Limits...
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.

Show MeSH

Related in: MedlinePlus

Wound margin H2O2 production in zebrafish larvae. (a) Experimental procedure. (b) HyPer imaging in an injured zebrafish larva. [H2O2] is inferred from the YFP500/YFP420 excitation ratio of HyPer. Greyscale scaling is adjusted to improve contrast. (c) Temporal [H2O2] profile in a ∼10−30 μm broad region of interest along the wound margin. Arrival of first leukocyte at wound (solid red line) ± SD (dashed red line). (d) [H2O2] line profile normal to the wound margin. (e) Imaging of leukocyte recruitment and [H2O2] in a lysC::DsRED210 fish line. Coloured lines: superimposed leukocyte tracks. Scale bars: 100 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2803098&req=5

Figure 1: Wound margin H2O2 production in zebrafish larvae. (a) Experimental procedure. (b) HyPer imaging in an injured zebrafish larva. [H2O2] is inferred from the YFP500/YFP420 excitation ratio of HyPer. Greyscale scaling is adjusted to improve contrast. (c) Temporal [H2O2] profile in a ∼10−30 μm broad region of interest along the wound margin. Arrival of first leukocyte at wound (solid red line) ± SD (dashed red line). (d) [H2O2] line profile normal to the wound margin. (e) Imaging of leukocyte recruitment and [H2O2] in a lysC::DsRED210 fish line. Coloured lines: superimposed leukocyte tracks. Scale bars: 100 μm.

Mentions: We measured H2O2 by expressing HyPer, a genetically encoded ratiometric sensor that is highly selective for H2O2 over other ROS1. HyPer consists of the bacterial H2O2-sensitive transcription factor OxyR fused to a circularly permuted YFP. Cysteine oxidation of the OxyR part induces a conformational change that increases emission excited at 500 nm (YFP500) and decreases emission excited at 420 nm (YFP420). This change is rapidly reversible within the reducing cytoplasmic environment, allowing dynamic monitoring of intracellular H2O2 concentration. We introduced HyPer by mRNA injection into zebrafish embryos to induce global cytoplasmic expression (Figure 1a) and confirmed that HyPer ratios respond to externally added H2O2 (Supplementary Figure S1a).


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

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

Wound margin H2O2 production in zebrafish larvae. (a) Experimental procedure. (b) HyPer imaging in an injured zebrafish larva. [H2O2] is inferred from the YFP500/YFP420 excitation ratio of HyPer. Greyscale scaling is adjusted to improve contrast. (c) Temporal [H2O2] profile in a ∼10−30 μm broad region of interest along the wound margin. Arrival of first leukocyte at wound (solid red line) ± SD (dashed red line). (d) [H2O2] line profile normal to the wound margin. (e) Imaging of leukocyte recruitment and [H2O2] in a lysC::DsRED210 fish line. Coloured lines: superimposed leukocyte tracks. Scale bars: 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2803098&req=5

Figure 1: Wound margin H2O2 production in zebrafish larvae. (a) Experimental procedure. (b) HyPer imaging in an injured zebrafish larva. [H2O2] is inferred from the YFP500/YFP420 excitation ratio of HyPer. Greyscale scaling is adjusted to improve contrast. (c) Temporal [H2O2] profile in a ∼10−30 μm broad region of interest along the wound margin. Arrival of first leukocyte at wound (solid red line) ± SD (dashed red line). (d) [H2O2] line profile normal to the wound margin. (e) Imaging of leukocyte recruitment and [H2O2] in a lysC::DsRED210 fish line. Coloured lines: superimposed leukocyte tracks. Scale bars: 100 μm.
Mentions: We measured H2O2 by expressing HyPer, a genetically encoded ratiometric sensor that is highly selective for H2O2 over other ROS1. HyPer consists of the bacterial H2O2-sensitive transcription factor OxyR fused to a circularly permuted YFP. Cysteine oxidation of the OxyR part induces a conformational change that increases emission excited at 500 nm (YFP500) and decreases emission excited at 420 nm (YFP420). This change is rapidly reversible within the reducing cytoplasmic environment, allowing dynamic monitoring of intracellular H2O2 concentration. We introduced HyPer by mRNA injection into zebrafish embryos to induce global cytoplasmic expression (Figure 1a) and confirmed that HyPer ratios respond to externally added H2O2 (Supplementary Figure S1a).

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