Plane Of First Cleavage Research Articles

An investigation of the origin and significance of bilateral symmetry of the pronuclear zygote in the mouse

Preliminary observations revealed that advanced zygotes of the PO strain mouse are often bilaterally symmetrical, and suggested that both the plane of first cleavage and features of the blastocyst bear a consistent relationship to the zygote's bilateral plane. Spaced oil drops were injected into the zona pellucida to delineate the bilateral plane in pronuclear zygotes, and a distinct cluster of drops then placed over the second polar body. Such non-invasive marking was combined with gelation of the perivitelline space to prevent rotation of the zygotes within the zona pellucida. Nearly two-thirds of advanced pronuclear stage zygotes were bilaterally symmetrical and, regardless of whether first cleavage was meridional, it was almost invariably orthogonal to the bilateral plane. Moreover, both the axis of polarity and bilateral plane of the blastocyst bore a consistent relationship to the zygote's bilateral plane. Haploid parthenotes also exhibited bilateral symmetry, although in the absence of fertilization, first cleavage was less consistently orthogonal to the bilateral plane. Bilateral symmetry may be an intrinsic property of the oocyte that is induced by its activation and, from the reproducible way it maps on both the 2-cell conceptus and blastocyst, seems to play a role in early patterning.

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.

Is the plane of first cleavage related to the point of sperm entry in the mouse?

The notion that pattern formation cannot depend on cues that are already present in the zygote has dominated thinking about early development in mammals for more than 3 decades. However, recent studies in the mouse have rendered this notion untenable. The first pertinent study provided evidence that the axis of bilateral symmetry of the early blastocyst was aligned with the animal–vegetal axis of the zygote (Gardner, 1997). Subsequently, it was found that the embryonic–abembryonic axis and equatorial plane of the blastocyst were normally related to the plane of first cleavage, being approximately orthogonal and parallel to this plane respectively (Gardner, 2001; Piotrowska and Zernicka-Goetz, 2001; Piotrowska et al., 2001). Collectively, these findings argue that both axes of the blastocyst are already specified before cleavage in the mouse, and therefore raise the question whether the asymmetries necessary for their specification are intrinsic to the egg or imposed on it by the fertilizing spermatozoon. Using phytohaemagglutinin (PHA)-treated fluorescent microspheres (beads) as markers, Piotrowska and Zernicka-Goetz (2001) claim to have established a patterning role for the spermatozoa by demonstrating that the sperm entry point (SEP) consistently maps to the plane of first cleavage.

The plane of first cleavage is not related to the distribution of sperm components in the mouse.

Marking experiments using phytohaemagglutinin (PHA) and Concanavalin A (ConA) have suggested that the first cleavage plane is related to the point of sperm entry. Because of concerns about the specificity of lectin binding, the distribution of sperm components has been investigated directly. The sperm tail could be identified in cleaving zygotes and early 2-cell stages following their permeabilization and exposure to Oregon Green Paclitaxel. At neither stage did the anterior end of the tail, which lies initially at the site of sperm entry, bear a consistent relationship to the first cleavage plane, even when it had clearly retained its original location. Moreover, using artificial insemination with MitoTracker-labelled sperm, the midpiece was found to remain associated with anterior end of the tail through to the 2-cell stage. Lectins showed no discernible binding to the fertilization cone of mechanically denuded zygotes and very strong binding to the zona pellucida. Moreover, after general labelling of zygotes with either ConA or PHA, persisting surface lectin tended to be concentrated towards the cleavage plane. The present findings challenge the claim that the sperm specifies the plane of first cleavage, and also question the methodology on which it was based.

Specification of embryonic axes begins before cleavage in normal mouse development.

Studies on the development of aggregated, isolated and rearranged blastomeres have engendered the view that in mammals, unlike most other animals, egg organization has no role in the genesis of asymmetries that are essential for cellular diversification and the specification of embryonic axes. Such asymmetries are assumed to arise post-zygotically through interactions between initially naive cells. However, various findings are difficult to reconcile with this view. Here, a consistent relationship between the structure of the blastocyst and the two-cell stage in the mouse has been found using a strictly non-invasive marking technique: injection of small oil drops into the substance of the zona pellicuda. This has revealed that both the embryonic-abembryonic axis of the blastocyst and its plane of bilateral symmetry are normally orthogonal to the plane of first cleavage. This relationship was also seen when denuded two-cell conceptuses were prevented from rotating during subsequent cleavage by immobilizing them in a gel. Therefore, during normal mouse development the axes of the blastocyst, which have been implicated in establishing those of the fetus, are already specified by the onset of cleavage.

The Unique Developmental Program of the Acoel Flatworm, Neochildia fusca

Acoel embryos exhibit a unique form of development that some investigators argue is related to that found in polyclad turbellarians and coelomate spiralians, which display typical quartet spiral cleavage. We generated the first cell-lineage fate map for an acoel flatworm, Neochildia fusca, using modern intracellular lineage tracers to assess the degree of similarity between these distinct developmental programs. N. fusca develops via a “duet” cleavage pattern in which second cleavage occurs in a leiotropically oblique plane relative to the animal–vegetal axis. At the four-cell stage, the plane of first cleavage corresponds to the plane of bilateral symmetry. All remaining cleavages are symmetrical across the sagittal plane. No ectomesoderm is formed; the first three micromere duets generate only ectodermal derivatives. Endomesoderm, including the complex assemblage of circular, longitudinal, and oblique muscle fibers, as well as the peripheral and central parenchyma, is generated by both third duet macromeres. The cleavage pattern, fate map, and origins of mesoderm in N. fusca share little similarity to that exhibited by other spiralians, including the Platyhelminthes (e.g., polyclad turbellarians). These findings are considered in light of the possible evolutionary origins of the acoel duet cleavage program versus the more typical quartet spiral cleavage program. Finally, an understanding of the cell-lineage fate map allows us to interpret the results of earlier cell deletion studies examining the specification of cell fates within these embryos and reveals the existence of cell–cell inductive interactions in these embryos.

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.

The first cleavage plane and the embryonic axis are determined by separate mechanisms in Xenopus laevis: I. Independence in undisturbed embryos

We examined the spatial relationships between the meridian of sperm entry the plane of first cleavage, and the embryonic axis (defined by the neural groove) in eggs of Xenopus laevis. Direct measurement of the angular separations between these embryonic structures in gelatin-embedded eggs confirmed the classical conclusion that the sperm entry point and neural groove tend to form on opposite sides of the egg, and also revealed that the first cleavage plane has a nearly random orientation with respect to the neural groove. We next examined the distortion of the first cleavage plane that results from the normal processes of convergence and extension during gastrulation and neurulation. We permanently marked the first cleavage plane by injecting one blastomere of the two-cell embryo with a fluorescent lineage marker. At the start of gastrulation, the interface between the labeled and unlabeled regions was almost randomly oriented relative to the dorsal blastopore lip, confirming our first set of observations. In embryos with the interface <60° to the plane passing through the midline of the dorsal lip, convergent movements of cells produced a confrontation of labeled and unlabeled cells along much of the dorsal midline. Thus, although the first cleavage plane and the bilateral plane were frequently not congruent, the morphogenetic movements of gastrulation and neurulation brought about an apparent congruence in many half-labeled embryos.

OBSERVATIONS ON REPRODUCTION, PREMATURATION, AND FERTILIZATION IN SABELLARIA VULGARIS

1. Sabellaria vulgaris, a polychæte annelid inhabiting sand tubes on dead Echinarachnius and other shells in Bryozoa nodules, and on stones, occurs abundantly in the vicinity of Woods Hole, Mass., and gametes may be secured throughout the summer months. Shedding takes place immediately following the removal of the animals from their tubes.2. The early development is described from shedding to early cleavage and a comparison made with Sabellaria alveolata as described by Fauré-Fremiet. In most respects the early developmental phenomena of these two species are strikingly similar.3. After shedding the eggs become spherical, the vitelline membrane elevates, leaving fine protoplasmic processes between membrane and egg cortex, and the contents of the germinal vesicle form a clear area at the animal pole. Visible polarity appears during pre-maturation which ceases at the metaphase of the first maturation spindle.4. It is said that fertilization occurs normally during May and June but a second and shorter time has been found during the early part of August. During July a high percentage of fertilization may be secured with slightly alkaline sea water (8.6).5. The highest percentage of cleavage is obtained when the gametes are shed together. Eggs may be fertilized after seven hours, but the fertilizing capacity of the spermatozoa is considerably shorter (about one hour).6. The fertilization phenomenon is described. The protoplasmic strands serve to transmit the stimulus of sperm contact. No fertilization membrane appears and the first indication that a spermatozoön has entered is the withdrawal of the processes.7. The fertilization cone is enormous and changes shape. Spermatozoa may enter at any point on the periphery but more frequently in the animal hemisphere. The polar bodies are large and the first one frequently divides.8. Various factors such as increase of pH, shaking, and standing are sufficient to disturb the equilibrium of the pre-maturation spindle with the resultant formation of one or two polar bodies. This is followed by lobular fragmentation and no swimming embryos develop. Old unfertilized eggs stratify into three zones.9. Plane of first cleavage bears no fixed relationship to sperm entrance point and passes to one side of pole as determined by the position of the polar bodies. The polar bodies lie on the smaller blastomere while the larger one contains the polar lobe material.