Live imaging in Drosophila: The optical and genetic toolkits.

Biological imaging based on light microscopy comes at the core of the methods that let us understanding morphology and its dynamics in synergy to the spatiotemporal distribution of cellular and molecular activities as the organism develops and becomes functional. Non-linear optical tools and superesolution methodologies are under constant development and their applications to live imaging of whole organisms keep improving as we speak. Genetically coded biosensors, multicolor clonal methods and optogenetics in different organisms and, in particular, in Drosophila follow equivalent paths. We anticipate a brilliant future for live imaging providing the roots for the holistic understanding, rather than for individual parts, of development and function at the whole-organism level.

Co-option of epidermal cells enables touch sensing.

The epidermis is equipped with specialized mechanosensory organs that enable the detection of tactile stimuli. Here, by examining the differentiation of the tactile bristles, mechanosensory organs decorating the Drosophila adult epidermis, we show that neighbouring epidermal cells are essential for touch perception. Each mechanosensory bristle signals to the surrounding epidermis to co-opt a single epidermal cell, which we named the F-Cell. Once specified, the F-Cell adopts a specialized morphology to ensheath each bristle. Functional assays reveal that adult mechanosensory bristles require association with the epidermal F-Cell for touch sensing. Our findings underscore the importance of resident epidermal cells in the assembly of functional touch-sensitive organs.

Photoablation at single cell resolution and its application in the Drosophila epidermis and peripheral nervous system.

Tissues contain diverse cell populations that, together, make up physiologically functional units. A remarkable example is the animal epidermis, where neuronal and non-neuronal cells intermingle to allow somatosensory perception. In the peripheral nervous system (PNS), the tight association between heterogenous cell types poses challenges when the structural and physiological contributions of neuronal and surrounding cells need to be dissected with suitable precision. When genetic tools for cell-specific, spatiotemporally controlled gene expression are not available, targeted cell ablation represents a considerable obstacle. Here, we describe an efficient method to overcome this limitation and demonstrate its application to the study of the differentiating Drosophila epidermis and PNS. This methodology relies on the use of near infrared (NIR) femtosecond (fs) laser pulses for ablation of the desired cells at the desired time. We show how to confine the photodamage to the targeted cell to induce its death, without harming neighbouring tissues or structures. We validated our approach in the Drosophila PNS by studying the responses of photo-ablated neurons, non-neuronal cells, and the surrounding epidermis. Diverse cellular behaviours including cell extrusion, cell rearrangements and cell shape changes can be monitored in vivo immediately after damage, as well as for several hours post-ablation with high optical resolution using confocal microscopy. This methodology provides a flexible tool to ablate individual cells with high precision and study morphological responses to cell loss in targeted areas or neighbouring structures. We anticipate that this protocol can be easily adapted to other model systems and tissues.

ECM degradation in the Drosophila abdominal epidermis initiates tissue growth that ceases with rapid cell-cycle exit.

During development, multicellular organisms undergo stereotypical patterns of tissue growth in space and time. How developmental growth is orchestrated remains unclear, largely due to the difficulty of observing and quantitating this process in a living organism. Drosophila histoblast nests are small clusters of progenitor epithelial cells that undergo extensive growth to give rise to the adult abdominal epidermis and are amenable to live imaging. Our quantitative analysis of histoblast proliferation and tissue mechanics reveals that tissue growth is driven by cell divisions initiated through basal extracellular matrix degradation by matrix metalloproteases secreted by the neighboring larval epidermal cells. Laser ablations and computational simulations show that tissue mechanical tension does not decrease as the histoblasts fill the abdominal epidermal surface. During tissue growth, the histoblasts display oscillatory cell division rates until growth termination occurs through the rapid emergence of G0/G1 arrested cells, rather than a gradual increase in cell-cycle time as observed in other systems such as the Drosophila wing and mouse postnatal epidermis. Different developing tissues can therefore achieve their final size using distinct growth termination strategies. Thus, adult abdominal epidermal development is characterized by changes in the tissue microenvironment and a rapid exit from the cell cycle.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages.

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

The Dachsous/Fat/Four-Jointed Pathway Directs the Uniform Axial Orientation of Epithelial Cells in the Drosophila Abdomen.

The achievement of the final form of an individual requires not only the control of cell size and differentiation but also integrative directional cues to instruct cell movements, positions, and orientations. In Drosophila, the adult epidermis of the abdomen is created de novo by histoblasts. As these expand and fuse, they uniformly orient along the anteroposterior axis. We found that the Dachsous/Fat/Four-jointed (Ds/Ft/Fj) pathway is key for their alignment. The refinement of the tissue-wide expression of the atypical cadherins Ds and Ft result in their polarization and directional adhesiveness. Mechanistically, the axially oriented changes in histoblasts respond to the redesign of the epithelial field. We suggest that the role of Ds/Ft/Fj in long-range oriented cell alignment is a general function and that the regulation of the expression of its components will be crucial in other morphogenetic models or during tissue repair.

High-speed flow of interacting organic polaritons.

The strong coupling of an excitonic transition with an electromagnetic mode results in composite quasi-particles called exciton polaritons, which have been shown to combine the best properties of their individual components in semiconductor microcavities. However, the physics and applications of polariton flows in organic materials and at room temperature are still unexplored because of the poor photon confinement in such structures. Here, we demonstrate that polaritons formed by the hybridization of organic excitons with a Bloch surface wave are able to propagate for hundreds of microns showing remarkable third-order nonlinear interactions upon high injection density. These findings pave the way for the study of organic nonlinear light-matter fluxes and for a technologically promising route of the realization of dissipation-less on-chip polariton devices operating at room temperature.