Adhesion-regulated junction slippage controls cell intercalation dynamics in an Apposed-Cortex Adhesion Model.

Cell intercalation is a key cell behaviour of morphogenesis and wound healing, where local cell neighbour exchanges can cause dramatic tissue deformations such as body axis extension. Substantial experimental work has identified the key molecular players facilitating intercalation, but there remains a lack of consensus and understanding of their physical roles. Existing biophysical models that represent cell-cell contacts with single edges cannot study cell neighbour exchange as a continuous process, where neighbouring cell cortices must uncouple. Here, we develop an Apposed-Cortex Adhesion Model (ACAM) to understand active cell intercalation behaviours in the context of a 2D epithelial tissue. The junctional actomyosin cortex of every cell is modelled as a continuous viscoelastic rope-loop, explicitly representing cortices facing each other at bicellular junctions and the adhesion molecules that couple them. The model parameters relate directly to the properties of the key subcellular players that drive dynamics, providing a multi-scale understanding of cell behaviours. We show that active cell neighbour exchanges can be driven by purely junctional mechanisms. Active contractility and cortical turnover in a single bicellular junction are sufficient to shrink and remove a junction. Next, a new, orthogonal junction extends passively. The ACAM reveals how the turnover of adhesion molecules regulates tension transmission and junction deformation rates by controlling slippage between apposed cell cortices. The model additionally predicts that rosettes, which form when a vertex becomes common to many cells, are more likely to occur in actively intercalating tissues with strong friction from adhesion molecules.

The tricellular vertex-specific adhesion molecule Sidekick facilitates polarised cell intercalation during Drosophila axis extension.

In epithelia, tricellular vertices are emerging as important sites for the regulation of epithelial integrity and function. Compared to bicellular contacts, however, much less is known. In particular, resident proteins at tricellular vertices were identified only at occluding junctions, with none known at adherens junctions (AJs). In a previous study, we discovered that in Drosophila embryos, the adhesion molecule Sidekick (Sdk), well-known in invertebrates and vertebrates for its role in the visual system, localises at tricellular vertices at the level of AJs. Here, we survey a wide range of Drosophila epithelia and establish that Sdk is a resident protein at tricellular AJs (tAJs), the first of its kind. Clonal analysis showed that two cells, rather than three cells, contributing Sdk are sufficient for tAJ localisation. Super-resolution imaging using structured illumination reveals that Sdk proteins form string-like structures at vertices. Postulating that Sdk may have a role in epithelia where AJs are actively remodelled, we analysed the phenotype of sdk null mutant embryos during Drosophila axis extension using quantitative methods. We find that apical cell shapes are abnormal in sdk mutants, suggesting a defect in tissue remodelling during convergence and extension. Moreover, adhesion at apical vertices is compromised in rearranging cells, with apical tears in the cortex forming and persisting throughout axis extension, especially at the centres of rosettes. Finally, we show that polarised cell intercalation is decreased in sdk mutants. Mathematical modelling of the cell behaviours supports the notion that the T1 transitions of polarised cell intercalation are delayed in sdk mutants, in particular in rosettes. We propose that this delay, in combination with a change in the mechanical properties of the converging and extending tissue, causes the abnormal apical cell shapes in sdk mutant embryos.

Clones of cells switch from reduction to enhancement of size variability in Arabidopsis sepals.

Organs form with remarkably consistent sizes and shapes during development, whereas a high variability in growth is observed at the cell level. Given this contrast, it is unclear how such consistency in organ scale can emerge from cellular behavior. Here, we examine an intermediate scale, the growth of clones of cells in Arabidopsis sepals. Each clone consists of the progeny of a single progenitor cell. At early stages, we find that clones derived from a small progenitor cell grow faster than those derived from a large progenitor cell. This results in a reduction in clone size variability, a phenomenon we refer to as size uniformization. By contrast, at later stages of clone growth, clones change their growth pattern to enhance size variability, when clones derived from larger progenitor cells grow faster than those derived from smaller progenitor cells. Finally, we find that, at early stages, fast growing clones exhibit greater cell growth heterogeneity. Thus, cellular variability in growth might contribute to a decrease in the variability of clones throughout the sepal.

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.

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.

Extracting Subcellular Fibrillar Alignment with Error Estimation: Application to Microtubules.

The order and orientation of cortical microtubule (CMT) arrays and their dynamics play an essential role in plant morphogenesis. To extract detailed CMT alignment structures in an objective, local, and accurate way, we propose an error-based extraction method that applies to general fluorescence intensity data on three-dimensional cell surfaces. Building on previous techniques to quantify alignments, our method can determine the statistical error for specific local regions, or the minimal scales of local regions for a desired accuracy goal. After validating our method with synthetic images with known alignments, we demonstrate the ability of our method to quantify subcellular CMT alignments on images with microtubules marked with green fluorescent protein in various cell types. Our method could also be applied to detect alignment structures in other fibrillar elements, such as actin filaments, cellulose, and collagen.

Meristem size contributes to the robustness of phyllotaxis in Arabidopsis.

Using the plant model Arabidopsis, the relationship between day length, the size of the shoot apical meristem, and the robustness of phyllotactic patterns were analysed. First, it was found that reducing day length leads to an increased meristem size and an increased number of alterations in the final positions of organs along the stem. Most of the phyllotactic defects could be related to an altered tempo of organ emergence, while not affecting the spatial positions of organ initiations at the meristem. A correlation was also found between meristem size and the robustness of phyllotaxis in two accessions (Col-0 and WS-4) and a mutant (clasp-1), independent of growth conditions. A reduced meristem size in clasp-1 was even associated with an increased robustness of the phyllotactic pattern, beyond what is observed in the wild type. Interestingly it was also possible to modulate the robustness of phyllotaxis in these different genotypes by changing day length. To conclude, it is shown first that robustness of the phyllotactic pattern is not maximal in the wild type, suggesting that, beyond its apparent stereotypical order, the robustness of phyllotaxis is regulated. Secondly, a role for day length in the robustness of the phyllotaxis was also identified, thus providing a new example of a link between patterning and environment in plants. Thirdly, the experimental results validate previous model predictions suggesting a contribution of meristem size in the robustness of phyllotaxis via the coupling between the temporal sequence and spatial pattern of organ initiations.