The dorsoventral axis is specified prior to first cleavage in the direct developing sea urchin Heliocidaris erythrogramma

Previous fate mapping studies as well as the culture of isolated blastomeres have revealed that the dorsoventral axis is specified as early as the 2-cell stage in the embryos of the direct developing echinoid, Heliocidaris erythrogramma. Normally, the first cleavage plane includes the animal-vegetal axis and bisects the embryo between future dorsal and ventral halves. Experiments were performed to establish whether the dorsoventral axis is set up prior to the first cleavage division in H. erythrogramma. Eggs were elongated and fertilized in silicone tubes of a small diameter in order to orient the cleavage spindle and thus the first plane of cell division. Following first cleavage, one of the two resulting blastomeres was then microinjected with a fluorescent cell lineage tracer dye. Fate maps were made after culturing these embryos to larval stages. The results indicate that the first cleavage division can be made to occur at virtually any angle relative to the animal-vegetal and dorsoventral axes. Therefore, the dorsoventral axis is specified prior to first cleavage. We argue that this axis resides in the unfertilized oocyte rather than being set up as a consequence of fertilization.     

Allocation of cells in mouse blastocyst is not determined by the order of cleavage of the first two blastomeres

The second cleavage of the mouse embryo is asynchronous. Some recent investigators have proposed that the sequence of division of blastomeres in two-cell embryos may predict the ultimate location of the descendants of these blastomeres within the blastocyst. To verify this model, we tracked the cells derived from two-cell stage blastomeres using tetramethylrhodamine-conjugated dextran as a lineage tracer. In the first variant of the experiment, we labeled one of two blastomeres in two-cell embryos and subsequently recorded which blastomere cleaved first. In the second variant of the experiment, fluorescent dextran was injected at the three-cell stage into the blastomere that had not yet cleaved. Subsequently, the fate of the progeny of labeled and unlabeled blastomeres was followed up to the blastocyst stage. Our results suggest that allocation of cells into the embryonic and abembryonic parts of the blastocyst is not determined by the order of cleavage of the first two blastomeres.     

Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development

Two independent studies have recently suggested similar models in which the embryonic and abembryonic parts of the mouse blastocyst become separated already by the first cleavage division. However, no lineage tracing studies carried out so far on early embryos provide the support for such a hypothesis. Thus, to re-examine the fate of blastomeres of the two-cell mouse embryo, we have undertaken lineage tracing studies using a non-perturbing method. We show that two-cell stage blastomeres have a strong tendency to develop into cells that comprise either the embryonic or the abembryonic parts of the blastocyst. Moreover, the two-cell stage blastomere that is first to divide will preferentially contribute its progeny to the embryonic part. Nevertheless, we find that the blastocyst embryonic-abembryonic axis is not perfectly orthogonal to the first cleavage plane, but often shows some angular displacement from it. Consequently, there is a boundary zone adjacent to the interior margin of the blastocoel that is populated by cells derived from both earlier and later dividing blastomeres. The majority of cells that inhabit this boundary region are, however, derived from the later dividing two-cell stage blastomere that contributes predominantly to the abembryonic part of the blastocyst. Thus, at the two-cell stage it is already possible to predict which cell will contribute a greater proportion of its progeny to the abembryonic part of the blastocyst (region including the blastocyst cavity) and which to the embryonic part (region containing the inner cell mass) that will give rise to the embryo proper.     

The Establishment of Embryonic Axial Properties in the Nemertean,Cerebratulus lacteus

Experiments were performed to determine whether the first cleavage division plays a role in setting up the dorsoventral axis in embryos of the equal-cleaving nemerteanCerebratulus lacteus.Fertilized eggs were compressed to change the orientation of the first cleavage spindle, and thus the plane of the first cleavage division. One cell of the resulting two-celled embryos was then injected with lineage tracer to determine whether the first cleavage plane always maintains its normal relationships to the median and frontal planes or whether new relationships (and thus, novel cell lineages) could be created. Many of these compressed embryos gave rise to normal-appearing pilidium larvae in which the first cleavage plane had taken on various oblique angular relationships relative to the plane of bilateral symmetry and the dorsoventral axis of the larva. These findings indicate that the first cleavage plane can be dissociated from its normal relationships to these axial properties. Thus, the first cleavage division is not causally involved in the establishment of the dorsoventral and bilateral axes. We argue that the dorsoventral axis is specified prior to the first cleavage division.     

Establishment of the dorsoventral axis in nemertean embryos: Evolutionary considerations of spiralian development

The Nemertea represent one of a number of invertebrate phyla that display a highly conserved pattern of cell division known as spiral cleavage. The fates of the early blastomeres are known for representatives of some spiralian phyla (i.e., molluscs and annelids) and in these species there appears to be a high degree of conservation in the ultimate fates of particular embryonic cells. The first two cleavage planes bear an invariant relationship to the symmetry properties of the future larval and adult body plan. To investigate whether these properties of spiralian embryo-genesis are shared (conserved) amongst members of other spiralian phyla, individual blastomeres in two- and four-cell embryos of the nemertean, Nemertopsis bivittata, were microinjected with bi-otinylated dextran lineage tracers. N. bivittata is a direct-developing hoplonemertean that forms a nonfeeding larva. When individual blastomeres are injected at the two-cell stage, two sets of complementary labeling patterns (a total of four different patterns) were observed in the ectoderm of the larvae. When cells were injected at the four-cell stage, four different patterns were observed that represented subsets of the four patterns observed in the previous experiment. Unlike the case in the annelids and molluscs, in which the first cleavage plane bears a strict 45° angular relationship to the future dorsoventral axis, the first cleavage plane in N. bivittata can bear one of two different relationships relative to the larval/adult dorsoventral axis. In half the cases examined, the first cleavage plane corresponded roughly to the plane of bilateral symmetry, and in the rest, it lay along a frontal plane. A similar result was observed for the embryos of the indirect-developing heteronemertean, Cerebratutus lacteus. These results indicate that the fates of the four cell quadrants in nemerteans are not directly homologous to those in other spira-lians, such as the annelids and molluscs. For instance, no single cell quadrant appears to contribute a greater share to the formation of ectoderm, as is the case in the formation of the post-trochal region by the D-cell quadrant in annelids and mol-luscs. Rather, two adjacent cell quadrants contribute nearly equally to the formation of dorsal or ventral ectoderm in the larvae. Possible explanations for the determination of dorsoventrality in nemerte-ans, as well as implications of these findings regarding the evolution of spiralian development, are discussed. © 1994 Wiley-Liss, Inc.     

Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591-5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.     

Deuterostome evolution: early development in the enteropneust hemichordate, Ptychodera flava

SUMMARY Molecular and morphological comparisons indicate that the Echinodermata and Hemichordata represent closely related sister‐phyla within the Deuterostomia. Much less is known about the development of the hemichordates compared to other deuterostomes. For the first time, cell lineage analyses have been carried out for an indirect‐developing representative of the enteropneust hemichordates, Pty‐ chodera flava. Single blastomeres were iontophoretically labeled with DiI at the 2‐ through 16‐cell stages, and their fates followed through development to the tornaria larval stage. The early cleavage pattern of P. flava is similar to that of the direct‐developing hemichordate, Saccoglossus kowalevskii, as well as that displayed by indirect‐developing echinoids. The 16‐celled embryo contains eight animal “mesomeres,” four slightly larger “macromeres,” and four somewhat smaller vegetal “micromeres.” The first cleavage plane was not found to bear one specific relationship relative to the larval dorsoventral axis. Although individual blastomeres generate discrete clones of cells, the appearance and exact locations of these clones are variable with respect to the embryonic dorsoventral and bilateral axes. The eight animal mesomeres generate anterior (animal) ectoderm of the larva, which includes the apical organ; however, contributions to the apical organ were found to be variable as only a subset of the animal blastomeres end up contributing to its formation and this varies from embryo to embryo. The macromeres generate posterior larval ectoderm, and the vegetal micromeres form all the internal, endomesodermal tissues. These blastomere contributions are similar to those found during development of the only other hemichordate studied, the direct‐developing enteropneust, S. kowalevskii. Finally, isolated blastomeres prepared at either the two‐ or the four‐cell stage are capable of forming normal‐appearing, miniature tornaria larvae. These findings indicate that the fates of these cells and embryonic dorsoventral axial properties are not committed at these early stages of development. Comparisons with the developmental programs of other deuterostome phyla allow one to speculate on the conservation of some key developmental events/mechanisms and propose basal character states shared by the ancestor of echinoderms and hemichordates.     

The effects of altering the position of cleavage planes on the process of localization of developmental potential in ctenophores.

During the transition from the four- to the eight-cell stage in ctenophore embryos, each blastomere produces one daughter cell with the potential to form comb plate cilia and one daughter cell that does not have this potential. If the second cleavage in a two-cell embryo is blocked, at the next cleavage these embryos frequently form four blastomeres which have the configuration of the blastomeres in a normal eight-cell embryo. At this division there is also a segregation of comb plate-forming potential. By compressing a two-cell embryo in a plane perpendicular to the first plane of cleavage it is possible to produce a four-cell blastomere configuration that is identical to that produced following the inhibition of the second cleavage. However, under these circumstances the segregation of comb plate potential does not occur. These results suggest that the appropriate plane of cleavage must take place for a given cleavage cycle, in order for localizations of developmental potential to be properly positioned within blastomeres.     

Unbiased contribution of the first two blastomeres to mouse blastocyst development

Several recent studies have proposed a model that the organization of the mouse blastocyst is determined by the pattern of early cleavages: the plane of first cleavage divides the two-cell embryo into embryonic (Em) and abembryonic (Ab) halves, while the timing of the second cleavages specifies which blastomere becomes the Em half. This model is still controversial because of conflicting observations in various studies. Here, we investigated the possibility that the difference between mouse strains contributed to the discrepancy of the findings of different experiments regarding the relationship between the first two cleavages and the blastocyst axial pattern. First, we showed by using a lipophilic, fluorescent tracer that the plane of the first cleavage bears no consistent spatial relationship to the Em-Ab axis of the blastocyst regardless of the genotypic background. Secondly, the order of the second cleavage does not correlate with the Em-Ab polarity of the blastocyst. This was demonstrated by tracing the lineage of the early- and later-dividing two-cell stage blastomeres in the whole embryo as well as by comparing the developmental potential of isolated early- and later-dividing blastomeres and chimeras made entirely of early- or later-dividing blastomeres. These results suggest that contrary to recent studies, the differences between the early- and later-dividing blastomeres of the two-cell embryo are not functionally evident and do not define the Em-Ab polarity of the blastocyst. The significance of these findings is discussed in relation to human assisted reproduction and preimplantation genetic diagnosis.     

Experimental change of cytoplasmic composition can convert determination of blastomeres inPlatynereis dumerilii (Annelida,Polychaeta)

Summary Early development ofPlatynereis dumerilii is characterized by an extremely constant cleavage pattern in which the volumes and cytoplasmic contents of the blastomeres show remarkably little variability (Dorresteijn 1990). In order to test the necessity of a precise partitioning of the cytoplasm, we have stratified the ooplasm by mild centrifugation (10 min at 300 g) after completion of meiosis but before first cleavage. The cytoplasm of the zygote stratifies randomly with respect to the pre-existing animal-vegetal axis, but first cleavage follows the animal-vegetal axis dividing the plasm before it has rearranged to its normal distribution. As usual, first cleavage is unequal in the majority of centrifuged eggs. Different sorts of cytoplasm are always distributed abnormally in comparison to normal two-cell stages. Under two circumstances this leads to the formation of double trunk structures in the young worm. Such double monsters either originate from zygotes whose clear cytoplasm has been distributed equally to the two daughter blastomeres at first cleavage, or from unequal two-cell embryos whose larger blastomere cleaved equally at second cleavage forming blastomeres with equal lots of clear cytoplasm. Cell-lineage could be followed in an individual embryo of the latter category and showed the existence of two adjacent D-quadrants, giving rise to a double monster with a forked trunk. Embryos of the former category give rise to two opponent D-quadrants and double monsters with a four-sided trunk. As in the normal embryo, the amount of clear cytoplasm in a blastomere is positively correlated with the speed of its cell cycle, and endows the cell with D-quadrant developmental capacities as can be judged by the cleavage pattern.     

Reprograming of murine blastocoele formation

The present study shows that there is communication between reaggregated asynchronous cleavage stage blastomeres that regulates blastocoele formation. Individual blastomeres from eight-cell murine embryos were transferred to empty zonae pellucidae, intact two-cell embryos, or enucleated two-cell embryos, and were examined over a period of 75 hours for development of cavitation. It was found that the isolated blastomeres cavitated concurrently with intact control eight-cell embryos, while intact control two-cell embryos cavitated 24 hours later. However, the embryos resulting from combining a two-cell embryo and a blastomere from an eight-cell embryo cavitated at a time in between the eight- and two-cell controls.     

Archenteron-Forming Capacity in Blastomeres Isolated from Eight-Cell Stage Embryos of the Starfish, Asterina pectinifera

Starfish blastomeres are reported to be totipotent up to the 8-cell stage. We reinvestigated the development of blastomeres of 8-cell stage embryos with a regular cubic shape consisting of two tiers of 4 blastomeres. On dissociation of the embryo by disrupting the fertilization membrane at the 8-cell stage, each of the 4 blastomeres of the vegetal hemisphere gave rise to an embryo that gastrulated, whereas blastomeres from the animal hemisphere did not. By injection of a cell lineage tracer into blastomeres of 8-cell stage embryos, we found that only those of the vegetal hemisphere formed cells constituting the archenteron. Next, we compressed 4-cell stage embryos along the animal-vegetal axis so that all the blastomeres in the 8-cell stage were in a single layer. When these 8 blastomeres were then dissociated, an average of 7 of them developed into gastrulae. By cell lineage analysis, all the blastomeres in single-layered embryos at the 8-cell stage were shown to have the capacity to form cells constituting an archenteron. Taken together, these findings indicate that the fate to form the archenteron is specified by a cytoplasmic factor(s) localized at the vegetal hemisphere, and that isolated blastomeres that have inherited this factor develop into gastrulae.     

Spatial alignment of the mouse blastocyst axis across the first cleavage plane is caused by mechanical constraint rather than developmental bias among blastomeres

The embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst has been found in several studies to align orthogonal to the first cleavage plane, raising the possibility that a developmental prepattern already exists at the two-cell stage. However, it is also possible that such alignment is not due to any developmental disparity between the two-cell stage blastomeres, but rather is caused by an extrinsic mechanical constraint that is conferred by an irregular shape of the zona pellucida (ZP). Here, we conducted a series of experiments to distinguish between these possibilities. We showed that the shape of the ZP at the two-cell stage varied among embryos, ranging from near spherical to ellipsoidal, and that the ZP shape did not change until the blastocyst stage. In those embryos with an ellipsoidal ZP, the Em-Ab axis tended to lie orthogonal to the first cleavage plane, while in those embryos with a near spherical ZP, there was no such relationship. The clonal boundary between the descendants of the two-cell stage blastomeres tended to lie orthogonal to the Em-Ab axis when the rotation of the embryo within the ZP was experimentally prevented, while the control embryos did not exhibit such tendency. These results support the possibility that an apparent correlation between the first cleavage plane and the blastocyst axis can be generated by the mechanical constraint from the ZP but not by a developmental prepattern. Moreover, recent reports indicate that the vegetal blastomere of the four-cell stage embryo that had undergone a specific type of second cleavages is destined to contribute to the abembryonic side of the blastocyst. However, our present study shows that in spite of such specific second cleavages, the vegetal blastomere did not preferentially give rise to the abembryonic side. This result implicates that the lineage of the four-cell stage blastomere is not restricted even when embryos undergo a specific type of second cleavages.     

Morphogenetic tissue movement and the establishment of body plan during development from blastocyst to gastrula in the mouse

In many animal species, the early development of the embryo follows a stereotypic pattern of cell cleavage, lineage allocation and generation of tissue asymmetry leading to delineation of the body plan with three primary embryonic axes. The mammalian embryo has been regarded as an exception and primary body axes of the mouse embryo were thought to develop after implantation. However, recent findings have challenged this view. Asymmetry in the fertilised oocyte, as defined by the position of the second polar body and the sperm entry point, can be correlated with the orientation of the animal-vegetal and the embryonic-abembryonic axes in the preimplantation blastocyst. Studies of the pattern of morphogenetic movement of cells and genetic activity in the peri-implantation embryo suggest that the animal-vegetal axis of the blastocyst might presage the orientation of the anterior-posterior axis of the gastrula. This suggests that the asymmetry of the zygote that is established at fertilisation and early cleavage has a lasting impact on the delineation of body axes during embryogenesis.     

Prospective fate of early blastomeres in Hydractinia echinata (Cnidaria,Hydrozoa)

Summary Blastomeres of two-cell, four-cell, and eight-cell embryos of Hydractinia echinata were injected with horseradish-peroxidase (HRP) or fluorescein isothiocyanate (FITC)-dextran. The fate of the descendants of the injected blastomeres was followed until the planula larva had developed. The results obtained after HRP or FITC-dextran injection were essentially the same. Blastomeres are equivalent up to the four-cell stage, i.e. half-blastomeres produce half of the ectoderm of the planula larva and quarter-blastomeres give rise to one quarter of the larval ectoderm. During normal embryogenesis, the larval anterior-posterior axis corresponds to the animal-vegetal axis of the zygote. Thus, the labelled areas of larvae consisting of the progeny of injected half or quarter blastomeres normally stretch along the larval anterior-posterior axis. Normally, material giving rise to anterior or posterior larval parts, respectively, is separated at the third cleavage. Irrespective of the type of experiment, the progeny of injected blastomeres always contributed to endoderm formation, i.e. in larvae resulting from injected embryos the endoderm was more or less uniformly labelled. Application of vital stains locally to the exterior of zygotes and following these markers through first and second cleavage, produced evidence that in the vast majority of cases, the second cleavage is meridional. Offprint requests to: A. Schlawny     

Multiple,alternative cleavage patterns precede uniform larval morphology during normal development of Dreissena polymorpha (Mollusca,Lamellibranchia)

In this study we reinvestigate the early development of the freshwater mussel Dreissena polymorpha, previously studied by Meisenheimer (1901). The data include video time-lapse recordings of living embryos and bisbenzimide stains of fixed embryos as well as morphometry on fixed, serially-sectioned embryos. We present the cell lineage and cell cycle durations up to the first indication of symmetrization within this embryo. We show that early cell cycles last approximately 1h. A dramatic extension of cell cycle duration and a concomitant asynchrony among the various cell lines was observed starting at the fifth cleavage. Short cell cycles, like those of early blastomeres, were a constant property of the largest descendants of the 2d-cell line only. In contrast to Meisenheimer's observations and our experiences with other spiralian embryos, the cleavage pattern proved to follow multiple alternatives. The embryonic quadrants A-D were arranged in either a clockwise or counter-clockwise fashion and the chirality of the third cleavage was either dextral or sinistral irrespective of the arrangement of the quadrants. As a consequence, four different blastomere configurations were encountered and the dorsoventral axis could take four different angles with respect to the plane of first cleavage. The dorsal side was most easily recognized by the position of the 2d-micromere at the 16-cell stage. The fact that all of such embryos could develop into normal, uniform larvae is interpreted as the result of cell-cell interactions in morphogenetic regulation.     

Determinative development in the polyclad turbellarian,Hoploplana inquilina

Summary

Blastomere deletion experiments at the two- and four-cell stages were carried out on the embryo of the polyclad turbellarian Hoploplana inquilina to further examine the relationship between spiral cleavage and early embryonic determination in primitive spiralians. Deletion of one cell at the two-cell stage resulted in “half” larvae that were abnormal in body shape, lobe development, and behavior. Deletion of one cell at the four-cell stage produced less abnormal “three-quarter” larvae which were still underdeveloped in one of the quadrants. A 3:1 ratio of one-eyed to two-eyed larvae implies that deletion of any one of three blastomeres results in loss of an eye, with two constituting the eye lineage and the third controlling the development of two eyes. The results demonstrate that the polyclad embryo is determined early in development, though significant cell interactions occur during cleavage, and suggest that determinative development and quartet spiral cleavage are always associated and probably represent a primitive, strongly conserved evolutionary condition.     

A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida,Polychaeta)

Summary Early development of Platynereis massiliensis was studied in serial sections of fixed embryos and in living or fixed embryos whose nuclei had been made visible with a fluorescent label. The unfertilized egg is an ellipsoid with three axes of differing length. The longest axis corresponds to the dorsoventral axis of the developing embryo. Egg volume is ten times that in the sibling species, P. dumerilii, mainly due to increased yolk content. The timing and spatial pattern of cleavage were observed from first cleavage to the 62-cell stage. Volumes of the blastomeres, their nuclei, their yolk-free cytoplasm and their yolk were determined from serial sections up to the 29-cell stage. In the P. massiliensis embryo, cell cycles are on average 3.7 times longer than in P. dumerilii; volume proportions among the blastomeres also differ and the macromeres containing the bulk of yolk are particularly large, but otherwise the cleavage patterns, differential segregation of yolk and yolk-free cytoplasm, and the histogenetic fates of the blastomeres are the same as in P. dumerilii. This equivalence of cell lineage and of cytoplasmic segregation mechanisms in both species, maintained in spite of the different appearance of the embryos, suggests functional importance of and selective constraint on these developmental features. The relatively accelerated divisions of the 2d cell line in P. massiliensis may be interpreted as the precocious development of cell lines which give rise to adult structures. Several structures, obviously functional in developing P. dumerilii, have lost their function in P. massiliensis: the egg contains few cortical granules, giving rise to only a moderate egg jelly layer in the zygote; prototroch cells develop cilia, but the heavy embryo is unable to swim; the larva develops three pairs of parapodia but, unlike the corresponding stage in P. dumerilii, is not capable of coordinate locomotion. This loss of motility is related to the brooding habit of the species developing inside the parental tube and is explained as the result of a switch from pelagic to benthic, protected reproduction in P. massiliensis. Offprint requests to: A.W.C. Dorresteijn     

LOCALIZATION AND SEGREGATION OF MATERNAL RNA'S DURING EARLY CLEAVAGE OF XENOPUS LAEVIS EMBRYOS

The localization and segregation of maternal RNA's during early cleavage of Xenopus laevis embryos were studied. Blastomeres and hemispheres of eggs and early embryos were separated manually and the amounts of ribosomal RNA and poly(A) ⁺RNA extracted from each blastomere and hemisphere were determined by optical density measurement and by ³H-poly(U) hybridization, respectively. It was found that both kinds of the maternal RNA's were more abundant (two-thirds of the total) in the animal hemisphere (cells), while they were evenly distributed between the dorsal and ventral halves. This pattern of localization remained unchanged from the egg to the blastula stage, indicating that these maternal RNA's were segregated into blastomeres quite simply by cell division. Gel electrophoresis showed that the size distributions of poly(A) ⁺RNA and poly(A) sequences obtained from different blastomeres of 8-cell embryos did not differ greatly. It was also found that cytoplasmic polyadenylation of maternal RNA, which occurs during early cleavage and blastulation, took place equally in all regions of the cleaving embryos, suggesting no regional difference in the localization of maternally inherited nonpolyadenylated RNA. These observations are discussed in relation to previous findings on differences along the animal-vegetal and dorsal-ventral axes of the early amphibian embryo.     

Dyssymmetrical cytotomy and orientation of the spindles in early isolated blastomeres of gastropod molluscs]

While removing the vitelline membrane mechanically or by trypsinization, one or two blastomeres were isolated from two-, four- and eight-celled embryos Lymnaea stagnalis, Physa fontinalis and Ph. acuta. If a pair of blastomeres was isolated prior to the formation of the III and IV cleavage spindles, it became similar to a two-cell embryo; the spindles oriented in parallel to each other and the contact zone of blastomeres preserving the previous localization of the nuclei and the subsequent division was equal. If a pair of blastomeres was isolated in metaphase, the localization of spindles did not change and the relative size of sister blastomeres after the subsequent division resembled the normal size (in particular, the III division was unequal). In the course of division of isolated single blastomeres, as well as pairs of blastomeres with parallel spindles, mutual turns of sister cells along the plane of new furrow, always dexiotropic in Lymnaea and laetropic in Physa, were observed in all cycles. The ability of dissymmetrical invariant turns during cytotomy, shown earlier when studying the whole embryos, is, thus, inherent to each blastomere irrespective of the neighbour ones but is supressed during the normal development after the III division mechanically due to the dense cellular packing projected by the orientation of spindles.