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R R Markwald

Publications and source records attributed to R R Markwald.

At least 73 records · Page 4Linked to original sources
PubMed1985

The distribution and spatial organization of the extracellular matrix encountered by mesencephalic neural crest cells.

Cephalic neural crest (NC) cells enter a cell-free space (CFS) that contains an abundant extracellular matrix (ECM). Numerous in vitro investigations have shown that extracellular matrices can influence cellular activities including NC cell migration. However, little is known about the actual ECM composition of the CFS in vivo, how the components are distributed, or the nature of NC cell interactions with the CFS matrix. Using ultrastructural, autoradiographic, and histochemical techniques we analyzed the composition and spatial organization of the ECM found in the CFS and its interaction with mesencephalic NC cells. We have found that a specific distribution of glycoproteins and sulfated polyanions existed within the CFS prior to the translocation of NC cells and that this ECM was modified in areas occupied by NC. The interaction between the ECM components and the NC cells was not the same for all NC cells in the population. Subpopulations of the NC cell sheet became associated with ECM of the ectoderm (basal lamina) while other NC cells became associated with the ECM of the CFS. Trailing NC cells (NC cells that emerge after the initial appearance of NC cells) encountered a modified ECM due to extensive matrix modifications by the passage of the initial NC cell population.

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PubMed1985

Histological analysis of limb regeneration in postmetamorphic adult Ambystoma.

Previous investigation into the regenerative ability of postmetamorphic adult land phase Ambystoma has revealed that these species have the capacity to completely regenerate a limb, given optimal environmental conditions, and the gross morphological characteristics of limb regeneration in these species compared favorably with the external regeneration morphology of aquatic phase forms. The present study concerns a histological and histochemical examination of the regenerating limb tissues and their respective extracellular and intracellular tissue matrices. Postmetamorphic adult Ambystoma were amputated through the forearm, placed within optimal environmental conditions, and allowed to regenerate. The tissues were harvested at designated intervals after amputation and prepared for light microscopic examination. The limb tissues were assayed histologically for similarities to and differences from previously established regeneration morphologies. It was noted that specific correlations (i.e., apical epidermal cap formation, but outgrowth and elongation, palette formation, and digit formation) existed between regeneration histologies in these species and those previously reported for the aquatic urodeles, newt, axolotl, and larval salamander. By utilizing the histological and histochemical characteristics of the tissue, the regenerate limb was divided into five tissue units: epidermal, blastemal, soft, hard, and neuro/vascular. Based on the unique morphology of their extracellular matrices and respective histochemical staining patterns, four distinct blastemal regions were delineated within the blastemal units: subregenerate epidermal blastema, soft-tissue blastema, hard-tissue blastema, and core blastema. Histochemically, changing patterns of highly sulfated, weakly sulfated, and carboxylated polysaccharides and glycosylated compounds were located within both the extra- and intracellular stump and regenerate tissue matrices during regeneration. In addition, these patterns of intra- and extracellular macromolecular material correlated to previous reports of similar-type compounds assayed during regeneration in aquatic urodeles. With this in mind, the adult land phase Ambystoma can be considered an appropriate model system for studies concerning normal limb regeneration.

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PubMed1985

Protein extracts from early embryonic hearts initiate cardiac endothelial cytodifferentiation.

Prior to the formation of multiple chambers, the embryonic heart consists of two epithelial tubes, one within the other. As development proceeds, portions of the inner epithelium, i.e., the endothelium, undergo a morphological transformation into a migrating mesenchymal cell population. Our results show that this transformation is affected by proteins secreted by the outer epithelium, i.e., the myocardium, into the extracellular matrix between these two tissues. This conclusion is based on tissue autoradiographic studies of whole embryo cultures with 3H-amino acids. Continuous labeling conditions generated an apparent gradient of proteins extending away from the myocardium and contacting the endothelium just prior to the formation of mesenchyme, i.e., activation of the transformation sequence. Pulse/chase studies confirmed this directional movement of matrix protein. By performing sequential extractions of preactivation staged embryonic hearts with EDTA and testicular hyaluronidase followed by ammonium sulfate precipitation we obtained an enriched preparation of cardiac extracellular matrix. This fraction was capable of eliciting several of the events characteristic of endothelial activation in vitro. These events included: (i) cell-cell separation, (ii) lateral cell mobility, and (iii) hypertrophy and polarization of intracellular PAS staining (Golgi apparati). The biological activity of the extract was sensitive to heat denaturation: a homogenate of the remaining extracted tissue would not substitute for the matrix extract. Morphologically the extracted hearts appeared intact, however, the extracellular matrix space was significantly diminished. No more than 6% of the total lactic dehydrogenase activity, a cytosolic enzyme, was found in the extract. Preliminary electrophoretic characterization of the extract (metabolically labeled with 14C-amino acids) indicated that it may contain as many as 35 proteins or subunits. The relationship of ECM to endothelial differentiation in cardiac morphogenesis is discussed as a model for other developmental systems.

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PubMed1984

Effects of two glycosaminoglycans on seeding of cardiac cushion tissue cells into a collagen-lattice culture system.

A collagen-lattice culture model of developing heart valves was utilized to test two glycosaminoglycans, normally found in the cardiac jelly matrix of developing heart valve primordia, for their effects on the capability of mesenchymal derivatives of cardiac cushion endothelial cells to enter the substrate from the surface. Treatment with hyaluronate increased the rate of cell seeding to 2.04 times that of untreated control cultures and 1.82 times that of chondroitin sulfate-treated cultures. Scanning electron microscopic studies suggested that the increased rate was due to an enhanced disruption of intercellular junctions, influenced by hyaluronate, permitting disengagement of cells from the surface population and migration as mesenchymal cells into the collagen matrix. The results of this study correlate well with the presence of high hyaluronate concentrations in the cardiac jelly matrix beneath the cushion endothelium at periods of active seeding of cushion tissue cells.

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PubMed1984

Selected views of early heart development by scanning electron microscopy.

This tutorial on cardiac development is designed to acquaint the novice student of embryology with the key events that occur during cardiogenesis. Each of the following events is depicted through a series of scanning electron micrographs which convey the spatial relationships between minute, yet essential structures: fusion of paired heart tubes; looping; partitioning of the common atrium, the atrioventricular canal, the primitive ventricle and the outflow tract. The cellular and biochemical mechanisms responsible for these events cannot be adequately determined nor illustrated by SEM and therefore will not be considered in detail herein.

Animals↗
PubMed1983

Endocytic activity in embryonic cardiac cushion mesenchyme in vivo and in collagen gel lattices.

Mesenchymal cells, termed cushion tissue (CT) cells, are the principal cellular elements in atrioventricular (AV) endocardial cushions, the latter constituting AV septal and valvular primordia in the embryonic heart. Atrioventricular canals of 2 1/2 day chick embryo hearts were explanted onto collagen gel lattices, wherein cushion endothelial cells acquired the characteristics of CT cells and invaded the gel. The endocytic activity of in situ CT cells was compared to that of gel-cultured CT cells by exposing appropriate preparations to horseradish peroxidase (HRP), cationized ferritin (CF) or to lysozyme. Stimulation by these exogenous proteins resulted in phagocytosis. In addition, all three markers were associated with coated pits, smooth surfaced vesicles (45-60 nm), C- or cup-shaped structures and other lysosomal elements, but were excluded from Golgi cisternae and their entire vesicle population. In CT cells, HRP does not act solely as a "content" marker that reflects fluid-phase uptake. Instead it appears to follow adsorptive endocytic pathways. The endocytic behavior of in vivo and in vitro cushion tissue cells appears to be the same. Endogenous endocytic activity may reflect in part membrane retrieval, which counterbalances the extensive exocytosis observed in these cells.

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PubMed1983

Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue.

In normal heart development the endothelium of the atrioventricular canal, but not the ventricle, produces mesenchymal cells which seed (invade) into the intervening extracellular matrix toward the myocardium at around 64-69 hr of development. We have utilized three-dimensional collagen substrates to examine the initiation of seeding by atrioventricular canal endothelia in vitro and to compare and contrast the responses of the ventricular endothelia. Explants of atrioventricular canals and ventricles from staged embryos were placed on the surfaces of collagen gels prior to the onset of seeding in situ. At varied intervals of incubation, the explant was removed, leaving behind a monolayer on the surface of the gel which consisted of endothelial cells. Subsequently, the endothelial outgrowths were examined for seeded cells. The results confirm the regional endothelial differences seen in vivo. They also show that invasion of the collagen gels is due to an alteration in phenotype mediated by interaction with other components of embryonic heart explant. Lastly, the time course of this tissue interaction in vitro mimics the onset of seeding in vivo.

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PubMed1983

Use of 6-diazo-5-oxo-L-norleucine to study interaction between myocardial glycoconjugate secretion and endothelial activation in the early embryonic chick heart.

The glutamine analog, 6-diazo-5-oxo-L-norleucine (DON), a glycoconjugate inhibitor, was used to probe the relationships between myocardial secretion of extracellular matrix and endothelial differentiation and formation of cushion mesenchyme (primordia of A V values). When DON was given to stage 12 chick embryos maintained in shell-less culture, the myocardial secretion gradient of glucose- and sulfate-labeled matrix was blocked. Concomitantly, the endothelium failed to complete activation but continued to divide and incorporate thymidine. By varying DON concentration, two distinct phases of endothelial differentiation were identified: the first (labile to 0.5 micrograms) involved hypertrophy, the second (labile to 0.25 micrograms) acquisition of migratory appendages with resultant mesenchyme formation. Glucosamine + DON (but not inosine, glucose, or glutamine) restored the matrical secretion gradient and to varying degrees both phases of endothelial activation. Endothelia totally suppressed from forming mesenchyme in situ acquired this capacity when explanted into three-dimensional collagen gel culture. The capacity was enhanced by glucosamine given in situ as an inhibitory override, dependent upon serum concentration, inhibited by heat-inactivated serum or by adding DON to the medium, but unaffected by hyaluronate. These results were compared to those obtained by co-culturing endothelium and myocardium and discussed in terms of the hypothesis that cushion mesenchyme formation results from an epithelial interaction mediated by glycoconjugates.

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PubMed1981

Structural response of adult rat Sertoli cells to peritubular fibroblasts in vitro.

Sertoli cells were harvested from sexually mature rats and maintained in vitro for up to ten days (Sertoli cell-enriched cultures) or co-cultured with rat peritubular fibroblasts. Cultures were examined by differential light microscopy and by scanning or transmission electron microscopy. The presence of peritubular fibroblasts in co-culture greatly enhanced plating efficiency and viability of adult Sertoli cells. Sertoli cell aggregates preferentially adhered to peritubular cells, and by ten days had flattened and spread across these cells. Sertoli cells retained their characteristic ultrastructural features. Early in co-culture a collagen-like extracellular material was seen bridging Sertoli and peritubular cells, and its appearance was coincident with the presence of swollen rough endoplasmic reticulum in peritubular cells. Interperitubular cell spaces became engorged with a fibrillar material morphologically similar to the basal lamina of the seminiferous tubule wall. In the absence of peritubular cells, in culture medium "conditioned" by prior incubation with peritubular cells but not containing them, or when cultured with other fibroblastic cells, plating efficiency was low; Sertoli cells never flattened and cell ultrastructure progressively degenerated. The results indicated that peritubular cells support adult Sertoli cells in culture, possibly via extracellular material derived from peritubular cells, and suggest that Sertoli cells have an inductive effect on peritubular cell secretory activity.

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PubMed1981

Endocardial cushion tissue development: structural analyses on the attachment of extracellular matrix to migrating mesenchymal cell surfaces.

The progressive growth and eventual fusion of the atrioventricular (AV) endocardial cushions is of critical importance to normal embryonic heart development. Failure to do so would result in septal and AV valvular defects. A central feature in initial cushion growth is the migration of cushion tissue (CT) cells through an heterogeneous extracellular matrix (ECM) which has previously been shown (in particular hyaluronate) to modify migratory behavior. Attention was directed to migrating CT cells to determine if (1) their surfaces physically attach to or bind ECM and (2) are modified to suggest a morphological basis for cell:matrix interaction. The migratory appendages (filopodia) of CT cells maintained in organ culture attached both to collagenous microfibrils coated with polyanionic material and hyaluronate (HA) enriched ECM. The cell:matrix associations were of sufficient strength to restrain the cell from contracting following freezing procedures and were labile to mild trypsin treatment. HA enriched matrix persisted at the cell surface even after treatments which removed most free ECM, but was readily removed by hyaluronidase and trypsin digestion. Freeze fracture analyses revealed 16-18 nm particles elevated above the plane of the filopodial surface which closely interfaced with ECM components. These particles were variably distributed, ranging from almost homogenous dispersion to focalized clusters, but were absent on surrounding non-migratory (myocardial) cells. Results are consistent with a model in which cell attachment to its migratory substratum is mediated by polyanions (probably sulfated glycosaminoglycan and fucosylated glycoprotein) and detachment by hyaluronate.

Animals↗
PubMed1981

Structural analysis of extracellular matrix prior to the migration of cephalic neural crest cells.

Cephalic neural crest cells enter cell free areas containing abundant extracellular matrix (ECM). Previous histochemical studies have identified both sulfated and non-sulfated glycosaminoglycans within this matrix. In the present study, ultrastructural examination of the ECM demonstrated an anastomosing network of pleomorphic, cetyl pyridinium chloride-dependent strands within cell free spaces and in association with the basement membrane of the surface ectoderm. Thin section analysis revealed that the strands consisted of three components: (1) 3-5 nm filament meshwork; (2) electron dense amorphous material and (3) 30 nm granules. In contrast, the ECM associated with the basement membrane consisted principally of a continuum of electron dense, amorphous material. The molecular ordering of ECM within crest cell pathways was compared to the well-characterized, hyaluronate-rich, premigratory matrix of cardiac jelly.

Animals↗
PubMed1980

A histochemical analysis of polyanoinic compounds found in the extracellular matrix encountered by migrating cephalic neural crest cells.

Neural crest cells destined to form craniofacial primordia initially are "seeded" into and subsequently migrate through the extracellular matrix (ECM) of a cell free space (CFS) between the surface ectoderm and the underlying mesoderm. Utilizing histochemical procedures for polyanionic compounds, we have demonstrated that both sulfated and nonsulfated glycosaminoglycans (GAG) are present in the CFS of the cephalic region of the chick embryo and that their distribution and structural organization vary with the passage of neural crest or mesodermally derived (MD) mesenchymal cells through it. In stages 7 and 8 embryos a predominance of fine filamentous strands composed primarily on nonsulfated, carboxyl-rich GAG is seen spanning intercellular spaces between adjacent tissues and MD mesenchymal cells. In older embryos (stages 9 and 10) much of the filamentous material is replaced by coarse fibrillar strands or amorphous material which coats the surfaces of MD mesenchymal and neural crest cells as they invade the CFS. Using enzymatic digestions (Streptomyces and testicular hyaluronidase) and the critical electrolyte concentration procedure, data suggest that the fine filamentous matrix onto which the neural crest cells migrate consists mainly of hyaluronate with lesser amounts of chondroitin and some sulfated GAG present. The coarse fibrillar matrix that appears after passage of either neural crest or MD mesenchymal cells through the original CFS contains strongly sulfated polyanionic material, predominantly chondroitin sulfates A, C. Since GAG is located ubiquitously within the ECM of embryos at various stages, the role of GAG, if any, in the transfer of developmental information may be of a general nature (ie. stimulus of motility) rather than of specific morphogenetic cues (for specific differentiation into craniofacial primordia).

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PubMed1980

Sialic acid: regulation of electrogenesis in cultured heart cells.

Removal of up to 50% of the sialic acid from aggregates of 7-day chick embryo heart cells during incubation in a highly purified preparation of neuraminidase resulted in hyperpolarization of the maximum diastolic potential, reduction in the slope of diastolic depolarization leading to slowing of beating, negative shifts of threshold potential and the voltage at which upstroke velocity was maximal, and an initial increase in the action potential overshoot. These effects were opposite to those induced by phospholipase C, a possible contaminant. The modification of electrical properties by neuraminidase is suggested to result from an enhanced influx of calcium ions that might "screen" or bind specifically to internal negative fixed charges and thereby shift the voltage dependence of conductance and kinetic parameters to more negative potentials. This hypothesis is supported by the results above and by greater uptake of 45Ca following release of sialic acid, augmentation of enzyme-induced changes in elevated Ca2+, and the similarity of effects produced by A23187, and inophore which also increased 45Ca uptake. However, other mechanisms cannot be ruled out at the present time.

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