NanoIndentation, an ImageJ Plugin for the Quantification of Cell Mechanics.

Growth and morphogenesis in plants depend on cell wall mechanics and on turgor pressure. Nanoindentation methods, such as atomic force microscopy (AFM), enable measurements of mechanical properties of a tissue at subcellular resolution, while confocal microscopy of tissues expressing fluorescent reporters indicates cell identity. Associating mechanical data with specific cells is essential to reveal the links between cell identity and cell mechanics. Here we describe an image analysis protocol that allows us to segment AFM scans containing information on tissue topography and/or mechanics, to stitch several scans in order to reconstitute an entire region of the tissue investigated, to segment the scans and label cells, and to associate labeled cells to the projection of confocal images. Thus all mechanical data can be mapped to the corresponding cells and to their identity. This protocol is implemented using NanoIndentation, a plugin that we are developing in the Fiji distribution of ImageJ.

3D cell neighbour dynamics in growing pseudostratified epithelia.

During morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organise their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organisation in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, that is, Euler's polyhedron formula, Lewis' law, and Aboav-Weaire's law, at all times and across the entire tissue thickness. This arrangement minimises the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organisation in growing epithelia emerges from simple physical principles.

The biomechanical basis of biased epithelial tube elongation in lung and kidney development.

During lung development, epithelial branches expand preferentially in a longitudinal direction. This bias in outgrowth has been linked to a bias in cell shape and in the cell division plane. How this bias arises is unknown. Here, we show that biased epithelial outgrowth occurs independent of the surrounding mesenchyme, of preferential turnover of the extracellular matrix at the bud tips and of FGF signalling. There is also no evidence for actin-rich filopodia at the bud tips. Rather, we find epithelial tubes to be collapsed during early lung and kidney development, and we observe fluid flow in the narrow tubes. By simulating the measured fluid flow inside segmented narrow epithelial tubes, we show that the shear stress levels on the apical surface are sufficient to explain the reported bias in cell shape and outgrowth. We use a cell-based vertex model to confirm that apical shear forces, unlike constricting forces, can give rise to both the observed bias in cell shapes and tube elongation. We conclude that shear stress may be a more general driver of biased tube elongation beyond its established role in angiogenesis. This article has an associated 'The people behind the papers' interview.

Cellular Heterogeneity in Pressure and Growth Emerges from Tissue Topology and Geometry.

Cell-to-cell heterogeneity prevails in many systems, as exemplified by cell growth, although the origin and function of such heterogeneity are often unclear. In plants, growth is physically controlled by cell wall mechanics and cell hydrostatic pressure, alias turgor pressure. Whereas cell wall heterogeneity has received extensive attention, the spatial variation of turgor pressure is often overlooked. Here, combining atomic force microscopy and a physical model of pressurized cells, we show that turgor pressure is heterogeneous in the Arabidopsis shoot apical meristem, a population of stem cells that generates all plant aerial organs. In contrast with cell wall mechanical properties that appear to vary stochastically between neighboring cells, turgor pressure anticorrelates with cell size and cell neighbor number (local topology), in agreement with the prediction by our model of tissue expansion, which couples cell wall mechanics and tissue hydraulics. Additionally, our model predicts two types of correlations between pressure and cellular growth rate, where high pressure may lead to faster- or slower-than-average growth, depending on cell wall extensibility, yield threshold, osmotic pressure, and hydraulic conductivity. The meristem exhibits one of these two regimes, depending on conditions, suggesting that, in this tissue, water conductivity may contribute to growth control. Our results unravel cell pressure as a source of patterned heterogeneity and illustrate links between local topology, cell mechanical state, and cell growth, with potential roles in tissue homeostasis.

Heterogeneity and Robustness in Plant Morphogenesis: From Cells to Organs.

Development is remarkably reproducible, producing organs with the same size, shape, and function repeatedly from individual to individual. For example, every flower on the Antirrhinum stalk has the same snapping dragon mouth. This reproducibility has allowed taxonomists to classify plants and animals according to their morphology. Yet these reproducible organs are composed of highly variable cells. For example, neighboring cells grow at different rates in Arabidopsis leaves, sepals, and shoot apical meristems. This cellular variability occurs in normal, wild-type organisms, indicating that cellular heterogeneity (or diversity in a characteristic such as growth rate) is either actively maintained or, at a minimum, not entirely suppressed. In fact, cellular heterogeneity can contribute to producing invariant organs. Here, we focus on how plant organs are reproducibly created during development from these highly variable cells.

Mechanical Shielding of Rapidly Growing Cells Buffers Growth Heterogeneity and Contributes to Organ Shape Reproducibility.

A landmark of developmental biology is the production of reproducible shapes, through stereotyped morphogenetic events. At the cell level, growth is often highly heterogeneous, allowing shape diversity to arise. Yet, how can reproducible shapes emerge from such growth heterogeneity? Is growth heterogeneity filtered out? Here, we focus on rapidly growing trichome cells in the Arabidopsis sepal, a reproducible floral organ. We show via computational modeling that rapidly growing cells may distort organ shape. However, the cortical microtubule alignment along growth-derived maximal tensile stress in adjacent cells would mechanically isolate rapidly growing cells and limit their impact on organ shape. In vivo, we observed such microtubule response to stress and consistently found no significant effect of trichome number on sepal shape in wild-type and lines with trichome number defects. Conversely, modulating the microtubule response to stress in katanin and spiral2 mutant made sepal shape dependent on trichome number, suggesting that, while mechanical signals are propagated around rapidly growing cells, the resistance to stress in adjacent cells mechanically isolates rapidly growing cells, thus contributing to organ shape reproducibility.

Variable Cell Growth Yields Reproducible OrganDevelopment through Spatiotemporal Averaging.

Organ sizes and shapes are strikingly reproducible, despite the variable growth and division of individual cells within them. To reveal which mechanisms enable this precision, we designed a screen for disrupted sepal size and shape uniformity in Arabidopsis and identified mutations in the mitochondrial i-AAA protease FtsH4. Counterintuitively, through live imaging we observed that variability of neighboring cell growth was reduced in ftsh4 sepals. We found that regular organ shape results from spatiotemporal averaging of the cellular variability in wild-type sepals, which is disrupted in the less-variable cells of ftsh4 mutants. We also found that abnormal, increased accumulation of reactive oxygen species (ROS) in ftsh4 mutants disrupts organ size consistency. In wild-type sepals, ROS accumulate in maturing cells and limit organ growth, suggesting that ROS are endogenous signals promoting termination of growth. Our results demonstrate that spatiotemporal averaging of cellular variability is required for precision in organ size.

A Mechanical Feedback Restricts Sepal Growth and Shape in Arabidopsis.

How organs reach their final shape is a central yet unresolved question in developmental biology. Here we investigate whether mechanical cues contribute to this process. We analyze the epidermal cells of the Arabidopsis sepal, focusing on cortical microtubule arrays, which align along maximal tensile stresses and restrict growth in that direction through their indirect impact on the mechanical anisotropy of cell walls. We find a good match between growth and microtubule orientation throughout most of the development of the sepal. However, at the sepal tip, where organ maturation initiates and growth slows down in later stages, microtubules remain in a configuration consistent with fast anisotropic growth, i.e., transverse, and the anisotropy of their arrays even increases. To understand this apparent paradox, we built a continuous mechanical model of a growing sepal. The model demonstrates that differential growth in the sepal can generate transverse tensile stress at the tip. Consistently, microtubules respond to mechanical perturbations and align along maximal tension at the sepal tip. Including this mechanical feedback in our growth model of the sepal, we predict an impact on sepal shape that is validated experimentally using mutants with either increased or decreased microtubule response to stress. Altogether, this suggests that a mechanical feedback loop, via microtubules acting both as stress sensor and growth regulator, channels the growth and shape of the sepal tip. We propose that this proprioception mechanism is a key step leading to growth arrest in the whole sepal in response to its own growth.