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
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