Cytochalasin B (CB) was applied to early developmental stages of the egg of the squid Loligopealei and defects in cleavage and/or organogenesis were produced. If CB was applied in relatively high dosages (2·0 μg/ml) or for long periods (30 min) to embryos in early cleavage the existing furrows irreversibly disappeared. If precleavage embryos were similarly treated the streaming pattern which forms the blastodisc was interrupted and patches of clear cytoplasm appeared on the zygote surface. Short low dosage pulses (0·2 μg/ml for 10 min) produced cleavage effects correlated with the time of pulsing. If pulsed within 30 min of first cleavage the first furrow did not appear in the majority of cases but the second furrow appeared synchronously with the controls and appeared normal in all respects. If pulsed approximately 30 min or longer before cleavage the first furrow usually appeared on time as a weak surface line. Second cleavage appeared normally. However, the asymmetrical pattern of third cleavage was ‘equalized’ and the blastomeres tended to be of equal size. It was concluded that there are subtle cytoplasmic movement patterns, sensitive to CB, which position the nuclei before each cleavage and thereby determine the cleavage pattern.

If embryos were pulsed (0·2 μg/ml for 10 min) at various times during early development, anomalies in differentiation at organogenesis (3–4 days later) were produced which correlated with the time of pulsing. Pulses after germinal layer formation (stage 11) produced no noticeable effects and pulses before the cytoplasmic streaming which produces the blastodisc similarly had very little or no effect. However, pulses during blastodisc formation or early cleavage produced severely affected embryos in which organ displacement, poor tissue and organ differentiation, or organ deficiencies were common. Pulsed embryos were scored and the time of pulsing correlated with the severity of effects. The nuclei of the inductive yolk epithelium were abnormal, frequently being rounded, clumped and/or containing clumped or abnormally diffuse chromatin. It was concluded that the streaming pattern which forms the blastodisc in some way fixes or enhances a pre-existing pattern of developmental information which resides in or at the surface of the egg. Once this pattern is established it is insensitive to CB but it can still be demonstrated by other techniques. This informational pattern apparently influences the nuclei of the yolk epithelium that come to lie in specific regions and controls the expression of their genome so that specific organs are programmed.

Recently the importance of the role of the egg cortex in embryonic development has been re-emphasized by the work of Brachet & Hubert (1972), who produced aneuploidy by wounding the grey crescent area of Xenopus laevis zygotes. Curtis (1960, 1965), also using Xenopus, has demonstrated that the grey crescent region can be transplanted and will induce a secondary blastopore. In the Mollusca the work of Verdonk (1968) on Dentalium, Clement (1962, 1966) on Hyanassa, Raven (1963, 1970) on Limnaea and Arnold (1968 a) on cephalopod embryos has demonstrated a significant contribution of developmental information from localized regions of the egg cortex. In general, the theory thus far evolved is that a specialized informational pattern arises, probably in the ovarian follicle, and produces or organizes specialized cytoplasmic patterns which are segregated in development and eventually influence and/or control the genome of the cells of the embryo. By centrifugation Verdonk (1968), Clement (1968) and many others have been able to demonstrate that the obviously different cytoplasmic regions are relatively unimportant in organ determination but that the egg surface and its associated non-moving gel, i.e. the egg cortex, contains the developmentally significant information. This paper is an attempt to elaborate upon the establishment of the cortical informational pattern and to offer evidence in support of a suggestion of how the cortical influence is exerted in development.

Because of recently published information it is necessary to discuss briefly the effect of cytochalasin B (CB), the major tool used in these experiments. Wessels et al. (1971) reviewed the cytological effects on both the light- and electron-microscope level and implied that CB had a more or less specific effect on microfilaments, causing them to disappear and certain types of cytoplasmic movements (e.g. streaming, contraction of specific regions and cytokinesis) to be inhibited. Sanger & Holtzer (1972), Zigmond & Hirsch (1972), Kletzien, Perdue & Springer (1972) and Estensen & Plagemann (1972), however, have found a rapid and profound effect of CB on carbohydrate metabolism. Glucose utilization appears to be quickly and significantly reduced apparently because it is not transported across the plasma membrane. Furthermore, Plagemann & Estensen (1972) reported a competitive inhibition of nucleoside transport in tissue culture cells. Obviously this would have far reaching effects on the cell in general. Carter (1967) found that cytokinesis was inhibited by low dosages of CB, Schroeder (1969, 1970) has demonstrated that the microfilaments responsible for formation of cleavage furrows are selectively destroyed by this treatment. Among the other CB-sensitive cellular processes that Wessels et al. (1971) described were cessation and disappearance of cleavage furrows, stoppage of single cell movements, prevention of blood clot retraction, a gradual stoppage of beating cultured heart cells, prevention of specialized cellular contractions, and a cessation of cytoplasmic streaming. In all these cases microfilaments have been implicated in a causative role (Wessels et al. 1971). In the case of the Xenopus egg cortex, the calcium-activated surface contraction can be inhibited by CB treatment (Gingell, 1970). At this time it is not possible to state specifically what the total effects of the cytochalasins are on cells, but for the purposes of this paper such information is highly interesting but not vital.

The normal development of the cephalopods has recently been reviewed (Arnold, 1971b) but a very brief description of normal development is necessary for the convenience of the reader. The egg of Loligo pealei is large, about 1·6 by 1·0 mm, telolecithal, ovate, covered with a transparent chorion about 30–40 μm thick and contained in a finger-like jelly egg capsule. Each of these egg capsules is laid one at a time and because fertilization is simultaneous, development is highly synchronous for all of the 150–200 embryos in each capsule. About 40 min after sperm penetration the first polar body is given off and a general surface cytoplasmic movement toward the animal pole is evident. A blastodisc is thus established and subsequent early development occurs there. The second polar body forms about 40 min after the first. By the time cleavage occurs 3 h after fertilization (ca. 22 °C) a large blastodisc of clear cytoplasm has been established, the egg has undergone a slight rounding up, and the chorionic space has enlarged. The cleavage of the blastodisc follows a regular and rigid pattern (Arnold, 1971 a) and a band of CB-sensitive filaments is present at the base of each furrow (Arnold, 1968 b, c; Arnold & Williams-Arnold, 1970). Eventually three layers of cells are produced: an inner syncytial yolk epithelium ( = periblast) which is digestive in function and morphogenetic in significance, and two layers of outer cells, the inner of which was originally derived by a peripheral delamination of the edge of the early blastoderm. These three layers spread peripherally and eventually enclose the entire central yolk mass about the time of organogenesis (stage 18; Arnold, 1965 a). The yolk epithelium induces the overlying cells to form various organ primordia (Arnold, 1965b) in accordance with a pattern present in the egg cortex at least at the time of first cleavage (Arnold, 1968a). An understanding of the above two papers is necessary to interpret the current work. Essentially there is a pattern of developmental information which can be disrupted by microbeam irradiation or ligature but not by low speed centrifugation. This pattern of morphogenetic information is also present at the time of organogenesis when it resides in the yolk epithelium. The yolk epithelium at that time interacts with the relatively indifferent overlying cells to produce the organ primordia. By deletion, isolation, stripping off the outer layers of cells from the yolk epithelium, and by grafting, it has been possible to demonstrate that this interaction fits the criteria of classical embryonic induction (Arnold, 1965b)

The embryos used in this study were all derived from naturally laid egg capsules. Only in this way could maximum synchrony be obtained and would all the eggs be coated with egg jelly which is necessary for development beyond stage 10. A sample of eggs was removed from the newly laid capsules and examined with Nomarski and polarizing optics to determine the exact stage of development. When the proper stages were found, they were mechanically separated from almost all of their egg jelly with great care since excessive disturbance will frequently result in asynchrony. However, each egg had at least some egg jelly still attached to the chorion, because without it the chorion fails to swell away from the growing embryo and development ceases at about stage 10 due to mechanical restriction. The eggs were separated into small clutches of a few to ten or twelve but in each egg some of the chorion was exposed directly to the sea water. The clutches to be treated were randomly selected but all were from the same egg capsule. Control embryos invariably came from the same egg capsule as the treated embryos. After treatment the embryos were transferred through a rinse of sea water, put into a new dish and after observation incubated at 21 – 22 °C on a running sea water table. They were checked daily and compared with the controls, photographed frequently, and their water was changed daily. In almost all of the control embryos completely normal development followed this handling procedure. At first a portion of each capsule was retained with the embryos still in place in their jelly but no differences between the in-jelly controls and those partially dejellied was ever apparent so this practice was discontinued to allow greater numbers of embryos to be used in each experiment. The CB solutions were made up according to the method of Carter (1967). The CB was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/ml, and 0·1 ml aliquots were put into small vials and stored at – 4 °C to – 20 °C until use. Every 10 days to 2 weeks a new DMSO-CB solution was made up. Just before use the CB-DMSO solution was diluted with filtered sea water to the desired strength (usually 0·2 μg/ml) and a control solution of DMSO sea water was made. A dose-response curve was worked out and it was found that the early embryos of L. pealei were quite sensitive to CB treatment. A treatment at 2·0 μg/ml was irreversible but a 20 min pulse with 0·2 μg/ml appeared to be reversible as far as immediate observable effects were concerned. In the majority of experiments reported here the embryos were pulsed for 10 min in 0·2 μ g/ml solution, rinsed in sea water and transferred to normal filtered sea water. The chorion is known to be quite permeable to small molecules although it is assumed there was a certain time factor involved in penetration of the chorion as well as in leaching out the embryo during reversal. It is not possible to estimate how long it took for the CB to be lost from the eggs but if cleavage eggs were treated with 2·0 μ g/ml, solution effects on the cleavage furrows were observable in 3-5 min. In embryos treated with solutions of 0·2 μ g/ml before the time of first cleavage, a complete second furrow appeared on time, 1 h after first cleavage appeared in controls.

During and for a few hours after treatment the embryos were continually observed, the effects of treatment recorded and photographed, and continual comparisons made with the controls. The length of cleavage delay, if any, was carefully noted and altered cleavage patterns were recorded. After about 5 days of development, when the controls had reached stage 24 or 25, the experiments were terminated by carefully observing, photographing, and fixing in Bouin’s fluid all of the embryos. In a few cases embryos were also fixed in Palade’s 1% OsO4, with an adjusted osmolarity and embedded for electron microscopy in Epon. The Bouin’s fixed embryos were later carefully scored, embedded in Epon, serially sectioned at 1 – 2 μ m, stained and examined for histological detail. A system of scoring the developmental effects using six arbitrary classes of anomalies was devised and is given in the figure caption of Fig. 1. A total of 1320 embryos were each individually assigned a score according to the severity of effects and the totals per experiment were averaged and compared.

Figure 1

Fig. 1. Graphic representation of the effects of CB (0·2 μ g/ml for 10 min) pulses at various times during development. The classes of anomalies are defined as follows: Class 1 = normal development; class 2=all organs present, but reduced in size, either bilaterally, unilaterally, or dorsoventrally; class 3 = some organs missing and/or organs in abnormal positions; class 4=few recognizable organs regardless of position; class 5=no recognizable organs but presence of embryonic axis; class 6=no organ differentiation and embryo spherical with all tissue layers present. Each experimental batch is represented by a vertical line. The circled number represents the total number of embryos in an experiment which reached an interpretable stage of development. The position of the circle represents an average derived by multiplying the number of embryos in each class (small numbers) by the class number and dividing by the total number of interpretable embryos. This produced a weighted score which could be compared graphically. Solid vertical lines indicate that there were embryos in a particular class while the dashed vertical lines indicate that there were no embryos in that particular class. It is evident that the maximum sensitivity to CB occurs in the period prior to cleavage (half of stage 2 and all of stage 3). A minor peak of sensitivity also occurred just before formation of the germ layers (stage 9). (PB = polar body.)

Figure 1

Fig. 1. Graphic representation of the effects of CB (0·2 μ g/ml for 10 min) pulses at various times during development. The classes of anomalies are defined as follows: Class 1 = normal development; class 2=all organs present, but reduced in size, either bilaterally, unilaterally, or dorsoventrally; class 3 = some organs missing and/or organs in abnormal positions; class 4=few recognizable organs regardless of position; class 5=no recognizable organs but presence of embryonic axis; class 6=no organ differentiation and embryo spherical with all tissue layers present. Each experimental batch is represented by a vertical line. The circled number represents the total number of embryos in an experiment which reached an interpretable stage of development. The position of the circle represents an average derived by multiplying the number of embryos in each class (small numbers) by the class number and dividing by the total number of interpretable embryos. This produced a weighted score which could be compared graphically. Solid vertical lines indicate that there were embryos in a particular class while the dashed vertical lines indicate that there were no embryos in that particular class. It is evident that the maximum sensitivity to CB occurs in the period prior to cleavage (half of stage 2 and all of stage 3). A minor peak of sensitivity also occurred just before formation of the germ layers (stage 9). (PB = polar body.)

In several other experiments embryos in early cleavage (2 – 32 cells) were subjected to regional application of CB by a modification of the classical agar block technique used for vital staining. Small ball-tipped glass rods were coated with agar and soaked to saturation in a solution of 2·0 μ g/ml CB to which 0·05% phenol red had been added to serve as a tracer dye. The embryos were oriented on a wax surface and the CB soaked agar was pushed against the dejellied chorion with a micromanipulator. It was possible to rather precisely orient the embryo because of the bilateral nature of the cleavage pattern. Thus specific organogenetic areas of the egg surface were treated according to the regional informational pattern demonstrated earlier (Arnold, 1968a). In each experiment the CB soaked agar was taken from the CB solution and quickly pressed to the embryo to minimize loss to the sea water in the treatment dish. The phenol red color disappeared from the agar in 5 – 6 min. Although a similar rate of diffusion cannot be assumed for the CB molecules the dye did give an idea of the region treated. The total time of treatment was 10 min. Each embryo was either sketched or photographed during regional treatment and the exact position of agar contact recorded for later comparison with the resultant embryo. Controls from the same egg string were similarly dejellied and subjected to sham manipulations.

Treating embryos during early stages of development with 2·0 μg/ml solutions of CB had an irreversible effect as did long-term treatments (40 min or longer) with a concentration of 0·2 μ g/ml. If embryos were placed in a 2·0 μ g/ml solution of CB for 10 min at the time of first meiosis the first polar body extrusion was inhibited and cytoplasmic streaming to form the blastodisc stopped within 3–5 min. Gradually, small accumulations of clear cytoplasm began to appear, apparently randomly, over the egg surface but not near or on the blastodisc (Fig. 2A). Eventually the blastodisc underwent shape changes and indented at the edge but never increased in size. These eggs did not undergo the slight ‘rounding up’ shape change seen in the controls and frequently retained the indentation which is occasionally present in newly laid or ovarian eggs. Placing embryos in 2·0 μg/ml CB at the time of second meiosis had much the same effect except the blebs appear only at the vegetal pole region of the egg and since the blastodisc is much larger, the cessation of cytoplasmic streaming is less noticeable (Fig. 2B). Excised pieces of ovary placed in sea water will frequently shed mature oocytes by contraction of the follicle. The oocytes thus released frequently had indentations in their surface as a consequence of the compression by the follicle wall during extrusion. If similar pieces of ovary were put into 2·0 μg/ml of CB the oocytes were shed but did not undergo the characteristic rounding up despite the fact that the follicular walls were able to contract and shed the oocyte (Fig. 3). If the embryos were put into a 2·0 μg/ml concentration of CB during cleavage, within 8–11 min the furrows became irregular and the ends appeared to shorten and become less definite. In 11–20 min the furrows had disappeared but in their place the surface was now slightly irregularly convoluted because the membrane between the blastomeres had risen to the surface (Fig. 4). The microfilaments were no longer evident at the base of the cleavage furrows but areas of granular material could be found in association with the former furrow area.

Fig. 2

Failure of formation of the blastodisc in embryos treated with 2-0 μ g/ml CB before first cleavage. (A) Treatment at the time of the extrusion of the first polar body. The blastodisc is quite small (M) and blebs of clear cytoplasm are randomly distributed over the yolk (arrows). (B) Treatment just after second polar body extrusion. The blastodisc is almost normal size for this stage but blebs of clear cytoplasm have appeared only at the vegetal pole of the egg (arrows). These photographs were taken just after first cleavage had occurred in the controls, ca. x 20.

Fig. 2

Failure of formation of the blastodisc in embryos treated with 2-0 μ g/ml CB before first cleavage. (A) Treatment at the time of the extrusion of the first polar body. The blastodisc is quite small (M) and blebs of clear cytoplasm are randomly distributed over the yolk (arrows). (B) Treatment just after second polar body extrusion. The blastodisc is almost normal size for this stage but blebs of clear cytoplasm have appeared only at the vegetal pole of the egg (arrows). These photographs were taken just after first cleavage had occurred in the controls, ca. x 20.

Fig. 3

Shedding of the oocytes in 2 0 μ g/ml CB. Although the oocyte was extruded by the contraction of the follicular cells the characteristic rounding up of the oocyte did not occur. (A) Oocyte completely surrounded by the follicular cells. (B) Oocyte extruded from the follicle (/) but the indentation present earlier still remains. ca. x 28.

Fig. 3

Shedding of the oocytes in 2 0 μ g/ml CB. Although the oocyte was extruded by the contraction of the follicular cells the characteristic rounding up of the oocyte did not occur. (A) Oocyte completely surrounded by the follicular cells. (B) Oocyte extruded from the follicle (/) but the indentation present earlier still remains. ca. x 28.

Figure 4

Disappearance of the first cleavage furrow after treatment with 0-2 μ g/ml CB.

(A) Normal furrow base after completion of cleavage. Note the band of microfilaments associated with the base of the furrows and the tubular network (tn) in the longitudinal folds (If), ca. × 33200.

(B) Complete disappearance of the first furrow evident 11 min after treatment began. The cell membrane formerly between the two blastomeres has returned to the surface of the embryos and is evident as a broad convoluted line. ca. ×1900.

(C) The former furrow base is evident at the surface to the right of the area shown in (B). The tubular network (tn) is evident and some suggestion of the folds remain but the organized band of microfilaments is not present. There is some suggestion of bunches of microfilaments in a few regions (mfT). ca. 27200.

Figure 4

Disappearance of the first cleavage furrow after treatment with 0-2 μ g/ml CB.

(A) Normal furrow base after completion of cleavage. Note the band of microfilaments associated with the base of the furrows and the tubular network (tn) in the longitudinal folds (If), ca. × 33200.

(B) Complete disappearance of the first furrow evident 11 min after treatment began. The cell membrane formerly between the two blastomeres has returned to the surface of the embryos and is evident as a broad convoluted line. ca. ×1900.

(C) The former furrow base is evident at the surface to the right of the area shown in (B). The tubular network (tn) is evident and some suggestion of the folds remain but the organized band of microfilaments is not present. There is some suggestion of bunches of microfilaments in a few regions (mfT). ca. 27200.

When held in a solution of 2·0 μg/ml CB, development of all the embryos ceased although nuclear division did continue. The nuclei were evenly distributed throughout the cytoplasm of the blastodisc and did not assume the distribution typical of the normal cleavage pattern. This point will be elaborated upon below.

The majority of experiments were performed using 10 min pulses at 0·2 μg/ml CB from stage 2 (formation of the first polar body) until stage 15 (yolk mass one-half cellulated). For convenience in presentation the results of these treatments on cleavage, cellulation and organogenesis will be discussed separately and times of treatment will be presented in chronological order.

Effects on cleavage

In all, 20 batches of embryos were pulsed at some time before first cleavage. Rather than present each experiment they have been grouped into three time ranges: treatment about the time of first polar body extrusion; treatment about the time of second polar body extrusion; and precleavage ( = during first mitosis) treatment. The actual times before first cleavage varied from −149 min to −5 min with the majority of cases being between −60 and −5 min because the variability was greatest during this period.

Those embryos pulsed about the time of first polar body extrusion showed little effect on first cleavage. The major cytoplasmic streaming to form the blastodisc had not yet begun so effects on the blastodisc were not noticeable. The blastodisc formed in synchrony with the controls and first cleavage usually occurred on time or at most was delayed by a few minutes. In more than 90% of the embryos the subsequent pattern of cleavage was quite regular but in the remaining cases there was a tendency to equalize the position of the anterior and posterior third cleavage furrows.

When embryos were treated at the time of second polar body formation, most of the streaming that forms the blastodisc had already occurred. Frequently the first furrow was reduced and appeared only as a surface line or was completely missing. In those eggs treated late in this period there was a tendency for the first furrow to be lacking entirely. Second cleavage occurred normally and on time in all but a few cases in which it was slightly delayed but normal. At third cleavage there was a marked tendency for the anterior and posterior third furrows to be equally distributed in the blastoderm and the nuclei showed a symmetrical arrangement rather than the typical bilateral placement in which the posterior furrow parallels the first furrow while the anterior third furrow goes off at an angle of approximately 30° (Fig. 5A,B).

Figure 5

Alteration of the cleavage pattern by 10 min pulses of 0-2 μ g/ml CB.

(A) Control embryos in fourth cleavage. Note the bilaterally symmetrical pattern in which the anterior third furrows go off at an angle.

(B) Embryo was pulsed 77 min before first cleavage (2nd polar body appeared about 30 min earlier). The posterior third furrows are at an angle to the first furrow.

(C) Embryo pulsed 5 min before cleavage. The partial first furrow appeared as a faint surface line 37 min after cleavage in the controls. Note the position of the nuclei (arrows).

(D) Embryo pulsed 48 min before cleavage in the controls. Note that the first furrow does not divide two of the posterior cells and the cleavage pattern is atypical.

(E) Embryo pulsed 10 min before cleavage occurred in the controls. The cleavage pattern is quite symmetrical. Third furrows were still forming when this photograph was taken.

(F) Later stage of an embryo similar to (E). The first furrow never appeared in any of the embryos which lacked it by the time of second cleavage. This photograph was taken at an angle to show the base of the furrows as well as the surface indentation, ca. x 100.

Figure 5

Alteration of the cleavage pattern by 10 min pulses of 0-2 μ g/ml CB.

(A) Control embryos in fourth cleavage. Note the bilaterally symmetrical pattern in which the anterior third furrows go off at an angle.

(B) Embryo was pulsed 77 min before first cleavage (2nd polar body appeared about 30 min earlier). The posterior third furrows are at an angle to the first furrow.

(C) Embryo pulsed 5 min before cleavage. The partial first furrow appeared as a faint surface line 37 min after cleavage in the controls. Note the position of the nuclei (arrows).

(D) Embryo pulsed 48 min before cleavage in the controls. Note that the first furrow does not divide two of the posterior cells and the cleavage pattern is atypical.

(E) Embryo pulsed 10 min before cleavage occurred in the controls. The cleavage pattern is quite symmetrical. Third furrows were still forming when this photograph was taken.

(F) Later stage of an embryo similar to (E). The first furrow never appeared in any of the embryos which lacked it by the time of second cleavage. This photograph was taken at an angle to show the base of the furrows as well as the surface indentation, ca. x 100.

When embryos were pulsed within one hour of first cleavage, there was a strong tendency for the furrow to be missing or to appear only as a surface line at times later than the appearance of the first furrow in the controls. Thus six-cell stages resulted when second and third furrows were formed; the inner two cells being binucleate and the outer four uninucleate (Fig. 5). Rarely sevencell stages could be found because of the unilateral inhibition of the first furrow. The posterior third furrow in all these cases was shifted to a position at an angle to the first furrow making the blastomeres more equal in size.

When pulsed very close to the time of first cleavage the first furrow never formed and the embryos appeared extremely sensitive to the drug. Although most did form second furrows, they were often shallow furrows or only a surface line with no depth.

Pulsing after the first furrow had appeared caused it to slowly regress and the second furrow was delayed and weakened. Only a few of these first furrow experiments were performed and because the survival rate was so low, the results are not included in the compilation of the data. This low survival rate is discussed elsewhere.

Treatment at later cleavage (4–64 cells) also caused formed furrows to regress to some degree. After the formation of the fourth furrow the four central cells are completely separated from those peripheral to them and they are unaffected by the pulse in that their furrows do not regress. The furrows of the peripheral cells are still continuous with the surrounding surface and they tended to regress so that an embryo resulted which was formed of a population of free cells in the center with a more or less syncytial peripheral ring at the edge of the blastoderm. Since the undercutting furrow was also affected the number of cells the embryo had at pulsing determined the size of the area of free cells.

In all of the above cases, once formation of a furrow was totally inhibited, it never reformed. Often, partially inhibited furrows were formed but did not completely cut the cytoplasm between the two daughter nuclei. Also furrows were found that existed only as a surface line and never seemed to increase in length or depth except in regions crossed by later furrows. Typically, however, these surface lines regressed.

Effects on cellulation

By the time cellulation of the yolk occurred and spreading of the blastoderm began there were no effects apparent from a 10 min pulse before or about the time of first cleavage. However, if the undercutting furrow had been destroyed cellulation was irregular and the germinal layers were improperly formed. Pulsing at the time of germinal layer formation caused anomalous embryos which did not cellulate properly. In infrequent cases the embryos arrested (77 of 1397) during cellulation but since this is also the result of incorrect handling and was evident in the controls (albeit less common) it is not considered further here.

Effects on organogenesis

The effects on cleavage seen immediately after precleavage pulsing were followed by normal development until the time of organogenesis (stage 18) when in the majority of the embryos anomalies appeared. The first noticeable effects were a general retardation of development and frequently but not invariably an irregular or unusual shape change (Fig. 6). Normally in stage 18 embryos, a slight waist had appeared which indicated the margin between the organogenetic areas and the external yolk sac. In the experimental batches, however, affected embryos rounded up or become somewhat elongate. The frequency and severity of these changes correlated with the time of precleavage pulsing. The appearance and placement of the organ primordia were abnormal and in severe cases some or all organ primordia were absent. As development continued the anomalies became more pronounced and six arbitrary classes of anomalies (Fig. 1 caption) were set up to classify the range of effects. The result of this classification plotted against time is given in Fig. 1 and pictorially in Fig. 6.

Figure 6

Effects of pulses of 0-2 μ g/ml CB on organogenesis.

(A-F) Examples of classes of developmental anomalies. (A) Class 1, normal control embryos at stage 25. (B) Class 2, all organs present but reduced in size, either bilaterally, unilaterally, or dorsoventrally. (C) Class 3, some organs and/or organs in abnormal positions. (D) Class 4, few recognizable organs regardless of position. (E) Class 5, no recognizable organs but presence of embryonic axis. (F) Class 6, no organ differentiation, embryo spherical with all tissue layers present. (G) Embryo pulsed for 10 min at the 32-cell stage. This was the most severely affected embryo of all those treated at this age. Note that the organs are present in their normal positions but that the embryo has been retarded in general. The control embryos were in stage 25 at the time of this photograph. (H) Embryo pulsed for 10 min at stage 13. It is essentially normal. (I) Control embryo for stage 13 treated embryo shown in (H). ca. × 37.

Figure 6

Effects of pulses of 0-2 μ g/ml CB on organogenesis.

(A-F) Examples of classes of developmental anomalies. (A) Class 1, normal control embryos at stage 25. (B) Class 2, all organs present but reduced in size, either bilaterally, unilaterally, or dorsoventrally. (C) Class 3, some organs and/or organs in abnormal positions. (D) Class 4, few recognizable organs regardless of position. (E) Class 5, no recognizable organs but presence of embryonic axis. (F) Class 6, no organ differentiation, embryo spherical with all tissue layers present. (G) Embryo pulsed for 10 min at the 32-cell stage. This was the most severely affected embryo of all those treated at this age. Note that the organs are present in their normal positions but that the embryo has been retarded in general. The control embryos were in stage 25 at the time of this photograph. (H) Embryo pulsed for 10 min at stage 13. It is essentially normal. (I) Control embryo for stage 13 treated embryo shown in (H). ca. × 37.

Although there seemed to be no specific organogenetic deficiency associated with a given time of precleavage pulsing, the general severity of effect correlated quite well with the time of drug application. In each experiment all of the embryos were individually judged by one of us (L.D.W.A.) and spot confirmed by the other. The range of class members was then averaged and compared to similar pulse treatments applied at other times during development. These results are presented graphically in Fig. 1 which shows not only the range of classes in each experiment but also the average effect (circled number). It is obvious that there is a period of maximum sensitivity just before the time of first cleavage and prior to this time (stage 1 and 2) or after (stage 7 and 8) the embryos were less sensitive. There seems to be a minor peak of sensitivity just before the germinal layers are formed but embryos beyond stage 11 are only slightly affected or normal.

The histological appearance of these embryos correlated well with the class of anomaly. For example, in the class 6 embryos (Fig. 7B) the outer layers of cells which normally form the organ primordia appear undifferentiated and have an irregular surface. There seemed to be little organization other than the separation of the cells into an irregular layer with a series of protrusions and random folds which were usually present in one place and covered about one-third of the surface of the embryo. In older embryos of this class, there was frequently evidence of pycnosis in the center of these folds as there is no established circulatory system. The histological appearance of class four embryos is shown in Fig. 7C and D. The rudiment of a single eye has been formed at the apex of the embryonic axis but the organization and tissue differentiation is quite incomplete. A number of cells were pycnotic and a region of ‘bubbly’ acellular material, possibly a hemal space, is present in the center of the embryo.

Figure 7

Histological appearance of the pulsed embryos.

(A) Eye region of a normal stage 22 embryo, ca. × 400. Note the yolk epithelium nuclei (arrow) are widely spaced and flattened and that the cytoplasm is quite thin and uniform.

(B) Section of a class 6 embryo pulsed for 10 min with 0-2 μ g/ml CB 41 min before cleavage, ca. × 400. The outer layers of cells have formed meaningless folds and there is no evidence of any organ differentiation. The yolk epithelium is greatly altered so that the cytoplasm is concentrated in areas beneath the cellular folds. The yolk epithelium nuclei have a variable appearance and tend to be clustered. Most are rounded and contain clumps of chromatin. A few are abnormally large and their chromatin quite diffuse except for a few areas of dense concentration.

(C, D, and inset) Sections of a class 4 embryo (pulsed after second polar body extrusion). The inset shows the position of the single apical eye in the embryo. ca. × 16.

(C) Shows histological detail of the eye. The whole organ is greatly reduced in size with the retina (r) composed of fewer cellular layers and the outer wall of the optic vesicle greatly thickened. There are many pycnotic nuclei (py) in the surrounding tissue because no circulatory system was established ca. × 100.

(D) A higher magnification of the same embryo. The general level of organ differentiation is very poor. The cytoplasm of the yolk epithelium is thickened and the nuclei seem far more abundant. The nuclei are rounded and tend to be clustered beneath the organogenic areas of the embryo, ca. × 400.

Figure 7

Histological appearance of the pulsed embryos.

(A) Eye region of a normal stage 22 embryo, ca. × 400. Note the yolk epithelium nuclei (arrow) are widely spaced and flattened and that the cytoplasm is quite thin and uniform.

(B) Section of a class 6 embryo pulsed for 10 min with 0-2 μ g/ml CB 41 min before cleavage, ca. × 400. The outer layers of cells have formed meaningless folds and there is no evidence of any organ differentiation. The yolk epithelium is greatly altered so that the cytoplasm is concentrated in areas beneath the cellular folds. The yolk epithelium nuclei have a variable appearance and tend to be clustered. Most are rounded and contain clumps of chromatin. A few are abnormally large and their chromatin quite diffuse except for a few areas of dense concentration.

(C, D, and inset) Sections of a class 4 embryo (pulsed after second polar body extrusion). The inset shows the position of the single apical eye in the embryo. ca. × 16.

(C) Shows histological detail of the eye. The whole organ is greatly reduced in size with the retina (r) composed of fewer cellular layers and the outer wall of the optic vesicle greatly thickened. There are many pycnotic nuclei (py) in the surrounding tissue because no circulatory system was established ca. × 100.

(D) A higher magnification of the same embryo. The general level of organ differentiation is very poor. The cytoplasm of the yolk epithelium is thickened and the nuclei seem far more abundant. The nuclei are rounded and tend to be clustered beneath the organogenic areas of the embryo, ca. × 400.

In all of the abnormal embryos the yolk epithelium showed degrees of disorganization correlated with the class of anomaly. Normally, the yolk epithelium is a thin syncytial layer over the entire yolk sac with the nuclei approximately evenly distributed throughout the thin cytoplasmic layer. In the class 6 embryos the yolk epithelium showed striking disorganization with a few large concentrations of vesicular cytoplasm containing randomly distributed nuclei in some areas, while in other areas the cytoplasm was quite thin and the nuclei were widely scattered. In the concentrations of vesicular cytoplasm the nuclei were quite irregular: some were clumped into small groups, others had densely condensed chromatin or were diffuse in appearance. These areas of cytoplasmic concentration were frequently, but not exclusively, found associated with areas of protrusions and folds as described above. Class 5 embryos were intermediate between class 6 and class 4. In the class 4 embryos the yolk epithelium had a more regular appearance but the nuclei were far more frequent than in the normal embryos and tended to be densely packed in some regions beneath the partially formed organs. Where organs were lacking, the yolk epithelium was thinner and the nuclei were sparsely distributed. Class 3 embryos were intermediate between class 4 and class 2. In the class 2 embryos both the outer tissue layers and the yolk epithelium appeared essentially normal except occasionally pycnotic cells were seen where they normally would not occur.

The modification of the nuclear morphology was striking in the pulsed embryos. In the normal embryos, the nuclei of the yolk epithelium were sparsely distributed and elongate with a thin surrounding layer of cytoplasm. In the treated embryos, the nuclei were more rounded, clustered into groups and frequently of abnormal size (Fig. 7 B). The chromatin appeared to be clumped into one or two large central masses within the nuclear envelope. Mitotic figures were not observed but dividing nuclei are rare in the yolk epithelium of normal embryos at this stage. The modification of nuclear morphology in response to CB pulsing will be discussed in detail elsewhere.

In the 38 embryos which were regionally treated with the agar soaked in CB, the results correlated with the position of application and the phase of the division cycle. Embryos treated just before or during cytokinesis tended to lack the furrow in or close to the region of CB application while the rest of the blastoderm appeared unaffected. If the undercutting furrow was eliminated in one part of the blastoderm there was no cavity into which the germinal layers could separate and the blastoderm in that region did not expand. Therefore, blastoderms were formed which covered only part of the embryonic surface and partial embryos resulted (Fig. 8 A). In embryos treated when cytokinesis was not imminent or active the furrows appeared more stable and remained permanent, or at worst, became surface lines. Embryos treated in regions well away from the blastodisc did not have their furrows affected and formation of the blastoderm and cellulation of the yolk surface proceeded normally. These later embryos are of considerable interest here. During organogenesis defects or deficiencies appeared in 27% of the cases which correlated quite precisely with the area of CB application. Fig. 8B shows an example of such an embryo treated at stage 5. The region exposed to the drug was the posterior lateral animal half of the egg peripheral to the blastoderm. Note the funnel on the treated side is missing but on the untreated side the embryo is quite normal. Similar examples can be seen in Fig. 8C,D. When the future eye region and arm region was treated, the resultant embryos had those organs reduced or lacking. In this series of experiments 33% of the embryos had no noticeable abnormality, 6% had general defects which did not particularly correlate with the position of the CB application, 19·5% died and 14+% failed to develop due to various other causes.

Fig. 8

Results of regional treatment of early cleavage embryos with CB in agar. In (A) the blastoderm was inhibited from spreading over half of the egg surface and a half embryo formed from the cellulated region. Note the eye (e) on one side only, the arms (a), and the half mantle (m). These embryos did not survive beyond stage 20 because the uncellulated surface was fragile and eventually burst. (B-D) Retardation or inhibition of organs due to regional application of CB during early cleavage. In B the side of the egg in the equatorial region was treated during second cleavage and the arms on that side are reduced. In (C) the treated region was the anterior quarter of the animal pole (at the 16-cell stage) and the embryo correspondingly lacks the anterior organs. In (D) a small lateral portion of the animal half was treated at the 16-cell stage and the resultant embryo lacks the funnel on one side and has a partially reduced mantle and somewhat reduced eye. (E) A section through the embryo shown in (D), showing the lack of organization of the embryo in the treated region. (A-D) ca. x 37; (E) ca. × 100.

Fig. 8

Results of regional treatment of early cleavage embryos with CB in agar. In (A) the blastoderm was inhibited from spreading over half of the egg surface and a half embryo formed from the cellulated region. Note the eye (e) on one side only, the arms (a), and the half mantle (m). These embryos did not survive beyond stage 20 because the uncellulated surface was fragile and eventually burst. (B-D) Retardation or inhibition of organs due to regional application of CB during early cleavage. In B the side of the egg in the equatorial region was treated during second cleavage and the arms on that side are reduced. In (C) the treated region was the anterior quarter of the animal pole (at the 16-cell stage) and the embryo correspondingly lacks the anterior organs. In (D) a small lateral portion of the animal half was treated at the 16-cell stage and the resultant embryo lacks the funnel on one side and has a partially reduced mantle and somewhat reduced eye. (E) A section through the embryo shown in (D), showing the lack of organization of the embryo in the treated region. (A-D) ca. x 37; (E) ca. × 100.

For the purposes of discussion, the effect of CB pulsing at various times in early development will be covered separately and then a general scheme of the mechanism of cortical-nuclear interaction will be postulated.

Effects of CB on the cleavage pattern

The irreversible effects of CB caused by long term treatment or high concentration pulses were probably related to the sensitivity of cytokinesis to this drug and/or the large amount of yolk which could function in retention of CB and its gradual release over a protracted period of time. The observable changes in the position of the third cleavage furrows which resulted from low-dosage pulse treatment two or more hours before the formation of that furrow are of interest because they imply a cytochalasin sensitive predetermined cleavage pattern. In effect it appears that the position of the nuclei (hence the mitotic apparatus and, therefore, the furrow) within the blastodisc is determined by subtle cytoplasmic forces involving cytoplasmic streaming in a predetermined pattern. Possibly this nuclear positioning is related to a microfilament pattern or structure (or at least, an architecture sensitive to CB) and not to the microtubules of the asters or mitotic apparatus. The exact nature of this nuclear positioning force is open to speculation since no hard facts other than the observable events are available. It might be possible that there exists, or arises, a pattern of subtle movements which position the mitotic apparatus during division or that during the formation of the blastodisc a relatively rigid controlling pattern arises. This is very reminiscent of the classically discussed cytoskeleton (Wilson, 1925). Obviously more data are needed before extensive speculation is warranted.

In light of the work of Schroeder (1969) and Arnold (1969, 1971 a) it is not surprising that the formed furrows disappear when treated with CB. The delay or blockage of furrowing after the CB treatment is most likely related to a gradual leaching out rather than a direct effect on an already existing but not yet contracted furrow. No evidence of such an uncontracted furrow exists from fine structural analysis (unpublished data). Because the cytokinetic microfilaments of the second furrow do form normally and in proper position it can be assumed the leaching out of the CB is completed within less than 1 h; therefore the positions of the third furrow would not be affected directly by this low level of CB. However, if the position of the nuclei and their resultant furrowinducing spindles were determined by a cytochalasin sensitive prepattern in the blastodisc the abnormal ‘equalized division pattern’ would be expected.

Effects of CB on cellulation of the embryo

With regard to cellulation of the embryo, CB pulsing before cleavage or during development subsequent to germ-layer formation had little noticeable effect and development continued unhampered. If the undercutting furrow which separates the cytoplasm from the yolk subsequent to second cleavage is destroyed by the CB the space into which the germinal layers separate is not formed and subsequent development is disturbed or completely prevented. This appears to be a mechanical effect because in half embryos the unaffected half has normal appearing blastodermal spreading and organogenesis proceeds as normally as can be expected until stage 20 when constriction of the cellular portion of the embryo causes the plasma-membrane-only covered area of the yolk to burst. If the faulty formation of the germinal layers was caused by a systemic effect such half embryos would not be expected. The CB sensitivity noted at the time of germinal layer formation is probably related to the inhibition of divisions from which the middle cellular layer arises.

Effects of CB on organogenesis

The developmental anomalies that occur during organogenesis are strongly reminiscent of Ranzi’s (1928) work with LiCl on Loligo vulgaris. However, the regular pattern of cyclopia was not observed and since the effects of lithium are general and unknown these results are not directly comparable. Recently Elbers (1969) has studied the effects of LiCl on the fine structure of the egg of Limnaea and concluded it must affect the plasma membrane since no other organelles seemed to be affected. It is evident from the data presented here that the cytoplasmic streaming pattern is critical for the establishment of the cortical pattern which determines the eventual position of the organ primordia. Raven (1967) demonstrated that certain subcortical accumulations appeared in the egg of Limnaea as it passed through the genital tract of the parent and postulated that these subcortical accumulations in association with specific sites on the plasma membrane play a causal role in development. Logically, it would appear that these specific sites would have to arise in the ovary and be located in a non-moving component, possibly the plasma membrane. It would follow, then, that there are two steps in the determination of the egg cortex – the origin of the primary pattern in the plasma membrane during oogenesis, and later, subsequent to fertilization, the fixation of the informational component by specialized cytoplasmic rearrangements. In the case presented here the cytoplasmic movements are CB-sensitive and express themselves later in development via the inductive pattern in the yolk epithelium (see below).

The results of the regional application of CB indicate the CB effects are regional rather than systemic. Since treatments outside the blastoderm had an organogenetic effect, they also indicate the effects were essentially on the egg surface rather than an indirect protracted mechanism some way related to changes in the cleavage furrows or cleavage pattern. The regional nature of the cortical pattern correlates exactly with the pattern reported earlier by other techniques (Arnold, 1968 a).

Of considerable interest is the interaction of the pattern of information present in the egg cortex at and before cleavage and the ‘inductive morphogenetic map’ apparent in the yolk epithelium during organogenesis. Although the exact relationship of these two cytoplasmic informational patterns is, as yet, incompletely understood a few generalizations can be drawn. (1) There seems to be a rather precise positional relationship of organ forming potential in both the egg cortex (Arnold, 1968 a) and the yolk epithelium (Arnold, 1965b). (2) The pattern of the major organ forming primordia is determined as early as first cleavage and is present in the egg surface through cellulation and is then transcribed into the syncytial yolk epithelium. (3) During organogenesis the yolk epithelium induces the overlying cells to differentiate in accordance with its latent pattern. (4) The outer layers of cells seem to be able to change their fate in accordance with instructions derived from the yolk epithelium and appear to be quite labile so long as they remain in contact with the yolk epithelium during stages 17–19 (histodifferentiation of the major organ primordia). (5) Differentiation of the outer layers of cells, once induced, can proceed independently of further contact with the yolk epithelium (Marthy, 1973). (6) Modification of the informational pattern in the cortex by brief pulses of CB results in changes in nuclear morphology (e.g. enlarged nuclei) in the yolk epithelium which is expressed as faulty inductive interaction and the resultant organogenesis is incomplete, positionally misplaced, or lacking.

This would indicate that the egg cortex, i.e. the cell surface, has a role in determination of the expression of the yolk epithelium nuclei and their subsequent role in the control of embryonic induction of the labile outer layers of cells. Although premature, it is interesting to speculate on the site of informa-tional pattern in the cell surface and its possible nature. Raven (1961) discussed at length the theoretical possibilities of the plasma membrane being the site of informational molecules. Because the reported effects of CB seem to be both on the surface in blocking transport of carbohydrate substrates and other metabolites (Zigmond & Hirsch, 1972; Estensen & Plagemann, 1972; Plagemann & Estensen, 1972; Sanger & Holtzer, 1972) as well as on the microfilaments in the cytoplasm it would seem possible to separate these two locales as sites of the developmental informational pattern. However, because the data presented here support the concept of two steps being necessary for determination of the egg cortex information, the plasma membrane becomes a likely site of the primary information pattern. In the case presented here it would seem most likely that the effect of a 10 min pulse of CB at 0·2 μg/ml would not permanently affect the transport of substances across the membrane to an extent that days later nuclei which invade the region would be adversely affected. Also pulses at very early or later stages had no effect on organogenesis. If it were a case of a permanent, irreversible, blockage or a vital transport site being lost, then the later treatment would have an effect similar to a precleavage pulse. It would seem, rather, that the CB effect is on the cytoplasm immediately underlying the plasma membrane, possibly by disruption of a microfilament directed pattern of cytoplasmic information (in Raven’s terminology, a subcortical accumulation). That such a pattern is possible is illustrated by the altered pattern of third cleavage following low dose pulses of the drug 3 h before furrowing begins. It would seem, then, that the target of CB is the cytoplasm that becomes associated with a primary plasma membrane, since it does not take part in the cytoplasmic streaming that forms the blastodisc, and centrifugation which displaced the blastodisc had no effect on the location of the organ primordia (Arnold, 1968a). The fact that CB has no pattern disrupting effect on later stages, although presence of a pattern can be demonstrated by ligation experiments (Arnold, 1968a) could indicate that once in place the determining factors maintain their position without microfilaments. It might be that the primary information pattern lies between the leaflets of the plasma membrane as special particle arrays as suggested by Branton & Beamer (1972).

Exactly how a cytoplasmic structure such as the cell surface controls nuclear expression is obviously worthy of considerable effort and further attention.

The authors would like to thank Mr C. T. Singley and Dr Michael G. Hadfield for reading and criticizing this manuscript and Miss Frances Horiuchi for preparing it for publication.

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After this paper had been returned by the reviewers and revised, a paper by Marthy (1973, J. Embryol. exp. Morph.) appeared which casts some doubt on the induction hypothesis of the senior author (Arnold, 1965b). However, several important differences in technique and interpretation exist between these two papers. Only by reading the references would one realize two different species of Loligo were used. The differences in developmental time and the size of the embryo are not trivial (L. pealei develops to hatching in 12–14 days, L. vulgaris takes longer than 21 days). Marthy removed a considerable amount of yolk from his embryos which not only reduced the size of the whole embryo but also changed the size and spatial relationships of the individual organs. Marthy ‘cultured’ his embryos and tissues in sterile sea water while I used a simple nutrient medium which has successfully maintained adult cephalopod tissues for 10 months (Szabo & Arnold, 1963). The conclusion that ‘nutritive conditions’ must be sufficient is therefore questionable because Marthy provided a nutritive environment only when the cells were attached to the embryo and not when isolated in sterile sea water. In his introduction Marthy states complete removal of the eye region, with the yolk epithelium left intact, resulted in a lack of an eye. However, in several of my cases one-third of the surface cells were removed (1965b, p. 76) and the wound closed from the surrounding cells and an eye was formed. It seems unlikely that one eye primordium is larger than one-third of the embryos surface. Also smaller organs (e.g. otocyst), which would have a correspondingly smaller primordium, were completely formed. In all cases the area removed was larger than that shown in Marthy’s fig. 7. Marthy removed the whole eye rudiment with the underlying yolk epithelium and found no eye formed but removal of as large an area as the eye in L. pealei results in extensive yolk loss and this presents a mechanical block to wound closure. My yolk epithelium extirpation was done on the otocyst primordium, a considerably smaller organ, and this wound closes quickly and completely and no otocyst is formed. Marthy apparently did not duplicate the grafting experiment using labeled dissociated-reaggregated embryonic cells. Unpublished experiments in our own laboratory have indicated that once a tissue has been ‘induced’ the cells continue in a single course of organ development. Randomly reaggregated cells taken from stage 16 or 17 embryos do not differentiate into any recognizable organs unless they are in contact with denuded yolk epithelium. Finally, Marthy invokes special, hypothetical ‘contractile elements’ as having a causal role in morphogenesis but neither defines them nor presents a testable hypothesis of how they might function in embryonic differentiation. The hypothesis put forth in my 1965(b) paper is no doubt too simple and open to some question. However, Marthy’s experiments on another species under considerably different conditions do not disprove the hypothesis, nor does he suggest a workable alternative hypothesis. Therefore, the hypothesis presented in my 1965(b) paper is used in the current paper.