From cells to form: A roadmap to study shape emergence in vivo.

Organogenesis arises from the collective arrangement of cells into progressively 3D-shaped tissue. The acquisition of a correctly shaped organ is then the result of a complex interplay between molecular cues, responsible for differentiation and patterning, and the mechanical properties of the system, which generate the necessary forces that drive correct shape emergence. Nowadays, technological advances in the fields of microscopy, molecular biology, and computer science are making it possible to see and record such complex interactions in incredible, unforeseen detail within the global context of the developing embryo. A quantitative and interdisciplinary perspective of developmental biology becomes then necessary for a comprehensive understanding of morphogenesis. Here, we provide a roadmap to quantify the events that lead to morphogenesis from imaging to image analysis, quantification, and modeling, focusing on the discrete cellular and tissue shape changes, as well as their mechanical properties.

Multi-sample SPIM image acquisition, processing and analysis of vascular growth in zebrafish.

To quantitatively understand biological processes that occur over many hours or days, it is desirable to image multiple samples simultaneously, and automatically process and analyse the resulting datasets. Here, we present a complete multi-sample preparation, imaging, processing and analysis workflow to determine the development of the vascular volume in zebrafish. Up to five live embryos were mounted and imaged simultaneously over several days using selective plane illumination microscopy (SPIM). The resulting large imagery dataset of several terabytes was processed in an automated manner on a high-performance computer cluster and segmented using a novel segmentation approach that uses images of red blood cells as training data. This analysis yielded a precise quantification of growth characteristics of the whole vascular network, head vasculature and tail vasculature over development. Our multi-sample platform demonstrates effective upgrades to conventional single-sample imaging platforms and paves the way for diverse quantitative long-term imaging studies.

Active T1 transitions in cellular networks.

In amorphous solids as in tissues, neighbor exchanges can relax local stresses and allow the material to flow. In this paper, we use an anisotropic vertex model to study T1 rearrangements in polygonal cellular networks. We consider two different physical realizations of the active anisotropic stresses: (i) anisotropic bond tension and (ii) anisotropic cell stress. Interestingly, the two types of active stress lead to patterns of relative orientation of T1 transitions and cell elongation that are different. Our work suggests that these two realizations of anisotropic active stresses can be observed in vivo. We describe and explain these results through the lens of a continuum description of the tissue as an anisotropic active material. We furthermore discuss the energetics of the dynamic tissue and express the energy balance in terms of internal elastic energy, mechanical work, chemical work and heat. This allows us to define active T1 transitions that can perform mechanical work while consuming chemical energy.

Frame, metric and geodesic evolution in shape-changing nematic shells.

Non-uniform director fields in flat, responsive, glassy nematic sheets lead to the induction of shells with non-trivial topography on the application of light or heat. Contraction along the director causes metric change, with, in general, the induction of Gaussian curvature, that drives the topography change. We describe the metric change, the evolution of the director field, and the transformation of reference state material curves, e.g. spirals into radii, as curvature develops. The non-isometric deformations associated with heat or light change the geodesics of the surface, intriguingly even in regions where no Gaussian curvature results.

Core PCP mutations affect short-time mechanical properties but not tissue morphogenesis in the Drosophila pupal wing.

How morphogenetic movements are robustly coordinated in space and time is a fundamental open question in biology. We study this question using the wing of Drosophila melanogaster, an epithelial tissue that undergoes large-scale tissue flows during pupal stages. Previously, we showed that pupal wing morphogenesis involves both cellular behaviors that allow relaxation of mechanical tissue stress, as well as cellular behaviors that appear to be actively patterned (Etournay et al., 2015). Here, we show that these active cellular behaviors are not guided by the core planar cell polarity (PCP) pathway, a conserved signaling system that guides tissue development in many other contexts. We find no significant phenotype on the cellular dynamics underlying pupal morphogenesis in mutants of core PCP. Furthermore, using laser ablation experiments, coupled with a rheological model to describe the dynamics of the response to laser ablation, we conclude that while core PCP mutations affect the fast timescale response to laser ablation they do not significantly affect overall tissue mechanics. In conclusion, our work shows that cellular dynamics and tissue shape changes during Drosophila pupal wing morphogenesis do not require core PCP as an orientational guiding cue.

Collective cell migration during optic cup formation features changing cell-matrix interactions linked to matrix topology.

Cell migration is crucial for organismal development and shapes organisms in health and disease. Although a lot of research has revealed the role of intracellular components and extracellular signaling in driving single and collective cell migration, the influence of physical properties of the tissue and the environment on migration phenomena in vivo remains less explored. In particular, the role of the extracellular matrix (ECM), which many cells move upon, is currently unclear. To overcome this gap, we use zebrafish optic cup formation, and by combining novel transgenic lines and image analysis pipelines, we study how ECM properties influence cell migration in vivo. We show that collectively migrating rim cells actively move over an immobile extracellular matrix. These cell movements require cryptic lamellipodia that are extended in the direction of migration. Quantitative analysis of matrix properties revealed that the topology of the matrix changes along the migration path. These changes in matrix topologies are accompanied by changes in the dynamics of cell-matrix interactions. Experiments and theoretical modeling suggest that matrix porosity could be linked to efficient migration. Indeed, interfering with matrix topology by increasing its porosity results in a loss of cryptic lamellipodia, less-directed cell-matrix interactions, and overall inefficient migration. Thus, matrix topology is linked to the dynamics of cell-matrix interactions and the efficiency of directed collective rim cell migration during vertebrate optic cup morphogenesis.

Nonlinear rheology of cellular networks.

Morphogenesis depends crucially on the complex rheological properties of cell tissues and on their ability to maintain mechanical integrity while rearranging at long times. In this paper, we study the rheology of polygonal cellular networks described by a vertex model in the presence of fluctuations. We use a triangulation method to decompose shear into cell shape changes and cell rearrangements. Considering the steady-state stress under constant shear, we observe nonlinear shear-thinning behavior at all magnitudes of the fluctuations, and an even stronger nonlinear regime at lower values of the fluctuations. We successfully capture this nonlinear rheology by a mean-field model that describes the tissue in terms of cell elongation and cell rearrangements. We furthermore introduce anisotropic active stresses in the vertex model and analyze their effect on rheology. We include this anisotropy in the mean-field model and show that it recapitulates the behavior observed in the simulations. Our work clarifies how tissue rheology is related to stochastic cell rearrangements and provides a simple biophysical model to describe biological tissues. Further, it highlights the importance of nonlinearities when discussing tissue mechanics.

Fiji plugin for annotating movies with custom arrows.

Annotation of time-lapse data provides an important tool to highlight dynamic processes. Particularly, arrows, circles and arrowheads are useful to pinpoint a specific process, stationary or evolving over time. Here, we describe a user-friendly Fiji plugin to facilitate annotation of movies with arrows, arrowheads and circles. The plugin also enables saving and loading of annotated tracks.This article has an associated First Person interview with the first author of the paper.

Cell and tissue morphology determine actin-dependent nuclear migration mechanisms in neuroepithelia.

Correct nuclear position is crucial for cellular function and tissue development. Depending on cell context, however, the cytoskeletal elements responsible for nuclear positioning vary. While these cytoskeletal mechanisms have been intensely studied in single cells, how nuclear positioning is linked to tissue morphology is less clear. Here, we compare apical nuclear positioning in zebrafish neuroepithelia. We find that kinetics and actin-dependent mechanisms of nuclear positioning vary in tissues of different morphology. In straight neuroepithelia, nuclear positioning is controlled by Rho-ROCK-dependent myosin contractility. In contrast, in basally constricted neuroepithelia, a novel formin-dependent pushing mechanism is found for which we propose a proof-of-principle force generation theory. Overall, our data suggest that correct nuclear positioning is ensured by the adaptability of the cytoskeleton to cell and tissue shape. This in turn leads to robust epithelial maturation across geometries. The conclusion that different nuclear positioning mechanisms are favored in tissues of different morphology highlights the importance of developmental context for the execution of intracellular processes.

Geometrical frustration in two dimensions: idealizations and realizations of a hard-disk fluid in negative curvature.

We examine a simple hard-disk fluid with no long-range interactions on the two-dimensional space of constant negative Gaussian curvature, the hyperbolic plane. This geometry provides a natural mechanism by which global crystalline order is frustrated, allowing us to construct a tractable, one-parameter model of disordered monodisperse hard disks. We extend free-area theory and the virial expansion to this regime, deriving the equation of state for the system, and compare its predictions with simulations near an isostatic packing in the curved space. Additionally, we investigate packing and dynamics on triply periodic, negatively curved surfaces with an eye toward real biological and polymeric systems.

Negative Gaussian curvature from induced metric changes.

We revisit the light or heat-induced changes in topography of initially flat sheets of a solid that elongate or contract along patterned in-plane director fields. For radial or azimuthal directors, negative Gaussian curvature is generated-so-called "anticones." We show that azimuthal material displacements are required for the distorted state to be stretch free and bend minimizing. The resultant shapes are smooth and asterlike and can become reentrant in the azimuthal coordinate for large deformations. We show that care is needed when considering elastomers rather than glasses, although the former offer huge deformations.

Structural self-assembly and avalanchelike dynamics in locally adaptive networks.

Transport networks play a key role across four realms of eukaryotic life: slime molds, fungi, plants, and animals. In addition to the developmental algorithms that build them, many also employ adaptive strategies to respond to stimuli, damage, and other environmental changes. We model these adapting network architectures using a generic dynamical system on weighted graphs and find in simulation that these networks ultimately develop a hierarchical organization of the final weighted architecture accompanied by the formation of a system-spanning backbone. In addition, we find that the long term equilibration dynamics exhibit behavior reminiscent of glassy systems characterized by long periods of slow changes punctuated by bursts of reorganization events.

Disclination-mediated thermo-optical response in nematic glass sheets.

Nematic solids respond strongly to changes in ambient heat or light, significantly differently parallel and perpendicular to the director. This phenomenon is well characterized for uniform director fields but not for defect textures. We analyze the elastic ground states of a nematic glass in the membrane approximation as a function of temperature for some disclination defects with an eye toward reversibly inducing three-dimensional shapes from flat sheets of material, at the nanoscale all the way to macroscopic objects, including nondevelopable surfaces. The latter offers a paradigm to actuation via switchable stretch in thin systems.

Hard disks on the hyperbolic plane.

We examine a simple hard disk fluid with no long range interactions on the two-dimensional space of constant negative Gaussian curvature, the hyperbolic plane. This geometry provides a natural mechanism by which global crystalline order is frustrated, allowing us to construct a tractable model of disordered monodisperse hard disks. We extend free-area theory and the virial expansion to this regime, deriving the equation of state for the system, and compare its predictions with simulation near an isostatic packing in the curved space.