected in a drastic decrease of ChAT activity in cultures treated from day 4 to day 6 but not from 6 to 8. In contrast to morphine, exposure to 10(-6) M methadone from day 4 to day 6 resulted in reduced ChAT activity but the growth pattern of the cultures remained morphologically intact. We suggest that morphine exerts a general neurotoxic effect whereas methadone may affect some specific cholinergic function.     

Enhancement of performance by methyl β-carboline-3-carboxylate, in learning and memory tasks

Benzodiazepines are known to induce a profound anterograde amnesia in man. In this report, it is shown that methyl β-carboline-3-carboxylate (β-CCM), an inverse agonist of the benzodiazepine receptor, has the opposite effect; it enhances performance in learning and memory tasks. Three different learning models were used: habituation to a new environment and passive avoidance in mice and imprinting in chicks. The opposite effects of both β-CCM and the benzodiazepine diazepam were blocked by administration of the benzodiazepine receptor antagonist Ro 15-1788, provicling evidence that the benzodiazepine receptor is involved in these effects.     

Chronotropic and inotropic effects of histamine in developing chick heart: differential mechanisms before and after hatching

Chronotropic and inotropic effects of histamine were examined in isolated atrial and ventricular preparations from embryonic and hatched chicken hearts. Histamine produced positive chronotropic and inotropic responses both in embryonic and hatched hearts. The responses to histamine in middle embryonic myocardia, which were observed in the micromolar range, were antagonized by H₂ antagonists but not by H₁, H₃ antagonists and propranolol. Isobutylmethylxantine, an inhibitor of phosphodiesterase, produced a leftward shift of the concentration-response curve for the chronotropic effect of histamine in the embryo. The responses to histamine in myocardia from hatched chicks, which were observed in the milimolar range, appeared concurrently with the responses to tyramine during development and were antagonized by beta adrenoceptor antagonists but not by any of the histamine antagonists. The positive inotropic response to histamine in hatched ventricular preparations were greatly attenuated by reserpine pretreatment or in the presence of desipramine. Thus, we demonstrated that exogenously applied histamine produces positive chronotropic and inotropic responses in developing chicken hearts and that the mechanisms are different between embryonic and hatched chicks: direct action on H₂ receptors in the embryonic heart and release of norepinephrine from sympathetic nerve terminals in hatched hearts.     

Scanning and transmission electron microscopy of venous sphincters in the rat lung

The rat pulmonary microvasculature was studied using scanning and transmission electron microscopy of vascular corrosion casts and tissue sections. Special emphasis was placed on small pulmonary venous vessels. The shape of vascular casts was analyzed and interpreted concerning the wall composition of corresponding vessels studied in tissue sections. On the casts of pulmonary venules and small pulmonary veins, narrow or wider annular constrictions were regularly observed. Within these constrictions, marks of circularly running grooves were seen as an additional structural detail, which obviously mimic impressions of single or grouped smooth muscle cells. The depth of the constrictions varies; it may be more or less pronounced, occasionally narrowing down the luminal diameter to approximately 50%. These constrictions are caused by muscular sphincters. In tissue sections of small pulmonary veins, sphincter regions were identified as abruptly appearing single or grouped true smooth muscle cells. Smooth muscle cells may be arranged side by side in a group or bundle or even staked in two or three layers. Between the sphincter regions, the venous wall consists merely of endothelium and an accompanying connective tissue layer. The smooth muscle cells of a sphincter are regularly positioned between endothelial layer and elastic lamina. The smooth muscle cells next to the endothelium form myoendothelial junctions. Autonomic nerves near the sphincters were never seen. The venous sphincters described are suggested to be effective devices involved in blood flow regulation. Blood-borne substances or local tissue hormones might govern sphincter function. © 1992 Wiley-Liss, Inc.     

Mathematical modelling of the growth processes in the developing chick wing bud

This paper illustrates how a simple geometric model resembling the shape of the chick wing bud at an early growth stage can be mathematically expanded to simulate subsequent growth characteristics of the developing bud. The model was tested against several sets of experimental data and gave an acceptable representation of growth over the range considered. Representing growth patterns in this form enables the determination of differential growth characteristics in different parts of the bud and provides boundary constraints which will play an important part in the eventual evaluation of internal growth mechanisms.     

Expression of TGFβ1/β3 during early chick embryo development

We have used an antibody against a TGFβ peptide fragment to localize this growth factor in the early chick embryo from laying to the ten-somite stage of development. Western blotting showed that the antibody reacted with both mammalian TGFβ1 and chicken TGFβ3. By immunocytochemistry we find that at the earliest developmental stage (stage X of Eyal-Giladi and Kochav) immunoreactivity to this antibody is primarily located in the cells of the area opaca and marginal zone, as well as in the most peripheral edge cells of the blastoderm. The yolk is non-reactive, except in a highly localized region subjacent to the edge cells. This pattern persists at stage XII, and at both stages individual isolated cells in the epiblast and hypoblast are also reactive. By the time to gastrulation, reactivity in the epiblast is polarized to the ventral extremity of the cells, and again some isolated cells in this layer are intensely immunoreactive. At this stage also, the endoderm cells, particularly those underlying the primitive streak, are positive, as are the mesoderm cells lateral to the streak. At somite stages, the neuroepithelium is not reactive but the ectoderm lateral to it is strongly positive. At the caudal primitive streak levels of early somite embryos, the ectoderm and endoderm are immunoreactive while the mesoderm loses the reactivity it showed at the early gastrulation stages. The neuroepithelial cells later show reactivity at their apical poles, and, as at the earlier stages, individual cells show intense labelling. These results indicate that TGFβ1 and/or TGFβ3 immunoreactivity is developmentally regulated from very early stages of morphogenesis in the chick, and together with data from earlier functional studies, suggest that this factor has roles in embryonic axis formation and in blastoderm expansion. © 1994 Wiley-Liss, Inc.