C.elegans野生型和par突变体早期胚胎卵裂的观察

不对称分裂在动植物的发育中起到了非常重要的作用。Caenorhabditis elegans（C．elegans）胚胎最早的两次卵裂为研究控制不对称分裂的机制提供了很好的机会。用普通光学显微镜观察了野生型胚胎早期卵裂和par-1、par-2、par-3、par-4突变体胚胎的早期卵裂。野生型胚胎最早的分裂是不等的,产生了两个不同大小的子细胞。两个子细胞又以不同的方向进行第二次分裂。在C．elegans中任意一个par基因的缺失会使胚胎的第一次卵裂丧失不对称性。这会导致一些发育调控因子不能在特定的胚胎细胞中准确地定位,造成细胞分裂纺锤体方向的异常。par类基因参与不对称性的建立,这种不对称性决定了C．elegans身体的前后轴。     

Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo

In the unperturbed development of the mouse embryo one of the 2-cell blastomeres tends to contribute its progeny predominantly to the embryonic and the other to the abembryonic part of the blastocyst. However, a significant minority of embryos (20-30%) do not show this correlation. In this study, we have used non-invasive lineage tracing to determine whether development of blastocyst pattern shows any correlation with the orientation and order of the second cleavage divisions that result in specific positioning of blastomeres at the 4-cell stage. Although the orientation and order of the second cleavages are not predetermined, in the great majority (80%) of embryos the spatial arrangement of 4-cell blastomeres is consistent with one of the second cleavages occurring meridionally and the other equatorially or obliquely with respect to the polar body. In such cleaving embryos, one of the 2-cell stage blastomeres tends to contribute to embryonic while the other contributes predominantly to abembryonic part of the blastocyst. Thus, in these embryos the outcome of the first cleavage tends to correlate with the orientation of the blastocyst embryonic-abembryonic axis. However, the order of blastomere divisions predicts a specific polarity for this axis only when the earlier 2-cell blastomere to divide does so meridionally. In contrast to the above two groups, in those embryos in which both second cleavage divisions occur in a similar orientation, either meridionally or equatorially, we do not observe any tendency for the 2-cell blastomeres to contribute to specific blastocyst parts. We find that all these groups of embryos develop to term with similar success, with the exception of those in which both second cleavage divisions occur equatorially whose development can be compromised. We conclude that the orientations and order of the second cleavages are not predetermined; they correlate with the development of blastocyst patterning; and that the majority, but not all, of these cleavage patterns allow equally successful development.     

The second polar lobe of theSabellaria cementarium embryo plays an inhibitory role in apical tuft formation

Summary The embryo ofSabellaria cementarium (Polychaeta) forms a polar lobe at each of the first two cleavage divisions which becomes absorbed into one of the blastomeres at the end of the division. Lobe removal experiments show that the polar lobe preceding first cleavage is necessary for the development of the apical tuft and the posttrochal region of the trochophore larva. The polar lobe preceding second cleavage is smaller than the first polar lobe and is necessary only for post-trochal region development. In blastomere isolation experiments, isolates containing the C but not the D blastomere form apical tufts. Isolates containing the D but not the C blastomere do not form apical tufts. When the polar lobe preceding second cleavage is removed and the C and D blastomeres are separated and raised in isolation, each can form an apical tuft. When the second cleavage is equalized with sodium dodecyl sulfate (SDS) such that both the C and the D blastomeres receive second polar lobe material, no apical tuft is formed. These results suggest that apical tuft determinants are distributed to both the C and D blastomeres at second cleavage but that the second polar lobe contains an inhibitor for apical tuft formation which is shunted to the D blastomere after the completion of second cleavage.     

The pie-1 and mex-1 genes and maternal control of blastomere identity in early C. elegans embryos.

During C. elegans embryogenesis an 8-cell stage blastomere, called MS, undergoes a reproducible cleavage pattern, producing pharyngeal cells, body wall muscles, and cell deaths. We show here that maternal-effect mutations in the pie-1 and mex-1 genes cause additional 8-cell stage blastomeres to adopt a fate very similar to that of the wild-type MS blastomere. In pie-1 mutants one additional posterior blastomere adopts an MS-like fate, and in mex-1 mutants four additional anterior blastomeres adopt an MS-like fate. We propose that maternally provided pie-1(+) and mex-1(+) gene products may function in the early embryo to localize or regulate factors that determine the fate of the MS blastomere.     

Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions

Setting aside pluripotent cells that give rise to the future body is a central cell fate decision in mammalian development. It requires that some blastomeres divide asymmetrically to direct cells to the inside of the embryo. Despite its importance, it is unknown whether the decision to divide symmetrically versus asymmetrically shows any spatial or temporal pattern, whether it is lineage-dependent or occurs at random, or whether it influences the orientation of the embryonic-abembryonic axis. To address these questions, we developed time-lapse microscopy to enable a complete 3D analysis of the origins, fates and divisions of all cells from the 2- to 32-cell blastocyst stage. This showed how in the majority of embryos, individual blastomeres give rise to distinct blastocyst regions. Tracking the division orientation of all cells revealed a spatial and temporal relationship between symmetric and asymmetric divisions and how this contributes to the generation of inside and outside cells and thus embryo patterning. We found that the blastocyst cavity, defining the abembryonic pole, forms where symmetric divisions predominate. Tracking cell ancestry indicated that the pattern of symmetric/asymmetric divisions of a blastomere can be influenced by its origin in relation to the animal-vegetal axis of the zygote. Thus, it appears that the orientation of the embryonic-abembryonic axis is anticipated by earlier cell division patterns. Together, our results suggest that two steps influence the allocation of cells to the blastocyst. The first step, involving orientation of 2- to 4-cell divisions along the animal-vegetal axis, can affect the second step, the establishment of inside and outside cell populations by asymmetric 8- to 32-cell divisions.     

Asymmetrical rotations of blastomeres in early cleavage of gastropoda

Summary The movements of blastomere surfaces marked with carbon particles during cytokinesis of the Ist–IVth cleavage divisions in the eggs of the gastropodsLymnaea stagnalis, L. palustris, Physa acuta and Ph. fontinalis have been studied by time-lapse cinematographic methods. The vitelline membrane was removed with trypsin. At 2- and 4-cell stages shifts of nuclei have also been studied.Symmetrical as well as asymmetrical surface movements (in respect to the furrow plane) have been revealed. Symmetrical surface movements at the beginning of cytokinesis consist mainly in contraction of the furrow zone and in expansion of the more peripheral regions; between these there is a stationary zone. After the end of cytokinesis the furrow region expands.Considerableasymmetrical surface movements have also been observed in all four divisions. From anaphase until the end of cytokinesis each of the two sister blastomeres rotates with respect to the other in such a way, that if viewed along the spindle axis, the blastomere nearest to the observer rotates dexiotropically in a dextral species and laeotropically in a sinistral species (primary rotations). After the completion of cytokinesis the blastomeres may rotate in a reverse direction. The latter rotations are less pronounced in the IInd and IIIrd divisions and most pronounced in the IVth division. Blastomeres with the vitelline membrane intact retain a slight capacity for primary rotations. In normal conditions nuclei of the first two blastomeres shift mainly laeotropically in dextral species, but dexiotropically in sinistral species, being carried along by the reverse surface rotations.The invariable primary asymmetrical rotations of blastomeres seem to be the basis of enantiomorphism in molluscan cleavage. They are assumed to be determined by an asymmetrical structure of the contractile ring carrying out the cytokinesis.     

D quadrant specification in the leech Helobdella: Actomyosin contractility controls the unequal cleavage of the CD blastomere

The unequal division of the CD blastomere at second cleavage is critical in establishing the second embryonic axis in the leech Helobdella, as in other unequally cleaving spiralians. When CD divides, the larger D and smaller C blastomeres arise invariantly on the left and right sides of the embryo, respectively. Here we show that stereotyped cellular dynamics, including the formation of an intercellular blastocoel, culminate in a morphological left-right asymmetry in the 2-cell embryo, which precedes cytokinesis and predicts the chirality of the second cleavage. In contrast to the unequal first cleavage, the unequal second cleavage does not result from down-regulation of one centrosome, nor from an asymmetry within the spindle itself. Instead, the unequal cleavage of the CD cell entails a symmetric mitotic apparatus moving and anisotropically growing rightward in an actomyosin-dependent process. Our data reveal that mechanisms controlling the establishment of the D quadrant differ fundamentally even among the monophyletic clitellate annelids. Thus, while the homologous spiral cleavage pattern is highly conserved in this clade, it has diverged significantly at the level of cell biological mechanisms. This combination of operational conservation and mechanistic divergence begins to explain how the spiral cleavage program has remained so refractory to change while, paradoxically, accommodating numerous modifications throughout evolution.     

The Caenorhabditis elegans par-5 gene encodes a 14-3-3 protein required for cellular asymmetry in the early embryo.

The establishment of anterior-posterior polarity in the Caenorhabditis elegans embryo requires the activity of the maternally expressed par genes. We report the identification and analysis of a new par gene, par-5. We show that par-5 is required for asynchrony and asymmetry in the first embryonic cell divisions, normal pseudocleavage, normal cleavage spindle orientation at the two-cell stage, and localization of P granules and MEX-5 during the first and subsequent cell cycles. Furthermore, par-5 activity is required in the first cell cycle for the asymmetric cortical localization of PAR-1 and PAR-2 to the posterior, and PAR-3, PAR-6, and PKC-3 to the anterior. When PAR-5 is reduced by mutation or by RNA interference, these proteins spread around the cortex of the one-cell embryo and partially overlap. We have shown by sequence analysis of par-5 mutants and by RNA interference that the par-5 gene is the same as the ftt-1 gene, and encodes a 14-3-3 protein. The PAR-5 14-3-3 protein is present in gonads, oocytes, and early embryos, but is not asymmetrically distributed. Our analysis indicates that the par-5 14-3-3 gene plays a crucial role in the early events leading to polarization of the C. elegans zygote.     

Early patterning of the mouse embryo--contributions of sperm and egg

The first cleavage of the fertilised mouse egg divides the zygote into two cells that have a tendency to follow distinguishable fates. One divides first and contributes its progeny predominantly to the embryonic part of the blastocyst, while the other, later dividing cell, contributes mainly to the abembryonic part. We have previously observed that both the plane of this first cleavage and the subsequent order of blastomere division tend to correlate with the position of the fertilisation cone that forms after sperm entry. But does sperm entry contribute to assigning the distinguishable fates to the first two blastomeres or is their fate an intrinsic property of the egg itself? To answer this question we examined the distribution of the progeny of early blastomeres in embryos never penetrated by sperm - parthenogenetic embryos. In contrast to fertilised eggs, we found there is no tendency for the first two parthenogenetic blastomeres to follow different fates. This outcome is independent of whether parthenogenetic eggs are haploid or diploid. Also unlike fertilised eggs, the first 2-cell blastomere to divide in parthenogenetic embryo does not necessarily contribute more cells to the blastocyst. However, even when descendants of the first dividing blastomere do predominate, they show no strong predisposition to occupy the embryonic part. Thus blastomere fate does not appear to be decided by differential cell division alone. Finally, when the cortical cytoplasm at the site of sperm entry is removed, the first cleavage plane no longer tends to divide the embryo into embryonic and abembryonic parts. Together these results indicate that in normal development fertilisation contributes to setting up embryonic patterning, alongside the role of the egg.     

SPD-3 is required for spindle alignment in Caenorhabditis elegans embryos and localizes to mitochondria

During the development of multicellular organisms, cellular diversity is often achieved through asymmetric cell divisions that produce two daughter cells having different developmental potentials. Prior to an asymmetric cell division, cellular components segregate to opposite ends of the cell defining an axis of polarity. The mitotic spindle rotationally aligns along this axis of polarity, thereby ensuring that the cleavage plane is positioned such that segregated components end up in individual daughter cells. Here we report our characterization of a novel gene required for spindle alignment in Caenorhabditis elegans. During the first mitosis in spd-3(oj35) embryos the spindle failed to align along the anterior/posterior axis, leading to abnormal cleavage configurations. spd-3(oj35) embryos had additional defects reminiscent of dynein/dynactin loss-of-function possibly caused by the mislocalization of dynactin. Surprisingly, we found that SPD-3GFP localized to mitochondria. Consistent with this localization, spd-3(oj35) worms exhibited slow growth and increased ATP concentrations, which are phenotypes similar to those described for other mitochondrial mutants in C. elegans. To our knowledge, SPD-3 is the first example of a link between mitochondria and spindle alignment in C. elegans.     

Presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo

The present study examines the indispensability of a nucleus or nucleus-deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)-damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV-damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV-damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV-damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos.     

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.     

OOC-3, a novel putative transmembrane protein required for establishment of cortical domains and spindle orientation in the P(1) blastomere of C. elegans embryos

Asymmetric cell divisions require the establishment of an axis of polarity, which is subsequently communicated to downstream events. During the asymmetric cell division of the P(1) blastomere in C. elegans, establishment of polarity depends on the establishment of anterior and posterior cortical domains, defined by the localization of the PAR proteins, followed by the orientation of the mitotic spindle along the previously established axis of polarity. To identify genes required for these events, we have screened a collection of maternal-effect lethal mutations on chromosome II of C. elegans. We have identified a mutation in one gene, ooc-3, with mis-oriented division axes at the two-cell stage. Here we describe the phenotypic and molecular characterization of ooc-3. ooc-3 is required for the correct localization of PAR-2 and PAR-3 cortical domains after the first cell division. OOC-3 is a novel putative transmembrane protein, which localizes to a reticular membrane compartment, probably the endoplasmic reticulum, that spans the whole cytoplasm and is enriched on the nuclear envelope and cell-cell boundaries. Our results show that ooc-3 is required to form the cortical domains essential for polarity after cell division.     

Chromatin diminution and early cleavage in Parascaris univalens (Nematoda)

Summary In Parascaris developmental commitment to the germ line and somatic lineages is indicated by the orientation of the mitotic spindle in blastomeres, the topology of cells in the embryo, and chromatin diminution in presomatic blastomeres. Using three different experimental techniques: transient pressure treatment, application of cytochalasin B, and isolation of blastomeres, we have succeeded in uncoupling several developmental processes during cleavage of P. univalens. The following results were obtained: (1) Following mitotic nondisjunction we observed identical behavior of all chromatids in each blastomere. Thus chromosome differentiation by differential replication does not occur. (2) Chromosome fragments obtained by pressure treatment of egg cells underwent chromatin diminution. Thus this process does not require an intact germ-line chromosome. However, chromosomes immobilized on a monopolar spindle did not undergo chromatin diminution. Thus diminution appears to require segregation of chromatids. (3) Blastomeres that completely lacked chromosomes as a result of mitotic nondisjunction underwent normal early cleavage divisions. (4) Pressure treatment or prolonged treatment with cytochalasin B caused egg cells or germ line blastomeres to lose their germ line quality, as deduced from the coincident occurrence of symmetrical (presomatic-like) cleavage and chromatin diminution. (5) Isolated blastomeres from 2-cell embryos, i.e. 1/2 blastomeres, usually cleaved according to their prospective fates in the whole embryo. However, in some partial embryos derived from such blastomeres, chromatin diminution was delayed for either one or two cleavage mitoses. An activation model as an alternative to a prelocalization model is presented, which can account for early blastomere topogenesis and chromatin diminution.     

Mutations in ooc-5 and ooc-3 disrupt oocyte formation and the reestablishment of asymmetric PAR protein localization in two-cell Caenorhabditis elegans embryos.

The early development of Caenorhabditis elegans embryos is characterized by a series of asymmetric divisions in which the mitotic spindle is repeatedly oriented on the same axis due to a rotation of the nuclear-centrosome complex. To identify genes involved in the control of spindle orientation, we have screened maternal-effect lethal mutants for alterations in cleavage pattern. Here we describe mutations in ooc-5 and ooc-3, which were isolated on the basis of a nuclear rotation defect in the P(1) cell of two-cell embryos. These mutations are novel in that they affect the asymmetric localization of PAR proteins at the two-cell stage, but not at the one-cell stage. In wild-type two-cell embryos, PAR-3 protein is present around the entire periphery of the AB cell and prevents nuclear rotation in this cell. In contrast, PAR-2 functions to allow nuclear rotation in the P(1) cell by restricting PAR-3 localization to the anterior periphery of P(1). In ooc-5 and ooc-3 mutant embryos, PAR-3 was mislocalized around the periphery of P(1), while PAR-2 was reduced or absent. The germ-line-specific P granules were also mislocalized at the two-cell stage. Mutations in ooc-5 and ooc-3 also result in reduced-size oocytes and embryos. However, par-3 ooc double-mutant embryos can exhibit nuclear rotation, indicating that small size per se does not prevent rotation and that PAR-3 mislocalization contributes to the failure of rotation in ooc mutants. We therefore postulate that wild-type ooc-5 and ooc-3 function in oogenesis and in the reestablishment of asymmetric domains of PAR proteins at the two-cell stage.     

Orientation of the cleavage spindles in pulmonate mollusks. II. The role of the architecture of the intercellular contacts in III and IV cleavage spindle orientation

In the normal development of pulmonate molluscs, the variety of orientations of the III and IV cleavage spindles markedly reduces in the process of transition from pro- to meta- and anaphase. Even prior to the completion of spindle growth in these cleavage divisions the external faces of blastomeres become asymmetrical due to intercellular interactions but the whole system of external intercellular contacts (edges) is characterized by a certain symmetry. This symmetry coincides partially or fully with that of the system of definitive spindles. In the artificially obtained four-celled common pond snail embryos with the chain-like position of blastomeres, the III cleavage spindles were oriented at right angles to the polar axis, rather than in parallel with it (as in the normal development). The eight-celled embryos with symmetrical external faces of macromeres were also obtained. The variety of orientation of the IV cleavage definitive spindles in such embryos was markedly widened and in the macromeres with inverse asymmetry the inversion of the sign of declination of the spindles was observed. The spindle orientation depends, thus, on the form of adjacent region of the external face. This form as a whole is determined by the mutual position of blastomeres, curvature of surface and relative length of the face edges.     

Effects of hydrostatic pressure on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Indirect immunofluorescence staining was used to detect cytological changes of isolated blastodisks during mitosis of flounder haploid eggs treated with hydrostatic pressure. Changes in microtubule structure and expected cleavage suppression were observed from blastodisk formation to the third cell cycle, with obvious differences between treated and control eggs. In most eggs, microtubules were disassembled and the nucleation capacity of the centrosome was temporarily inhibited after pressure treatment. Within 15-20 min after treatment, the nucleation capacity of the centrosome began to gradually recover, with slow regeneration of microtubules; approximately 25 min after treatment, the nucleation capacity of the centrosome recovered completely, regenerated distinct bipolar spindles, and the first mitosis ensued. During the second cell cycle, approximately 61% of the embryos were at the two-cell stage, with a monopolar spindle in each blastomere; that treatment was effective was based on second cleavage blockage. Approximately 15% of the eggs still remained at the one-cell stage and had a monopolar spindle (treatment was effective, according to the general model of first cleavage blockage). However, treatment was ineffective in approximately 15% of the embryos (bipolar spindle in each blastomeres) and in another 8% (bipolar spindle in one of the two blastomeres and a monopolar spindle in the other; both mechanisms operating in different parts of the embryo). This is the first report elucidating mitotic gynogenetic diploid induction by hydrostatic pressure in marine fishes and provides a cytological basis for developing an efficient method of inducing mitotic gynogenesis in olive flounder.     

The oral-aboral axis of a sea urchin embryo is specified by first cleavage

Several lines of evidence suggest that the oral-aboral axis in Strongylocentrotus purpuratus embryos is specified at or before the 8-cell stage. Were the oral-aboral axis specified independently of the first cleavage plane, then a random association of this plane with the blastomeres of the four embryo quadrants in the oral-aboral plane (viz. oral, aboral, right and left) would be expected. Lineage tracer dye injection into one blastomere at the 2-cell stage and observation of the resultant labeling patterns demonstrates instead a strongly nonrandom association. In at least ninety percent of cases, the progeny of the aboral blastomeres are associated with those of the left lateral blastomeres and the progeny of the oral blastomeres with the right lateral ones, respectively. Thus, ninety percent of the time the oral pole of the future oral-aboral axis lies 45 degrees clockwise from the first cleavage plane as viewed from the animal pole. The nonrandom association of blastomeres after labeling of the 2-cell stage implies that there is a mechanistic relation between axis specification and the positioning of the first cleavage plane.     

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.     

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.