Investigating the Mechanisms Behind Symmetry Breaking and Cell Fate Specification in the Mouse Embryo

In all animal species, the initially symmetric embryo undergoes symmetry breaking, where parts of the embryo gain different identities and form first cell fates. In the mouse embryo, symmetry is broken by the blastocyst stage (day 4) during pre-implantation development, where three distinct cell types have formed: trophectoderm (TE), primitive endoderm (PE) and epiblast (EPI) - the latter two forming the inner cell mass (ICM). Unlike other model systems, such as fly and worm model systems, the symmetry breaking event is not determined by a molecular gradient laid down in the egg, but rather is a more complicated event. The current model proposes that: cells of the embryo gain apical-basal polarity at the 8-cell stage, with subsequent cell divisions generating an outer polar population, and an inner apolar population. This biases cell fate to allocate the first asymmetric lineages, as the polar cells are biased towards TE and the apolar cells towards ICM.

My project focuses on forming a deeper understanding of this story.

First, I am looking at the possible role played by cells that gain apical-basal polarity early: while majority of the cells polarise after an event called 'compaction' at the 8-cell stage, some polarise before. This heterogeneity has important consequences for cell fate decisions, as cells that polarise early contribute preferentially to TE. I am investigating whether factors relating to heterogeneity at the 4-cell stage, such as CARM1 (an arginine methyltransferase known to be heterogeneous between cells of the 4-cell embryo), bias cells towards polarising early and thus have an influence on symmetry breaking in this manner. This can be accomplished using techniques such as overexpression and RNAi knockdown. If this study shows a link between early heterogeneity and early polarisation, I will attempt to identify the molecular pathway and cell machinery leading to early polarisation. There is evidence that normal polarisation is triggered by PKC-Rho GTPase signalling, so I will look at this in the context of early polarisation and 4-cell stage heterogeneity e.g. testing whether CARM1 activity would affect PKC and/or Rho GTPase activity. This can be accomplished using techniques such as a Forster resonance energy transfer (FRET) based RhoA sensor.

I am also looking at the factors that determine the division pattern of cells, which enable distribution of polarity factors during the 8 - 16 cell stage division. Some cells divide symmetrically to form two polar cells, and some asymmetrically to form one polar and one apolar cell. I will investigate how various factors, such as cell shape and apical domain size, affect division orientation, and how early polarising cells are biased towards TE through symmetric cell division. I can investigate this using fluorescent labelling of spindle and apical domains, as well as measurements and perturbations of cell shape and size.

My proposed work will significantly advance our understanding of symmetry breaking in the mouse embryo. Moreover, due to the high similarities between mouse and human pre-implantation development, the results yielded from this proposed work can also shed light on the beginnings of human life. This may have clinical benefits relating to pregnancy loss which occurs frequently during pre-implantation development. M

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