Dynamic Mechanical Multi-Cellular Response in Embryo Early Development

Introduction::
Mechanotransduction is a crucial process that converts mechanical forces into biochemical signals, which is essential for tissue development and maintaining normal physiology. Drosophila embryos are an excellent model system to study mechanotransduction in an endogenous context and are especially important for understanding the way that collective behavior of cells are able to sense and response to external stimuli. Drosophila development is fundamentally linked to mechanical force, which opens mechanosensitive ion channels (MSCs), resulting in a wave of Ca²⁺ through the oocyte that activates the egg. The process of epithelial wound healing in Drosophila embryos has also been shown to involve MSCs in a Ca²⁺-dependent manner. Furthermore, cellular Ca²⁺ spiking has been observed at early stages of development, such as during gastrulation, and later during midgut morphogenesis in muscle cells at sites of tissue constrictions. In this study, we investigated the relationship between Ca²⁺ spiking and mechanical stimulation of epithelial cells during gastrulation in Drosophila embryos with the ability to resolve individual and collective cell responses simultaneously giving us tremendous insight into the overall biosystem functioning.




Materials and Methods:: Time-lapse microscopy was utilized to observe Ca²⁺ spiking events during embryogenesis. A fly stock was generated to express both a calcium sensor, GCaMP6s, and a cortical actin cytoskeleton marker, moe-Scarlet. Three-dimensional, time-lapse, confocal microscopy recordings of uncompressed or compressed (20% laterally) embryos immobilized in custom designed and fabricated microfluidic channels were captured (z-stacks were collected every 10 seconds over a 20 minute period). The Ca²⁺spikes were annotated with ImageJ.

Results, Conclusions, and Discussions::
Results: The overall finding of this work was to investigate calcium response in living multi-cellular embryos through our novel microfabricated system that is able to mechanically stimulate entire embryos while imaging them simultaneously. Ca²⁺ spiking events were observed throughout numerous stages of embryogenesis after cellularization. Here, we focus on the mechanically intensive process of dorsal germband extension during stage 6 (Fig 1A). There is a concentration of Ca²⁺ spikes along the dorsal midline in proximity of the ATF and PTF (Fig1 B & C). Compressed embryos displayed approximately 40 spikes over the 20-minute period, similarly to uncompressed embryos. However, the distribution of Ca²⁺ spike was more centrally located in compressed embryos, which is the region of maximal compression (Fig 1B & C). Also, this compression caused a significant increase in the maximum spike intensity (Fig 1D).









Conclusion: This study shows that externally applied, mechanical stress affects Ca²⁺ signaling during early embryogenesis. The observed concentration of Ca²⁺ spiking events around the transverse furrows suggests that endogenous cellular deformation stimulates Ca²⁺ spikes. Additionally, the increase in maximum spike intensity upon compression suggests that spiking is sensitive to the magnitude of mechanical stress. Collectively, these findings suggest that Ca²⁺ spiking is significant during embryogenesis and that mechanical stress plays a critical role in regulating Ca²⁺ signaling events.









Discussion: These results support the role of mechanotransduction in embryonic development. The increased maximum spike intensity under compression and the localization of spikes around invagination sites suggests that mechanical strain can enhance Ca²⁺ signaling in cells. These findings shed light on the complex interplay between mechanical forces and biochemical signals during embryonic development, raising questions about the molecular sensors of this force and the cellular response to Ca²⁺ signaling as well as the extremely important intercellular collective behavior of multi-cellular systems. These findings have implications for human health and regenerative medicine, as there is a growing appreciation for the role of mechanically activated Ca²⁺ spikes in cancer metastasis and stem-cell differentiation. Further investigations are needed to understand the widespread role of mechanical regulation and Ca²⁺ signaling in development and more complex biological systems.




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