Analysis of the “Time Spent Outside” Hypothesis using Fluorescent Tracking of Ingressing Cells

Early embryogenesis is a self-organised process with remarkable plasticity. This early development is crucial in forming distinct cell lineages with varying levels of developmental potential. The first lineage decision transitions totipotent cells into the inner cell mass and trophoblast tissues. The second lineage decision of pre-implantation development segregates the inner cell mass into epiblast and hypoblast progenitors. The variation of this development between mammals is not fully understood, as most of what is known about mammalian cell-fate decisions has been extrapolated from the embryonic murine model. Embryonic development in mice differs greatly from other mammalian models, including humans and cattle. Insight into early lineage regulation is key in agricultural settings that require the application of artificial reproductive technologies. These industries require species-specific understanding that is not yet available. Cattle provide a new model of early mammalian embryogenesis that may prove more appropriate for human application.

Early models of mammalian embryonic development proposed that the location of a cell would determine its fate; inside cells will become epiblast progenitors and outside cells will become hypoblast progenitors. Termed the ‘Inside-Outside’ model, this hypothesis was disregarded as the understanding of signalling pathways key events such as compaction and polarisation evolved. However, recent investigations in mice determined that morphological events such as compaction, polarisation, and internalisation play a key role in early embryonic development. As cells of the compact embryo form a blastocyst, cells move into the inner cell mass in multiple, distinct waves. Fibroblast growth factor 4 is shown to mediate differential signalling of MAPK in early and late ingressing cells, through receptors Fgfr1 and Fgfr2. Late ingressing cells show increased expression of Fgfr1/2 that predispose them to the hypoblast lineage. This effect of a cell’s history on differential expression and lineage contribution is known as ‘The Time Spent Outside Hypothesis’.

The research in thesis aimed to determine if this mechanism of second lineage determination may be analogous in cattle. Outer cells of the compact morula were traced using lipophilic membrane stains, DiI and DiO, on Day 5 and Day 6 of development. Early ingressing cells were positive for DiI, while late ingressing cells were positive for DiI and DiO. Analysis with confocal microscopy revealed that inner cell mass cells stained with both DiI and DiO showed positive expression of the hypoblast marker SOX17. These results indicate an apparent lineage bias towards hypoblast commitment in cells that internalise to the inner cell mass at a later stage.