Three‐dimensional reconstruction of tetraploid↔diploid chimaeric mouse blastocysts

Abstract

Studies of tetraploid diploid (4n 2n) mouse chimaeras have demonstrated unequal contributions of 4n cells to different tissues of the midgestation conceptus. Such a pattern has also been reported in chimaeras as early as E3.5d, which show an enhanced contribution of 4n cells to the mural trophectoderm . In this study, sectioned 4n 2n and 2n 2n control chimaeric blastocysts were digitised and reconstructed in 3 dimensions (3-D). The 3-D images revealed only limited mixing of cells from the 2 contributing embryos of individual blastocysts in both chimaera groups. Consequently, the distribution pattern of the 2 cell types was dependent on the spatial relationship between the orientation of the blastocyst and the boundary between the 2 clusters of cells. The distribution patterns observed were not strikingly different for 4n 2n and 2n 2n chimaeras, each showing some transgenic positive cell contribution in all 3 identifiable developmental lineages. It was notable, however, that in all 4n 2n blastocysts at least some 4n cells were located adjacent to the blastocyst cavity. Such a consistent pattern was not evident in 2n 2n chimaeras. This study has demonstrated the value of 3-D reconstructions for the analysis of spatial relationships of 2 cell populations in chimaeric mouse blastocysts.Key words : Tetraploidy ; blastocyst ; chimaera ; chimera ; embryo. Tetraploid cells (4n) have been shown to be distributed nonrandomly in mouse postimplantation tetraploid\ diploid (4n\2n) mosaics (Tarkowski et al. 1977) and 4n 2n chimaeras (Lu & Markert, 1980 ;Nagy et al. 1990 ;James et al. 1995), readily contributing to the extraembryonic tissues but rarely to the fetus itself. Studies at E12.5 and E7.5 have revealed that 4n cells are not simply restricted to extraembryonic tissues but more specifically to trophectoderm and primitive endoderm derivatives (James et al. 1995). We also demonstrated that, as a group, 4n 2n blastocysts show a statistically significant greater contribution of 4n cells to the mural trophectoderm than to the inner cell mass or polar trophectoderm . This phenomenon appears to be related to ploidy not cell size at aggregation and Three-dimensional (3-D) reconstruction is potentially an extremely powerful tool for visualising the distribution of cell types within embryos. We produced 4n 2n aggregation chimaeras, in which the tetraploid component was hemizygous for the β-globin transgene TgN(Hbb-1)83Clo (Lo et al. 1987), and analysed them at the blastocyst stage by computer-aided 3-D reconstruction of histological sections. The aims were (1) to evaluate the use of 3-D reconstruction for the analysis of cell distributions in chimaeras and (2) to test whether any consistent pattern could be distinguished for the distribution of 4n cells in individual E3.5 4n 2n chimaeric blastocysts. Preliminary results from this study have been published in abstract form .

Tetraploid Cells of Enhanced Green Fluorescent Protein Transgenic Mice in Tetraploid/Diploid-Chimeric Embryos

“…Everett and West [12] and Everett et al [13] reported that 4n-cells made a greater contribution to the TE than to the ICM of E3.5 chimeras; in the present experiments, however, we did not find a high population of 4n-cells in the TE of E3.5 chimeras (data not shown), and chimeric embryos did not eliminate 4n-cells until E4.5. The results of the BrdU incorporation assay revealed that the capabilities of cell division and DNA synthesis of 4n cells were almost the same as those of 2n-cells until the 4-cell-compaction stage.…”

“…Because of this characteristic of 4n-cells, tetraploid (4n) blastocysts are utilized as host embryos to produce embryonic stem cells that contribute effectively to the fetus [9][10][11]. Moreover, it has been reported that 4n↔2n-chimeric embryos display a restricted distribution of 4n-cells among the mural trophectoderm even at an embryonic age of 3.5 days (E3.5) [12,13], and the contribution of 4n-cells to 4n↔2n-embryos decreases from E3.5 to E4.5 [14].…”

The case for prepatterning in the mouse

“…2B in Chroscicka et al, 2004). Such an extraordinarily high rate of seemingly precocious breakdown of coherent clonal growth, particularly in the trophectoderm lineage, has not been reported in previous lineage studies on either standard conceptuses or morula aggregation chimeras (Garner and McLaren, 1974; Pedersen et al, 1986; Dvorak et al, 1995; Gardner, 1997; Everett et al, 2000). Consequently, the possibility that it may relate to the injection‐related depression of cell number needs to be considered.…”

Tetraploid development in the mouse

“…The tendency of tetraploid cells to colonize the trophectoderm occurs at the blastocyst stage. This colonization may be accompanied by selection against tetraploid cells in the ICM, although at 4.5 days of development, the tetraploid cells may still be present at levels of 13% West, 1996, 1998;Everett et al, 2000). At days 7.5, 10.5, 12.5, 13.5, 16.5, and 17.5 of development, the tetraploid cells are observed to contribute to progressively smaller amounts of the ICM-derived tissues (Tarkowski et al, 1977;Nagy et al, 1990;James et al, 1995;Wang et al, 1997;Tang and West, 2000;Goto et al, 2002).…”