Cell allocation in GFP chimeric blastocysts by confocal microscopy

Abstract

This study combined confocal microscopy with the use of a tau-GFP (green fluorescent protein) transgenic mouse strain to study cell fate in two main types of chimeric blastocysts. In each case one component of the chimera was known to contribute poorly to the fetal lineage at later stages. The experiments were designed to test whether this was due to non-random allocation to different tissues at the blastocyst stage.

The initial part of this thesis involved the establishment of confocal microscopy techniques and the characterisation of two novel tau-GFP transgenic mouse strains. A method of culturing embryos on the confocal microscope was established for use in further studies. Two tau-GFP transgenic mouse lines, TgTP6.3 and TgTP6.4, were evaluated for their use in following chimera studies by assessing the timing of the onset of GFP expression during preimplantation development and the viability of heterozygote and homozygote mice.

The remaining studies involved the use of tau-GFP chimeric embryos. Mouse tetraploid«-*diploid chimeras have previously been used as a model of confined placental mosaicism (CPM). Approximately 2% of human conceptuses investigated by chorionic villus sampling contain chromosomally abnormal cells that are confined to the placenta. This condition, known as human CPM, can lead to incorrect prenatal diagnosis.

Animal models would be useful for investigating the mechanisms responsible for the exclusion of abnormal cells from the fetus. As spontaneous chromosomal mosaicism is rare in mouse embryos, mouse aggregation chimeras have been used as a model. Previous results have shown that tetraploid cells are excluded from the epiblast derivatives, including the fetus, of mid gestation tetraploid*-* diploid chimeras.

Tetraploid cells have been shown to be preferentially allocated to the trophectoderm, in particular the mural trophectoderm, of mouse tetraploid-^diploid blastocysts. However, tetraploid cells are present within the inner cell mass region of the blastocyst. Therefore, the current study used tau-GFP tetraploid*-*diploid aggregation chimeras to determine if tetraploid cells are present within the epiblast and lost later or are excluded from the epiblast region by preferential allocation to the hypoblast. Tetraploid<-*diploid chimeras were produced using TgTP6.3 embryos. Analysis of these chimeras at E3.5 and E4.5 has confirmed that tetraploid cells are preferentially allocated to the mural trophectoderm. However, tetraploid cells were present within the region of the blastocyst that forms the epiblast. Analysis of expanded chimeric blastocysts at E5.5 and E7.5, produced by transferring them to delayed implantation females, also showed that tetraploid cells were present within the epiblast region. This suggests that tetraploid cells are initially present within the epiblast region but lost from the epiblast later by some mechanism of cell selection against tetraploid cells.

Embryos from some inbred strains, such as BALB/c, also tend to contribute poorly to chimeras, so producing 'unbalanced chimeras'. Therefore unbalanced BALB/c chimeras could be a possible model of CPM. BALB/c^GFP aggregation chimeras were analysed using the established time-lapse technique. This was to determine if BALB/c cells are underrepresented in mid-gestation BALB/c chimeras by preferential allocation of BALB/c cells to the mural trophectoderm. These results showed that BALB/c cells were not preferentially allocated to the mural trophectoderm and indicate that a general cell selection mechanism takes place.