[Studies on the localization of NPY-like immuno-reactivity in the nervous system during development of Cynops orientalis].

By immuno-cytochemical method the localization of neuropeptide Y (NPY) in the nervous system during embryonic development of Cynops orientalis was studied. The results revealed that NPY was first localized in the peripheral nervous system (late tail-bud stage), and later appeared in the central nervous system (larval stage) where it appeared with the appearance of glial cells. Very probably with the migration of neural crest cells NPY appeared first in the peripheral nervous system and then distributed to the central nervous system.

[The antagonistic effect of naloxone on the epidermal conductivity of amphibian embryos].

The immunocytological work revealed that in amphibia beta-endorphin is distributed in embryonic epidermis during development with prominent expression at late tail bud stage which coincides with the stage of epidermal conductivity of full strength. To see whether any relationship exists between the presence of beta-endorphin and the epidermal conductivity of amphibian embryos, naloxone of 1 and 2 mumol/L was used. It has been demonstrated that after 45-60 minutes in the naloxone solution the embryonic epidermal conductivity disappeared and 90-120 minutes after the beginning of naloxone treatment the epidermal conductivity was again recovered. It was suggested that the opiate peptides, such as beta-endorphin, play some role in the epidermal conductivity of amphibian embryos.

[Intercellular relationship of notochord determination of Xenopus laevis].

During the process of determination, the presumptive notochord is situated beneath neuroepithelium, flanked at two sides by presumptive somites and underlain with archenteron roof ventrally. Among these neighbouring embryonic tissues, presumptive somites were found to exert the main influence on notochord determination. By electron microscopic observations, the presumptive notochord and somite cells were seen to situate either close to each other (plate I, Fig. 1) or connected by cytoplasmic processes forming intercellular lumen (plate I, Fig. 5). Coated pits and coated vesicles appeared at the outer surface of both types of cells (plate I, Figs. 1-4). For the presumptive somite cells, spherical bodies of different sizes and variable contents were observed either near or protruding from the outer surface (plate II, Figs. 6-10). The spherical bodies were also found in the intercellular lumen (plate III, Fig. 11). These spherical bodies were mainly composed of granules, loosely scattered or densely packed. The granules were of similar size and similar shade of electron staining as those of ribosomes of the presumptive somite cells. For the presumptive notochord cells, no spherical bodies of the above mentioned type were found, but phenomenon of engulfing luminal material was observed (plate III, Fig. 12). The significance of the appearance of these spherical bodies in the determination of notochord cells has been discussed.

Determination of notochord cells of Xenopus laevis.

In amphibians, numerous works of influences of the notochord on neighbouring tissues have been accumulated. However, on the contrary, scarcely any work is known about how the notochord is influenced by its neighbouring tissues and how it is determined. By using the experimental method of explantation and culturing in vitro, how the notochord is determined in the early development and whether the neighbouring tissues exert influences on it have been investigated. The results showed that the determination of notochord is a progressive process and the presumptive notochord of Xenopus appears to be a very good material to study influences of neighbouring tissues on the determination of the notochord.

The effect of a phorbol ester on amphibian myogenesis.

The effect of a phorbol ester (TPA) on myogenesis of urodelian embryo has been investigated. Presumptive somites were extirpated from mid-neurula, the stage when they have already attained certain degree of determination. The explants were treated with TPA (10 ng/ml in Steinberg solution with the addition of 10% Leibovitz's L-15) for 4 days, the period during which the frequency and size of gap junctions in normal myogenic cells are at the maximum. After the treatment many cells became dispersed and isolated while others spread out and remained connected in the form of a sheet. During further culture for 6 days in the solution without TPA myogenesis was blocked. Both the dispersed cells and the cells in a sheet remained at the earliest stage of muscle differentiation with irregular shape or in the form of myoblast, while cells of the control series formed myotubes containing bundles of myofibrils. The result suggests that the blockage of myogenesis is the consequence of interruption of junctional communication evoked by TPA treatment.

Effect of antibodies against the gap junction protein on differentiation of induced embryonic cells.

Microinjections of antibodies against gap junction protein to early blastomeres of Xenopus laevis were succeeded by the treatments of mesodermal inductor. It was found that induction was not affected by the injection of the antibodies but the differentiation of notochord and muscle was impeded at different degrees. Cell interactions via gap junction communication seem to be involved in the differentiation of the induced cells.

Ultrastructural changes of nuclei after treatment with mesodermal inducing substance.

Ultrastructural changes occur in the nuclei of the presumptive ectoderm cells of the early gastrula of Cynops orientalis after treatment with a mesodermal inducing substance (guinea pig bone marrow extract). The large condensed chromatin masses in the nuclei of the control series are no longer found; they become more or less dispersed. The successive changes of chromatin from condensed to dispersed have been observed. And in connection with the observations on neural induction after Ca-free treatment, the transformation from heterochromatin to euchromatin as the initiation of cell differentiation has been discussed.