Fujisue M et al. (1993), Occurrence of dorsal axis-inducing activity aro...

XB-ART-39216

Development 1993 May 01;1181:163-70.

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Occurrence of dorsal axis-inducing activity around the vegetal pole of an uncleaved Xenopus egg and displacement to the equatorial region by cortical rotation.

Fujisue M , Kobayakawa Y , Yamana K .

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Specification of the dorsoventral axis is a subject of great importance in amphibian embryogenesis. We have found that cytoplasm of the vegetal dorsal cells of a 16-cell embryo of Xenopus laevis, when injected into the ventral vegetal cells of a recipient at the same stage, can induce formation of a second axis. In the present experiments,using the same assay procedure, we found that the cytoplasm around the vegetal pole of an egg before cortical rotation is also active in inducing a second axis,that the activity decreases throughout the second half of the cell cycle and appears in a presumptive dorsal equatorial region at the 2- to 16-cell stages. This is the first demonstration of the localization of dorsal forming activity in any specific region of an egg. After UV irradiation,a treatment that is known to block cortical rotation and thereby inhibit axis specification, the activity remains near the vegetal pole beyond the first cell cycle and does not appear in an equatorial region, at least at the 16-cell stage. This suggests that cortical rotation ora related force is in some way involved in changes in distribution of the activity.We also found that UV-irradiated 8-cell embryos can rescue dorsal development when they are cut into halves along the first cleavage plane. Histological examination revealed that the rescued embryos have a neural tube and notochord. In the half embryo, the animal and vegetal regions came into contact during wound healing, an event that enables the activity to localize in the new equator of an embryo. Therefore this rescue suggests that, if the activity is distributed only in the equatorial region, dorsal specification occurs. In fact, the dorsal side of the rescued embryos seems to correspond to the plane through which the embryos have been cut.Based on our results, we propose (1) that a determinant that carries axis-inducing activity is first present around the vegetal pole, (2) that the determinant shifts from the vegetal pole to an equatorial region by or in close association with cortical rotation and (3) that occurrence of the determinant in the equatorial region is a prerequisite for axis specification.

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	Fig. 1. (A) A 16-cell-stage recipient embryo. It was denuded, the darkly pigmented side turned up. Then cytoplasm was injected at the upper end of the vegetal cells. (B) A 16-cell stage donor embryo. The cytoplasm was withdrawn from the dorsalmost vegetal cells. (C) A grade 4 double embryo obtained by injection of cytoplasm from the vegetal pole region of an uncleaved egg. Bar: 0.5 mm. (D) A fertilized egg was stained with Nile blue, turned upside down and cytoplasm was sucked out. Note that the cytoplasm stained with Nile blue comes together around the tip of a micropipette. Bar: 0.5 mm. In A, B, D same magnification.
	Fig. 2. Histogram and percentage of second axis formation showing the effect of UV irradiation on change in distribution of the activity. Note the axis-inducing activity of the cytoplasm from the vegetal pole (light column) and from the dorsal or anti-SEP side equatorial region (dark column). At start of cell cycle, the activity was restricted to around the vegetal pole, in both unirradiated and irradiated eggs. At the 16-cell stage, the activity decreased around the vegetal pole and increased at the dorsal equatorial region in unirradiated embryos, while no such changes occurred in irradiated embryos.
	Fig. 3. Rescue of UV-irradiated embryos by bisection. (A-D) Development after bisection at the 8-cell stage. (A) Soon after bisection. Cut side is right. (B) About 19 hours after insemination (at 17°C). Invagination occurs simultaneously at many points of a circle. (C) About 21 hours after insemination. The previous cut side, now dorsal side, is right. (D) Upper, an UV-irradiated embryo; middle, an embryo developed from the same as shown in A-C; lower, unirradiated control. (E) Embryos obtained by bisection of irradiated 8-cell embryos. Right uppermost is the UV-irradiated embryo and left uppermost is an unirradiated one. 6 of 10 embryos have one or two eyes. This group of embryos is obtained from a batch of eggs different from those in A-D and F. Bar: 0.5 mm in A-E. (F) Transverse section of a grade 3 embryo, derived from an irradiated embryo cut at the 8-cell stage. nt, notochord; n, neural tube. Bar: 0.1 mm.