PMC

(A) Design of the experiment shown in (B).
(B–E) Left column: 3-D representations of the 64-AB cell stages of various experiments. The eight AB-derived regions are colour-coded independently of their fate to show the topology of each region. Middle column: “Löwe projections” (see A for explanation). Right column: 3-D representations from which the contribution of cell movements was removed (“mitoses only”, see legend to ). In all experiments presented, these “mitoses only” representations resemble the original embryonic fragment, which suggests that the shape of the embryonic fragments is mainly due to cleavage orientation.
(B) Combination of an isolated AB blastomere (ABa) and a blastomere which touched P₂ transiently (ABp). Four of the eight AB-derived regions of the ABp blastomere (right side of embryonic fragment; blue, violet, dark yellow, and light yellow) elongate comparable to regions in the experiment where a single isolated AB transiently touched P₂ (see D). These regions align in a V-shape configuration, possibly because their elongation is hampered by the descendants of the other AB blastomere (left side of embryonic fragment). The “Löwe projections” show that the cells of the ABp blastomere (right) divide mainly in the direction of the point where P₂ was transiently added. Additionally, the cleavages of the originally nonpolarised ABa blastomere (left) are mainly directed towards the added polarised ABp blastomere (which is here represented by the centre of the target area).
(C) Combination of two isolated AB blastomeres. Both cells have never been exposed to a polarising signal, similar to the ABa blastomere in (B). A round structure with nonelongated regions forms (, row K). The direction of cleavages appears to be nonpolarised compared to (B).
(D) Single AB blastomere that transiently touched P₂. Cells mainly divide in the direction where the point of contact between AB and P₂ has been. A nicely elongated structure forms (, row Q). This elongation resembles the behaviour of the polarised ABp blastomere in (B).
(E) Experiment in which a mom-2 P₂ blastomere touched a wild-type AB blastomere transiently. The ABp fate is induced by P₂ but, because of the absence of a polarising signal, cells behave in a nonpolarised way compared to the experiment where a wild-type P₂ blastomere was added transiently to AB-derived blastomeres (D).

c-kit gene targeted Knock-in analysis by single blastomeres. (A) The ratio of each modified blastomere of 36 nt ssODN and CRISPR/nuclease injection KI embryos is shown. Circles represent an injected embryo in each condition, and each color indicates the blastomere sequence. Red indicates a precise KI homozygous blastomere, blue indicates heterozygous blastomere of precise KI and wild type, green represents a heterozygous precise KI and KO (indel) mutation blastomere, and yellow indicates homozygous or heterozygous KO blastomeres. Embryo number (no) s in this illustration are correlated to the Embryo no. in Supplementary Tables  and . KI: knock-in, KO: knock-out. (B) Representative Sanger sequencing chromatograms of modified c-kit and putative amino acid sequences derived from blastomere sequence analyses of c-kit in the KI experiment correlate with the blastomere samples listed in Supplemental Table . (a) Wild-type blastomere (Embryo 1, 36nt-AS and CRISPR/nuclease injection). (b) KI blastomere (Embryo 2, 36nt-S and CRISPR/nuclease injection). Blue and red arrows indicate knock-in of donor ssODN, red arrows and letters indicate mutations contributing to E579K mutant (W³⁷). (c) KI/wild-type heterozygous blastomere (Embryo 3, 36nt-S and CRISPR/nuclease injection). (d) Imprecise KI blastomere (Embryo 1, 36nt-S and CRISPR/nuclease injection). The top sequences indicate the KI donor ssODN. The insertion (black arrows) caused a frameshift mutation and introduced a stop codon (*).

Figure 4. Oct4 and Cdx2 expression in developmentally arrested blastomere and blastocyst derived from uncut blastomere (One blastomere of the 2-cell embryo was isolated and cultured as control), and time-dependent caspase-3 staining in early embryos. (A) Oct4 and Cdx2 expression in blastocyst derived from uncut blastomere was similar to that of the control, while developmentally arrested cut blastomere showed faint staining of Oct4 and Cdx2 (arrow); (B) Cleaved caspase-3 staining was detected in the cut blastomere at the blastocyst stage; (C) Caspase-3 staining was evident 48 h after microcut in the cut blastomere (white arrow), and was increasing by 72 h. The caspase-3 staining was located in the nuclear region of the cut blastomere, and later extended to the whole cell as indicated in (bar = 50 μm).

Copy number variation detection in three blastomere samples. (a,c,e) Genome-wide CNV detection of blastomere A (a), blastomere E (c) and blastomere G (e) from embryo 20 before channel clone normalization. (b,d,f) Genome-wide CNV detection of blastomere A (b), blastomere E (d) and blastomere G (f) from embryo 20 after channel clone normalization. The x-axis represents the coordinate range from chromosome 1 to × and the y-axis represents the log2 ratios. The blue line represents the CBS segmentation line. The green regions represent the duplication region and red regions represent the deletion region called by the CGHcall program.