Recoil Measurements in Drosophila Embryos: from Mounting to Image Analysis

Background

Tissue morphogenesis requires that cells change their individual shapes and rearrange with one another to form novel structures. Most often, these processes depend on tension and force propagation to induce cell shape changes (Barrera-Velázquez and Ríos-Barrera, 2021; Inman and Smutny, 2021). Forces can act within the same cell or specific subcellular compartments, but can also act in neighboring cells with different biochemical and mechanical properties (Bhide et al., 2021; Ríos-Barrera and Leptin, 2022).

Cytoskeletal elements like actin are often the molecular basis of force generation. Actin fibers interact with motors and crosslinkers to generate force that induces cell autonomous and non-autonomous shape changes (Belmonte et al., 2017; Miao and Blankenship, 2020). Nevertheless, the sole presence of actin meshworks within a cell or tissue may not be indicative of tissue tension. To show that a given structure is under tension, one must manipulate or measure the mechanical properties of the tissue of interest. Different tools have been developed to do this, with particular advantages and caveats. Optogenetics (Izquierdo et al., 2018; Krueger et al., 2019) has a high temporal and spatial specificity but requires the incorporation of genetic tools that are sensitive to light, requiring special handling conditions and restricting imaging possibilities. Atomic force microscopy (Haase and Pelling, 2015) provides a direct readout of tissue properties but uses dedicated equipment and only works in direct contact with the specimen of interest. Fluorescent tension sensors (Lemke et al., 2019) do not require external manipulations but depend on complex image analyses to obtain quantitative information. Laser microdissection was among the first tools that enabled probing mechanical properties of tissues. It consists in focusing high-frequency photons within a region of interest (ROI), which will then disrupt and destabilize actin fibers (Nishimura et al., 2006). Severing the actin cytoskeleton using laser microdissection alters the force distribution in the cell or tissue of interest, resulting in a recoil in the direction in which the tissue is being stretched. Initial recoil speed of the tissue is directly proportional to the tension of the sample immediately before the laser procedure (Rauzi and Lenne, 2014; Shivakumar and Lenne, 2016). Therefore, by measuring the recoil speed, it is possible to infer the tension exerted in the sample of interest. This provides a quantitative approximation of the forces participating in the process of interest and can be used to compare tension generation in different directions (anisotropy), in different regions and stages of embryo development, or in varying genetic conditions (genetic gain- and loss-of-function studies).

There are several ways in which one can measure recoil speed [for an alternative approach, see Bio-protocol by Liang et al. (2016)]. In this protocol, we use particle image velocimetry (PIV) analyses, a straightforward approach that does not rely on image segmentation or manual annotation of the signal of interest (Tseng et al., 2012). Instead, PIV compares the spatial distribution of the fluorescent signal within two consecutive time points by comparing the signal within small interrogation windows within the ROI. A shift in the signal in an interrogation window is converted into a vector, which depicts the strength and direction of recoil, if there is any.

As an example of this procedure, we use tracheal terminal cells of the Drosophila respiratory system, which uses actin to coordinate their direction of migration with the formation of a subcellular tube (Ríos-Barrera and Leptin, 2022). Nevertheless, this approach can be used to study a wide range of developmental processes in the fruit fly and in other model systems.