Involvement of Myt1 kinase in the G2 phase of the first cell cycle in Xenopus laevis.

During cleavage of Xenopus laevis, the first mitotic cell cycle immediately following fertilization is approximately 90 min and consists of S, G2, and M phases. In contrast, the subsequent eleven cell cycles are approximately 30 min and consist mostly of S and M phases. The balance between Cdc25 and Wee1A/Myt1 is thought to be crucial for Xenopus first cell cycle progression; however, the role of Myt1 in this period has not been fully investigated. In this study, we examined the roles of Myt1, Wee1A, and Cdc25A in the first cell cycle of Xenopus laevis. Inhibition of Cdc25A with antisense morpholino oligonucleotides lengthened the duration of the first cell cycle to some extent, whereas it was slightly shortened by ectopic Cdc25A expression, suggesting that the low concentration of Cdc25A during the first cell cycle does not fully account for the long duration of this cycle. Using the Wee1A antisense morpholino oligonucleotide and neutralizing antibody against Myt1, we found that Myt1 phosphorylates and inhibits Cdk1 much more effectively than Wee1A during the first cell cycle in Xenopus. Taken together, these results suggest that the activity of Myt1 is predominantly responsible for the duration of the long G2 phase in the first mitotic cell cycle in Xenopus.

Transgenic frogs expressing the highly fluorescent protein venus under the control of a strong mammalian promoter suitable for monitoring living cells.

To easily monitor living cells and organisms, we have created a transgenic Xenopus line expressing Venus, a brighter variant of yellow fluorescent protein, under the control of the CMV enhancer/chicken beta-actin (CAG) promoter. The established line exhibited high fluorescent intensity not only in most tissues of tadpoles to adult frogs but also in germ cells of both sexes, which enabled three-dimensional imaging of fluorescing organs from images of the serial slices of the transgenic animals. Furthermore, by using this transgenic line, we generated chimeric animals by brain implantation and importantly, we found that the brain grafts survived and expressed Venus in recipients after development, highlighting the boundary between fluorescent and nonfluorescent areas in live animals. Thus, Venus-expressing transgenic frogs, tadpoles, and embryos would facilitate their use in many applications, including the tracing of the fluorescent cells after tissue/organ transplantation.

Integration of multiple signal transducing pathways on Fgf response elements of the Xenopus caudal homologue Xcad3.

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. We investigate the nature of Fgf response element (FRE) in a posterior neural gene, Xcad3 (Xenopus caudal homologue) that plays a crucial role of posterior neural development. We provide evidence that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s).

Nodal signaling in Xenopus gastrulae is cell-autonomous and patterned by beta-catenin.

The classical three-signal model of amphibian mesoderm induction and more recent modifications together propose that an activin-like signaling activity is uniformly distributed across the vegetal half of the Xenopus blastula and that this activity contributes to mesoderm induction. In support of this, we have previously shown that the activin-response element (DE) of the goosecoid promoter is uniformly activated across the vegetal half of midgastrula-stage embryos. Here, we further examine the nature of this activity by measuring DE activation by endogenous signals over time. We find that the spatiotemporal pattern of DE activation is much more dynamic than was previously appreciated and also conclude that DE(6X)Luc activity reflects endogenous nodal signaling in the embryo. Using both the DE(6X)Luc construct and endogenous Xbra and Xgsc expression as read-outs for nodal activity, and the cleavage-mutant version of Xnr2 (CmXnr2) to regionally suppress endogenous nodal activity, we demonstrate that nodal signals act cell-autonomously in Xenopus gastrulae. Nodal-expressing cells are unable to rescue either reporter gene activation or target gene expression in distant nodal-deficient cells, suggesting that nodals function at short range in this context. Finally, we show that DE activation by endogenous signals occurs in the absence of dorsal beta-catenin-mediated signaling, but that the timing of dorsal initiation is altered. We conclude that nodal signals in Xenopus gastrulae function cell autonomously at short ranges and that the spatiotemporal pattern of this signaling along the dorsoventral axis is regulated by maternal Wnt-like signaling.

Expression of FTZ-F1alpha in transgenic Xenopus embryos and oocytes.

Fushi tarazu transcription factor-1 (FTZ-F1) was originally found as a regulator of fushi tarazu gene expression in Drosophila. The frog homologue (FTZ-F1alpha) and the 3.5 kb 5'-flanking region of the FTZ-F1alpha gene have been cloned, and it has been shown by reverse transcription-polymerase chain reaction that FTZ-F1alpha expression begins in embryos at stage 11 and becomes stronger after that. By in situ hybridization analysis, the FTZ-F1alpha mRNA was also found in immature frog oocytes. In this study, immunohistology revealed that the product of FTZ-F1alpha was localized in the cytoplasm of the immature oocyte. To analyze the promoter activity of the Rana rugosa FTZ-F1alpha gene, transgenic Xenopus were produced carrying the fusion construct, consisting of truncated 5'-flanking regions (3.0, 1.8 and 0.3 kb) of the FTZ-F1alpha gene and the green fluorescent protein (GFP) open reading frame. The 0.3 kb 5'-flanking region could drive GFP expression in Xenopus embryos at stage 20 and in immature oocytes in the ovary 2 months after metamorphosis. Gel mobility shift assay was used to test whether proteins in extracts from Xenopus embryos and ovaries bound to the 0.3 kb DNA. The extract from embryos at stage 11 formed one retarded band. The extract from ovaries formed a different retarded band. The results, taken together, indicate that production of transgenic Xenopus is very useful for the analysis of the promoter activity of genes in amphibians. The results also suggest that at least two proteins (one in the embryo and the other in the ovary of 2-month-old postmetamorphosing Xenopus) bind the 0.3 kb 5'-flanking region of the FTZ-F1alpha gene. These proteins may be involved in the regulation of FTZ-F1alpha gene expression in amphibians.

Regulation of the Dorsal Axial Structures in Cell-Deficient Embryos of Xenopus laevis: (regulation/Xenopus laevis/cell-deficient embryo/dorsal axial structures).

To study the regulation of the dorsal axial structures, we removed the right animal dorsal and the right vegetal dorsal cells from an 8-cell embryo of Xenopus laevis. Most of the right dorsal cell-deficient embryos developed to normally proportioned tailbud embryos. No detectable delay was observed in their development. Examinations of serial sections revealed that they had restored bilateral symmetry. The cell numbers of the somite and the notochord had recovered to more than 90% and 70%, respectively, those of controls. Since the right dorsal cell-deficient embryo retained roughly three-quarters of the prospective region for the somites and half of that for the notochord, respectively, the cell number was more than that expected from the remaining prospective regions. Cell lineage analyses showed that progeny of the right ventral cells had formed almost all of the right dorsal axial structures, which are normally formed by the progeny of the right dorsal cells. However, almost all the notochord cells had been derived from the remaining left dorsal cells. These results indicate that some quantitative aspects of regulation as expressed in terms of the cell number were different between the two tissues examined.