Blastomere Cells Research Articles

Alternative routes for the establishment of surface polarity during compaction of the mouse embryo

During the process of compaction, mouse 8-cell blastomeres flatten upon each other and polarize along an axis perpendicular to cell contacts. If the process of flattening is prevented, polarization can still occur, but does so in a lower proportion of cells than for control populations, and without the normal contact-directed orientation. We compared contact-directed and noncontact-directed processes to see if they involve common mechanisms. In nonflattened cells, surface polarization was favored in cells with nuclei located close to the cell surface, and the positions of surface poles and of nuclei tended to coincide. We present evidence that microtubules are involved in the development of microvillous poles associated with nuclei. In contrast it is known that polarization of microvilli occurs in the absence of microtubules if blastomeres are allowed to flatten. We conclude that surface polarization of mouse blastomeres can be accomplished by at least two alternative routes. One requires flattening but is independent of microtubules, and another can occur without flattening but involves a microtubule-mediated interaction between the nucleus and the cell cortex. It seems that both these pathways operate in the undisturbed embryo.

Influence of Nuclear and Cytoplasmic Activity on the Development in Vivo of Sheep Embryos after Nuclear Transplantation

Reconstituted sheep embryos have been produced by electrofusion-mediated nuclear transplantation in which single cells derived from embryos at the 16-cell (Day 4) and the inner cell mass (ICM) of early blastocyst stage (Day 6) were fused to unfertilized enucleated secondary oocytes. Electrofusion rates were higher with 16-cell blastomeres (82%) than with ICM cells (47%) and when an alternating current (a.c.) preceded the direct current (d.c.) fusing pulse (88% vs. 47%). The addition of cytochalasin B (7.5% micrograms/ml) to the medium for 1 h after electrofusion significantly improved the development to morula-blastocyst stage of reconstituted embryos derived from both 16-cell blastomeres (11% vs. 35%) and ICM cells (0% vs. 56%), indicating that the cytoskeletal mechanisms operating at the time of oocyte activation may affect the developmental potential of exogenous nuclei. Transplantation of 22 reconstituted morulae and blastocysts from both groups into the uteri of recipient ewes led to the development of four lambs (18%) with the phenotype of the nuclear donor breed. These findings indicate that at least some nuclei derived from transcriptionally active embryos are totipotent and able to be reprogrammed to support full-term development when fused to enucleated secondary oocytes.

Evolutionary modification of cell lineage in the direct-developing sea urchin Heliocidaris erythrogramma

The sea urchin Heliocidaris erythrogramma undergoes direct development, bypassing the usual echinoid pluteus larva. We present an analysis of cell lineage in H. erythrogramma as part of a definition of the mechanistic basis for this evolutionary change in developmental mode. Microinjection of fluoresceinated tracer dye and surface marking with vital dye are used to follow larval fates of 2-cell, 8-cell, and 16-cell blastomeres, and to examine axial specification. The animal-vegetal axis and adult dorsoventral axis are basically unmodified in H. erythrogramma. Animal cell fates are very similar to those of typically developing species; however, vegetal cell fates in H. erythrogramma are substantially altered. Radial differences exist among vegetal blastomere fates in the 8-cell embryo: dorsal vegetal blastomeres contribute proportionately more descendants to ectodermal and fewer to mesodermal fates, while ventral vegetal blastomeres have a complementary bias in fates. In addition, vegetal cell fates are more variable than in typical developers. There are no cells in H. erythrogramma with fates comparable to those of the micromeres and macromeres of typically developing echinoids. Instead, all vegetal cells in the 16-cell embryo can contribute progeny to ectoderm and gut. Alterations have thus arisen in cleavage patterns and timing of cell lineage partitioning during the evolution of direct development in H. erythrogramma.

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos.

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.

Analysis of the fifth cell cycle of mouse development

The 5th cell cycle of mouse development was analyzed to determine the lengths of each cell cycle phase. The DNA content of Feulgen-stained blastomere nuclei was measured at various times throughout the cell cycle by microdensitometry. To achieve precise timing of the start of the 5th cell cycle, experiments utilized isolated 16-cell blastomeres and cell pairs obtained by in-vitro division of isolated 8-cell blastomeres. The following estimates were made for a mixed population of polar and apolar 16-cell blastomeres: G1, less than or equal to 2 h; S, 8-9 h; G2 + M, 2 h. No significant difference was found in the timing of DNA synthesis between polar and apolar cells or between cell pairs and whole embryos.

The influence of cell contact on the division of mouse 8-cell blastomeres.

The pattern of division of polarized 8-cell blastomeres with respect to the axis of cell polarity has been compared (i) for cells dividing alone with cells dividing in pairs, and (ii) for early and late dividing cells within a pair. Cell interactions do not seem to influence significantly the overall pattern of division within the population. The only significant difference found was that the second dividing cell in a pair tended to divide in the same way as its earlier dividing companion slightly more frequently than expected. These results suggest that cell interactions immediately prior to and during division do not influence strongly the orientation and position of the division plane. In contrast, interactions between the cells within an intact early 8-cell embryo, which is subsequently disaggregated to singletons or pairs, do influence the type of progeny generated at division to the 16-cell stage, and seem to do so via an effect on the size of the microvillous region generated at the cell apex.

Cytocortical organization during natural and prolonged mitosis of mouse 8-cell blastomeres.

Late 8-cell blastomeres were harvested within the first 45 min after entering mitosis. Some mitotic cells were analysed within the ensuing 2 h for the organization of their surface in relation to their progress through mitosis. Whereas in most late interphase cells microvilli were restricted to a discrete polar region, in mitotic cells at all stages from early metaphase to immediately postcytokinesis microvilli were found to be present over more of the cell surface. Other mitotic cells were placed in nocodazole to arrest them in M-phase for up to 10 h. They were found to show an even more extensive distribution of microvilli over the whole surface, the longer periods of incubation yielding more extended coverage such that many cells no longer appeared to have any residual surface polarity. Removal from nocodazole at all time points from 1 to 10 h resulted in most cells completing mitosis to yield pairs of cells which, in most cases, resembled pairs derived from nonarrested blastomeres and in which a defined polar area of microvilli was restored. However, the percentage of differentiative divisions decreased after 6 h arrest. If, instead of removing cells from nocodazole, they were placed in both nocodazole and cytochalasin D (CCD) for periods of up to 3 h, most microvilli retracted to reveal a tight polar zone of CCD-resistant microvilli. This result suggests that a heterogeneity of cytocortical organization may still exist within the arrested mitotic cell. We propose a model to explain the origin of this heterogeneity of organization and its relationship to the generation of cell diversity.

Nuclear cytoplasmic interactions following nuclear transplantation in mouse embryos.

We have investigated the development of reconstituted embryos in which enucleated 1- or 2-cell embryos received various advanced nuclei. Enucleated 1-cells developed to the blastocyst stage only when an early 2-cell donor nucleus was transferred but very rarely if the donor nucleus was derived from a late 2-cell, early 4-cell or mid 8-cell embryo. Although an 8-cell nucleus could only support development of an enucleated zygote to the 2-cell stage, it did express the hsp 68/70 X 10(3) Mr proteins that are characteristic of the first embryonic gene activity. These polypeptides were absent in enucleated zygotes that did not receive a donor nucleus. Moreover, an 8-cell nucleus transferred to an enucleated late 2-cell blastomere could also support preimplantation development provided that the nuclear:cytoplasmic ratio was maintained as in intact 2-cell blastomeres. 8-cell nuclei transferred to zygotes that retained at least one pronucleus were able to support development to the blastocyst stage provided that the pronucleus was both fully transcriptionally active and present beyond the late 1-cell stage. This study suggests an active and continued helper role of the resident pronucleus for the participation by an 8-cell nucleus in reconstituted eggs.

Cytokeratin filament assembly in the preimplantation mouse embryo

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse embryo.

A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst

The allocation of cells to the trophectoderm and inner cell mass (ICM) in the mouse blastocyst has been examined by labelling early morulae (16-cell stage) with the short-term cell lineage marker yellow-green fluorescent latex (FL) microparticles. FL is endocytosed exclusively into the outside polar cell population and remains autonomous to the progeny of these blastomeres. Rhodamine-concanavalin A was used as a contemporary marker for outside cells in FL-labelled control (16-cell stage) and cultured (approximately 32- to 64-cell stage) embryos, immediately prior to the disaggregation and analysis of cell labelling patterns. By this technique, the ratio of outside to inside cell numbers in 16-cell embryos was shown to vary considerably between embryos (mean 10.8:5.2; range 9:7 to 14:2). In cultured embryos, the trophectoderm was derived almost exclusively (over 99% cells) from outside polar 16-cell blastomeres. The origin of the ICM varied between embryos; on average, most cells (75%) were descended from inside nonpolar blastomeres with the remainder derived from the outside polar lineage, presumably by differentiative cleavage. In blastocysts examined by serial sectioning, polar-derived ICM cells were localised mainly in association with trophectoderm and were absent from the ICM core. In nascent blastocysts with exactly 32 cells an inverse relationship was found between the proportion of the ICM descended from the polar lineage and the deduced size of the inside 16-cell population. From these results, it is concluded that interembryonic variation in the outside to inside cell number ratio in 16-cell morulae is compensated by the extent of polar 16-cell allocation to the ICM at the next division, thereby regulating the trophectoderm to ICM cell number ratio in early blastocysts.

Endocytic traffic in trophectoderm and polarised blastomeres of the mouse preimplantation embryo.

Endocytosis from apical and basolateral cell membranes of mouse blastocyst trophectoderm was examined morphologically by using unconjugated horseradish peroxidase (HRP, fluid phase marker), cationized ferritin (membrane marker, bound ionically), and protein A-HRP conjugate (membrane marker, identifying antigens recognised by antimouse species serum). The markers were applied in single and double labelling procedures designed to reveal the derivation, sorting site, and fate of all the major endocytic pathways. Endocytosis at the apical surface led to the obligate fusion of labelled elements with prelysosomal endosomes prior to the redistribution of membrane into lysosomal, transcellular, or recycling pathways, and to the passage of internalised fluid into lysosomes only. Complementary routes appear to operate following endocytosis at the basolateral domain. Thus, endocytic trafficking within the trophectoderm, regulated by "sorting" mechanisms localised at the endosome compartment, may be responsible for the maintenance of polarised membrane domains. The polarised transcellular pathway involving obligatory endosome fusion is present in cleavage-stage, peripheral 16-cell blastomeres prior to zonular tight junction formation. Nocodazole treatment to depolymerize microtubules in many cases induced a bypass of the endosomal sorting compartment during transcytosis, indicating that microtubules contribute to the spatial organization of endocytic membrane traffic.

The generation of cell surface polarity in mouse 8-cell blastomeres: the role of cortical microfilaments analysed using cytochalasin D

The mechanism by which a surface pole of microvilli is generated in mouse 8-cell blastomeres has been investigated. 4-cell and 8-cell embryos (or cell couplets) were incubated for precise times during their respective cell cycles in medium containing cytochalasin D (CCD) to disrupt the microfilament system. The blastomeres were analysed immediately for the distribution and state of organization of their microvilli, using three morphological techniques. The results indicate that the surface pole, characterized by microvilli containing CCD-resistant core filaments, is not generated by the gradual segregation of stable microvilli to the apical surface. An alternative model is proposed, based upon the stabilization of the apical cytocortex prior to the elongation of apical microvilli and the destabilization of cytocortical elements in contact areas and the shortening and loss of basolateral microvilli.

The role of cell adhesion in the synchronization and orientation of polarization in 8-cell mouse blastomeres

A detailed investigation into the activity of the homotypic, Ca2+-dependent cell-cell adhesion system (CDS) in the early mouse embryo has revealed its involvement in the synchronizing of the time of polarization of 8-cell blastomeres, and the orienting of the axis of polarization. Since polarization marks an important and early event in the process of cell diversification in the mouse embryo, it is concluded that the CDS provides an important component of the system by which the temporal and spatial elements of normal development are integrated.

A dissection of the mechanisms generating and stabilizing polarity in mouse 8- and 16-cell blastomeres: the role of cytoskeletal elements

Pairs of 8-cell or 16-cell blastomeres were cultured for up to 9 h after their formation from isolated 1/4 or 1/8 blastomeres respectively. Blastomeres were examined for the incidence and orientation of their surface polarity, as assessed by binding of FITC-Con A and by distribution of microvilli, and of their cytoplasmic polarity, as assessed by distribution of cytoplasmic actin, clathrin and a 100 kD antigen associated with the lysosomal/acid vesicle fraction of membranous organelles. The effect on polarity of incubating the pairs of cells in taxol, nocodazole, cytochalasin D or in a combination of nocodazole plus cytochalasin D for different parts of the incubation period was examined. Neither the development nor the stability of the surface polarity in 8-cell blastomeres was blocked by any treatment and only the use of CCD in combination with nocodazole affected the incidence of surface polarity appreciably. However, with some treatments, the form and position of the surface poles were modified. In the presence of microtubule inhibitors surface poles extended over a larger area of the cell surface, while exposure to CCD led to poles that were not opposite to the contact point between cells. In contrast to surface polarity, the development of cytoplasmic polarity was suppressed by both microtubule- and microfilament-inhibiting drugs, which also reversed it rapidly. In polar 16-cell blastomeres surface polarity was influenced in a similar manner to that of 8-cell blastomeres, only the combined use of cytochalasin D and nocodazole having any major effect. Polarization of clathrin in polar 16-cell blastomeres was inhibited almost completely by all drug treatments applied including cytochalasin D. The focal concentration of lysosomal antigen that occurs during the 16-cell stage was reduced only in the continuous presence of nocodazole plus cytochalasin D, but once established was not reversed appreciably by any drug. However, the localization of the lysosomal antigen to the basal region of polarized cells did not seem to occur in the presence of any drug. The dissociation of surface and cytoplasmic polarity revealed in these experiments leads us to conclude that either surface polarity is a prerequisite for the organization of cytoplasmic polarity, and mediates the latter via the cytoskeleton, or surface and cytoplasmic polarity develop by parallel but separate mechanisms.

Changes in the distribution of membranous organelles during mouse early development

The unfertilized oocyte, fertilized egg and early embryo (2-cell to 16-cell) of the mouse have been examined immunocytochemically for the distribution of antigens associated with the endoplasmic reticulum, the lysosomal and acidic vesicle fraction (100 kD antigen), Golgi apparatus (135 kD antigen) and coated vesicles (clathrin). The distribution of these antigens has also been examined in isolated 8-cell and 16-cell-stage blastomeres of various ages and phenotypes. Endoplasmic reticulum is detected only weakly in the oocyte and egg, but is seen abundantly at later stages both in association with the nuclear membrane and evenly distributed throughout the cytoplasm, except in regions of cell:cell apposition from which it is excluded. Intracellular clathrin is associated with the spindle in mitotic and meiotic cells. During interphase, clathrin is distributed throughout the cell until the mid-8-cell stage when it is concentrated into the apical region of the cell under the region of membrane at which a surface pole of microvilli will form subsequently. Thus, the cytoplasmic polarization of clathrin precedes overt polarization at the surface. At mitosis, the clathrin relocates to the spindle and is distributed to both daughter cells. It resumes an apical location beneath the surface pole of microvilli in polar daughter 1/16 cells, but remains dispersed in apolar daughter 1/16 cells. Both the lysosomal and Golgi antigens are distributed throughout the cytoplasm until the early 16-cell stage. In pairs of 16-cell blastomeres both antigens aggregate in a single cluster and do so whether the surface phenotype of the blastomeres is polar or apolar. The position of this cluster is not consistently related to the point of contact with the other cell in the pair but there is a suggestion that in cells with a polar surface phenotype the polar foci of Golgi/lysosomal antigens are located between the nucleus and the surface pole at earlier time points, but shift to a position between the basolateral membrane and the nucleus at the later time point. In intact 16-cell embryos also, the aggregated Golgi/lysosomal antigens of polar cells appear to localize to the basal region. The distributions of these various organelles in embryonic cells reported here show a number of differences from those reported previously for mature, differentiated cells.

Electrofusion of mouse blastomeres

Fusion of blastomeres of 2-cell mouse embryos with an intact zona pellucida can be induced with electric pulses. Fusion was most frequent with the field strength of 1 kV/cm and direct current pulses of 100–250 μsec duration. An electrolyte solution (PBS) can be used instead of a non-electrolyte solution (0.3 M mannitol). The viability of blastomeres fused in these two types of solution is similar. Fused 2-cell blastomeres develop into tetraploid blastocysts but die after implantation. Embryos in which blastomeres failed to fuse despite the treatment (diploid controls) can develop till term. The technique can also be applied to 3- and 4-cell embryos and to zona-free oocytes and blastomeres.

The distribution of cytoplasmic actin in mouse 8-cell blastomeres

Three non-homogeneous patterns of cytoplasmic actin distribution have been demonstrated in pairs of 8-cell blastomeres. Newly formed blastomeres showed an actin distribution associated with the remnant of the previous mitotic spindle. Subsequently blastomeres showed a zonal clearing of actin from regions of intercellular contact, the extent of the clearing increasing with the extent of contact. A polarized distribution of actin was evident from the early to mid 8-cell stage and coincided with the movement of nuclei towards the point of intercellular contact. The detection of polar actin preceded by 2-4 h the detection of a surface polarity as assessed by the FITC-Con A binding pattern and the distribution of cortical microvillous actin. However, once a surface pole of microvilli had formed, it persisted under conditions which led to loss of polar cytoplasmic actin. Incubation in cytochalasin D (CCD) resulted in a dispersed homogeneous pattern of actin distribution but did not prevent the formation of surface poles as assessed both by the Con A binding pattern and detection of polar microvilli. However, the poles formed were less clearly defined and the density and length of microvilli within them was variable. Moreover, when CCD was added early during the 8-cell stage the position of the poles was frequently not on an axis perpendicular to the point of intercellular contact. Cytochalasin D also affected the movement of the nucleus that occurs during the process of polarization. On the basis of these experiments, we conclude that actin is likely to be involved in the events of polarization, but that its precise role remains to be determined.

The fifth cell cycle of the mouse embryo is longer for smaller cells than for larger cells

Newly-formed pairs of 16-cell blastomeres were collected by periodic observation of isolated 8-cell blastomeres. Any pairs formed by division were recovered and classified as being composed of 1/16 blastomeres that differed in size or were of similar size. All of the latter and some of the former were then cultured for periods of up to 20h. The remaining pairs of different-sized blastomeres were disaggregated to larger or smaller cells. Some of these were reaggregated as smaller: smaller or larger: larger pairs, and these, together with the remaining isolated smaller and larger blastomeres were also cultured for up to 20h. At hourly intervals, cultured cells were sampled and analysed for incidence of division. It was found that larger cells divided on average after 12h whereas smaller cells divided on average after 14h.

The roles of phenotype and position in guiding the fate of 16-cell mouse blastomeres

Newly formed 16-cell blastomeres were typed as larger or smaller, labelled with the short-term lineage marker FITC, and aggregated in various spatial arrays with 15 other age-matched unlabelled 16-cell blastomeres. The aggregates were cultured for 8 or 24 hr and the fluorescently labelled progeny identified. In all but 6 of 185 cases, the progeny developed as a physically coherent patch. Labelled larger cells placed on the outside of the aggregate generated mainly trophectoderm; when placed on the inside or randomly they always generated at least one trophectodermal offspring and in some cases also contributed cells to the inner cell mass (ICM). Labelled smaller cells placed on the inside of the aggregate generated mainly ICM; when placed on the outside or randomly they generated cells in the ICM alone, in trophectoderm alone, or in both tissues. From these results we conclude that phenotype is of major importance in determining the fate of larger cells whereas position strongly influences the fate of smaller cells.

Induction of polarity in mouse 8-cell blastomeres: specificity, geometry, and stability.

We studied the cellular mechanisms underlying the induction of polarity in individual blastomeres of the 8-cell mouse embryo. The ability to induce polarity is lacking in the membranes of unfertilized and newly fertilized mouse eggs, then develops during the 2-cell stage, and is present in membranes of cells from 4-, 8-, and 16-cell stages. The axis of polarity takes 3-5 h to become established and thereafter appears to be stable. Multiple cell contacts affect the orientation of the axis of polarity, and no polarity develops in cells which are totally surrounded. Polarized cells show evidence of an limited capacity for slight adjustments in their position relative to other cells. The implications of these results for the mechanisms by which a blastocyst is generated are discussed briefly.