Epithelial cells connect via cell-cell junctions to form sheets of cells with separate cellular compartments. These cellular connections are essential for the generation of cellular forms and shapes consistent with organ function. Tissue modulation is dependent on the fine-tuning of mechanical forces that are transmitted in part through the actin connection to E-cadherin as well as other components in the adherens junctions. In this report we show that p100 amotL2 forms a complex with E-cadherin that associates with radial actin filaments connecting cells over multiple layers. Genetic inactivation or depletion of amotL2 in epithelial cells in vitro or zebrafish and mouse in vivo, resulted in the loss of contractile actin filaments and perturbed epithelial packing geometry. We further showed that AMOTL2 mRNA and protein was expressed in the trophectoderm of human and mouse blastocysts. Genetic inactivation of amotL2 did not affect cellular differentiation but blocked hatching of the blastocysts from the zona pellucida. These results were mimicked by treatment with the myosin II inhibitor blebbistatin. We propose that the tension generated by the E-cadherin/AmotL2/actin filaments plays a crucial role in developmental processes such as epithelial geometrical packing as well as generation of forces required for blastocyst hatching.A central question during development is how single cells form functional multi-cellular organ structures. The high reproducibility indicates intricate synchronization of cellular processes such as migration, proliferation and cell shape changes. Much attention has been focused on how growth factors form biochemical gradients that govern some of these processes 1-3 . However, less is known regarding how mechanical signals or forces modulate cell shape and control cellular expansion 4, 5 . Cells perceive and respond to exogenous mechanical forces via different points of contact in the outer membrane. Forces exerted on the extra-cellular matrix are detected by epithelial cells via integrins in focal adhesions which transfer tension from the extracellular matrix to the cytoskeleton 6 . Low rigidity in the extra-cellular matrix transfers less extracellular force and thereby promotes the formation of organ-like epithelial structures in vitro whereas increased force or stiffness in the matrix causes loss of tissue architecture associated with tumor progression and promotes cell proliferation [7][8][9][10] . Recent evidence has also shown that actomyosin contractility is transmitted via the adherens junctions. External forces applied to cadherins have indicated a mechanical coupling between the cytoplasmic domain of cadherin and the actin cytoskeleton 11 . Cellular interactions and the force-mediated morphological changes are also important for the processes involved in organ development. One example is apical contraction where the

The assembly of individual epithelial or endothelial cells into a tight cellular sheet requires stringent control of cell packing and organization. These processes are dependent on the establishment and further integration of cellular junctions, the cytoskeleton and the formation of apical-basal polarity. However, little is known how these subcellular events are coordinated. The (Angiomotin) Amot protein family consists of scaffold proteins that interact with junctional cadherins, polarity proteins and the cytoskeleton. In this report, we have studied how these protein complexes integrate to control cellular shapes consistent with organ function. Using gene-inactivating studies in zebrafish and cell culture systems in vitro, we show that Par3 to be essential for localization of AmotL2 to cellular junctions to associate with VE/E-cadherin and subsequently the organization of radial actin filaments. Our data provide mechanistic insight in how critical processes such as aortic lumen expansion as well as epithelial packing into hexagonal shapes are controlled.