Holowacz T and Elinson RP (1993), Cortical cytoplasm, which induces dorsal axis f...

XB-ART-22249

Development 1993 Sep 01;1191:277-85.

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Cortical cytoplasm, which induces dorsal axis formation in Xenopus, is inactivated by UV irradiation of the oocyte.

Holowacz T , Elinson RP .

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Localized maternal determinants control the formation of dorsal axial structures in Xenopus embryos. To examine the spatial distribution of dorsal determinants, we injected cytoplasm from various regions of the egg and 16-cell embryo into the ventral vegetal cells of a 16-cell recipient embryo. Cortical cytoplasm from the egg vegetal surface induced the formation of a secondary dorsal axis in 53% of recipients. In contrast, animal cortical, equatorial cortical and vegetal deep cytoplasm never induced secondary axis formation. We also compared the axis-inducing ability of animal versus vegetal dorsal cortical cytoplasm from 16-cell embryos. Significantly more dorsalizing activity was found in vegetal dorsal cytoplasm compared to animal dorsal cytoplasm at this stage. Previous work has shown that UV irradiation of the vegetal surface of either prophase I oocytes, or fertilized eggs, leads to the development of embryos that lack dorsal structures. Egg vegetal cortical cytoplasm was capable of restoring the dorsal axis of 16-cell recipient embryos derived from UV-irradiated oocytes or fertilized eggs. We also tested the axis inducing ability of cytoplasm obtained when UV-irradiated oocytes and eggs were treated as donors of cytoplasm. While vegetal cortical cytoplasm from UV-irradiated fertilized eggs retains its dorsalizing activity, cytoplasm obtained from eggs, UV irradiated as oocytes, does not. The egg vegetal cortex provides a suitable source for the isolation of maternal dorsal determinants. In addition, since UV irradiation of the oocyte vegetal surface destroys the dorsalizing activity of transferred cytoplasm, UV can be used to further restrict possible candidates for such determinants.

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	Fig. 1. Secondary axis formation by injection of dorsal cortical cytoplasm. All 16-cell stage embryos are viewed laterally with ventral blastomeres evident by the accumulation of pigment. (A) Cytoplasm was drawn from the cortex of two dorsal vegetal blastomeres and microinjected (hatched arrow) into two ventral vegetal blastomeres. Recipient embryos were capable of developing a secondary axis. (B) Controls in which ventral cortical cytoplasm was transferred developed normally. D, dorsal; V, ventral; stippled regions, location of putative dorsal activity; hatched arrows, transfer of cytoplasm.
	Fig. 2. Tailbud embryos with a secondary axis resulting from the injection of dorsal cortical cytoplasm. (A) Embryo with secondary axis, indicated by arrowheads. The DAI value of the secondary axis is 1. Bar, 1 mm. (B) Section of embryo that has a secondary spinal cord. Primary and induced secondary notochords (n, n’), neural tubes (nt, nt’) and sets of somites (s, s’) are indicated. t, tail; Bar, 100 mm. (C) Embryo with complete secondary axis including well formed head structures. The DAI value of the secondary axis is 5. Bar, 1 mm. (D) Control embryo that was injected with ventral cytoplasm appears normal. Since no secondary axis is observed, the DAI value of the induced axis is 0. Bar, 1 mm.
	Fig. 3. Superior dorsalizing activity in dorsal vegetal blastomeres compared to dorsal animal blastomeres. (A) To compare animal versus vegetal dorsal cytoplasm, one donor embryo was used as a source of cytoplasm for each pair of recipient embryos. Cortical cytoplasm from two dorsal animal blastomeres was microinjected (hatched upper arrow) into the two ventral vegetal blastomeres of a recipient embryo. Cortical cytoplasm from the two dorsal vegetal blastomeres was microinjected (hatched lower arrow) into the two ventral vegetal blastomeres of another recipient. The pair of recipients was allowed to develop to tailbud stage and the degree of secondary dorsal axis formation was expressed as a DAI value. (B) Secondary axis formation in paired recipients of animal and vegetal dorsal cytoplasm. DAI of the vegetal dorsal recipient is plotted on the horizontal axis and that of the animal dorsal recipient is plotted on the vertical axis. Note that each point represents a pair of recipients that received cytoplasm from the same donor embyro. The 12 points that lie on the origin (no secondary dorsal axis in either recipient) are not shown. The straight line denotes the location of points that would be observed if cytoplasm from vegetal and animal dorsal blastomeres had equivalent dorsalizing activity. Most of the points lie below the line and closer to the horizontal axis, and thus the dorsalizing activity of vegetal dorsal cytoplasm is significantly superior to that of animal dorsal cytoplasm (P < 0.01, Wilcoxon Signed Rank Test, paired, twotailed).
	Fig. 4. Dorsalizing activity from UV-irradiated prophase I oocytes versus UV-irradiated fertilized eggs. (A) UVirradiated fully grown prophase I oocytes were matured, prick activated, and used as donors of vegetal cortical cytoplasm. Cytoplasm was transferred to two ventral vegetal blastomeres of a 16-cell embryo (hatched arrow). Recipients of cytoplasm did not develop a secondary dorsal axis and thus appeared normal. A subset of oocytes (lower curved arrow) was matured, implanted into the body cavity of a host female and fertilized. These embryos developed with dorsoanterior deficiencies. (B) UV irradiated fertilized eggs were used as donors of vegetal cortical cytoplasm. Cytoplasm was transferred to two ventral vegetal blastomeres of a 16-cell embryo (hatched arrow). Recipients developed a secondary dorsal axis. Sibling donor embryos (upper curved arrow) developed dorsoanterior deficiencies. V, ventral blastomeres; D, dorsal blastomeres; hatched arrow, transfer of cytoplasm; stippled regions, location of putative dorsal activity.
	Fig. 5. Rescue of dorsal axis formation in embryos derived from UV-treated oocytes and UV-fertilized eggs. (A) The vegetal surface of prophase I oocytes was exposed to UV light prior to maturation, transfer and fertilization. At the 16-cell stage, the ventral vegetal blastomeres were microinjected with cortical cytoplasm from the vegetal pole of a donor egg (hatched arrow). The donor was previously UV-treated after fertilization. Many of the recipient embryos developed normally. Uninjected embryos (lower curved arrow) developed with dorsoanterior deficiencies. (B) UV-irradiated fertilized eggs were grown to 16-cell stage and used as recipients. Injection of vegetal cortical cytoplasm (hatched arrow) rescues dorsal development and many embryos develop with dorsal axial structures. Uninjected sibling embryos (upper curved arrow) developed dorsoanterior deficiencies. Note that pigmentation differences shown in the animal hemisphere of recipient 16-cell embryos are due to the sperm entry point and do not reflect dorsoventral polarity. V, ventral blastomeres; D, dorsal blastomeres; stippled regions, location of putative dorsal activity; hatched arrow, injection of cytoplasm.