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Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction ...

Imperial College London

The Francis Crick Institute

Organ morphogenesis involves dynamic changes of tissue properties while cells adapt to their mechanical environment through mechanosensitive pathways. How mechanical cues influence cell behaviors during morphogenesis remains unclear. Here, we studied the formation of the zebrafish atrioventricular canal (AVC) where cardiac valves develop. We show that the AVC forms within a zone of tissue convergence associated with the increased activation of the actomyosin meshwork and cell-orientation changes. We demonstrate that tissue convergence occurs with a reduction of cell volume triggered by mechanical forces and the mechanosensitive channel TRPP2/TRPV4. Finally, we show that the extracellular matrix component hyaluronic acid controls cell volume changes. Together, our data suggest that multiple force-sensitive signaling pathways converge to modulate cell volume. We conclude that cell volume reduction is a key cellular feature activated by mechanotransduction during cardiovascular morphogenesis. This work further identifies how mechanical forces and extracellular matrix influence tissue remodeling in developing organs.

Extracellular mechanical forces drive endocardial cell volume decrease during zebrafish cardiac valve morphogenesis.

During vertebrate development, cells must proliferate, move, and differentiate to form complex shapes. Elucidating the mechanisms underlying the molecular and cellular processes involved in tissue morphogenesis is essential to understanding developmental programmes. Mechanical stimuli act as a major contributor of morphogenetic processes and impact on cell behaviours to regulate tissue shape and size. Specifically, cell extrinsic physical forces are translated into biochemical signals within cells, through the process of mechanotransduction, activating multiple mechanosensitive pathways and defining cell behaviours. Physical forces generated by tissue mechanics and the extracellular matrix are crucial to orchestrate tissue patterning and cell fate specification. At the cell scale, the actomyosin network generates the cellular tension behind the tissue mechanics involved in building tissue. Thus, understanding the role of physical forces during morphogenetic processes requires the consideration of the contribution of cell intrinsic and cell extrinsic influences. The recent development of multidisciplinary approaches, as well as major advances in genetics, microscopy, and force-probing tools, have been key to push this field forward. With this review, we aim to discuss recent work on how tissue shape can be controlled by mechanical forces by focusing specifically on vertebrate organogenesis. We consider the influences of mechanical forces by discussing the cell-intrinsic forces (such as cell tension and proliferation) and cell-extrinsic forces (such as substrate stiffness and flow forces). We review recently described processes supporting the role of intratissue force generation and propagation in the context of shape emergence. Lastly, we discuss the emerging role of tissue-scale changes in tissue material properties, extrinsic forces, and shear stress on shape establishment.

Mechanical control of tissue shape: Cell-extrinsic and -intrinsic mechanisms join forces to regulate morphogenesis.

Expansion microscopy of millimeter-large mechanically heterogeneous tissues, such as whole vertebrate embryos, has been limited, particularly when combined with post-expansion immunofluorescence. Here, we present a protocol to perform ultrastructure expansion microscopy of whole vertebrate embryos, optimized to perform post-expansion labeling. We describe steps for embedding and denaturing zebrafish larvae or mouse embryos. We then detail procedures for hydrogel handling and mounting. This protocol is particularly well suited for super-resolution imaging of macromolecular protein complexes in situ but does not preserve lipids. For complete details on the use and execution of this protocol, please refer to Steib et al..

TissUExM protocol for ultrastructure expansion microscopy of zebrafish larvae and mouse embryos.

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease.

Christina Vagena-Pantoula

Department of Bioengineering

Imperial College London

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

Christina Vagena-Pantoula

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Bioengineering

Imperial College London

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

Department of Bioengineering