Abstract
After a tissue is wounded, cells surrounding the wound adopt distinct wound-healing behaviors to repair the tissue. Considerable effort has been spent on understanding the signaling pathways that regulate immune and tissue-resident cells as they respond to wounds, but these signals must ultimately originate from the physical damage inflicted by the wound. Tissue wounds comprise several types of cellular damage, and recent work indicates that different types of cellular damage initiate different types of signaling. Hence to understand wound signaling, it is important to identify and localize the types of wound-induced cellular damage. Laser ablation is widely used by researchers to create reproducible, aseptic wounds in a tissue that can be live-imaged. Because laser wounding involves a combination of photochemical, photothermal and photomechanical mechanisms, each with distinct spatial dependencies, cells around a pulsed-laser wound will experience a gradient of damage. Here we exploit this gradient to create a map of wound-induced cellular damage. Using genetically-encoded fluorescent proteins, we monitor damaged cellular and sub-cellular components of epithelial cells in living Drosophila pupae in the seconds to minutes following wounding. We hypothesized that the regions of damage would be predictably arrayed around wounds of varying sizes, and subsequent analysis found that all damage radii are linearly related over a 3-fold range of wound size. Thus, around laser wounds, the distinct regions of damage can be estimated after measuring any one. This report identifies several different types of cellular damage within a wounded epithelial tissue in a living animal. By quantitatively mapping the size and placement of these different types of damage, we set the foundation for tracing wound-induced signaling back to the damage that initiates it.
Highlights
The field of wound repair and regeneration has long sought to connect the signals emanating from wounds to the behavioral changes undertaken by cells around the wound
We identify several more types of cells that are damaged by laser wounds; we monitored initial laser-induced rupture, delayed cell lysis, nuclear membrane damage, plasma membrane damage, chromatin disruption, Ecadherin loss, and a calcium expansion outward from the wound site
This study analyzed wounds made by pulsed-laser ablation, which creates reproducible and orderly wounds, with the regions of damage arranged in a reproducible manner
Summary
The field of wound repair and regeneration has long sought to connect the signals emanating from wounds to the behavioral changes undertaken by cells around the wound. The signals emanating from wounds must be derived from the damage itself; only a few studies have characterized the damaged tissue on a cellular/sub-cellular level to understand the distinct types of damage created by wounds [10,11,12,13]. Even before cytokine signaling is evident, cells with a different type of damage–torn plasma membranes–initiate calcium signaling, as extracellular calcium floods in through damaged membranes and out through gap junctions to undamaged neighboring cells [15]. Together, these studies suggest that understanding the origin of wound-induced signaling requires identifying and categorizing different types of cellular damage induced by wounding. We attempt to solve this problem by classifying various zones of damage around epithelial wounds visualized using genetically-encoded fluorophores, and mathematically defining the relationships between these zones of damage to create a map of the characteristic cellular damage experienced by a tissue following a wound
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