Cell identity is specified in the early mammalian embryo by the generation of precursors for two cell lineages: the pluripotent inner cell mass and differentiating trophectoderm. Here we identify Angiomotin as a key regulator of this process. We show that the loss of Angiomotin, together with Angiomotin-like 2, leads to differentiation of inner cell mass cells and compromised peri-implantation development. We show that Angiomotin regulates localization of Yap, and Yap-binding motifs are required for full activity of Angiomotin. Importantly, we also show that Angiomotin function can compensate for the absence of Lats1/2 kinases, indicating the ability of Angiomotin to bypass the classical Hippo pathway for Yap regulation. In polarized outside cells, Angiomotin localizes apically, pointing to the importance of cell polarity in regulating Yap to promote differentiation. We propose that both Hippo pathway-dependent and Hippo pathway-independent mechanisms regulate Yap localization to set apart pluripotent and differentiated lineages in the pre-implantation mouse embryo.
The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the mouse embryo beyond the blastocyst stage in vitro. We established two types of medium to be applied sequentially, and we used a substrate permitting high-resolution imaging of the transition from blastocyst to egg cylinder. We developed two variants of this protocol: the first starts with intact early blastocysts that upon zona removal can attach to the substrate and develop into egg cylinders after 5 d, and the second starts with late blastocysts that upon dissection of the mural trophectoderm form egg cylinders in only 3 d. This method allows observation of a previously hidden period of development, and it provides a platform for novel research into peri-implantation embryogenesis and beyond.
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