John G. Bluemink

Ligand-mediated osmium binding: Its application in coating biological specimens for scanning electron microscopy

The osmium-bridging properties of thiocarbohydrazide have been used to bind additional osmium to a variety of soft tissue surfaces before critical-point drying. The procedure gives satisfactory protection to specimens under a scanning electron beam at accelerating voltages between 5 and 25 kV and provides the microscopist with a simple, efficient, and economical alternative to evaporative coating with other heavy metals prior to analysis in the scanning electron microscope.

Intimate cell contacts at the epithelial/mesenchymal interface in embryonic mouse lung

The epithelial-mesenchymal interface in 11-, 12-, and 13-day lung was analyzed at the ultrastructural level with emphasis on extracellular matrix organization and the occurrence of cell contacts. The basal lamina is intact in 11-day lung (primary bronchus), but occasionally cell surface extensions establish intimate cell contacts between epithelium and mesenchyme in the distal region of the primordium. In 12- and 13-day lung the basal lamina is partly absent along distal outgrowths and sometimes the domains of epithelium and mesenchyme are difficult to define. Intimate cell contacts between epithelium and mesenchyme cells (50–90 A separation) are regularly seen. Collagen fibers (130 A thick) having a 30 A striation pattern are found mainly along quiescent regions. In general, extracellular matrix material is very scanty along distal outgrowths. Aggregates of electron-dense vacuoles are found almost exclusively in the basal cytoplasm of epithelial cells. A relationship with RER is shown. These organelles exhibit morphological characteristics reminiscent of collagen-secreting vacuoles. The findings are discussed in connection with current ideas regarding branching morphogenesis. It is suggested that transient intimate cell contacts between epithelium and mesenchyme may be instrumental in the initiation of bifurcation.

The first cleavage of the amphibian egg. An electron microscope study of the onset of cytokinesis in the egg of Ambystoma mexicanum

At the animal pole of the first-cleavage zygote of Ambystoma mexicanum (Amphibia) the plane of furrowing is first indicated by a stripe of pigmentation, which some minutes later splits into two. The area between the stripes deepens and widens, and on both sides stress marks appear. The surface smoothes out again when a shallow groove develops. These surface changes that mark the onset of cytokinesis were investigated by electron microscopy. It was shown that the egg surface in the region of the single and the double stripe is greatly enlarged by surface protrusions. In the cortex previously nonoriented filaments become polarized and aggregate. Light vesicles that apparently arise at the expense of stacked membranes from the diastema form a cortical layer. The pigment granules undergo changes, and in the subcortical cytoplasm yolk granules undergo degradation. These structural alterations are thought to represent the mobilization of cytoplasmic pools, giving rise to the appearance of multivesicular bodies and dense bodies. As a result of constringent surface movements, wrinkles and folds come into being. Cytoplasmic areas packed with osmiophilic stacked lamellae are found exclusively in that region of the primary furrow which is known to contribute directly to the formation of new surface. A possible functional relationship between the light vesicles and the layer of filaments was discussed in relation to the mechanism by which the karyokinetic event initiates furrow formation.

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity become extreme upon fertilization

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-(N-hexadecanoyl)-aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching indicating the existence of lipidic microdomains. In the unfertilized egg the mobile fraction of plasma membrane lipids (approximately 50%) has a fivefold smaller lateral diffusion coefficient (D = 1.5 X 10(-8) cm2/sec) in the animal than in the vegetal plasma membrane (D = 7.6 X 10(-8) cm2/sec). This demonstrates the presence of an animal/vegetal polarity within the Xenopus egg plasma membrane. Upon fertilization this polarity is strongly (greater than 100X) enhanced leading to the formation of two distinct macrodomains within the plasma membrane. At the animal side of the egg lipids are completely immobilized on the time scale of FPR measurements (D less than 10(-10) cm2/sec), whereas at the vegetal side D is only slightly reduced (D = 4.4 X 10(-8) cm2/sec). The immobilization of animal plasma membrane lipids, which could play a role in the polyspermy block, probably arises by the fusion of cortical granules which are more numerous here. The transition between the animal and the vegetal domain is sharp and coincides with the boundary between the presumptive ecto- and endoderm. The role of regional differences in the plasma membrane is discussed in relation to cell diversification in early development.

Cortical wound healing in the amphibian egg: an electron microscopical study

Changes following injury of the animal pole cortex of fertilized uncleaved eggs of Xenopus laevis were studied with the electron microscope. In the course of the healing process the egg surface bordering the exovate protruding from a tear wound is thrown into folds. Pigment granules aggregate in the region below the edges of the damaged area. Concomitantly filament arrays come into being there. Fuzzy material is present in a diffuse and in a condensed form. It coats the membrane in the region of the surface folds as well as membrane surfaces inside the exovate. Parts of the exovate surface are in the form of so-called “crenelated layer.” Probably this layer has a transitory sealing function and is homologous to the “new membrane” formed in wounded amoebae (18). The morphological alterations are interpreted as manifestations of membrane growth, active or passive contraction of filament arrays, and cytoplasmic coagulation. In this context Holtfreters concept of a “surface coat” and his interpretation of cortical wound healing is reexamined.

The action of cytochalasin B during egg cleavage in Xenopus laevis: dependence on cell membrane permeability.

Abstract By exposing Xenopus eggs during the first cleavage to cytochalasin B (CCB) for successive periods of 4 min, it has been shown that CCB sensitivity becomes manifest approximately 7 min after the onset of furrow formation. However, even before this time furrow regression can be induced by the injection of CCB under the membrane in the furrow. This shows that during the first 7 min of cleavage the operative contractile system is CCB sensitive. Using microelectrode techniques, electrical membrane characteristics (membrane potential and resistance) were measured continuously in normally cleaving eggs and in cleaving eggs injected with CCB. It was found that the onset of sensitivity to externally applied CCB coincides with a rapid alteration of the membrane potential and resistance. We have concluded that externally applied CCB can only enter the egg when the membrane permeability increases. No evidence has been found that CCB alters the ionic permeability of preexisting cell membrane.

An ultrastructural analysis of cell and matrix differentiation during early limb development in Xenopus laevis

Abstract Early development of the hind limb of Xenopus (stages 44–48) has been analyzed at the level of ultrastructure with emphasis on differentiation of extracellular matrix components and intercellular contacts. By stages 44–45, mesenchyme is separated from prospective bud epithelium by numerous adepidermal granules in a subepithelial compartment (the lamina lucida), a continuous basal lamina and several layers of collagen (the basement lamella). Tricomplex stabilization of amphoteric phospholipid demonstrates that each adepidermal granule consists of several membranelike layers (electron-lucent band 25–30 A; electron-dense band 20–40 A), which are usually parallel to the basal surface of adjacent epithelial cells. Collagen fibrils are interconnected by filaments (35 A in diameter) which stain with ruthenium red. Epithelial cells possess junctional complexes at their superficial borders, numerous desmosomes at apposing cell membranes and hemidesmosomes at their basal surface. Mesenchymal cells predominantly exhibit close contacts (100–150 A separation) with few focal tight junctions at various areas of their surface. By stages 47–48, adepidermal granules are absent beneath bud epithelium and layers of collagen in the basement lamella lose filamentous cross-linking elements. Filopodia of mesenchymal cells penetrate the disorganized matrix and abut the basal lamina. Hemidesmosomes disappear at the basal surface of the epidermis and mesenchymal cells immediately subjacent to epithelium exhibit focal tight junctions and gap junctions at their lateral borders. These structural changes may be instrumental in the epitheliomesenchymal interactions of early limb development. Degradation of oriented collagenous lamellae permits direct association of mesenchymal cell surfaces (filopodia) with surface-associated products of epithelial cells (organized into the basal lamina). Development of structural pathways for intercellular ion and metabolite transport in mesenchyme may coordinate events specific to limb morphogenesis.

The effect of injected cytochalasin B on filament organization in the cleaving egg of Xenopus laevis.

By injecting cytochalasin B (CCB) under the membrane in the furrow of Xenopus eggs, local surface contraction can be abolished almost immediately. Thus, the contractile filament system is CCB-sensitive. Electron microscopy of the regressed furrow shows that the organization of arrayed filaments is lost. Instead of the previous distinct filament layer, small bundles of filaments as well as single filaments are seen. It is concluded that injected CCB affects the contractile system, as a result of which filaments become disarranged but not necessarily degraded.

The plasma-membrane IMP pattern as related to animal/vegetal polarity in the amphibian egg

Freeze-fracture electron microscopy of the plasma membrane of the fertilized, uncleaved Xenopus egg shows that intramembranous particles (IMPs) range in size from ca. 50 to 200 A and that more IMPs are attached to the E-face than to the P-face. The overall IMP densities of the animal and the vegetal hemisphere do not differ significantly. IMP-free regions (∅, ca. 0.1 μm) on the tips of surface protrusions were irregularly distributed in the animal and the vegetal half (E-face) occupying ca. 8.5 and 2%, respectively of the free area. The relative densities for 16 different IMP sizes have been compared, on the basis of seven animal and seven vegetal halves, counting (E-faces only) ca. 10,000 IMPs in each hemisphere. For IMP sizes of ≤81 A, a significant difference (P < 0.0005) was found, more small IMPs being present in the animal half. Some evidence for IMP-associated thin elements was found. These findings are discussed in relation to plasma membrane anisotropy and the morphogenetic role of the egg cortex.

Animal-vegetal polarity in the plasma membrane of a molluscan egg: a quantitative freeze-fracture study.

Using freeze-fracture electron microscopy, the numerical particle distribution in the fertilized Nassarius egg plasma membrane has been analyzed in four areas at different positions along the animal-vegetal axis of the egg. These areas can be distinguished by distinct microvilli patterns and differences in microvilli densities. In all areas, more IMPs (intramembrane particles) are present on the P face than on the corresponding E face. The ratio of the number of IMPs present on E and P face is similar in all areas (0.48-0.55) except for the most animal part of the vegetal hemisphere, where relatively more IMPs remain attached to the exterior half of the fractured membrane (E/P ratio = 0.88). The IMP density at the vegetal pole of the egg is considerably higher than in the animal hemisphere and in the animal part of the vegetal hemisphere. This difference is due to an increased number of IMPs in all size classes (4-18 nm). In the area adjacent to the vegetal pole the density of particles is also higher than in the two more animal areas, but here the difference is exclusively due to the smaller IMP size classes (4-8 nm). Statistical analysis of our data reveals that the area adjacent to the vegetal pole patch is significantly different from the other areas with respect to the distribution of the IMPs over the different IMP size classes. These results demonstrate the polar organization of the Nassarius egg plasma membrane. The possible role of this surface heterogeneity in the spatial organization of the egg cell and the later embryo is discussed.