Immunocytochemical studies of the interactions of cadherins and catenins in the early Xenopus embryo.

Linkage of cadherins to the cytoskeleton is crucial for their adhesive function. Since alpha- and beta-catenin play a key role in this linkage, these proteins are possible targets for processes that control cell-cell adhesion. To achieve a better understanding of the regulation of cell-cell adhesion in embryonic morphogenesis, we used immunohistology to investigate how in Xenopus blastomeres catenins respond to disturbances in the expression of maternal cadherins. Overexpression of myc-tagged maternal cadherin leads to a proportionate increase of the level of beta-catenin. The two proteins colocalize in the endoplasmic reticulum, in cytoplasmic vesicles, and along the cell membrane, indicating that the beta-catenin binds to overexpressed cadherin early in its passage to the plasma membrane. Expression of cadherin is essential for the stable presence of beta-catenin, as depletion from maternal cadherin mRNA leads to a complete loss of beta-catenin from the blastomeres. alpha-Catenin behaves differently. Overexpression of cadherin leaves the amount and localization of alpha-catenin largely unaffected, and additional cadherin inserts itself into the membrane without a proportionate rise in the level of membrane-bound alpha-catenin. However, cadherin mRNA depletion leads to a redistribution of alpha-catenin from the membrane to the cytoplasm. Thus, cadherin is required to localize alpha-catenin to the membrane, but the amount of alpha-catenin along the membrane seems to be restricted to a certain level which cannot be exceeded. The relevance of these observations for the regulation of cadherin-mediated cell adhesion in the Xenopus embryo is discussed. Additionally, we demonstrate that plakoglobin, like beta-catenin an armadillo repeat protein, shows neither accumulation after overexpression nor colocalization with the overexpressed cadherin.

The homeobox-containing gene XANF-1 may control development of the Spemann organizer.

At the beginning of gastrulation the homeobox-containing gene, XANF-1, is expressed at a low level throughout the animal hemisphere of Xenopus laevis embryos, with a local maximum of expression in the region of the dorsal blastopore lip. By the end of gastrulation expression ceases everywhere except in the most anterior part of the neurectoderm. We have investigated the functions of this gene by microinjecting XANF-1 mRNA in the blastomeres of the 32-cell stage embryo and have observed the following effects. First, microinjections of the mRNA in the animal blastomeres and the blastomeres of the marginal zone elicited massive migration of cells to the interior of the embryo at the early gastrula stage. Second, overexpression of XANF-1 in the ventral marginal zone (VMZ) resulted in the appearance of an additional centre of gastrulation movements and the formation of a secondary axis. In addition we showed that synthetic XANF-1 mRNA was able to cause dorsal-type differentiation in VMZ explants extirpated from the microinjected embryos at the beginning of gastrulation. These results suggest that XANF-1 may control the main functions of cells of the Spemann organizer.

[A new method of comparative analysis of gene expression and identification of differentially expressed mRNA]. / Novyi metod sravnitel'nogo analiza ékspressii genov i identifikatsii differentsial'no ékspressiruiushchikhsiia mRNK.

A novel method of comparative gene expression analysis is proposed which is based on representing a population of expressed mRNA sequences in the form of a set of discrete cDNA restriction fragments. By means of selective isolation of 3' terminal cDNA fragments, each mRNA species is represented by no more than one cDNA fragment of specific length and sequence. Populations of cDNA fragments from different cell types are separated by high-resolution gel electrophoresis, and the separation patterns are compared. Using the proposed approach, fragments of two genes differentially expressed in murine thymus and spleen were identified and cloned. One of the genes was found to encode the terminal deoxynucleotidyltransferase, the there is apparently a heretofore unknown gene.

[The role of mechanical stresses in the formation and orientation of vessel-like structures in explants of clawed toad embryos]. / Rol' mekhanicheskikh natiazhenii v obrazovanii i orientatsii sosudopodobnykh struktur v éksplantatakh émbrionov shportsvoi liagushki.

The role of mechanical tensions in the formation of blood vessel-like tubes and lumens in embryonic Xenopus laevis mesenchyme was studied using ectomesoderm explanted from the area of ventral blood islands. The mechanical tensions in extracellular matrix of the explanted mesenchyme were caused by slightly squeezing the explants between two parallel coverslips. Long-term deformations (50 min and more) followed by incubation for 40 min resulted in the formation of vessel-like tubes and circular structures by mesenchymal cells oriented along the plane of stretching. The circular structures were similar to those formed by angioblasts during development of the blood vessel lumen. The comparison between long- and short-term (3 min) stretching experiments demonstrated that these effects represent the result of active cell reactions rather than of a rapid passive cellular response to stretching of the substrate. It is suggested that similar morphogenetic reactions take place during the vessel tube formation in the course of embryonic vasculogenesis.