Two distinct types of axis lacking embryos resulted from partial deletion of the vegetal part of early one-cell-stage embryos. When the deleted volume was 20-40% (relative surface area), the embryos underwent ventral-type gastrulation and formed ventral mesodermal tissues. When the deleted volume was more than 60%, the embryo did not gastrulate nor make mesodermal structures (M. Sakai, 1996, Development 122, 2207-2214). We have designated these two types of embryos as "gastrulating nonaxial embryos (GNEs)" and "permanent blastula-type embryos (PBEs)," respectively. Using these embryos as recipients, a series of Einsteck transplantation experiments were carried out to investigate mechanisms controlling anteroposterior patterning during early Xenopus development. GNEs receiving dorsal marginal zone (DMZ) transplants (GNE/DMZs) elongated and formed posteriorized phenotypes, which had muscle cells, melanocytes, and tail fins. In contrast, PBE/DMZs did not elongate but formed cement glands and brain-like structures showing strong anteriorization. Simultaneous transplantation of the cells from various regions of normal embryos with the DMZ into PBEs revealed that the entire vegetal half of normal embryos, except for the DMZ, showed posteriorizing activity. These results strongly suggest that anteroposterior patterning in Xenopus is not achieved solely by the dorsal marginal zone (the Spemann organizer), but instead by a synergistic mechanism of the dorsalizing domain (DMZ) and the posteriorizing domain (the entire vegetal half except for the DMZ).

Ablation of vegetal cytoplasm from newly fertilized Xenopus eggs results in the development of permanent blastula-type embryos (PBEs). PBEs cleave normally and develop into a very simple tissue consisting only of atypical epidermis. We tried to restore complete embryonic development in PBEs by cytoplasmic transplantation or by mRNA injection. We show a two-step reconstruction of the body plan. In the first step, PBEs injected with either marginal cytoplasm or synthetic VegT RNA restored gastrulation and mesoderm formation, but not axial patterning. Injection of Xwnt8 mRNA (acting upstream of β-catenin and thus substitutes for the dorsal determinant)did not restore axial development in PBEs. Simultaneous injections of Xwnt8 and VegT into PBEs resulted in dorsal axis development, showing the synergy of these molecules in axial development. These results suggest that the mixing of two cytoplasmic determinants, i.e. the dorsal determinant in the vegetal pole and the endo-mesodermal determinant in the whole vegetal half, triggers the early axial developmental process in Xenopus embryos.

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.