Sakai M (1996), The vegetal determinants required for the Spema...

XB-ART-18039

Development 1996 Jul 01;1227:2207-14. doi: 10.1242/dev.122.7.2207.

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The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle.

Sakai M .

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Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.

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Species referenced: Xenopus laevis

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	Fig. 1. Development of an early vegetal-deleted fragment (deletion started at time 0.30, A-C) and a late-vegetal-deleted fragment (D-F, time 0.83). (A,D) Just after placing a glass rod. (B,E) Vegetal view at gastrulation. (C,F) At the tadpole stage. Bar in A for A, B, D and E and bar in C for C and F: 1 mm.
	Fig. 2. Diagrams showing the method of bisection.
	Fig. 3. Effect of early-vegetal and early-vertical (anti-SEP) deletion. Data from individual embryos (DAI of the embryo) are plotted against the deleted area (% of the whole surface). Data from embryos deleted before time 0.5 (start of deletion) are shown. Embryos which completed separation after the first cleavage (time 1.0) were omitted. Open triangles: vegetal deletion. Black triangles: vertical (anti-SEP) deletion. NG denotes the embryos that did not gastrulate, while DAI 0 denotes embryos that did gastrulate.
	Fig. 4. Histological sections of an early-deleted embryo and a late-deleted embryo. (A-C) An early-deleted embryo deleted 20% of the egg surface at time 0.30. This is the same embryo as shown in Fig. 5. (D-F) A late-deleted embryo deleted 35% of the egg surface at time 0.86. Note striated muscles in F, which was not observed in the early-deleted embryos (see for example, C). Bar in A for A and D: 1 mm. Bar in B for B and E: 1 mm. Bar in C for C and F: 10 mm.
	Fig. 5. Cortical rotation in a vegetal-deleted embryo. At time 0.30, the vegetal pole (20% of the egg surface) was deleted. The embryo was then stained with small pieces of Nile red crystal (Kirschner and Hara, 1980) and loaded with minute carbon particles (Norit, âSX Plusâ). The movement of the Nile blue spots were photographed with a Nikon inverted epifluorescence microscope equipped with the filter sets for rhodamine. (A) At time 0.58. (B) At time 1.03. Note the movement of the Nile blue spot relative to the egg surface, which is indicated by the carbon particles (three black dots can be seen in both panels). In this case, the angular distance traveled by the dye spots was 12Â°. Bar: 1 mm.
	Fig. 6. Lithium treatment and bisection at the first cleavage. (A) Two examples of bisected early-vegetal-deleted embryos at control stage 35. No axis (DAI 0). (B) An UV-ventralized embryo (DAI 0). (C) An UV-bisected embryo, which has two small eyes and a sucker (DAI 4). (D) An example of a hyperdorzalized early-vegetal-deleted embryo obtained by lithium treatment. (E) A lithium-treated whole embryo. Bar in A for A-C and bar in D for D and E: 1 mm.
	Fig. 7. Injection of vegetal cytoplasm restores dorsal structure. All embryos of one experimental series are shown. (A) A control embryo. (B-H) Vegetal-deleted embryos injected with vegetal pole cytoplasm. The DAI was 5, 5, 5, 5, 2, 2, 2 respectively. (I-K) Vegetal-deleted embryos injected with animal cytoplasm. All have no axis (DAI 0). Bar: 1 mm.
	Fig. 8. Schematic illustration of the present results and the suggested model for the initial step of dorsal axis formation. (A) Early vegetal deletion. (B) Injection of the cytoplasm of a vegetal fragment after A. (C) Injection of the cytoplasm of animal fragment after A. (D) Late vegetal deletion. (E) Proposed model for normal axis formation. Hatched areas, gastrulating region (blastopore-forming region); stippled areas, dorsal determinants (DDs); black areas, organizer region: this is a part of the gastrulating region that receives the DDs.