Oocyte shuttle, a recombinant protein transporting donor DNA into the Xenopus oocyte in situ.

The newly developed oocyte shuttle protein contains a streptavidin moiety that tightly binds biotinylated DNA. Injected intravenously into adult Xenopus females, the protein-DNA complex is rapidly transported through the bloodstream and, within the ovary, the vitellogenin ligand present in the protein binds to the receptors at the surface of the oocytes. The bound complex is internalized and translocates into the oocyte nucleus thanks to an SV40 nuclear localization signal, enhanced by an adjacent casein kinase phosphorylation site. Functioning of the shuttle protein is documented by transporting DNA molecules that, upon intramolecular homologous recombination within the oocyte nucleus, express easily traceable markers such as green fluorescence or tetracycline resistance.

Xenopus helveticus, an endangered species?

This review traces the history of Xenopus research in Switzerland, its worldwide beginnings and British chapters having been summarised previously (Gurdon and Hopwood, 2000). As in other countries, Xenopus was initially used in the pharmaceutical industry at Basel for pregnancy testing. Developmental biologists became interested in this peculiar amphibian because it may be induced to ovulate all year round. Swiss Xenopus research is reviewed over 50 years, from the introduction of Xenopus by Rudolf Weber to the University of Bern, the return from Great Britain of two Swiss expatriates, Michail Fischberg and Max Birnstiel through the numerous pupils of the founder labs to the independently arisen Xenopus research units in the country. Besides developmental biology, Swiss Xenopus research engaged in immunology, genetics and cell biology, the latter focusing mainly on the oocyte. It set highlights in molecular biology by isolating some of the first eukaryotic genes and analysing their transcriptional regulation and post-transcriptional modifications through 'surrogate genetic' approaches in the oocyte system. An important line of research applied this system to study nuclear trafficking. Presently, functional testing mainly serves to characterise the function of proteins produced from expression vectors injected into the oocyte. A main accent of developmental studies was, from the early beginnings and still today, set on molecular characterisation of gene function in the embryo.

Retinal patterning by Pax6-dependent cell adhesion molecules.

Long-standing evidence gained from Pax6 mutant embryos pointed to an involvement of Pax6-dependent cell adhesion molecules in patterning the central nervous system and, in particular, the retina. However, direct evidence for such pathways remained elusive. We here present direct evidence that knockdown of Pax6 expression by morpholino antisense molecules in Xenopus embryos and knockdown of maternal N-cadherin (mNcad), N-cadherin (Ncad) and neural cell adhesion molecule (NCAM) produce similar phenotypes. Eye formation is reduced and retinal lamination is heavily disorganized. In Pax6 knockdown embryos, the levels of mRNAs coding for these cell adhesion molecules are markedly reduced. Overexpression of Pax6 efficiently rescues the phenotype of Pax6 knockdown embryos and restores expression of these putative target genes. Rescue of Pax6-deficiency by the putative target gene mNcad moderately rescues eye formation. The promoters of the genes coding for cell adhesion molecules contain several putative Pax6 binding sites, as determined by computer analysis. Chromatin immunoprecipitation shows that, in embryonic heads, Pax6 binds to promoter regions containing such predicted binding sites. Thus, several cell adhesion molecules are direct target genes of Pax6 and cooperate in retinal patterning.

Expression of alphasmooth muscle actin in lens epithelia from human donors and cataract patients.

In order to re-evaluate functional implications of alphasmooth muscle actin (alphaSMA) expression in lens epithelial cells (LECs), we assessed its presence in donor lenses without visible opacities (DON), lenses with mature cataract (CAT), and cataractous lenses with posterior subcapsular opacities (PSO) or anterior subcapsular fibrosis (ASF). The levels of alphaSMA and transforming growth factor-beta2 (TGFbeta2) mRNAs were measured by classical and real-time PCR. Expression and structural organisation of alphaSMA protein and beta-catenin were monitored by Western blotting and confocal microscopy. All DON analysed contained measurable amounts of alphaSMA mRNA. In CAT without and with PSO, mRNA expression was increased and, again more than doubled in ASF. TGFbeta2 mRNA expression varied widely between the individual samples but was slightly increased in ASF. No correlation existed between alphaSMA or TGFbeta2 expression and the age of the donors in any of the lens categories. Confocal microscopy revealed that, in DON and CAT, alphaSMA was preferentially expressed in a simple granular pattern in single or small clusters of LECs within a normally shaped cobblestone epithelium. Locally, the granules were merged into short stretches at the cell margin. In CAT, a few abnormally shaped cells contained polygonal alphaSMA structures and short stress fibres. In CAT with PSO and ASF, polygons and stress fibre bundles predominated in spindle-shaped cells. Expression patterns of different complexity were often present in the same epithelium. Apical polygons and basal stress fibres were detected within the same cell and may reflect instability of the interface between epithelium and cortical fibres and changes in adhesion to the capsule, respectively. High levels of betacatenin mRNA and protein were present in all lens types. However, with increasing complexity of alphaSMA organisation, betacatenin staining disappeared from the cell margin and basal infoldings and was shifted towards the cytoplasm and nucleus. The presence of alphaSMA in DON, the absence of any correlation between mRNA level and age, and the modest increase in complexity of alphaSMA-containing structures in CAT argue against an inevitable link between alphaSMA expression and the development of age-related cataract. Low levels of alphaSMA expression may reflect repair of normal wear and tear. In pathologic situations such as PSO and ASF, persisting stimulation and additional incentives may induce increased alphaSMA expression and more elaborate patterning, eventually leading to completion of EMT.

Antisense inhibition of Xbrachyury impairs mesoderm formation in Xenopus embryos.

Expression of the Xbrachyury (Xbra) gene was inhibited by antisense RNA synthesized in situ from an expression vector read by RNA polymerase III, injected into the fertilized egg or the 2-cell stage embryo of Xenopus laevis. Antisense-treated embryos had markedly reduced levels of Xbra mRNA and protein, and showed deficiencies in mesodermal derivatives and axis formation. In particular, organization of the posterior axis was affected, but often the anterior axis was also reduced. Some embryos failed to form mesoderm altogether and remained amorphous. The antisense effect is dose-dependent and may be "rescued" by overexpression of Xbra. In Xbra-deficient embryos, expression of several mesodermal genes (Xvent, pintallavis, Xlim, Xwnt-8 and noggin) was reduced to varying degrees, whereas goosecoid levels remained normal. The modified expression levels were partly normalized when Xbra deficiency was rescued. The observation that antisense inhibition yields slightly different phenotypes from dominant-negative inhibition suggests the recommendation of using several surrogate genetic approaches to determine the functional role of a gene in Xenopus development.