Blastomeres of the mouse embryo lose totipotency after the fifth cleavage division: expression of Cdx2 and Oct4 and developmental potential of inner and outer blastomeres of 16- and 32-cell embryos

Sixteen inner or outer blastomeres from 16-cell embryos and 32 inner or outer blastomeres from 32-cell embryos (nascent blastocysts) were reaggregated and cultured in vitro. In 24 h old blastocysts developed from blastomeres derived from 16-cell embryos the expression of Cdx2 protein was upregulated in outer cells (new trophectoderm) of the inner cells-derived aggregates and downregulated in inner cells (new inner cell mass) of the external cells-derived aggregates. After transfer to pseudopregnant recipients blastocysts originating from both inner and outer blastomeres of 16-cell embryo developed into normal, fertile mice, but the implantation rate of embryos formed from inner cell aggregates was lower. The aggregates of external blastomeres derived from 32 cell embryo usually formed trophoblastic vesicles accompanied by vacuolated cells. In contrast, the aggregates of inner blastomeres quickly compacted but cavitation was delayed. Although in the latter embryos the Cdx2 protein appeared in the new trophectoderm within 24 h of in vitro culture, these embryos formed only very small outgrowths of Troma1-positive giant trophoblastic cells and none of these embryos was able to implant in recipient females. In separate experiment we have produced normal and fertile mice from 16- and 32-cell embryos that were first disaggregated, and then the sister outer and inner blastomeres were reaggregated at random. In blastocysts developed from aggregates, within 24 h of in vitro culture, the majority of inner and outer blastomeres located themselves in their original position (internally and externally), which implies that in these embryos development was regulated mainly by cell sorting.     

Asynchronous CDX2 expression and polarization of porcine trophoblast cells reflects a species‐specific trophoderm lineage determination progress model

Upregulation of Cdx2 expression in outer cells is a key event responsible for cell lineage segregation between the inner cell mass and the trophoderm (TE) in mouse morula‐stage embryos. In TE cells, polarization can regulate Hippo and Rho‐associated kinase (Rho‐ROCK) signaling to induce the nuclear location of YAP, which has been demonstrated to further induce the expression of Cdx2. However, we found that CDX2 expression could not be detected in the outer cells of porcine morula‐stage embryos but only in some TE cells at the early blastocyst stage. The biological significance and the regulation mechanism of this species‐specific CDX2 expression pattern have still not been determined. We show here that an asynchronous CDX2 expression pattern exists in porcine TE cells during the development of the blastocyst. We demonstrate that CDX2 expression in porcine TE cells depends on the nuclear localization of YAP and polarization of the embryo through Y27632 treatment. We found that the polarization process in the morula to the late blastocyst stage porcine embryos was asynchronous, which was revealed by the apical localization of phosphorylated EZRIN staining. Artificially enhancing the number of polarized blastomeres by culturing the separated blastomeres of four‐cell stage porcine embryos resulted in increased CDX2‐positive cell numbers. These results indicate that the mechanism of CDX2 expression regulation is conserved, but the polarization progress is not conserved between the pig and the mouse, and results in a species‐specific trophoblast determination progress model.     

Par‐aPKC‐dependent and ‐independent mechanisms cooperatively control cell polarity,Hippo signaling,and cell positioning in 16‐cell stage mouse embryos

In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par‐aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE‐specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16‐cell stage embryos. Similar to 32‐cell stage embryos, disruption of the Par‐aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32‐cell stage embryos, 16‐cell stage embryos with a disrupted Par‐aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p‐ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16‐cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8‐cell stage embryos form polar–apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16‐cell stage is regulated by both Par‐aPKC‐dependent and ‐independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling.     

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.     

Archenteron-Forming Capacity in Blastomeres Isolated from Eight-Cell Stage Embryos of the Starfish, Asterina pectinifera

Starfish blastomeres are reported to be totipotent up to the 8-cell stage. We reinvestigated the development of blastomeres of 8-cell stage embryos with a regular cubic shape consisting of two tiers of 4 blastomeres. On dissociation of the embryo by disrupting the fertilization membrane at the 8-cell stage, each of the 4 blastomeres of the vegetal hemisphere gave rise to an embryo that gastrulated, whereas blastomeres from the animal hemisphere did not. By injection of a cell lineage tracer into blastomeres of 8-cell stage embryos, we found that only those of the vegetal hemisphere formed cells constituting the archenteron. Next, we compressed 4-cell stage embryos along the animal-vegetal axis so that all the blastomeres in the 8-cell stage were in a single layer. When these 8 blastomeres were then dissociated, an average of 7 of them developed into gastrulae. By cell lineage analysis, all the blastomeres in single-layered embryos at the 8-cell stage were shown to have the capacity to form cells constituting an archenteron. Taken together, these findings indicate that the fate to form the archenteron is specified by a cytoplasmic factor(s) localized at the vegetal hemisphere, and that isolated blastomeres that have inherited this factor develop into gastrulae.     

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.     

Determinative mechanisms in secondary muscle lineages of ascidian embryos: development of muscle-specific features in isolated muscle progenitor cells

Muscle cells of the ascidian larva originate from three different lines of progenitor cells, the B-line, A-line and b-line. Experiments with 8-cell embryos have indicated that isolated blastomeres of the B-line (primary) muscle lineage show autonomous development of a muscle-specific enzyme, whereas blastomeres of the A-line and b-line (secondary) muscle lineage rarely develop the enzyme in isolation. In order to study the mechanisms by which different lines of progenitors are determined to give rise to muscle, blastomeres were isolated from embryos of Halocynthia roretzi at the later cleavage stages when conspicuous restriction of the developmental fate of blastomeres had already occurred. Partial embryos derived from B-line muscle-lineage cells of the 64-cell embryo (B7.4, B7.5 and B7.8) showed autonomous expression of specific features of muscle cells (acetylcholinesterase, filamentous actin and muscle-specific antigen). In contrast, b-line muscle-lineage cells, even those isolated from the 110-cell embryo (b8.17 and b8.19), did not express any muscle-specific features, even though their developmental fate was mainly restricted to generation of muscle. Isolated A-line cells from the 64-cell embryos (A7.8) did not show any features of muscle differentiation, whereas some isolated A-line cells from the 110-cell embryos (A8.16) developed all three above-mentioned features of muscle cells. This transition was shown to occur during the eighth cell cycle. These results suggest that the mechanism involved in the process of determination of the secondary-lineage muscle cells differs from that of the primary-lineage muscle cells. Interaction with cells of other lineages may be required for the determination of secondary precursors to muscle cells. The presumptive b-line and A-line muscle cells that failed to express muscle-specific features in isolation did not develop into epidermal cells. Thus, although interactions between cells may be required for muscle determination in secondary lineages, the process may represent a permissive type of induction and may differ from the processes of induction of mesoderm in amphibian embryos.     

Reproductive cell specification during Volvox obversus development

Asexual spheroids of the genus Volvox contain only two cell types: flagellated somatic cells and immotile asexual reproductive cells known as gonidia. During each round of embryogenesis in Volvox obversus, eight large gonidial precursors are produced at the anterior extremity of the embryo. These cells arise as a consequence of polarized, asymmetric divisions of the anteriormost blastomeres at the fourth through nine cleavage cycles, while all other blastomeres cleave symmetrically to yield somatic cell precursors. Blastomeres isolated from embryos at any point between the 2-cell and the 32-cell stage cleaved in the normal pattern and produced the same complement and spatial distribution of cell types as they would have in an intact embryo. This result indicates that intrinsic features control the cleavage patterns and developmental potentials of blastomeres, and rules out any significant role for cell-cell interactions in gonidial specification. When substantial quantities of anterolateral cytoplasm were deleted from uncleaved gonidia or 4-cell stage blastomeres, the cell fragments frequently regulated and embryos were produced with the expected number of asymmetrically cleaving cells and gonidial precursors at their anterior ends. However, when anterior cytoplasm was deleted from 8-cell stage blastomeres, the depleted cells frequently failed to cleave asymmetrically and produced no gonidial precursors. Furthermore, when compression was used to reorient cleavage planes at the fourth division cycle, so that anterior cytoplasm was transmitted to more than the normal number of cells, those cells receiving a significant amount of such cytoplasm cleaved asymmetrically to produce supernumerary gonidial precursors. Together, these last two experiments indicate that blastomeres in the V. obversus embryo acquire (at least by the end of the third cleavage cycle) a polarized organization in which anterior cytoplasm plays a causal role in the process of reproductive-cell specification.     

Changes in the distribution of vinculin during preimplantation mouse development

It has been proposed that vinculin is a microfilament bundle-membrane linking cytoskeletal protein. We used double-fluorescence microscopy to study the distribution of vinculin and F-actin in mouse oocytes and preimplantation embryos. In oocytes and in the cells of cleavage- and blastocyst-stage embryos, vinculin exhibited a diffuse cytoplasmic distribution and was concentrated in a submembranous layer. The presence of vinculin in oocytes was confirmed by immunoblotting. In oocytes, a distinct concentration of actin was observed above the second metaphase spindle. During the 8-cell stage, compacting blastomeres exhibited partial polarization of cortical vinculin and actin toward their outward-facing surfaces. In precompaction-stage blastomeres, the submembranous layer of vinculin contained a ring-like concentration in the most peripheral region of each intercellular contact area. During later development, the amount of vinculin localized in the areas of intercellular contacts became modified. In embryos ranging from the compacted 8-cell stage to the mid-morula stage, the vinculin-specific fluorescence was only intense in some intercellular contacts, being indistinct in most contact areas. In late morulae, the flattened outer cells increasingly exhibited concentration of vinculin in contact areas. In contrast, actin-specific fluorescence was clearly evident in most intercellular contacts throughout the morula stage. At the early blastocyst stage, all contacts of the trophectoderm (TE) cells again regularly exhibited concentration of both components. At the late blastocyst stage, the staining pattern changed once again: the contact-associated concentration of vinculin-specific fluorescence was not observed in polar TE cells, while remaining clear in mural TE cells. In blastocyst outgrowths, TE cells displayed typical vinculin plaques at the peripheries of the cells. The continuous changes in the distribution of vinculin and actin suggest that these components are involved in the control of cellular relationships during early development. Immunoelectron microscopy and experiments using cytochalasin were performed in an attempt to relate the distribution of vinculin to the ultrastructural features of embryo cells.     

Roles of ERα during mouse trophectoderm lineage differentiation: revealed by antagonist and agonist of ERα

During mouse early embryogenesis, blastomeres increase in number by the morula stage. Among them, the outer cells are polarized and differentiated into trophectoderm (TE), while the inner cells remain unpolarized and give rise to inner cell mass (ICM). TE provides an important liquid environment for ICM development. In spite of extensive research, the molecular mechanisms underlying TE formation are still obscure. In order to investigate the roles of estrogen receptor α (ERα) in this course, mouse 8‐cell embryos were collected and cultured in media containing ERα specific antagonist MPP and/or agonist PPT. The results indicated that MPP treatment inhibits blastocyst formation in a dose‐dependent manner, while PPT, at proper concentration, promotes the cavitation ratio of mouse embryos. Immunofluorescence staining results showed that MPP significantly decreased the nuclear expression of CDX2 in morula, but no significant changes of OCT4 were observed. Moreover, after MPP treatment, the expression levels of the genes related to TE specification, Tead4, Gata3 and Cdx2, were significantly reduced. Overall, these results indicated that ERα might affect mouse embryo cavitation by regulating TE lineage differentiation.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.