The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothelial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by noninvasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells. We demonstrate that alterations in shear stress, ciliogenesis, or expression of the calcium channel PKD2 impair the endothelial calcium level and both increase and perturb vascular morphogenesis. Altogether, these results demonstrate that endothelial cilia constitute a highly sensitive structure that permits the detection of low shear forces during vascular morphogenesis.

Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an essential role in this process by generating a directional fluid flow inside the vertebrate left-right organizer. Detailed characterization of how fluid flow dynamics modulates laterality is lacking. We used zebrafish genetics to experimentally generate a range of flow dynamics. By following the development of each embryo, we show that fluid flow in the left-right organizer is asymmetric and provides a good predictor of organ laterality. This was tested in mosaic organizers composed of motile and immotile cilia generated by dnah7 knockdowns. In parallel, we used simulations of fluid dynamics to analyze our experimental data. These revealed that fluid flow generated by 30 or more cilia predicts 90% situs solitus, similar to experimental observations. We conclude that cilia number, dorsal anterior motile cilia clustering, and left flow are critical to situs solitus via robust asymmetric charon expression.

Fluid flows generated by motile cilia are guiding the establishment of the left-right asymmetry of the body in the vertebrate left-right organizer. Competing hypotheses have been proposed: the direction of flow is sensed either through mechanosensation, or via the detection of chemical signals transported in the flow. We investigated the physical limits of flow detection to clarify which mechanisms could be reliably used for symmetry breaking. We integrated parameters describing cilia distribution and orientation obtained in vivo in zebrafish into a multiscale physical study of flow generation and detection. Our results show that the number of immotile cilia is too small to ensure robust left and right determination by mechanosensing, given the large spatial variability of the flow. However, motile cilia could sense their own motion by a yet unknown mechanism. Finally, transport of chemical signals by the flow can provide a simple and reliable mechanism of asymmetry establishment.DOI: http://dx.doi.org/10.7554/eLife.25078.001

Cells need to sense their mechanical environment during the growth of developing tissues and maintenance of adult tissues. The concept of force-sensing mechanisms that act through cell-cell and cellmatrix adhesions is now well established and accepted. Additionally, it is widely believed that force sensing can be mediated through cilia. Yet, this hypothesis is still debated. By using primary cilia sensing as a paradigm, we describe the physical requirements for cilium-mediated mechanical sensing and discuss the different hypotheses of how this could work. We review the different mechanosensitive channels within the cilium, their potential mode of action and their biological implications. In addition, we describe the biological contexts in which cilia are actingin particular, the left-right organizerand discuss the challenges to discriminate between cilium-mediated chemosensitivity and mechanosensitivity. Throughout, we provide perspectives on how quantitative analysis and physics-based arguments might help to better understand the biological mechanisms by which cells use cilia to probe their mechanical environment.

Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics. In addition, we show that the calcium dynamics associated with heart contraction are impaired and are associated with sarcoplasmic reticulum dilatation as well as abnormal subcellular distribution of Ryanodine receptors. Our results demonstrate that desminopathies are associated with perturbed excitation-contraction coupling machinery and that aggregates are more detrimental than Desmin loss of function. Additionally, we show that pharmacological inhibition of aggregate formation and Desmin knockdown revert these phenotypes. Our data suggest alternative therapeutic approaches and further our understanding of the molecular determinants modulating Desmin aggregate formation.

Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.

Highlights d KV cilia display asymmetric orientation between the right and left sides d Cilia orientation depends on PCP, but does not directly follow its direction d Impact of asymmetric cilia orientation on left-right symmetry breaking is unclear d Asymmetric cilia orientation is independent of left-right signaling pathway or flow

Left-right patterning and asymmetric morphogenesis arise from a complex set of molecular and cellular interactions that are particularly dynamic and associated with mechanical forces. How do mechanical forces translate into tissular asymmetries? Are these forces asymmetrical de novo, or do they build up from pre-existing asymmetries? Advances in developmental genetics, live imaging and cell biology have recently shed light on the origins of mechanical forces generated at the cell scale and their implication in asymmetric patterning and morphogenesis is now emerging. Here we ask when and how, molecular asymmetries and mechanical forces contribute to left-right patterning and organ asymmetries.