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This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.
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.
To achieve their roles, epithelial tissues fully rely on the spatial organization of their cellular components. In most epithelia, cells are not only packed against each other to create a precise cobblestone layer but they orient themselves relative to the body axes.
The functional importance of precise tissue organization is obvious in sensory epithelia, such as the vertebrate inner ear and retina. In the first case, hair and supporting cells align in a specific axial direction to efficiently sense mechanical inputs such as sound and motion1,2. Similarly, photoreceptor cell spatial....
NOTE: This protocol is divided into five steps: (1) staging the pupae, (2) preparing the pupae for imaging, (3) live imaging of the growing abdominal epithelia, (4) generation of genetic mosaics, (5) data processing and analysis (including sections describing how to analyze cell orientation dynamics from cell junction outlines and growth dynamics from cell clones).
1. Staging of Drosophila pupae before imaging
The protocol described above covers the preparation of Drosophila pupae for long-term live imaging and the procedures for the analysis of cell orientation and growth dynamics of the abdominal epidermis. By applying this methodology it is possible to generate high-resolution movies of the developing pupae for periods of up to 48 h without significant photobleaching or phototoxicity. Snapshots depicting the abdominal epidermis (e.g., histoblasts and LECs) at different time points a.......
Long-range order is an essential characteristic of most functional physiological units. During morphogenesis, order is achieved through the integration of complex instructions implemented with high temporal and spatial precision. Multiple and multilevel constrains are integrated into stereotyped tissue arrangements.
Polarity and directionality are critical to ordered spatial arrangement during development. Polarity implies symmetry breaking during development. The achievement of asymmetry is n.......
We would like to thank members of the Martín-Blanco laboratory for helpful discussions. We also thank Nic Tapon (The Crick Institute, London, UK), the Bloomington Stock Center (University of Indiana, USA) and FlyBase (for Drosophila gene annotation). Federica Mangione was supported by a JAE-CSIC predoctoral fellowship. The Martín-Blanco laboratory was funded from the Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia (BFU2014-57019-P and BFU2017-82876-P) and from the Fundación Ramón Areces.
....Name | Company | Catalog Number | Comments |
Analysis Software | - | ImageJ | Analyzing data |
Drosophila | Atpa::GFP | - | Strains employed for data collection |
Drosophila | hsflp1.22;FRT40A/FRT40A Ubi.RFP.nls | - | Strains employed for data collection |
Dumont 5 Forceps | FST | 11251-20 | 1.5 mm diameter for dissection |
Glass Bottom Plates | Mat Tek | P35G-0.170-14-C | Mounting pupae for data collection |
Halocarbon Oil 27 | Sigma-Aldrich | 9002-83-9 | mounting pupae |
Inverted Confocal microscope | Zeiss | LSM700 | Data collection |
Stereomicroscope | Leica | DFC365FX | Visualization of the pupae during dissection |
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