Experimental reversal of polarity in chick embryo epiblast sheets in vitro.
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Animals↗
Biomedical subjects
C D Stern
Publications and source records attributed to C D Stern.
Cell-substrate contacts in cultured chick embryonic cells: an interference reflection study.
Cell-substrate contacts in explants of different regions of early chick tissues were investigated using the technique of interference reflection microscopy. All the explants spread as epithelial sheets. During initial spreading a peripheral zone of 2-3 cells formed broad contacts with the substrate. In spread explants some cells in the centre made broad substrate contacts. A mat of extracellular material containing fibronectin was found under the explants. Focal contacts and focal adhesions increased in number during culture, and stress fibres were associated with them. These changes in cell contacts appeared more quickly in some tissues than in others. After 24 h, explants of hypoblast and definitive endoblast could easily be distinguished but by 7 days they were very similar. In the absence of serum, specialized cell contacts developed more quickly; in higher concentrations of serum, more slowly. Confrontations between explants were also examined. The most conspicuous feature was that cells in invading explants normally underlapped invaded cells. Invasion from above by an unspread explant could occur even if the invaded explant had formed many focal adhesions.
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An integrated experimental study of endoderm formation in avian embryos.
The formation of the endoderm during primitive streak stages in avian embryos was studied by combining several of the following techniques for each embryo. These included microsurgery, time-lapse filming, use of chick-quail chimaeras, tritiated thymidine autoradiography and a novel technique for identifying the morphology of the cells after small pieces of tissue from known areas had been maintained in culture for 24 h. Using these techniques we have confirmed that the ventral layer of the early chick embryo receives contributions from both the marginal and the central regions of the area pellucida. The former seems to consist of yolky cells derived from the germ wall, whilst the latter consists of smaller, less yolky cells derived from the more dorsal layers of the embryo. The movement of the lower layer anteriorly during these stages appears to be dependent upon mechanical constraints imposed upon it by the expanding tissue in more caudal regions. The extent of each of the two contributions to the lower layer was determined as a function of stage and presence or absence of a lower layer, and the findings are discussed in the light of the existing literature.
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Behaviour and motility of cultured chick mesoderm cells in steady electrical fields.
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Animals↗
The behaviour of embryonic chick and quail tissues in culture.
Pieces of tissue were dissected from early chick and quail embryos (Stages XIII and XIV of Eyal-Giladi & Kochav, 1976; and stages 3-5 of Hamburger & Hamilton, 1951). These tissues were taken from three different regions of the early embryos, and from eight different regions of the older ones, and were derived mainly from the lower layer. Epiblast tissues were also used. The experiments were designed to test the ability of one tissue to penetrate another. A single tissue was grown in culture in a Falcon dish for 18-24 h until it had formed a coherent sheet of cells (Explant I). A second tissue was then combined with it in one of two ways: (a) A small piece of tissue (Explant II) was explanted on top of Explant I. In most cases Explant II penetrated through Explant I and spread on the Falcon dish. (b) Another small piece of tissue (Explant III) was explanted beside (in confrontation with) Explant I. Usually, Explant III penetrated into Explant I rather than vice versa. The results were analysed to see if there were any variations in behaviour of the different tissues. The main result was that important differences were found to exist between certain types of chick and quail cells when grown in culture; the implications of this finding for the widely used technique of xenoplastic grafting are mentioned. Another result was that Explant I was more likely to be penetrated when the second tissue was placed on top of it (Explant II) than when it was confronted with it (Explant III). The significance of these results is discussed.
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Strong electrical currents leave the primitive streak of chick embryos.
The electrical fields above chick embryos were explored with a vibrating probe. These fields indicate that steady currents with exit densities of the order of 100 microamperes per square centimeter leave the whole streak and return elsewhere through the epiblast. The epicenter of these strong exit currents lies near Hensen's node. They are probably pumped into the intraembryonic space by the epiblast and then leak out of the streak because it is a zone of junctional disruption.
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A re-examination of mitotic activity in the early chick embryo.
As a result of extensive mitotic index analysis in colchicine-arrested chick embryos during gastrulation, it was ascertained that the primitive streak is a region of elevated mitotic index as compared to the surrounding tissue. Along the cephalo-caudal axis, the embryo displays two large peaks of mitotic index, one at the posterior end of the primitive streak and the other just anterior to Hensen's node. The length of the various phases of the mitotic period was determined in vitro by time-lapse filming, and the colchicine-arrested mitotic indices in vivo and in vitro were determined and compared for various regions. Some observations regarding the orientation of mitotic spindles and abnormal mitosis in vitro are also included, and the relevance of the above observations to early embryonic development is discussed.
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Waves and periodic events during primitive streak formation in the chick.
Morphogenetic movements occurring during formation of the primitive streak in the chick embryo are of a periodic nature, with a mean frequency of one pulse every 2.6 min. The period of the oscillatory movement is shown to be temperature-dependent. The onset of these pulses of movement can be seen as a slow wave starting at the posterior end of the embryo and making its way towards the anterior end. An interpretation of this behaviour is discussed.
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Segmentation in the vertebrate nervous system.
Although there is good evidence that growing axons can be guided by specific cues during the development of the vertebrate peripheral nervous system, little is known about the cellular mechanisms involved. We describe here an example where axons make a clear choice between two neighbouring groups of cells. Zinc iodide-osmium tetroxide staining of chick embryos reveals that motor and sensory axons grow from the neural tube region through the anterior (rostral) half of each successive somite. 180 degrees antero-posterior rotation of a portion of the neural tube relative to the somites does not alter this relationship, showing that neural segmentation is not intrinsic to the neural tube. Furthermore, if the somitic mesoderm is rotated 180 degrees about an antero-posterior axis, before somite segmentation, axons grow through the posterior (original anterior) half of each somite. Some difference therefore exists between anterior and posterior cells of the somite, undisturbed by rotation, which determines the position of axon outgrowth. It is widespread among the various vertebrate classes.
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Evolution of vertebrate forebrain development: how many different mechanisms?
Over the past 50 years and more, many models have been proposed to explain how the nervous system is initially induced and how it becomes subdivided into gross regions such as forebrain, midbrain, hindbrain and spinal cord. Among these models is the 2-signal model of Nieuwkoop & Nigtevecht (1954), who suggested that an initial signal ('activation') from the organiser both neuralises and specifies the forebrain, while later signals ('transformation') from the same region progressively caudalise portions of this initial territory. An opposing idea emerged from the work of Otto Mangold (1933) and other members of the Spemann laboratory: 2 or more distinct organisers, emitting different signals, were proposed to be responsible for inducing the head, trunk and tail regions. Since then, evidence has accumulated that supports one or the other model, but it has been very difficult to distinguish between them. Recently, a considerable body of work from mouse embryos has been interpreted as favouring the latter model, and as suggesting that a 'head organiser', required for the induction of the forebrain, is spatially separate from the classic organiser (Hensen's node). An extraembryonic tissue, the 'anterior visceral endoderm' (AVE), was proposed to be the source of forebrain-inducing signals. It is difficult to find tissues that are directly equivalent embryologically or functionally to the AVE in other vertebrates, which led some (e.g. Kessel, 1998) to propose that mammals have evolved a new way of patterning the head. We will present evidence from the chick embryo showing that the hypoblast is embryologically and functionally equivalent to the mouse AVE. Like the latter, the hypoblast also plays a role in head development. However, it does not act like a true organiser. It induces pre-neural and pre-forebrain markers, but only transiently. Further development of neural and forebrain phenotypes requires additional signals not provided by the hypoblast. In addition, the hypoblast plays a role in directing cell movements in the adjacent epiblast. These movements distance the future forebrain territory from the developing organiser (Hensen's node), and we suggest that this is a mechanism to protect the forebrain from caudalising signals from the node. These mechanisms are consistent with all the findings obtained from the mouse to date. We conclude that the mechanisms responsible for setting up the forebrain and more caudal regions of the nervous system are probably similar among different classes of higher vertebrates. Moreover, while reconciling the two main models, our findings provide stronger support for Nieuwkoop's ideas than for the concept of multiple organisers, each inducing a distinct region of the CNS.
Amphibians↗
Analysis of neural crest cell lineage and migration.
In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.
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