Figure 5.

mTORi does not affect secondary ICM founder cell generation and ICM cell numbers recover during blastocyst maturation (E3.5–E4.5) after prior mTORi from the late 8-cell stage. (a) Experimental scheme and quantification of inner cell numbers at 32-cell stage, as contributed by primary and secondary ICM cells, +/− Torin1. Primary ICM cell count was estimated by fixation of some embryos at 16-cell stage and quantification of their number of inner cells, to allow for the number of such primary and secondary founders to be determined at the 32-cell stage, when deducting this number and allowing for the extra cell division. (b) Experimental scheme and quantification of total cell number, ICM cell number and the numbers of NANOG + GATA4− (EPI), NANOG− GATA4+ (PrE) and NANOG + GATA4+ ICM cells, +/− Torin1 (E4.5). (c) Experimental scheme and quantification of the proportion of apoptotic ICM cells (positive for cleaved Caspase-3 IF staining), +/− Torin1 (E4.0 + 7 h). In all graphs, numbers of analysed embryos are shown.