C. elegans PAR-3 and PAR-6 are required for apicobasal asymmetries associated with cell adhesion and gastrulation

PAR proteins distribute asymmetrically across the anterior-posterior axis of the 1-cell-stage C. elegans embryo, and function to establish subsequent anterior-posterior asymmetries. By the end of the 4-cell stage, anteriorly localized PAR proteins, such as PAR-3 and PAR-6, redistribute to the outer, apical surfaces of cells, whereas posteriorly localized PAR proteins, such as PAR-1 and PAR-2, redistribute to the inner, basolateral surfaces. Because PAR proteins are provided maternally, distinguishing apicobasal from earlier anterior-posterior functions requires a method that selectively prevents PAR activity after the 1-cell stage. In the present study we generated hybrid PAR proteins that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but showed defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appeared between lateral surfaces of cells that are normally tightly adherent, cells that ingress during gastrulation failed to accumulate nonmuscle myosin at their apical surfaces and ingression was slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the first few cell cycles of embryogenesis.     

Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

The cytoskeleton and associated proteins during cleavage, compaction and blastocyst differentiation in the pig

Abstract. The organization of the cytoskeleton during early pig embryogenesis was investigated by using fluorescence and electron microscopy. The early morphogenesis of the pig embryo differed from that of the mouse, the standard model of the early mammalian development. In the pig, both compaction and polarization were gradual, and definitive polarization of cell surface microville occurred first shortly before blastocyst formation; the compaction and polarization of the mouse embryo are completed as early as at the 8 cell stage. Furthermore, the pig morula undergoes cycles of compaction and decompaction throughout its development. Distinct changes in the distribution of actin and the actin-associated proteins α-fodrin, vinculin and E-cadherin coincided with these events. In the pig, all these molecules were evenly distributed at all aspects of the blastomeres during early cleavage and then gradually accumulated in regions of intercellular contacts toward the blastocyst stage; microfilaments in trophectoderm cells formed a cortical meshwork associated with apical microvilli and adherent junctions (zonula adherens). In the mouse, the corresponding changes occur earlier, at the 8 cell stage. Microtubules formed a network-like cortical layer beneath the microvilli at the free outer surfaces of pig blastomeres. Cytokeratin bundles were not observed until the early blastocyst, where they characteristically associated with newly formed desmosomes.
In both species a close correlation between morphologically defined developmental stages and the organization of the cytoskeleton: actin and actin-associated proteins are involved in polarization and compaction, whereas the appearance of intermediate filament bundles coincides with the building of the first epithelium, the trophectoderm; it is in the timing of events that a contrast between species is observed.     

The cytoskeleton and associated proteins during cleavage, compaction and blastocyst differentiation in the pig

Abstract. The organization of the cytoskeleton during early pig embryogenesis was investigated by using fluorescence and electron microscopy. The early morphogenesis of the pig embryo differed from that of the mouse, the standard model of the early mammalian development. In the pig, both compaction and polarization were gradual, and definitive polarization of cell surface microville occurred first shortly before blastocyst formation; the compaction and polarization of the mouse embryo are completed as early as at the 8 cell stage. Furthermore, the pig morula undergoes cycles of compaction and de-compaction throughout its development. Distinct changes in the distribution of actin and the actin-associated proteins α-fodrin, vinculin and E-cadherin coincided with these events. In the pig, all these molecules were evenly distributed at all aspects of the blastomeres during early cleavage and then gradually accumulated in regions of intercellular contacts toward the blastocyst stage; microfilaments in trophectoderm cells formed a cortical meshwork associated with apical microvilli and adherent junctions (zonula adherens). In the mouse, the corresponding changes occur earlier, at the 8 cell stage. Microtubules formed a network-like cortical layer beneath the microvilli at the free outer surfaces of pig blastomeres. Cytokeratin bundles were not observed until the early blastocyst, where they characteristically associated with newly formed desmosomes.
In both species a close correlation between morphologically defined developmental stages and the organization of the cytoskeleton: actin and actin-associated proteins are involved in polarization and compaction, whereas the appearance of intermediate filament bundles coincides with the building of the first epithelium, the trophectoderm; it is in the timing of events that a contrast between species is observed.     

Fucosylated glycoconjugates in mouse preimplantation embryos

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.     

Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo

The preimplantation development of the mouse embryo leads to the divergence of the first two cell lineages, the inner cell mass and the trophectoderm. The formation of a microvillus pole during compaction at the eight-cell stage and its asymmetric inheritance during mitosis are key events in the emergence of these two cell populations. Ezrin, a member of the ERM protein family, seems to be involved in the formation and stabilization of this apical microvillus pole. To further characterize its function in early development, we mutated the key residue T567, which was reported to be essential for regulation of ezrin function through phosphorylation. Here, we show that expression of ezrin mutants in which the COOH-terminal threonine T567 was replaced by an aspartate (to mimic a phosphorylated residue; T567D) or by an alanine (to avoid phosphorylation; T567A) interferes with E-cadherin function and disrupts the first morphogenetic events of development: compaction and cavitation. The active mutant ezrin-T567D induces the formation of numerous and abnormally long microvilli at the surface of blastomeres. Moreover, it localizes all around the cell cortex and inhibits cell-cell adhesion and cell polarization at the eight-cell stage. During the following stages, only half of the embryos are able to compact and finally to cavitate. In those embryos, the amount of ezrin-T567D decreases in the basolateral areas, while the proportion of adherens junctions increases. The reverse inactive mutant ezrin-T567A is mainly cytoplasmic and does not perturb compaction at the eight-cell stage. However, at the 16-cell stage, it relocalizes at the basolateral cortex, leading to a strong decrease in the surface of adherens junctions, and finally, embryos abort development. Our results show that ezrin is directly involved in the formation of microvilli in the early mouse embryo. Moreover, they indicate that maintenance of ezrin in basolateral areas prevents microvilli breakdown and inhibits the formation of normal cell-cell contacts mediated by E-cadherin, thereby impairing blastomeres polarization and morphogenesis of the blastocyst.     

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.     

MEX-5 and MEX-6 function to establish soma/germline asymmetry in early C. elegans embryos

An asymmetrical network of cortically localized PAR proteins forms shortly after fertilization of the C. elegans egg. This network is required for subsequent asymmetries in the expression patterns of several proteins that are encoded by nonlocalized, maternally expressed mRNAs. We provide evidence that two nearly identical genes, mex-5 and mex-6, link PAR asymmetry to those subsequent protein asymmetries. MEX-5 is a novel, cytoplasmic protein that is localized through PAR activities to the anterior pole of the 1-cell stage embryo. MEX-5 localization is reciprocal to that of a group of posterior-localized proteins called germline proteins. Ectopic expression of MEX-5 is sufficient to inhibit the expression of germline proteins, suggesting that MEX-5 functions to inhibit anterior expression of the germline proteins.     

Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner.

Present studies were performed to investigate what factors affect the morphogenesis of preimplantation mouse embryos, and to find the action mechanism of that factor by using cytoplasm removal and its reconstitution from a different developmental stage embryo. Half (HP group) or one-third of cytoplasm (TP group) was removed from 1-cell mouse embryos by micromanipulation, and their morphogenesis and genome expression were compared with sham-operated embryos (SP group). The compaction and blastocoel formation of embryos in both the HP and TP groups were accelerated in time and cell stage when compared with those of the SP group. However, the total activity and time of RNA synthesis, and gene expression of ZO-1alpha+ isoform were not different. To change the cytoplasm composition without altering the nucleus/cytoplasmic ratio, half a 1-cell embryo with both pronuclei was reconstituted with the half enucleated cytoplasm of 1-cell embryo (P + P group), 2-cell (P + 2 group) or 4-cell (P + 4 group) by electrofusion. Embryonic compaction, timing of RNA synthesis, and stage-specific gene expression of the ZO-1alpha(+) isoform in the P + 2 and P + 4 groups were accelerated in time and cell stage than that in the P + P group, but not different between the P + 2 and P + 4 groups. In addition, a blastomere of 2-cell embryo was reconstituted with the enucleated cytoplasm of 1-cell embryo (2 + P group) or 2-cell (2 + 2 group) in equal volume by electrofusion. Also, the karyoplast of 2-cell was fused with the enucleated 1-cell embryo (2 + PP group). Embryonic development, total activity of RNA synthesis, and gene expression of the ZO-1alpha(+) isoform of embryos in the 2 + P and 2 + PP groups were delayed when compared with those of the 2 + 2 group. Also, the phenomena of compaction and blastocoel formation were delayed in the development time and cell stage. From these results, the nucleus/cytoplasm ratio was found to have no direct effect on the regulation of embryonic morphogenesis, although it accelerated compaction and blastocoel formation. However, cytoplasmic factors that altered between 1- and 2-cell stages regulate embryonic morphogenesis, especially compaction, of preimplantation mouse embryos in concentration-dependent manner.     

Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis

Cell-cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin-catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell-cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5'UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and beta-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.     

Gap junctional communication in the preimplantation mouse embryo.

In this study, we examined cell-to-cell communication via gap junctional channels between the cells of the early mouse embryo from the 2-cell stage to the preimplantation blastocyst stage. The extent of communication was examined by monitoring for the presence of ionic coupling, the transfer of injected fluorescein (molecular weight 330) and the transfer of injected horseradish peroxidase (molecular weight 40,000). In the 2-cell, 4-cell and precompaction 8-cell embryos, cytoplasmic bridges between sister blastomeres were responsible for ionic coupling and the transfer of injected fluorescein as well as the transfer of injected horseradish peroxidase.In contrast, no communication was observed between blastomeres from different sister pairs. Junction-mediated intercellular communication was unequivocably detected for the first time in the embryo at the early compaction stage (late 8-cell embryo). At that stage, ionic coupling was present and fluorescein injected into one cell spread to all eight cells of the embryo. Injected horseradish peroxidase was passed to only one other cell, however, again indicating the presence of cytoplasmic bridges between sister blastomeres. Junctional communication with respect to both ionic coupling and dye transfer was retained between all the cells throughout compaction. At the blastocyst stage, trophoblast cells of the blastocyst were linked by junctional channels to other trophoblast cells as well as to cells of the inner cell mass, as indicated by the spread of injected fluorescein. In addition, the extent of communication between the cells of the inner cell mass was examined in inner cell masses isolated by immunosurgery; both ionic coupling and the complete spread of injected fluorescein were observed.     

Inactivation of aPKCλ Reveals a Context Dependent Allocation of Cell Lineages in Preimplantation Mouse Embryos

Background

During mammalian preimplantation development, lineage divergence seems to be controlled by the interplay between asymmetric cell division (once cells are polarized) and positional information. In the mouse embryo, two distinct cell populations are first observed at the 16-cell stage and can be distinguished by both their position (outside or inside) and their phenotype (polarized or non-polarized). Many efforts have been made during the last decade to characterize the molecular mechanisms driving lineage divergence.

Methodology/Principal Findings

In order to evaluate the importance of cell polarity in the determination of cell fate we have disturbed the activity of the apical complex aPKC/PAR6 using siRNA to down-regulate aPKCλ expression. Here we show that depletion of aPKCλ results in an absence of tight junctions and in severe polarity defects at the 16-cell stage. Importantly, we found that, in absence of aPKCλ, cell fate depends on the cellular context: depletion of aPKCλ in all cells results in a strong reduction of inner cells at the 16-cell stage, while inhibition of aPKCλ in only half of the embryo biases the progeny of aPKCλ defective blastomeres towards the inner cell mass. Finally, our study points to a role of cell shape in controlling cell position and thus lineage allocation.

Conclusion

Our data show that aPKCλ is dispensable for the establishment of polarity at the 8-cell stage but is essential for the stabilization of cell polarity at the 16-cell stage and for cell positioning. Moreover, this study reveals that in addition to positional information and asymmetric cell divisions, cell shape plays an important role for the control of lineage divergence during mouse preimplantation development. Cell shape is able to influence both the type of division (symmetric or asymmetric) and the position of the blastomeres within the embryo.     

Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR

Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 +/- 0.0282 in the oocyte, 0.0102 +/- 0.0036 in the zygote, 0.0007 +/- 0.0003 in the 2-cell embryo, 0.0031 +/- 0.0017 in the 4-cell embryo, 0.0084 +/- 0.0024 in the 8-cell embryo, 0.0537 +/- 0.0121 in the morula and 0.0392 +/- 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.