Pluripotency deficit in clones overcome by clone-clone aggregation: epigenetic complementation?

Abstract

Abnormal gene expression patterns in somatic cell clones and their attrition in utero are commonly considered a consequence of errors in nuclear reprogramming. We observe that mouse clone blastocysts have less than half the normal cell number, and that higher cell number correlates with correct expression of Oct4, a gene essential for peri-implantation development and embryonic pluripotency. To increase the cell number, we aggregated genetically identical clones at the 4-cell stage. Clone-clone aggregates did not form more blastocysts, but the majority expressed Oct4 normally and had higher rates of fetal and postnatal development. Fertilized blastocysts with low cell numbers, induced by removal of two blastomeres at the 4-cell stage, did not exhibit abnormal Oct4 expression, indicating that improved gene expression and post-implantation development of clone-clone aggregates is not a consequence of increased cell number. Rather, we propose that complementation of non-cell-autonomous defects of genetically identical, but epigenetically different, embryos results in improved gene expression in clone-clone aggregates.

Fig. 1. Distribution of total cell number grouped into intervals of 10 cells for clone, fertilized and clone–clone aggregate embryos at 96 h of development. Top, distribution of total cell counts in clone and fertilized mouse embryos. Bars indicate the proportion (%) of embryos with counts that fall within each interval. The 50th percentile of the fertilized embryo distribution (80–89 total cells) coincides with the 98th percentile inclusive of the clone embryo distribution. Clones, n = 136; fertilized embryos, n = 225. Bottom, comparison of total cell counts of single clones and 2X and 3X clone–clone aggregates at 96 h. 1X clones, n = 16; 2X clones, n = 70; 3X clones, n = 45.

Fig. 2. Spatial Oct4 expression in blastocysts and outgrowths of 1X, 2X and 3X clones and fertilized embryos. Top, Oct 4 mRNA distribution in blastocyst-stage cumulus cell clones, clone–clone aggregates and fertilized embryos was assessed by ISH. Outgrowths were analyzed for expression of Oct4–GFP. Bottom, analysis of Oct4 mRNA distribution and cell number in single and aggregate clone embryos. Subsequent to ISH and evaluation of Oct4 mRNA distribution, individual embryos were mounted on slides in a squash preparation (left) and stained with propidium iodide (right) for determination of the total cell number.

Fig. 3. Development of clone–clone aggregates to the blastocyst stage and full-term. (A) Blastocyst-stage fertilized (top) and clone–clone (bottom) 1X, 2X and 3X aggregation chimeras. (B) Development of 2X clone–clone aggregates to 19.5 d.p.c. Uterus from female receiving 29 blastocysts of 2X blastocyst-stage clones, with four fetuses. (C) Four 2X clone–clone chimeras after Caesarean section.

Fig. 4. Total cell number and spatial Oct4 expression in clones, controls and bisected embryos. Embryos at 96 h of development were analyzed by ISH with an Oct4-mRNA-specific antisense riboprobe and subsequently stained with propidium iodide to determine cell counts in squashed preparations (see Figure 2, bottom).