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E J Sanders

Publications and source records attributed to E J Sanders.

At least 109 records · Page 6Linked to original sources
PubMed1984

Intercellular relationships during cavitation of aggregates of extraembryonic endoderm cells from gastrulating chick embryos.

Extraembryonic endoderm cells from gastrulating chick embryos undergo epiboly and change from a multilayered cell group to a single cell layer surrounding the yolk. Single cell suspensions from this cell layer can aggregate in vitro to form aggregates that cavitate. To study the stages of cavitation aggregates were harvested after different times in culture, and fixed and processed for light and electron microscopy. In aggregates harvested at 75 min of culture cell contact consisted of areas of parallel and close membrane apposition and interdigitation. Desmosomes were occasionally observed. Aggregates in the early stages of cavitation (24 h) contained numerous intercellular spaces bordered by irregularly shaped cells which appeared to be digesting their yolk and releasing material extracellularly. Long cytoplasmic projections were extended into these spaces. In addition to regions of parallel membrane apposition and interdigitation, desmosomes and adherens junctions were observed. Cells closer to the periphery of the aggregates displayed fewer cell projections and also showed signs of release of material extracellularly. After 48 h of culture, a single smooth-walled central cavity was present and cells still exhibited signs of extracellular release of material. These same cell shapes and intercellular junctions were also observed when area opaca tissue dissected from gastrulating embryos was examined. Aggregates of different sizes were created and cultured. The results suggest that a critical tissue mass may be important for cavitation.

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PubMed1984

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

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.

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PubMed1983

The effects of microtubule dissociating agents on the physiology and cytology of the sensory neuron in the femoral tactile spine of the cockroach, periplaneta americana L.

Microtubules are prominent cellular components of the mechanosensory and chemosensory sensilla associated with the insect cuticle, and a range of hypotheses have been proposed to account for their role in sensory transduction. Chemical agents such as colchicine and vinblastine, which dissociate microtubules, also interfere with transduction in these sensilla, and this has been attributed to their anti-microtubule activity. We have now examined the dynamic properties of sensory transduction in the mechanosensitive neuron of the cockroach femoral tactile spine, after the application of colchicine, vinblastine and lumicolchicine. Concurrently we have examined the ultrastructure of the same sensory ending by transmission electron microscopy. All of the drugs reduced the mechanical sensitivity o the receptor. Colchicine and vinblastine achieved this reduction without altering the dynamic properties of the receptor but lumicolchicine changed the dynamic response, and increased the relative sensitivity to rapid movements. Conduction velocity, another measure of neuronal function, which relies upon ionic currents flowing through the membrane, was reduced by all three drugs. The effects of the drugs upon the ultrastructure of the sensory ending were also disparate. In the case of colchicine there was complete dissociation of microtubules in the tubular body and distal dendrite before a total loss of mechanical sensitivity. Vinblastine was less effective in dissociating microtubules, although more effective in the reduction of mechanical sensitivity. With lumicolchicine the dominant morphological effect was a severe disruption of the dendritic membrane. We conclude from these experiments that microtubules are not essential in the transduction of mechanical stimuli by cuticular receptors and that the effects of these drugs upon mechanosensitivity are not directly related to their dissociation of the microtubules in the tubular body, but are more likely to arise from actions upon the cell membrane. These actions could include effects upon tubulin in the membrane or upon other membrane components.

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PubMed1983

The culture of chick embryo mesoderm cells in hydrated collagen gels.

Chick embryo mesoderm cells are various stages of differentiation were cultured in three-dimensional matrices of hydrated collagen. The tissues used were: stage 5 mesoderm from regions adjacent to the primitive streak; stage 12 mesoderm, comprising somitic, unsegmented (segmental plate) and lateral plate mesoderm; and stage 18 sclerotome. Explants were examined by phase contrast microscopy, including time-lapse, and scanning and transmission electron microscopy. The cells showed an increased ability to adhere to, and move in, the collagen gel with advancing stage. Of the stage 12 tissues, the unsegmented mesoderm was initially the slowest to grow out of the explant. Sclerotome cells showed by far the greatest ability to move within the gel. Where the collagen fibrils were randomly oriented, the cell morphology was polypodial and advancing lamellipodia showed clear undulations at their leading edges. A distinction was drawn between these undulations and the classical major ruffles which are seen in two-dimensional culture to uplift and pass back along the cell surface. The latter were not seen in the collagen matrix and were presumably suppressed by the three-dimensional culture configuration while the leading edge undulations were not. Ultrastructural examination showed that the cells possessed patches of amorphous material on their surface, which was sometimes interposed between the plasma membrane and collagen fibrils. Addition of hyaluronic acid (2 mg/ml) had an effect only the segmented mesoderm, where outgrowth was enhanced. Although the addition of plasma fibronectin (50 micrograms/ml) to the cultures did not affect any of the tissues, the removal of this substance, by antifibronectin antiserum or by the use of fibronectin depleted serum, inhibited outgrowth in most cases. The only tissue not reproducibly inhibited in this way was sclerotome. Alignment of the collagen fibres by the explants was observed, accompanied by an elongation of the outgrowing cells which, in bipolar form, preferentially moved up and down the aligned tracts. Scanning electron microscopy suggested that cell processes attached to, and presumably exerted tension on, bundles of fibrils thereby pulling them into line. Cell-to-cell contact was not accompanied by contact paralysis as judged by time-lapse micrography.

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PubMed1983

Cationized ferritin and phosvitin uptake by coated vesicles of the early chick embryo.

The endocytosis of cationized ferritin and of a phosvitinferritin conjugate by cells of the chick embryo area pellucida has been examined. Cationized ferritin was bound mainly to the free surface of the epiblast but was absent from the region of the primitive streak. The binding was patchy and experiments suggest that the anionic sites which bind cationized ferritin are themselves naturally clustered. Uptake of cationized ferritin was exclusively by coated pits. The resulting coated vesicles delivered the cationized ferritin to membrane-bound sites of accumulation in the cytoplasm and to the close vicinity of Golgi bodies. The cationized ferritin was frequently found to share intracellular vacuoles with yolk granules. The uptake was not affected by the presence of microfilament or microtubule-inhibiting agents. Native ferritin, even at concentrations forty times that of cationized ferritin, was not bound or endocytosed. Coated pits in the epiblast were often associated with overlying extracellular yolk granules. This suggested that the yolk might be inducing the formation of the coated pits. The yolk protein phosvitin was coupled to ferritin and this conjugate was found to be endocytosed by coated pits. This uptake was inhibited in the presence of an excess of free phosvitin but not by albumin, indicating some selectivity for phosvitin over other proteins. The phosvitin conjugate was also found sharing intracellular vacuoles with yolk. We conclude that the cells of the area pellucida, and in particular those of the epiblast, have an active coated vesicle uptake system which may be able to selectively endocytose yolk or yolk protein.

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PubMed1983

The ultrastructure of polygonal networks in chick embryonic cells in vitro.

Polygonal networks in cultured chick endoderm cells are ordered arrays of actin microfilaments situated just beneath the dorsal cell surface. Each strut is formed from a bundle of microfilaments and 5-7 bundles intersect at each node. Dense bodies are seen in nodes and some struts. At its periphery the network is attached to the substrate at the termini of long radial struts. Most of the network is resistant to detergent extraction. Sliding microfilaments can explain the observed behaviour of networks in live cells.

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PubMed1983

Recent progress towards understanding the roles of the basement membrane in development.

A brief description of the structure of basement membranes is presented including results of immunocytochemical investigations. A number of developmental events are considered in which the basement membrane has been implicated in the regulation of morphogenesis. One of the clearest developmental functions appears to be the maintenance of the lobular morphology of glandular epithelia, where the basement membrane supplies a supportive scaffold. The contributions of the glycoproteins laminin and fibronectin to basement membrane function are discussed with reference to potential roles for this structure as a substratum for morphogenetic cell migrations and as a mediator of inductive interactions in embryogenesis. Examples used to illustrate these proposals include the mesodermal migrations of gastrulation, kidney tubule morphogenesis, and limb bud development. Evidence from these and other sources lend support to current view for an active and changing role for the basement membrane in the epithelial-mesenchymal interactions of embryonic development.

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PubMed1982

Inability of mesoderm cells to locomote on the modified free surface of epithelial cell sheets in vitro.

Previous work has shown that when chick embryo mesoderm tissue is seeded onto the free, dorsal surface of established sheets of embryonic epithelial endoblast, the former penetrates the latter and spreads on the underlying artificial substratum. In this work, the surface charge on the epithelial sheet has been altered, prior to seeding the mesoderm, to ascertain whether such a change could alter the behavior of the mesoderm with respect to the free surface of the epithelium. Charge alteration was accomplished using the polycations, poly-L-lysine. Surface charge characteristics were examined ultrastructurally using cationized and anionized ferritin. Results showed that although surface charge changes were detectable, there was no difference in the behavior of the mesoderm with respect to the endoblast. Neuraminidase did not detectably affect the epithelial surface charge. These results are consistent with the view that changes in substratum surface charge are not necessarily correlated with changes in adhesiveness.

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PubMed1982

Ultrastructural immunocytochemical localization of fibronectin in the early chick embryo.

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.

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PubMed1981

The mechanosensory apparatus of the femoral tactile spine of the cockroach, Periplaneta americana.

Tactile spines are large cuticular sense organs that appear to provide insects with a sense of touch which is spatially coarse but of great sensitivity. Cockroach legs have a number of these spines on each leg and a particularly prominent spine on the end of each femur, the femoral tactile spine. The ease of recording afferent activity from this spine during mechanical stimulation has made it one of the most thoroughly studied insect mechanoreceptors and yet it has never been examined by electron microscopy. We report here the results of an examination of the femoral tactile spine by both scanning and transmission electron microscopy, as well as by light microscopy. The spine is shown to be innervated by a single sensory bipolar neuron with its soma located in the base of the spine. A canal through the wall of the spine leads to the outside and emerges just above the junction between the base of the spine and its articulating socket membrane. The sensory dendrite of the neuron passes from the soma through this canal and forms a modified ciliary sensory ending with the typical dendritic sheath and dense tubular body that is characteristic of insect mechanosensory cuticular sensilla. The tubular body is embedded in a cuticular terminal plug which closes the exterior end of the canal but appears to be fastened to the spine by a very flexible ring of cuticle. This plug is connected to the socket membrane by a specialized socket attachment which presumably serves to move the plug relative to the wall of the spine during movement of the spine within the socket. The morphology of this sensillum is discussed in terms of the possible ways in which it is stimulated by movements of the spine and also in light of the dynamic behaviour of the receptor which is now very well described.

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PubMed1981

Cell junctions in explanted tissues from early chick embryos.

Hypoblast and definitive endoblast derived from young chick embryos were explanted and grown for 24 h in culture. The junctional complexes which characterise these tissues were studied on freeze-fracture replicas and thin sections. Cell membranes of the hypoblast displayed tight junctions only, disposed in randomly arranged strands or narrow belts which included many discontinuous strands. The definitive endoblast showed tight and gap junctions as well as desmosomes in close association with the tight junctions. It is suggested that the differences between the two types of tissue may be related to cell cohesiveness, which appears to be relatively low in the hypoblast and high in the definitive endoblast.

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PubMed1981

Morphological differentiation of an embryonic epithelium in culture.

This communication reports the results of a morphological study of three-day old cultures of epiblast tissue from the early chick embryo. The most striking feature of these cultures was the appearance of "domes" or elevated blister-like structures, composed of a single layer of cells which were morphologically distinct from the remaining cells in the culture. The domes arose in high-density areas of the culture. Their roofs were lined by basal laminae that did not develop in other areas of the culture. In several morphological respects, the cells of the dome roof closely resembled the epiblast in vivo. This was in contrast to the cells spread on the substratum in sparse regions of the culture, which did not. Each dome was surrounded by a dense ring of multilayered ruffling cells which appeared to give rise to both the dome roof and to fibroblast-like cells that spread on the substratum beneath the dome. Fibroblast-like cells also developed in discrete patches in other regions of the culture. In other tissues, dome formation has been attributed to fluid transport by the epithelium; in the present case it is also possible to invoke the capacity of the epiblast to fold, as contributing to the mechanism of dome formation.

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PubMed1981

The behaviour of embryonic chick and quail tissues in culture.

Pieces of tissue were dissected from early chick and quail embryos (Stages XIII and XIV of Eyal-Giladi & Kochav, 1976; and stages 3-5 of Hamburger & Hamilton, 1951). These tissues were taken from three different regions of the early embryos, and from eight different regions of the older ones, and were derived mainly from the lower layer. Epiblast tissues were also used. The experiments were designed to test the ability of one tissue to penetrate another. A single tissue was grown in culture in a Falcon dish for 18-24 h until it had formed a coherent sheet of cells (Explant I). A second tissue was then combined with it in one of two ways: (a) A small piece of tissue (Explant II) was explanted on top of Explant I. In most cases Explant II penetrated through Explant I and spread on the Falcon dish. (b) Another small piece of tissue (Explant III) was explanted beside (in confrontation with) Explant I. Usually, Explant III penetrated into Explant I rather than vice versa. The results were analysed to see if there were any variations in behaviour of the different tissues. The main result was that important differences were found to exist between certain types of chick and quail cells when grown in culture; the implications of this finding for the widely used technique of xenoplastic grafting are mentioned. Another result was that Explant I was more likely to be penetrated when the second tissue was placed on top of it (Explant II) than when it was confronted with it (Explant III). The significance of these results is discussed.

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PubMed1980

The effect of fibronectin and substratum-attached material on the spreading of chick embryo mesoderm cells in vitro.

Endoblast and hypoblast tissue, dissected from early chick embryos, was explanted and cultured on glass or plastic substrata. These tissues grew rapidly to form epithelial sheets. Under the same conditions, mesoderm, dissected without the aid of dissociating agents, grew poorly. After 24 h in culture, the mesoderm explants consisted of a sparse outgrowth of fibroblast-like cells. When pieces of mesoderm were seeded onto the dorsal surface of the epithelia, however, the cells penetrated the sheet and rapidly spread on the substratum within 4 h. If the epithelial sheet was detached from the substratum and the mesoderm then seeded onto areas of substratum previously occupied by epithelium, similar rapid spreading occurred. This effect could be produced in the absence of serum. The method used to remove the epithelium (EDTA, detergent or manual dissection) did not influence the result. When the substratum-attached material (SAM) was examined by scanning electron microscopy, 2 types of material were seen. One type appeared to be the remains of detached filopodia and cytoplasmic lamellae, while the other appeared to be of extracellular origin. Both these types reacted positively by immunofluorescence using anti-fibronectin serum. SAM derived from mesoderm reacted negatively. When mesoderm was cultured in the presence of plasma fibronectin on unmodified plastic or glass, spreading was complete in 4-5 h and thus was similar to mesoderm seeded onto SAM. The morphology of mesoderm explants on SAM or in the presence of plasma fibronectin was more epithelial than on untreated substratum in normal medium. Hypoblast and endoblast cultured in the presence of anti-fibronectin serum failed to spread normally, apparently being unable to attach to the substratum. Mesoderm did not spread rapidly on SAM in the presence of this antiserum. Cycloheximide reversibly inhibited the spreading of hypoblast and endoblast, and this effect could be eliminated, at least for hypoblast, by the addition of plasma fibronectin. Covering attachment sites on the substratum with bovine serum albumin, thereby preventing the attachment of SAM or fibronectin, also inhibited spreading. It is proposed that mesoderm cells have low levels of surface fibronectin in comparison with endoblast and hypoblast, and that this results in a comparatively low adhesiveness, which is important for its morphogenetic activity within the embryo.

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PubMed1980

Behavioural properties of chick somitic mesoderm and lateral plate when explanted in vitro.

Tissue culture, time-lapse cinematographic and electron microscopic techniques have been used to study the properties of chick mesoderm at several stages of differentiation. Lateral plate, unsegmented mesoderm (segmental plate), and newly formed somites were dissected from stage-12 embryos, whilst dermo-myotomes and sclerotomes were dissected from stage-18 embryos. Each type of mesoderm was found to exhibit a characteristic pattern of behaviour. The explants from the unsegmented mesoderm from the newly formed somites and from the older embryos could be placed in a developmental sequence; with increasing differentiation they settled and spread on the substrate more readily, whether explanted as pieces of tissue or as individual cells, and it was concluded that this implied an increased adhesion to the substrate. Similarly, with increasing differentiation, the cells segmented at a faster rate. No significant differences could be discerned in the internal structure of the different types of cells, although differences in the general shape were apparent. The lateral plate mesoderm cells, which bear some resemblances to the unsegmented mesoderm cells in the embryo, also show some morphological resemblances to them in vitro. However, the lateral plate cells had a much greater success in attaching to glass or platic substrates. They were also found to have the highest speed of locomotion of all the tissues studied, whereas the unsegmented had the lowest. It is concluded therefore, that although cells may look similar to one another morphologically, their behaviour may differ greatly, probably because they are already partially determined.

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PubMed1979

Development of the basal lamina and extracellular materials in the early chick embryo.

Chick embryos at developmental stages up to primitive streak formation were fixed in a mixture of tannic acid and glutaraldehyde. A basal lamina was present in the unincubated embryo and consisted of a lucent lamina interna and a lamina densa. At the primitive streak stage the lamina densa showed a periodicity of stained elements. Densely stained materials were present on the cell surfaces lining the cavity between the epiblast and endoblast, and on the mesoderm cells within this cavity. Considerable amounts of extracellular material were observed in the cavity. Hyaluronidase treatment removed the cell surface and extracellular material, indicating that hyaluronic acid is a major component. This enzyme disrupted the basal lamina, leaving a fibrillar remnant with no periodic structure. It is therefore suggested that the dense periodicities consist of glycosaminoglycan built on an enzyme-resistant framework which is probably collagen. Enzyme-resistant fibrils, presumably collagen precursors, are present elsewhere within the tissue spaces.

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