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Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

ABSTRACT: In multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

SUBMITTER: Maemura M

PROVIDER: S-EPMC8160171 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Totipotency of mouse zygotes extends to single blastomeres of 4-cell stage embryos

Project description:In multicellular organisms, heterogametes of oocytes and sperms are fertilized and resulting zygotes give rise to new individuals. The ability of zygotes that produce a fully formed individual from single cell when placed in a supportive environment is defined as totipotency. Given that totipotent cells are the source of all multicellular organisms, better understanding of totipotency has a profound effect on not only biology but also our society. However, the exact distribution of totipotent cells in mammals remains elusive, although zygotes and single blastomeres at 2-cell stage embryos has been thought to be only mouse cells to be totipotent. We now show that a single blastomere isolated from 2- and 4-cell stage embryos gives rise to a fertile adult individual when it placed in a uterus, although isolation of blastomeres at these stage results in the disturbance of transcriptome in single blastomere derived embryos. Single blastomeres from 8-cell and morula stage embryos that were separately cultured in vitro exhibited severe defects in the formation of epiblast and primitive endoderm in the inner cell mass and the development to blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of 4-cell stage embryos.

2021-05-27 | GSE163427 | GEO

Totipotency segregates between the sister blastomeres of two-cell stage mouse embryos.

Project description:Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. This model of totipotency was inferred from a minority of cases in which blastomeres produced monozygotic twins in mice. Was this due to experimental limitation or biological constraint? Here we removed experimental obstacles and achieved reliable quantification of the prevalence of dual totipotency among mouse two-cell stage blastomeres. We separated the blastomeres of 1,252 two-cell embryos, preserving 1,210 of the pairs. Two classes of monozygotic twins became apparent at the blastocyst stage: 27% formed a functional epiblast in both members (concordant), and 73% did so in only one member of the pair (discordant) - a partition that proved insensitive to oocyte quality, sperm-entry point, culture environment and pattern of cleavage. In intact two-cell embryos, the ability of sister blastomeres to generate epiblast was also skewed. Class discovery clustering of the individual blastomeres' and blastocysts' transcriptomes points to an innate origin of concordance and discordance rather than developmental acquisition. Our data place constraints on the commonly accepted idea that totipotency is allocated equally between the two-cell stage blastomeres in mice.

| S-EPMC5557898 | biostudies-literature

Totipotency of mouse zygotes extends to single blastomeres of 4-cell stage embryos

Project description:Totipotency of mouse zygotes extends to single blastomeres of 4-cell stage embryos

| PRJNA686092 | ENA

Developmental capacity is unevenly distributed among single blastomeres of 2-cell and 4-cell stage mouse embryos.

Project description:During preimplantation development, mammalian embryo cells (blastomeres) cleave, gradually losing their potencies and differentiating into three primary cell lineages: epiblast (EPI), trophectoderm (TE), and primitive endoderm (PE). The exact moment at which cells begin to vary in their potency for multilineage differentiation still remains unknown. We sought to answer the question of whether single cells isolated from 2- and 4-cell embryos differ in their ability to generate the progenitors and cells of blastocyst lineages. We revealed that twins were often able to develop into blastocysts containing inner cell masses (ICMs) with PE and EPI cells. Despite their capacity to create a blastocyst, the twins differed in their ability to produce EPI, PE, and TE cell lineages. In contrast, quadruplets rarely formed normal blastocysts, but instead developed into blastocysts with ICMs composed of only one cell lineage or completely devoid of an ICM altogether. We also showed that quadruplets have unequal capacities to differentiate into TE, PE, and EPI lineages. These findings could explain the difficulty of creating monozygotic twins and quadruplets from 2- and 4-cell stage mouse embryos.

| S-EPMC8563712 | biostudies-literature

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos.

Project description:BACKGROUND: Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS: Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS: Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT-PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION: We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.

| S-EPMC2763129 | biostudies-literature

Transcriptome asymmetry within mouse zygotes but not between early embryonic sister blastomeres.

Project description:Transcriptome regionalization is an essential polarity determinant among metazoans, directing embryonic axis formation during normal development. Although conservation of this principle in mammals is assumed, recent evidence is conflicting and it is not known whether transcriptome asymmetries exist within unfertilized mammalian eggs or between the respective cleavage products of early embryonic divisions. We here address this by comparing transcriptome profiles of paired single cells and sub-cellular structures obtained microsurgically from mouse oocytes and totipotent embryos. Paired microsurgical spindle and remnant samples from unfertilized metaphase II oocytes possessed distinguishable profiles. Fertilization produces a totipotent 1-cell embryo (zygote) and associated spindle-enriched second polar body whose paired profiles also differed, reflecting spindle transcript enrichment. However, there was no programmed transcriptome asymmetry between sister cells within 2- or 3-cell embryos. Accordingly, there is transcriptome asymmetry within mouse oocytes, but not between the sister blastomeres of early embryos. This work places constraints on pre-patterning in mammals and provides documentation correlating potency changes and transcriptome partitioning at the single-cell level.

| S-EPMC3101998 | biostudies-literature

Transcript profiling of individual twin blastomeres derived by splitting two-cell stage murine embryos.

Project description:In invertebrates and amphibians, informational macromolecules in egg cytoplasm are organized to provide direction to the formation of embryonic lineages, but it is unclear whether vestiges of such prepatterning exist in mammals. Here we examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. mRNA from 26 blastomeres derived from 13 embryos approximately mid-way through their second cell cycle was subjected to amplification. Twenty amplified samples were hybridized to arrays. Of those samples that hybridized successfully, 12 samples in six pairs were used in the final analysis. Probes displaying normalized values >0.25 (n = 4573) were examined for consistent bias in expression within blastomere pairs. Although transcript content varied between both individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes, with only 178 genes displaying a >1.4-fold difference in expression across all six pairs. Although class discovery clustering showed that blastomere pairs separated into two distinct groups in terms of their differentially expressed genes, when the data were tested for significance of asymmetrical expression, only 39 genes with >1.4-fold change ratios in six of six blastomere pairs passed the two-sample t-test (P < 0.05). Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were among the most abundant, differentially distributed mRNA, suggesting that a stochastically based lack of synchrony in cell cycle progression between the two cells might explain at least some and possibly all of the asymmetries in transcript composition.

| S-EPMC3043130 | biostudies-literature

Single-cell RNA-seq of blastomeres from 2- to 32-cell stage mouse embryos

Project description:Transcriptomes were determined for all blastomeres of 28 embryos at the 2- (n=8), 4- (n=16) and 8-cell (n=4) stages, and for individual cells taken from 16- (n=6) and 32- (n=6) cell stage embryos. We also carefully monitored the 2- to 4-cell divisions noting whether the cleavage plane was meridional (M, along the Animal-Vegetal (AV) axis marked by the second attached polar body) or equatorial (E, bisecting the AV axis) and the order in which such divisions occurred. This resulted in four groups of 4-cell stage embryos: ME, EM, MM and EE, which were all collected 10 hours after the first 2- to 4-cell division.

2016-03-23 | E-MTAB-3321 | biostudies-arrayexpress

Developmental bias in cleavage-stage mouse blastomeres.

Project description:The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing.When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development.The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.

| S-EPMC3543519 | biostudies-literature

Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.

Project description:Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1?400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ?0.8 ?g (16-cell embryo) to ?0.2 ?g (50-cell embryo), the number of protein group identifications declined from 1?466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32-, and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development.

| S-EPMC4940028 | biostudies-literature

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