ventral polarity of the amphibian embryo: Use of ultraviolet irradiation and egg rotation as probes

DEVELOPMENTAL

BIOLOGY

80.120-133

(1980)

Establishment of the Dorsal/Ventral Use of Ultraviolet Irradiation

Polarity of the Amphibian Embryo: and Egg Rotation as Probes

HAE-MOONCHUNGANDGEORGE

M. MALACINSKI

Cell and Molecular Biology Program, Department of Biology, Indiana University, Bloomington, Indiana 47401 Received January 16, 1980; accepted April i4, 1980 Ultraviolet irradiation and egg rotations were employed as probes for the study of the establishment of the dorsal/ventral polarity of the amphibian embryo. Ultraviolet irradiation was discovered to alter the natural position of the doral lip and to modify the pigmentation pattern of the early embryo. Rotation of the uncleaved egg was found to succeed in relocating the dorsal lip to a new site in the embryo. Also, rotation of the egg was capable of preventing the characteristic defects associated with irradiation of the uncleaved egg. A combination of these probes was employed, and the results were interpreted in terms of models for the role of the egg surface and the internal cytoplasm in the establishment of the dorsal/ventral polarity of the egg.

rearrangements and new cellular interactions occurs there. In order to gain insight into the mechanisms which may be involved in the development of primary embryonic organizer activity, various experimental approaches have been taken (see recent reviews by Nakamura and Toivonen, 1978; Gerhart, 1979). These include extensive studies on the use of ultraviolet irradiation (uv) of uncleaved eggs as a probe into the components of the uncleaved egg cytoplasm which may be involved in later neural morphogenesis (Grant and Wacaster, 1972; Malacinski et al., 1977, Youn and Malacinski, 1979). During the course of recent studies of irradiated eggs it was discovered that the site of dorsal lip formation was altered in irradiated eggs. Rather than forming on the side of the egg opposite from the sperm entry point, a marked tendency for the lip of irradiated embryos to form on the same side as the sperm pit was discovered. Also, it has been recently reported that a simple rotation of the uncleaved egg results in an

INTRODUCTION

The establishment of the dorsal/ventral polarity is perhaps the first morphogenetic event which follows fertilization of the amphibian egg. Prior to fertilization the egg is polarized into animal (pigmented) and vegetal halves. In anuran eggs, the point of sperm entry defines the dorsal/ventral axis of the egg. It is on the side of the egg opposite from the sperm entry point that pigmentation changes related to the formation of the gray crescent occur (Roux, 1887; Elinson, 1975; Palaecek et al., 1979). Although the cortex of the egg associated with the gray crescent was considered to be the site of localization of morphogens (Curtis, 1962; Tompkins and Rodman, 1971), recent evidence indicates that such a relationship probably does not, in fact, exist (Malacinski et al., 1979). Nevertheless, the gray crescent provides a useful morphological marker, for it is on that side of the egg that primary embryonic organizer activity develops (Spemann, 1938;Malacinski et al., 1979). Eventually the dorsal lip forms on alteration of the site of dorsal lip formation the “gray crescent side” of the embryo, and (Kirschner et al., 1979). That sort of rotainvolution which brings about dramatic cell tion has been found to prevent the appear120 ool2-1606/so/l30120-14$02.~/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ante of the defects in neural development which characterize irradiated eggs (Gerhart, personal communication). The present studies were initiated with the goal of combining these two probesirradiation and egg rotation-in order to study the early events involved in primary embryonic induction. The experiments described in this report have generated new information about the mechanism of action of uv and the manner in which gravity affects dorsal lip formation. A model has been generated which should lead to further experimentation on the establishment of dorsal/ventral polarity of the amphibian egg. MATERIALS

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Collection of eggs and uv irradiation. Xenopus laevis eggs were obtained and irradiated according to previously published methods (Malacinski et al., 1975). Unless stated otherwise (e.g., Fig. lA), ah irradiations were carried out within 30 min after fertilization, that is, during the period of greatest sensitivity to uv (Malacinski et al., 1978). Egg rotations. Irradiated eggs were immersed in dechlorinated tap water which contained 5% (W/V) Ficoll (Type 400, Sigma, St. Louis). That treatment shrinks the perivitelline space and permits the convenient rotation of the egg onto its side (Kirschner et al., 1979). The vitelline membrane is left intact. All the rotations described in this report were at 90“ and were carried out for approximately 35 min (3040 min), unless stated otherwise. The eggs were rotated in small wells cut out of a 2% agar bed. It was possible, by orienting the eggs in agar wells, to follow their development carefully through gastrulation and score the position of the dorsal lip with respect to the direction (sperm pit up or down) of rotation of the uncleaved egg. Characteristics of the uv syndrome. The effects of uv on neural morphogenesis were ascertained at the tail bud stage of development (Stages 30-35, Nieuwkoop and Far-

FIG. 1. Summary of the individual experimental designs which employed irradiation as a probe. The questions posed in the Results were used to formulate these designs. In those experiments in which the sperm entry point was located, the sperm pit is shown as a small open circle on the egg surface. Figure 10 contains a photograph of typical sperm entry points on the surface of uncleavedXenopus eggs. In each experiment except Expt A, the egg was fertilized prior to irradiation.

ber (1956) staging series). The scoring system which has been used in previous studies (e.g., Malacinski et al., 1977) was employed to provide a semiquantitative estimate of the extent of irradiation-induced damage to primary embryonic induction. The following notations are employed: 0, no apparent damage; +l, microcephalic; +2, extremely microcephalic; +3, short axis; +4, acephalic; +5, aneural. Embryos which manifested any of the defects associated with these +l to +5 notations are stated as displaying the “uv syndrome.” Evidence from histological analyses has indicated that the gross morphological features associated with +l to +5 embryos are also related to defects in the cytological features of the neural tube and notochord (e.g., Malacinski et al., 1975). More recent evidence from our laboratory has substantiated the validity of this scoring system. Assays for the enzyme acetylcholinesterase indicate

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that the specific activity of that enzyme is proportional to the extent to which embryos exhibit normal gross neural morphologies (Youn and Malacinski, 1979). The “average uv effect” was obtained by summing the number of embryos multiplied by the numerical notation of uv damage and dividing by the total number of embryos in the group. As pointed out in previous publications from this laboratory, various Xenopus spawnings show somewhat different sensitivities to uv. It was considered necessary, therefore, to complete an individual experiment with the eggs from a single spawning. Only in those instances in which the data from several FIG. 2. Summary of experimental designs in which experiments were identical was a summary egg rotation, in some instances preceded by irradiatable of the data compiled. Likewise, the tion, was employed. If the egg was rotated without capacity for egg rotation to prevent the uv regard for the location of the sperm pit (random rotation), no open circle representing the sperm entry syndrome was discovered during the pre- point is shown. liminary stages of this investigation to vary somewhat from spawning to spawning (e.g., Fig. 12). A similar precaution of using only (e.g., Malacinski et al., 1977, 1978; Chung et eggs from a single spawning was also ad- al., 1977). Most of the experimental protocols were designed to provide information hered to in the egg rotation experiments. which would permit the development of a RESULTS model to account for the main observations These experimental analyses were orga- on neural defects in irradiated embryos. nized in terms of a series of questions. This Is the infertile egg sensitive to uv (Expt approach permitted a critical evaluation of A)? Eggs were stripped from an ovulated the significance of each set of results and female directly onto a quartz slide. They facilitated the formulation of a model to were oriented vegetal hemisphere down account for the experimental observations. and irradiated. Immediately after irradiaThe design of each experiment is included tion the eggs were fertilized by pipetting a in Figs. 1 and 2. A summary of the data sperm suspension directly onto the quartz follows. The first set of experiments deals slide. The eggs were permitted to develop with the effects of uv irradiation alone on and scored in the usual manner for the uv the development of the early embryo. Then syndrome. Eggs irradiated prior to fertilithe results of a set of experiments on ori- zation displayed exactly the same types of entation of the uncleaved egg are described. gross morphological defects in neural deFinally, results which were derived from velopment as eggs irradiated following ferthe use of both uv irradiation and egg ro- tilization. The sensitivity to uv of the untation are presented. fertilized egg is, however, somewhat lower than that of fertile eggs. A uv dose approxUltraviolet Irradiation as a Probe imately twofold greater than usually emThe rationale for these studies was de- ployed was required to obtain the same rived mainly from the previous series of “average uv effect” in unfertilized eggs as observations on the effects of uv on eggs in fertile control eggs. Future experiments which have emanated from our laboratory will be designed to determine whether the

CHUNG AND MALACINSKI

presence of the jelly layer contributes to the relative insensitivity of unfertilized eggs. Does the dorsal lip form at its natural site in irradiated eggs (Expt B)? Fertile eggs were irradiated and then oriented in agar wells re the location of the sperm entry point (sperm pit). At the early gastrula stage the location of the dorsal lip in relation to the original sperm entry point was scored. As the data in Fig. 3 demonstrate, the dorsal lip formed on the side of the embryo opposite the original sperm entry point in virtually all (92%) of the unirradiated cases.This observation is consistent with the previous results of Elinson (1975) on the site of sperm entry in Rana eggs. Irradiated eggs, however, displayed a tendency to form the dorsal lip on the side of the egg where the sperm pit was originally located. Only 14% of the eggs exhibited dorsal lips opposite the sperm pit, while 40% of the embryos showed dorsal lips on the same side of the egg as the original sperm entry point. As previously observed (Malacinski et al., 1977), a significant number of instances (30%) were detected in uv’d eggs in which the lip lacked a definite dorsal/ventral polarity. It would appear, therefore, that one of the effects of uv irradiation of the uncleaved egg is the location of the dorsal lip at a novel orientation with regard to the sperm entry point. This observation is actually consistent with the results of previous studies on the effects of uv on the location opera’ion

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amphibian egg (Malacinski et al., 1975;also see Discussion). Do uv’d embryos which form their dorsal lip at a novel site display a more or less severe uv syndrome (Expt C)? Irradiated embryos were sorted at gastrulation according to the position of their dorsal lip and later scored for the severity of their uv syndrome. The results of two experiments are shown in Fig. 4. The average uv effect did not vary substantially among embryos which differed with regard to the location of the dorsal lip. Does uv irradiation alter the surface pigmentation pattern of the early embryo (Expt D)? Xenopus laevis eggs typically display a dramatic alteration in the pigmentation pattern of the animal hemisphere at the four-cell stage. Those blastomeres located on the future dorsal side of the embryo are usually more lightly pigmented than the blastomeres on the ventral side (Nieuwkoop and Farber, 1956). Preliminary studies of Gerhart (unpublished observation) and Elinson (unpublished observation) indicated that irradiation of the vegetal hemisphere of uncleaved eggs may alter the pigmentation pattern of the early embryo. An experiment was therefore designed to determine whether uv alters the

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FIG. 3. Effects of uv on the location of the dorsal lip (summary of several experiments.). * The location of the original sperm entry point on the uncleaved egg is shown as a small dot. t Embryos with dorsal lips which lacked a distinct dorsal/ventral polarity.

FIG. 4. Severity of the “uv syndrome” in irradiated embryos which formed their dorsal lips at various sites re the original sperm entry point. The “average uv effect” was calculated by grouping the embryos into categories based upon the severity of the effects of uv on neural structure development (0 = no effect; +5 = aneural).

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early pigmentation pattern. Typically, a heterogeneity in pigmentation pattern is observed in a large clutch of normal (unirradiated) embryos. While some embryos exhibit a dramatic dorsal/ventral distinction in the animal hemisphere pigmentation pattern, others show no pigmentation differentiation whatsoever. For the purpose of these experiments, embryos were grouped into three categories according to the extent of dorsal/ventral pigmentation differentiation (Fig. 5). Eggs were irradiated and observed at the four-cell stage. The data in Fig. 6 demonstrate that uv does indeed alter the pigmentation pattern of the early embryo. Compared with unirradiated eggs, uv’d eggs only very seldom display a distinct dorsal/ventral pigmentation difference. Among irradiated embryos, those which lack a marked pigmentation separation (groups B and C) predominate. It would appear, therefore, that one of the

first effects of irradiation is an alteration in the pattern of pigmentation changes which normally accompany the first few cleavage divisions. Further experiments were designed to determine whether these early alterations in pigmentation are correlated with later effects of irradiation on gastrulation and neural induction. Is the severity of the uu syndrome correlated with the effects of uv on the early (four-cell-stage) pigmentation pattern (Expt E)? Irradiated embryos were sorted at the four-cell stage according to their pigmentation pattern (Fig. 5). Later they were scored for the severity of their uv syndrome. The results are presented in Fig. 7. From the data included in the six experiments summarized in Fig. 7, a consistent picture emerged. The few embryos which displayed a distinct dorsal/ventral pigmentation pattern (group A) exhibited the least severe uv syndrome. Embryos in groups B

FIG. 5. Embryos (unirradiated) at the four-cell stage which show heterogeneity of animal hemisphere pigmentation pattern. Embryos with a distinct dorsal/ventral pigmentation differentiation were sorted into group A. Those with a less distinct differentiation were sorted into group B, while those which displayed no polarity were sorted into group C. No contrast-enhancing falters were employed in the production of this photograph.

CHUNG AND MALACINSKI

FIG. 6. Effects of uv on the pigmentation pattern of the early embryo. Fertile eggs were irradiated and, at the four-cell stage, sorted into three groups, according to the pigmentation pattern in the animal hemisphere (Fig. 5). The results from three separate experiments are presented.

and C showed a more severe uv syndrome. Most of the embryos in each of the experiments were sorted into either of these two groups (see also Fig. 6). A less dramatic difference in the severity of the uv syndrome was observed between embryos in these two groups. It would appear, therefore, that those few irradiated embryos which manifest a relatively normal pigmentation pattern tend to show a markedly diminished uv syndrome. From time to time during the course of these experiments, additional studies were carried out

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in order to assess the relationship of pigmentation alterations to previously characterized (Malacinski et al., 1975) aspects of the uv syndrome. Is there a correlation between uv effects on the early pigmentation pattern and the tendency of uv’d embryos to form dorsal lips at novel sites (Expt F)? Irradiated eggs were sorted at the four-cell stage according to their pigmentation pattern. Later, at early gastrulation, they were observed for the location of the dorsal lip. The lip site was scored in relation to the sperm entry point. The data from a typical experiment are summarized in Fig. 8. There appeared to be no obvious correlation between pigmentation pattern and dorsal lip site. This interpretation of the data is consistent with the former observation concerning the lack of a relationship between lip site and severity of the uv syndrome (Expt C; Fig. 4). That is, group C embryos display a more severe uv syndrome than group B embryos (Fig. 7). Yet the site of the dorsal lip was observed with approximately equal frequency in the various locations (Fig. 8). Egg Rotation as a Probe The use of egg rotation as a probe was stimulated by a recent report of Kirschner et al. (1979) and unpublished observations

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FIG. 8. Relationship between uv effect on pigmentation pattern and location of the dorsal lip. Irradiated eggs were sorted into groups A, B, and C. At the early gastrula stage they were scored for the location of the dorsal lip in relation to the sperm entry point. Data from a single experiment are presented. * Embryos in group A are very rare, and none were observed in this experiment. In another experiment, 11 group A embryos were observed. The sperm pit was, however, obscure, so they were not scored as above. All 11 did, however, form their dorsal lip on the lightly pigmented side.

of Gerhart and colleagues (personal communication). Those authors demonstrated that a simple rotation of the uncleaved egg could reorient the dorsal lip site to a novel location, including the side containing the sperm entry point. The initial set of questions concerning the use of egg rotation as a probe was directed toward independently verifying the original observations of Kirschner et al. (1979). Then further characterizations of the general parameters associated with the use of this probe were established.

forming on the side which opposed gravity during the rotation. The site of the lip is determined in rotated embryos not by the sperm entry point, but rather by the relation of the egg to gravity. That is, the lip forms on the side opposing gravity.

Does rotation of the egg alter the position of the gray crescent (Expt H)? The gray crescent area of the uncleaved Xenopus egg is generally not easily visualized in most clutches of eggs. Occasionally, however, a spawning will provide eggs which do

Can a brief (e.g., 30-min) rotation of a normal egg onto its side-prior to first cleavage-alter the location of the dorsal lip (Expt G)? Fertile eggs were bathed in 5% Ficoll, then rotated 90” in agar wells according to the location of the sperm pit. These rotations, as well as others to be described, were carried out during the middle-third time interval between fertilization and first cleavage. At the end of the 30-min rotation period the eggs were returned to their natural orientation in which the equator of the egg is parallel to the earth’s surface. When the embryos had developed to the early gastrula stage, they were scored for dorsal lip location. The data given in Fig. 9 demonstrate that a brief rotation of the egg does indeed result in the dorsal lip

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FIG. 9. Relocation of the site of the dorsal lip by a brief (30-min) 90” rotation of the uncleaved egg. Fertile eggs were bathed in 5% Ficoll and rotated in relation to the sperm pit. At gastrulation the location of the dorsal lip was scored. * Oriented without regard to the positon of the sperm pit. Data given in terms of the side of the egg which was “up” (opposing gravity) during the rotation period. That is, the dorsal lip formed on the up side in 31 of 36 cases.

CHUNG AND MALACINSKI

display very distinct gray crescents. One such spawning was employed to track the gray crescent in rotated eggs. More than three dozen eggs in each rotational configuration (sperm pit up, sperm pit down, or random) were followed. It was observed that the gray crescent uniformly appeared on the side opposing gravity of rotated eggs (data not shown). Combined Use of uv and Egg Rotation as Probes

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UV effect Stimulated by discussions with J. C. GerFIG. 10. Prevention of the uv syndrome in irradihart (Berkeley), a combination of egg roated eggs by rotation prior to first cleavage. Irradiated tations and uv irradiations was designed to embryos displayed a typical spectrum of uv effects analyze egg cytoplasmic components which (bottom), while rotated embryos showed normal may be required for primary embryonic neural morphologies (top). induction. Can rotation of the uv’d egg for a brief tern (A or C) was not significantly modified period (e.g., 30 min) prior to first cleavage by any of the rotation configurations (Fig. prevent defects later in neural morphogen- 12). esis (Expt I)? Irradiated eggs were rotated A simple rotation of the egg which serves 90” in a random configuration. After 30 min to prevent neural defects does not, thereon their sides, the eggs were reoriented to fore, correct the uv effects on pigmentation their normal disposition and later scored pattern. It would appear that these data for their uv syndrome. The results of a establish the important fact that uv effects single experiment are shown in Fig. 10, and on the early pigmentation pattern can be the results of a series of several experiments experimentally dissociated from uv effects are summarized in Fig. 11. Those data re- on subsequent primary embryonic inducveal that rotation provided a dramatic cor- tion. It remains as an important observarection of the irradiation-induced damage tion, however, that pigmentation pattern in to neural morphogenesis. The average uv irradiated, nonrotated eggs is a reliable preeffect was calculated for each experiment dictor of the severity of the uv syndrome and a “rescue index” derived by simple (Fig. 7). division of the average uv effect of nonroDoes irradiation alter the ability of a tated (control) embryos by the average uv brief rotation to relocate the dorsal lip to effect of rotated embryos. The higher the the “up” side (Expt K)? Irradiated eggs rescue index, the more dramatically rota- were rotated in various configurations prior tion served to prevent defects in neural to cleavage, reoriented, and later scored for morphogenesis. the location of their dorsal lips. The data Does an egg rotation which prevents the from two experiments are summarized in uv syndrome also prevent alterations in Fig. 13. Control (nonrotated) embryos disthe pigmentation pattern (Expt J)? Irradiplayed their usual tendency (see also Fig. ated eggs were rotated in various configu- 3) to form the dorsal lip on the side of the rations and sorted at the four-cell stage egg which contained the sperm entry point. according to their pigmentation pattern Rotation was, however, able to overcome into groups A, B, and C (Fig. 5). The pro- that tendency. For example, the dorsal lip portion of the embryos which displayed consistently formed on the up side in those either of the extremes of pigmentation pat- eggs which were rotated so that the sperm

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FIG. 11. Summary of results from several experiments designed to determine whether rotation of the irradiated uncleaved egg could prevent abnormal neural morphogenesis. Eggs were rotated 9W’, without regard for the sperm entry point (random rotation). Not only were relatively minor uv defects (e.g., Expt III; average = 2.4) prevented, but also major defects (e.g., Expt II; average = 4.2) were prevented. con. = control; no rotation,

pit was down. If uv prevented the relocation of the dorsal lip, many of those eggs would have displayed their dorsal lips on the “down” side. DISCUSSION

The results of these analyses provide further insight into the nature of the egg cytoplasmic components which are involved in the complex process of primary embryonic induction. Recent studies reveal that uv probably does not penetrate very deep beneath the egg cortex (Youn and Malacinski, 1979). The effects on surface pigmentation verify the prediction that uv probably acts either at the egg surface or in the subcortical cytoplasm. The effects of uv on locating the dorsal lip at a novel site (same side as the sperm pit) suggest that the mechanism for the establishment of the dorsal/ventral polarity of the egg is altered. Previous investigations of the effects of irradiation of a localized region of the vegetal hemisphere are consistent with the observations reported here. Those earlier experiments generated groups of embryos which displayed a tendency to form a dorsal lip at a novel site (Malacinski et al., 1975, Fig. 5). Ultraviolet-induced lesions confined to the surface of the egg are probably not the single cause of defects in neural morpho-

genesis. As observed in Expt J, egg rotations which prevented the uv syndrome did so without preventing the uv-induced alterations in pigmentation pattern. Although the uv-damaged egg surface is probably involved in generating the uv syndrome (see model below), the internal egg cytoplasm is most likely involved as well. Despite earlier reports (Curtis, 1962; TompNo

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FIG. 12. Dissociation of the effects of uv on pigmentation pattern from the uv effects on primary embryonic induction. Various rotation configurations which prevent the uv syndrome in irradiated embryos were employed in an attempt to prevent alterations in the early pigmentation pattern. None of the rotations was successful in maintaining a normal pigmentation pattern.

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kins and Rodman, 1971) recent evidence from our laboratory indicates that the egg cortex alone probably does not possesstrue morphogenetic information (Malacinski et al., 1979). The egg cortex-tested in a variety of bioassay systems-was found to be completely devoid of the “inducer” activity described by Curtis (1962). Perhaps the internal cytoplasm is affected by rotation of the uncleaved egg. Weyer et al. (1978) have proposed a model for the diffusion of morphogens as a mechanism for mesoderm induction. Alteration, by rotation, of a concentration gradient could conceivably alter the polarity of normal uncleaved eggs.Likewise, rotation of irradiated eggs may reorient cytoplasmic components in such a way as to repair or reconstitute the polarity determining mechanism of the egg. Despite the ability of egg rotation to prevent the uv syndrome, the rescue effect varied somewhat from one spawning to another (e.g., Fig. 11). As well, the magnitude of the uv syndrome for a given dose of irradiation varies among spawnings. Other effects of uv, such as an effect on cleavage division, have also been reported to vary between batches of eggs (Malacinski et al., 1977). Beal and Dixon (1975) have, therefore, called for caution in the interpretation of data derived from irradiation studies. In the present studies, such caveats were ac-

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commodated by employing only moderate doses of uv (e.g., 2.8 x lo4 ergs/cm’) and by performing all the operations included in one experiment with the eggs from a single frog. The data generated by the experiments described in this report, when interpreted in terms of both the classical literature on amphibian egg polarity and the more recent insights obtained from irradiation experiments, suggest several models to account for egg symmetrization. These models are summarized in Fig. 14 and are discussed below. Symmetrization of the egg is no doubt the result of the fertilization reaction. There is no evidence to support the existence of a predetermined sperm entry point on the surface of Xenopus eggs. Once activated by sperm attachment, the egg undergoes a cytoplasmic segregation which leads to a displacement of some of the cytoplasmic components to the future dorsal side of the egg (Herkovits and Ubbels, 1979). A single-component model (Fig. 14a) can be developed which accounts for many, if not all, of the descriptions of the effects of uv and egg rotation on the pattern of early embryogenesis. In that model, the internal segregation of the yolk-free cytoplasm (Ubbels, 1978) is postulated to require the normal surface contraction-movements which result in the appearance of the gray

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FIG. 13. Location of the dorsal lip in irradiated, rotated embryos. Irradiation configurations from relocating the dorsal lip to novel locations.

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crescent on the future dorsal side of the egg. Rotation re gravity perhaps causes the symmetrization to occur in a plane which is perpendicular to the earth’s surface. Hence, the gray crescent and the dorsal lip consistently appear on the side of the egg which opposes gravity, even when that side coincides with the sperm entry point (Expt H). By affecting the surface contraction process, uv may actually prevent the normal movements of the egg cytoplasm so that symmetrization is altered. Rotation of irradiated eggs perhaps succeeds in redistributing the internal cytoplasm so that even in the absence of normal surface contractions, the symmetrization of the egg cytoplasm which is a prerequisite for normal development takes place. These features of the model are consistent with classical observations on egg symmetry (reviewed by Brachet, 1977) and the recent findings of Elinson and Manes (1979) which demonstrate that gray crescent formation in Rana pipiens eggs is suppressed by uv. The effects of uv on the surface contraction mechanism of the egg were monitored in the

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present studies by observing the pigmentation pattern of four-cell-stage embryos. The results of several experiments (e.g., D, E, F) reveal that the Xenopus egg surface is affected by uv. Yet those effects on pigmentation can be experimentally dissociated from uv effects on subsequent primary embryonic induction (Expt J, Fig. 12). Further experimentation on the effects of uv on surface movements will, therefore, be required in order to develop substantial confidence in this model. A double-component model can be devised which also employs a surface component. But in this model the role of the surface component is considered to be independent of the surface contractions which are central to the single component model. The salient features of this model are based upon a variety of earlier observations recorded in the literature of classical embryology. For example, Penners and Schleip (1928) carefully observed the formation of double embryos in inverted (rotated) eggs and concluded that the flow of yolk from the vegetal to the animal hemi-

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sphere caused the double axes. Pasteels (1938) later speculated that in inverted eggs the position of the dorsal lip is determined by an interaction of two factors: migration of yolk and some influence from the gray crescent area. It was further postulated that a cortical field accounts for the capacity of the dorsal lip to form at novel sites. The gray crescent area would contain the highest concentration of a cortical component which interacts with the internal cytoplasm. Other areas of the cortex should contain reduced, albeit significant, amounts of that cortical component. Rotation of the egg would succeed in locating the dorsal lip to a new site, by virtue of that cytoplasmcortex interaction. Dalcq and Pasteels (1937) offered a “double gradient” theory to account for those observations. That theory has recently been reviewed by Brachet (1977). The results of the most recent experiments on egg rotation (Kirschner et al., 1979) are entirely consistent with the hypothesis of Dalcq and Pasteels. The “double-component” model (Fig. 14) is derived from those previous ideas. In Fig. 14b a model is diagrammed in which the surface component (peripheral coupler) activates the internal cytoplasmic component. Once activated, the dorsal ventral polarity of the egg is established. It is postulated that the “peripheral couplers” constitute the uv target, and the internal cytoplasm is the essential object of the egg rotation. Prior to fertilization the peripheral couplers are not organized close to the egg surface, which is consistent with the observation that the unfertilized egg is somewhat less sensitive to uv than the fertile egg (Expt A). Rather, during activation the peripheral couplers become situated close to the egg surface on the side opposite the sperm entry point. The results of recent irradiation experiments reveal that the uv target is indeed located close to the egg surface (Youn and Malacinski, 1979). The future dorsal side of the egg is the most uvsensitive area (Malacinski et al., 1975), and the sensitivity of the target diminishes rap-

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idly after fertilization (Malacinski et al., 1978). Likewise, the main features of the model are consistent with the data included in this report. Rotation of irradiated eggs presumably brings the internal cytoplasm into contact with the peripheral couplers which, by virtue of their incomplete association with the egg surface, remained undamaged by the vegetal hemisphere irradiation treatment. The location of the dorsal lip at novel sites in irradiated embryos (Fig. 3) was predicted from earlier studies, which employed localized irradiation and generated large numbers of uniquely oriented eggs (see Fig. 5 of Malacinski et al., 1975). An alternative double-component model is also shown in Fig. 14 (Fig. 14~). In that version the peripheral couplers are an integral component of the unfertilized egg surface. The surface contraction (movement) serves to concentrate the peripheral couplers on the side opposite the sperm entry point. Otherwise, the salient features are similar to those which characterize the model diagrammed in Fig. 14b. This model, like the “single-component” model, calls for further experimentation on the nature of the effects of uv on the surface contraction process which normally accompanies fertilization. Each of the above models deals with the “reconstitution” of the normal symmetrization mechanism of the irradiated and/or rotated egg. A formal alternative to those models, however, exists. Perhaps in response to rotation a repair mechanism is activated which corrects the irradiation damage. Evidence for the ability of irradiated Rana eggs to correct the uv damage at low temperatures (cryoreversion) is available (Malacinski et al., 1974). A repair model, although not necessarily as amenable to direct testing as the other models, should, however, be evaluated by further experimentation. Several of the characteristics of the model can be tested by further experimentation. As further studies are carried out these models will no doubt undergo major

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revisions. Yet as a point of departure, they should facilitate the conceptualization of future experimental designs. The authors would like to express appreciation to S. Scharf, S. Black, and J. Gerhest (Berkeley). They generously shared with us the details of their discovery-prior to its publication-that egg rotation corrects the uv syndrome. R. Elinson (Toronto) provided two critical reviews of the manuscript, including suggestions concerning the models, for which the authors are grateful. Several other colleagues also provided valuable reviews of the manuscript and are hereby acknowledged. B. -W. Youn contributed assistance with the design of several experiments, and D. Malacinski supplied technical expertise. NSF PCM 77-04457 and a joint NSF (INT-7827366) Korean Science and Engineering Foundation grant furnished financial assistance. REFERENCES BEAL, C. M., and DIXON, K. E. (1975). Effect of UV on cleavage of Xenopus laevis J. Exp. 2001. 192, 277-283. BRACHET, J. (1977). An old enigma: The gray crescent of amphibian eggs. Curr. Topics Develop Biol. 11, 133-186. CHUNC, H. M., MALACINSKI, G. M., and KIM, B.-G. (1977). Developmental lesions in amphibian embryos induced by ultraviolet irradiation of the fertile egg. Korean J. 2001.20,109-121. CURTIS, A. S. G. (1962). Morphogenetic interactions before gastrulation in the amphibian, Xenopus laevis-The cortical field. J. Embryol. Exp. Morphol. 33, 147-157. DALCQ, A., and PASTEELS, J. (1937). Une conception nouvelle des bases physiologiques de la morphog&&se. Arch. Biol. 48,669-710. ELINSON, R. P. (1975). Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipiens. Develop. Biol. 47, 257268. ELINSON, R. P., and MANES, M. E. (1979). Grey crescent formation and its inhibition by ultraviolet light. J. Cell Biol. 83, 213a (abstr.). GERHART, J. C. (1980). Mechanisms regulating pattern formation in the amphibian egg and early embryo. In “Biological Regulation and Development” (R. F. Godberger, ed.). Plenum, New York (in press). GRANT, P., and WACASTER, J. F. (1972). The amphibian gray crescent region-A site of developmental information? Develop. Biol. 28.454-471. HERKOVITS, J., and UBBELS, G. A. (1979). The ultrastructure of the dorsal yolk-free cytoplasm and the immediately surrounding cytoplasm in the symme-

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