Mechanics of the cellular microenvironment as probed by cells in vivo during zebrafish presomitic mesoderm differentiation.

Tissue morphogenesis, homoeostasis and repair require cells to constantly monitor their three-dimensional microenvironment and adapt their behaviours in response to local biochemical and mechanical cues. Yet the mechanical parameters of the cellular microenvironment probed by cells in vivo remain unclear. Here, we report the mechanics of the cellular microenvironment that cells probe in vivo and in situ during zebrafish presomitic mesoderm differentiation. By quantifying both endogenous cell-generated strains and tissue mechanics, we show that individual cells probe the stiffness associated with deformations of the supracellular, foam-like tissue architecture. Stress relaxation leads to a perceived microenvironment stiffness that decreases over time, with cells probing the softest regime. We find that most mechanical parameters, including those probed by cells, vary along the anteroposterior axis as mesodermal progenitors differentiate. These findings expand our understanding of in vivo mechanosensation and might aid the design of advanced scaffolds for tissue engineering applications.

Embryonic Tissues as Active Foams.

The physical state of embryonic tissues emerges from non-equilibrium, collective interactions among constituent cells. Cellular jamming, rigidity transitions and characteristics of glassy dynamics have all been observed in multicellular systems, but it is unclear how cells control these emergent tissue states and transitions, including tissue fluidization. Combining computational and experimental methods, here we show that tissue fluidization in posterior zebrafish tissues is controlled by the stochastic dynamics of tensions at cell-cell contacts. We develop a computational framework that connects cell behavior to embryonic tissue dynamics, accounting for the presence of extracellular spaces, complex cell shapes and cortical tension dynamics. We predict that tissues are maximally rigid at the structural transition between confluent and non-confluent states, with actively-generated tension fluctuations controlling stress relaxation and tissue fluidization. By directly measuring strain and stress relaxation, as well as the dynamics of cell rearrangements, in elongating posterior zebrafish tissues, we show that tension fluctuations drive active cell rearrangements that fluidize the tissue. These results highlight a key role of non-equilibrium tension dynamics in developmental processes.

Mechanical control of tissue shape and morphogenetic flows during vertebrate body axis elongation.

Shaping embryonic tissues into their functional morphologies requires cells to control the physical state of the tissue in space and time. While regional variations in cellular forces or cell proliferation have been typically assumed to be the main physical factors controlling tissue morphogenesis, recent experiments have revealed that spatial variations in the tissue physical (fluid/solid) state play a key role in shaping embryonic tissues. Here we theoretically study how the regional control of fluid and solid tissue states guides morphogenetic flows to shape the extending vertebrate body axis. Our results show that both the existence of a fluid-to-solid tissue transition along the anteroposterior axis and the tissue surface tension determine the shape of the tissue and its ability to elongate unidirectionally, with large tissue tensions preventing unidirectional elongation and promoting blob-like tissue expansions. We predict both the tissue morphogenetic flows and stresses that enable unidirectional axis elongation. Our results show the existence of a sharp transition in the structure of morphogenetic flows, from a flow with no vortices to a flow with two counter-rotating vortices, caused by a transition in the number and location of topological defects in the flow field. Finally, comparing the theoretical predictions to quantitative measurements of both tissue flows and shape during zebrafish body axis elongation, we show that the observed morphogenetic events can be explained by the existence of a fluid-to-solid tissue transition along the anteroposterior axis. These results highlight the role of spatiotemporally-controlled fluid-to-solid transitions in the tissue state as a physical mechanism of embryonic morphogenesis.

Decoupling the Roles of Cell Shape and Mechanical Stress in Orienting and Cueing Epithelial Mitosis.

Distinct mechanisms involving cell shape and mechanical force are known to influence the rate and orientation of division in cultured cells. However, uncoupling the impact of shape and force in tissues remains challenging. Combining stretching of Xenopus tissue with mathematical methods of inferring relative mechanical stress, we find separate roles for cell shape and mechanical stress in orienting and cueing division. We demonstrate that division orientation is best predicted by an axis of cell shape defined by the position of tricellular junctions (TCJs), which align with local cell stress rather than tissue-level stress. The alignment of division to cell shape requires functional cadherin and the localization of the spindle orientation protein, LGN, to TCJs but is not sensitive to relative cell stress magnitude. In contrast, proliferation rate is more directly regulated by mechanical stress, being correlated with relative isotropic stress and decoupled from cell shape when myosin II is depleted.

Physical control of tissue morphogenesis across scales.

During embryogenesis, tissues and organs are progressively shaped into their functional morphologies. While the information about tissue and organ shape is encoded genetically, the sculpting of embryonic structures in the 3D space is ultimately a physical process. The control of physical quantities involved in tissue morphogenesis originates at cellular and subcellular scales, but it is their emergent behavior at supracellular scales that guides morphogenetic events. In this review, we highlight the physical quantities that can be spatiotemporally tuned at supracellular scales to sculpt tissues and organs during embryonic development of animal species, and connect them to the cellular and molecular mechanisms controlling them.

Xenopus as a model for studies in mechanical stress and cell division.

We exist in a physical world, and cells within biological tissues must respond appropriately to both environmental forces and forces generated within the tissue to ensure normal development and homeostasis. Cell division is required for normal tissue growth and maintenance, but both the direction and rate of cell division must be tightly controlled to avoid diseases of over-proliferation such as cancer. Recent studies have shown that mechanical cues can cause mitotic entry and orient the mitotic spindle, suggesting that physical force could play a role in patterning tissue growth. However, to fully understand how mechanics guides cells in vivo, it is necessary to assess the interaction of mechanical strain and cell division in a whole tissue context. In this mini-review we first summarise the body of work linking mechanics and cell division, before looking at the advantages that the Xenopus embryo can offer as a model organism for understanding: (1) the mechanical environment during embryogenesis, and (2) factors important for cell division. Finally, we introduce a novel method for applying a reproducible strain to Xenopus embryonic tissue and assessing subsequent cell divisions.

Otolith tethering in the zebrafish otic vesicle requires Otogelin and α-Tectorin.

Otoliths are biomineralised structures important for balance and hearing in fish. Their counterparts in the mammalian inner ear, otoconia, have a primarily vestibular function. Otoliths and otoconia form over sensory maculae and are attached to the otolithic membrane, a gelatinous extracellular matrix that provides a physical coupling between the otolith and the underlying sensory epithelium. In this study, we have identified two proteins required for otolith tethering in the zebrafish ear, and propose that there are at least two stages to this process: seeding and maintenance. The initial seeding step, in which otolith precursor particles tether directly to the tips of hair cell kinocilia, fails to occur in the einstein (eis) mutant. The gene disrupted in eis is otogelin (otog); mutations in the human OTOG gene have recently been identified as causative for deafness and vestibular dysfunction (DFNB18B). At later larval stages, maintenance of otolith tethering to the saccular macula is dependent on tectorin alpha (tecta) function, which is disrupted in the rolling stones (rst) mutant. α-Tectorin (Tecta) is a major constituent of the tectorial membrane in the mammalian cochlea. Mutations in the human TECTA gene can cause either dominant (DFNA8/12) or recessive (DFNB21) forms of deafness. Our findings indicate that the composition of extracellular otic membranes is highly conserved between mammals and fish, reinforcing the view that the zebrafish is an excellent model system for the study of deafness and vestibular disease.

The role of hair cells, cilia and ciliary motility in otolith formation in the zebrafish otic vesicle.

Otoliths are biomineralised structures required for the sensation of gravity, linear acceleration and sound in the zebrafish ear. Otolith precursor particles, initially distributed throughout the otic vesicle lumen, become tethered to the tips of hair cell kinocilia (tether cilia) at the otic vesicle poles, forming two otoliths. We have used high-speed video microscopy to investigate the role of cilia and ciliary motility in otolith formation. In wild-type ears, groups of motile cilia are present at the otic vesicle poles, surrounding the immotile tether cilia. A few motile cilia are also found on the medial wall, but most cilia (92-98%) in the otic vesicle are immotile. In mutants with defective cilia (iguana) or ciliary motility (lrrc50), otoliths are frequently ectopic, untethered or fused. Nevertheless, neither cilia nor ciliary motility are absolutely required for otolith tethering: a mutant that lacks cilia completely (MZovl) is still capable of tethering otoliths at the otic vesicle poles. In embryos with attenuated Notch signalling [mindbomb mutant or Su(H) morphant], supernumerary hair cells develop and otolith precursor particles bind to the tips of all kinocilia, or bind directly to the hair cells' apical surface if cilia are absent [MZovl injected with a Su(H)1+2 morpholino]. However, if the first hair cells are missing (atoh1b morphant), otolith formation is severely disrupted and delayed. Our data support a model in which hair cells produce an otolith precursor-binding factor, normally localised to tether cell kinocilia. We also show that embryonic movement plays a minor role in the formation of normal otoliths.