When and how does cell division order influence cell allocation to the inner cell mass of the mouse blastocyst?

Aggregate 8-cell embryos were constructed from four 2/8 pairs of blastomeres, one of which was marked with a short-term cell lineage marker and was also either 4 h older (derived from an early-dividing 4-cell) or 4 h younger (derived from a late-dividing 4-cell) than the other three pairs. The aggregate embryos were cultured to the 16-cell stage, at which time a second marker was used to label the outside cell population. The embryos were then disaggregated and each cell was examined to determine its labelling pattern. From this analysis, we calculated the relative contributions to the inside cell population of the 16-cell embryo of older and younger cells. Older cells were found to contribute preferentially. However, if the construction of the aggregate 8-cell embryo was delayed until each of the contributing 2/8 cell pairs had undergone intercellular flattening and then had been exposed to medium low in calcium to reverse this flattening immediately prior to aggregation, the advantage possessed by the older cells was lost. These results support the suggestion that older cells derived from early-dividing 4-cell blastomeres contribute preferentially to the inner cell mass as a result of being early-flattening cells.