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.

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 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)

Ultrastructural immunocytochemical localization of fibronectin in the early chick embryo

Development ◽  
1982 ◽  
Vol 71 (1) ◽  
pp. 155-170
Author(s):  
E. J. Sanders
Keyword(s):  
Chick Embryo ◽  
Basal Lamina ◽  
Regional Differences ◽  
Developmental Stages ◽  
Primitive Streak ◽  
Extracellular Material ◽  
Cell Surfaces ◽  
Mesoderm Cell ◽  
Stage Of Development ◽  
Primary Location

Fibronectin was localized using peroxidase and biotin-avidin-ferritin techniques in developmental stages of the chick embryo from laying to primitive streak formation. The primary location of fibronectin at all stages examined was the basal lamina of the epiblast and its associated extracellular material. The remaining tissues showed little or no immunochemical deposit. At the primitive streak stage of development there were some regional differences in the density of the deposit on the basal lamina. Closely adjacent to the primitive streak, where the basal lamina is fragmentary, deposit was sparse or absent. In this, and other regions, mesoderm cell surfaces did not stain except where they closely approached the stained basal lamina or interstitial bodies. Staining was variable in the basal lamina anterior to the primitive streak, but in a number of cases particularly heavy deposit was noted overlying the crescent of late hypoblast. This area seemed to correspond with the anterior fibronectin- rich band reported by others using immunofluorescence localization.

scholarly journals AUTORADIOGRAPHIC STUDIES OF THE UTILIZATION OF S35-SULFATE BY THE CHICK EMBRYO

1957 ◽  
Vol 3 (2) ◽  
pp. 231-238 ◽  
Author(s):  
Perry M. Johnston ◽  
Cyril L. Comar
Keyword(s):  
Chick Embryo ◽  
Basement Membrane ◽  
Chondroitin Sulfate ◽  
Developmental Stages ◽  
Primitive Streak ◽  
Intracellular Accumulation ◽  
Histological Differentiation ◽  
Membrane Area ◽  
Thin Ring ◽  
Inorganic Sulfate

From studies of autoradiograms of various developmental stages of the chick embryo containing S35 given us sulfate it was determined that as early as Stages 3+ and 4 there is a selective utilization or accumulation of sulfate by the various parts. The earliest accumulation site is the axial portion of the primitive streak and the floor of the groove. Later S35 was found in the head process, Hensen's node, notochord, amniocardiac vesicle, wall of the omphalomesenteric vein, endocardium, subendocardial jelly, mesenchyme destined to become cartilage, basement membrane area of the gut, and a mucopolysaccharide layer formed on the free surface of the stomach. The early notochordal localizations of S35 coincide with the region in which a thin ring of chondroitin sulfate is subsequently laid down. However, it is apparent that there is an intracellular accumulation of inorganic sulfate by the chondroitin-forming cells prior to the time they produce sufficient chondroitin sulfate to be demonstrable histochemically. It was interesting to note that the endocardium appears to concentrate sulfate that later apparently finds its way into the subendocardial jelly. The fact that those mesenchymal cells which later form chondroblasts begin to utilize sulfate selectively before histological differentiation is apparent was determined. In addition, the presence of sulfate-containing substances in the forming basement membrane of the gut would seem to indicate that sulfate is important in the histological differentiation of this membrane.

Epithelial and basement membrane responses to chick embryo primitive streak grafts

1986 ◽  
Vol 18 (4) ◽  
pp. 233-242 ◽  
Author(s):  
E.J. Sanders ◽  
S. Prasad
Keyword(s):  
Chick Embryo ◽  
Basement Membrane ◽  
Primitive Streak

Identification of a novel growth factor with transforming activity secreted by individual chick embryos

Development ◽  
1990 ◽  
Vol 109 (4) ◽  
pp. 905-910 ◽  
Author(s):  
J. Smith ◽  
J.C. McLachlan
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Chick Embryo ◽  
Primitive Streak ◽  
Soft Agar ◽  
Heat Stable ◽  
Stage Of Development ◽  
Transforming Activity ◽  
Embryo Tissue ◽  
Differential Responsiveness

Previously we have developed a microassay for anchorage independent growth (AIG) of fibroblasts in soft agar, which can detect very small quantities of transforming growth factors (TGFs). Using this assay, we have shown that small pieces of dissected chick embryo tissue will stimulate AIG of both NR6 and NRK 49f cells, and that this property can be used to map production of growth factors with transforming activity in individual early embryos. We now show that this activity can be transferred to conditioned medium (CM) prepared using chick embryo tissue. Using two cell lines with differential responsiveness to TGFs, and by coincubating normal and heat-treated CM with trypsin, Con-A and neutralising antibodies, we show that CM contains at least two different growth factors with transforming activity. One of these is heat-stable, and stimulates colony formation in NRK 49f cells in the presence of EGF, but not in its absence. This activity corresponds to a TGF beta-like molecule. The other component is a heat-labile glycoprotein, which has TGF alpha-like properties, but does not appear to behave like known TGFs with these properties. It therefore appears to be a novel growth factor. Both activities are present from the intermediate primitive streak stage of development.

Fates and migratory routes of primitive streak cells in the chick embryo

Development ◽  
1996 ◽  
Vol 122 (5) ◽  
pp. 1523-1534 ◽  
Author(s):  
D. Psychoyos ◽  
C.D. Stern
Keyword(s):  
Chick Embryo ◽  
Primitive Streak ◽  
Final Position ◽  
Fate Map ◽  
Early Chick Embryo ◽  
Carbocyanine Dyes ◽  
Early Stages ◽  
Migratory Routes ◽  
Pass Through ◽  
Different Tissues

We have used carbocyanine dyes to fate map the primitive streak in the early chick embryo, from stages 3+ (mid-primitive streak) to 9 (8 somites). We show that presumptive notochord, foregut and medial somite do not originate solely from Hensen's node, but also from the anterior primitive streak. At early stages (4- and 4), there is no correlation between specific anteroposterior levels of the primitive streak and the final position of their descendants in the notochord. We describe in detail the contribution of specific levels of the primitive streak to the medial and lateral halves of the somites. To understand how the descendants of labelled cells reach their destinations in different tissues, we have followed the movement of labelled cells during their emigration from the primitive streak in living embryos, and find that cells destined to different structures follow defined pathways of movement, even if they arise from similar positions in the streak. Somite and notochord precursors migrate anteriorly within the streak and pass through different portions of the node; this provides an explanation for the segregation of notochord and somite territories in the node.

Radiation Damage, Contrast and Statistics in High Resolution Biological Electron Microscopy

1970 ◽  
Vol 28 ◽  
pp. 260-261
Author(s):  
Robert M. Glaeser
Keyword(s):  
High Resolution ◽  
Particle Flux ◽  
Unit Area ◽  
Signal To Noise ◽  
Resolution Image ◽  
High Resolution Image ◽  
Pass Through ◽  
Counting Statistics ◽  
The Relationship ◽  
Picture Element

It is well known that a large flux of electrons must pass through a specimen in order to obtain a high resolution image while a smaller particle flux is satisfactory for a low resolution image. The minimum particle flux that is required depends upon the contrast in the image and the signal-to-noise (S/N) ratio at which the data are considered acceptable. For a given S/N associated with statistical fluxtuations, the relationship between contrast and “counting statistics” is s131_eqn1, where C = contrast; r2 is the area of a picture element corresponding to the resolution, r; N is the number of electrons incident per unit area of the specimen; f is the fraction of electrons that contribute to formation of the image, relative to the total number of electrons incident upon the object.

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