The question of how dorsal-ventral polarity is established in vertebrates is central to our understanding of their early development. Several lines of evidence suggest that wnt-signaling is involved in the induction of dorsal-specific gene expression in the Spemann Organizer of amphibians. Here, we show that beta-catenin, acting as a component of the wnt-pathway, transiently accumulates in nuclei on the dorsal side of Xenopus and zebrafish blastulae. The spatially restricted nuclear translocation of beta-catenin precedes the expression of dorsal-specific genes. In experimentally ventralized frog embryos the dorsal ventral pattern of beta-catenin nuclear staining is abolished; in contrast, embryos hyperdorsalized by Li-ions or by injection of Xwnt8 mRNA exhibit an enhanced nuclear accumulation of beta-catenin. The results show that translocation of beta-catenin into nuclei in the wake of wnt-signaling is an early step in the establishment of the dorsal-ventral axis in frog and fish embryos.

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An enzyme present in extracts from calf thymus degrades specifically the RNA moiety of DNA-RNA hybrids. Other nucleic acids, such as single- or double-stranded DNA and single- or double-stranded RNA, are not affected to a comparable degree. If prepared free of the hybrid-degrading enzyme, RNA polymerase from calf thymus shows a fivefold increase in activity on denatured DNA as compared to native DNA.

Extracts from calf thymus contain an enzyme degrading the RNA moiety of DNA-RNA hybrids. An assay for this enzyme and several characteristics of the enzyme reaction are described. The specificity of the enzyme for DNA-RNA hybrids is indicated by several lines of evidence : The RNA of the hybrid is rendered resistant to the action of this enzyme upon heating. Nucleic acids other than DNA-RNA hybrids are not affected to a comparable degree. The enzyme rapidly degrades polyribouridylic acid if it is hybridized to polydeoxyriboadenylic acid, but not if it is hybridized to polyriboadenylic acid. Experiments with double labelledDNA-RNA hybrids show that specifically the RNA moiety of the hybrid and not the DNA is attacked by the enzyme.I n experiments on the purification of RNA polymerase from calf thymus we observed that RNA synthesis on denatured DNA is possible only when highly purified enzyme preparations are used, whereas crude extracts are inactive in this respect. Since both preparations are effectively synthesizing RNA on native DNA, this result indicated that a factor is present in the crude extract which specifically interferes with RNA synthesis on denatured DNA. Further experiments showed that the crude extracts contained an enzyme which rapidly degrades the RNA synthesized on denatured DNA but leaves the product of the polymerase reaction on native DNA virually unaffected. Since it is known that the RNA synthesized by RNA polymerase on denatured DNA forms a hybrid with the template [I-31, the above observations indicated the presence of an enzyme which specifically degrades RNA if the latter is hybridized to DNA. This specificity was confirmed in different experiments described in this communication. An enzyme with these properties has, to the best of our knowledge, not been described before. For this reason we propose the name ribonuclease H (H standing for hybrid) for this enzyme. A preliminary report of these observations has already been published [4]. these preparations was purified from calf thymus by a method described in the accompa.nying paper [5]. The purification procedure included extraction of the enzyme at low salt concentration ; precipitation of deoxyribonucleoprotein by 0.1 M (NH,),SO,; precipitation of the enzyme by (NH,),SO, and chromatography on a DEAE-cellulose column. All characteristics of the purified enzyme were similar to those described for other preparations from bacterial or mammalian sources. For some experiments DNA-RNA hybrids were prepared using RNA polymerase from Escherichia coli. Their behaviour was in all respect identical to that of hybrids obtained with calf thymus RNA polymerase. MATERIALS AND METHODS SubstratesFor the synthesis of hybrid I00 pg of calf thymus DNA were denatured by heating to 100" for I0 min followed by rapid cooling. The DNA was incubated together with 2 pmoles of ATP, GTP, and CTP, I pC [14C]UTP (56 pC/pmole) and 250 pg of RNA polymerase in 10 ml of 2 mM MnCl,, 0.1 M (NH4),S04, 0.03 M Tris pH 7.8, 5 mM mercaptoethanol. After 30 min at 37" sodium d...

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Tight junctions (TJs) perform a critical role in the transport functions and morphogenetic activity of the primary epithelium formed during Xenopus cleavage. Biogenesis of these junctions was studied by immunolocalization of TJ-associated proteins (cingulin, ZO-1 and occludin) and by an in vivo biotin diffusion assay. Using fertilized eggs synchronized during the first division cycle, we found that membrane assembly of the TJ initiated at the animal pole towards the end of zygote cytokinesis and involved sequential incorporation of components in the order cingulin, ZO-1 and occludin. The three constituents appeared to be recruited from maternal stores and were targeted to the nascent TJ site by different pathways. TJ protein assembly was focused precisely to the border between the oolemma-derived apical membrane and newly-inserted basolateral membrane generated during cytokinesis and culminated in the formation of functional TJs in the two-cell embryo, which maintained a diffusion barrier. New membrane formation and the generation of cell surface polarity therefore precede initiation of TJ formation. Moreover, assembly of TJ marker protein precisely at the apical-basolateral membrane boundary was preserved in the complete absence of intercellular contacts and adhesion. Thus, the mechanism of TJ biogenesis in the Xenopus early embryo relies on intrinsic cues of a cell autonomous mechanism. These data reveal a distinction between Xenopus and mammalian early embryos in the origin and mechanisms of epithelial cell polarization and TJ formation during cleavage of the egg.

The Xenopus blastula consists of two morphologically distinct cell types. Polarized epithelial cells build up the embryonic surface and fence off an inner non-polarized cell population. We examined the establishment of this early functional cell diversification in the embryo by single cell analysis, in vitro cell culture, and transplantation experiments. Single blastomeres from a 64-cell embryo (1/64 cells) exhibit several features of polarized cells. The plasma membrane of 1/64 cells consists of an apical domain, which is inherited from the original egg membrane, and a basolateral domain derived from newly formed membrane during cleavage. These are inherent, cell-autonomous properties of the blastomeres, as they form and are maintained in blastomeres raised in the absence of any cell interactions in calcium free medium. Upon in vitro culture a single 1/64 cell gives rise to an aggregate of two different cell types. Cells carrying a part of the former egg membrane domain differentiate into polarized epithelial cells, whereas cells lacking this membrane domain are not polarized. These results demonstrate that the inclusion of the egg membrane, rather than external signals related to the position of a cell in the intact embryo, is required for the apicalhasolateral differentiation of the surface epithelium. This view is supported by cell transplantation studies. A single 1/64 cell was implanted into the blastocoel of a stage 8 blastula embryo. The progeny of the implanted cell proliferate within the host embryo and split into two morphologically distinct populations with different cell behaviours. Cells incorporating a part of the egg membrane form coherent patches of polarized epithelial cell sheets in the interior of the host embryo. In contrast, cells lacking egg membrane do not exhibit any characteristics of polarized cells and eventually spread into different regions of the host embryo. Our results show that the egg membrane and/or components of the submembrane cortex play a determinative role in the formation of the blastula epithelium.

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