Cell surface alterations in embryonic tissues exposed to RGD-peptides: selective expression.

Whole animal studies have implicated cell adhesion molecules in a diverse array of developmental processes. The present study reports on the morphological effects of RGD-related peptides on the cell surface of various living chick embryonic tissues. We report a novel and characteristic plasma membrane reaction that is caused by treatment with different RGD-peptides. Not only does each peptide evoke a response in certain tissues and not in others, but each brings about a specific type of plasma membrane reaction (bleb). Although the mechanisms are unknown, the specificity of this phenomenon suggests that it could provide a window into new surface interactions in morphogenetic systems.

The primitive streak.

The emphasis of this review is on the primitive streak of the chick embryo, collated with such information as is available on the mouse embryo. Little modern work has been published on any reptile primitive streak. The following topics are considered: evolutionary significance; formation of the primitive streak; ingression and de-epithelialisation; the basal lamina; migration from the primitive streak of the endoderm and mesoderm; the role of the extracellular matrix; changes in cell adhesiveness; regression of the primitive streak and its role in body patterning; the primitive streak and induction.

Early mesoderm differentiation in the chick embryo.

Two groups of experiments were carried out. In the first group, grafts of quail mesoderm whose presumptive fate was to form somites or heart tissues, were taken from quail embryos (stage 4-5 of Hamburger and Hamilton 1951) and inserted beneath the ectoderm of chick embryos of stage 3-4 immediately lateral to the primitive streak. Whilst most grafts contributed to the somites and or the heart, 22 out of a total of 46 were found to have contributed also to the pharyngeal endoderm. Although three of these grafts were known to have included some quail endoderm cells, the remainder were considered to consist of mesoderm alone. It is concluded that mesoderm at the primitive streak stages is still capable of forming endoderm. In the second group of experiments, grafts of quail somites (stage 10-14) were inserted beneath the ectoderm of chick embryos of stage 3-4. In 18 out of 23 cases the graft cells were found in somitic tissue, but they were also found in the endoderm (4 specimens), lateral plate (3 specimens) and endothelium (4 specimens). It is concluded that even at stages 10-14, the somite-derived cells are still not completely determined to form somite derivatives. In those cases where the grafted somites differentiated further, sclerotome cells which migrated from them did not necessarily move towards the host notochord.

Differentiation of the neural plate and neural tube in the young chick embryo. A study by scanning and transmission electron microscopy.

The differentiation of the presumptive neural plate, the neural plate and the neural tube have been investigated in the chick embryo by SEM, TEM and histochemical techniques. The relationship of these tissues to neighbouring structures, including extracellular materials, has also been studied. When SEM micrographs of primitive streak stage embryos were examined in stereo, it was found that cells which had been invaginating at the time of fixation were similar in shape to fibroblasts migrating in vitro. It was concluded that SEM stereo pairs could provide evidence about the mode and direction of cell migration. Many more mid-bodies have been found associated with the developing neural tissue than with the lateral ectoderm. It was found possible to recognise mid-bodies not only by TEM but also by SEM. It is therefore proposed that SEM montages may be used for assessing which regions of a tissue have recently undergone extensive mitosis. The beads on the specialised threads seen in the early stages of development are now considered to be formed from mid-bodies. Similar, but unbeaded threads have been described which span the gap between the neural folds just prior to the dorsal closure of the neural tube and it seems probably that these threads help to close the neural tube. It is suggested that the beaded threads arise by incomplete separation of two daughter cells at mitosis, whereas the unbeaded threads form by outgrowth of cell processes.

Placodes of the chick embryo studied by SEM.

The otic, the lens and the nasal placodes have been examined in chick embryos between stages 10 and 18 of Hamburger and Hamilton. At the stage when each placode first becomes visible conspicuous differences have been seen in the surface morphology between those cells which will invaginate and form the placode and those which will remain on the surface of the head, forming the epidermis. The differences become more pronounced with increasing development. The placode cells possess many surface projections whilst the epidermal cells do not. These differences in surface morphology are related to other differences which are visible in TEM sections, the placode cells being highly columnar and extending the full depth of the placode, whilst the epidermal cells are cuboidal or even squamous. This modification in cell shape of the placode cells is correlated with the presence of longitudinally orientated microtubules. The mechanism of invagination is discussed and evidence is presented which supports the idea that there is a migration of cells into the placode from one side. Such a phenomenon would help to explain the asymmetrical structure of the placode, including the presence of the overhanging lip.

Somitomeres in the chick tail bud: an SEM study.

In the chick embryo the final number of somites is achieved at about stage 22 of Hamburger and Hamilton. By this time the neural tube and notochord have reached the tip of the tail bud but some paraxial mesoderm remains unsegmented. In this study using scanning electron microscopy we show that somitomeres are present in this mesoderm. This indicates that the terminal paraxial mesoderm of the tail bud may have the potential to form supplementary somites, though we do not as yet know what prohibits the completion of segmentation to the tip.

Mitosis and cell death in the tail of the chick embryo.

Although somites develop from the mesoderm in the tail of the chick embryo, they do not form to the tip of the tail. Previous work has shown that this terminal mesoderm possesses many of the characteristics of the segmental plate mesoderm which gives rise to the somites in the trunk. This investigation is aimed therefore at understanding why the terminal mesoderm fails to form somites. Mitotic and pyknotic rates have been obtained for the tail region of chick embryos between stages 13 and 27. Embryos were treated with colchicine, so that the mitoses were blocked in metaphase, and counts were made on serial sections. The overall mitotic rates were highest between stages 15 and 18. Regions of high mitotic rate, which are an indication of cell synchrony, were found in the tail bud mesoderm though not in a consistent location, and only infrequently near the anterior end of the tail segmental plate. In the trunk however (Stern and Bellairs 1984) a single peak of cell synchrony was routinely found near the cranial end of the segmental plate. It is concluded that the cells of the tail mesoderm are less synchronised in preparation for somitogenesis than are the corresponding mesoderm cells in the trunk. A further conclusion is that the tail bud is not per se a region of high proliferation, though there are patches of high mitotic rate. The overall pyknotic rate reached a maximum at stage 25; peaks of pyknosis corresponded initially with the mitotic peaks and were associated with the ventral ectodermal ridge and the tail gut. By stage 25 however, the high levels of cell death were restricted mainly to the tip of the tail.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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 code named 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.

Mitotic activity during somite segmentation in the early chick embryo.

The mitotic activity of the somites, segmental plate and posterior mesoderm were investigated in colchicine-treated and untreated chick embryos at st. 7-14. The mitotic figures in the somites are restricted to the proximity of the lumen and have their spindles orientated predominantly tangentially to the cavity. In the segmental plate there is no pattern in terms of the position or orientation of the mitotic spindles, but there is a single region, often found close to the cranial end of the segmental plate, with an elevated mitotic index. This may indicate a certain degree of synchrony among groups of segmental plate cells. These results are discussed in relation to the process of somite segmentation.

Is chemotaxis a factor in the migration of precardiac mesoderm in the chick?

The chick heart is formed from bilateral patches of presumptive cardiac mesoderm cells which migrate over the endoderm and fuse in the midline. We have tested the possibility that this migration is controlled, at least in part, by a chemotactic substance exuded by the anterior end of the endoderm. We have used chick/quail combinations to follow naturally marked cells during the course of their migration. Chimaeric embryos were formed by fusing together parts of chick and quail embryos of stage 5-6. Each embryo possessed two pairs of precardiac regions, the quail pair lying immediately anterior to that of the chick. These chimaeras were then explanted in embryo culture. In the event of chemotaxis, cells from the posterior end of the quail precardiac mesoderm might be expected to invade the chick area. Samples of explants and chimaeras were examined at intervals from 2 to 24 h, but in no case were cells found to have changed their direction of migration as a result of the proximity of anterior endoderm. It is concluded that this work does not provide evidence for a chemotactic attraction by the anterior end of the endoderm.

An experimental and morphological analysis of the tail bud mesenchyme of the chick embryo.

In the chick embryo, the tail bud reaches its maximum length at about stage 22 of Hamburger and Hamilton, after which it starts to regress. By this stage the neural tube and notochord extend right to the tip of the tail, but the somites do not do so, the terminal tail bud mesoderm never becoming segmented. The investigation is concerned with analysing why this mesoderm fails to segment. When tail buds were explanted to the chorio-allantoic membrane, they continued to form somites only until the "correct" number had segmented, i.e., the tail bud formed no more somites when isolated from the embryo than it would have formed if undisturbed. Morphological studies suggest that in the normal embryo massive cell death overtakes the tail bud mesoderm before it can segment. It is suggested therefore that cell death may be a contributory factor in preventing segmentation.

Evidence for the involvement of receptors for fibronectin in the promotion of chick tail segmentation.

In the chick embryo the paraxial mesoderm forms about 50-53 pairs of somites, the precise number depending on the extent to which segmentation proceeds along the tail. However, the terminal mesoderm of the tail fails to segment despite the fact that it appears to contain a reservoir of potential somites. Why does this mesoderm not segment? Some clues can be obtained by comparing this non-segmenting region with the segmental plate in the trunk. We and others have shown that in the trunk region of the chick, cell adhesion plays a major role in somitogenesis and that this increased cell adhesion is associated with compaction of segments of mesoderm immediately prior to segmentation. This compaction can be brought about prematurely by fibronectin and by the specific adhesion peptide GRGDS. The terminal mesoderm in the tail resembles the segmental plate mesoderm in the trunk in undergoing compaction in response to fibronectin and GRGDS. The tail mesoderm differs from the segmental plate mesoderm in that it can also respond to peptides closely related to GRGDS. The response suggests that, whereas the integrin receptors for fibronectin and GRGDS appear to be specific in the presomitic trunk mesoderm, responding only to the specific adhesion-peptide GRGDS, the tail mesoderm may contain more heterogeneous sets of receptors within the integrin/VLA family that respond to a wider variety of ligands. Coincident with these differences is the phenomenon of regional cell death in the tail bud mesoderm. All of these factors are thought to play a role in the extent of segmentation in the paraxial mesoderm of the embryonic chick.

Endpoints in the development of chick embryos.

Although the use of mammalian embryos must remain the ideal for most teratological studies, non-mammalian embryos offer certain advantages. Their independence from the mother makes it possible to study the effects of reagents directly on the embryo without having to take possible placental effects into consideration, and it enables manipulations to be carried out more easily. In the early embryonic stages the cells of Xenopus and the chick are larger than those of the mouse, and these non-mammalian embryos tend to be bigger than those of the mouse at comparable stages of development. Attention is focused on the chick embryo as a useful experimental animal in the study of teratological effects. Consideration is given to some of the critical stages in embryogenesis as well as to the culture techniques available.

Experiments on embryos in space: an overview.

The question is, "Does gravity play an essential role in the normal development of an embryo?". Experiments on Earth which have disturbed the position of the embryo relative to the gravitational force, have implied that it does. But the critical tests are those in which the embryo is maintained in conditions of microgravity. The problems, both practical and conceptual, in conducting these experiments in Space, are considered, together with a brief discussion of selected achievements to date and a look at the problems to be tackled in the future.

Fertilization and early embryonic development in poultry.

There are two major controllers of development in the early stages of bird embryos. These are: 1) gravity, probably acting through the distribution of yolk and its components, which lays down the initial plans for polarity that are later established firmly through the genes; and 2) the primitive streak, which controls the orderly ingression of the cells and imposes a pattern on the developing tissues.

The mechanism of somite segmentation in the chick embryo.

The segmentation of somites in the chick embryo has been studied by transmission and scanning electron microscopy (stages 8-14). The segmental plate mesoderm consists of loosely arranged mesenchymal cells, whereas the newly formed somites are composed of elongated, spindle-shaped cells arranged radially around a lumen, the myocoele. The diamter of each somite is thus two cells plus the myocoele. Two major factors appear to be responsible for the change in cell shape at segmentation: (1) Each prospective somite cell becomes anchored at one end to the adjacent epithelia (i.e. the neural tube, the notochord, the ectoderm, the endoderm or the aorta) by means of collagen fibrils. These fibrils are already present in the segmental plate before the somites begin to form. (2) A change in cell-to-cell adhesiveness causes the free ends of these cells to adhere to one another. (Bellairs, Curtis & Sanders, 1978). This adhesion is then supplemented by the development of tight junctions proximally in the somite. Because it is anchored at both ends, each somite cell is under tension in much the same way as a fibroblast cell in tissue culture is under tension. Each somite cell therefore becomes elongated and the somite as a whole accommodates its general shape to that of the space available between the adjacent tissues. The arrangement of the cells in the more differentiated somites (stages 17-18) has also been examined and it has been found that the chick resembles Xenopus in that the myotome cells undergo rotation and become orientated in an anteroposterior direction.

An experimental analysis of somite segmentation in the chick embryo.

In a previous paper it was suggested that collagen fibrils play an important role in the process of somite segmentation. This paper was designed mainly to test that concept. In one series of experiments, embryos were treated with either alpha, alpha'-dipyridyl or L-azetidine-2-carboxylic acid, which are analogues that interfere with the formation of normal collagen. The reagents led to a reduction in the numbers of somites that formed, as well as to the production of other anomalies such as overall diminution in size and retardation. The older the embryo at the time of treatment, the further posteriorly were the major anomalies located. It is concluded that these results lend some support to the concept. In a second series of experiments an incision was made along one side of the neural tube and notochord to separate it from the segmental plate on one side. The result was that many more somites formed on the unoperated (control) side of the embryo than on the operated side. It is concluded that these results also lend support to the concept; but that they are of interest also in relation to the mechanisms involved in the control of somite numbers. In a third group of experiments, attempts were made to obtain somites in the absence of endoderm. Although this was not possible using surgery, it was achieved by treating the young embryos with U.V. irradiation. It was concluded that the presence of endoderm is not essential for the segmentation of mesoderm.

Midbodies and beaded threads.

When the dorsal surface of the young chick embryo is examined by SEM, long threads are visible, each of which appears to connect pairs of cells; these cells may be separated from each other by several intervening cells. Many of the threads possess a bead-like structure about half way along their length. When sections of the beads are examined by TEM they are found to resemble midbodies. Furthermore, the threads possess longitudinally arranged structures within them, which are probably the remnants of the microtubules which were part of a mitotic spindle. It is concluded that each bead is a midbody and that each beaded thread is the remains of a telophase bridge connecting two daughter cells which were incompletely separated after mitosis had taken place. The possible function of the beaded threads is considered.

Experimental analysis of control mechanisms in somite segmentation in avian embryos. I. Reduction of material at the blastula stage in Coturnix coturnix japonica.

The blastulae of unincubated eggs of the quail, Coturnix coturnix japonica, have been bisected in ovo, using the technique of Lutz (1949). Some embryos were harvested after 24 h and found to possess two primitive streaks. Most were fixed at 48 h or 72 h. Some were found to have regulated to form almost normal single axes, whilst others had developed into duplicitas anterior embryos, separate twins or collided axes. All three types of twinned embryos were smaller than the control embryos. The number of somites was not however reduced in the shorter embryos. This finding corresponds with a similar result obtained by Cooke (1975) who reported that if a Xenopus blastula is reduced in size, it nevertheless develops the correct number of somites. The quail however adjusts the shape of the individual somites so that they fit into the reduced body length, whereas Xenopus reduces the size of somites. No miniaturized somites were ever seen in these quail embryos. As a result of the present experiments, it was concluded that the length of incubation time does not directly control the rate of somite formation, because different numbers of somites were found in twins which possessed identical genomes and had developed in almost the same environment for identical periods. In addition, the size of the area pellucida does not appear to control somite formation. Probably, the most important influence is the regression of the node.