Destruction of components of the neural induction system of the amphibian egg with ultraviolet irradiation

DEVELOPMENTAL

BIOLOGY

Destruction

GEORGE

56, 24-39

(1977)

of Components of the Neural Induction System of the Amphibian Egg with Ultraviolet Irradiation M.

MALACINSKI,

Department

ANN

of Zoology, Received

JANICE

Indiana June

BROTHERS,

University,

1,1976;

accepted

AND

Bloomington, August

HAE-MOON

Indiana

CHUNG

47401

30,1976

Ultraviolet irradiation of the vegetal hemisphere of the fertilized amphibian (Xenopus laeuis) egg prior to first cleavage results in the embryo developing an incomplete set of neural structures. The effects of irradiation on various morphogenetic processes, including cell division, formation of the dorsal lip, invagination at gastrulation, and neural induction by the primary organizer, were examined. A decrease in the capacity for invagination during gastrulation and a diminution in the neural inducing capacity of the primary organizer were found to account for defective neurulation in irradiated embryos. Consequently, irradiation of the uncleaved egg leads to interference with the events of both gastrulation and neurulation.

lished that some of the cytoplasmic components which are involved in the formation of the primary axis of the embryo are present in the amphibian egg prior to cleavage division (Spemann, 1938; Curtis, 1962; Nieuwkoop, 1973). The experiments which have employed uv further indicate that at least one of those components is sensitive to irradiation and is localized on the future dorsal side of the egg (Malacinski et al ., 1975). The destruction of that component results in a diminution of the inducing capacity of the dorsal lip, as measured by embryological assays. The apparent specificity of the developmental lesion produced by uv was demonstrated by tissue grafting experiments. Replacement of the dorsal lip area of an irradiated embryo with a normal lip area promoted normal neural morphogenesis (Chung and Malacinski, 1975). Although those previous grafting experiments clearly demonstrated that uv affects the inducing capacity of the dorsal lip, they did not establish which of the morphogenetic events of neural induction were affected. An effect on any of a number of cellular processes, including cell division, cell movements of invagination during gastrulation, or the actual cell in-

INTRODUCTION

Control of the morphogenetic pattern of early amphibian development is generally understood to be achieved by an interaction between cytoplasmic components and the nuclei of the embryo. Although various lines of evidence support this concept, there exists only a limited number of experimental approaches which permit direct insight into either the nature of the active components or the mechanisms whereby they exert their effects. Maternal effect mutations, which lead to specific cytoplasmic deficiencies, provide one important experimental approach (reviewed by Briggs, 1973). Alternative approaches include the use of manipulative techniques such as surgical operations (Hennen, 1973), localized ultraviolet (uv) irradiation (Grant and Wacaster, 1972), and metabolic inhibitors (Cooke, 1973). The usefulness of each of those methods is directly related to the extent to which their effects can be shown to be exerted on single, specific morphogenetic processes such as the pattern of cleavage, the cell movements of gastrulation, or the construction of the embryonic axis. Several of those analyses have estab24 Copyright All rights

Q 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0012-1606

MALACINSKI,

BROTHERS

AND

CHUNG

teractions which promote the differentiation of neural structures from presumptive ectoderm, could lead to defective neurulation. In an attempt to better understand which of these cellular processes are affected by irradiation of the uncleaved egg, a detailed analysis of those separate morphogenetic processes was undertaken. By carefully controlling the experimental conditions, it was possible to select for manipulation groups of embryos which displayed a uniform response to uv. This permitted a systematic analysis of the effect of uv on cell division, dorsal lip formation, cell movements of invagination, and the primary embryonic organizer. The results of these investigations revealed that uv apparently diminishes the embryo’s capacity for neural development through an inhibition of cell movements and a diminution of the actual inducing capacity of the primary embryonic organizer. MATERIALS

AND

METHODS

Collection of eggs. Xenopus laevis eggs were artificially inseminated according to published methods (Wolf and Hedrick, 1971). The eggs were chemically dejellied within 0.5 hr after fertilization in a 2% cysteine-HCl solution (pH 7.8) and irradiated within 1 hr after fertilization. All obse&ions and surgical operations were carried out at 20°C. uv irradiation. The method of irradiation of the vegetal hemisphere of the uncleaved egg and the characteristics of the uv source (2537 A) are described in a previous publication (Malacinski et al., 1975). Control eggs were treated in an identical fashion to the irradiated eggs, except that uv irradiation was omitted. Figure 1 displays the range of effects on the formation of neural structures observed when eggs from a single clutch are irradiated with 12,000 erg/mm*. The embryos are scored according to the extent of uv-induced damage. Surgical operations. The dorsal lip

Destruction

of Neural

Induction

System

25

grafts and the primary organizer transplants followed methods recently described by Hennen (1973) and modified by Chung and Malacinski (1975). In all of the experiments, the primary embryonic organizer was operationally defined as the area above the dorsal lip which promoted the development of a secondary axis in the implantation assay. Following the experimental design described by Spenmann (1938), the dorsal lip itself was not included in the tissue used in the bioassays for primary organizer activity (see Discussion). Implantation of the primary organizer was performed under the same conditions established for the grafting experiments. The primary organizer was inserted directly into the blastocoel through a small hole cut in the roof of the blastocoel. Organ cultures. Primary organizer tissue was inserted into the presumptive ectoderm of the blastocoel roof of stage 7.5 embryos. Control cultures of ectoderm from that stage of development did not differentiate neural tissue without the insertion of primary organizer tissue. The preparation of the organ cultures was carried out in 100% Steinberg’s solution + 2 X Ca*+ 400 mg of and Mg*+, pH 6.9, containing penicillin-G and 50 mg of Gentomycin (Schering Corp.)/liter. After approximately 2 hr, the cultures were transferred to a 20% Steinberg’s solution, pH 7.4, containing antibiotics. All operations and cultures were kept in petri dishes on a layer of 2% agar. Histological analyses. The organ cultures were fixed in Masui’s fixative (Masui, 1973) embedded, sectioned, and stained with the Feulgen procedure and counterstained with Light Green (Moore, 1940). Embryos were fixed in 5% neutralized formalin solution, embedded, sectioned, and stained. For scanning electron microscopy, embryos were fixed in glutaraldehyde solution (Kalt, 1971) and dried and coated by standard critical-point

26

DEVELOPMENTAL

BIOLOGY

VOLUME

56, 1977

FIG. 1. Ultraviolet-induced lesions in Xenopus Lueuis embryos irradiated at the vegetal hemisphere prior to the first cleavage. In a single clutch of eggs, the extent of damage to neural morphogenesis varies from embryo to embryo. A scoring system with the following notations is employed to provide a semiquantitative estimate of the effect of uv: 0, no apparent damage; + 1, microcephalic; +2, extreme microcephalic; +3, short axis; +4, acephalic; +5, aneural.

drying and coating methods (Anderson, 1951). Dried embryos for scanning were set onto sample platforms which were coated with a bed of fast drying epoxy resin cement. EXPERIMENTAL

RESULTS

Effects of uv on Cleavage The site of invagination of control (nonirradiated) and irradiated embryos was examined under the scanning electron microscope. Embryos irradiated at either a low dose of uv (12,000 erg/mm2) or a high dose of uv (18,000 erg/mm*) were analyzed. It was observed in embryos which received the low dose of uv that cleavage was apparently normal. That is, at both stage 10 (early dorsal lip) and stage 11.5 (yolk plug), the size and number of cells were normal, when compared with control em-

bryos of the same stage (Figs. 2A and 2B). Embryos which received a high dose of uv displayed a diminution in the number of cells in the area of invagination, as well as a corresponding increase in the size of the cells in that area (Fig. 20. Despite the increase in cell size in embryos which were irradiated with high doses of uv, a dorsal lip formed, invagination proceeded, and an incomplete yolk plug was observed. A random sample (5-10 embryos) of control, low-dose irradiated, and high-dose irradiated embryos was examined at 880x magnification. With the aid of a template, the mean number of cells occupying a given area (5 x 6 cm) in the vicinity of the site of invagination on the display screen of the scanning microscope was measured. Control embryos contained 18.7 L 4.7 cells, low-dose irradiated embryos contained

MALACINSKI,

BROTHERS

AND CHUNG

Destruction

of

Neural

Induction

27

System

F IG. 2. EXfects of uv on cleavage. Embryos at the dorsal lip and yolk plug stages w el -e fixed and exam an ed in the scanning electron microscope at 84 x (A) Control (unirradiated) e In Ibryos; whit :h we re irradiated with 12,000 erg/mm*; (0 embryos irradiated with 18,000 erg/m LIlP. Eml dose of uv displayed a more severe effect upon cytokinesis in the regetal with tF Le higher Lisphe :re

I, slladowed, (B) embryos xyt LX irradi(ir radiated)

28

DEVELOPMENTAL

BIOLOGY

FIGURE

18.4 + 1.4 cells, and high-dose irradiated embl70s contained 11.0 + 4.0 cells in that area of the field of the embryo. These resuits indicate that, when the uv dose is

VOLUME

56, 1977

2B.

increased from 12,000 to 18,000 erg/n 1m2, the rate of cleavage is diminished in the vegetal hemisphere, leading to the increased size of the irradiated blastome!res.

MALACINSKI,

BROTHERS

AND

Destruction

CHUNG

FIGURE

Examination of the embryos displayed in Fig. 2 reveals that cells in the animal hemisphere of irradiated embryos (seen in the equator region of embryos at the yolk

of Neural

Induction

System

29

2C.

normal in plug stage) were apparently size. For the later experiments on embryo ‘nit induction, the low dose of uv (:12,000 e!rg/

30

DEVELOPMENTAL

BIOLOGY

During the course of velopment of the several bryos employed in the scribed in this report, it

Lip

observing the dehundreds of emexperiments dewas noticed that TABLE

ANALYSES Description of tissue combinations tested in the organ cultures

(1) Primary organizer from early gastrula (stage 10) implanted into early blastula (stage 7.5) ectoderm (2) Ventral region from early gastrula (stage 10) implanted into early blastula (stage 7.5) ectoderm (3) Isolated early blastula ectoderm (stage 7.5) (4) Controls-incision made around the primary organizer from early gastrula (stage 10) piece left in place (5) uv-irradiated (low dose = 12,000 erg/mm2) early gastrula primary organizer implanted into early blastula (stage 7.5) ectoderm (6) uv-irradiated (high dose = 18,000 erg/mm*) early gastrula primary organizer implanted into early blastula (stage 7.5) ectoderm

OF HISTOLOGICAL

56, 1977

irradiated embryos frequently displayed a delay in the time of appearance of the dorsal lip. This observation has been noted previously (Grant and Wacaster, 1972; Malacinski et al., 19751, but has not been examined in detail. An analysis of the relationship between the delay in the formation of the site of invagination and the ultimate extent of neural morphogenesis was carried out. Among different batches of eggs there appeared to be substantial variability in the duration of the delay in dorsal lip formation in irradiated embryos. In each of several experiments, irradiated

mm21 was used exclusively, unless mentioned otherwise (e.g., Table 1). The effects on cleavage, although not necessarily involved in the lesion which contributes to the development of microcephalic embryos (see Discussion), could be eliminated with the use of the low dose. Effects of uv on Time of Dorsal Formation

VOLUME

1

SECTIONS

Combined Neural tube plus notochord

Abnorma1 neural tube (no closure) plus notochord

15

OF ORGAN

results

of three

CULTURES different

experiments

Notochord only

Neural plate

No neural differentiation

Total number cultures

9

2

1

0

27

0

0

0

0

22

22

0

0

0

0

28

28

20

1

0

0

0

21

0

3

6

9

0

18

0

0

0

4

16

20

of

MALACINSKI,

BROTHERS

AND

CHUNG

Destruction

of Neural

Induction

31

System

embryos were grouped according to the length of the delay in lip formation and scored with regard to the extent of neural morphogenesis. The results of three such experiments are summarized in Fig. 3. Embryos which displayed a delay of 1 hr or longer consistently developed severe abnormalities in their neural morphologies. Conversely, a delay in the formation of the dorsal lip was not a prerequisite for the development of neural defects. Frequently, embryos which displayed no delay in dorsal lip formation developed extreme neural defects (e.g., Fig. 3, Expt 3). The reverse was, however, always true. A delay in the formation of the dorsal lip provided a reliable indication that the embryo would exhibit severe defects in neural morphology. This observation was considered very significant, for it provided a solution to the problem of the variation of response to irradiation of individual embryos from a single batch of eggs. The morphology of the lip in embryos which displayed the delay in the formation of the site of invagination was frequently not as well defined as control embryos. The lip often lacked the usual pigment markings and was much broader and thin-

ner in appearance than control lips. These features are also included in the data of Fig. 3.

The wide range of response to uv of individual embryos from a single clutch of eggs is displayed in Fig. 4. At the relatively low doses of uv which were employed in these experiments to circumvent effects on cleavage (Fig. 2), some embryos developed normal neural morphologies, while others displayed severe effects on neural development. Furthermore, the range of response varied from one batch of eggs to another (Fig. 4, insert). By selecting embryos which exhibited a slight delay in the formation of the dorsal lip, it was possible, however, to obtain from a single clutch of irradiated eggs a group of embryos which displayed a uniform response to uv. This selection method was employed in subsequent experiments on invagination and primary embryonic induction. By

FIG. 3. Relationship between delay in dorsal lip formation and development of neural structures. Small batches of eggs were irradiated and grouped according to the duration of the delay in dorsal lip formation. Their development was observed at stage 25 as normal, microcephalic, acephalic, or aneural. *Min. delay compared with control, unirradiated embryos (20°C). Nieuwkoop and Faber (1956) stage series was used.

FIG. 4. Response of a single clutch of eggs to irradiation. A wide range of neural morphologies was displayed by individual embryos. Insert: results of a separate experiment. In both experiments, embryos which displayed a 60-90-min lag in lip formation were selected to provide embryos which would display a uniform response to uv. *A description of the scoring system of (0) to (+ 5) has been previously published (Malacinski et al., 1975).

Variation

I

in Response

to uv

1

32

DEVELOPMENTAL

selecting embryos which displayed relatively brief lags in dorsal lip formation, and embryos which displayed long lags, embryos which displayed uniform microcephaly or acephaly, respectively, could be employed. Effects of uv on Morphogenetic of Gustrulation

VOLUME

BIOLOGY

Movements

Despite the apparent normal morphology of the yolk plug of irradiated embryos (Fig. 2), dissection revealed that the cell movements of gastrulation were incomplete. That is, some embryos displayed negligible invagination, and other embryos displayed partial invagination. A systematic analysis of invagination in irradiated embryos was carried out by employing a vital dye to trace the extent of cell movements. The design of the experimental procedure is given in Fig. 5. The time of formation of the dorsal lip on irradiated embryos was observed and embryos were grouped according to the length of the lag. This procedure ensured a uniform response to uv by individual embryos in each group. These embryos, as well as control (unirradiated) embryos, were microinjetted with approximately 2-5 nl of a 5% nile blue sulfate solution just at the edge of the site of invagination. Several of the injected embryos were dissected at the neural fold/plate stage, while others from the same batch, but which had not received a dye injection, were scored for neural morphology at the muscular response stage. The extent to which the dye had moved toward the anterio-ventral region was indicated on a schematic map of a saggital section of a neural plate stage embryo. The data in Fig. 5 reveal that embryos which displayed a 90-min lag in dorsal lip formation failed to accomplish normal morphogenetic movements of the site of invagination toward the anterior region of the embryo. Embryos from the same group which were permitted to develop demonstrated a microcephalic syn-

56, 1977

CONTROL

EXPERIMENT

STEP irradiation Of egg=

e

STEP

\I

timing: dye injection Of some embryos

0 A

0 hrs

STEP

3

diy& (St. 13)

I

al

. Es3 I 10 “cwtn~l

1; hrs I

2Q t-m I

@i&i& I I

T microcwhalic, acephkic

I mic;ocephalic. 8 acephalic

FIG. 5. Experimental procedure for measuring the extent of morphogenetic movements in irradiated embryos. The time of dorsal lip formation was observed in both control (unirradiated) and irradiated embryos (20°C). Some of these embryos were microinjected with nile blue sulfate dye, while others were maintained separately so that the extent of neural morphology of the various batches of embryos could be recorded. The uv-irradiated embryos develop slightly slower than normal embryos. Each group of injected embryos was allowed to develop to neural plate stage before fixation in 5% neutral formalin. The position of the dye at the time of injection and at the time of dissection is shown by a x. The x marks shown in the saggital sections at step 3 represent the position of the dye in each of the individual embryos which were dissected. The embryos shown in this figure represent the results of a single experiment with one batch of eggs. *Embryos irradiated with uv were sorted according to the length of the lag in lip formation.

drome. Embryos which displayed a 165 min lag in dorsal lip formation demonstrated even less internal movement of the site of invagination and displayed a more severe effect on neural development (acephal y) . The results of a more extensive experiment are summarized in Fig. 6. There appeared to be a direct correlation between the duration of the delay in dorsal lip formation, the extent to which normal cell movements were accomplished, and the resulting severity of the neural defect syn-

MALACINSKI,

BROTHERS

AND CHUNG

Destruction

of Neural

Induction

System

33

P control, non UV’d

Delay O-10

in

dorsal

min.

50

lip min.

appearance 90

min.

250

min.

,QOQQ~BFIG. 6. Extent of morphogenetic movements of invagination in irradiated embryos. A batch of embryos was irradiated and divided into groups based upon the length of delay in dorsal lip appearance. They were injected and scored as described in Fig. 5. The uv-irradiated embryos are somewhat slower in development when compared to normal embryos. Each group of embryos was allowed to develop to midneurulation before fixation. These embryos represent the results of a single experiment.

drome. In this experiment, embryos which displayed a 0-lo-min delay in dorsal lip appearance developed a microcephalic syndrome, while those which displayed a 90-250-min delay developed an aneural morphology. In another experiment, embryos were given a double injection of dye. Nile blue sulfate was injected into the dorsal lip area, and neutral red (3%) was injected into the area of the ventral lip of the same embryos. Invagination of the ventral area marked with red dye was consistently more normal than invagination of the dorsal area marked with blue dye (unpublished observation). Occasionally, microcephalic embryos were observed in batches of embryos which displayed apparently normal movements of invagination. That observation suggested that a diminution in the cell movements of gastrulation alone might not be sufficient to account for incomplete neural induction in irradiated embryos. Accordingly, experiments were designed to examine the effect of uv on the primary embryonic organizer. Effects of uu on the Primary Organizer

Embryonic

Previous experiments from this laboratory (Chung and Malacinski, 1975) have demonstrated that irradiation of the vegetal hemisphere of Rana pipiens eggs diminished the neural inducing capacity of the dorsal lip area at gastrulation. In con-

L FIG. 7. Experimental procedure for demonstrating that uv diminishes the inducing capacity of the dorsal lip area of the embryo. At the early gastrula stage, the dorsal lip and a small portion of the area above it (primary organizer region) were grafted to the ventral region of nonirradiated recipient gastrulae. At the muscular response stage, the extent of secondary induction was scored.

sideration of the results of the preceding experiments, which demonstrated that uv diminishes the ability of the cells in the area of the lip to invaginate, it was of importance to determine whether uv also affected the inducing capacity of the primary organizer of the embryo. In those previous experiments (Chung and Malacinski, 19751, the “dorsal lip area” employed in the grail included both the dorsal lip and a small portion of the primary embryonic organizer. Figure 7 displays the experimental protocol which was employed to demonstrate that uv diminishes the inducing capacity of the lip area. Since a different species of amphibian, Xenopus Zaeuis, was employed in the present studies, those grafting experiments were repeated as a starting point for a careful analysis of the effect of uv on the primary

34

DEVELOPMENTAL BIOLOGY

organizer itself. The results are summarized in Fig. 8 and clearly demonstrate that irradiation diminishes the inducing capacity of the dorsal lip area in X. laevis embryos. Of the 27 experimental grafts which were prepared, none displayed a significant secondary induction. In the control series, however, all 11 of the grafts displayed substantial secondary inductions. Two experimental approaches were employed to assay the neural inducing capacity of the primary organizer. An in vivo implantation assay and an organ culture assay were used. Both of these assay systems provided a test of the inducing capacity of the primary organizer which did not depend upon the dorsal lip’s capacity for invagination. That is, the effect of uv on the primary organizer was assayed in a system which permitted a test which was independent of the effects of uv on the cell movements of gastrulation. The implantation assay procedure is diagrammed in Fig. 9. The region of the embryo just above the dorsal lip was implanted into the blastocoel of midblastula stage embryos. Care was taken to ensure that the dorsal lip itself was not included

>:, ml51411

0

/

0

/

22

1 19

FIG. 8. Results of the dorsal lip area grafts (Fig. 7) are displayed in the top two lines of the chart. Results of the primary organizer implantation experiments (Fig. 9) are summarized in the bottom four lines of the chart.

VOLUME 56, 1977

FIG. 9. Bioassay for the inducing capacity of the primary embryonic organizer of irradiated embryos. The area above the dorsal lip was surgically implanted into the blastocoel of a midblastula stage normal host embryo. As neural morphogenesis proceeded, the host was examined for the presence of a secondary embryonic axis. In the experimental series (top row), embryos which displayed a lag (approximately 1 hr) in lip formation were employed.

in the implanted tissue. As neurulation proceeded, a second set of axial structures frequently developed in the control series. The extent of secondary induction in the tissue implantation assay was usually not as great as in the dorsal lip graft assay. For each set of implants, histological sections were prepared and examined for the presence of a secondary axis. Examination of those histological sections verified the scoring, based on gross morphology, presented in Fig. 8. When ventral marginal zone tissue was used as the implant, a second set of axial structures never developed. In the experimental series, in which the primary organizer of an irradiated embryo was implanted, significant amounts of secondary induction were not observed. These observations are recorded in Fig. 8. For each of the experiments listed in Fig. 8, less than 10% of the operated embryos failed to develop. The data in Fig. 8 represent >90%, for each experiment, of the embryos on which the implantation operation was performed. The interpretation of the data in Fig. 8 was not, therefore, obscured by a failure of some of the operated embryos to develop and be omitted from the tabulations. Representative embryos

MALACINSKI,

BROTHERS

AND

CHUNG

from each experiment were fixed, sectioned, and examined. The microscopic analysis supported the visual observations of the neural morphologies in that a second set of axial structures (neural tube, notochord, etc.) was present in those embryos which were scored as having a secondary axis and was lacking in those embryos which were scored as not displaying a secondary induction. These data indicate that the inducing capacity of primary organizers from irradiated embryos is diminished. That interpretation was further examined by the “organizer replacement” experiment described in Fig. 10. The primary organizer of a normal (unirradiated) host was removed and replaced by the organizer from an irradiated embryo. At the muscular response stage, the neural morphology of the grafted embryos was scored, and the total lengths of the embryos were

Destruction

of Neural

Induction

System

35

measured (Fig. 11). These results indicated that embryos which contained a primary organizer from an irradiated embryo, as compared with control embryos in which a normal organizer replaced the host’s organizer, displayed both microcephaly and a shortened axial length. In the alternative experimental approach, described in Fig. 12, the primary organizer (stage 10) was explanted into the presumptive ectoderm of an early-mid blastula (stage 7.5) stage embryo. The recipient ectoderm folded over the primary organizer and was maintained as an organ culture for 2 days. The presence of neural structures was usually easily observed in these organ cultures. Histological sections were prepared and examined for neural differentiation. The results of three series of experiments are summarized in Table 1. The results indicate that the inducing capacity of the primary organizers from irra-

replace normal primary organizer wth U.V.U organizer

side

view

FIG. 10. Experimental procedure for the primary organizer replacement grafts. The primary organizer of a normal early gastrula stage host was replaced with the organizer from an irradiated embryo. In a control experiment, the organizer from a normal host was replaced by another normal organizer. At the muscular response stage, the extent of neural development and the axial lengths of the experimental and control embryos were compared (Fig. 11).

FIG. 11. Results of the primary organizer organizer from an irradiated embryo displayed axial structures.

replacement microcephaly

graft experiment. and a diminution

Embryos which in the average

contained an length of the

36

DEVELOPMENTAL

BIOLOGY

, earlv-mid

ectoderm

culture

FIG. 12. Experimental procedure for preparing an organ culture for the measurement of the inducing capacity of primary organizers. The primary organizer (from stage 10) was implanted into the presumptive ectoderm of an early-mid blastula stage (stage 7.5) embryo. After 2 days in culture, the organ culture was examined for the presence of neural structures.

diated embryos is reduced and that the degree of reduction is dependent upon the uv irradiation dose. The implants of organizers taken from donors which received a high dose (18,000 erg/mm2) of uv exhibited a more severe reduction in inducing capacity than the organizer implants from donors which had received a low dose (12,000 erg/mm? of uv. DISCUSSION

Ultraviolet irradiation of the amphibian egg results in several effects on early embryological development. Baldwin (1915), and, more recently, Grant and Wacaster (1972) and Malacinski et al. (19751, have observed that microcephalic embryos resulted from irradiation of the uncleaved egg. In addition, irradiation has occasionally been observed to display other effects, such as an effect on cell division (Grant and Youngdahl, 1974; Z&t and Dixon, 1975) and a somewhat variable effect on gastrulation (Malacinski et al., 1975). The experiments reported in this paper represent a systematic analysis of the development of the irradiated egg in order to determine which of the various effects of uv contribute to the embryo’s inability to undergo normal neural morphogenesis.

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The effects of uv on cleavage were not entirely unexpected. Preliminary experiments (H. Benford, unpublished observations) indicated that, at sufficiently high doses of uv (e.g., 20,000 erg/mm2>, all cleavage at the vegetal hemisphere could be inhibited. For the present studies, it was important that a dose of uv could be employed which generated microcephalic embryos without having substantial effects on cleavage. The lower dose of uv (12,000 erg/mm2), applied within 1 hr after fertilization (approximately 1 hr before the first cleavage division), had no apparent lasting effects on cleavage, as measured by the size of the blastomeres in the vegetal hemisphere (Fig. 2B). Ultraviolet irradiation has also been observed to delay cytokinesis during early cleavage stages, rather than to inhibit all cleavage, since cell membranes did form during blastula stages (Zust and Dixon, 1975). Although minor effects of uv on cleavage might have gone undetected in our studies, a variety of evidence indicates that a small residual effect on cleavage in the vegetal hemisphere of the amphibian egg could not alone account for the embryo’s diminished capacity for neural induction. The inhibition of cleavage with metabolic inhibitors, for example, showed that cell division during gastrulation plays no necessary role in morphogenesis (Cooke, 1973). In addition, replacement of the dorsal lip area of irradiated embryos with a normal lip served to repair the effect of uv on neural induction (Chung and Malacinski, 1975). In those replacement grafts, only a relatively small portion of the dorsal side of the embryo was replaced. Vegetal hemisphere cells which might have been encountering difficulty in cleaving did not interfere with a normal dorsal lip’s ability to promote neural induction. In order to circumvent severe effects on cytokinesis in the vegetal hemisphere, the lower dose of uv (12,000 erg/mm21 was routinely em-

MALACINSKI,

BROTHERS

AND CHUNG

ployed in these experiments. The effects of irradiation on formation and invagination of the dorsal lip were carefully examined. Many batches of embryos displayed a small delay in the time of appearance of the dorsal lip. The dorsal lip frequently was not as well defined with regard to its size and pigmentation characteristics in irradiated embryos as in control embryos. The delay in lip formation was not, however, a prerequisite for the formation of incomplete or defective neural structures. Embryos containing lips which dis-

played a normal temporal appearance and morphology frequently displayed severe defects in neural induction (Fig. 3). These observations on the delay in lip formation permitted the selection from a single clutch which would otherwise display a broad response to uv (ranging from normal neural development to aneural development), embryos which consistently displayed a defect in neural development (Fig. 4). A uniform response to uv was considered a prerequisite for the bioassay of the inducing capacity of the primary organizer. The only alternatives to working with embryos which would display a uniform response to uv were either (a) employing very large numbers of embryos in each experiment-an impractical consideration for such experiments as the primary organizer replacement grafts (Fig. lo&or (b) employing large doses of uvwhich would usually result in severe effects on cleavage, frequent developmental arrest prior to the completion of gastrulation, and severe aneural syndromes. Measurements of the extent of invagination of cells in the area of the dorsal lip revealed that cell movements in irradiated embryos were usually incomplete (Figs. 5 and 6). These studies provided a clear-cut demonstration that uv irradiation of the uncleaved egg results in a diminished invagination capacity of the cells in the vicinity of the dorsal lip. The major effect of

Destruction

of

Neural

Induction

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37

uv on neural induction in irradiated embryos might, therefore, be attributed to the embryo’s inability to complete the normal movements of gastrulation. Apparently, morphogenetic movements are necessary for differentiation. Further experiments were designed to determine whether or not uv has the additional effect of destroying the neural inducing capacity of the primary embryonic organizer. Preliminary observations (unpublished) on the delay in lip formation and invagination revealed that, occasionally, embryos which displayed virtually no lag in dorsal lip formation, and almost normal invagination movements (e.g., Fig. 3, Expt 3), displayed severe defects in neural induction. A variety of tests of the inducing capacity of the primary organizer were employed. The primary organizer was, in these investigations, given a purely operational definition. It was defined as the area of the future dorsal side of the embryo which, when implanted into the blastocoel, successfully organized the host tissue to form a second primary embryonic axis. That same region, when implanted into ectoderma1 tissue as an organ culture, also induced the formation of axial structures. A clear distinction between the dorsal lip and the primary organizer tissue may be unwarranted, because cells included in the lip area might have some primary organizer activity. Conversely, cells in the region of the primary organizer might have some capacity for cell movements (Cooke, 1975). But for the purpose of minimizing the potential role of cell movements in these bioassays, the dorsal lip itself was not included in the implanted tissue. In a series of control experiments, neither sham operations nor the implantation of the ventral marginal zone displayed inducing activity. The results of the implantation assays, of the primary organizer replacement grafts, of and the organ cultures indicate that the primary organizer

38

DEVELOPMENTAL

BIOLOGY

activity of irradiated embryos is diminished by uv. In each of these experiments, between 90 and 100% of the embryos on which operations were performed healed successfully, developed normally, and were included in the various tabulations. Ultraviolet irradiation of the uncleaved egg can be considered, therefore, to result in at least two effects which could account for the diminution in the extent of neural morphogenesis: The cell movements of gastrulation are inhibited, and the inducing capacity of the primary organizer is reduced. Among individual eggs, the relative sensitivities of these two effects could possibly vary. Although no clear-cut evidence on this point is available, the possibility exists that the extent to which neural induction is diminished in an irradiated egg represents a balance between the damage to the cell movements of invagination and the destruction of the primary organizer. Wahn et al. (1976) have shown that both induction and cell migration are required for neural differentiation. Irradiated embryos which did not display a lag in lip formation were not used in the embryological assays. It remains as a formal possibility, therefore, that embryos which exhibit completely normal morphogenesis through gastrulation could also display a diminution in the inducing capacity of the primary organizer. The data in Fig. 3 (Expt 3) support that possibility. Those observations on invagination and neurulation permit the design of further experiments on the mechanism of the uv damage to morphogenesis. Various methods which permit a correction of the uv lesion (Malacinski et al., 1974) can now be examined with regard to these various effects of uv. Furthermore, it should be possible to design experiments which permit a biochemical and/or more quantitative analysis of the effects of uv now that it is known, for example, that uv actually reduces the inducing capacity of the primary organizer.

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56, 1977

Valuable discussions with S. Hennen (Milwaukee) and reviews of the manuscript provided by R. Briggs and A. Mahowald are gratefully acknowledged. This research was supported, in part, by NSF Grant No. GB36973.

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