Previous work has shown that ultraviolet (UV) irradiation of fertilized frog eggs yields embryos that lack dorsal and anterior structures. The eggs fail to undergo the cortical/cytoplasmic rotation that specifies dorsoventral polarity, and they lack an array of parallel microtubules associated with the rotation. These eggs can be rescued by tilting with respect to gravity, and normal dorsoanterior development occurs. We find here that UV irradiation of Xenopus prophase I oocytes or Rana metaphase I oocytes also causes the dorsoanterior deficient syndrome, but the UV target is different from that in fertilized eggs. Tilting eggs, irradiated as oocytes, with respect to gravity, does not rescue dorsoanterior development, although lithium treatment does. The UV dose required to produce dorsoanterior deficiency for Rana metaphase I oocytes is much less than that for fertilized eggs, and the oocytes can form the array of parallel microtubules and undergo the cortical/ cytoplasmic rotation after fertilization. Despite these features of normal development, no dorsoanterior structures form. While the UV target in fertilized eggs is thought to be the parallel microtubules (Elinson & Rowning, 1988; Devi Biol. 128, 185–197), the UV target in the oocytes may be a dorsal determinant.

The dorsoventral axis of the frog embryo is specified during the first cell cycle by a rotation of the egg cortex relative to the cytoplasm (Ancel & Vintemberger, 1948; Vincent et al. 1986). Through early cleavage, a few blastomeres contain dorsal information, and these cells induce others to become notochord and dorsal mesoderm (Nieuwkoop, 1969; Gimlich & Gerhart, 1984; Yamana & Kageura, 1987). A transforming growth factor activity has been implicated in the induction of dorsal mesoderm development (Kimelman & Kirschner, 1987; Rosa et al. 1988; Smith et al. 1988).

It is possible to interfere experimentally with dorsal specification in a variety of ways. Embryos with excessive dorsoanterior development can be produced by treating 32-cell embryos with lithium (Kao et al. 1986). All of the mesoderm is converted to dorsal mesoderm and differentiates into notochord (Kao & Elinson, 1988). Lithium appears to act on animal cells, so that when they are induced by vegetal cells, they form dorsal mesoderm (Slack et al. 1988; Kao & Elinson, 1989; Cooke et al. 1989). The extreme embryo produced by lithium has radial symmetry with radial dorsoanterior structures including eye, cement gland, notochord and heart.

Conversely, radial ventral embryos lacking all dorsoanterior structures are produced by irradiation of the vegetal half of the fertilized egg with ultraviolet (UV) light (Malacinski et al. 1975; Scharf & Gerhart, 1980). The critical period for this effect is the first half of the first cell cycle, prior to the cortical/cytoplasmic rotation that specifies dorsoventral polarity (Manes & Elinson, 1980; Vincent & Gerhart, 1987). UV irradiation prevents the rotation as well as the formation of an array of parallel microtubules associated with the rotation. We have suggested that the parallel microtubules are part of a mechanism for driving the cortical/cytoplasmic rotation, and their absence would account for the UV inhibition of rotation (Elinson & Rowning, 1988).

UV-irradiated eggs can be rescued by tilting the eggs with respect to gravity (Scharf & Gerhart, 1980). Gravity causes a cytoplasmic rearrangement which mimicks some aspect of the cortical/cytoplasmic rotation (Ubbels et al. 1983; Vincent & Gerhart, 1987) and the embryos develop the normal complement of dorsoanterior structures. Rescue of UV-irradiated eggs by gravity demonstrates that no essential component for dorsoanterior development is destroyed by UV, although the normal means for activating those components via the cortical/cytoplasmic rotation has been blocked by UV.

Recently, Holwill et al. (1987) found that UV irradiation of prophase I oocytes led later to dorsoanterior deficient embryos. UV irradiation up to two days prior to fertilization also had this effect, so it seems likely that the targets for UV in the prophase I oocyte and in the fertilized egg are different, even though they both result in the dorsoanterior deficient syndrome. We present evidence here showing that is the case.

Animate and embryos

Procedures for maintaining Xenopus laevis, induction of ovulation, insemination and dejellying were described by Kao & Elinson (1988). Comparable procedures for Rana pipiens were described by Elinson (1983). Normalized time was used to compare events in the first cell cycle of fertilized eggs. Insemination is time 0, first cleavage is 1·0, and intervening times are given as decimal fractions.

UV irradiation and the dorsoanterior index

Vegetal halves of oocytes and eggs were irradiated by UV through a quartz slide using a short-wave Mineralight Lamp (UVP, San Gabriel, CA). Exposure times were determined empirically to give embryos that cleaved normally and gastrulated but which lacked dorsoanterior development. Dosage of UV was measured with a Blak-Ray Short Wave UV Meter (UVP).

The various abnormalities were scored using the Dorsoanterior Index or DAI (Kao & Elinson, 1988). Embryos with no dorsoanterior development are DAI 0 while DAI 1 through DAI 4 represent progressively more dorsoanterior development. Normal embryos are DAI 5 while embryos with enhanced dorsoanterior development receive scores of DAI 6 to DAI 10. The DAI 10 embryo is radially symmetric with a radial retina and cement gland and large amounts of notochord. The dorsoanterior deficient embryos (DAI 1–4) are produced by UV, while the dorsoanterior enhanced embryos (DAI 6–10) are produced by lithium.

Oocyte transfer

For Xenopus, the oocyte transfer procedures of Holwill et al. (1987) were used. Pieces of ovary were removed surgically from a pregnant mare serum-primed female into OR-2 (Wallace et al. 1973) adjusted to pH 7-8 and containing 400 μg ml-1 bovine serum albumen and 50 μg ml-1 penicillin. Oocytes were dissected from ovarian follicles using watch-maker’s forceps and stored in modified OR-2 at 15 °C until use. When sufficient numbers of oocytes were collected, some were UV-irradiated, and then all were induced to mature with a 10min treatment in 10 μg ml-1 progesterone in OR-2. Host females were injected with human chorionic gonadotropin at the same time. After oocyte maturation, donor oocytes were stained with 0·00125 % Nile blue or 0·0025 % neutral red for 15 min and transferred surgically to host females anesthetized with MS222. Our usual schedule was progesterone treatment of donor oocytes at 10p.m. at 18°C, hCG injection of host females at 10p.m. at 15°C, and transfer of donor oocytes to host females at 9–10a.m. the next day. Recovery and fertilization of the jellied donor oocytes occurred between noon and 3p.m. at 22 ± 1°C.

For Rana, modifications of previous oocyte transfer procedures were used (Arnold & Shaver, 1962). A donor female was anesthetized with MS222, and incisions were made on her sides, about one cm posterior to each arm. The anterior part of each oviduct was pulled out and ligated as close to the ostial opening as possible. Both donor and host females were induced to ovulate by injection of pituitaries and progesterone. Host females were sometimes injected intraperitoneally with 10 μg of a luteinizing hormone releasing hormone (LH-RH, Sigma L4513) in place of pituitaries. A few hours after ovulation had begun, about 16–19h at 18°C after injection, the donor female was pithed, her body cavity was opened, and the hundreds of trapped body cavity oocytes were spilled into 200% Steinberg’s solution. Maturing oocytes were UV-irradiated and stained for one minute in 0-013 % Nile blue or 0·05 % neutral red in 200 % Steinberg’s or for two minutes in 0·2% Bismarck brown Y. Several hundred oocytes were transferred surgically to the body cavity of a host female via a ventral incision. All host oocytes in the ovisac were stripped out at the time of transfer and about half of the host’s ovary was sometimes removed in order to increase the relative numbers of donor to host oocytes. Hosts were placed at 15–18°C, and fertilization was performed the next day.

Gravity and lithium rescue

Rescue of dorsoanterior deficient (DAI 0) embryos has been accomplished by tilting eggs with respect to gravity (Scharf & Gerhart, 1980) and by treating early embryos with lithium (Kao et al. 1986). For tilting, fertilized eggs were dejellied and placed in 5–10% Ficoll to shrink the perivitelline space (Kirschner & Hara, 1980). They were placed in small agar wells and held with an equatorial point uppermost. This position is 90° off the egg’s natural position with their animal pole uppermost. Tilting was begun at 0·3–0·45 of the first cell cycle, and the eggs were freed from the wells at first cleavage (1·0).

For lithium treatment, fertilized eggs were dejellied and raised to the 32-cell stage. The embryos were treated for 6–10 min with LiCl in 20% Steinberg’s at 0·3 M for Xenopus and 0·4–0·5 M for Rana. The embryos were thoroughly rinsed and raised in 20 % Steinberg’s.

Examination of fixed eggs

To obtain histological sections, oocytes or embryos were fixed in Smith’s Solution, embedded in Paraplast, and stained by the Feulgen procedure (Kao & Elinson, 1988). To measure grey crescents, eggs at the time of first cleavage, were fixed in 3 % glutaraldehyde in 100 mm-phosphate buffer, pH 7·4. The greatest height of the grey crescent between the black animal and the white vegetal region was measured using an ocular micrometer on a dissecting microscope (Manes & Elinson, 1980). To detect microtubules immunochemically, eggs were fixed in methanol (–20°C) and processed according to Elinson & Rowning (1988). The primary antibody was directed against β (-tubulin (N.357, Amersham, diluted 1/500).

Meiotic stage and UV-induced dorsoanterior deficiencies

Holwill et al. (1987) reported that UV irradiation of full-grown Xenopus oocytes at prophase I caused them to develop as dorsoanterior deficient embryos following oocyte maturation and fertilization. We have repeated this experiment and have confirmed their results (Table 1). Prophase I oocytes were usually UV-irradiated 0·5–6 h before progesterone treatment and 16–22 h (at 18°C) before insemination. Most of the UV-irradiated prophase I oocytes lacked dorsoanterior structures (average DAI = 1·3). The most extreme embryos lacked an axis and appeared identical externally to DAI 0 embryos. Large numbers of red blood cells could be seen pooled in the living embryo, and a small clump of striated muscle was revealed by histological examination.

Table 1.

Gravity rescue of UV-irradiated Xenopus oocytes and eggs

Gravity rescue of UV-irradiated Xenopus oocytes and eggs
Gravity rescue of UV-irradiated Xenopus oocytes and eggs

We have extended the analysis from Xenopus prophase I oocytes to Xenopus metaphase II oocytes and to Rana oocytes. In order to UV irradiate Xenopus oocytes at metaphase II, jellied oocytes were stripped from the female’s ovisac into 200% Steinberg’s to preserve fertilizability. They were oriented with the vegetal half down on a quartz slide, UV-irradiated vegetally, and then inseminated. There were several difficulties with the procedure. The jelly absorbed UV and underwent partial dissolution, particularly over the vegetal half. Sometimes, the jelly change altered the oocyte’s orientation. More importantly, however, was the fact that UV irradiation activated the oocytes, rendering them subsequently unfertilizable. The time of UV irradiation had to be balanced between that necessary to cause dorsoanterior deficiencies, with that insufficient to activate the oocyte. This compromise meant that it was possible to obtain groups of embryos with an average DAI of 2–3 (Table 1), but difficult to obtain large numbers of DAI 0 embryos. Nonetheless, as Chung & Malacinski (1980) previously mentioned, UV irradiation of Xenopus metaphase II oocytes caused dorsoanterior deficiencies (Table 1).

For Rana, oocytes were collected from the female’s body cavity shortly after ovulation. These oocytes were at prometaphase I or metaphase I as confirmed by cytological sections. Following UV irradiation, the oocytes were transferred to host females and usually inseminated after one day at 15–18°C. The resulting embryos exhibited the usual dorsoanterior deficiencies, and a DAI of less than one was easily obtained (Table 2).

Table 2.

Gravity rescue of UV-irradiated Rana oocytes and eggs

Gravity rescue of UV-irradiated Rana oocytes and eggs
Gravity rescue of UV-irradiated Rana oocytes and eggs

Rana oocytes were also collected from donors about 8–10h later than the metaphase I oocytes. These oocytes had given off the first polar body and were at metaphase II, as shown cytologically. A variable fraction, from 10% to 70% in different experiments, activated in response to a needle prick. This suggests that the Rana metaphase II oocytes used here were not fully mature, and therefore slightly younger developmentally than the jellied Xenopus metaphase II oocytes described above. As with Rana metaphase I oocytes, UV irradiation of Rana metaphase II oocytes led to dorsoanterior deficient embryos (Table 2).

In summary, UV irradiation caused later dorsoanterior deficiencies when applied to Xenopus prophase I, Xenopus metaphase II, Rana metaphase I, and Rana metaphase II oocytes. The dorsoanterior deficient syndrome is similar to that produced by UV irradiating Xenopus or Rana fertilized eggs in the first half of the first cell cycle.

Gravity rescue of dorsoanterior deficiencies

The dorsoanterior deficiencies caused by UV irradiation of Xenopus fertilized eggs can be prevented by tilting the irradiated eggs with respect to gravity during the first cell cycle (Scharf & Gerhart, 1980; Table 1). We have asked whether oocytes damaged by UV at earlier stages can be rescued by gravity.

Oocytes were UV-irradiated as described in the previous section, fertilized and tilted 90° with respect to gravity. Tilting was usually begun at 0·3–0·4 of the first cell cycle and stopped at first cleavage (1·0). Xenopus eggs, UV-irradiated at prophase I, were not rescued later by gravity (Table 1). This result suggests that the UV target is different in the prophase I oocyte compared to the fertilized egg.

Unlike those irradiated at prophase I, Xenopus eggs, UV-irradiated at metaphase II, were rescued by gravity (Table 1). This result can be interpreted in several ways, since the untilted eggs had some dorsoanterior development (average DAI = 2·6). If the UV target were a dorsal determinant, a DAI of 2·6 would imply that the determinant was only partially destroyed. Tilting might generate a sufficiently concentrated localization of the determinant, so that the embryos would develop normally. This was not the case with prophase I oocytes, since batches with intermediate DATs of T8 and 2·3 showed little rescue with tilting (Table 1). The UV target in the metaphase II oocyte, therefore, appears different from that in the prophase I oocyte.

The dorsoanterior deficiency following UV irradiation of Rana fertilized eggs can also be prevented by tilting 90° with respect to gravity (Table 2). Tilting was usually begun at 0·35–0·45 of the first cell cycle and stopped at first cleavage (TO). Rana eggs, UV-irradiated at metaphase I, were not rescued later by tilting (Table 2), similar to results with Xenopus prophase I oocytes. Rana eggs, UV-irradiated at metaphase II, were also not rescued by tilting, unlike the results with Xenopus metaphase II oocytes. This difference may be a species one, but further experiments would be necessary using Xenopus eggs with DAI 0–1 and ensuring that the eggs of the two species were the same developmental age. As discussed earlier, the Rana metaphase II oocytes may have been UV-irradiated at a slightly earlier stage compared to the Xenopus metaphase II oocyte.

Dorsoanterior development is normally specified by a rotation of the egg cortex relative to the cytoplasm. With Rana eggs, the grey crescent is a clear marker for the movement of the cytoplasm relative to the cortex. The failure of tilting to rescue UV-irradiated Rana metaphase I oocytes was not due to a failure of gravity-induced cytoplasmic movement, since all of the tilted eggs had a clear grey crescent on the up side. These eggs failed to produce dorsoanterior structures even though a cytoplasmic rearrangement occurred.

The gravity rescue results are summarized in Fig. 1.

Fig. 1.

Effect of UV irradiation at different times. Oocytes arrested at prophase (pro I) are stimulated to mature by progesterone (prog). They proceed to metaphase I (meta I), give off the first polar body, and arrest at metaphase II (meta II). The metaphase II arrest is broken by sperm entry, and the sperm pronucleus surrounded by a large aster migrates inward (0·4). By 0·8, the egg reaches metaphase of the first cell cycle. UV irradiation of the vegetal half causes later axis (dorsoanterior) deficiency (+) at the times indicated, but has no detrimental effect (—) when applied at 0·8. Tilting eggs in the first cell cycle with respect to gravity can rescue dorsoanterior development (+) of previously UV-irradiated oocytes and eggs in some cases but not in others (—). Lithium treatment of 32-cell embryos always rescues dorsoanterior development (+)

Fig. 1.

Effect of UV irradiation at different times. Oocytes arrested at prophase (pro I) are stimulated to mature by progesterone (prog). They proceed to metaphase I (meta I), give off the first polar body, and arrest at metaphase II (meta II). The metaphase II arrest is broken by sperm entry, and the sperm pronucleus surrounded by a large aster migrates inward (0·4). By 0·8, the egg reaches metaphase of the first cell cycle. UV irradiation of the vegetal half causes later axis (dorsoanterior) deficiency (+) at the times indicated, but has no detrimental effect (—) when applied at 0·8. Tilting eggs in the first cell cycle with respect to gravity can rescue dorsoanterior development (+) of previously UV-irradiated oocytes and eggs in some cases but not in others (—). Lithium treatment of 32-cell embryos always rescues dorsoanterior development (+)

UV dose and gravity rescue

The UV doses used for most of the experiments in Tables 1 and 2 were determined empirically as the dose that yielded an average DAI of less than one when applied to Xenopus or Rana fertilized eggs. Holwill et al. (1987) mentioned that lower doses produce dorsoanterior deficiency in Xenopus prophase I oocytes compared to Xenopus fertilized eggs, so the doses that we used may be high. Accordingly, we examined Rana metaphase I oocytes with respect to the UV dose required for dorsoanterior deficiency.

A UV dose of 3·4×10−4J mm-2 was sufficient to yield an average DAI of less than one when applied to Rana metaphase I oocytes (5 experiments), while a dose of 1·7×10−4J mm-2 had little effect (4 experiments). In contrast, a UV dose of 10–16×10−4 J mm-2 was required to produce an average DAI of less than one for Rana fertilized eggs (5 experiments). These results indicate that 3–4 times more UV is necessary to produce dorsoanterior deficiency for Rana fertilized eggs compared to Rana metaphase I oocytes.

Use of high UV on Rana metaphase I oocytes, however, was not the reason that gravity failed to rescue them. The last three entries for Rana metaphase I oocytes in Table 2 are based on doses of 5·1, 3·4 and 3·4×10−4J mm-2, respectively; yet gravity rescue was unsuccessful.

We also considered that UV irradiation of fertilized eggs made it easier for gravity to rearrange the cytoplasm and to rescue development. In two experiments, Rana fertilized eggs, previously irradiated at metaphase I with 3·4× 10−4J mm-2, were irradiated again with 13·8×10−4J mm-2 prior to tilting. The second UV irradiation did not help, and all of the tilted embryos lacked dorsoanterior development.

Lithium rescue of dorsoanterior deficiencies

Dorsoanterior deficient Xenopus embryos can also be rescued by treatment with lithium at the 32-cell stage (Kao et al. 1986). We wanted to see whether lithium could rescue dorsoanterior development in Xenopus eggs, UV-irradiated earher at prophase I.

Xenopus prophase I oocytes were UV-irradiated, progesterone-treated and later fertilized. When they reached the 32-cell stage, they were treated with 0·3 M-LiCl in 20% Steinberg’s for 6–10 min. In three experiments, lithium promoted enhanced dorsoanterior development (Table 3). Most embryos approached the radial extreme with radial pigmented retina, a radial cement gland, and a reduced or absent tail and trunk (DAI 8–10). There was no rescue in one experiment, perhaps due to a lower sensitivity to lithium found in some batches of eggs.

Table 3.

Lithium rescue of UV-irradiated oocytes

Lithium rescue of UV-irradiated oocytes
Lithium rescue of UV-irradiated oocytes

Lithium also caused dorsoanterior development in Rana. In preliminary experiments, Rana fertilized eggs were UV-irradiated and treated with Li+ at early cleavage stages to determine a dose. Patterns of enhanced dorsoanterior development, similar to those described for Xenopus (Kao & Elinson, 1988), were obtained by treating Rana 32-cell embryos with 0·4–0·5M-Li+ for 6 min. The Li+ concentration is higher than the 0·3M-Li+ usually used for Xenopus embryos.

Rana metaphase I oocytes were UV-irradiated, transferred to a host female and later inseminated. When they reached the 32-cell stage, they were treated with 0·3–0·5M-Li+ for 6 min. Most of the embryos had enhanced dorsoanterior development (Table 3), and twins, Janus twins and radial proboscis embryos were produced. Dorsoanterior development was strongly evoked by lithium even though tilting of Rana eggs, UV-irradiated at metaphase I, did not rescue dorsoanterior structures.

The lithium rescue results are summarized in Fig. 1.

Grey crescent formation in UV-irradiated oocytes

UV irradiation of fertilized eggs prevents the cortical/ cytoplasmic rotation that produces the grey crescent. Rana eggs, UV-irradiated at metaphase I, were examined for grey crescents at the time of first cleavage in order to see whether UV treatment of oocytes had the same effect as treatment of fertilized eggs. Grey crescents were clear on transferred control eggs and measured 0·52±0·13mm at their maximum height between animal and vegetal halves (Table 4). This height corresponds to a 35° rotation of the cortex relative to the cytoplasm, similar to the 30° found on Xenopus eggs by the dye imprinting method (Vincent et al. 1986).

Table 4.

Grey crescents on UV-irradiated Rana metaphase I oocytes

Grey crescents on UV-irradiated Rana metaphase I oocytes
Grey crescents on UV-irradiated Rana metaphase I oocytes

Grey crescents were also clear on eggs, UV-irradiated at metaphase I, although there was a 25% reduction in the height of the grey crescent (Table 4). The UV dose was sufficient to inhibit dorsoanterior development completely, since sibling eggs developed with an average DAI of less than one. The embryos lacked dorsoanterior structures even though they had undergone most of the normal rotation of the cortex relative to the cytoplasm.

Higher UV doses led to greater inhibition of grey crescent formation. The lamp output was not measured in these experiments, but the doses were similar to those used for fertilized eggs. In the most extreme experiments, two thirds of the eggs (17/26) lacked grey crescents. UV irradiation of metaphase I oocytes can inhibit later grey crescent formation, but at a dose greater than that required to eliminate dorsoanterior development.

Parallel microtubules in UV-irradiated oocytes

A transient array of parallel microtubules appears at the vegetal surface of eggs undergoing the cortical/ cytoplasmic rotation, and UV irradiation of fertilized eggs eliminates the array (Elinson & Rowning, 1988). We have asked whether fertilized eggs, developing from UV-irradiated Rana metaphase I oocytes, have parallel microtubules. Oocytes were irradiated with low doses of UV (2·6–6·9×10−4J mm-2), sufficient to eliminate dorsoanterior development. After fertilization, the eggs were fixed during the first cell cycle for immunocytochemistry of microtubules. UV-irradiated eggs had parallel microtubules (Fig. 2A), comparable to those found in unirradiated eggs. Eggs fixed at 0·73, about midway in the rotation period, had nice patterns, while in three of four experiments, irradiated eggs fixed at 0·62 had microtubules that were often less organized than unirradiated eggs. Despite the possibility of a later formation, UV irradiation of metaphase I oocytes permits the appearance of parallel microtubules, even though dorsoanterior development is inhibited.

Fig. 2.

Immunofluorescent detection of microtubules in the vegetal cortex. (A) This Rana egg was UV-irradiated at metaphase I with 4·6×10∼-4J mm-2. After maturation and fertilization, it was fixed at 0·69 normalized time and stained to reveal the characteristic array of parallel microtubules. Sibling eggs had no dorsoanterior development (DAI 0, 27 eggs), and grey crescents were present but reduced to 62 % of control values. (B) This Rana fertilized egg (0 · 64) received a higher UV dose at metaphase I than the egg in A. A cobweb-like meshwork of microtubules is present. Scale lines = 25 μm

Fig. 2.

Immunofluorescent detection of microtubules in the vegetal cortex. (A) This Rana egg was UV-irradiated at metaphase I with 4·6×10∼-4J mm-2. After maturation and fertilization, it was fixed at 0·69 normalized time and stained to reveal the characteristic array of parallel microtubules. Sibling eggs had no dorsoanterior development (DAI 0, 27 eggs), and grey crescents were present but reduced to 62 % of control values. (B) This Rana fertilized egg (0 · 64) received a higher UV dose at metaphase I than the egg in A. A cobweb-like meshwork of microtubules is present. Scale lines = 25 μm

With higher doses of UV, more disruption of the parallel array was found. Many eggs had cobweb-like meshworks of microtubules (Fig. 2B), or areas of meshwork with patches of parallel microtubules. The parallel patches were often near the equator. We suspect that the higher UV dose is causing a general disruption of the cortical area, and this prevents the organization of microtubules into a parallel array.

UV irradiation of the vegetal halves of frog oocytes and eggs eliminates dorsoanterior development whether applied at prophase I, metaphase I, or just after fertilization. The targets for UV inactivation appear to differ depending on the stage of the egg. UV irradiation of fertilized eggs stops the cortical/cytoplasmic rotation leading to grey crescent formation (Manes & Elinson, 1980; Vincent & Gerhart, 1987) and prevents the formation of an array of parallel microtubules associated with the rotation (Elinson & Rowning, 1988). Since these eggs can be rescued by gravity, the UV lesion is a failure of cytoplasmic rearrangement rather than the destruction of an essential dorsal determinant (Scharf & Gerhart, 1980; Chung & Malacinski, 1980).

In contrast, UV irradiation of Xenopus prophase I or Rana metaphase I oocytes inhibits later dorsoanterior development in a different way. This conclusion is derived from three results. First, the dose required to eliminate dorsoanterior development is much less for Xenopus prophase I (Holwill et al. 1987) and Rana metaphase I oocytes than for fertilized eggs. Second, eggs, irradiated as oocytes, cannot be rescued by a gravity-induced cytoplasmic rearrangement. Third, irradiated Rana eggs exhibiting parallel microtubules and grey crescent formation, nonetheless failed to develop dorsoanterior structures. There was a 25 % reduction in the amount of cortical/cytoplasmic rotation, raising the possibilty that an essential small movement was inhibited. This seems unlikely since gravity rescue failed and since a reduction by as much as 70 % of the rotation following UV irradiation of fertilized Xenopus eggs permitted dorsoanterior development (Vincent & Gerhart, 1987).

The UV target in the oocyte is more sensitive than that in the fertilized egg. This indicates that the oocyte target must be moved or changed during oocyte maturation. Otherwise, the higher dose of UV used on fertilized eggs would inactivate the oocyte target as well, and gravity rescue would not be possible.

About 70 % of the UV is absorbed within 60 μm of the egg surface (Youn & Malacinski, 1980). The vegetal cytoplasm within this region of Xenopus prophase I oocytes is special as it contains high concentrations of Vg1 mRNA, tubulin mRNA, and poly A(+) RNA (Melton, 1987; Larabell & Capeo, 1988). Vgl mRNA is a localized mRNA, which may be involved in dorsoanterior development. It codes for a TGF β-like protein (Weeks & Melton, 1987), and TGF βcan induce animal cells in vitro to develop dorsal mesoderm (Kimelman & Kirschner, 1987; Rosa et al. 1988). Vgl mRNA is localized close to the vegetal cortex in prophase I oocytes but disperses through the vegetal cytoplasm by the time of fertilization (Weeks & Melton, 1987), a property expected of the oocyte UV target. One hypothesis for the effect of UV on oocytes is that it alters an mRNA like Vgl mRNA, preventing its later utilization in dorsal development.

If UV irradiation of prophase I or metaphase II oocytes inactivates a mRNA or other dorsal factor, embryos would lack dorsoanterior development even with a normal or gravity-driven cytoplasmic rearrangement, as was found. Lithium, however, rescued dorsoanterior development, indicating that lithium affects a step downstream from the action of the factor. Lithium causes animal cells to interpret a ventral mesodermal signal as a dorsal signal (Slack et al. 1988; Kao & Elinson, 1989; Cooke et al. 1989). The dorsal factor could be a signalling molecule which acts through the same intracellular messenger system that lithium affects. In this way, eggs lacking the dorsal factor would be rescued by lithium but not by gravity.

UV is a relatively nonspecific agent with many targets; yet, it produces very specific effects on development. UV prevents dorsoanterior development by two different routes depending on the time of application. When applied to fertilized eggs, UV prevents the cortical/cytoplasmic rotation, likely due to an effect on microtubules. When applied to oocytes, UV permits the later cortical/cytoplasmic rotation but inactivates a factor required for dorsal development. In addition, UV activates unfertilized eggs and eliminates primordial germ cells. In a sense, UV behaves like many growth factors, hormones, or other signals in development. Specific signals are used to activate precise responses, but the response is a reflection more of the responding system than of the signal’s identity.

We thank Jinjong Chung for help with histology. This work was supported by grants from NSERC and MRC, Canada.

Ancel
,
P.
&
Vintemberger
,
P.
(
1948
).
Recherches sur le déterminisme de la symétrie bilatérale dans l’oeuf des amphibiens
.
Bull. Biol. Fr. Belg. suppl
.
31
,
1
182
.
Arnold
,
J. F.
&
Shaver
,
J. R.
(
1962
).
Interfemale transfer of eggs and ovaries in the frog
.
Expl Cell Res
.
27
,
150
153
.
Chung
,
H.-M.
&
Malacinski
,
G. M.
(
1980
).
Establishment of the dorso/ventral polarity of the amphibian embryo: use of ultraviolet irradiation and egg rotation as probes
.
Devi Biol
.
80
,
120
133
.
Cooke
,
J.
,
Symes
,
K.
&
Smith
,
E. J.
(
1989
).
Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor
.
Development
105
,
549
558
.
Elinson
,
R. P.
(
1983
).
Cytoplasmic phases in the first cell cycle of the activated frog egg
.
Devi Biol
.
100
,
440
451
.
Elinson
,
R. P.
&
Rowning
,
B.
(
1988
).
A transient array of parallel microtubules: potential tracks for a cortical/cytoplasmic rotation that forms the grey crescent of frog eggs
.
Devi Biol
.
128
,
185
197
.
Gimlich
,
R. L.
&
Gerhart
,
J. G.
(
1984
).
Early cellular interactions promote embryonic axis formation in Xenopus laevis
.
Devi Biol
.
104
,
117
130
.
Holwill
,
S.
,
Heasman
,
J.
,
Crawley
,
C. R.
&
Wylie
,
C. C.
(
1987
).
Axis and germ line deficiencies caused by u.v. irradiation of Xenopus oocytes cultured in vitro
.
Development
100
,
735
743
.
Kao
,
K. R.
&
Elinson
,
R. P.
(
1988
).
The entire mesodermal mantle behaves as Spemann’s organizer in dorsoanterior enhanced Xenopus laevis embryos
.
Devi Biol
.
127
,
64
77
.
Kao
,
K. R.
&
Elinson
,
R. P.
(
1989
).
Dorsalization of mesoderm induction by lithium
.
Devi Biol
.
132
,
81
90
.
Kao
,
K. R.
,
Masui
,
Y.
&
Elinson
,
R. P.
(
1986
).
Lithium-induced respecification of pattern in Xenopus laevis embryos
.
Nature, Lond
.
322
,
371
373
.
Kimelman
,
D.
&
Kirschner
,
M.
(
1987
).
Synergistic induction of mesoderm by FGF and TGF-/? and the identification of an mRNA coding for FGF in the early Xenopus embryo
.
Cell
51
,
869
877
.
Kirschner
,
M. W.
&
Hara
,
K.
(
1980
).
A new method for local vital staining of amphibian embryos using Ficoll and “crystals” of Nile Red
.
Mikroskopie (Wien)
36
,
12
15
.
Larabell
,
C. A.
&
Capco
,
D. G.
(
1988
).
Role of calcium in the localization of maternal poly(A)+ RNA and tubulin mRNA in Xenopus oocytes
.
Wilhelm Roux’s Arch Devi Biol
.
197
,
175
183
.
Malacinski
,
G. M.
,
Benford
,
H.
&
Chung
,
H.-M.
(
1975
).
Association of an ultraviolet irradiation sensitive cytoplasmic localization with the future dorsal side of the amphibian egg
.
J. exp. Zool
.
191
,
97
110
.
Manes
,
M. E.
&
Elinson
,
R. P.
(
1980
).
Ultraviolet light inhibits grey crescent formation on the frog egg
.
Wilhelm Roux’s Arch Devi Biol
.
189
,
73
76
.
Melton
,
D. A.
(
1987
).
Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes
.
Nature, Lond
.
328
,
80
82
.
Nieuwkoop
,
P. D.
(
1969
).
The formation of the mesoderm in urodelean amphibians. II. The origin of the dorso-ventral polarity of the mesoderm
.
Wilhelm Roux’s Arch. EntwMech. Org
.
163
,
298
315
.
Rosa
,
F.
,
Roberts
,
A. B.
,
Danielpour
,
D.
,
Dart
,
L. L.
,
Sporn
,
M. B.
&
Dawid
,
I. B.
(
1988
).
Mesoderm induction in amphibians: the role of TGF-/12-like factors
.
Science
239
,
783
785
.
Scharf
,
S. R.
&
Gerhart
,
J. C.
(
1980
).
Determination of the dorso-ventral axis in eggs of Xenopus laevis: complete rescue of UV-impaired eggs by oblique orientation before first cleavage
.
Devi Biol
.
79
,
181
198
.
Slack
,
J. M. W.
,
Isaacs
,
H. V.
&
Darlington
,
B. G.
(
1988
).
Inductive effects of fibroblast growth factor and lithium ion on Xenopus blastula ectoderm
.
Development
103
,
581
590
.
Smith
,
J. C.
,
Yaqoob
,
M.
&
Symes
,
K.
(
1988
).
Purification, partial characterization and biological effects of the XTC mesoderminducing factor
.
Development
103
,
591
600
.
Ubbels
,
G. A.
,
Hara
,
K.
,
Koster
,
C. H.
&
Kirschner
,
M. W.
(
1983
).
Evidence for a functional role of the cytoskeleton in determination of the dorsoventral axis in Xenopus laevis eggs
.
J. Embryol. exp. Morph
.
17
,
15
37
.
Vincent
,
J.-P.
&
Gerhart
,
J. C.
(
1987
).
Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification
.
Devi Biol
.
123
,
526
539
.
Vincent
,
J.-P.
,
Oster
,
G. F.
&
Gerhart
,
J. C.
(
1986
).
Kinematics of gray crescent formation in Xenopus eggs: the displacement of subcortical cytoplasm relative to the egg surface
.
Devi Biol
.
113
,
484
500
.
Wallace
,
R. A.
,
Jared
,
D. W.
,
Dumont
,
J. N.
&
Sega
,
M. W.
(
1973
).
Protein incorporation by isolated amphibian oocytes. III. Optimum incubation conditions
.
J. exp. Zool
.
184
,
321
334
.
Weeks
,
D. L.
&
Melton
,
D. A.
(
1987
).
A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-/3
.
Cell
51
,
861
868
.
Yamana
,
K.
&
Kageura
,
H.
(
1987
).
Re-examination of the “regulative development” of amphibian embryos
.
Cell Diff
.
20
,
3
10
.
Youn
,
B. W.
&
Malacinski
,
G. M.
(
1980
).
Action spectrum for ultraviolet irradiation inactivation of a cytoplasmic component(s) required for neural induction in the amphibian egg
.
J. exp. Zool
.
211
,
369
377
.