Benoît Aigouy

The Influence of Cell Mechanics, Cell-Cell Interactions, and Proliferation on Epithelial Packing

BACKGROUND Epithelial junctional networks assume packing geometries characterized by different cell shapes, neighbor number distributions and areas. The development of specific packing geometries is tightly controlled; in the Drosophila wing epithelium, cells convert from an irregular to a hexagonal array shortly before hair formation. Packing geometry is determined by developmental mechanisms that likely control the biophysical properties of cells and their interactions. RESULTS To understand how physical cellular properties and proliferation determine cell-packing geometries, we use a vertex model for the epithelial junctional network in which cell packing geometries correspond to stable and stationary network configurations. The model takes into account cell elasticity and junctional forces arising from cortical contractility and adhesion. By numerically simulating proliferation, we generate different network morphologies that depend on physical parameters. These networks differ in polygon class distribution, cell area variation, and the rate of T1 and T2 transitions during growth. Comparing theoretical results to observed cell morphologies reveals regions of parameter space where calculated network morphologies match observed ones. We independently estimate parameter values by quantifying network deformations caused by laser ablating individual cell boundaries. CONCLUSIONS The vertex model accounts qualitatively and quantitatively for the observed packing geometry in the wing disc and its response to perturbation by laser ablation. Epithelial packing geometry is a consequence of both physical cellular properties and the disordering influence of proliferation. The occurrence of T2 transitions during network growth suggests that elimination of cells from the proliferating disc epithelium may be the result of junctional force balances.

Interplay of cell dynamics and epithelial tension during morphogenesis of the Drosophila pupal wing

How tissue shape emerges from the collective mechanical properties and behavior of individual cells is not understood. We combine experiment and theory to study this problem in the developing wing epithelium of Drosophila. At pupal stages, the wing-hinge contraction contributes to anisotropic tissue flows that reshape the wing blade. Here, we quantitatively account for this wing-blade shape change on the basis of cell divisions, cell rearrangements and cell shape changes. We show that cells both generate and respond to epithelial stresses during this process, and that the nature of this interplay specifies the pattern of junctional network remodeling that changes wing shape. We show that patterned constraints exerted on the tissue by the extracellular matrix are key to force the tissue into the right shape. We present a continuum mechanical model that quantitatively describes the relationship between epithelial stresses and cell dynamics, and how their interplay reshapes the wing. DOI: http://dx.doi.org/10.7554/eLife.07090.001

Mechanics and remodelling of cell packings in epithelia

Abstract.Epithelia are sheets of cells that are dynamically remodelled by cell division and cell death during development. Here we describe the cell shapes and packings as networks of polygons: stable and stationary network configurations obey force balance and are represented as local minima of a potential function. We characterize the physical properties of this vertex model, including the set of ground states, and the energetics of topological rearrangements. We furthermore discuss a quasistatic description of cell division that allows us to study the mechanics and dynamics of tissue remodelling during growth. The biophysics of cells and their rearrangements can account for the morphology of cell packings observed in experiments.

Establishment of Global Patterns of Planar Polarity during Growth of the Drosophila Wing Epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.

Time-lapse and cell ablation reveal the role of cell interactions in fly glia migration and proliferation

Migration and proliferation have been mostly explored in culture systems or fixed preparations. We present a simple genetic model, the chains of glia moving along fly wing nerves, to follow such dynamic processes by time-lapse in the whole animal. We show that glia undergo extensive cytoskeleton and mitotic apparatus rearrangements during division and migration. Single cell labelling identifies different glia: pioneers with high filopodial, exploratory, activity and, less active followers. In combination with time-lapse, altering this cellular environment by genetic means or cell ablation has allowed to us define the role of specific cell-cell interactions. First, neurone-glia interactions are not necessary for glia motility but do affect the direction of migration. Second, repulsive interactions between glia control the extent of movement. Finally, autonomous cues control proliferation.

Glial Chain Migration Requires Pioneer Cells

The migration of glial chains along the nerve entails directional and coordinated movement. Despite its importance in the formation of the nervous system, this process remains poorly understood, because of the difficulty of manipulating identified cells. Using confocal time-lapse and cell ablation in the whole animal, we provide direct evidence for a discrete number of Drosophila peripheral glial cells acting as pioneers and guiding the rest of the migratory chain. These cells are in direct contact with several follower cells through a very long and stable cytoplasmic extension. The presence of pioneer cells and homotypic interactions at the tip of the chain allows coordinated movement and the formation of a continuous sheath around the nerve. These in vivo data open novel perspectives for understanding the cellular bases of vertebrate glial migration in physiological and pathological conditions.

TissueMiner: A multiscale analysis toolkit to quantify how cellular processes create tissue dynamics

Segmentation and tracking of cells in long-term time-lapse experiments has emerged as a powerful method to understand how tissue shape changes emerge from the complex choreography of constituent cells. However, methods to store and interrogate the large datasets produced by these experiments are not widely available. Furthermore, recently developed methods for relating tissue shape changes to cell dynamics have not yet been widely applied by biologists because of their technical complexity. We therefore developed a database format that stores cellular connectivity and geometry information of deforming epithelial tissues, and computational tools to interrogate it and perform multi-scale analysis of morphogenesis. We provide tutorials for this computational framework, called TissueMiner, and demonstrate its capabilities by comparing cell and tissue dynamics in vein and inter-vein subregions of the Drosophila pupal wing. These analyses reveal an unexpected role for convergent extension in shaping wing veins. DOI: http://dx.doi.org/10.7554/eLife.14334.001

Imaging Drosophila Pupal Wing Morphogenesis

Drosophila pupal (P) wing development entails a series of dynamic developmental events, such as epithelial and glial morphogenesis, that are of outstanding interest to cell biologists. Here, we first describe how to prepare P and prepupal (PP) wings for immunofluorescence microscopy. This protocol has been optimized to visualize wing epithelial architecture, such as polarized cortical domains of planar cell polarity proteins. We then provide a protocol to prepare pupae for whole mount live imaging of P wings. This procedure has allowed us to live-image glial cell migration and proliferation along wing sensory nerves.

UV laser mediated cell selective destruction by confocal microscopy

Analysis of cell-cell interactions, cell function and cell lineages greatly benefits selective destruction techniques, which, at present, rely on dedicated, high energy, pulsed lasers and are limited to cells that are detectable by conventional microscopy. We present here a high resolution/sensitivity technique based on confocal microscopy and relying on commonly used UV lasers. Coupling this technique with time-lapse enables the destruction and following of any cell(s) in any pattern(s) in living animals as well as in cell culture systems.

Segmentation and Quantitative Analysis of Epithelial Tissues

Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.