In the mouse embryo, asymmetric divisions during the 8-16 cell division generate two cell types, polar and apolar cells, that are allocated to outer and inner positions, respectively. This outer/inner configuration is the first sign of the formation of the first two cell lineages: trophectoderm (TE) and inner cell mass (ICM). Outer polar cells become TE and give rise to the placenta, whereas inner apolar cells become ICM and give rise to the embryo proper and yolk sac. Here, we analyze the frequency of asymmetric divisions during the 8-16 cell division and assess the relationships between cell polarity, cell and nuclear position, and Hippo signaling activation, the pathway that initiates lineage-specific gene expression in 16-cell embryos. Although the frequency of asymmetric divisions varied in each embryo, we found that more than six blastomeres divided asymmetrically in most embryos. Interestingly, many apolar cells in 16-cell embryos were located at outer positions, whereas only one or two apolar cells were located at inner positions. Live imaging analysis showed that outer apolar cells were eventually internalized by surrounding polar cells. Using isolated 8-cell blastomeres, we carefully analyzed the internalization process of apolar cells and found indications of higher cortical tension in apolar cells than in polar cells. Last, we found that apolar cells activate Hippo signaling prior to taking inner positions. Our results suggest that polar and apolar cells have intrinsic differences that establish outer/inner configuration and differentially regulate Hippo signaling to activate lineage-specific gene expression programs.

The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.

LKB1/PAR-4 is essential for the earliest polarization steps in Caenorhabditis elegans embryos and Drosophila oocytes. Although LKB1 (also known as STK11) is sufficient to initiate polarity in a single mammalian intestinal epithelial cell, its necessity in the formation and maintenance of mammalian epithelia remains unclear. To address this, we completely remove LKB1 from mouse embryos by generating maternal-zygotic Lkb1 mutants and find that it is dispensable for polarity and epithelia formation in the early embryo. Instead, loss of Lkb1 leads to the extrusion of cells from blastocyst epithelia that remain alive and can continue to divide. Chimeric analysis shows that Lkb1 is cell-autonomously required to prevent these extrusions. Furthermore, heterozygous loss of Cdh1 exacerbates the number of extrusions per blastocyst, suggesting that LKB1 has a role in regulating adherens junctions in order to prevent extrusion in epithelia.

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The aggregation and accumulation of amyloid beta peptides is one of the pathological hallmarks of Alzheimer's disease (AD). Amyloid beta is produced by sequential proteolytic cleavage of Amyloid Precursor Protein (APP) by beta‐ and gamma‐secretase, which are plausible molecular targets for AD treatment. Thus, identification of modulators of these secretases could lead to new therapeutics for this prevalent and debilitating disease. SH‐SY5Y are human neuroblastoma cells that express APP and the various secretases that cleave this membrane‐bound protein into sAPPβ, sAPPα and amyloid beta peptides. We have developed new highly sensitive and specific AlphaLISA kits that allow the detection of endogenous amounts of these various APP metabolites. Specific inhibitors of alpha‐, beta‐ and gamma‐secretase were used to modulate the relative activities of these enzymes. Application of these inhibitors resulted in characteristic signature levels of product sAPPα, sAPPβ, Aβ 1–40 and Aβ x‐40 (defined as the combined amount of both Aβ 1–40 and Aβ 17–40) from SH‐SY5Y cell culture supernatants. These results show that the new AlphaLISA kits have sufficient sensitivity and specificity to study this pathway, even in wild‐type cells that do not overexpress APP or BACE. The Z′‐factor obtained for these cell‐based secretase assays was greater than 0.6, indicating reproducible and robust assays for HTS. In summary, these new AlphaLISA detection kits can accurately quantitate endogenous APP cleavage products. This will in turn result in a better understanding of the APP degradation pathway and facilitate the identification of novel therapeutic drugs for the treatment of Alzheimer's disease.