In a small percentage of normal embryos and in a higher percentage of embryos centrifuged prior to the first cleavage the positions of the polar bodies and the site of the first cleavage furrow do not coincide. These cases have been used to establish whether polar body formation sites or first cleavage initiation sites correlate best with the oral-aboral axis of the embryo. In all cases when the first cleavage is initiated at a site different from the site where the polar bodies were given off, the pattern of the first four cleavages is normal, the segregation of comb plate potential at these stages is normal, and the larvae that form are normal. The extent to which comb plate potential is localized along the oral-aboral axis of the embryo prior to the first cleavage, during the first cleavage and at the 2-cell stage was also examined. These experiments demonstrate that the oral-aboral axis is established at the time of the first cleavage, that cleavage plays a causal role in setting up the axis, and that comb plate-forming potential begins to be localized in the aboral region of the embryo at this time.

Developmental biologists traditionally argue that many aspects of the pattern of cleavage and the localization of cytoplasmic factors which play a role in specifying different types of cell differentiation are determined by a ‘pro-morphological scaffold’ which is laid down during oogenesis (Wilson, 1925). This scaffold is generally thought of from a formal standpoint as one or more axial coordinates with the property of polarity; however, at present its structural basis is obscure.

Most of the evidence for a promorphological scaffold is based upon studies which have demonstrated a correlation between one or more structural features of the oocyte and a symmetry property of the embryo. There have been only a few cases in which the significance of this kind of correlation has been tested experimentally. One example frequently cited in support of this notion, on both correlative and experimental grounds, is based upon studies of embryogenesis in ctenophores (Wilson, 1925; Schliep, 1929).

Figure 1 outlines the pattern of early cleavages in ctenophore embryos and indicates how this pattern is related to the symmetry properties of the larva. The early cleavages in these animals are unipolar; the furrow is initiated at a circumscribed site and spreads out from there passing through the cell. There are several reports which indicate that the first cleavage originates at or near the site where the polar bodies are given off. Marking experiments show that this site corresponds to the future oral region of the larva; the apical organ will form opposite this site (Reverberi & Ortolani, 1963). These two loci define the oral-aboral axis of the larva.

Fig. 1.

Diagrams of the first cleavage, the 2-, 4-, 8-, and 16-cell stage embryos and the larvae. All stages are shown from the side in the tentacular plane; the oral pole is marked by a polar body. The first cleavage is initiated at the future oral pole and passes through what will be the sagittal plane of the larvae. The second cleavage also begins at the future oral pole of the embryo, and passes through what will be the future tentacular plane of the larvae. The third cleavage begins in the oral region ; it divides each of the four blastomeres along a plane oblique to the first division plane. The cells at the ends of the tentacular axis are referred to as ‘E’ blastomeres while the more oral cells are referred to as ‘M’ blastomeres. During the fourth cleavage each blastomere gives off an ‘e’ or an ‘m’ micromere toward the aboral pole.

Fig. 1.

Diagrams of the first cleavage, the 2-, 4-, 8-, and 16-cell stage embryos and the larvae. All stages are shown from the side in the tentacular plane; the oral pole is marked by a polar body. The first cleavage is initiated at the future oral pole and passes through what will be the sagittal plane of the larvae. The second cleavage also begins at the future oral pole of the embryo, and passes through what will be the future tentacular plane of the larvae. The third cleavage begins in the oral region ; it divides each of the four blastomeres along a plane oblique to the first division plane. The cells at the ends of the tentacular axis are referred to as ‘E’ blastomeres while the more oral cells are referred to as ‘M’ blastomeres. During the fourth cleavage each blastomere gives off an ‘e’ or an ‘m’ micromere toward the aboral pole.

There are several environmental factors that could act upon the developing oocyte to set up an oral-aboral axis in it. Oogenesis in ctenophores is mediated by an oocyte-nurse cell system, a syncytial complex containing one central oocyte connected by intercellular bridges with three nurse cell clusters (Dunlap, 1966; Pianka, 1974). During oocyte maturation polar bodies are released from the end of the oocyte where the nucleus has been located throughout oogenesis. This region bears a consistent relationship with the three intercellular bridges connecting the nurse cell clusters to the oocyte. There is also a consistent spatial arrangement between the oocyte-nurse cell complex and the tissues surrounding it during oogenesis.

Several investigators have claimed, on the basis of experimental studies, that factors specifying comb plate cilia formation are localized in the ctenophore egg prior to the 2-cell stage (Driesch & Morgan, 1895; Fischel, 1903; Yatsu, 1912a). This claim implies some kind of promorphological organization. Unfortunately these studies are difficult to evaluate because certain key pieces of information are missing (see Discussion).

The purpose of this study is to test the validity of the notion that the uncleaved ctenophore egg has a promorphological scaffold in the form of an oral-aboral axis. This problem has been approached in two ways: (1) The correlation between the position of the polar bodies and the site where the first cleavage is initiated was examined. In a small percentage of normal embryos and in a higher percentage of experimentally manipulated embryos the positions of the polar bodies and the site of origin of the first cleavage furrow do not coincide. These cases have been used to establish whether polar body sites or first cleavage initiation sites correlate best with the oral-aboral axis of the embryo. (2) The extent to which comb plate-forming potential is localized along the ‘oral-aboral’ axis of the embryo prior to the first cleavage, during the first cleavage, and at the 2-cell stage was examined.

These experiments demonstrate that the oral-aboral axis is established at the time of first cleavage, that cleavage plays a causal role in setting up the axis, and that comb plate-forming potential begins to be localized in the aboral region of the embryo at this time.

Three species were used in this study: Bolinopsis microptera, Pleurobrachia pileus and Pleurobrachia bachei. The descriptions of Dunlap (1966) and Kozloff (1974) have been used as a basis for identification. Fertile eggs were obtained through natural spawnings. These species are in reproductive condition at Friday Harbor from the last part of April to the first part of June. During this season only about half of the adults of each species will contain eggs. Adults were generally collected the day before they were to be used to obtain gametes. Individual animals were placed in finger bowls. During the evening hours the finger bowls were placed in the dark in an incubator at 10-11 °C. The next morning they were removed from the dark and examined at frequent intervals in order to detect animals preparing to spawn; the eggs of these animals will be visible in their spawning sinuses. There is a time interval when individuals of each species are most apt to spawn after being brought into the light; these time intervals are 1 –1 ·5 h for P. pileus, 1 ·5 –2 h for Bolinopsis and 4 –5 h for P. bachei. Individuals of a given species which do not spawn at the expected time will frequently spawn at some other time. Spawning also occurs with a high frequency when the animals are in the dark. Individual Bolinopsis will sometimes spawn more than once during the 12 h period after they have been removed from the dark. During spawning sperm are released first and eggs a few minutes later (Dunlap, 1966). In P. bachei large numbers of sperm are frequently released; under these conditions fertilization is invariably poly-spermie and the eggs develop abnormally. This condition can be mitigated by transferring an animal that has begun to release sperm to a large volume of sea water and stirring the sea water so that the sperm will disperse.

The distances between different sites on the egg surface were measured using a dissecting microscope at 150 × with a goniometer eye piece. Eggs were orientated with the two sites to be measured at the periphery, the egg centered with respect to the eye piece, and the angle between the two sites was then measured. Two sources of error put limits on the accuracy of these measurements: (1) If a series of measurements are made between two sites on a given egg the range over which the measurements will vary is about 5°. This error is probably related to slight differences in the orientation of the egg from measurement to measurement. (2) When an egg is in the process of dividing it flattens perpendicular to the plane of cleavage. If these eggs are oriented in certain ways the angle between two sites being measured will be slightly different from what it would be if the egg was a perfect sphere. These two sources of error place the accuracy of these goniometric measurements in the range of ± 5 –10°.

The procedures used to remove egg envelopes, for marking selected regions of the egg surface with carbon particles, for cutting uncleaved eggs or blasto-meres into parts, and for isolating blastomeres have been described previously (Freeman, 1976; Freeman & Reynolds, 1973). One new experimental procedure involved centrifugation of uncleaved eggs. A sucrose cushion (0 ·5 ml of 1-1 molal sucrose) was placed in a conical centrifuge tube. About 20 eggs were layered on top of the cushion in 0 ·2 –0 ·3 ml of sea water and centrifuged for 5 min at ca. 5000 g in an IEC table top clinical centrifuge with a horizontal rotor. The eggs were then washed several times to remove the sucrose. Intact embryos in their envelopes were reared in millipore filtered pasteurized sea water in the wells (1-5 ml vol.) of glass spot plate dishes. Isolated blastomeres and eggs with their envelopes removed were reared in the same way, except that the wells were lined with a coat of 2 % agar.

The embryos and isolated blastomeres were raised at 10 –12 °C. Under these conditions the time interval from spawning to initiation of the first cleavage is about 60 min for P. bachei, 80 min for P. pileus, and 130 min for Bolinopsis. The time interval between the two-cell stage and the first appearance of comb plate cilia in these embryos is 13 –14 h for P. bachei, 15 –16 h for P. pileus, and 19 –20 h for Bolinopsis. In some of the experiments reported here the presence or absence of comb plate cilia was monitored; these observations were always made 3 –6 h after these cilia first appeared (see Freeman, 1976 for procedure). In other experiments the normality of larvae was assayed; these observations were usually made 12 –24 h after comb plate cilia first appeared.

The cytological methods and procedures used to make reconstructions of eggs from serial sections have been described previously (Freeman, 1976; Freeman & Reynolds, 1973).

I. The correlation between the site of polar body formation and the origin of the first cleavage furrow

Normal development

During the process of oocyte maturation in ctenophores, the oocyte moves into a spawning sinus and then to the exterior of the animal via a gonopore (Dunlap, 1966). The oocyte gives off the first polar body in the spawning sinus (Dunlap, 1966). The second polar body is either present at the time the oocyte emerges from the gonopore, or is given off within a few minutes after emergence. The release of the second polar body occurs on schedule, even in unfertilized eggs. The region of the oocyte where the polar bodies are given off is the first region that passes through the gonopore. Fertilization probably takes place while the oocyte passes through the gonopore or very shortly after it is liberated into the sea water. As the oocyte passes through the gonopore a fluid filled space forms between it and the vitelline membrane which surrounds it.

The polar bodies can adhere to the surface of the egg, float in the space between the egg and the vitelline membrane, or adhere only to the vitelline membrane. Twenty per cent of the P. bachei eggs, 75 % of the P. pileus eggs and 45 % of the Bolinopsis eggs have one or both polar bodies attached. This average figure is somewhat misleading, since for a given species in three out of four spawnings only a small percentage of eggs may have one or more polar bodies attached, while in the fourth spawning over 75 % of the eggs may have at least one polar body attached. This variability in the percentage of eggs from a given spawning with at least one polar body attached is probably related to the timing of polar body formation relative to the raising of the vitelline membrane. An animal that has spawned eggs in which a high percentage of cases have at least one polar body attached may subsequently spawn eggs in which only a low percentage of the eggs have at least one polar body attached. If only one polar body adheres to the egg it is always the second one. The first polar body can almost always be distinguished from the second one because the former usually divides to form two polocytes which remain attached to each other. There are a number of cases, especially for P. pileus, in which both polar bodies are attached to the egg. Since they are always within 10° of each other, they are probably given off at the same site. There is no indication that the polar bodies change their relative distance from each other.

The relationship between the region where the polar bodies are given off and the site where the first cleavage is initiated was examined by measuring the distance between these two sites before the first cleavage furrow was half way across the egg. These measurements (Table 1) show that the first cleavage furrow can originate in any quadrant of the egg with reference to the site of polar body formation. Figure 2 shows a case for each species in which the site of the first cleavage furrow does not correspond to the region where the polar bodies were given off. Nevertheless the most probable site of furrow formation is in the quadrant nearest the site of polar body formation; as the distance from this site increases the probability of furrow formation decreases. Table 1 also indicates that there are differences among the species in the degree of correlation between the site of polar body formation and the site where the first cleavage is initiated. This correlation is quite strong in P. bachei but only of moderate strength in Bolinopsis and P. pileus. In those cases in which the first cleavage furrow originated between 45° and 135° from the site of polar body formation, the plane of the first cleavage does not correlate with the site of the polar body formation.

Table 1.

The relationship between the site of polar body formation and the site of origin of the first cleavage furrow

The relationship between the site of polar body formation and the site of origin of the first cleavage furrow
The relationship between the site of polar body formation and the site of origin of the first cleavage furrow
Fig. 2.

Photographs of eggs in which the site of polar body formation does not correlate with the origin of the first cleavage furrow: (A) Bolinopsis, (B) P. pileus, (C) P. bachei. All photographs are at the same magnification. The bar indicates 50 μm. The arrow indicates the polar body.

Fig. 2.

Photographs of eggs in which the site of polar body formation does not correlate with the origin of the first cleavage furrow: (A) Bolinopsis, (B) P. pileus, (C) P. bachei. All photographs are at the same magnification. The bar indicates 50 μm. The arrow indicates the polar body.

The suitability of using attached polar bodies as surface landmarks has been studied by applying a carbon mark to the surface of the egg within 10° of the site where the polar body is and measuring the distance between the mark and the polar body at frequent intervals up until the third cleavage. Five Bolinopsis, eight P. pileus and three P. bachei eggs were marked within 10 min after spawning. There were no cases in which the distance between the carbon mark and the polar body changed prior to the first cleavage; if the mark or the polar body was not near a cleavage furrow the distance between the mark and the polar body was not changed during cleavage (see Freeman, 1976 for observations on the behavior of carbon marks near cleavage furrows). In two of these Bolinopsis and five of the P. pileus eggs the origin of the first cleavage furrow was over 45° from the polar body and the carbon mark. Observations that have been made on these eggs in the course of cutting experiments indicate that the polar bodies adhere firmly to the egg. This evidence suggests that the polar bodies remain at the site where they originate and indicates that they are a stable landmark.

The role of nuclear events in establishing the site of the first cleavage has been studied by measuring the positions of male and female pronuclei relative to the site of polar body formation at various time intervals prior to the first cleavage. Even though the ctenophore egg is translucent it is difficult to make judgements about the movement of the male and female pronuclei in living eggs. The description presented here is based upon P. pileus eggs which have been fixed and sectioned for cytological study. For each spawning a sample of 20 eggs was monitored in order to establish the percentage that cleaved and the synchrony of the first cleavage. The eggs from a given spawning were used for cytological analysis if at least 75 % of the eggs cleaved and at least 75 % of the cleaving eggs initiated their first cleavage within 10 min of the same event in the first egg.

The ctenophore egg has a centrolecithal organization : it is composed of an inner endoplasmic zone of yolk spheres and a thin outer layer of basophilic cortical cytoplasm which surrounds the endoplasm. The male and female pronuclei always reside in the cortical cytoplasm just under the cell membrane. Observations on the relative positions of the male and female pronuclei in eggs as a function of time after spawning are summarized in Table 2. In eggs fixed 5 min after spawning the male pronucleus is highly condensed (average diameter ca. 2 μm) while the female pronucleus is not condensed (average diameter ca. 61 μm). The female pronucleus is almost always directly under the region of the egg surface where the polar bodies are. The male pronucleus can be found in any quadrant of the egg but is found most frequently in the quadrant where the polar bodies are. This observation suggests that the sperm most often fuses with the egg near the site where the polar bodies are given off. At 15 min after fertilization the male pronucleus is less condensed (average diameter ca. 4 m). There is no change in the morphology of the female pronucleus. The distribution of the male pronuclei is roughly the same as it was 5 min after spawning; the distribution of female pronuclei is much less uniform. In two cases the female pronuclei moved more than 45° from the site of the polar body emission. In most cases the female pronucleus appears to have moved toward the site where the male pronucleus is located. Figure 3 shows three egg reconstructions which document this point. At 25 min the pronuclei have fused in the majority of cases. In those cases where the pronuclei have fused the zygote nucleus is generally in the quadrant between 0° and 45° from the site where the polar bodies have been given off. If the pronuclei have not fused they are generally in close proximity to each other, but some distance from the site where the polar bodies have been given off. The results of similar observations on Bolin-opsis eggs indicate that the female pronucleus behaves in essentially the same way in this species. These cytological studies suggest that in undisturbed eggs, the female pronucleus is initially at the site where the polar bodies were given off, but that it will migrate some distance in order to fuse with the male pronucleus if the latter is not in the immediate vicinity.

Table 2.

The positions of the pronuclei or zygote nucleus relative to the site of polar body formation at different times after fertilization in P. pileus

The positions of the pronuclei or zygote nucleus relative to the site of polar body formation at different times after fertilization in P. pileus
The positions of the pronuclei or zygote nucleus relative to the site of polar body formation at different times after fertilization in P. pileus
Fig. 3.

Reconstruction of three P. pileus eggs fixed 15 min after spawning. In each case the female pronucleus has moved from the site where the polar bodies were given off toward the male pronucleus.

Fig. 3.

Reconstruction of three P. pileus eggs fixed 15 min after spawning. In each case the female pronucleus has moved from the site where the polar bodies were given off toward the male pronucleus.

Centrifugation experiments

While in some eggs the region where the first cleavage furrow originates bears no relationship to the site of polar body formation, in many cases these sites closely coincide. However, one can alter this correlation by centrifuging eggs prior to the first cleavage. In each centrifugation experiment about 20 eggs were set aside as a control. These eggs were treated in the same way as the experimentals were except that they were not centrifuged. They were used to establish the per cent cleavage and the synchrony of the first cleavage in each spawning. Centrifugation always took place at least 20 min prior to initiation of the first cleavage in controls. In most cases the eggs were probably centrifuged after pronuclear fusion. In eggs centrifuged with sufficient force the cortical cytoplasm takes up a centripetal position while the endoplasm takes up a centrifugal position (Fig. 4). Examination of these eggs with phase optics indicates that the zygote nucleus ends up in the cortical cytoplasmic mass in the centripetal region of the egg. In certain batches of eggs from all three species, especially for P. pileus, some centrifuged eggs formed blebs on their surfaces; these eggs were always discarded. After centrifugation the cortical cytoplasm slowly envelops the endoplasm re-establishing the original centrolecithal organization. The time this process takes depends both upon the centrifugal force used and the species. Within 30-45 min it is frequently difficult to identify the centripetal pole of P. pileus eggs, while this pole can be identified for 90-120 min in uncleaved eggs of Bolinopsis and P. bachei.

Fig. 4.

Photographs showing eggs approximately 15 min after centrifugation. Note the polar bodies and the stratification of the egg cytoplasm: (A) Bolinopsis, (B) P. pileus, (C) P. bachei. All photographs are at the same magnification. The bar indicates 50 μm. The arrow indicates the polar body.

Fig. 4.

Photographs showing eggs approximately 15 min after centrifugation. Note the polar bodies and the stratification of the egg cytoplasm: (A) Bolinopsis, (B) P. pileus, (C) P. bachei. All photographs are at the same magnification. The bar indicates 50 μm. The arrow indicates the polar body.

Table 3 summarizes observations on the relationship between the centripetal pole of the centrifuged eggs and the site where the polar bodies formed. These results indicate that the centripetal end of the centrifuged egg bears no relationship to the region of polar body extrusion. Eight P. pileus eggs were marked with carbon within 10° of the polar body prior to centrifugation. Centrifugation did not change the distance between the mark and the polar body. These centrifugation experiments provide another way of demonstrating that the polar bodies are a stable marker.

Table 3.

The relationship between the site of polar body formation and the centripetal pole of centrifuged eggs

The relationship between the site of polar body formation and the centripetal pole of centrifuged eggs
The relationship between the site of polar body formation and the centripetal pole of centrifuged eggs

Figure 5 shows a set of centrifuged eggs with an attached polar body in which the first cleavage is beginning. Table 4 examines the site of origin of the first cleavage furrow as a function of distance from the centripetal end of the egg for subpopulations of eggs in which the site of polar body formation is at different distances from the centripetal region. This table defines the most centripetal end of the egg as 0° while the most centrifugal end is 180°; 0-45° is entirely within the cortical cytoplasmic region of the centrifuged egg. The boundary between the cortical and the endoplasmic region of the egg is somewhere between 45° and 90°, while the region between 90° and 180° contains endoplasm. In almost every case the site of origin of the first cleavage furrow occurs within or just at the edge of the cortical cytoplasm. If the first cleavage furrow originated near the interface between the cortical and endoplasmic layers the plane of cleavage was always parallel to this interface. Table 5 examines the site of origin of the first cleavage furrow as a function of distance from the site of polar body formation for subpopulations of eggs in which the site of polar body formation is different distances from the centripetal pole. A comparison of Tables 5 and 1 indicates that the site of origin of the first cleavage furrow, with reference to the site where the polar bodies are given off, can be altered substantially by centrifugation especially when the polar bodies are some distance from the centripetal pole.

Table 4.

The relationship between the origin of the first cleavage furrow and the centripetal pole of centrifuged eggs

The relationship between the origin of the first cleavage furrow and the centripetal pole of centrifuged eggs
The relationship between the origin of the first cleavage furrow and the centripetal pole of centrifuged eggs
Table 5.

The relationship between the origin of the first cleavage furrow and the polar bodies of centrifuged eggs

The relationship between the origin of the first cleavage furrow and the polar bodies of centrifuged eggs
The relationship between the origin of the first cleavage furrow and the polar bodies of centrifuged eggs
Fig. 5.

Photographs showing the origin of the first cleavage in centrifuged eggs. Note the axis along which the egg is stratified and the polar bodies: (A) Bolinopsis, (B) P. pileus, (C) P. bachei. All photographs are at the same magnification. The bar indicates 50 μm. The arrow indicates the polar body.

Fig. 5.

Photographs showing the origin of the first cleavage in centrifuged eggs. Note the axis along which the egg is stratified and the polar bodies: (A) Bolinopsis, (B) P. pileus, (C) P. bachei. All photographs are at the same magnification. The bar indicates 50 μm. The arrow indicates the polar body.

The observations made on normal eggs and the centrifugation experiments reported here make it clear that the first cleavage furrow does not have to originate at the site of polar body formation. At this point the developmental consequences of having the first cleavage originate at a site other than the region where the polar bodies have been given off will be examined. One way that this problem has been approached is by comparing the cleavage pattern in embryos in which the site of the origin of the first cleavage furrow corresponds to the site of polar body emission with embryos in which it does not. The other approach to this problem has examined the way in which developmental potential is segregated during cleavage and the normality of the larvae that develop from these two classes of embryos.

In those ctenophore eggs where the site of origin of the first cleavage differs from that of polar body formation there may be a change in the sites where one or more subsequent cleavage furrows originate. Such an effect has been demon-strated in other kinds of embryos (e.g. Morgan & Spooner, 1909). The first four cleavages (see Fig. 1) were carefully monitored for at least 15 eggs from each species in which the first cleavage furrow originated between 90° and 180° from the region where the polar bodies were given off. These cases were selected either from batches of untreated eggs or from eggs that had been centrifuged. A carbon mark was placed on each egg at the site where the first cleavage was initiated and the origin of the subsequent cleavages was examined with reference to this mark and to the site where the polar bodies were given off. While in some cases a given cleavage was blocked or delayed, especially after centrifugation, there was no indication of any other changes in cleavage pattern. In every case the micromeres were given off during the fourth cleavage at a region which corresponded to the pole of the uncleaved egg directly opposite the site of origin of the first cleavage furrow.

The segregation of comb plate-forming potential in embryos where the origin of the first cleavage furrow differs from the site of polar body formation has been studied by isolating blastomeres at the 8- and 16-cell stages and monitoring their ability to differentiate. Blastomere isolation experiments delimit the following sequence for the segregation of comb plate-forming potential during cleavage in normal embryos. At the 4-cell stage each blastomere has the ability to give rise to an isolate which subsequently differentiates comb plate cilia cells, at the 8-cell stage only the E macromere inherits this capability, while only the e micromere inherits it at the 16-cell stage (see Reverberi, 1971 for a review). If one supposes that the factors specifying comb plate cilia formation are localized at the aboral pole of the uncleaved egg and that this pole can be identified by using the site of polar body formation as a marker, a disturbance in the segregation of developmental potential might result if the sites of origin of the first cleavage furrow and polar body formation differ. In each species a number of eggs were selected in which (1) the origin of the first cleavage furrow was within 20° of the site of polar body emission, (2) the origin of the first cleavage furrow was within 80-100° of this region, or (3) the origin of the first cleavage furrow was within 160-180° of this site. These cases were selected from batches of both untreated and centrifuged eggs. When eggs were operated on at the 8-cell stage each blastomere that made up an E and M macromere pair derived from the same progenitor cell at the 4-cell stage was isolated and its ability to differentiate comb plate cilia was monitored. When the eggs were operated on at the 16-cell stage each blastomere that made up an e micromere E macromere pair derived from an E macromere progenitor cell at the 8-cell stage was isolated and its ability to differentiated comb plate cilia was monitored. These results are summarized in Table 6. Only normally developing isolates are reported in this table; if an isolate lost more than 10 % of its cells, or if there was evidence that a cleavage block had occurred it was not included. Twelve per cent of the isolates were discarded for these reasons; almost all of these cases originated from centrifuged eggs. If one isolate from any pair was not suitable for analysis the other member of the pair was also discarded. The results show that, regardless of the site of origin of the first cleavage furrow with reference to the region of polar body formation, the segregation of comb plate cilia-forming potential was nearly always normal. However, both the blastomeres that make up a pair formed comb plates in four cases; Fig. 6 documents the history of each case. This figure shows that the region of cytoplasm sampled in these cases did not bear a consistent relationship to the hypothetical oral-aboral axis marked by the region where the polar bodies were given off.

Table 6.

The segregation of comb plate-forming potential in eggs where the origin of the first cleavage furrow corresponds to the site of polar body formation and at different distances from this site

The segregation of comb plate-forming potential in eggs where the origin of the first cleavage furrow corresponds to the site of polar body formation and at different distances from this site
The segregation of comb plate-forming potential in eggs where the origin of the first cleavage furrow corresponds to the site of polar body formation and at different distances from this site
Fig. 6.

The early developmental history of embryos in which there was an inappropriate segregation of comb plate-forming potential during the 8- or 16-cell stage. The top part of each figure indicates the site of origin of the first cleavage furrow and the site where the polar bodies were given off. A hypothetical distribution for the factors which specify comb plates, as defined by the position of the polar bodies is indicated by x’s. In those cases in which the egg has been centrifuged the stratification of cortical cytoplasm is indicated by shading. The bottom part of each figure shows the location of the blastomeres from these embryos in which there was not an appropriate segregation of comb plate-forming potential at the 8- or 16-cell stage. (A) A P. bachei embryo, all four of the E, M blastomere pairs were assayed. An E, M blastomere pair in which neither blastomere formed comb plates is on the same side of the sagittal plane as the E, M pair in which both blastomeres formed comb plates. (B) A P. pileus embryo, three of the E, M blastomere pairs were assayed. One of the E, M pairs for which there is a normal segregation of comb plate potential is on the same side of the sagittal plane as the E, M pair in which both blastomeres formed comb plates. (C) A P. pileus embryo, all four of the E, M blastomere pairs were assayed. Three of these pairs showed a normal segregation of developmental potential. (D) A P. pileus embryo, only the blastomere pair indicated was assayed.

Fig. 6.

The early developmental history of embryos in which there was an inappropriate segregation of comb plate-forming potential during the 8- or 16-cell stage. The top part of each figure indicates the site of origin of the first cleavage furrow and the site where the polar bodies were given off. A hypothetical distribution for the factors which specify comb plates, as defined by the position of the polar bodies is indicated by x’s. In those cases in which the egg has been centrifuged the stratification of cortical cytoplasm is indicated by shading. The bottom part of each figure shows the location of the blastomeres from these embryos in which there was not an appropriate segregation of comb plate-forming potential at the 8- or 16-cell stage. (A) A P. bachei embryo, all four of the E, M blastomere pairs were assayed. An E, M blastomere pair in which neither blastomere formed comb plates is on the same side of the sagittal plane as the E, M pair in which both blastomeres formed comb plates. (B) A P. pileus embryo, three of the E, M blastomere pairs were assayed. One of the E, M pairs for which there is a normal segregation of comb plate potential is on the same side of the sagittal plane as the E, M pair in which both blastomeres formed comb plates. (C) A P. pileus embryo, all four of the E, M blastomere pairs were assayed. Three of these pairs showed a normal segregation of developmental potential. (D) A P. pileus embryo, only the blastomere pair indicated was assayed.

In those centrifuged eggs in which the plane of the first cleavage parallels the boundary between the endoplasm and the cortical cytoplasm two blastomere lineages result in which there are quantitative differences in cytoplasmic composition of the cells. One of these lineages is rich in endoplasm but poor in cortical cytoplasm while the other lineage is cortical cytoplasm rich but endoplasm poor. In most of these cases the segregation of comb plate-forming potential is normal regardless of the cytoplasmic composition of the blastomere pair (see Freeman & Reynolds, 1973 for a description of the behavior of egg fragments made up exclusively of cortical cytoplasm).

Larvae were also allowed to develop from intact embryos selected from untreated and centrifuged eggs in which the first cleavage originated at varying distances from the site of polar body formation. These cases were carefully examined after the mouth, the apical organ, comb rows and tentacle pouches could be clearly identified. If one discounts the cases in which cleavage was abnormal the embryos from all three species developed into normal larvae regardless of the relationship between the site of origin of the first cleavage furrow and the site of the polar body formation (Table 7).

Table 7.

The degree of normality for larvae where the origin of the first cleavage furrow corresponds to or is different from the site of polar body formation

The degree of normality for larvae where the origin of the first cleavage furrow corresponds to or is different from the site of polar body formation
The degree of normality for larvae where the origin of the first cleavage furrow corresponds to or is different from the site of polar body formation

These observations on normal and centrifuged eggs have made it clear that the site of origin of the first cleavage furrow does not have to correspond to the region where the polar bodies were given off. In those cases where the origin of the first cleavage does not correspond to the region of polar body formation, the oral-aboral axis of the embryo is clearly related to the site of origin of the first cleavage furrow.

II. The localization of comb plate-forming potential in the uncleaved egg, during the first cleavage, and at the 2-cell stage

At some point in development prior to the 16-cell stage the factors which specify comb plate differentiation must become localized at the aboral pole of the ctenophore embryo. Studies which have mapped the distribution of comb plate-forming potential in Mnemiopsis have indicated that there is a moderate localization of these factors at the aboral pole of the blastomeres in the 2-cell stage embryo (Freeman, 1976). In this study similar procedures have been used to establish whether comb plate potential is localized in the uncleaved egg.

A known volume of cytoplasm was excised from the ‘aboral’ region of either the uncleaved egg, an egg in which the first cleavage is occurring, or a blastomere of a 2-cell stage embryo. The ability of the remaining nucleated fragment to differentiate comb plate cilia was then assayed. If the factors responsible for specifying comb plate cilia differentiation are not localized in the future aboral region of the embryo when the operation is done, one would expect the remaining isolate to form comb plate cilia. If these factors are localized exclusively in the aboral region at the time of the operation, the isolates derived from the nucleated fragment remaining after the operation would not form comb plate cilia. As a control another set of operations was done in which cytoplasmic regions of comparable volume were removed, but where the fragment that remained after the operation contained cytoplasm of both presumptive oral and aboral regions of the embryo.

Most of these operations were performed on the eggs and embryos of P. pileus’, these cases are supplemented by a smaller group of operations that were performed on P. bachei. In uncleaved eggs the polar bodies were used to mark the oral pole. They are a reliable marker in P. bachei but are less reliable in P. pileus (Table 1). The poor correlation between the site of polar body formation and the oral-aboral axis for P. pileus was mitigated to a certain extent by analyzing separately those cases where the origin of the first cleavage furrow corresponded to the site of polar body formation. When an egg is cut into an oral and an aboral half, usually only the oral half cleaves; however, there are some cases in which the aboral half cleaves. By way of analogy with other eggs that have been cut into two parts, it is reasonable to suppose that the division center of the male pronucleus is necessary for cleavage and that the fragment which cleaves is either haploid or diploid. For the operations on eggs that were going through their first cleavage, and the 2-cell stage embryos, the origin of the first cleavage furrow was used to mark the oral-aboral axis. Only the oral half of embryos cut at these stages will continue to cleave. These operations are outlined in Fig. 7. When the developmental stage was reached which was equivalent to the 4-cell stage, the blastomeres were separated and raised in isolation in order to assay their ability to differentiate comb plate cilia. These results are summarized in Table 8.

Table 8.

The effects of removing defined cytoplasmic regions from uncleaved eggs, cleaving eggs, and 2-cell blastomeres on the differentiation of comb plate cilia by their isolated EM blastomere derivatives*

The effects of removing defined cytoplasmic regions from uncleaved eggs, cleaving eggs, and 2-cell blastomeres on the differentiation of comb plate cilia by their isolated EM blastomere derivatives*
The effects of removing defined cytoplasmic regions from uncleaved eggs, cleaving eggs, and 2-cell blastomeres on the differentiation of comb plate cilia by their isolated EM blastomere derivatives*
Fig. 7.

Description of the operations on uncleaved eggs, eggs undergoing their first cleavage and the blastomere of the 2-cell embryo. (A) Operation in which a cut is made along the presumptive equatorial plane of the uncleaved egg separating the presumptive oral region from the presumptive aboral region. (B) Operation in which a cut is made along the presumptive oral-aboral axis of the uncleaved egg generating two fragments which contain presumptive oral and aboral cytoplasmic regions. After operation ‘A’ or ‘B’ the diameter of the blastomere fragments were measured. (Note: In each case the diameter of the fragment that did not cleave was measured; in addition, in several cases the diameter of the cleaving fragment was measured before cleavage began. In those cases in which only the diameter of the uncleaved fragment was measured, this value was used to estimate the volume of the cleaving fragment by subtracting the volume of the uncleaved fragment from the average volume of the eggs for the species used. The diameter of the P. pileus egg is 171 ± S.D. 7 · 6 μ m; the diameter of the P. bachei egg is 140 ± S.D. 7 · 4 μ m). Usually only one fragment will cleave following an operation. This fragment is allowed to generate four blastomeres, the blastomeres are then separated and each is raised in isolation. Operations ‘A’ and ‘B’ were done prior to pronuclear fusion in some cases and after fusion in other cases. The time span between the operation and the initiation of cleavage did not influence the results. (C) Operation in which a cut is made along the equatorial plane during the first cleavage separating the nucleated oral region from the enucleated aboral region of the egg. In effecting this operation the cut was always made through the uncleaved portion of the egg prior to the advance of the furrow through that area. After this operation the enucleated fragment was measured. The cleaving fragment was allowed to generate four blastomeres, the blastomeres were then separated and raised in isolation. (D) Operation in which a cut is made along the developing first cleavage furrow to give two nucleated fragments each containing presumptive oral and aboral cytoplasmic regions. Each ‘blastomere’ was allowed to divide once more, the four blastomeres were separated and each raised in isolation. (Note: this operation is not strictly comparable to operation ‘C’ in that the volume of the blastomeres isolated at the 4-cell stage is about twice the volume of the blastomeres from the experimentals). (E) Operation in which a cut is made along the equatorial plane separating a nucleated oral region from an enucleated aboral region of one blastomere at the 2-cell stage. These eggs were orientated prior to the operation by marking the site where the first cleavage furrow originated. (F) Operation in which a cut is made along the oral-aboral axis in the tentacular plane of one blastomere at the 2-cell stage to give a nucleated fragment containing oral and aboral cytoplasmic regions and an enucleated fragment of similar composition. These eggs were orientated prior to the operation by marking one end of the oral-aboral axis and the tentacular end of the blastomere. The blastomeres of the embryo were separated and the marked blastomere was cut. After operations ‘E’ and ‘F’ the enucleated fragment was measured, the nucleated fragment was allowed to cleave once and the two blastomeres were separated and raised in isolation.

Fig. 7.

Description of the operations on uncleaved eggs, eggs undergoing their first cleavage and the blastomere of the 2-cell embryo. (A) Operation in which a cut is made along the presumptive equatorial plane of the uncleaved egg separating the presumptive oral region from the presumptive aboral region. (B) Operation in which a cut is made along the presumptive oral-aboral axis of the uncleaved egg generating two fragments which contain presumptive oral and aboral cytoplasmic regions. After operation ‘A’ or ‘B’ the diameter of the blastomere fragments were measured. (Note: In each case the diameter of the fragment that did not cleave was measured; in addition, in several cases the diameter of the cleaving fragment was measured before cleavage began. In those cases in which only the diameter of the uncleaved fragment was measured, this value was used to estimate the volume of the cleaving fragment by subtracting the volume of the uncleaved fragment from the average volume of the eggs for the species used. The diameter of the P. pileus egg is 171 ± S.D. 7 · 6 μ m; the diameter of the P. bachei egg is 140 ± S.D. 7 · 4 μ m). Usually only one fragment will cleave following an operation. This fragment is allowed to generate four blastomeres, the blastomeres are then separated and each is raised in isolation. Operations ‘A’ and ‘B’ were done prior to pronuclear fusion in some cases and after fusion in other cases. The time span between the operation and the initiation of cleavage did not influence the results. (C) Operation in which a cut is made along the equatorial plane during the first cleavage separating the nucleated oral region from the enucleated aboral region of the egg. In effecting this operation the cut was always made through the uncleaved portion of the egg prior to the advance of the furrow through that area. After this operation the enucleated fragment was measured. The cleaving fragment was allowed to generate four blastomeres, the blastomeres were then separated and raised in isolation. (D) Operation in which a cut is made along the developing first cleavage furrow to give two nucleated fragments each containing presumptive oral and aboral cytoplasmic regions. Each ‘blastomere’ was allowed to divide once more, the four blastomeres were separated and each raised in isolation. (Note: this operation is not strictly comparable to operation ‘C’ in that the volume of the blastomeres isolated at the 4-cell stage is about twice the volume of the blastomeres from the experimentals). (E) Operation in which a cut is made along the equatorial plane separating a nucleated oral region from an enucleated aboral region of one blastomere at the 2-cell stage. These eggs were orientated prior to the operation by marking the site where the first cleavage furrow originated. (F) Operation in which a cut is made along the oral-aboral axis in the tentacular plane of one blastomere at the 2-cell stage to give a nucleated fragment containing oral and aboral cytoplasmic regions and an enucleated fragment of similar composition. These eggs were orientated prior to the operation by marking one end of the oral-aboral axis and the tentacular end of the blastomere. The blastomeres of the embryo were separated and the marked blastomere was cut. After operations ‘E’ and ‘F’ the enucleated fragment was measured, the nucleated fragment was allowed to cleave once and the two blastomeres were separated and raised in isolation.

If the presumptive aboral region was removed from a 2-cell stage blastomere of P. pileus or P. bachei, 33 – 37 % of the isolates did not differentiate comb plate cilia. When control operations were done at this stage that gave blastomere fragments in which both the presumptive oral and aboral regions were present, only 12 – 21 % of the isolates failed to differentiate comb plate cilia. Figures 8 and 9 show the sizes of the nucleated fragments obtained after these operations and the distribution of cases that did not form comb plate cilia. As the size of the aboral cytoplasmic region removed gets larger, the ability of the nucleated fragment that remains to support comb plate cilia differentiation declines (Figs. 8 A and 9 A). When both oral and aboral cytoplasmic regions are present (Figs. 8 B and 9B) the failure to form comb plate cilia is not related to the volume of the nucleated fragment. This comparison between nucleated fragments of comparable size which either have or do not have an aboral region indicates that comb plate-forming potential is partially localized in the aboral region of these 2-cell stage embryos.

Fig. 8.

The size distribution of the nucleated fragments produced following operations on the uncleaved egg, the cleaving egg, and the 2-cell embryo of P. pileus (Fig. 8) and P. bachei (Fig. 9). Each isolate that developed from a blastomere fragment was assigned the relative volume of the fragment. The size distribution of the fragments which produced comb plates are white and those that did not are colored black. (A) The size distribution of fragments produced after an equatorial cut removed the presumptive aboral region from a blastomere of a 2-cell stage embryo (Fig. 7E). Note the size distribution of the isolates that did not form comb plate cilia. (B) The size distribution of the fragments produced after a cut was made along the tentacular plane of a blastomere from a 2-cell embryo giving a blastomere fragment with both oral and aboral cytoplasmic regions (Fig. 7F). (C) The size distribution of the fragments produced after an equatorial cut removed the presumptive aboral region from the cleaving egg (Fig. 7C). (D) The size distribution of the fragments produced after an equatorial cut removed the presumptive aboral region from the uncleaved egg (Fig. 7 A).

Fig. 8.

The size distribution of the nucleated fragments produced following operations on the uncleaved egg, the cleaving egg, and the 2-cell embryo of P. pileus (Fig. 8) and P. bachei (Fig. 9). Each isolate that developed from a blastomere fragment was assigned the relative volume of the fragment. The size distribution of the fragments which produced comb plates are white and those that did not are colored black. (A) The size distribution of fragments produced after an equatorial cut removed the presumptive aboral region from a blastomere of a 2-cell stage embryo (Fig. 7E). Note the size distribution of the isolates that did not form comb plate cilia. (B) The size distribution of the fragments produced after a cut was made along the tentacular plane of a blastomere from a 2-cell embryo giving a blastomere fragment with both oral and aboral cytoplasmic regions (Fig. 7F). (C) The size distribution of the fragments produced after an equatorial cut removed the presumptive aboral region from the cleaving egg (Fig. 7C). (D) The size distribution of the fragments produced after an equatorial cut removed the presumptive aboral region from the uncleaved egg (Fig. 7 A).

Fig. 9.

The size distribution of the nucleated fragments produced following operations on the uncleaved egg, the cleaving egg, and the 2-cell embryo of P. pileus (Fig. 8) and P. bachei (Fig. 9). Each isolate that developed from a blastomere fragment was assigned the relative volume of the fragment. The size distribution of the fragments which produced comb plates are white and those that did not are colored black. (A) The size distribution of fragments produced after an equatorial cut removed the presumptive aboral region from a blastomere of a 2-cell stage embryo (Fig. 7E). Note the size distribution of the isolates that did not form comb plate cilia. (B) The size distribution of the fragments produced after a cut was made along the tentacular plane of a blastomere from a 2-cell embryo giving a blastomere fragment with both oral and aboral cytoplasmic regions (Fig. 7F). (C) The size distribution of the fragments produced after an equatorial cut removed the presumptive aboral region from the cleaving egg (Fig. 7C). (D) The size distribution of the fragments produced after an equatorial cut removed the presumptive aboral region from the uncleaved egg (Fig. 7 A).

Fig. 9.

The size distribution of the nucleated fragments produced following operations on the uncleaved egg, the cleaving egg, and the 2-cell embryo of P. pileus (Fig. 8) and P. bachei (Fig. 9). Each isolate that developed from a blastomere fragment was assigned the relative volume of the fragment. The size distribution of the fragments which produced comb plates are white and those that did not are colored black. (A) The size distribution of fragments produced after an equatorial cut removed the presumptive aboral region from a blastomere of a 2-cell stage embryo (Fig. 7E). Note the size distribution of the isolates that did not form comb plate cilia. (B) The size distribution of the fragments produced after a cut was made along the tentacular plane of a blastomere from a 2-cell embryo giving a blastomere fragment with both oral and aboral cytoplasmic regions (Fig. 7F). (C) The size distribution of the fragments produced after an equatorial cut removed the presumptive aboral region from the cleaving egg (Fig. 7C). (D) The size distribution of the fragments produced after an equatorial cut removed the presumptive aboral region from the uncleaved egg (Fig. 7 A).

If the presumptive aboral region was removed from an uncleaved egg or an egg that was undergoing its first cleavage, almost all of the isolates derived from the nucleated fragment will develop comb plate cilia. In those cases in which comb plate cilia did not develop, there was a less pronounced relationship between the size of the aboral region removed from the egg and its ability to differentiate (Figs. 8C, D and 9C, D). A comparison of these operations with the control operations and with the operations done on uncleaved eggs in which the presumptive aboral region cleaved shows that a comparable percentage of isolates differentiated comb plate cilia in all cases. Of special interest are two uncleaved P. bachei eggs which were cut into presumptive oral and aboral halves; in each of these cases both halves cleaved. Yatsu (1912a) has described a similar case for Beroë; presumably these eggs were fertilized by two sperm. All eight isolates from both halves of one egg were successfully raised in isolation. Only one of these isolates, from the aboral half, did not differentiate comb plate cilia. In the other case only three isolates from the oral half and three isolates from the aboral half were raised successfully in isolation. All six of these isolates developed comb plate cilia. These comparisons suggest that the factors which specify comb plates are not yet localized exclusively in the presumptive ‘aboral’ region of the uncleaved egg or in the uncleaved portion of the egg which is undergoing its first cleavage.

This study demonstrates that the oral-aboral axis of the ctenophore embryo is set up as a consequence of the first cleavage. This demonstration is based upon the observation that the location of this axis is determined by the site where the first cleavage furrow originated for eggs in which this site is different from the site of polar body formation. The observations made here which indicate that there is not always a one to one correspondence between the site of polar body formation and the origin of the first cleavage furrow in normal eggs do not agree with the published reviews on ctenophore development which imply that there is a strict one to one correspondence (Wilson, 1925; Schliep, 1929; Korschelt, 1936; Reverberi, 1971 ; Pianka, 1974). If one looks beyond these reviews to their primary sources one sees that there is almost no data on the relationship between the site of polar body formation and the origin of the first cleavage furrow (Yatsu, 1912b; Komai, 1922; Reverberi & Ortalani, 1963). In fact, Komai (1922) shows a 2-cell embryo in which the first cleavage furrow clearly originated more than 45° from the site of polar body formation.

The fact that the first cleavage can originate some distance from the point of polar body extrusion in normal eggs indicates that the frequently observed coincidence of these two sites is probably due only to chance. I suspect that the site of origin of the first cleavage furrow corresponds to the site where the sperm fuses with the egg for this group of animals. Further studies on the fertilization process and measurements of any male pronuclear migration are needed to validate this suggestion. Several other studies have demonstrated that there can be a close correlation between the site of fertilization and the positioning of cleavage furrows (see Morgan, 1927 and Guerrier, 1971 for reviews). During the process of spawning in ctenophores the first part of the oocyte surface exposed to the sea water is the pole where the polar bodies are given off (Dunlap, 1966). Consequently, the probability of sperm-egg fusion will be highest at this pole which would then be translated into a high probability of furrow initiation at the same site. While the site of fertilization or zygote nucleus formation may determine the placement of the oral-aboral axis, the actual establishment of the axis appears to be related to the act of cytokinesis. The centrifugation experiments presented here show that if the zygote nucleus is moved to a new location, the cleavage furrow which forms at this new site corresponds to the oral-aboral axis of the embryo.

This study has also examined the extent to which the potential for comb plate cilia differentiation is localized in the presumptive aboral region of uncleaved eggs, cleaving eggs and 2-cell stage embryos. These experiments suggest that the potential for comb plate differentiation begins to become localized in the aboral region of the embryo as a consequence of the first cleavage. This judgement does not agree with the conclusions reached by Driesch & Morgan (1895), Fischel (1903) and Yatsu (1912a) who all argued that there was some localization of comb plate-forming potential prior to the 2-cell stage.

Experiments analogous to those described here in which eggs were cut into parts prior to the initiation of the first cleavage have been done by Driesch & Morgan (1895) and Yatsu (1912a) on the egg of Beroë ovata. In Yatsu’s experiments eggs were cut either before or after polar body formation. Beroë ovata seems to differ from other ctenophores in that both polar bodies are reportedly given off after fertilization. The eggs cut prior to polar body formation could not be oriented; the size of the fragments produced was not indicated. Thirteen cases developed to the point where they could be analyzed; 11 formed eight rows of comb plates while two cases formed seven rows. In the experiments which were performed after polar body formation 12 fragments were produced. Three developed normally, two cases were missing from two to six rows of comb plates, while the other seven cases were classified as ‘more or less defective as Fischel, 1903 has found out’ (almost all of Fischel’s defective cases had eight rows of comb plates, but had a smaller number of plates in some rows). In his 1911 paper Yatsu indicates how these eggs were cut and presents diagrams showing the relative size of the fragments; however, he does not relate the origins of the egg fragments to the quality of the resulting larvae. Driesch & Morgan (1895) studied 16 eggs cut at random; the time of the operation with reference to the initiation of the first cleavage was not recorded. Six of their cases developed into normal larvae; eight had four normal rows on one side, and only one or two comb rows which were frequently rudimentary on the other side. In the last two cases, the egg was cut into a large and a small fragment and the small fragment cleaved; these did not form comb plate cilia. These experiments suggest that a change occurs in the Beroë egg during polar body formation which makes it more difficult for an egg fragment to develop normally. However, it is not clear that the change involves the localization of comb plate-forming potential at a particular site.

Eggs have also been cut into parts at the beginning of the first cleavage by Fischel (1903) and Zeigler (1898) working on Beroë and by Freeman & Reynolds (1973) working on Mnemiopsis. Fischel removed variable amounts of egg cytoplasm from the aboral region (six cases), while in six similar operations Zeigler, and Freeman & Reynolds removed up to 50 % of the egg cytoplasm. All of these cases developed into normal larvae. The experiments reported here confirm these findings. Fischel also removed variable amounts of cytoplasm from the oral region to one side of the developing furrow. He reports that this operation causes defects in comb plate differentiation. I have tried to repeat this experiment on P. pileus (Freeman, unpublished results). While I don’t feel that I have done enough operations to make a definitive statement, the results at hand do not indicate that the operation causes a decrease in the number of comb plates.

The fact that there is a moderate localization of comb plate-forming potential in the aboral region of the 2-cell stage blastomeres of P. pileus and P. bachei confirms the results of similar experiments which have been done on Mnemiopsis (Freeman, 1976). The failure to detect this localization prior to the 2-cell stage provides another line of evidence which is consistent with the argument that an oral-aboral axis does not exist in the embryo prior to this stage. The increase in comb plate-forming potential in the aboral region of the embryo after the first cleavage furrow has passed through that region, suggest that the process of cytokinesis itself, or the local cytoplasmic movements which accompany it may play a role in setting up this localization of developmental potential.

I feel that there has been a tendency on the part of developmental biologists to try to explain too many features of early development on the basis of some kind of hypothetical spatial organization which is supposed to be laid down during oogenesis. Clearly, there are many kinds of animal eggs in which some kind of promorphological organization exists. However, this study and published work on spiralians (Tadano, 1962; Guerrier, 1968) show that eggs from some groups of animals have essentially no promorphological organization at the end of oogenesis and acquire this organization only as a consequence of the initiation of embryonic development. The Fucus egg has been one of the main models used in analyzing the intracellular events that accompany the development of polarity in an unstructured system (Quatrano, 1973). The eggs of ctenophores and certain spiralians may be excellent material for similar studies on animal cells.

This work was supported by Grant GM 20024-03 from the National Institutes of Health. I want to thank Dr Dennis Willows, the director of the Friday Harbor Laboratories, for facilitating my work there, and Drs Antone Jacobson and Helen Pianka for reading this manuscript.

Driesch
,
H.
&
Morgan
,
T.
(
1895
).
Zur Analysis der ersten Entwicklungsstadien des Ctenophoreneies. II. Von der Entwickelung ungefurchter Eier mit Protoplasmadefkten
.
Arch. EntwMech. Org.
2
,
216
224
.
Dunlap
,
H.
(
1966
).
Oogenesis in the Ctenophora. Ph.D. thesis, University of Washington, Seattle
.
Fischel
,
A.
(
1903
).
Entwicklung und Organdifferenzierung
.
Arch. EntwMech. Org.
15
,
679
-
750
.1
Freeman
,
G.
(
1976
).
The role of cleavage in the localization of developmental potential in the ctenophore Mnemiopsis leidyi.
Devi Biol.
49
,
143
177
.
Freeman
,
G.
&
Reynolds
,
G.
(
1973
).
The development of bioluminescence in the ctenophore Mnemiopsis leidyi.
Devi Biol.
31
,
61
100
.
Guerrier
,
P.
(
1968
).
Origine et stabilité de la polarité animale végétative chez quelques spiralia
.
Annls Embryol. Morph.
1
,
119
139
.
Guerrier
,
P.
(
1971
).
La polarisation cellulaire et les caracteres de la segmentation au cours de la morphogenese spirale
.
Année Biol. Fr.
10
,
151
192
.
Komai
,
T.
(
1922
).
Studies on two aberrant ctenophores Coeloptana and Gastrodes.
Kyoto
:
privately published
.
Korschelt
,
E.
(
1936
).
Vergleichende Entwicklungsgeschichte der Tiere.
Jena
:
Gustav Fischer
.
Kozloff
,
E.
(
1974
).
Keys to the Marine Invertebrates of Puget Sound, the San Juan Archipelago and Adjacent Regions.
Seattle
:
University of Washington Press
.
Morgan
,
T.
(
1927
).
Experimental Embryology.
New York
:
Columbia University Press
.
Morgan
,
T.
&
Spooner
,
G.
(
1909
).
The polarity of the centrifuged egg
.
Arch. EntwMech. Org.
28
,
104
117
.
Pianka
,
H.
(
1974
).
Ctenophora
. In
Reproduction of Marine Invertebrates, vol. 1
(ed.
A.
Giese
&
J.
Pearse
), pp.
201
265
.
New York
:
Academic Press
.
Quatrano
,
R.
(
1973
).
Separation of processes associated with differentiation of two-celled Fucus embryos
.
Devi Biol.
30
,
209
213
.
Reverberi
,
G.
(
1971
).
Ctenophores
. In
Experimental Embryology of Marine and Freshwater Invertebrates
(ed.
G.
Reverberi
), pp.
85
103
.
Amsterdam
:
North-Holland
.
Reverberi
,
G.
&
Ortolani
,
G.
(
1963
).
On the origin of ciliated plates and the mesoderm of ctenophores
.
Acta Embryol. Morph, exp.
6
,
175
190
.
Schleip
,
W.
(
1929
).
Die Determination der Primitiventwicklung.
Leipzig
:
Academische Verlaggesellschaft
.
Tadano
,
M.
(
1962
).
Artificial inversion of the primary polarity in Ascaris eggs
.
Jap. J. Zool.
13
,
229
255
.
Wilson
,
E.
(
1925
).
The Cell in Development and Heredity, 3rd
ed.
New York
:
Macmillan
.
Yatsu
,
N.
(
1911
).
Observations and experiments on the ctenophore egg: II. Notes on early cleavage stages and experiments on cleavage
.
Annotnes zool. Japon.
7
,
333
346
.
Yatsu
,
N.
(
1912a
).
Observations and experiments on the ctenophore egg: Ill. Experiments on germinal localization of the egg of Beroë ovata
.
Annotnes zool. Japon.
8
,
5
13
.
Yatsu
,
N.
(
1912b
).
Observations and experiments on the ctenophore egg: I. The structure of the egg and experiments on cell division
.
J. Coll. Sci. imp. Univ. Tokyo
32
,
1
21
.
Zeigler
,
H.
(
1898
).
Experimentelle Studien fiber Zelltheilung. III. Die Furchungszellen von Beroë ovata
.
Arch. EntwMech. Org.
7
,
34
64
.
1

A verbatim English translation of this paper is available on request.