Berenika Plusa

Downregulation of Par3 and aPKC function directs cells towards the ICM in the preimplantation mouse embryo

Generation of inside cells that develop into inner cell mass (ICM) and outside cells that develop into trophectoderm is central to the development of the early mouse embryo. Critical to this decision is the development of cell polarity and the associated asymmetric (differentiative) divisions of the 8-cell-stage blastomeres. The underlying molecular mechanisms for these events are not understood. As the Par3/aPKC complex has a role in establishing cellular polarity and division orientation in other systems, we explored its potential function in the developing mouse embryo. We show that both Par3 and aPKC adopt a polarized localization from the 8-cell stage onwards and that manipulating their function re-directs cell positioning and consequently influences cell fate. Injection of dsRNA against Par3 or mRNA for a dominant negative form of aPKC into a random blastomere at the 4-cell stage directs progeny of the injected cell into the inside part of the embryo. This appears to result from both an increased frequency by which such cells undertake differentiative divisions and their decreased probability of retaining outside positions. Thus, the natural spatial allocation of blastomere progeny can be over-ridden by downregulation of Par3 or aPKC, leading to a deceased tendency for them to remain outside and so develop into trophectoderm. In addition, this experimental approach illustrates a powerful means of manipulating gene expression in a specific clonal population of cells in the preimplantation embryo.

The first cleavage of the mouse zygote predicts the blastocyst axis

One of the unanswered questions in mammalian development is how the embryonic–abembryonic axis of the blastocyst is first established. It is possible that the first cleavage division contributes to this process, because in most mouse embryos the progeny of one two-cell blastomere primarily populate the embryonic part of the blastocyst and the progeny of its sister populate the abembryonic part. However, it is not known whether the embryonic–abembryonic axis is set up by the first cleavage itself, by polarity in the oocyte that then sets the first cleavage plane with respect to the animal pole, or indeed whether it can be divorced entirely from the first cleavage and established in relation to the animal pole. Here we test the importance of the orientation of the first cleavage by imposing an elongated shape on the zygote so that the division no longer passes close to the animal pole, marked by the second polar body. Non-invasive lineage tracing shows that even when the first cleavage occurs along the short axis imposed by this experimental treatment, the progeny of the resulting two-cell blastomeres tend to populate the respective embryonic and abembryonic parts of the blastocyst. Thus, the first cleavage contributes to breaking the symmetry of the embryo, generating blastomeres with different developmental characteristics.

First cleavage of the mouse embryo responds to change in egg shape at fertilization.

Although mouse development is regulative, the cleavage pattern of the embryo is not random. The first cleavage tends to relate to the site of the previous meiosis. Sperm entry might provide a second cue, but evidence for and against this is indirect and has been debated. To resolve whether sperm entry position relates to the first cleavage, we have followed development from fertilization by time-lapse imaging. This directly showed cytokinesis passes close to the site of the previous meiosis and to both the sperm entry site and trajectory of the male pronucleus in a significant majority of eggs. We detected asymmetric distribution of Par6 protein in relation to the site of meiosis, but not sperm entry. Unexpectedly, we found the egg becomes flattened upon fertilization in an actin-mediated process. The sperm entry position tends to lie at one end of the short axis along which cleavage will pass. When we manipulated eggs to change their shape, this repositioned the cleavage plane such that eggs divided along their experimentally imposed short axis. Such manipulated eggs were able to develop to term, emphasizing the regulative nature of their development.

Site of the previous meiotic division defines cleavage orientation in the mouse embryo.

The conservation of early cleavage patterns in organisms as diverse as echinoderms and mammals suggests that even in highly regulative embryos such as the mouse, division patterns might be important for development. Indeed, the first cleavage divides the fertilized mouse egg into two cells: one cell that contributes predominantly to the embryonic part of the blastocyst, and one that contributes to the abembryonic part. Here we show, by removing, transplanting or duplicating the animal or vegetal poles of the mouse egg, that a spatial cue at the animal pole orients the plane of this initial division. Embryos with duplicated animal, but not vegetal, poles show abnormalities in chromosome segregation that compromise their development. Our results show that localized factors in the mammalian egg orient the spindle and so define the initial cleavage plane. In increased dosage, however, these factors are detrimental to the correct execution of division.

Embryology: Does prepatterning occur in the mouse egg? (Reply)

Hiiragi et al. compare our model of the developing mouse egg with theirs. They seem to present patterning as equivalent to determination, but this is confusing as patterning does not have to mean determination. We have never stated that mouse embryo development is determined. Mouse development is regulative rather than determinative, and this can be explained in two ways: first, development could be entirely unbiased, generating identical cells; second, there could be some developmental bias or, in other words, pattern from the beginning that is not constraining. There is evidence for early bias from several laboratories but this does not mean that cells have localized determinants fixing their fates. Regulative development does not exclude bias, which indicates inclination, not determination.