Making the blastocyst: lessons from the mouse (original) (raw)
Early cleavage and zygotic genome activation. The fertilized egg first undergoes a series of early cleavage divisions, producing increasing numbers of progressively smaller cells, known as blastomeres, without changing the overall size of the embryo (Figure 1). As in other types of organisms, protein synthesis in the mammalian zygote initially relies on a deposit of maternally loaded mRNA (1). Transcription of mRNA coded by the zygotic genome begins during the first few cleavage divisions, and this transition from maternal to zygotic transcripts is known as zygotic genome activation (ZGA). ZGA takes place quite early in the mouse: there is an initial burst of zygotic transcription at the end of the one-cell stage, followed by a second, larger burst at the two-cell stage (2, 3). This second burst is accompanied by degradation of maternal transcripts (4, 5). In humans, ZGA occurs later than in the mouse, at the four- to eight-cell stage (6). This is the first of several indications that the timing of events in human and mouse preimplantation development may differ. Although maternal mRNAs may be degraded, proteins that have been synthesized from these transcripts during oogenesis can persist into later development. The presence of such “maternal” proteins can confound the analysis of gene function during preimplantation development in mouse studies, often requiring the generation of maternal and zygotic loss-of-function mutants (7, 8). Given the difference in timing of ZGA between mice and humans, the relative roles of maternal and zygotic transcripts may be somewhat different in mouse and human embryos.
Stages of mouse and human preimplantation development. (A) In the mouse, the fertilized egg undergoes three rounds of cleavage, producing an eight-cell embryo that then undergoes compaction. From the eight-cell stage onward, cell divisions produce two populations of cells, those that occupy the inside of the embryo and those that are located on the outside. The blastocoel cavity begins to form inside the embryo beginning at the 32-cell stage and continues to expand as the embryo grows and matures into the late blastocyst stage. Cdx2 becomes upregulated in outside, future TE cells, starting at the 32-cell stage, while Oct4 expression becomes limited to the ICM in the early blastocyst stage. By the late blastocyst stage, while continuing to express Oct4 ubiquitously, the ICM contains a population of _Nanog_-positive EPI cells and a population of _Gata6_-positive PE cells. (B) Development is similar in the early human embryo, although compaction occurs at the 16-cell stage and the mutually exclusive expression patterns of CDX2 and OCT4 are not established until the late blastocyst stage. The expression patterns of NANOG and GATA6 in the human preimplantation embryo have not yet been characterized.
Compaction and polarization. The early cleavage divisions produce an eight-cell embryo that subsequently undergoes an increase in intercellular adhesion known as compaction, causing all cells to adopt a more flattened morphology (Figure 1). This process of compaction is essential for later morphogenetic events and for the proper segregation of the three embryonic lineages. In the mouse, compaction is associated with the formation of adherens and, later, tight junctions between cells. E-cadherin, a major component of adherens junctions, becomes localized to regions of cell-cell contact at the eight-cell stage (9), and disruption of E-cadherin–mediated cell adhesion, by removal of Ca2+ ions or addition of E-cadherin–specific antibodies to embryo culture media, inhibits compaction (10–12). E-cadherin–knockout embryos do compact normally at the eight-cell stage because of the presence of E-cadherin protein inherited from the egg, but they fail to maintain proper cell adhesion into the blastocyst stage (7, 8). Conversely, embryos deficient in the maternal supply of E-cadherin fail to compact at the eight-cell stage, but they are rescued by zygotic expression of the paternal allele and compact by the 16-cell stage (13).
It remains unclear how the process of compaction is initiated. A simple increase in the level of expression of E-cadherin or its intracellular binding partners α- and β-catenin cannot account for the change, as all are present in the mouse embryo from fertilization onward (9, 14). In fact, compaction can occur even when mRNA synthesis is blocked from the early four-cell stage onward (15), and is actually induced prematurely by culturing four-cell–stage embryos in the presence of inhibitors of protein synthesis (16). This indicates that all the components required for compaction have been synthesized by the time the embryo reaches the early four-cell stage. Notably, culture of embryos with small molecules that activate PKC also causes premature compaction (17, 18). This suggests that posttranslational mechanisms play an important role in the induction of compaction, possibly by maintaining the E-cadherin complex in an inactive state, although how this might occur remains to be elucidated. In support of this theory, both E-cadherin and β-catenin become phosphorylated at the time of compaction (19, 20). The Rho family GTPases also play a role in this process (21, 22). In cultured cells, IQ motif–containing GTPase-activating protein 1 (IQGAP1) can disrupt cadherin/catenin complexes by preventing the binding of α-catenin to β-catenin and E-cadherin until it is bound and inactivated by the Rho family GTPases Rac1 and Cdc42 (23). Changes in the subcellular distribution of IQGAP1 and Rac1 protein before and during compaction suggest that a similar relationship exists in the preimplantation mouse embryo (22). This has led to the hypothesis that IQGAP1 prevents premature compaction until the eight-cell stage, when Rac1 and Cdc42 are activated, although this has not yet been tested experimentally (24).
Blastomeres do not show any signs of intracellular polarity until compaction but, concomitant with the increase in cell adhesion at this stage, all cells rapidly polarize along the axis perpendicular to cell contact such that outward facing (apical) regions become distinct from inward facing (basolateral) regions (Figure 2). The cytoplasm becomes reorganized: cell nuclei move basolaterally (25), while the endosomes, previously distributed randomly, become localized apically (26). Actin accumulates apically, as do most microtubules, although a smaller population of more stable acetylated microtubules becomes localized basolaterally (27, 28). Microvilli that were equally distributed on the cell surface prior to compaction accumulate at the apical pole and are almost completely eliminated basolaterally (29). As is the case in other polarized cell types, the membrane protein ezrin (30), the polarity proteins Par3 and Par6 (31, 32), and atypical PKC (aPKC) (33) all become localized to the apical domain, while the polarity proteins Par1 and lethal giant larva homolog (Lgl) accumulate basolaterally (32).
Polarity in the mouse preimplantation embryo. (A) At the eight-cell stage, all blastomeres polarize along the axis of cell contact, forming outward, apical domains and inward-facing basolateral domains. (B) As the embryo grows from eight to 16 cells, blastomeres that divide parallel to the inside-outside axis produce two outside, polar cells. Blastomeres that divide perpendicular to the inside-outside axis produce one outside, polar daughter cell and one non-polar, inside daughter cell. This creates two populations of cells: outside, polar cells and inside, nonpolar cells. These two types of cell division also occur as the embryo grows from 16 to 32 cells.
It is unclear how polarization is initiated de novo at the eight-cell stage. Based on the close temporal link between compaction and polarization, one hypothesis is that cell contact is somehow important for the establishment of the apical and basolateral domains. Multiple studies have shown that cellular interactions are involved in setting up the orientation of polarity, as apical poles tend to form in positions that are as far away as possible from locations of cell contact (34, 35). However, polarization can occur in blastomeres that have been isolated from cell contact or prevented from compacting, albeit at a lower frequency than usual (36, 37). Thus it appears that cell contact is partially responsible for the establishment of polarity but that there are other mechanisms involved, one of which is dependent on nucleus-microtubule-cortex interactions (32, 36). Regardless of how polarity is established, it is likely maintained, as in other systems, by the mutual antagonism of apical and basal protein complexes containing the various PAR proteins, aPKC, and Lgl (38).
Symmetric versus asymmetric cell divisions up to the 32-cell stage. Once the eight-cell embryo has compacted and polarized, it undergoes two further rounds of cleavage, growing from eight cells to 16, and from 16 cells to 32. During these divisions, inheritance of the polarized state is influenced by the orientation of the cleavage plane of the blastomere (Figure 2). If a cell undergoes mitosis at an angle perpendicular to its axis of polarity (that is, parallel to its inside-outside axis), its two daughter cells will both be polar and will remain on the outside of the embryo. However, cells can also divide parallel to their axis of polarity, producing one polarized outside daughter cell and one apolar cell that is located on the inside of the embryo (39, 40). In this way the preimplantation embryo, which was previously composed of a uniform population of cells, has now generated two separate groups of cells: apolar inside cells and polar outside cells. Cell polarity and cell position are both important in defining these two populations, as experimentally manipulating the position of a cell in the embryo can alter its polarity (41–43) and changing the polarity of a cell can in turn affect its position (31). From the 32-cell stage onward, these two cell populations have distinct developmental fates: cells on the outside of the embryo contribute to the TE lineage, while inside cells contribute to the inner cell mass (ICM), the group of cells that further diverges into the EPI and PE lineages (see below).
The processes of compaction, polarization, and asymmetric division have not been well studied in mammalian species other than the mouse. Studies of human preimplantation embryos developing in vitro indicate that their development closely resembles that of mouse embryos at the gross morphological level. One notable exception is the observation that compaction often occurs later in the human embryo than in the mouse, at the 16-cell stage (44–46), although it has also been reported to begin earlier, at the four- to eight-cell stage in some embryos (47). It is unclear how the different timing of compaction might affect polarization and asymmetric cell divisions in the human embryo.
Blastocoel formation. Starting at the 32-cell stage, as the outside cells of the embryo are becoming fully committed to the TE lineage (48, 49), a fluid-filled cavity known as the blastocoel begins to form (Figure 1). The presence of a blastocoel is essential for proper development of the ICM (49). During blastocoel formation water may enter the embryo via an osmotic gradient, as a result of Na+/K+ ATPases that produce an accumulation of Na+ on the basolateral side of the TE (50). Water movement may also be facilitated by aquaporins, which are present in the TE and functional by the 32-cell stage (51). Once it begins to form, maintenance of the blastocoel depends on the epithelial character of the TE. As early as one hour after compaction, and continuing for another full day, tight junction components such as occludin (52), zona occludens 1 (ZO-1) and ZO-2 (53, 54), and cingulin (55, 56) begin to assemble in outside cells, until functional tight junctions are fully formed by the 32-cell stage (52, 54). These tight junctions form a seal, preventing water leakage. With the formation of the blastocoel at E3.5, the mouse embryo is now considered a blastocyst. It continues to mature for an additional 24 hours and is ready to implant into the uterine wall by E4.5. Although it has not been studied extensively, blastocoel formation in the human embryo appears to take place at approximately E4.5 (44).