Re-evaluation of the causes of variation among mouse aggregation chimaeras.

The composition of adult mouse aggregation chimaeras is much more variable than X-inactivation mosaics. An early theoretical model proposed that almost all the extra variation in chimaeras arises, before X-inactivation occurs, by spatially constrained, geometrical allocation of inner cell mass (ICM) cells to the epiblast and primitive endoderm (PrE). However, this is inconsistent with more recent embryological evidence. Analysis of published results for chimaeric blastocysts and mid-gestation chimaeras suggested that some variation exists among chimaeric morulae and more variation arises both when morula cells are allocated to the ICM versus the trophectoderm (TE) and when ICM cells are allocated to the epiblast versus the PrE. Computer simulation results were also consistent with the conclusion that stochastic allocation of cells to blastocyst lineages in two steps, without the type of geometrical sampling that was originally proposed, could cause a wide variation in chimaeric epiblast composition. Later allocation events will cause additional variation among both chimaeras and X-inactivation mosaics. We also suggest that previously published U-shaped frequency distributions for chimaeric placenta composition might be explained by how TE cells are allocated to the polar TE and/or the subsequent movement of cells from polar TE to mural TE.

Selection against BALB/c strain cells in mouse chimaeras.

It has been shown previously that BALB/c strain embryos tend to contribute poorly to mouse aggregation chimaeras. In the present study we showed that BALB/c cells were not preferentially allocated to any extraembryonic lineages of mouse aggregation chimaeras, but their contribution decreased during the early postimplantation period and they were significantly depleted by E8.5. The development of BALB/c strain preimplantation embryos lagged behind embryos from some other strains and the contribution that BALB/c and other embryos made to chimaeras correlated with their developmental stage at E2.5. This relationship suggests that the poor contribution of BALB/c embryos to aggregation chimaeras is at least partly a consequence of generalised selection related to slow or delayed preimplantation development. The suitability of BALB/c embryos for maximising the ES cell contribution to mouse ES cell chimaeras is also discussed.

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing.

BACKGROUND: The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/- hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3. RESULTS: Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/- hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/- hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex. CONCLUSIONS: Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.

Fate of tetraploid cells in 4n<-->2n chimeric mouse blastocysts.

Previous studies have shown that tetraploid (4n) cells rarely contribute to the derivatives of the epiblast lineage of mid-gestation 4n<-->2n mouse chimeras. The aim of the present study was to determine when and how 4n cells were excluded from the epiblast lineage of such chimeras. The contributions of GFP-positive cells to different tissues of 4n<-->2n chimeric blastocysts labelled with tauGFP were analysed at E3.5 and E4.5 using confocal microscopy. More advanced E5.5 and E7.5 chimeric blastocysts were analysed after a period of diapause to allow further growth without implantation. Tetraploid cells were not initially excluded from the epiblast in 4n<-->2n chimeric blastocysts and they contributed to all four blastocyst tissues at all of the blastocyst stages examined. Four steps affected the allocation and fate of 4n cells in chimeras, resulting in their exclusion from the epiblast lineage by mid-gestation. (1) Fewer 4n cells were allocated to the inner cell mass than trophectoderm. (2) The blastocyst cavity tended to form among the 4n cells, causing more 4n cells to be allocated to the hypoblast and mural trophectoderm than the epiblast and polar trophectoderm, respectively. (3) 4n cells were depleted from the hypoblast and mural trophectoderm, where initially they were relatively enriched. (4) After implantation 4n cells must be lost preferentially from the epiblast lineage. Relevance of these results to the aetiology of human confined placental mosaicism and possible implications for the interpretation of mouse tetraploid complementation studies of the site of gene action are discussed.

Evaluation of the mouse TgTP6.3 tauGFP transgene as a lineage marker in chimeras.

The mouse TgTP6.3 transgene, encoding a tauGFP fusion protein, is becoming widely used but has yet to be fully characterized and evaluated as suitable lineage marker. The aim of the present study was to investigate the phenotype of TgTP6.3(+/+) homozygotes and TgTP6.3(+/-) hemizygotes, characterize the expression of the TgTP6.3 transgene in different tissues and critically evaluate its use as a lineage marker. TgTP6.3(+/+) homozygotes died between embryonic day 14.5 and weaning, whereas TgTP6.3(+/-) hemizygotes were mostly viable and fertile but smaller than non-transgenic siblings. TgTP6.3 expression began in the late two-cell stage, persisted in most fetal and adult tissues and was uniformly expressed in many (but not all) tissues. TgTP6.3(+/-) cells were readily identified in many chimeric tissues and their contribution appeared to be quantitatively and spatially normal. Overall, tauGFP expression in hemizygous TgTP6.3(+/-) cells fulfils the main criteria of a good lineage marker for many tissues. It provides a useful lineage marker, which should be particularly suitable for axons, blood vessels and pre-implantation embryos.