Peter Hausen

β-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos

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.

Changes in the nuclear lamina composition during early development of Xenopus laevis

Changes in protein composition of the nuclear lamina were monitored during early development in Xenopus. Lamin LIII, the only lamin present in oocyte nuclei, serves as a lamin pool for the formation of pronuclei and early cleavage nuclei. It is present in embryos up to the tail bud stages. Lamins LI and LII, the lamins originally found in adult cell nuclei, appear at characteristic times in development. LI first appears at the midblastula transition (MBT), and LII at the gastrula. Tryptic peptide analysis revealed that all three lamin forms found in the embryo are identical with the adult lamins. De novo synthesis of LIII and LI, observed at MBT, is independent of transcription and must therefore be due to activation of maternal mRNAs. These results are discussed in relation to other nuclear changes occurring during early development.

Enzyme from Calf Thymus Degrading the RNA Moiety of DNA-RNA Hybrids: Effect on DNA-Dependent RNA Polymerase

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.

Fates and states of determination of single vegetal pole blastomeres of X. laevis

Vegetal pole cells of Xenopus morulae contribute progeny to all three germ layers, but from the midblastula stage onward they contribute only to the endoderm. We have investigated whether this restriction in fate reflects cell determination by implanting labeled vegetal pole cells into the blastocoels of host embryos and asking which structures later include labeled progeny. Single vegetal pole cells from the morula and also from the midblastula stage can contribute progeny to all germ layers. At the early gastrula stage the cells can contribute only to the endoderm. Thus the restriction of fate in the midblastula does not reflect cell determination. However, the cells do become determined by the beginning of gastrulation.

The Use of Polyacrylamide as an Embedding Medium for Immunohistochemical Studies of Embryonic Tissues

Fixed amphibian embryos and tadpoles were impregnated with acrylamide. After polymerization the gels were frozen and sectioned on a cryostat. The preservation of gross and fine morphological features in these sections is comparable to conventional paraffin sections and they can be stained by immunohistochemical methods without affecting the quality of the preparations.

The fate of oocyte nuclear proteins during early development ofXenopus laevis

SummaryThe localization and movements of four nuclear proteins, originally contained in the germinal vesicle ofXenopus oocytes, were followed through early development from cleavage to late neurula. The study made use of monoclonal antibodies directed against germinal vesicle proteins. Biochemical methods showed that all proteins persist in the embryo without a change in molecular size or gross concentration. At early stages the proteins are localized preferentially in the cytoplasm of the animal hemisphere. They shift from the cytoplasm to the nucleus at stages specific for the individual proteins. During mitosis the proteins are released from the nucleus into the cytoplasm.

Two-dimensional gel analysis of the fate of oocyte nuclear proteins in the development of Xenopus laevis.

The amphibian oocyte nucleus is thought to provide a maternal store of protein required in embryogenesis. The fate of germinal vesicle proteins has been studied by comparing polypeptide patterns of oocytes, embryos, and several adult organs of Xenopus laevis on two-dimensional gels. A combination of silver staining and fluorography of radiolabeled protein on gels was used to analyze maternal and newly synthesized polypeptides in embryogenesis. Comparison of protein patterns was facilitated and corroborated by application of monoclonal antibodies against several germinal vesicle proteins. These were characterized by immunoblotting from two-dimensional gels, and polypeptides of identical structure were recognized in oocyte nuclei, embryos, and tadpoles. The following conclusions were drawn: (1) Almost all prevalent germinal vesicle proteins can be continuously traced in embryos up to swimming tadpole stages, although their patterns of new synthesis are greatly different, some are not radiolabeled in the embryo but solely provided by the maternal store. (2) Many of the polypeptides occurring in oocyte nuclei are also found in one or several organs of the adult. (3) Tissue specificities of germinal vesicle proteins, previously detected by immunocytochemistry with monoclonal antibodies, could be confirmed by independent biochemical methods. (4) As has been previously shown by immunohistological methods, oocyte nuclear antigens are shed into the cytoplasm of the maturing egg, and are reaccumulated in the nuclei of the embryonic cells, each at a characteristic developmental stage. These shifts between intracellular compartments are not accompanied by a change of the covalent structure of the antigen.

Pre-MBT patterning of early gene regulation in Xenopus: the role of the cortical rotation and mesoderm induction

Patterning events that occur before the mid-blastula transition (MBT) and that organize the spatial pattern of gene expression in the animal hemisphere have been analyzed in Xenopus embryos. We present evidence that genes that play a role in dorsoventral specification display different modes of activation. Using early blastomere explants (16-128-cell stage) cultured until gastrula stages, we demonstrate by RT-PCR analysis that the expression of goosecoid (gsc), wnt-8 and brachyury (bra) is dependent on mesoderm induction. In contrast, nodal-related 3 (nr3) and siamois (sia) are expressed in a manner that is independent of mesoderm induction, however their spatially correct activation does require cortical rotation. The pattern of sia and nr3 expression reveals that the animal half of the 16-cell embryo is already distinctly polarized along the dorsoventral axis as a result of rearrangement of the egg structure during cortical rotation. Similar to the antagonistic activity between the ventral and the dorsal mesoderm, the ventral animal blastomeres can attenuate the expression of nr3 and sia in dorsal animal blastomeres. Our data suggest that no Nieuwkoop center activity at the blastula stage is required for the activation of nr3 and sia in vivo.

Tissue specific nuclear antigens in the germinal vesicle ofXenopus laevis oocytes

SummaryA library of hybridoma cell lines has been established which produce monoclonal antibodies against antigens from the germinal vesicle ofXenopus laevis oocytes. Many of the antigens are also found in the nuclei ofXenopus embryonic cells in culture. The fate of two of these antigens during embryogenesis was traced by immunofluorescence on embryo and tadpole sections. Early in development these antigens appear to be evenly distributed in the nuclei of all cells. In later stages they gradually disappear from most embryonic structures but are strongly accumulated in the nuclei of some specific cell types and organs.

Integrin α5 during early development of Xenopus laevis

Abstract The full length sequence of the Xenopus integrin α 5 subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of α 5 mRNA occurs in the embryo. Furthermore, a variant form of the α 5 mRNA is expressed which encodes an integrin α 5 subunit with a truncated cytoplasmic domain. Integrin α 5 mRNA and protein are expressed in oocytes, eggs and throughout development. Spatial expression of α 5 mRNAs is first detected by whole mount in situ hybridization in presumptive neural crest cells and in the somitic mesoderm from the midgastrula stage onwards. In contrast, the α 5 protein is present on newly formed plasma membranes beginning at first cleavage. During neurulation, the integrin α 5 subunit disappears from the outer layer of the ectoderm, the notochord and the neural tube and accumulates in the sensorial layer of the ectoderm, the somites and the neural crest cells. These results provide evidence for the position specific regulation of α subunit expression in early vertebrate embryos.