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Biomedical subjects

F Harrisson

Publications and source records attributed to F Harrisson.

At least 55 records · Page 3Linked to original sources
PubMed1987

Immunohistochemical localization of S-100 protein, glial fibrillary acidic protein, and neuron-specific enolase in the pars distalis of quail, rat, and human hypophyses.

In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.

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PubMed1987

Differential distribution of cell protrusions on the ventral surface of the deep layer in gastrulating quail and chick embryos.

The ventral surface of the deep layer of gastrulating quail and chick embryos was examined using scanning electron microscopy. On the basis of cell protrusions, three or four different cell types were recognized. Cells covered with microplicae were found in the caudal region of the germ and as a narrow band extending along the lateral and anterior borders of the area pellucida. Cells covered with microvilli were found in a horseshoe-shaped zone in the anterior part of the germ. Beneath the rostral end of the primitive streak, the flattened deep-layer cells exhibited intercellular ridges and few microvilli. This area was surrounded by cells that usually had extended microvilli. The pattern of these cell types is discussed in relation to the formation of the different tissues that compose the deep layer in gastrulating embryos.

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PubMed1986

Microinjection of glycosaminoglycan-degrading enzymes in the chicken blastoderm. An ultrastructural study.

The relationship between the presence of glycosaminoglycans (GAGs) and the morphology of the middle layer or mesoblast was examined by performing transmission electron microscopy of chicken blastoderms microinjected with GAG-degrading enzymes. The controls included microinjections with saline or trypsin, as well as solid-phase assays for proteolytic activity in commercially available GAG-degrading preparations. The results indicate that, in normal as well as in saline-injected blastoderms, middle-layer cells are rounded or cuboidal in shape, and are linked to each other by small intercellular junctions in the primitive-streak region. As they migrate laterally along the basal lamina, they appear as typical mesenchymal cells, being separated by large intercellular spaces and covered by cell processes. The removal of hyaluronate (by the microinjection of hyaluronidases) led to compaction of the middle-layer cells in the area lateral to the primitive streak. These cells lost their mesenchymal aspect and retracted their processes, and intercellular junctions were observed. The presence of proteolytic activity in the enzyme preparations did not interfere with the results. On the basis of the results obtained using this microinjection technique, we were able to confirm at the ultrastructural level that hyaluronate, due to its space-creating properties, promotes the detachment of ingressed primitive-streak cells and preserves the mesenchymal aspect of the middle layer during the lateral migration of single cells along the basal lamina. Whether the presence of hyaluronate is necessary to allow positioning of the mesoblast could not be inferred using our experimental procedure. We present evidence that this molecule, as well as having physicochemical properties, is also involved in the modulation of tissue interactions during gastrulation.

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PubMed1986

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)

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PubMed1986

On the presence of proteolytic activity in glycosaminoglycan-degrading enzyme preparations.

A solid-phase protease assay has been used to screen different commercial preparations of glycosaminoglycan-degrading enzymes for the presence of proteolytic activity. Proteases cannot be detected in preparations of testicular hyaluronidase and of chondroitinase at the concentration used for histochemical purposes. Commercial Streptomyces hyaluronidase contains proteolytic contaminants detectable at the concentration used for histochemistry. At higher concentrations, all preparations appear to be contaminated with proteases. The results obtained using this assay suggest that addition of a mixture of proteinase inhibitors containing N-ethylmaleimide, EDTA, pepstatin, and phenylmethanesulfonylfluoride or soybean trypsin inhibitor has little effect on the proteolytic activity of the glycosaminoglycan-degrading enzyme preparations, irrespective of the pH used. Moreover, the use of EDTA in this mixture is questionable. This study also describes two testicular hyaluronidase preparations that may be particularly useful in functional studies of the living organism, as they are only slightly contaminated.

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PubMed1985

Fibronectin and its relation to the basal lamina and to the cell surface in the chicken blastoderm.

The ultrastructural distribution of fibronectin immunoreactivity was investigated in the chicken embryo during late gastrulation. Sites of binding of anti-fibronectin antibodies were ascribed to the basal lamina and associated structures, and to the cell surface. The fibronectin-rich basal lamina was resolved into a lamina densa, which appears as a continuous, dense sheet, a lamina lucida, consisting of anchoring cords between lamina densa and epithelial cells, and a lamina intima, closely juxtaposed to the cell surface. Cell-surface labelling was also observed in mesoblast cells, and along the dorsal side of the deep-layer cells. The ventral side of the latter cells was poorly stained in the endophyllic crescent, except in coated pits, and more regularly stained at the level of definitive endoblast. Some structures associated with the basal lamina reacted intensely with anti-fibronectin antibodies. These are the interstitial bodies, which are aggregates of extracellular material, and a kind of fibril or tubule, embedded in a fibronectin matrix and mainly found in the endophyllic crescent. Some intracellular labelling was found in most deep-layer cells, in few epiblast cells, never in mesoblast cells. These results extend previous studies on the localization of fibronectin, and correlate its presence and surface topology with its postulated role in migration of mesoblast cells on the basal lamina which, chemically, constitutes an appropriate substrate.

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PubMed1985

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms.

A chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of 3H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of 3H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1985

Masking of antigenic sites of fibronectin by glycosaminoglycans in ethanol-fixed embryonic tissue.

We studied the interaction between glycosaminoglycans (GAGs) and fibronectin in the basement membrane of the epiblast in the chicken blastoderm using testicular-hyaluronidase digestion of GAGs either on fixed tissue sections or in vivo after microinjection of the enzyme preparation prior to immunostaining for fibronectin. In the choice of fixatives, special attention was paid to their preservation of GAGs. The controls included alcian-blue staining of serial sections to test the efficiency of the digestion, and incubations in the presence of protease inhibitors to abolish contaminating proteolytic activity in the commercial hyaluronidase preparations. The results indicate that fixation in solutions which preserve GAGs, i.e. ethanolic solutions or aqueous solutions containing cetylpyridinium chloride, allows the immunocytochemical demonstration of fibronectin in the basement membrane of the epiblast at the level of the endophyllic crescent, but masks this glycoprotein at the epithelial-mesenchymal interface. As shown by both approaches, this masking of immunoreactivity is reversible. Moreover, the in vivo clearance of GAGs before fixation shows that the masking at the epithelial-mesenchymal interface is not an experimental peculiarity due to the use of a particular technique, but is the consequence of an interaction between GAGs and fibronectin in that particular area of the basement membrane that is used by mesoblast cells as a substrate for migration. The observation that fibronectin may be masked by GAGs in ethanol-fixed tissue--a commonly used fixation method--may require the re-evaluation of some negative results mentioned in the literature.

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PubMed1985

Cell junctions and locomotion of the blastoderm edge in gastrulating chick and quail embryos.

The blastoderm edge migrates by the active locomotion of a multilayer of epithelial cells, the so-called margin of overgrowth (MO), that uses the vitelline membrane as its substratum. The structural unity formed by the margin of overgrowth cells and their rapid migration suggest coordination of locomotion between individual cells. Using transmission electron microscopy of thin sections and freeze-fracture, we attempted to determine if the pattern of junctions of the migrating margin of overgrowth is related to the suggested cell--cell cooperation between individual cells in this region. In the leading edge there are large areas of closely apposed cell membranes. Incipient desmosomes and small gap junctions were observed. Tight junctions consisted of isolated strands or isolated networks of tight-junctional strands. In the proximal part of the margin of overgrowth the size of the gap junctions increased and the desmosomes were fully developed. Tight-junctional strands were either isolated or arranged into an isolated network. A broad belt of tight junctions was observed at the transition between margin of overgrowth and non-marginal cells. The distribution of the junctional elements in the MO suggests that junctions contribute to the maintenance of the structural and functional organization of the margin of overgrowth. Furthermore, the spatial distribution of the junctions might give information about the mechanism of locomotion of the margin of overgrowth.

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PubMed1984

Expression of different regional patterns of fibronectin immunoreactivity during mesoblast formation in the chick blastoderm.

The appearance and distribution of the extracellular material glycoprotein, fibronectin, was investigated in gastrulating chick embryos using affinity-purified anti-human plasma fibronectin antibodies. Preservation of tissue structure and immunoreactivity was carried out by ethanol/acetic acid fixation or by formaldehyde/glutaraldehyde fixation. Using the former fixation method, fibronectin immunoreactivity was detected (1) at the ventral surface of the upper layer or epiblast, mainly anterior and lateral to Hensen's node, in regions where middle-layer or mesoblast cells are not yet present, and (2) sparsely in extracellular spaces of the deep layer. Using the latter fixation method, fibronectin immunoreactivity was, moreover, found at the entire ventral surface of the upper layer, i.e., also at the epithelial-mesenchymal interface, where a basement membrane was previously described. At the light microscope level, we could not detect significant immunoreactivity in the middle layer. Treatment of sections of ethanol-fixed blastoderms with testicular hyaluronidase before immunostaining for fibronectin partially demasked the antigenic sites of this glycoprotein at the epithelial-mesenchymal interface. The present report indicates that the different regional patterns of fibronectin immunoreactivity in the basement membrane of the upper layer are spatially and temporally correlated with migration and positioning of mesoblast cells. These regional patterns are probably due to differences in the composition of fibronectin-associated material such as chondroitin sulfate A and/or C proteoglycans, and/or hyaluronate, before and after mesoblast expansion, rather than to differences in the distribution of fibronectin itself. In this respect. In this respect, it is noteworthy that the chemical composition of the basement membrane of an epithelium changes as mesenchyme cells migrate over it. The results also favor the idea that fibronectin is a structural component of the whole basement membrane which is used as a substrate for migration of mesenchymal cells.

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PubMed1984

Microinjection in the chick blastoderm. An improved method to study the extracellular matrix in the living organism.

A microinjection technique for the chick blastoderm is described. With a micropipette attached to a de Fonbrune micromanipulator, 25-45 nl of a reagent was injected into the entophyllic crescent of a chick blastoderm explanted in vitro according to New [7]. This procedure offers the advantage of eliminating the concentration variability which was observed after subblastodisc injection, and in contrast to the in ovo techniques, it allows one to stage the blastoderms properly. To check its applicability, testicular hyaluronidase was injected. On the basis of morphological and histochemical observations we ascertained that the experimental procedure itself did not interfere with the results. This method may provide a reliable experimental procedure with which to study the interactions between several macromolecules and the tissues during morphogenesis.

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PubMed1982

The relationship between the folliculo-stellate network and the thyrotropic cells of the avian adenohypophysis.

The folliculo-stellate network of the avian adenohypophysis consists of stellate cells surrounding colloid-containing follicular cavities into which cilia and microvilli project. Other identifying criteria are agranularity, junctional complexes at the apical pole, presence of cytoplasmic processes ramifying between adjacent secretory cells, and close appositions of plasma membranes linking folliculo-stellate cells and presumptive thyrotropic cells. Transmission electron microscopy reveals that TRH and L-DOPA induce simultaneous ultrastructural changes in the folliculo-stellate network and in the thyrotropic cells. TRH transforms at cell of the cephalic lobe into a highly hypertrophic cell in which enlargement of cisterns of rough endoplasmic reticulum containing secretory granules, development of a large Golgi complex, presence of newly synthesized secretory granules, and granulation of the cytoplasm are the main features. In the meantime, the follicular cavities become dilated by large amounts of homogeneous colloid. The administration of L-DOPA also leads to the development of dilated cisterns in presumptive thyrotropic cells of the cephalic lobe. Intracisternal granules, immature secretory granules, and large Golgi complexes, however, are not observed. Degranulation of the cytoplasm is obvious. The follicular cavities of both cephalic and caudal lobes are enlarged and filled with colloid in which granular elements are noted. The ultrastructural changes observed in thyrotropic cells and in the folliculo-stellate network reflect functional changes induced by the experimental manipulation. These changes may be related, directly or indirectly, or completely independent.

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PubMed1981

Peripheral avian yolk assemblage and its persistence in the blastoderm, studied by trypan blue-induced fluorescence.

Shortly after subcutaneous or intraperitoneal injection of nontoxic quantities of trypan blue into laying Japanese quails, red fluorescent yolk granules appear in the peripheral ooplasm of their oocytes at the end of the lampbrush stage or subsequently. Later a red fluorescence can be observed in the apical cytoplasm of the granulosa cells. The results obtained by this method confirm our previous results (Callebaut 1979) obtained by autoradiography after 3H-leucine administration and furnish interesting additional data. The trypan blue-induced fluorescence method gives a good indication of the permeability of the oocytal cortex and its derivative the germinal disc. The avian yolk which is, or has been peripherally assembled (primordial, true white and yellow yolk) can be characteristically labelled by the administration of trypan blue. The injection of higher, still nontoxic quantities of trypan blue has a prolonged "retarding" effect and permits the marking of a broader part of the germinal disc or eventually of the blastoderm which develops from it.

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