Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution

Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural observations on chick bone processed by quick-freezing and freeze-substitution

Summary The ultrastructure of newly formed bone was examined with the use of quick-freezing followed by freeze-substitution. Osteoblasts and young osteocytes were characterized by a smooth cell contour, whereas old osteocytes were irregular in shape. The plasma and intracytoplasmic membranes were clearly identifiable as trilaminar substructures. With the method described herein the tissue is handled in the anhydrous state. Thus mitochondrial granules could be demonstrated in all samples, since their preservation is not affected by non-aqueous solutions. The matrices of intact mitochondria were densely stained with poststaining. The contents of the Golgi complex, rough-surfaced endoplasmic reticulum (RER), nuclear envelope, vesicles, and vacuoles were stained to various degrees. Lacunar spaces were always filled with flocculent and filamentous materials, and the plasma membrane was in direct contact with them. Membrane-bounded matrix vesicles were clearly visible within the osteoid extracellular matrix which was the initial site of mineral crystal deposition. In heavily mineralized bone matrix, the periodic pattern of collagen fibrils was retained, and the electron density of mineralized matrix in freeze-substituted and unstained sections which had been floated on ethylene glycol was greater than that encountered in sections processed in aqueous reagents.     

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution

Summary Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of ⁴⁵Ca from prelabelled eggs. Exchangeable ⁴⁵Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.     

Glycosphingolipid biosynthesis in early chick embryos

Early chick embryos at the age of 22 h (neurula stage) and 48 h (20-25 somite stage) were explanted from eggs and cultured in vitro in the presence of radioactive sugar precursors. Metabolically labelled glycosphingolipids were isolated. Amongst these, neutral and acidic components, the latter including sulfatide and gangliosides, were identified. Cleavage by exoglycosidases, as well as immunostaining with antibodies on thin-layer plates, showed that at both embryonic stages glycosphingolipids were synthesized that belong to the globo series (globoside, Forssman glycolipid), the lacto series (lactoneotetraosylceramide, nLc4Cer, and two nLc4Cer-based gangliosides, a monosialo and a disialo species), and the ganglio series (ganglioside Gtet1a and higher sialylated derivatives).     

Presence of serotonin in early chick embryos

With biochemical analysis and with autoradiography based on injection of 5-[3H]hydroxytryptophan, it was possible to demonstrate the presence of serotonin (5-hydroxytryptamine) in early chick embryos as early as the pre-streak stage. The biochemical analysis which covered the early developmental period (0.5-6 days of incubation) revealed an elevated concentration of serotonin at gastrulation; from then it stayed at a lower and fairly even level. Autoradiographs of embryos at the pre-streak stage, the primitive streak stage, the head fold stage and the 4-6 somites stage indicated the presence of serotonin in intracellular yolk granules and in cell nuclei. Moreover, the amine appeared associated with microfilaments and microtubules, particularly in developing neural cells. Notably the elevated concentration of serotonin at gastrulation, but also the intracellular distribution of the amine during early organogenesis, indicates a prominent role for it in cell-shape changes and morphogenesis in the early chick embryo.     

Endogenous lectin secretion into the extracellular matrix of early embryos of Xenopus laevis

An endogenous galactoside-binding lectin with subunit molecular weight of 43,000-45,000, previously detected in unfertilized eggs of Xenopus laevis, persists at high levels in embryos through gastrulation. During embryonic development the lectin is found in cytoplasmic vesicles, and then is secreted into extracellular matrix which is prominent around the blastopore and on the roof of the blastocoel. The lectin is also found in the extracellular material in the developing neural fold. The presence of lectin at sites of active morphogenetic movements raises the possibility that it participates in the formation of an extracellular matrix that influences these processes.     

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution. Validity of the method

Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2 +/- 0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3 +/- 1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of 45Ca from prelabelled eggs. Exchangeable 45Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.     

Rearrangement of the morphological structure and degradation of the extracellular matrix in amphibian embryos after short-term disruption of cell contacts

The changes in cell structure of explants of presumptive early gastrula mesoderm of Xenopus laevis and dynamics of extracellular matrix formation has been studied for 6 h after their isolation. After 6 h, control explants of any dorsoventral level turned into dense spheric homogeneous cell clusters. Explants of dorsal and lateral sectors, treated with medium without Ca2+ and Mg2+ for 30 s, bent and separated into several layers, and in ventral sector explants, cavities, surrounded by polarized cells, arose. In control explants, the amount of extracellular matrix on the surface and in intercellular spaces increased, and in the experimental explants, extracellular matrix disappeared and was not observed for several hours. Previously described differential transformations of the treated explants (Georgiev, Belousov, 1986) are supposed to be determined by extracellular matrix degradation and the degradation-associated complications of cell structure of the explant.     

Development of extracellular matrix in chick paravertebral sympathetic ganglia

Alcian blue staining coupled with enzyme digestion or critical electrolyte staining revealed differences in the development of extracellular matrix (ECM) within sympathetic ganglia compared with the surrounding capsule. On day 5 of chick development (Hamburger-Hamilton stage 26) only hyaluronic acid (HA) could be detected in the ECM surrounding condensing primary ganglia. By day 7 (st 30) the ganglionic capsule contained HA, as well as sulfated glycosaminoglycans (GAGs), and this pattern continued into the adult stage. During the later stages of embryonic life (st 41-45) satellite cells appear, showing fine structural characteristics that point to their role in the secretion of intraganglionic ECM. Only during these stages could ECM be detected histochemically within ganglia, the same stages (days 15-19) when routine electron microscopic methods reveal collagen fibrils embedded in a granular ground substance. Thus, the intraganglionic environment appears as a separate compartment free of detectable amounts of GAG until late embryonic stages when ECM is secreted around satellite cells. This developmental pattern could represent a role of ECM in the histological stabilization of ganglia during the late stages of differentiation, since the appearance of intraganglionic ECM is correlated with the appearance of small dense-cored vesicles characteristic of adult neurons. The developmental pattern of ECM in differentiating sympathetic ganglia is compared with that of other tissues that undergo condensation and morphogenesis.     

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.     

An extracellular fibrillar matrix in gastrulating sea urchin embryos

Fine structural studies of fractured developing sea urchin embryos revealed the existence of a voluminous, fibrillar, extracellular matrix composed of fine filaments, twisting fibers and granules lining the blastocoel of midgastrula embryos. Glycine disaggregated embryos also exhibited this material. The fibrillar matrix is closely associated with the basal lamina of the ectodermal cells of the embryo and histochemical studies suggest it is composed mostly of sulfated glycosaminoglycans. The position of the matrix within the blastocoel as well as its organized association with embryonic cell surfaces is consistent with the hypothesis that it plays a major role in guiding the invaginating archenteron during gastrulation.     

Tendon collagen fibrillogenesis is a multistep assembly process as revealed by quick-freezing and freeze-substitution

The ultrastructure of chick embryo tendons has been examined after quick-freezing by liquid helium and freeze-substitution. Several stages of collagen assemblies were observed: intracellular packing of SLS-like aggregates surrounded by membrane containing areas with a clathrin coat; fine non cross-striated filaments connecting the cell membrane at 1 pole of the cells and collagen fibrils; tufts of filaments directly linked to collagen fibrils. This study reveals that some stages are more constant and abundant than supposed (the intracellular SLS-like aggregates) and that other extracellular assemblies that were hypothesized but usually badly preserved by conventional electron microscopy are clearly captured by the method.     

Cell junctions in explanted tissues from early chick embryos

Summary 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.