Sepaniac LA et al. (2023), Bring the pain: wounding reveals a transition f...

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Front Cell Dev Biol 2023 Jan 01;11:1295569. doi: 10.3389/fcell.2023.1295569.

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Bring the pain: wounding reveals a transition from cortical excitability to epithelial excitability in Xenopus embryos.

Sepaniac LA , Davenport NR , Bement WM .

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The cell cortex plays many critical roles, including interpreting and responding to internal and external signals. One behavior which supports a cell's ability to respond to both internal and externally-derived signaling is cortical excitability, wherein coupled positive and negative feedback loops generate waves of actin polymerization and depolymerization at the cortex. Cortical excitability is a highly conserved behavior, having been demonstrated in many cell types and organisms. One system well-suited to studying cortical excitability is Xenopus laevis, in which cortical excitability is easily monitored for many hours after fertilization. Indeed, recent investigations using X. laevis have furthered our understanding of the circuitry underlying cortical excitability and how it contributes to cytokinesis. Here, we describe the impact of wounding, which represents both a chemical and a physical signal, on cortical excitability. In early embryos (zygotes to early blastulae), we find that wounding results in a transient cessation ("freezing") of wave propagation followed by transport of frozen waves toward the wound site. We also find that wounding near cell-cell junctions results in the formation of an F-actin (actin filament)-based structure that pulls the junction toward the wound; at least part of this structure is based on frozen waves. In later embryos (late blastulae to gastrulae), we find that cortical excitability diminishes and is progressively replaced by epithelial excitability, a process in which wounded cells communicate with other cells via wave-like increases of calcium and apical F-actin. While the F-actin waves closely follow the calcium waves in space and time, under some conditions the actin wave can be uncoupled from the calcium wave, suggesting that they may be independently regulated by a common upstream signal. We conclude that as cortical excitability disappears from the level of the individual cell within the embryo, it is replaced by excitability at the level of the embryonic epithelium itself.

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Species referenced: Xenopus laevis
GO keywords: cell-cell junction [+]

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	FIGURE 1. Waves of F-actin in the Xenopus embryo cortex. (A) Single frame from a movie showing confocal F-actin waves in a Xenopus embryo. Red arrowhead indicates single wave. (B) Kymograph made form the movie in A, generated from the position indicated by black arrowhead in A. Red arrowhead indicates wave. (C) Plot of fluorescence intensity (in arbitrary units, AU) over time (in minutes) of 6 μm2 of cortex. (D) Montage of propagating waves of F-actin across a cortex, within a representative wave period.
	FIGURE 2. Impact of blastomere wounding on cortical F-actin waves. (A) Montage of blastomere surface, before and after wounding, with time (in minutes) indicated relative to wound initiation. Wound position labeled as “W”, and red arrows indicates direction of cortical flow to facilitate closure of the wound. (B) Kymograph of blastomere wounding. (B′) Enlarged portion of kymograph from (B), (enlarged region indicated via dashed white box in (B)), where wound initiation occurs at the black horizontal line and labeled via black arrowhead. Proximal regions of F-actin waves (indicated by star) are pulled inward, to the center of the wound (indicated by red arrowhead).
	FIGURE 3. Formation of a contractile structure between wounds and junctions. (A) Time lapse of a late-stage embryo, highlighting a region of the cortex nearby a cell-cell junction, with time (in minutes) reported relative to the induction of the first wound. Two wounds are introduced: Wound 1 (“W1”) appears further from the junction, while Wound 2 (“W2”) which is located closer to the junction, pulls this junction. (A′) Enlarged view of the cortex with Wound 2 and nearby cell junction, shown in (A). F-actin waves spanning the region between the wound edge and the proximal junction are indicated via star (‘*’), and these waves of F-actin are incorporated into the contractile array, creating a localized displacement of the junction as it is pulled toward the wound edge. (B) Kymograph of the wounding events shown in (A), where Wound 2 is shown to pull the nearby junction toward Wound 2, unlike the more distal Wound 1. (B) Kymograph of the wounding events shown in (A), where Wound 2 is shown to pull the nearby junction toward Wound 2, unlike the more distal Wound 1. Each wound initiation is shown by black horizontal line, and labeled via black arrowhead.
	FIGURE 4. A wave of wound-induced actin assembly in gastrulae. (A) Time lapse imaging of the cell cortex, with time relative to wound induction, showing a wave-like response of increased apical F-actin spreading beyond the wound and remaining to reinforce nearby cell junctions. (B) Plot of fluorescence intensity (in arbitrary units, AU) over time (in minutes) demonstrating increased apical F-actin response at different points across the cortex (corresponding to labeling in A): “W” at the wound site; “A” indicating a cell directly neighboring the wound; “B” indicating a neighboring cell positioned further from the wound. Increased apical F-actin is shown to occur at a similar time point and magnitude across these locations. (C) Kymograph of wound response, for example, shown in (A). Wound site labeled as “W” and timepoint of wound induction indicated by black horizontal line and emphasized via black arrowhead. Junctional actin increase is emphasized via white parallel lines, framing the increase in F-actin signal occurring at the junctions (labeled below). A “flash” of apical F-actin is highlighted, occurring almost immediately after wounding and spanning several neighboring cells (indicated to the right of the kymograph).
	FIGURE 5. Development of a wound-induced multicellular calcium wave. Wound site indicated as “W” in each example, described as follows. (A) Wounded early blastula results in a localized calcium response, mainly contained within the wounded cell, shown in greyscale. (B) Wounded late blastula/early gastrula results in a wave of calcium extending to neighbor cells. (C) Wounded gastrula results in a wave of elevated calcium, extending to surrounding neighbor cells. (C′) Wounded gastrula, with larger surface area depicted (note scale), leading to a broadened distance of cells exhibiting calcium wave response. (D) Plot of fluorescence intensity (in arbitrary units, AU), of calcium over time (in minutes), as indicated via the calcium sensor GCamp6, as measured at the wound site “W”, a neighboring cell, “A”, and a distal neighboring cell, “B”, where each of these locations are identified in (C′).
	FIGURE 6. Collisions of calcium waves result in auto-annihilation in late-stage gastrula. (A) Raw data for imaging of two opposing wavefronts of calcium, shown in grayscale with the direction of wave propagation indicated via red arrowhead. The wavefronts approach one another, resulting in rapid auto-inhibition of the wave. (A′) Difference movie of the opposing wavefronts, revealing auto-inhibition of the waves shown in (A).
	FIGURE 7. The long-distance, excitable calcium wave response in late-stage embryos is triggered independently of an apical actin response. (A) A gastrula stage embryo, expressing probes for both calcium (cyan) and F-actin (red), receives three successive wounds via ablation (wound locations indicated as: W1, W2, and W3). A nearby cell, marked with an asterisk, shows where calcium and F-actin responses are measured. (A′) Time course of calcium (top) and F-actin (bottom) response, following wounding, as measured in a single cell indicated in A (asterisk), where time is reported in minutes. W1 occurs at 1:40 min; W2 at 4:06 min; W3 at 10:30 min (A)″ Line scan for measured fluorescence intensity (arbitrary units, AU) for calcium and F-actin, respectively, following each wound, over time (seconds). (B) Late blastulae-early gastrula expressing probes for calcium and F-actin, wounded once (wound position labeled, W), with calcium and F-actin response measured in 5 cells, as indicated (labeled 1–5). (B′) Individual line intensity plots demonstrate the intensity of calcium and F-actin signals (arbitrary units, AU) reported over time (sec) after ablation.
	Supplementary Figure 2: F-actin wave consistently lags behind the calcium wave. (A) Representative images of a mid/late blastula co-expressing calcium and F-actin probes, with four representative regions of interest (yellow, boxed ROI labeled 1-4); regions encompass more than one cell per region. Timing relative to the time of wounding. (A’) Line traces for each of the four regions of interest, as labeled in A, with responding calcium and actin intensity (arbitrary units, AU) against time (seconds) after wounding (wound location not shown).