Interaction between epithelial basement membrane and migrating mesoblast cells in the avian blastoderm

We investigated the remodeling of glucosamine-containing basement-membrane components in chimaeric avian embryos during gastrulation. Epiblast grafts metabolically labelled with tritiated glucosamine were excised from gastrulating quail embryos and implanted orthotopically into chicken embryos at the same developmental stage. The chimaerae were allowed to develop in culture for 5-7 h before autoradiographic processing. The resulting autoradiographs not only showed the presence of silver grains in the grafted quail tissue and at the level of its basement membrane, but also revealed labelling in the basement-membrane region of the chicken tissue lateral to the graft, i.e. between the mesoblast and epiblast. This last labelling extended as far as at the edge of the area pellucida, i.e. in a region of chicken tissue situated more laterally than the initial position of the graft. No labelling was observed medial, anterior, or posterior to the graft. This observation argues against the interpretation that our results were due to diffusion of labelled compounds within the basement membrane. We also provide evidence to exclude the possibility that quail epiblast cells migrated on their own underlying basement membrane, leaving behind a carpet of labelled material. Taking into account, firstly, the morphogenetic movements that occur during gastrulation, i.e. the movement of epiblast cells towards the primitive streak where they ingress, and the migration of mesoblast cells along the basement membrane towards the periphery of the area pellucida, and secondly, the medial movement of the basement membrane, it is suggested that mesoblast cells picked up labelled compounds in the basement membrane of the graft and left these behind during their lateral migration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic evidence for the sliding of the upper layer over the basement membrane in chicken blastoderms during gastrulation.

An upper layer (epiblast) fragment taken laterally from the Anlage fields of neural plate or chordamesoderm of a quail blastoderm, labelled with 3H-glucosamine, was grafted isotopically (in a similar region), isochronically (at the similar stage of development) and isotropically (with the same caudocranial and dorsoventral polarity) in the epiblast of a mesoblast free area of a chicken blastoderm (St 4-5 Vakaet, 1970: full grown primitive streak). On the autoradiographs of the sections through such cultured blastoderms with fully integrated quail grafts, we observed a labelling of the basement membrane laterally and slightly cranially from the labelled graft in its final position. Since only the epiblast and its basement membrane are involved, the pattern of the observed labelling indicates that the grafted and integrated quail epiblast fragment glides in toto over the mediocaudally localized basement membrane, leaving behind a track of radioactivity. Sliding of whole groups of epiblast cells over the basement membrane seems thus to be a normal phenomenon during avian gastrulation.     

Behaviour of dissociated hypoblast cells on the basal lamina and on extracellular fibrils in the gastrulating chicken embryo.

The spreading behaviour of dissociated hypoblast cells on and besides a band of aligned fibrils associated with the basal lamina of the epiblast was investigated by the use of scanning electron microscopy. A horse-shaped band of aligned fibrils, first demonstrated by Wakely and England (1979), is present during the gastrulation stages of chicken embryos on the ventral side of the epiblast at the cranial and lateral borders of the area pellucida. The basal lamina of the area pellucida situated inside the fibrillar band enables the spreading and probably the locomotion of dissociated cells, which appeared as polarized cells. Numerous cells were also found on the fibrillar band, and these cells lacked distinct lamellae and a polarized shape. Extensions of the cells contacted the extracellular fibrils and, at these sites of contact, the pattern of the fibrils was frequently deformed. From these observations and from previous results emerged the concept that spreading and locomotion of dissociated hypoblast cells, as well as single mesoblast cells and healing hypoblast epithelium, are inhibited by the band of extracellular fibrils, which acts as a physical barrier. The cell biological basis of the mechanism by which extracellular fibrils associated with the basal lamina arrest the migration of hypoblast and mesoblast cells, but guide the migration of primordial germ cells, is discussed.     

Demonstration of the interaction between glycosaminoglycans and fibronectin in the basement membrane of the living chicken embryo

Summary A method utilizing microinjection of glycosaminoglycan-degrading enzymes in the chicken blastoderm prior to embryo culture and immunostaining for fibronectin have been applied to demonstrate an interaction between glycosaminoglycans and fibronectin in the basement membrane of the epiblast. Fixation of tissue in a mixture of formaldehyde and cetylpyridinium chloride allows detection of fibronectin only in those zones of the embryo that are not colonized by mesoblast cells. The epithelial-mesenchymal interface thus remains unstained. After degradation of glycosaminoglycans in the living organism, it is shown that this particular site, in fact, also contains fibronectin that is masked in vivo by, at least, hyaluronate. This interaction between both compounds is, during gastrulation, constantly correlated with mesoblast migration. Since previous studies have shown that the degradation of hyaluronate determines the behaviour of mesoblast cells, it is proposed that remodelling of the interaction between these compounds is necessary for mesoblast migration to occur.     

Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

Induction of the primitive streak in the chick blastoderm embryo: patterns of protein synthesis

Summary Induction of the primitive streak is correlated with specific qualitative and quantitative changes in protein synthesis in the component areas of chick blastoderm. Blastoderm embryos at the initial to intermediate primitive streak stage were labeled with L-[³⁵S] methionine. Radioactively labeled proteins separated by two-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed differences in the number and density of spots among the component areas of the epiblast and hypoblast. Protein patterns of the area opaca, marginal zone and central area of the epiblast are very similar qualitatively but show distinct quantitative differences. A comparison between any of the component areas of the epiblast and the hypoblast in chick blastoderm embryos, however, reveals both qualitative and quantitative differences. A protein with a molecular weight of 30,000 unique to the component areas of the epiblast, and proteins with a molecular weight of 22,000 and 37,000 unique to the hypoblast are prominent and seem to be related to the initial appearance of the primitive streak.     

Immunohistochemical analysis of the segregation process of the quail germ cell lineage

An antiserum against quail 7 day gonadal germ cells was found to react specifically with gonadal germ cells of both sexes. Transverse sections from a range of early quail developmental stages were submitted to the antibody PAP reaction. Blastodiscs from the earliest uterine stages (II to X E.G. & K) reacted very strongly, while the overall reaction gradually decreased in older blastoderms. At stage XIII both epiblast and hypoblast were weakly stained, but some large, PGC-like cells stained intensively. During gastrulation (PS formation) the reaction of the epiblast disappears quicker than that of the hypoblast. The newly formed mesoderm and entoderm do not react at all and the reaction gradually becomes limited mainly to the PGCs and somewhat to the primary hypoblast which is moving into the germinal crescent. The widely spread reaction at the early stages is thus gradually being restricted to the PGCs.     

Monensin inhibits the first cellular movements in early chick embryo

Summary In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.     

N-glycosylated proteins interfere with the first cellular migrations in early chick embryo.

Cell adhesion and migration properties which are known to play a crucial role in developmental events seem to be modulated by variations in glycosylation of glycoproteins. In the chick embryo, the extracellular matrix (ECM) appears as a loose meshwork of fibrillar material in the space between the epiblast and the hypoblast shortly before the first major cell migrations start. Chick embryos treated with tunicamycin (TN), a specific inhibitor of N-linked glycosylation of proteins, show little or no ECM, diminished cell adhesion and a dramatic alteration in the architecture of the epiblast and of the hypoblast. The first major cell migrations which signal the onset of PS and gastrula formation are inhibited irreversibly in these embryos. Tunicamycin induces a substantial change in the labeling pattern with change in mobility of some polypeptides and with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from these already present in the control blastoderm. The N-linked glycosylation of protein(s) that are synthesized during the interaction of the epiblast and of the hypoblast seem to play a critical role in cell adhesion and in the morphogenetic movements of gastrulation in the early chick embryo.     

Hypoblast cells of chick pre-streak stage embryos invaded basement membrane analogues in vitro: Implications for hypoblast layer formation

In early chick blastodermal morphogenesis, the hypoblast layer is organized beneath the epiblast and induces an axial structure. However, the origin of hypoblast cells and the mechanism of hypoblast layer formation are poorly understood. We hypothesized that the hypoblast layer is formed by an invasive process across the basement membrane of the juxtaposing epiblast, and tested the idea in vitro . Primary and secondary hypoblast cells from embryos at various pre-streak stages were dissociated into single cells and cultured on reconstituted basement membrane gel, laminin gel or fibronectin gel in the culture medium with or without serum for 24–48 h. As a result, we found that after 24 h of serum-supplemented culture, up to 35% of the hypoblast cells dissolved the gel and made holes on it. Similarly, up to 36% of the hypoblast cells showed invasiveness after 48 h in the serum-free culture. Furthermore, it was observed that Koller's sickle cells, which are regarded to be the progenitors of secondary hypoblast cells, penetrated those gels on which they were seeded. The posterior epiblast cells covering Koller's sickle were also invasive. These results suggest that the presumptive primary hypoblast cells that are known to mingle with epiblast cells invade through the basement membrane to form the hypoblast layer. Furthermore, the present results imply that invasion through the basement membrane may be involved in the formation of Koller's sickle, the anlage of secondary hypoblast.     

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp,Cyprinus carpio

Summary The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp. Correspondence to: P. Gevers     

The hypoblast (visceral endoderm): an evo-devo perspective

When amniotes appeared during evolution, embryos freed themselves from intracellular nutrition; development slowed, the mid-blastula transition was lost and maternal components became less important for polarity. Extra-embryonic tissues emerged to provide nutrition and other innovations. One such tissue, the hypoblast (visceral endoderm in mouse), acquired a role in fixing the body plan: it controls epiblast cell movements leading to primitive streak formation, generating bilateral symmetry. It also transiently induces expression of pre-neural markers in the epiblast, which also contributes to delay streak formation. After gastrulation, the hypoblast might protect prospective forebrain cells from caudalizing signals. These functions separate mesendodermal and neuroectodermal domains by protecting cells against being caught up in the movements of gastrulation.     

Intercellular bridges in vertebrate gastrulation

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.     

Dye-coupling and the formation and fate of the hypoblast in the teleost fish embryo, Barbus conchonius.

The present report describes Lucifer Yellow (LY) transfer between the syncytial layer of the yolk cell (YSL) and blastodermal cells during epiboly in the teleost fish Barbus conchonius. The fate of a group of labeled cells is described until germ layer formation. At the onset of epiboly, LY seems to be transferred from the YSL to all blastodermal cells. Between 10% and 40% epiboly, dye-coupling appears to be restricted to the marginal region. Within 60 min individually labeled cells are distributed among unlabeled cells within the blastoderm. Between 40% and 60% epiboly, we observed a ring-shaped group of labeled cells, which probably have involuted during early gastrulation. Consequently, this cell group may correlate with the leading edge of the hypoblast layer within the germ ring. At 60% epiboly and later, the blastodermal cells are dye-uncoupled from the YSL. A gradual translocation of the ring-shaped hypoblast towards a dorsally located bar-like structure is observed between 50% and 100% epiboly. At 100% epiboly, fluorescent cells were located in contact with the YSL within the embryo proper, with the brightest fluorescence in the future head region. The translocation is due to dorsalwards convergent cell movements during the gastrulation process. The appearance of the hypoblast as a dye-coupled cell layer may correlate with some restriction in cell fate since the hypoblast differs in fate from the epiblast.     

Histochemical demonstration of apoptotic cells in the chicken embryo using annexin V

This study describes the use of biotinylated annexin V for the histochemical detection of apoptotic cells in cultured chicken embryos during gastrulation. This method is based on the Ca2+-dependent binding of annexin V to phosphatidylserine, a negatively charged phospholipid, located at the inner leaflet of the cell membrane in living cells. However, in the early stages of apoptosis, phosphatidylserine is translocated to the outer layer of the cell membrane and can then be recognized by annexin V. Applying this method in cultured chicken embryos during gastrulation, we obtained labelling of apoptotic cells in the three germ layers. In the epiblast and mesoblast, labelling was predominantly present in the region lateral to the primitive streak. At the level of the germinal crescent, labelled cells were also found in the epiblast. Labelled cells in the deep layer, which is a heterogeneous tissue layer composed of endophyll, sickle endoblast and definitive endobl ast, were rather scarce. The distribution of cells, as observed in this study after labelling with annexin V in light microscopy and confocal laser scanning microscopy, is consistent with distributions reported by other authors using other approaches and with our previous observations made with the TUNEL technique and by electron microscopy after fixation in a tannic acid-based fixative. The main advantages of this method over other more sophisticated methods is its easiness and rapidity of execution and the fact that both early and late stages of apoptosis are detected. © 1998 Chapman & Hall     

Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin

Small groups of blastoderm cells were transplanted from wild-type donor embryos into genetically marked host embryos of the same age. Donor cells were injected either into an homologous or an ectopic region of the recipient, and both donor and recipient embryos were allowed to develop. Donor flies were examined for defects in external structures. Recipients were scored for patches of donor-type marked tissue derived from the injected cells. After ectopic transfer, the donor cells recovered in chimaeric recipients differentiated structures consistent with the donor site of cell removal. No apparent fate change was observed. In the rare cases when both individuals of a donor/host pair survived, a direct correspondence could be made between the deleted region in the donor and the chimaeric patch in the host. The results show that blastoderm cells are stably determined to within a segment.     

Intercellular contact in the unincubated chick embryo

Summary The unincubated chick blastoderm, which consists of a complete upper epithelial layer of one cell thickness (epiblast) and an incomplete lower layer (hypoblast), was examined with the electron microscope in order to define the types of cell contact present. The terminal contacts between the cells of the epiblast invariably involved several focal tight junctions, but only occasionally involved tight junctions. Desmosomes were not observed in these areas, but were encountered in various phases of development in the deeper contact regions between epiblast cells. This deeper region also showed sporadic focal tight junctions and frequent micropapillae. These micropapillae were also common on the surfaces of hypoblast cells. Intercellular spaces between epiblast and hypoblast cells and within the hypoblast were often wide, narrowing to occasional focal tight junctions. Tight junctions and desmosomes were not observed in association with hypoblast cells. Gap junctions were not observed in any region of the embryo.These observations are discussed in relation to the morphogenetic movements occurring in the forming hypoblast and also the influence of this layer on the subsequent development of the embryo. Comparisons are drawn between the contact morphology in the unincubated blastoderm and that in later stages of development.Supported by the Medical Research Council of Canada.