scholarly journals Invasion of a basement membrane matrix by chick embryo primitive streak cells in vitro

1989 ◽  
Vol 92 (3) ◽  
pp. 497-504 ◽  
Author(s):  
E.J. Sanders ◽  
S. Prasad
Keyword(s):  
Chick Embryo ◽  
Basement Membrane ◽  
Primitive Streak ◽  
Lamina Densa ◽  
Invasive Potential ◽  
Tissue Space ◽  
Epithelial Sheet ◽  
Mesenchymal Transformation

At the time of gastrulation in the chick embryo the upper epiblast layer penetrates its own basement membrane at the primitive streak so that its cells may invade the underlying tissue space. In so forming the primary mesoderm, the cells undergo a concomitant epithelial-to-mesenchymal transformation. In this study, epiblast tissue has been explanted onto a basement membrane gel in order to examine its invasive potential. Fully ingressed primary mesoderm cells were able to penetrate the gel as individual cells, during the course of which the texture of the gel was disrupted. By contrast, epiblast tissue taken from the immediate vicinity of the primitive streak penetrated the gel, but only as a coherent tongue of cells and without gel disruption. These tongues of cells did not undergo the epithelial-to-mesenchymal transformation, and consequently spread as a epithelial sheet when replated on glass. Thus, the absence of gel disruption correlated with the failure of transformation, suggesting that these two events may be linked and that they may require in situ cell interactions for their manifestation. Tissue from the lateral epiblast failed to penetrate the gel. Instead, this tissue either spread on the gel surface or rounded up into a hollow sphere with the basal surface of the cells innermost. In the former case, despite the cell spreading, no lamina densa was organized beneath the sheet, but in the latter case polarity reversal occurred with the formation of a new lamina densa at the cell-gel interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between mesoderm cells and the extracellular matrix following gastrulation in the chick embryo

1991 ◽  
Vol 99 (2) ◽  
pp. 431-441
Author(s):  
A.J. Brown ◽  
E.J. Sanders
Keyword(s):  
Extracellular Matrix ◽  
Chick Embryo ◽  
Basement Membrane ◽  
Primitive Streak ◽  
Cell Binding ◽  
Fab Fragment ◽  
Fibronectin Receptor ◽  
In Vitro Methods

In the gastrulating chick embryo, the mesoderm cells arise from the epiblast layer by ingression through the linear accumulation of cells called the primitive streak. The mesoderm cells emerge from the streak with a fibroblastic morphology and proceed to move away from the mid-line of the embryo using, as a substratum, the basement membrane of the overlying epiblast and the extracellular matrix. We have investigated the roles of fibronectin and laminin as putative substrata for mesoderm cells using complementary in vivo and in vitro methods. We have microinjected agents into the tissue space adjacent to the primitive streak of living embryos and, after further incubation, we have examined the embryos for perturbation of the mesoderm tissue. These agents were: cell-binding regions from fibronectin (RGDS) and laminin (YIGSR), antibodies to these glycoproteins, and a Fab' fragment of the antibody to fibronectin. We find that RGDS, antibody to fibronectin, and the Fab' fragment cause a decrease in the number of mesoderm cells spread on the basement membrane, and a perturbation of cell shape suggesting locomotory impairment. No such influence was seen with YIGSR or antibodies to laminin. These results were extended using in vitro methods in which mesoderm cells were cultured in fibronectin-free medium on fibronectin or laminin in the presence of various agents. These agents were: RGDS; YIGSR; antibodies to fibronectin, fibronectin receptor, laminin and vitronectin; and a Fab' fragment of the fibronectin antiserum. We find that cell attachment and spreading on fibronectin is impaired by RGDS, antiserum to fibronectin, the Fab' fragment of fibronectin antiserum, and antiserum to fibronectin receptor. The results suggest that although the RGDS site in fibronectin is important, it is probably not the only fibronectin cell-binding site involved in mediating the behaviour of the mesoderm cells. Cells growing on laminin were perturbed by YIGSR, RGDS and antibodies to laminin, suggesting that mesoderm cells are able to recognise at least two sites in the laminin molecule. We conclude that the in vivo dependence of mesoderm cells on fibronectin is confirmed, but that although these cells have the ability to recognise sites in laminin as mediators of attachment and spreading, the in vivo role of this molecule in mesoderm morphogenesis is not yet certain.

scholarly journals Unco-ordinated contractions caused by egg white and by alternations in the cation ratio of the medium in the heart of the chick embryo in vitro

1934 ◽  
Vol 115 (794) ◽  
pp. 380-402 ◽  
Keyword(s):  
Chick Embryo ◽  
Small Amplitude ◽  
Culture Medium ◽  
Primitive Streak ◽  
Egg White ◽  
Posterior Half ◽  
Cation Ratio ◽  
Made In ◽  
Area Pellucida

In May, 1932, some experiments were made in which fragments of chick embroys in primitive streak stages were explanted into crude white of egg as culture medium. The object was to study hæmatopoiesis, which occured in these explants (Murray, 1933). Only two of the cultures interest us here. Both were derived from embryos having pear-shaped areæ pellucidæ with primitive streaks but no head processes and each consisted of that part of the area pellucida of one side which lies opposite the posterior half or three-quarters of the primitive streak. Both cultures survived in the egg white and in each there was discovered, after two and five days incubation respectively, an area which contained actively contracting cells. The contractions were of small amplitude and there was no co-ordination between the cells. This activity persisted in one culture for 36 days. It was necessary to have some name by which this anarchic contractility could be designated; it resembled fibrillation, at least superficially, but it was thought best to avoid this term as the present phenomenon might prove entirely unconnected with fibrillation. The word "twitter", used as noun and verb, described the appearance rather well, and I have adopted it as a provisional name for this kind of activity.

scholarly journals Assembly of the exogenous extracellular matrix during basement membrane formation by alveolar epithelial cells in vitro

2000 ◽  
Vol 113 (5) ◽  
pp. 859-868 ◽  
Author(s):  
A. Furuyama ◽  
K. Mochitate
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Alveolar Epithelial Cells ◽  
Coarse Mesh ◽  
Lamina Densa ◽  
Alveolar Epithelial ◽  
Type Iv ◽  
Laminin 1

We found that immortalized alveolar type II epithelial cells (SV40-T2 cells) that were cultured on dense fibrillar collagen supplemented with Matrigel gel formed a thin and continuous lamina densa beneath them. Immunohistochemical analysis of laminin-1, type IV collagen, entactin (nidogen) and perlecan in the culture indicated that all these components were integrated into a sheet structure of basement membrane beneath the cells. Analysis of the temporal and spatial distribution of the basement membrane macromolecules revealed that the initial deposits of laminin-1 and entactin were significantly greater in area in the presence of Matrigel. These globular deposits and the coarse mesh of basement membrane macromolecules developed into a flat membranous basement membrane. In the absence of Matrigel, the SV40-T2 cells failed to form a continuous lamina densa, and the deposits stayed in the coarse mesh. The major biotinylated Matrigel components that were integrated into the basement membrane were laminin-1 and entactin. Furthermore, SV40-T2 cells supplemented with exogenous laminin-1 alone as well as laminin-1 contaminated with entactin formed a continuous lamina densa. These results indicate that the laminin-1 and entactin supplied from the Matrigel were incorporated into a basement membrane beneath the SV40-T2 cells, and contributed to the formation of basement membrane. Therefore, we concluded that the alveolar epithelial cells synthesize laminin-1, entactin, type IV collagen, and perlecan, but that they also needed to assemble exogenous laminin-1 into the basement membrane to complete its formation in vitro.

An analysis of the aggregation and morphogenesis of area opaca endoderm cells from the primitive-streak chick embryo

Development ◽  
1979 ◽  
Vol 51 (1) ◽  
pp. 121-135
Author(s):  
Nadine Milos ◽  
Sara E. Zalik ◽  
Robert Phillips
Keyword(s):  
Cell Adhesion ◽  
Chick Embryo ◽  
Intrinsic Property ◽  
Primitive Streak ◽  
Chick Embryos ◽  
Embryonic Cells ◽  
Thin Walls ◽  
Cell Layers ◽  
Aggregative Behaviour

The aggregative behaviour and subsequent morphogenesis of extra-embryonic endoderm cells from primitive-streak chick embryos have been investigated. A relatively pure population of area opaca endoderm cells was obtained by differential dissociation, which involves partial separation of epiblast and endoderm cell clumps by sieving through Nitex mesh. For aggregation studies cells were cultured in rotating flasks in Leibovitz (L-15) medium, in saline or in saline supplemented with glucose (1 mg/ml). Aggregation was monitored using the Coulter Counter. In these three media aggregation is rapid; by 10 min an average of 61% of the population had aggregated, to reach a plateau at 30 min when an average percent adhesion value of 83 % was obtained. The aggregates in L-15 medium were large and compact. After several days in culture, they cavitated and formed smooth hollow vesicles with thin walls composed of one or a few cell layers. Aggregates formed in PCS were smaller and looser in appearance; the addition of glucose resulted in a certain degree of compaction. Some morphogenesis occurred under these conditions with the aggregates developing numerous irregular cavities. These experiments suggest that some of the factors that affect cell adhesion in early embryonic cells can be studied in vitro. The results also indicate that the ability to cavitate is an intrinsic property of the endoderm cells of the area opaca since this occurs in the absence of epiblast or mesoderm.

Analysis of wound healing in an in vitro model: early appearance of laminin and a 125 × 10(3) Mr polypeptide during adhesion complex formation

1990 ◽  
Vol 96 (4) ◽  
pp. 651-660
Author(s):  
M.A. Kurpakus ◽  
E.L. Stock ◽  
J.C. Jones
Keyword(s):  
Wound Healing ◽  
Basement Membrane ◽  
In Vitro Model ◽  
Collagen Type ◽  
Basement Membrane Zone ◽  
Lamina Densa ◽  
Vitro Model ◽  
Collagen Type Vii ◽  
Adhesion Complex

The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K = 10(3) Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluorescence labeling could be detected with mAbHD before labeling with BP autoantibodies or collagen type VII antibodies. Laminin fluorescence was detected at the same time as mAbHD. Furthermore, the 125K polypeptide and laminin were located extracellularly prior to the appearance of BP antigen and collagen type VII. The appearance of the hemidesmosomal plaque at the electron microscope level succeeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evidence for the coordinated appearance of BP antigen, collagen type VII and laminin was observed in this model. We discuss the possibility that the 125K protein and laminin may play roles in the initiation of complex formation. Furthermore, although basement membrane zone components were detected early in adhesion complex re-formation, formation of the lamina densa region of the basement membrane zone followed the appearance of the hemidesmosomal plaque, indicating a role for the hemidesmosomal plaque in organizing the structure of the lamina densa.

Labelling of basement membrane constituents in the living chick embryo during gastrulation

Development ◽  
1984 ◽  
Vol 79 (1) ◽  
pp. 113-123
Author(s):  
Esmond J. Sanders
Keyword(s):  
Chick Embryo ◽  
Basement Membrane ◽  
Concanavalin A ◽  
Primitive Streak ◽  
Basal Surface ◽  
Stage Of Development ◽  
Pass Through ◽  
The Relationship

The basement membrane of the living chick embryo epiblast has been labelled with ultrastructural markers in order to study the movement and turnover of this structure during gastrulation. Two problems were addressed in these experiments. Firstly, to what extent does the basement membrane move medially with the epiblast during morphogenesis? Secondly, what is the relationship to the basement membrane of the so-called interstitial bodies? The ultrastructural markers used were concanavalin A conjugated to ferritin and fibronectin antibodies conjugated to peroxidase. Embryos were cultured using the technique of New, and the label was applied to the periphery of the basal surface of the epiblast through a hole in the endoblast at the early primitive streak stage of development. The embryos were then allowed to develop to the full primitive streak stage in the presence of the label. When the position of the label was determined after incubation, it was found to have accumulated in large amounts at the edge of the primitive streak at the point where the basement membrane is disrupted. This indicates that constituents of the basement membrane are transported medially with the epiblast cells and are sloughed off as the latter pass through the primitive streak. This movement of basement membrane constituents is counter to the direction of migration of the underlying mesoderm cells. When embryos are exposed to label for only 1 h, then washed and incubated for a further three hours, the marker was found in the interstitial bodies and not distributed throughout the basement membrane itself. This suggests that the interstitial bodies, which have been implicated in influencing the migration of the mesoderm cells, are turnover products of the basement membrane to which they are attached.

Synthesis of globin chains in the erythropoietic sites of the early chick embryo

Development ◽  
1983 ◽  
Vol 77 (1) ◽  
pp. 153-165
Author(s):  
L. Fucci ◽  
C. Cirotto ◽  
L. Tomei ◽  
G. Geraci
Keyword(s):  
Chick Embryo ◽  
Relative Concentration ◽  
The Other ◽  
Specific Mechanism ◽  
In Ovo ◽  
Globin Chains ◽  
Immunofluorescence Labelling ◽  
Α Chain

The synthesis of globins in the chick embryo before the onset of circulation has been studied in situ by specific immunofluorescence labelling of embryonic sections and by labelling newly synthesized proteins in ovo and in vitro in embryonic explants with [3H]leucine. The presence of major primitive haemoglobins is observed by 28 h of incubation. The minor primitive haemoglobins become detectable by immunofluorescence after 40 h of development, shortly before the onset of circulation. 3H-labelling shows that one definitive α chain is synthesized, though in low concentration, from the initial globin detection. The other definitive α chain is observed in embryos of at least 40 h of development. The relative concentration of the two definitive α chains changes rapidly with development indicating a specific mechanism of regulation. An erythropoietic site is observed in the wall of the dorsal aorta in embryos of about 45–50 h of development. From the initial detection, those cells contain all four primitive embryonic haemoglobins, in contrast to what is observed for the cells of the blood islands.

scholarly journals Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network.

1987 ◽  
Vol 105 (6) ◽  
pp. 2559-2568 ◽  
Author(s):  
P D Yurchenco ◽  
G C Ruben
Keyword(s):  
Basement Membrane ◽  
Type Iv Collagen ◽  
Thin Sheet ◽  
Great Part ◽  
Molecular Architecture ◽  
Branch Points ◽  
Collagen Network ◽  
Type Iv

To determine molecular architecture of the type IV collagen network in situ, the human amniotic basement membrane has been studied en face in stereo relief by high resolution unidirectional metal shadow casting aided by antibody decoration and morphometry. The appearance of the intact basement membrane is that of a thin sheet in which there are regions of branching strands. Salt extraction further exposes these strands to reveal an extensive irregular polygonal network that can be specifically decorated with gold-conjugated anti-type IV collagen antibody. At high magnification one sees that the network, which contains integral (9-11 nm net diameter) globular domains, is formed in great part by lateral association of monomolecular filaments to form branching strands of variable but narrow diameters. Branch points are variably spaced apart by an average of 45 nm with 4.4 globular domains per micron of strand length. Monomolecular filaments (1.7-nm net diameter) often appear to twist around each other along the strand axis; we propose that super helix formation is an inherent characteristic of lateral assembly. A previous study (Yurchenco, P. D., and H. Furthmayr. 1984. Biochemistry. 23:1839) presented evidence that purified murine type IV collagen dimers polymerize to form polygonal arrays of laterally as well as end-domain-associated molecules. The architecture of this polymer is similar to the network seen in the amnion, with lateral binding a major contributor to each. Thus, to a first approximation, isolated type IV collagen can reconstitute in vitro the polymeric molecular architecture it assumes in vivo.

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