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

Abstract

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.

Figure 1

Micromanipulation and culture of two-cell mouse embryos to generate monozygotic (MZ) twins. Embryos were held in a micromanipulation medium lacking calcium and phosphate. A holding pipette (right side in the picture of series A) applied negative pressure to hold a two-cell stage embryo at the equatorial region, aligning its cleavage axis with the pipette’s long axis. Using a spiked beveled pipette as a bisection tool (left side in the picture), a slit was made in the zona pellucida (ZP) at the upper pole of the embryo (A1). The upper blastomere was pushed through the slit by gently pressing the bisection pipette against the ZP at the equator, while the pressure in the holding pipette was slightly reduced (A2–A3). The second blastomere was released by pressing the ZP below the equator (A4–A5). Separated blastomeres recovered their round shape within a few minutes after bisection (A6). Upon culture in KSOM(aa) medium, the sister blastomeres cleaved and developed to the four-cell stage (planar conformation, (B1); tetrahedral conformation, (B2); open chain conformation, B3) and further to the blastocyst stage (B4). When MZ blastocysts were transferred as a pair to the same uterine horn, cases were recorded in which two fetuses were found in that horn (B5), suggesting that at least some two-cell embryos were totipotent in both blastomeres. Monozygotic twins formed blastocysts at the same rate (C1), comprising half the number of cells as unmanipulated blastocysts (C3). After transfer to the uterus, blastocysts obtained from bisected and then immediately reunited blastomeres yielded as many fetuses as unmanipulated controls (C2). The error bars associated with the diagram bars are standard deviations. Twin pairs that formed two blastocysts were equally frequent among the sister blastomeres, as were twin pairs that arrested at pre-blastocyst stages (C4).

Figure 2

Functional analysis of MZ twin pairs. Single pairs of MZ twin blastocysts were transferred to the uteri of pseudopregnant recipients (one pair per recipient), using DZ twins and reconstructed embryos (separated-reunited blastomeres) as controls (A). Single pairs of MZ twin blastocysts were transferred to a layer of feeder cells to enable outgrowth formation and ES cell derivation (B). Statistical comparisons were conducted on the pairs of MZ and DZ twins, and on the individual embryos of DZ twins and separated-reunited blastomeres (Fisher’s exact test). Gray-shaded areas visualize the comparison of MZ and DZ twins with each other; black-framed areas visualize the comparison of DZ twins and reconstructed controls with each other. n.a., not applicable. n.p., not performed. The functional status of the outgrowths was validated by scoring the expression of Oct4-GFP (C–E; “1” refers to light microscopy, “2” to fluorescence microscopy).

Figure 3

Cell lineage analysis of blastocysts. Immunoconfocal fluorescence images of representative MZ twin blastocysts, co-stained for cell lineage-specific markers: CDX2 for TE (A1,B1), SOX17 for pEnd (A2,B2) and NANOG for EPI (A3,B3). Microscope objective 20X, 0.75 N.A. Pairs were scored as either “concordant” (A) or “discordant” (B) based on the “9-2-2 criterion” (see main text). The proportions of concordant and discordant pairs were similar irrespective of culture medium, ovarian stimulation, co-culture, sperm-entry point and embryo conformation at the four-cell stage (tetrahedral, T; planar, P; open chain, O). The error bars associated with the proportions are standard deviations. (C). Linear correlation analysis (Pearson’s r) of the cells counted in the lineage compartments of each MZ pair shows that the main contributor to the discordance was the EPI lineage (D).

Figure 4

Tracing of the contribution of sister two-cell stage blastomeres to epiblast. Each blastomere in two-cell embryos were microinjected with fluorescent dextran beads of different colors (OGDB, OY568DB), followed by in vitro culture to blastocyst and NANOG immunofluorescence analysis (A). The quality of the microinjections was validated by survival rates of the blastomeres, blastocyst rates, and total cell numbers of blastocysts (B). The relative contribution of each two-cell blastomere to EPI (ratio from 0.5 to 1) and the absolute total number of EPI cells (•) are shown for non-bisected (C) and twin (D) embryos. Error bars in (B) are standard deviations. Black and gray bars in (C) and (D) indicate embryos cultured as pools and embryos cultured individually, respectively.

Figure 5

Transcriptome analysis of MZ twin pairs. Individual transcriptomes are shown as heat maps, with higher expression coded in red and lower expression coded in green, and linear correlation coefficient written on the heat maps (A,C). Constellation maps of the transcriptomes of individual blastocysts (B) and individual two-cell stage blastomeres (D) identified with both a number (for the original embryo subjected to bisection) and a letter (for the two members resulting from bisection). Unmanipulated embryos in B are labeled as controls (ctrl). The transcriptomes were subjected to hierarchical clustering analysis to see if they would return the original pair associations (e.g. 1a-1b, 7a-7b).