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Apoptosis and differentiation in presumptive neural retina (PNR) and presumptive retinal pigmented epithelium (PRPE) wert investigated during early retina development of toad, Bufo raddei Strauch. TUNEL staining was used to evaluate apoptotic cells and the immunohistochemistry was used to assess the expression levels ofglial fibrillary acidic protein (GFAP), RT97 and tyrosinase (Tyr) during early eye development respectively. The density of apoptotic cells and protein expression were quantitated with Image-Pro Plus 6.0. Apoptosis was found in both PNR and PRPE and the density of apoptotic profiles in PRPE was higher than that in PNR (most P<0.01) at the same stage during early eye development. The expression levels of GFAP and RT97 changed from low to high in PNR, but from high to low in PRPE, whereas the expression level of Tyr, was contrary to those of GFAP and RT97 in both PNR and PRPE. The point of intersection of these, increase and decrease respectively was found at 5-6 h after formation of optic vesicle (FOV). PRPE becomes thinner than PNR, one of the reasons might be due to higher density of apoptosis in PRPE than that in PNR during early eye development. Molecular differentiation, however, occurred after the contact of the optic vesicle outer wall with the overlying ectoderm which promotes the expression of specific molecules and inhibits the expression of non-specific molecules in PNR and PRPE respectively.

On the basis of the analysis of cDNA of three new homeobox-containing genes from human, chicken, and newt, a new class of homeobox genes Anf is characterized homologous to the Xanf-1 gene from Xenopus laevis, earlier cloned by us. These genes may be largely involved in the specification of embryonic subdivisions in the forebrain region of the embryo. The homeodomains of the proteins encoded by these genes differ greatly in the primary structure from all previously described homeobox genes. The high variability of the homeodomain sequences of the proteins of this class imply their rapid evolution.

Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see Mikhaĭlov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin.

The ability of various zones of the cephalic and trunk ectoderm to differentiate into adenohypophysis after the contact with the bottom of the prosencephalon was studied in tissue culture of chick embryos as the stage of 10-13 somites. Stomodeal presumptive lens ectoderm and lateral cephalic ectoderms were shown to be competent for development into adenohypophysis. In all cases adenohypophyseal cords were formed in the zones of ectoderm contact with the brain. The cords contained antigens A-2, A-3 specific for chicken adenohypophysis as well as ACTH and beta-lopotropin. Trunk ectoderm proved to be incapable to differentiate into adenohypophysis.

Cytochemical investigation on qualitative composition of polysaccharides in definitive and provisory anlagen of a normal 17-day-old human embryo "Krym" is performed. Basing on cytophotometric data, quantitation of glycogen and glycoproteins in the germ layers, their derivatives and in the chorionic membrane are analyzed. At early stages of differentiation glycogen and glycoproteins are demonstrated to predominate. Their greatest amount is noted in the ectoderm and its derivatives. In the chorionic membrane the total amount of glycogen and glycoproteins is prevailed in the epithelial components. Villose cytotrophoblast produces glycogen 3 times as great as the plasmodium. In the chorionic lamina there is noted a 2-fold increase of glycogen, respectively. At the same time, plasmodiotrophoblast increasingly produces glycoproteins to ensure, already at the given stage, the formation of the hemo-chorionic barrier in the system "mother--fetus" and production of gonadotropins. Studying biosynthesis of glycosaminoglycans, it is possible to suggest that at the stage of embryogenesis a young connective tissue differentiating at greater speed in the periepithelial zone is formed.

Spatial and temporal pattern and quantities of nicks in nuclear DNA during gastrulation and neurulation was studied using nick-translation in sections of Xenopus laevis embryos. Specific changes in the number of nicks in different mesoderm and ectoderm regions were detected during embryogenesis. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and inner ectoderm layer of early and middle gastrula. The gradient was inverted during transition from gastrula to neurula. At the same time dorso-ventral (in mesoderm) and ventro-dorsal (in outer ectoderm layer) gradients of nuclear labelling were increased. The intensity of nuclear labelling in all parts of embryo as a whole was remarkably higher during neurulation as compared with gastrulation. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and ectoderm during neurulation. A connection between the nicks and differentiation status of the cells during early embryogenesis in amphibians is suggested.