Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization.

Erg channel is critical in controlling cell volume during cell cycle in embryonic stem cells.

The cell cycle progression in mouse embryonic stem cells (mESCs) is controlled by ion fluxes that alter cell volume [1]. This suggests that ion fluxes might control dynamic changes in morphology over the cell cycle, such as rounding up of the cell at mitosis. However, specific channels regulating such dynamic changes and the possible interactions with actomyosin complex have not been clearly identified. Following RNAseq transcriptome analysis of cell cycle sorted mESCs, we found that expression of the K(+) ion channel Erg1 peaked in G1 cell cycle phase, which was confirmed by immunostaining. Inhibition of Erg channel activity caused loss of G1 phase cells via non-apoptotic cell death. Cells first lost the ability of membrane blebbing, a typical feature of cultured embryonic stem cells. Continued Erg inhibition further increased cell volume and the cell eventually ruptured. In addition, atomic force measurements on live cells revealed a decreased cortical stiffness after treatment, suggesting alterations in actomyosin organization. When the intracellular osmotic pressure was experimentally decreased by hypertonic solution or block of K(+) ion import via the Na, K-ATPase, cell viability was restored and cells acquired normal volume and blebbing activity. Our results suggest that Erg channels have a critical function in K(+) ion homeostasis of mESCs over the cell cycle, and that cell death following Erg inhibition is a consequence of the inability to regulate cell volume.

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra 'tissue movements' are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues.

Notch signalling defines critical boundary during budding in Hydra.

Boundary formation is an important mechanism of development and has been studied in a number of bilaterian model organisms where it is often controlled by Notch, FGF and Wnt signalling. Tissue boundaries are also formed in simple pre-bilaterian animals. The boundary between parent and bud during asexual reproduction in the fresh water polyp Hydra vulgaris is an example. The Hydra homolog of the FGF-receptor FGFR (kringelchen) and some components of the Wnt signalling pathway are expressed at this boundary, but their precise functions are unknown. In this work we have discovered an important role for Notch signalling at this boundary. Notch signalling is needed to sharpen the kringelchen expression zone during the final budding stages from an initially broad band into a clear line demarcating the boundary between bud and parent. Expression of the Notch target gene HyHes and the putative matrix metalloprotease MMP-A3 was observed at the boundary shortly before the bud began to constrict and differentiate foot cells. When Notch signalling was inhibited with the presenilin inhibitor DAPT the expression pattern for kringelchen changed dramatically into a diffused pattern. The expression of both HyHes and MMP-A3 was abolished. Moreover, morphogenesis of the bud was not completed and buds did not constrict, failed to form a foot and never detached from the parent. This resulted in the formation of two-headed animals. We suggest that the function of Notch signalling during budding in Hydra is in promoting the formation of two stripes of differing gene expression, which are needed to differentiate the foot of the bud and a progressing narrowing of the mesoglea on the side of the parent.

Wnt/beta-catenin and noncanonical Wnt signaling interact in tissue evagination in the simple eumetazoan Hydra.

In and evaginations of 2D cell sheets are major shape generating processes in animal development. They result from directed movement and intercalation of polarized cells associated with cell shape changes. Work on several bilaterian model organisms has emphasized the role of noncanonical Wnt signaling in cell polarization and movement. However, the molecular processes responsible for generating tissue and body shape in ancestral, prebilaterian animals are unknown. We show that noncanonical Wnt signaling acts in mass tissue movements during bud and tentacle evagination and regeneration in the cnidarian polyp Hydra. The wnt5, wnt8, frizzled2 (fz2), and dishevelled-expressing cell clusters define the positions, where bud and tentacle evaginations are initiated; wnt8, fz2, and dishevelled remain up-regulated in those epithelial cells, undergoing cell shape changes during the entire evagination process. Downstream of wnt and dsh expression, JNK activity is required for the evagination process. Multiple ectopic wnt5, wnt8, fz2, and dishevelled-expressing centers and the subsequent evagination of ectopic tentacles are induced throughout the body column by activation of Wnt/beta-Catenin signaling. Our results indicate that integration of axial patterning and tissue morphogenesis by the coordinated action of canonical and noncanonical Wnt pathways was crucial for the evolution of eumetazoan body plans.