A novel cell death phenotype in the developing mouse embryos

The pro-amniotic cavity is formed after implantation and expands up to the gastrulation stage in E7.5 embryos. We detected in the growing cavity high abundance of casapae3/9 activation co-localizing with fragmented nuclei, and strong positive TUNEL staining. A detailed analysis of Apaf1 knock out (KO) embryos revealed that activation of caspase 9/3 at these stages was Apaf1 dependent. Nevertheless, these embryos displayed the normal number and organization of the three epiblast layers and regular cavity size. We therefore looked for additional caspase-independent mechanisms that could either function in parallel or be induced exclusively in the Apaf1 KO to compensate for the attenuation of caspase activation. Strikingly, while analyzing in details the epiblast cells surrounding the cavity in WT E7.5 embryos, by transmission electron microscopy (TEM), we identified a novel cell death phenotype. Many of the cells in the epiblast layer facing the lumen were extremely small, they did not carry a nucleus, and some displayed increased number of mitochondria. Most importantly, these enucleated epiblasts were shedding parts of their content into the lumen including the different intracellular organelles and other cytosolic components, all surrounded by intact membranes. Notably, this gradual elimination of the epiblasts by shedding and enucleation was clearly caspase independent as was also identified in the KO Apaf1 embryos. We suggest that these tightly regulated death mechanisms evolved to control the epiblast cell number/layer organization and cavity size at these stages of embryonal development. These findings document a new and exciting cell death mechanism, which is unique for this stage of embryonic development.