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.     

Cellular distribution of RNA populations in 16-cell stage embryos of the sand dollar, Dendraster excentricus

RNA was extracted from pure preparations of micromeres and meso-plus macromeres isolated from 16-cell stage embryos of Dendraster excentricus. Molecular hybridization-competition experiments disclosed that the binding of 16-cell stage labeled RNA to denatured sperm DNA was competed equally well by micromere RNA, meso-plus macromere RNA, total 16-cell RNA and unfertilized egg RNA, indicating the egg-type populations were distributed almost equally in the different blastomeres. In contrast, experiments with ³H-RNA extracted from micromeres obtained from pulse-labeled 16-cell stage embryos showed qualitative differences when unfertilized egg RNA and total 16-cell stage RNA were used as competitors. Such differences in RNA populations could not be detected in ³H-RNA isolated from the meso-plus macromere fraction.     

Properties of polar and apolar cells from the 16-cell mouse morula

Summary The surface properties of newly formed, isolated 1/16 mouse blastomeres have been analyzed over the 10–12 h period prior to their division to 2/32 cells. Two populations of cells are formed at the 8- to 16-cell transition and their surface phenotypes vary with their relative position within the morula. Outer cells are polar, relatively non-adhesive and relatively large; inner cells are apolar, adhesive and smaller. The surface phenotypes of both inner and outer 1/16 cells are stable during culture for 11 h in isolation. However, the surface phenotypes can be induced to change by culture in combination with a second 1/16 cell, in a manner that is dependent upon the identity of the second cell. Two aggregated polar cells never flatten completely against each other, and both cells retain a clearly defined polar phenotype for 11–12 h. In aggregates of two apolar cells, cell outlines are lost as a result of intercellular flattening and microvilli are displaced away from areas of cell contact. However, if the two apolar cells are subsequently separated an even distribution of microvilli is restored. In most aggregates of an apolar and a polar cell, the polar cell envelops the apolar cell completely. These results are discussed in the context of the normal fate and potential of each cell type within the morula.     

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.     

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.     

Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme : II. The 16- and 32-cell stages

Cell lineages during development of ascidian embryos were analyzed by injecting horseradish peroxidase as a tracer enzyme into identified cells of the 16-cell and 32-cell stage embryos of Halocynthia roretzi. Most of the blastomeres of these embryos developed more kinds of tissues than have hitherto been reported, and therefore, the developmental fates of each blastomere are more complex. It has been thought that every blastomere of the 64-cell stage ascidian embryo gives rise to only one kind of tissues, but the finding that the several blastomeres at the 32-cell stage developed into at least three different kinds of tissues, clearly indicates that the stage at which the fates of every blastomere are determined to one tissue is later than the 64-cell stage. The results also clearly demonstrate that muscle cells are derived not only from B-line cells (B5.1, B5.2, B6.3, and B6.4) but also from A-line cells (A5.2 and A6.4) and b-line cells (b5.3 and b6.5). Based on the present analysis as well as other studies, complete cell lineages of muscle cells up to their terminal differentiation have been proposed. In addition, lineages of nervous system, notochord, and epidermis are also discussed.     

Fates of the blastomeres of the 16-cell stage Xenopus embryo

The fate of each of the blastomeres in the 16-cell stage Xenopus embryo which had been carefully selected for stereotypic cleavages was determined by intracellularly marking a single blastomere with horseradish peroxidase and identifying the labeled progeny in the tailbud embryo by histochemistry. Each blastomere populated all three primary germ layers. The progeny of each blastomere were distributed characteristically both in phenotype and in location. For example, most organs were populated by the descendants of particular sets of blastomeres. Furthermore, within an organ the progeny of a single blastomere were restricted to defined spatial addresses. This study describes the fates of identified 16-cell stage blastomeres and demonstrates that they are distinct and predictable if embryos are preselected for stereotypic cleavages.     

Regional differences of proteins in isolated cells of early embryos of Xenopus laevis

Regional differences of proteins were studied by two-dimensional gel electrophoresis in early embryos of Xenopus laevis. Pairs of blastomeres on the dorso-ventral axis were isolated from 16- and 32-cell embryos. Some dorso-ventral differences have been detected at 32-cell embryos. The proteins which were clearly detectable in the vegetal cells of the ventral marginal zone were only faintly detectable or undetectable in those of the dorsal marginal zone, and a regionally specific spot was detected in dorsal blastomeres.     

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.     

Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.     

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.     

Mesoderm formation by isolated and cultivated 8-cell stage blastomeres of the teleost, Leucopsarion ptersii (shiro-uo).

Isolation of cleavage-stage blastomeres and the study of their developmental potential has been used extensively for analyzing the mechanisms of embryogenesis in vertebrates, including amphibians and echinoderms. We devised a method to isolate 8-cell stage blastomeres in the teleost, shiro-uo, by utilizing its unique cleavage pattern of the horizontal 3rd cleavage plane. Removal of all the upper blastomeres at the 8-cell stage allowed almost normal embryogenesis from the remaining lower blastomeres and yolk cell mass. Isolated upper or lower blastomeres formed vesicles and spherical bodies, which later showed morphological changes during cultivation. Mesoderm formation was detected not only in the cultivated lower blastomeres or whole blastomeres but also in the upper blastomeres isolated from the yolk cell mass at the 8-cell stage, although at a lower frequency than the lower blastomeres. These results indicated the presence of very early signaling for mesoderm induction, which is independent from the currently postulated signals from the yolk syncytial layer at later stages. This also indicated non-equivalence or differentiation of the blastomeres from the very early cleavage stage in teleost embryos.     

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.     

Induction of murine 8-cell blastomere polarity by F9 embryonal carcinoma cells

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage of development. This polarity forms as a result of a specific cell-cell interaction that has been termed induction. The ability of embryonal carcinoma (EC) cells to induce 8-cell blastomere polarization has been investigated by aggregating nonpolar 8-cell blastomeres with various types of EC cells. F9, a nullipotent stem cell, induced polarization of a nonpolar 8-cell companion in 80% of the aggregates. Stimulation of differentiation of F9 cells with retinoic acid (RA), with or without dibutyryl cAMP, caused a reduction in the polarity-inducing ability of these cells. Other EC cells, PSA-1, NULLI-SCC1, 3TDM, C3HNE, and P10, all displayed less polarity-inducing activity than F9. In addition, it was observed that when any of these cell types assumed a more differentiated phenotype, either spontaneously or in response to specific stimuli, they displayed a decrease in their ability to induce 8-cell polarization. As a control, the inducing ability of cells from normal mouse tissues was examined. It was found that neither STO mouse fibroblasts nor primary cultures of mouse lymphocytes were able to induce significant polarization of 8-cell stage blastomeres. These data support the hypothesis that while undifferentiated stem cell populations retain the ability to induce 8-cell blastomere polarization, it is apparently lost upon cellular differentiation.     

Developmental autonomy of presumptive notochord cells in partial embryos of an ascidian

Summary

Ultrastructural features of larval notochord cell differentiation, sheath (membrane leaflets and filaments) and vacuoles of intracellular colloid, were found in some cells of certain partial embryos of the ascidian, Ciona intestinalis. As expected from established lineage fate maps, mature quarter-embryos developing from microsurgically isolated anterior-vegetal blastomeres (A4.1 pair) at the 8-cell stage had some cells with the notochord features. Such cells, however, also occurred in quarter-embryos resulting from the posterior-vegetal blastomere pair (B4.1) and in partial embryos derived from the B5.1 cell pair isolated at the next cleavage of the B4.1 blastomeres. These findings confirm a prediction of additional notochord cell fates from a recent revision of the ascidian lineage map based on cell marking with microinjected horseradish peroxidase. Partial embryos obtained from other lineages of the 8- and 16-cell stages did not develop notochord cells.     

Duration of competence and inducing capacity of blastomeres in notochord induction during ascidian embryogenesis

Notochord cells in ascidian embryos are formed by the inducing action of cells of presumptive endoderm, as well as neighboring presumptive notochord, at the 32-cell stage. Studies of the timing of induction using recombinations of isolated blastomeres have suggested that notochord induction must be initiated before the decompaction of blastomeres at the 32-cell stage and is completed by the 64-cell stage. However, it is not yet clear how the duration of notochord induction is strictly limited. In the present paper, the aim was to determine in detail when the presumptive notochord blastomeres lost their competence to respond, and when the presumptive endoderm blastomeres produced inducing signals for the notochord. Presumptive notochord blastomeres and presumptive endoderm blastomeres were isolated from early 32-cell embryos, and were heterochronously recombined at various stages ranging from the early 32-cell stage to the 64-cell stage. Presumptive notochord blastomeres could respond to inductive signals at the early 32-cell stage, and started to lose their responsiveness at the decompaction stage. By contrast, the presumptive endoderm blastomeres persisted in their inducing capacity even at the 64-cell stage. These observations suggest that the loss of competence in presumptive notochord blastomeres limits the duration of notochord induction in intact ascidian embryos.     

Delay of polarization event increases the number of Cdx2-positive blastomeres in mouse embryo

During preimplantation mouse embryo development expression of Cdx2 is induced in outer cells, which are the trophectoderm (TE) precursors. The mechanism of Cdx2 upregulation in these cells remains unclear. However, it has been suggested that the cell position and polarization may play a crucial role in this process. In order to elucidate the role of these two parameters in the formation of TE we analyzed the expression pattern of Cdx2 in the embryos in which either the position of cells and the time of polarization or only the position of cells was experimentally disrupted. Such embryos developed from the blastomeres that were isolated from 8-cell embryos either before or after the compaction, i.e. before or after the cell polarization took place. We found that in the embryos developed from polar blastomeres originated from the 8-cell compacted embryo, the experimentally imposed outer position was not sufficient to induce the Cdx2 in these blastomeres which in the intact embryo would form the inner cells. However, when the polarization at the 8-cell stage was disrupted, the embryos developed from such an unpolarized blastomeres showed the increased number of cells expressing Cdx2. We found that in such experimentally obtained embryos the polarization was delayed until the 16-cell stage. These results suggest that the main factor responsible for upregulation of Cdx2 expression in outer blastomeres, i.e. TE precursors, is their polarity.     

Transfer of nuclei from 8-cell stage mouse embryos following use of nocodazole to control the cell cycle

Mouse 2-, 4-, 8-, and 16-cell embryos were exposed to nocodazole in M16 culture medium. The effect of different concentrations and exposure times on the efficiency of cell cycle synchronization and the development of the treated embyros after release from the drug was determined. The minimum effective concentration (95% of arrested nuclei) for 4-, 8-, and 16-cell embryos was 5μM nocodazole. The effect upon subsequent development of mouse embryos depended upon both the stage of development of the embryo at treatment (P < 0.001) and the length of exposure to nocodazole (P < 0.001). Exposure to any concentration of nocodazole within the range 2.5–10 μM for 12 hr caused a reduction in the proportion of embryos that formed blastocysts. As the period of exposure to 5μM nocodazole increased from 12 to 24 hr, the proportion of embryos developing to the blastocyst stage decreased. The lower proportion of embyros developing to the blastocyst stage and to term (P < 0.01) suggests that the more advanced stages were more susceptible to damage as a result of exposure to nocodazole. The rate of development of 4-cell embryos to blastocysts was not affected when an exposure time of 9 hr was used. Together these results show that it is possible to use nocodazole to arrest mouse embryonic cells in mitosis but that it is not appropriate to culture the embryos in the presence of this drug for prolonged periods. Individual blastomeres completed mitosis at 60–90 min and started DNA synthesis at 120–150 min after release from nocodazole. Nuclei from blastomeres thus synchronized were used to conduct studies on the effect of the cell cycle on nuclear transfer. A signficant effect was found. When nuclei from 8-cell embryos in G1 or S-phase were used as nuclei donors, development to blastocyst was respectively 27% and none. ©Wiley-Liss, Inc.     

Cell surface interaction induces polarization of mouse 8-cell blastomeres at compaction

The development of the polarized surface binding of the fluoresceinated ligand concanavalin A (FITC-Con A) was studied in blastomeres of the early mouse embryo. Single 8-cell blastomeres, natural 8-cell couplets derived from the in vitro division of individual 4-cell blastomeres, and reaggregated couplets made from dissociated 8-cells were cultured for varying periods of time and on a variety of substrata. The development of surface polarity was found to be highly dependent upon cell contact. Over 50% of the cells in couplets were polarized after 4–5 hr in culture, with the smaller cell in the couplet usually more advanced in its polarization than the larger cell. The orientation of the poles of FITC-Con A binding was opposite the point of contact between cells in the couplets regardless of their previous orientation within the embryo or the plane of cleavage.