Relieving the Pressure on Tissue Development

Main Text

Understanding how tissue patterning emerges during embryogenesis is a long-standing goal of developmental biology. In particular, the complex interplay between extracellular and intracellular mechanical forces and biochemical cues that lead to cellular differentiation and ultimately adult tissue remains an active area of investigation. One of the main challenges has been that experimental methods that allow for the simultaneous measurement of mechanical force and biochemical signaling are limited. In this issue of Biophysical Journal, Narciso et al. (1) present an exciting experimental advance in their description of a microanalysis device that provides tight control of external mechanical stress in the immediate environment of a developing Drosophila organ while simultaneously allowing for imaging of intracellular biochemical signaling cues.

The common fruit fly Drosophila melanogaster has long been a model organism in developmental biology. Studies on development of the Drosophila wing imaginal disk have greatly contributed to our current understanding of how morphogen gradients and their associated genetic and biochemical regulatory networks contribute to cellular differentiation and pattern formation in developing tissues. Community resources such as FlyBase.org allow researchers to rapidly search sequenced genomes, identify mutant strains, perform gene ontology analysis, and access tools for RNaseq, RNA interference, and CRISPR implementation (2). Indeed, the availability of transgenic strains (over 22,000 are available), combined with CRISPR and RNA interference, allows for rapid screening of genes and biochemical pathways that contribute to cellular and tissue-level behaviors (3). These experimental tools, in combination with computational modeling of morphogen gradients (4) (reviewed in (5)), have greatly contributed to our current understanding of embryo development.

In addition to genetic and biochemical regulatory networks, mechanical cues have emerged as important fundamental regulators of cellular behavior. The ability of cells to both generate and respond to mechanical cues is critical for proper morphogenesis and tissue patterning (reviewed in (6)). Recent work has started to link mechanical stress with biochemical signaling in various model systems. For example, the combination of optogenetic and fluorescent biosensors with mechanical actuation and force-field generation has started to enable manipulation of cell signaling in real time (reviewed in (7)). Intracellular forces have been measured in early Drosophila embryos using microrheology enabled by high-speed video and particle tracking analysis (8). Moreover, intercellular Ca²⁺ waves (ICWs) propagating through tissues have been implicated in rapid actinomyosin cytoskeletal rearrangement in wounding models (9) and in tissue-wide responses to mechanical stress in vivo in the Drosophila imaginal wing disk (10, 11).

Building on this recent work, Narciso et al. (1) outline a new microanalytical device and corresponding methods for quantitatively studying the connections between extracellular mechanical forces and biochemical signaling. This work represents a significant technical advance in the ability to control and quantify the external pressure forces applied to a tissue. The device allows for simultaneous live cell/organotypic culture of relevant biological tissues, can deliver regulated mechanical compression to wing discs (or potentially other organotypic preparations) in culture, and allows for quantitative measurement of the resultant changes in the cells/tissues (e.g., displacement, Ca²⁺ wave propagation, and growth). Furthermore, the device accommodates possible adjustments to the culture medium or introduction of other chemical cues via microfluidics. Leveraging this device and tools of genetic perturbation, Narciso et al. clearly delineate the roles of biochemical and mechanical stimulation in the initiation of ICWs that propagate across the developing organ. They find that ICW propagation across the imaginal wing disk tissue is mediated via gap junctions and IP₃ receptors in what is likely a Ca²⁺-mediated Ca²⁺ release mechanism. Furthermore, these Ca²⁺ waves are initiated by the release of mechanical compression and depend on the pre-stress intercellular Ca²⁺ activity and not on the initiation, nor the duration of the mechanical stress. Additionally, Narciso et al. (1) uniquely combine a number of methods to quantitatively analyze both the performance of the device and the tissue under study. These are complemented by strong supplemental data and discussion of the methods that should allow for others to readily replicate and build upon this work.

Based on the results enabled by their microanalytical device, Narciso et al. propose that the spontaneous ICWs that occur during Drosophila larval development are a signature of stress dissipation in the wing disc as cells in the tissue grow and divide. This is an interesting hypothesis and an increasingly testable one. Techniques reported in Narciso et al. will enable new avenues for studying the connections between growth, extracellular mechanical forces, and biochemical signaling in developing tissue. Future work would connect these in a multiscale framework that also includes intracellular mechanical forces (actin-myosin flows and cytoskeletal rearrangement) and downstream biochemical and gene network regulation.

Editor: Leslie Loew.