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Biomedical subjects

E J Sanders

Publications and source records attributed to E J Sanders.

At least 91 records · Page 5Linked to original sources
PubMed1988

The effects of somite removal on vertebral formation in the chick.

We have examined the effects on vertebral development of various combinations of somite removal in two day old chick embryos as shown by vertebral formation after a further seven days of incubation. Each combination produced one of a variety of results ranging from completely normal vertebral formation, through fusion of various vertebral elements, to the absence of complete vertebral halves and the formation of hemivertebrae. Assessment of our operating ability showed that we were removing at least 90% of the somitic material and therefore these results suggest that there is a regulating mechanism available to the embryo, at least with regards to vertebral development. When two consecutive somites were removed, vertebrae frequently developed that were lacking certain elements. This suggests that the somitic cells are already determined with regards to formation of specific vertebral elements. Experiments involving the removal of a bilateral pair of somites (a repetitive unit) also provided evidence of a counting mechanism which ensures that the correct number of total vertebrae are present.

Animals↗
PubMed1988

Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices.

Very early embryonic mesoderm cells were taken from the primitive streak-stage chick embryo and cultured in a matrix of type I collagen in the presence of serum. Previous work has shown that under these conditions cells do not leave the explant and move in the collagen in the absence of supplemented avian transferrin. Cells explanted onto tissue culture plastic in the presence of serum do not require this transferrin supplement. These observations were investigated further by culturing cells in collagen in the presence of the lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), which can replace transferrin as an iron-delivery agent. Under conditions in which FePIH could effectively stimulate chick embryo myoblast growth, no such long-term stimulation was obtained with the early mesoderm cells in collagen. This suggested that for mesoderm cells, FePIH could not replace transferrin. Antibody to the transferrin receptor and to transferrin itself inhibited growth of myoblasts in collagen and on plastic, and of mesoderm cells in collagen. Mesoderm cells on plastic, however, were refractory to the presence of the antibody directed to the receptor and seemed to show a low dependency on transferrin-delivered iron under these conditions, inasmuch as antiserum to transferrin itself only caused a partial inhibition of outgrowth. The results suggest that mesoderm cells in collagen require transferrin for both iron uptake and for another unspecified function. It is consistent with the results to propose that transferrin binding might modulate the cells' attachment to collagen, thus influencing outgrowth. The distribution of the actin cytoskeleton in mesoderm cells actively migrating in collagen, such as in the presence of transferrin, suggests a stronger attachment to the collagen than nonmigrating cells.

Actins↗
PubMed1988

Extracellular matrix synthesis is required for the movement of sclerotome and neural crest cells on collagen.

During early embryogenesis cells of several different populations disperse by active cell movement from one location to another. Preexisting extracellular materials are major determinants of these dispersal patterns, but the cells are also able to modify their substrata by synthesizing and secreting extracellular matrix molecules as they move. In order to determine the contribution made by these deposited materials, several tissues from the early chick embryo have been cultured in the presence of inhibitors of extracellular matrix synthesis and secretion. The tissues examined were sclerotome cells from differentiated somites and neural crest cells. For comparison, undifferentiated somites were also cultured. The movement of these cells was compared in type I collagen gel culture and in conventional culture on artificial substrata. Inhibitors of collagen synthesis were used (cis-hydroxy proline and L-azetidine-2-carboxylic acid) in addition to a proteoglycan inhibitor (p-nitrophenyl-xylopyranoside) and a secretion inhibitor (monensin). Results indicate that sclerotome cells require collagen synthesis for movement in a collagen matrix. Reversal of the effects of collagen inhibitors, by proline and type II collagen, suggest that sclerotome cells normally condition the type I matrix in order to move in it. Inhibition of proteoglycan synthesis produced the greatest effect on the movement of neural crest cells regardless of the substratum, confirming an important role for these molecules in the crest migratory routes. The attachment of all cells to collagen was highly sensitive to the presence of monensin, which is known to reduce the deposition of glycosaminoglycans and fibronectin. These results suggest that conditioning of the extracellular matrix by newly synthesized material is required for cell attachment and movement during early development.

Animals↗
PubMed1988

The roles of epithelial-mesenchymal cell interactions in developmental processes.

This review surveys some recent trends in the study of the developmental interactions between epithelial and mesenchymal cells. The influence of such interactions on cell differentiation is considered with reference to kidney development, limb bud development, chondrogenesis and osteogenesis, and tooth development. Effects on epithelial morphogenesis are discussed, using salivary gland development as an example. The roles of humoral factors (hormones or growth factors) are considered, and the evidence for the participation of cell adhesion molecules is examined.

Animals↗
PubMed1988

The contribution made by a single somite to the vertebral column: experimental evidence in support of resegmentation using the chick-quail chimaera model.

The somitic involvement in the formation of the vertebral column was examined using the chick-quail chimaera model. Single cervical somites from quail donor embryos were transplanted into similarly staged chick host embryos. Following further incubation, serial sections of variously staged embryos were stained with the Feulgen reaction to distinguish the two cell populations. Quail cells were generally located within a delimited region in one half of each of the two adjacent vertebrae, as well as in the intervening disc. The horizontal plane of division through each vertebra passed approximately through the centre of the body and divided the neural arch into rostral and caudal halves through the rostral border of the caudal notch. These results give support to the controversial theory of resegmentation, in which it was suggested that there is an apparent realignment of segmentation between the somite stage and the subsequent vertebral stage of development.

Animals↗
PubMed1988

Effects of HNK-1 monoclonal antibody on the substratum attachment and survival of neural crest and sclerotome cells in culture.

The sclerotome portion of the differentiating embryonic chick somite becomes infiltrated by neural crest cells prior to its dispersal towards the embryonic axis. This means that sclerotome cells explanted into culture for the purpose of examining their interactions in vitro are contaminated with a proportion of neural crest cells. The purpose of this study was to explore the neural crest cell adhesion epitope recognized by the HNK-1 monoclonal antibody and ways in which this antibody can be used to eliminate neural crest cells from mixed culture by selective cytotoxicity. Immunofluorescence technique, under the conditions used here, indicated that the antibody appeared to stain all the mesenchymal (i.e. neural crest) cells emigrating from pieces of embryonic neural tube in culture. Examination of the effects of HNK-1 suggests that the antibody interacts with substratum-binding sites on the neural crest cell surface. On fibronectin-coated substrata the antibody tended to inhibit neurite outgrowth but left the cells relatively well spread, while on laminin substrata the effect was to discourage both neurite extension and cell spreading, causing cell retraction. These results suggest that the cell surface epitope recognized by HNK-1 influences neurite outgrowth, neurite adhesiveness or both. Failure of cell spreading on laminin suggests interaction with the laminin binding sites on the cell body. Elimination of the crest cells from mixed culture with sclerotome was achieved by culturing the cells in the presence of HNK-1 antibody and complement during the period required for complete cell outgrowth from the sclerotome explant.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Ethanol treatment inhibits mesoderm cell spreading in the gastrulating chick embryo.

Gastrulating chick embryos in culture were treated with ethanol solutions, following which the mesoderm cells migrating from the primitive streak were examined by scanning electron microscopy. Morphometric analysis of cell shape showed that mesoderm cells from treated embryos were significantly more rounded and therefore less well spread than controls, and showed fewer filopodial contacts with the overlying basement membrane. This result was only obtainable for cells leading the migration from the primitive streak, since the following cells in the mesodermal mass apparently did not show this difference. The ethanol concentration required to obtain a reliable effect was 5%, while lower concentrations produced highly variable results. The mesoderm cells were also examined for their in vitro responsiveness to ethanol by investigating their adhesiveness and cytoskeleton. No effect was observed on cell-glass adhesion as judged by interference reflection microscopy using up to 1% ethanol. This concentration did, however, disrupt the actin cytoskeleton of cultured cells when stained with NBD-phallacidin, but lower concentrations were ineffective. It is concluded that ethanol treatment of cultured embryos has a significant effect on the substratum relationships of some migrating mesoderm cells.

Animals↗
PubMed1987

Ultrastructural cytochemical localization of adenylate cyclase in the early chick embryo.

Adenylate cyclase activity was localized in various tissues of the early chick embryo using an ultrastructural histochemical technique. Reaction product was deposited on the lateral plasma membrane of all cells, but with a preferential localization at the apical terminal complex in the epiblast. There was no activity associated with the free surfaces of these or other cells in the embryo. Intracellular deposits were found in all cells associated with the endoplasmic reticulum, nuclear envelope and Golgi bodies. In the last organelle, the deposit was sometimes observed to be distributed through the stack in a non-uniform way, with the heaviest deposits occurring at the forming face. No clear difference could be detected between the cytochemical activity associated with cells in various regions of the embryo, or with embryos at different stages of early development.

Adenylyl Cyclases↗
PubMed1987

The effects of axotomy on bullfrog sympathetic neurones.

1. The effects of axotomy on the electrical properties of B cells in paravertebral sympathetic ganglia were studied using standard intracellular recording techniques. The effects were apparent after 1 week and persisted throughout the 47 days of study. 2. Action potential duration (spike width) and amplitude (spike height) were significantly increased in axotomized neurones. 3. The duration of the after-hyperpolarization which followed the action potential showed considerable scatter in control neurones (mean +/- S.E. of mean, 159.0 +/- 5.8 ms for 100 cells). Following axotomy, the duration was significantly reduced (50.9 +/- 2.3 ms for 97 cells). The amplitude of the after-hyperpolarization was also significantly smaller in axotomized neurones. 4. Changes in the characteristics of the action potential and the after-hyperpolarization in axotomized neurones were not due to alteration in resting membrane potential or input resistance which were unchanged after axotomy. Rheobase current was significantly increased. 5. There was neither a significant depression of the rate of rise or the amplitude of orthodromically evoked nicotinic e.p.s.p.s nor any obvious ultrastructural alteration following axotomy. 6. Despite the decrease in the duration of the after-hyperpolarization, the rate of discharge in response to constant current injection was little changed in axotomized neurones. 7. Although axotomy produces significant changes in several measurable electrophysiological parameters in bullfrog sympathetic ganglion cells, the present results imply that mature neurones are able to maintain relatively normal electrical activity despite injury.

Action Potentials↗
PubMed1987

Monensin inhibits secretion of extracellular matrix and the spreading of mesoderm cells in the early chick embryo.

Chick embryos in culture were treated with the secretion-inhibiting ionophore monensin at the gastrulation stage of development. After treatment for 5 h at a concentration of 10(-5) M the embryos showed a drastic reduction in the tissue space between the upper and lower epithelia, and reduced spreading of the mesoderm cells that occupied this space. The basement membrane, to which many mesoderm cells are normally attached, showed varying degrees of disruption, which permitted blebbing of the overlying epithelium. Intracellularly, the treatment caused extensive vacuolization in all tissues. These results were consistent with the effects expected by removal of hyaluronic acid from the extracellular space, and this was confirmed by demonstrating a sharp reduction in glucosamine incorporation in this region. Examination of the effects of monensin on isolated mesoderm cells in culture using interference reflection microscopy indicated that the spreading of these cells was reduced independently of the changes in the extracellular matrix of the embryo. That this was probably due to the inhibition of cell surface fibronectin secretion was shown by demonstrating severe changes in the distribution of this glycoprotein using the immunofluorescent technique. It is concluded that the effects of monensin on this intact, developing system are due primarily to the disruption of hyaluronic acid secretion, but that disrupted fibronectin synthesis contributes to the reduced spreading of mesoderm cells.

Animals↗
PubMed1986

Somitomeres in the chick tail bud: an SEM study.

In the chick embryo the final number of somites is achieved at about stage 22 of Hamburger and Hamilton. By this time the neural tube and notochord have reached the tip of the tail bud but some paraxial mesoderm remains unsegmented. In this study using scanning electron microscopy we show that somitomeres are present in this mesoderm. This indicates that the terminal paraxial mesoderm of the tail bud may have the potential to form supplementary somites, though we do not as yet know what prohibits the completion of segmentation to the tip.

Animals↗
PubMed1986

An experimental and morphological analysis of the tail bud mesenchyme of the chick embryo.

In the chick embryo, the tail bud reaches its maximum length at about stage 22 of Hamburger and Hamilton, after which it starts to regress. By this stage the neural tube and notochord extend right to the tip of the tail, but the somites do not do so, the terminal tail bud mesoderm never becoming segmented. The investigation is concerned with analysing why this mesoderm fails to segment. When tail buds were explanted to the chorio-allantoic membrane, they continued to form somites only until the "correct" number had segmented, i.e., the tail bud formed no more somites when isolated from the embryo than it would have formed if undisturbed. Morphological studies suggest that in the normal embryo massive cell death overtakes the tail bud mesoderm before it can segment. It is suggested therefore that cell death may be a contributory factor in preventing segmentation.

Allantois↗
PubMed1986

Epithelial and basement membrane responses to chick embryo primitive streak grafts.

Chick embryo primitive streak grafts, placed beneath the epiblast of host embryos, tend to result in the formation of either a neural plate in response to anterior streak grafts, or in de-epithelialization in response to posterior grafts. Ultrastructural and immunocytochemical examination shows that both reactions are preceded by basement membrane disruption and early removal of fibronectin therefrom. This disruption does not occur in response to non-streak grafts. It is suggested that the disruption, evoked by primitive streak cells, is a prerequisite first step, allowing direct graft-epiblast cell contact. This contact elicits a specific cytoskeletal reaction determining the epiblast response.

Animals↗
PubMed1986

The contribution of the primitive streak to the somites in the avian embryo.

Chick embryos were removed from the egg at stages 6-11 and explanted in culture. The greater part of the postnodal primitive streak of each embryo was replaced with a similar region taken from a corresponding quail embryo. The reciprocal experiment was also carried out, chick primitive streak being grafted in place of quail. After further incubation, the grafted primitive streak cells were found to contribute to lateral plate mesoderm, somites and intermediate cell mass. In an additional series of experiments, the postnodal primitive streak was extirpated and the embryo allowed to heal without a graft being inserted; after further incubation, many more somites formed in these embryos. It is concluded therefore that the contribution of cells from the primitive streak shown in the first experiment may not be essential for somite formation. It is suggested moreover that two major morphogenetic movements are taking place simultaneously in the mesoderm during this period: one is the mediolateral migration of cells after ingression through the streak, whilst the other is an anteroposterior movement associated with regression.

Animals↗
PubMed1986

Changes in the transferrin requirement of cultured chick embryo mesoderm cells during early differentiation.

Mesodermal tissue from the chick embryo at various stages of early differentiation was cultured in hydrated gels of type I collagen in the presence and absence of transferrin. Primary mesoderm explants from primitive-streak-stage embryos responded to the presence of avian transferrin by significantly improved outgrowth which appeared to be related to the ability of the cells to attach to, and migrate in, the collagen. No evidence was obtained which suggested that this observation was dependent on increased cell proliferation. This outgrowth enhancement was not duplicated by transferrin of human origin. The avian transferrin did not produce this effect on cells cultured on plastic substrata, suggesting that the species-specific effect involves modulation by the extracellular matrix. Mesoderm explants from somite stages of development showed no increase in outgrowth in the presence of either avian or human transferrin as judged by counting the number of outwandering cells. Ultrastructural immunocytochemistry indicated surface binding of transferrin by cells in the gels, and the presence of endogenous transferrin on the surfaces of mesoderm cells in situ and in their extracellular environment. It is suggested that by binding to cell surface receptors, transferrin may be able to influence the strength of cellular adhesion to collagen and hence the capacity for cell locomotion.

Animals↗
PubMed1984

Substratum attachment of embryonic mesoderm cells in culture.

The cell-substratum adhesive characteristics of cultured chick embryo primary mesoderm cells have been examined by interference reflection microscopy and transmission electron microscopy under various conditions. Correlations were drawn between the type of adhesion and the degree of motility shown by the cells. During the rapid spreading and motility of cells cultured on fibronectin-containing substrata, focal contacts (10 to 15-nm gap) were rare and close contacts (about 30-nm gap) were predominant. By contrast, when the cells were immobile, after 5 d in culture, extensive focal contacts were present, together with stress fibers. The results indicate that tight cell-substratum contact is incompatible with rapid cell motility and that fibronectin acts by inducing the formation of close contacts rather than focal contacts.

Animals↗