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A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.

Niethammer P, Grabher C, Look AT, Mitchison TJ - Nature (2009)

Bottom Line: Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling.Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor.This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm into the tail-fin epithelium as a decreasing concentration gradient.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02114, USA. Philipp_Niethammer@hms.harvard.edu

ABSTRACT
Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor. This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H(2)O(2) pattern, and the first evidence that H(2)O(2) signals to leukocytes in tissues, in addition to its known antiseptic role.

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Duox activity is required for wound margin H2O2 production and leukocyte recruitment. (a) Wound margin H2O2 after morpholino mediated duox knockdown (MO1-duox) or injection of a corresponding 5-misprime morpholino (5-MP) imaged 17 min pw. Inset: RT-PCR of a duox mRNA region flanking the targeted splice site. (b) Quantification of wound margin [H2O2]. (c) Injured tail fins of mpo::GFP9 larvae injected with MO1-duox, or 5-MP (42 min pw). Coloured lines: leukocyte tracks. (d) Quantification of leukocyte recruitment. Error bars: SEM of indicated number of larvae (brackets). ** P < 0.01, *** P < 0.001 (vs. control). Scale bars: 100 μm.
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Figure 3: Duox activity is required for wound margin H2O2 production and leukocyte recruitment. (a) Wound margin H2O2 after morpholino mediated duox knockdown (MO1-duox) or injection of a corresponding 5-misprime morpholino (5-MP) imaged 17 min pw. Inset: RT-PCR of a duox mRNA region flanking the targeted splice site. (b) Quantification of wound margin [H2O2]. (c) Injured tail fins of mpo::GFP9 larvae injected with MO1-duox, or 5-MP (42 min pw). Coloured lines: leukocyte tracks. (d) Quantification of leukocyte recruitment. Error bars: SEM of indicated number of larvae (brackets). ** P < 0.01, *** P < 0.001 (vs. control). Scale bars: 100 μm.

Mentions: We quantified leukocyte recruitment to the wound by imaging transmitted light and two different leukocyte-specific fluorescent tags, mpo::GFP9 (Figure 2d, 3c) and lysC::DsRED210 (Figure 1e). Some leukocytes were patrolling the fin at the time of wounding, while others were apparently recruited from the vasculature. Excluding occasional cases where a leukocyte was already present at the wound site, the first leukocyte arrived at the wound margin 17 ± 6 min pw (mean ± SD of n=14 larvae). This timing is superimposed on a typical H2O2 profile in Figure 1c. Wound margin H2O2 production clearly preceded recruitment of the first leukocyte in most cases (see also Figure 1e, Supplementary Figure S1d, Supplementary Movie 2), indicating that the source of H2O2 must be tail fin epithelial cells, not leukocytes. This finding runs counter to the prevailing view that ROS production during inflammatory responses originates from leukocyte oxidative bursts11.


A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.

Niethammer P, Grabher C, Look AT, Mitchison TJ - Nature (2009)

Duox activity is required for wound margin H2O2 production and leukocyte recruitment. (a) Wound margin H2O2 after morpholino mediated duox knockdown (MO1-duox) or injection of a corresponding 5-misprime morpholino (5-MP) imaged 17 min pw. Inset: RT-PCR of a duox mRNA region flanking the targeted splice site. (b) Quantification of wound margin [H2O2]. (c) Injured tail fins of mpo::GFP9 larvae injected with MO1-duox, or 5-MP (42 min pw). Coloured lines: leukocyte tracks. (d) Quantification of leukocyte recruitment. Error bars: SEM of indicated number of larvae (brackets). ** P < 0.01, *** P < 0.001 (vs. control). Scale bars: 100 μm.
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Figure 3: Duox activity is required for wound margin H2O2 production and leukocyte recruitment. (a) Wound margin H2O2 after morpholino mediated duox knockdown (MO1-duox) or injection of a corresponding 5-misprime morpholino (5-MP) imaged 17 min pw. Inset: RT-PCR of a duox mRNA region flanking the targeted splice site. (b) Quantification of wound margin [H2O2]. (c) Injured tail fins of mpo::GFP9 larvae injected with MO1-duox, or 5-MP (42 min pw). Coloured lines: leukocyte tracks. (d) Quantification of leukocyte recruitment. Error bars: SEM of indicated number of larvae (brackets). ** P < 0.01, *** P < 0.001 (vs. control). Scale bars: 100 μm.
Mentions: We quantified leukocyte recruitment to the wound by imaging transmitted light and two different leukocyte-specific fluorescent tags, mpo::GFP9 (Figure 2d, 3c) and lysC::DsRED210 (Figure 1e). Some leukocytes were patrolling the fin at the time of wounding, while others were apparently recruited from the vasculature. Excluding occasional cases where a leukocyte was already present at the wound site, the first leukocyte arrived at the wound margin 17 ± 6 min pw (mean ± SD of n=14 larvae). This timing is superimposed on a typical H2O2 profile in Figure 1c. Wound margin H2O2 production clearly preceded recruitment of the first leukocyte in most cases (see also Figure 1e, Supplementary Figure S1d, Supplementary Movie 2), indicating that the source of H2O2 must be tail fin epithelial cells, not leukocytes. This finding runs counter to the prevailing view that ROS production during inflammatory responses originates from leukocyte oxidative bursts11.

Bottom Line: Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling.Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor.This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm into the tail-fin epithelium as a decreasing concentration gradient.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02114, USA. Philipp_Niethammer@hms.harvard.edu

ABSTRACT
Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor. This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H(2)O(2) pattern, and the first evidence that H(2)O(2) signals to leukocytes in tissues, in addition to its known antiseptic role.

Show MeSH
Related in: MedlinePlus