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.     

Teratologic effects of LSD in explanted early chick embryos.

Chick embryos were explanted at stage; 4-7 and cultured for 20 h with or without LSD. At any stage 10 mug/ml LSD or higher caused abnormalities in axial structures, particularly somites, in over 50% of the embryos. LSD had no apparent effect on morphogenesis of the heart, but significantly lowered the pulse rate. Cellular degeneration occurred in severely affected structures, but LSD at embryotoxic doses caused alterations in neither cell morphology nor mitotic activity. The effects of LSD were not permanent, i.e., the embryos retained the ability to undergo normal morphogenesis when, after 4-5 h of treatment with 10 mug/ml LSD, they were subcultured on plain nutrient medium.     

Neural tube closure defects caused by papaverine in explanted early chick embryos.

Papaverine (50 micrograms/ml) preferentially inhibited uplifting of neural folds in explanted stage 8 chick embryos. Affected neuroepithelial cells often lost their wedge-shaped and elongated appearance. Also, luminal surfaces of most affected cells were smoother than usual as evidenced by the marked decrease in the number of cytoplasmic extensions, but the integrity of other structures (including cytoskeletal components) was not noticeably affected. The observed changes in cell surface topography were due, at least in part, to the imparied ability of apical microfilaments to contract and their eventual relaxation. The "relaxing" effect of papaverine on neural folds could be reversed by subsequent treatment with ionophore A23187. Since papaverine and ionophore A23187 are known to alter the normal distribution of intracellular Ca2+ and changes in cell surface topography are correlated with contractile activities of apical microfilaments, papaverine elicits neural tube closure defects by lowering intracellular free Ca2+ levels, thereby relaxing contracted apical microfilaments in neuroepithelial cells.     

Inhibition of neurulation and interkinetic nuclear migration by concanavalin A in explanted early chick embryos.

Effects of concanavalin A (Con A) were tested in chick embryos explanted at stages 4–7 and cultured for 24 hr. Con A (12–24 μg/ml) blocked neural tube formation by inhibiting interkinetic nuclear migration, irrespective of the stage at explanation. Somites, although less numerous than controls, were almost normal in appearance. Blastodermal expansion, heart development, and blood island formation usually were unaffected. Light microscopic and autoradiographic studies showed that the application of Con A (16 μg/ml or lower) caused no obvious variations in cell morphology, mitotic activity, and uptake of [³H]thymidine and [³H]uridine. The grafting experiments showed that Con A (16 μg/ml) strongly inhibited the differentiation of Hensen's node grafts, but not their neural inducing capacity. Furthermore, the inhibitory effect of Con A was reversible, i.e., embryos retained the ability to recover from sublethal effects of Con A when, after 4–5 hr of treatment, subcultured on plain nutrient medium.     

In vitro crystallization of ribosomes from chick embryos

A new two-dimensional ribosome crystal, having the tetragonal space group P42(1)2 (a = 593 A), has been grown from ribosome tetramers extracted from hypothermic chick embryos. It is of particular interest because of its larger size (up to 3 x 3 micron2) and greater stability compared to other related polymorphic forms, and because it can easily be grown in large amounts. X-ray diffraction shows the order in the crystal to extend to a resolution of at least 60 A. The crystalline ribosomes appear to contain a full complement of small and large ribosomal subunit proteins and an additional four proteins not characteristic of chick embryo polysomes.     

Ultrastructural analysis of chick embryo blastoderms explanted in vitro in absence of endoderm. Differentiation capacity of mesoderm

A study of the structural characteristics of stage 5 chick embryo blastoderms, cultured in vitro, was carried out after previous elimination of endoderm to analyse the differentiation capacity of mesoderm. Our results show that only the precardiac mesoderm is determined at this early stage and that it is able to differentiate cardiac tissue in the absence of the endoderm. The ultrastructural characteristics of this tissue are described. However, the degree of differentiation of cardiomyoblasts does not reach the expected level of organization and myofibrillar complexity.     

Differentiation of lens fibers in explanted embryonic chick lens epithelia

Central regions of explanted lens epithelia from 6-day-old chick embryos were maintained in tissue culture for 4 weeks to determine the extent to which lens fiber differentiation would progress in vitro. Cellular outgrowth from the explants created 3 distinct zones; namely, a thick central zone, a thicker annular zone and a flattened peripheral zone. Cells of the central and annular zones underwent morphological and biochemical changes which correspond to the differentiation of lens fibers in vivo. The mean cell length increased a minimum of 25-fold. The nuclei in the longer cells became pycnotic; DNA remained in the nuclei but accumulated single-strand breaks. The cytoplasm became filled with a homogeneous granular matrix. Organelle density decreased, but microtubules persisted, mostly along surface membranes; free ribosomal clusters were present. There were occasional desmosomes and infoldings of cell membranes. The proportion of ribosomal RNA synthesized decreased relative to the total RNA synthesized, especially in the central zone. Finally, the proportion of delta crystallin synthesized increased to 40–50% of the newly synthesized protein. These data suggest that the transformation of lens epithelial cells into fibers results from a programmed differentiation which can take place in tissue culture.     

An electron-microscopical analysis of embryonic chick tissues explanted in culture

Summary Three types of tissue (hypoblast, germ wall and epiblast) were dissected from early chick embryos and explanted on Falcon plastic dishes. After they had settled and spread, the explants were fixed, usually within 18–24 h after explantation, and sections were cut through the tissue and the Falcon dish. The closeness of the cells to the substrate varied even within the same explant, but the epiblast tended to be closer to the substrate than did the hypoblast or germ wall. Plaques were present in all three tissues in regions where the cell processes contacted the substrate. Extensive desmosomes were visible in the epiblast explants, small desmosomes were present in the germ wall explants, but desmosomes were never seen in hypoblast explants. These differences in cell/substrate and cell/cell morphology are discussed in relation to the different behavioural characteristics of the three tissues. Some mixed cultures were also examined by electron microscopy. When the epiblast was confronted with either hypoblast or germ wall, it underlapped them at the region of contact.