Our previous studies have shown that in
Our previous studies have shown that in a preimplantation mouse embryo the proportion of inner to outer cells (i.e. precursors of ICM and TE, respectively) is regulated by a different frequency of differentiative cell divisions at the 8- and 16-cell stages. Low number of inner blastomeres generated in the first round of differentiative divisions is compensated by the increased number of differentiative divisions in the second round (Krupa et al., 2014). Yet, it is not known how the pattern of blastomere divisions at the 8- and 16-cell stage is controlled, and how the number of inner cells arising in the first round influences the pattern of division during the next round. It seems plausible that the division pattern depends at least partially on spatial interactions between blastomeres. In this study we investigated how the modifications of these interactions, which are induced by the presence of additional exogenous cells in the embryo, influenced the pattern of divisions and, in consequence, affected the specification of TE and ICM. We used embryonic stem cells (ESCs), which are derived from ICM of a Teicoplanin and after re-introduction into the embryo contribute exclusively to the ICM of the resultant chimeric blastocyst and are predominantly incorporated into the EPI (Morgani et al., 2013), (Poueymirou et al., 2007). Thus, they exhibit “inner-like” character. We microsurgically introduced ESCs into 8-cell embryos and examined how their presence affects the proportion of differentiative divisions of the blastomeres at the 8-, 16-, and 32-cell stages and, in consequence, the number of embryo-derived cells in the ICM of the blastocysts. We show that the presence of additional inner-like ESCs leads to the emergence of a subsequent two-step mechanism regulating final proportions of TE and ICM cells within the blastocyst. First, we observed decrease in the frequency of differentiative divisions of the blastomeres at the 8- to 16-cell stage transition, while the frequency of differentiative divisions in the second round (at the 16-cell stage) was unaffected. The second step occurred in the early 32-cell blastocyst, probably due to the presence of ESCs-derived cells colonizing ICM. Hence, outer blastomeres in ESCs-containing embryos at this stage cleaved in a differentiative manner significantly less frequently, complementing arising ICM with smaller number of embryo-derived cells than in control embryos. Furthermore, we proved that contribution of host cells to EPI lineage within ICM of obtained chimeric blastocysts negatively correlated with the number of ESCs introduced into the 8-cell embryos. In line with this, we noticed increased number of host ICM cells in the PE lineage of chimeric embryos, suggesting that ESCs instructed embryo ICM cells to acquire PE rather than EPI fate.
Discussion The number of inner and outer cells in the cleaving mouse embryo, and in consequence the number of ICM and TE precursor cells, is controlled by the type of blastomere divisions (either conservative or differentiative in relation to the inheritance of the apical domain) between the 8- and 32-cell stage. It has been observed previously, although never directly proven, that the pattern of blastomere divisions and, in consequence, number of inner and outer cells, depend on the number of cells generated in the previous wave of differentiative divisions (Krupa et al., 2014). In the present study we showed experimentally that the supernumerary inner-like cells (i.e. ESCs injected into early 8-cell embryos) affect the pattern of the subsequent blastomere divisions. Blastomeres of the 8-cell embryo injected with 8–10 ESCs cleaved in a conservative manner more often, and consequently, produced more outer blastomeres than control embryos. Most importantly, we obtained similar results when proportions of polar and apolar blastomeres, instead of outer and inner ones, were compared between ESCs-injected and control embryos. Therefore, we excluded a possibility that our conclusions were inaccurate due to the fact that some apolar cells may be transiently positioned outside before adapting the final inner position (Anani et al., 2014). We also found that the supernumerary inner-like cells do not affect frequency of differentiative divisions during 16- to 32-cell stage transition, but they significantly decrease the number of nascent blastocysts, in which the third round of differentiative divisions (at 32- to 64-cell stage transition) occurs.