Endoderm regeneration in the chick embryo studied by SEM

Summary Regeneration of the area pellucida endoderm of the chick embryo was studied by scanning electron microscopy (SEM).A new endoderm was formed by in situ changes in the shape and relationships of mesoderm cells.Initially the cells flattened and lost their processes except along cell boundaries. Later even these processes were lost and an epithelium was formed. The area of regenerated endoderm coincided with the area of mesoderm at the time of endoderm removal, confirming the mesodermal origin of the new layer. Remnants of the original endoderm did not contribute to the regenerated layer. Contact inhibition was observed at the boundary between original and regenerated endoderms.     

Wound healing in the early chick embryo studied by scanning electron microscopy

Summary A simple incision was made in the early chick embryo (stages 3–5) area pellucida endoderm and its subsequent healing studied by scanning electron microscopy (SEM).Initially the wounded edges of the endoderm layer curl towards the ectoderm creating a gaping slit. The endoderm cells adjacent to the slit form large mounds probably in response to a loss of substrate and the trauma of the incision.Healing begins as the endoderm cells direct processes across the underlying cell layers and the two cut edges move towards one another. Many intervening mesoderm cells have cup-shaped processes.As the two endoderm edges meet in the corners of the wound, the wound outline changes to an oval shape.After 2 hours the wound outline is changed to a slit with the cut edges contacting in one or two areas. The cup-shaped mesoderm processes remain in the slit until the wound is healed primarily by endoderm cell movement.     

Scanning electron microscopy of the development of the mesoderm layer in chick embryos

Summary The distribution of mesoderm, the structure of mesoderm cells and relationship between mesoderm and ectoderm were examined by SEM in embryos at stages 3 to 5. The mesoderm was displayed by removal of the endoderm and by fracturing the embryos through mesoderm containing regions. Within the mesoderm layer four zones could be distinguished by their cell shape and arrangement — the primitive streak, a multilayered compact area around the margins of the area pellucida, multilayered and loosely arranged cells near the primitive streak and a flattened monolayer of cells around the advancing lateral and anterior edges of the mesoderm sheet. Secretion of basement membrane by the ectoderm was seen to precede the arrival of mesoderm cells. This suggests that ectoderm alone can synthesize basement membrane without mesodermal contribution.     

Natural wound formation: Endodermal responses in experimental primary neural induction in the chick embryo

Natural wound formation in experimental primary neural induction has been studied by SEM and in paraffin wax sections in embryos from 0 minutes to 10 hours of re-incubation. Stage 4 host and graft embryos were removed from hen's eggs and mounted as for New culture. Graft Hensen's nodes were transplanted into “pockets” created in the host area pellucida and re-incubated for up to 10 hours. Initially the cut edges of the graft establish contact with the host ectoderm layer. After 4 hours the cut edges of the graft move from the host ectoderm to the host endoderm layer. Several small openings form in the host endoderm over the graft tissue. By 6 hours, these openings join to form a single natural wound through which the underlying graft is exposed to the external environment. At 8 hours the graft forms a head-fold and neural folds are evident. During 8 to 10 hours of re-incubation the edges of the graft which attach to the edges of the host edoderm meet in the midline and close the opening in the host endoderm; simultaneously, the graft forms a neural tube. The endodermal wounds form by cell re-arrangement and by a minor contribution from cell loss. © 1993 Wiley-Liss, Inc.     

Scanning electron microscopy of developing primary endoderm during first 6 hours of incubation in the chick

Development of primary endoderm in the domestic fowl (Gallus domesticus) is described in scanning electron microscopy (SEM) supplemented by transmission electron microscopy (TEM). Although complicated by great variability, the ventral surface of the blastoderm reveals this process during the first 6 hours of incubation. Primary endoderm arises (1) from the hypoblast, (2) from the margin of the area pellucida, and (3) from intervening protions of the area pellucida. The early hypoblast becomes several cells thick while individual cells are still spherical. TEM reveals a variety of immature cell junctions. During subsequent flattening of these cells into primary endodermal epithelium, numerous filopodia arise from their surfaces. These are 0.20–0.25 μm in diameter. They become long and branched, attaching to each other and to other cell bodies. Similar filopodial processes are present less conspicuously among cells in the margin of the area pellucida. Here, there is pseudopodial evidence that cells or cell sheets creep along the ventral surface of the epiblast. The filopodia disappear as cell flattening proceeds. The ventral surface of the exposed epiblast delaminates cells that become free after their exploratory filopodia and lamellipodia are put forth. Lateral contacts among cell bodies from the above three sources increase until a continuous epithelium is formed. The primary endoderm of the embryo, a simple squamous epithelium that separates the connective tissue space above from the gastrocoele below, is generated by these developmental events. © Wiley-Liss, Inc.     

Placodes of the chick embryo studied by SEM

Summary 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.     

The effect of cytochalasin B on chick mesoderm cells as studied by scanning electron microscopy

Summary The effects of cytochalasin B (CCB) on chick mesoderm cells in vivo was examined by scanning electron microscopy (SEM). The embryos were mounted for New Culture and the mesoderm exposed by dissecting off the endoderm. Cytochalasin B was suspended in saline and the embryos flooded with the suspension. Control embryos were treated with saline alone. The embryos were reincubated for varying times at 37°C.In the treated embryos the mesoderm cells were rounded and separated from each other. Many had long branched processes and rough surfaces. These changes became more pronounced as treatment time was increased. They were also reversible on reincubating treated embryos in the absence of cytochalasin B. The morphological changes produced by CCB are thought to be due to an effect on the cytoskeleton, either a direct disruptive effect or detachment of skeletal microfilaments from the cell membrane. There may also be a direct removal of cell surface materials leading to the observed surface roughening of treated cells.     

Is extraembryonic angiogenesis in the chick embryo controlled by the endoderm?

Summary The area vasculosa of the chick embryo is subdivided into two concentric zones: the inner transparent area pellucida vasculosa (AVP) and the less transparent surrounding area opaca vasculosa (AOV). The different optical properties of these zones are caused by the different morphology of the endoderm, which consists of flat cells in the APV and of high-prismatic cells containing large yolk vacuoles in the AOV. The present study describes how this endodermal subdivision of the area vasculosa is related to the development of the extraembryonic vascular pattern. By injection of ink into the vascular system of chick embryos at stages 12 to 20 (Hamburger and Hamilton 1951 HH), it has been demonstrated that the vascular net of the area vasculosa from stage 14 (HH) onwards develops into different patterns in APV and AOV. The small loops of uniform capillary vessels of stage 13 (HH) are widened due to the rapid expansion of the extraembryonic mesoderm. In the AOV from stage 14 (HH) onwards numerous small blood vessels sprout into the enlarged intervascular spaces. This process is maximal at stage 17 (HH). In contrast, the blood vessels of the APV remain largely unbranched. These findings suggest that the development of the extraembryonic vascular pattern is controlled by the endodermal pattern. To test this hypothesis, both zones (APV and AOV) were examined by light microscopy, transmission and scanning electron microscopy, in vivo observations and by treatment with bromodeoxyuridine (BrdU). TEM examinations show that the ultrastructural organization of the APV mesoderm is different from that of the AOV: The splanchnopleuric cells of the APV form a continuous cover around the endothelial cells connected by numerous desmosomes, whereas the splanchnopleuric cells of the AOV are frequently separated by gaps. The largest gaps are seen in the small blood vessels at stage 17 (HH). These results should be considered in relation to the dynamic changes in the vascular pattern of the AOV. The endodermal cells of APV and AOV are two different populations. In vivo observation of the endodermal transition from APV to AOV detected no transformations of APV cells into AOV cells or vice versa. The borderline between the zones is stable.The AOV endoderm, having been overgrown by the expanding mesoderm, stops proliferating almost completely, whereas the proliferation of the APV endoderm is unaffected by contact with the mesoderm. The rate of its proliferation is approximately as high as that of the AOV prior to contact with the expanding mesoderm (results after treatment with BrdU). The contact of the basal side of the AOV endoerm with mesoderm is closer than that of the APV. In the AOV the basal compartments of endodermal cells show numerous small coated vesicles, probably exocytotic in nature.The transition between zones in the endoderm was found to be formed by small vaulted cells bearing microvilli on their surface. These cells probably are daughter cells of the primary hypoblast cells, which have been withdrawn to the margin of the APV by the invading endoblast.     

Scanning electron microscope study of the extracellular matrix between presumptive lens and presumptive retina of the chick embryo

Summary The three-dimensional architecture of the intercellular matrix contained in the interspace between the presumptive lens and optic vesicle of the chick embryo was examined by scanning electron microscopy. The fibrous structure of the basement membranes lining the space was demonstrated. The space was shown to be filled with a dense fibrous meshwork. The reaction of basement membranes and interspace contents to enzymic digestion is described. The functional significance of the arrangement of fibres in the interspace is discussed.     

Early mesoderm differentiation in the chick embryo

Summary 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.     

Morphogenetic processes involved in the remodeling of the tail region of the chick embryo

Summary Tail regions from chick embryos at three to ten days of incubation were examined by light and electron microscopy to determine what morphogenetic processes occur during transformation of the embryonic tail into the definitive type. The embryonic tail attained its maximum length (0.6 mm) between four and five days of incubation. Thereafter, the tip and base of the tail developed differently. The diameter of the tip of the tail decreased during four to six days of incubation, and many macrophages and presumptive necrotic cells appeared in this area. The tip of the tail then gradually increased in diameter during six to ten days of incubation, and changed in shape from conical to pyramidal. Only a few macrophages and presumptive necrotic cells were present during these latter stages. In most embryos during seven to ten days of incubation, the caudal end of the neural tube was cystic and small hemorrhagic zones were present in the ventral part of the tip of the tail. Near the end of this period, the ectoderm overlying the cystic portion of the neural tube often ruptured. The base of the tail increased gradually in breadth during all stages. Between six and ten days of incubation, as the leg buds elongated rapidly, the cranial part of the base of the tail became incorporated into the caudal part of the trunk. These results suggest that during morphogenesis of the tail region three morphogenetic events occur: differential growth, incorporation of part of the tail into the trunk, and cell death. The definitive tail of late incubation and posthatching stages is derived from mainly the distal portion of the base of the embryonic tail; the tip of the embryonic tail is mostly lost.     

A re-examination of mitotic activity in the early chick embryo

Summary 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.     

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.     

Edge cell migration in the extraembryonic mesoderm of the chick embryo

Summary The expansion of the extraembryonic mesoderm was investigated in chick embryos of 2 and 3 days incubation with special regard to the mesodermal edge cells. These cells are lying immediately distal to the sinus terminalis and have the shape of migrating cells. By SEM examination they appear to be linked together to form a uniform edge which extends numerous spike-like filopodia. The shape of these filopodia corresponds to their microtubule pattern, as shown by immunofluorescence staining. Filopodia contain strong bundles of microtubules. By in vivo observation at high magnification, the migration of edge cells was demonstrated, and the results of SEM and immunofluorescence studies could be confirmed. By local application of cytochalasin D, distal to the region of the sinus terminalis, the migration of edge cells was inhibited selectively. Subsequent to the inhibition of migration, the expansion of the mesoderm stopped although the interstitial growth of the mesoderm in drug-treated regions remained unaffected. Thus the edge cells have a promotor function in the expansive growth of the extraembryonic mesoderm. The proliferating mesoderm, located proximally to the edge cells, has no expansive tendency of its own. The selectivity of the cytochalasin effect was checked by examination of the phalloidin stained actin pattern. Furthermore, by in vivo observations at low magnification and by transplantation of endoderm from quail to chick it could be confirmed that the extraembryonic mesoderm spreads out invasively between ectoderm and endoderm separating the two sheets. The promotion of this invasion can be regarded as an additional function of the edge cells. An expansion of the mesoderm can also be observed after endoderm removal. In regions freed from endoderm the mesoderm expands faster than in adjacent regions still covered by endoderm. There is no promoting influence of endoderm on mesodermal expansion. On the contrary, expansion itself is facilitated, when the conditions for invasion are abolished by removing the endoderm.     

An integrated experimental study of endoderm formation in avian embryos

Summary 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.     

Glycoconjugate expression in the chick embryonic chorioallantoic membrane: Comparisons of the chorionic ectoderm and allantoic endoderm

Background: The chorioallantoic membrne (CAM) of the chick embryo expands during embryogenesis to meet the increased oxygen demands during growth and differentiation. Temporal and spatial glycosylation patterns of CAM ectodermal and endodermal proteins likely contribute to differentiation of the functional attributes of the CAM. Methods: Using lectins for light and electron microscopic observations, we studied the patterns of glycoconjugate expression on the ectoderm and endoderm of the chorioallantoic membrane (CAM) of the chick at days 4.5, 5.0, 6.0, and 10 of morphogenesis. For light microscopy, samples of unfixed CAM were incubated with the following FITC lectins: Con A, DBA, GSA-I, GSA-II, PNA, SBA, UEA-I, and WGA. Results: All lectins, except GSA-I and -II, gave positive results. The positive lectins, labeled with HRP, served to ultrastructurally localize PNA, SBA, and WGA, but not DBA binding to the luminal surface of the endoderm. UEA-I and Con A bound similarly except on day 10 when UEA-I no longer bound. On the ectodermal surface, only WGA bound at all times studied. PNA and SBA binding were present from days 5.0 to 6.0 but absent at days 4.5 and 10. DBA binding occurred through day 5.0 but was absent thereafter. UEA-I bound to the ectoderm at days 4.5, 5.0, and 10 but not days 5.5 and 6.0 Con A bound only on days 5.0 and 10. Conclusion: That the ultrastructurally similar ectoderm and endoderm of the CAM display functional differences conforms to the hypothesis that differential expression of glycoconjugate microdoains likely contributes to such functional specialization. © 1995 Wiley-Liss, Inc.     

Formation of extra-digits induced by surgical removal of the apical ectodermal ridge of the chick embryo leg bud in the stages previous to the onset of interdigital cell death

Summary In the present work we have studied the mechanism of formation and the possible morphogenetic significance of the process of ectopic chondrogenesis induced by surgical removal of AER of the interdigital spaces of the chick leg bud at stage 28–30 (Hurle and Gañan 1986). Our results show that ridge removal causes condensation and rounding of the underlaying mesenchymal cells followed by chondrogensis. The long-term study of the fate of these ectopic cartilages shows that in a high percentage of the cases the cartilages undergo morphogenesis taking by day 10 of incubation the appearance of the two distal phalanges of an extra-digit. These extra-digits lack tendons and are joined by thin interdigital membranes to the neighboring digits.     

A segmented pattern of cell death during development of the chick embryo

Summary During the early development of the chick embryo, specific groups of cells die in characteristic patterns. In this study, Nile Blue sulphate staining was used to reveal a novel pattern of segmentally repeated cell death in the paraxial mesoderm of the chick prior to stage 23. This pattern varies according to the developmental stage of the embryo and shifts rostrocaudally, corresponding to progressing somite differentiation. Initially, during early somite differentiation, cell death is restricted to the rostral half of the somite (the rostral pattern of cell death). After the somite has differentiated into dermomyotome and sclerotome, dead cells appear in superficial tissues in a pyramidal pattern which lies in register (rostrocaudally) with the central part of the sclerotome. Finally, small bands of dying cells are seen between the neural tube and the expanding sclerotome. This third pattern (the ventral path) lies in register with the rostral part of the caudal half of the sclerotome. We show by fluorescent labelling of the migrating neural crest that these patterns of cell death correspond to the routes of neural crest migration. In addition, serial sectioning of stage 23 chick embryos confirms that the position of dying cells correlates with the known routes of neural crest migration and with the sites of development of certain neural crest-derived tissues.     

Development of nucleolar apparatus in the chick primitive erythroid cells

The primitive erythroid line cells of chick embryos were studied during embryonic days 2–14 by means of a cytochemical method to investigate the appearance and frequency of the main nucleolar types. The populations of erythroblasts and erythrocytes were classified according to the presence of functionally dominant nucleoli in their nuclei. In the course of primitive erythroid cell differentiation and maturation, compact nucleoli and nucleoli with nucleolonemas (both supposed to be RNA biosynthetically active) were gradually replaced by ring-shaped nucleoli and finally by micronucleoli reflecting the reversible and irreversible inhibition of RNA synthesis, respectively. The occurence of the main nucleolar types and their values in primitive erythroid cells of the developing chick depend not only on the maturation stage of the blood cells, but also on the developmental stage of the chick embryo. In comparison with the definitive erythroid line of the post-hatching chick and hen, the cells of the chick embryonic primitive erythroid line possess relatively high values of active nucleolar types. These are still present in advanced maturation stages, and occur also as definitive erythroid lines of lower vertebrates.