Session B66: Morphogenesis II

Self-repairing symmetry in jellyfish

Standing on a British shore a century ago, D'Arcy Thompson wondered whether the shape of a medusa can be likened to the equilibrium form of a gelatinous drop. While many study how animals get their shapes during development, less is understood about how or whether animal shape is regulated in adulthood. We pursue this question of animal shape regulation in the moon jellyfish, Aurelia aurita. Using grafting and mechanical modulations, we found that Aurelia shape is governed by a morphogenetic system with multiple equilibria – such that we can make jellyfish with various stable shapes, including oval, rectangular, and triangular. Further, combining experimental and mathematical analyses, we found evidence that the shape of a medusa is governed as a dynamic equilibrium of the underlying tissue mechanics. Thus, as Thompson envisioned, belying its calm majesty, the shape of jellyfish is not statically encoded, but rather a continual balancing act.   

On "irreversible" torsion in early chick embryonic brain development

The rightward torsion of the chick embryonic brain tube is one of the earliest organ-level left-right asymmetry developmental events. Previous studies have shown that vitelline membrane (VM) exerts the necessary force on the chicken embryo brain that drives the torsion, and surface tension can replace the mechanical role of VM resulting in a similar degree of torsion at a comparable stage. However, recent experiments show that when the surface tension is removed the torsion does not fully reverse suggesting that there are other overlooked mechanical factors in this process. Here, we show through a combination of in vivo experiments and a physical model of the embryonic morphology that the twisting of the chick embryonic brain tube is partially reversed when the surface tension is removed and the deformation of the early brain can be path dependent. We also studied the effect of embryonic curvature and shape on the degree of torsion, and identified how buoyancy may play a mechanical role in in this “irreversible” brain torsion process.   

Motility and adhesion gradient induced vertebrate body axis elongation and somite formation

The body of vertebrate embryos forms by posterior elongation from a terminal growth zone called the Tail Bud (TB). The TB produces highly motile cells that eventually constitute the presomitic mesoderm (PSM), a tissue playing an important role in elongation movements. PSM cells establish an anterior-posterior cell motility gradient which parallels a gradient associated with the degradation of a specific cellular signal (Fgf8) known to be implicated in cell motility. As Fgf8 degrades over time, anteriorly positioned cells move less, before eventually coming to a rest as they aggregate into epithelial somites. We show that simple microscopic and macroscopic mechano-chemical models for tissue extension that couple Fgf activity, cell motility, cell density and tissue rheology at both the cellular and continuum levels suffice to capture the speed and extent of elongation. These model qualitatively capture the condensation of cells into somites due to the effect of adhesion in the anterior region. These observations explain how the continuous addition of cells that exhibit an increase in cell density and a gradual reduction in motility combined with lateral confinement can be converted into somite formation in the anterior region and an oriented movement and drive body elongation.   

Biomechanics of anteroposterior axis elongation in the chicken embryo

In vertebrate embryos, anteroposterior axis elongation is a crucial developmental process resulting in the establishment of the basic body plan and the growth of the embryo from the tail region. In chicken, it has been proposed that a gradient of random cell motility along the presomitic mesoderm (PSM), rather than directed cell movements, drives axis elongation (Bénazéraf et al, Nature 2010). In order to access the physical mechanisms that could explain this process, we study the rheological properties of the PSM and the forces produced due to posterior PSM expansion. Both micropipette aspiration, as well as rounding experiments, show a liquid-like behavior for the PSM, and allow us to measure its viscoelastic properties. In addition, we have developed a novel cantilever-based system to measure the force of axis elongation and we relate it to the cell movements along the axis. We demonstrate that an isolated PSM explant elongates autonomously and contributes to the total elongation force of the embryo, highlighting its role in axis elongation. Taken together, our results provide a first quantitative description of the mechanics of the tail region in the chicken embryo, which will be essential for future modeling of axis elongation.   

Actomyosin-mediated cytoplasmic flows synchronize the cell cycle in Drosophila embryos

The synchronous cleavage divisions of early embryogenesis require coordination of the cell cycle oscillator, the dynamics of the cytoskeleton and the cytoplasm. As yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective cell cycle dynamics. Biochemical oscillations are initiated by local Cdk1 inactivation close to the nuclei and spread through the activity of mitotic phosphatase PP1 to generate cortical myosin II gradients. These gradients generate cortical and cytoplasmic flows that control proper nuclear positioning. Despite being in the very low Reynolds number regime, we find that Stokes flow does not adequately describe these flows. We will describe the physical properties of the flows, its role in nuclear migration and synchronization of the cell cycle in Drosophila embryos.   

Quantifying the forces associated with body elongation in a chicken embryo

The role of mechanical forces in the context of morphogenesis and growth regulation in an embryo is now well accepted. Yet, it is still a challenge to perform mechanical measurements and manipulations on a whole embryo to obtain quantitative insights. Here we use chick embryos in the gastrulation stage to study how the tissues that form the body axis coordinate their rates of elongation. We found that the coordination and axis straightness are achieved via a positive feedback loop involving mechanics, as revealed by surgical implantation of soft gels and mechanical manipulation with magnetic pins. We also show that cell movement is required for the generation of, and can be a response to, the tissue forces. Finally, we deploy custom tools to apply force-clamps on the embryo over long times to affect both the growth and form of the embryo.   

Patterns make patterns: how hierarchical self-organization couples cell geometry to biochemical dynamics - Experiment

Many cellular and developmental processes rely crucially on self-organization of protein patterns in space and time. When these protein patterns are coupled to force generation pathways, they can precisely pattern mechanical stress during processes such as cell division or tissue folding. Importantly, these mechanical processes generate shape deformations and cytoplasmic flows, which can modulate intracellular reaction-diffusion dynamics. This suggests a close coupling between cell mechanics and biochemical dynamics. But how do these protein patterns respond to a mechanically changing environment? Here, we use the Rho GTPase driven surface contractions waves in starfish oocytes as a model system to study these effects. By constraining oocytes in microfabricated shape chambers, we found that the behavior of the Rho waves can be qualitatively modulated. Further experiments show that the upstream regulator Cdk1 forms a cytosolic gradient which is modulated by cell geometry, forming a template for downstream pattern formation. We demonstrate that the surface contraction wave is a result of a cascade of coupled protein patterns, which we call ‘hierarchical self-organization’.   

Patterns make patterns: how hierarchical self-organization couples cell geometry to biochemical dynamics - Theory

Many cellular and developmental processes rely crucially on self-organization of protein patterns in space and time. When these protein patterns are coupled to force generation pathways, they can precisely pattern mechanical stress during processes such as cell division or tissue folding. Importantly, these mechanical processes generate shape deformations and cytoplasmic flows which, in turn, can modulate intracellular reaction-diffusion dynamics. This suggests a close coupling between cell mechanics and biochemical dynamics. But how do these protein patterns respond to a mechanically changing environment?
Here, we use the Rho GTPase driven surface contractions waves in starfish oocytes as a model system. We combine experimental results with a reaction-diffusion model and show how a cascade of coupled protein patterns creates a feedback loop between the Rho membrane dynamics and the cell shape. We posit that such hierarchical self-organization is a general mechanism for cells to sense changes in its global geometry.   

Tuning biochemical patterns by dynamic mechanical deformations

Throughout embryonic development biochemical patterns are crucial for initiating and guiding vital cellular processes. As the geometry of the biological system evolves, patterns also adapt to reflect the new geometry, suggesting that patterning and geometrical deformations are closely coupled. Yet the mechanisms underlying this coordination are not fully understood. Here, we use cortical Rho activity in the oocytes of the starfish Patiria miniata as a model system to explore such coupling in evolving mechanochemical systems. In addition to being highly deformable, the oocyte exhibits versatile and tunable dynamical patterns on the membrane. Through the use of micropipette aspiration, we impose geometrical constraints on the oocyte that can be actively tuned. The evolution of the pattern in response to the changing geometry is probed in real time, revealing the underlying properties of the dynamics. This method provides a novel approach to studying the interplay between biochemical patterning and mechanically evolving biological systems.   

Active mechanics of starfish oocytes

During meiosis, starfish oocytes exhibit a dramatic surface contraction wave characterized by a band of large-scale deformation traveling from the vegetal pole to the animal pole. This inherently mechanical process is driven by active stresses generated by actomyosin contraction. What are the mechanical properties of these oocytes? How can modulating cytoskeletal organization affect the emergent mechanics and the contraction wave dynamics? Here, we present experimental results beginning to address these questions. Using micropipette aspiration, we find that the mechanical properties are well described by a modified Maxwell model, allowing us to quantitatively measure the viscosity, surface tension, and elastic modulus. These mechanical measurements are complemented by work combining quantitative light microscopy and pharmacological inhibition to assess cytoskeletal contributions to the emergent contraction wave dynamics. For example, using the actin inhibitor cytochalasin D, we find a dose dependent decrease in the magnitude of the contraction. This work represents a step towards understanding cytoskeletal contributions to the emergent dynamics and mechanics in starfish oocytes and in actin cortices more generally.
  

Steady-state contractile actin flow in Xenopus egg extract droplets

The actin cytoskeleton of eukaryotic cells is a highly dynamic viscoelastic “active material”. A typical cell maintains a cortex lining that supports the cell membrane, a polymer network consisting of actin, myosin motors and a plethora of regulatory proteins. Actin turns over between polymeric and monomeric forms on a time scale of minutes. Myosin motors generate active contractile stresses that can induce large-scale actin flow, which is essential for the transport of cytoplasmic components, locomotion as well as shape changes of cells. How exactly so many interacting biochemical processes result in static or dynamic steady states is unclear. Using water-in-oil droplet containing cytoplasmic extract of Xenopus laevis eggs as a model system for an active cytoskeleton, we could produce radially convergent continuous flow of polymerized actin that persist over time scales much longer than the turn-over time of a single actin filament. We mapped the spatiotemporal distribution of this contractile persistent actin flow. Interestingly, we found that macromolecular cargo present in the extract gets transported into the center of the droplet and compacted into a jammed state. We demonstrated this by tracking embedded IR fluorescent single-walled carbon nanotubes as mechanical probes.   

Mechanical competition leads to dynamic instabilities and heterogeneity in stem-cell derived cardiomyocytes

Cooperative and synchronized contraction of sarcomeres is important for the optimal function of cardiomyocytes. Theories of collective molecular motor dynamics, however, predict the possibility of emergent phenomena such as dynamic instabilities and spontaneous oscillatory motion due to non-monotonic force-velocity relations. We have tracked the contractions of individual sarcomeres in stem-cell derived cardiomyocytes by endogenous fluorescent labeling of α-actinin 2 using CRISPR/Cas9 technology. Cardiomyocytes were attached to micro-patterned elastic substrates with various Young’s moduli between 7 and 60 kPa. On soft substrates, sarcomeres in one cell contracted coherently, whereas contractions became increasingly incoherent and heterogeneous with increasing substrate stiffness. These findings suggest that competition between sarcomeres, enforced by rigid mechanical boundary conditions, perturbs dynamic coherence. Using a simple dynamic model, we show that elastic coupling of z-lines to the substrate in conjunction with a non-monotonic force-velocity relation can account for many of the observed features.
  

Cilia coordinated temporal-spatial flow in the mammalian brain

The walls of the ventricular system of mammalian brain are lined with ependymal cells, each of which sprouts a bundle of cilia that constantly beat and thereby maintain directional cerebrospinal fluid (CSF) flow. A transport network driven by coordinated motile cilia inside the ventral third ventricle (v3V) was reported recently. This network contains several CSF flow streams, generates flow patterns such as separatrix and whirl, and may coordinate the delivery of CSF components to different target sites within the ventricle. Particle tracking showed that in mouse brain this flow network locally differs between the two sides of the v3V and changes with age, which implies an age-dependent complex delivery system for CSF constituents. We also studied numerically the contribution of the temporal-spatial flow pattern to the overall CSF flow within the 3D ventricular cavity, and uncover likely physiological consequences of the flow pattern.