Zebrafish macrophages convert physical wound signals into rapid vascular permeabilization

Domain: neuroscience
Published: 2026-02-06
Editorial outcome: major_revision

Abstract
Blood vessels near injury sites rapidly dilate, become permeable, and release serum and leukocytes into the wounded tissue to support healing and regeneration. How the vasculature senses distant homeostatic tissue perturbations within seconds-to-minutes remains incompletely understood. Using high-speed imaging of live zebrafish larvae, we monitor two hallmark vascular responses to injury: vessel dilation and serum exudation. By genetic, pharmacologic, and osmotic perturbation along with leukocyte depletion, we show that the cPla2 nuclear membrane mechanotransduction pathway converts a ~50 μm/s osmotic wound signal into rapid vessel permeabilization via perivascular macrophages, 5-lipoxygenase (Alox5a), and leukotriene A4 hydrolase (Lta4h). By revealing a previously undescribed physiological function of nuclear membrane mechanotransduction, we provide real-time insights into the long-range communication of wounds and blood vessels in intact tissue.

Introduction
Real time imaging of zebrafish larvae allows studying the spatio-temporal regulation of wound detection in vivo at timescales inaccessible to conventional biochemical or molecular biology methods1,2. Previous work in zebrafish, flies, and frogs, revealed extracellular nucleotides, bioactive lipids, Ca2+ waves, reactive oxygen species (ROS), osmotic-, and ionic- gradients as physiological mediators of rapid wound detection3–12.

We previously showed that zebrafish detect integumental breaches via the osmotic shock that occurs when environmental freshwater enters their tissues from the outside. Consequently, immersing zebrafish larvae in isotonic salt or sugar solutions suppresses neutrophil recruitment and acute breach closure by epithelial cells. Compared to other isotonic treatments, NaCl has a stronger inhibitory effect4,5, which may be due to electric fields (EFs)7, or other, yet unknown salt-sensing mechanisms. Osmotic shock at the wound induces local cell swelling and nuclear deformation that stretches the inner nuclear membrane (INM)13. Along with Ca2+, INM tension (T_INM) recruits cPla2 to the INM to release arachidonic acid (AA). Lipoxygenases, cyclooxygenases, etc., further oxidize AA into bioactive lipids that control chemotaxis, cell differentiation, blood vessel tone and permeability, and many other processes14–18. Besides wound detection, the cPla2 nuclear membrane mechanotransduction/shape sensing pathway has been reported to control confined cell migration, dendritic cell chemotaxis, mesenchymal stem cell differentiation, and the cell cycle19–25.

At larval wounds and infection sites, downstream metabolites of cPla2 rapidly recruit leukocytes4,26,27. At least in part, these leukocytes come from the blood circuit. To release them nearby wound sites, blood vessels can apparently sense wounds as fast or even faster than leukocytes. Although a plethora of vascular regulators have been described, how local tissue injury is detected and relayed to nearby vessels remains little studied in situ.

In this work, real-time intravital imaging reveals that perivascular macrophages mediate rapid vessel permeabilization at acute osmotic wounds through the cPla2 nuclear membrane mechanotransduction pathway.

Results
Rapid blood vessel permeability is mediated by osmotic shock
To image rapid vessel responses to injury in vivo, we intracardially injected 2-3 days post fertilization (dpf) larvae with 70 kDa fluorescent dextran to label the circulating blood pool. Carefully avoiding damage to the vasculature, we amputated the tail fin tips of these larvae in E3 solution28 adjusted to isotonicity with sodium chloride (ISO_NaCl) to roughly match the composition of vertebrate interstitial fluid (Fig. 1a). We previously showed that ISO_NaCl prevents rapid leukocyte recruitment and epithelial wound closure in zebrafish larvae4,5,29. Wound detection can be triggered by transferring the injured larvae back to fresh water such as E3 (HYPO). This ISO→HYPO switch of bathing solutions, temporally synchronizes the wound response in animals injured and imaged at different times, facilitating statistical integration. The individual contributions of osmotic pressure and ionic cues to wound detection can be distinguished by shifting into isotonic solutions of different salts or sugar compositions (ISO*).

Using spinning disk confocal microscopy, we followed vessel permeability by measuring the fluorescence intensity (Iv(t)) of 70 kDa fluorescent dextran in a region of interest (ROI) adjacent to the caudal vein (Fig. 1b–e) after ISO_NaCl →HYPO, ISO_NaCl →ISO_NaCl, or ISO_NaCl →ISO* shifts. Besides permeabilization, we also measured vessel diameter (Dv(t)) within a ROI comprising a vein/artery subsection (Fig. 1f, g).

Vessel permeability drastically rose upon hypotonic but not isotonic switching (Fig. 1c). Both, dextran leakage from the vessel into the interstitium, and its loss through the wound may influence perivascular dextran intensity. We measured dextran’s wound leakage in a ROI next to the amputation site just outside the animal (Ib(t)) (Fig. 1b, e). Although wound leakage was very slow compared to vessel leakage (Rv0 ≫ Rb0) (Fig. 1c–e) presented a useful proxy for wound permeability/size.

To integrate the amount of dextran leaking from vessels over the course of the experiment, we summed up the respective, normalized Iv_norm intensity values (i.e., determined the areas under the curves in Supplementary Fig. 1a). Since Iv_norm is 1 when there is no wound-induced permeability change, a hypothetical Iv_tot=norm value of 3600 A.U. indicates that no net-leakage of dextran has occurred over the 3600 s of the experiment. In all the Iv bar-graphs, this theoretical baseline leakage is indicated by a dotted line. The measured baseline leakage in unwounded larvae (‘NW-HYPO’, Fig. 1d) is only slightly (~3%) higher than this theoretical minimum. Depending on which baseline is used for calculation, ISO/ISO* treatment blocks wound-induced vessel permeability by ~88/77%. All effect size approximations indicated below are based on the theoretical 3600 (arb. units.) Iv_tot=norm baseline.

For comparing wound closure and vessel dilation as a function of osmolyte treatment, we plotted normalized wound leakage (i.e., Ib_norm at 3600s; Fig. 1e) and the maximal vessel dilation (i.e., max(Dv_norm(t)); Fig. 1g). Consistent with earlier observations4,5, isotonic NaCl always showed the strongest inhibitory effect, very closely followed by the other non-ionic and ionic isotonicity treatments (Fig. 1d–e, 1g; Supplementary Fig. 1b–f; Supplementary Movie 1–3). Some of the differential osmolyte effects might reflect EF perturbation as previously proposed7.

Overall, our data suggested that the rapid surge of vessel permeability was primarily due to osmotic shock. The response was not sensitive to zebrafish strain (AB vs. Casper) or size of the injected dextran (500 kDa vs. 70 kDa) (Supplementary Fig. 1g–m; Supplementary Movie 4, 5). So, we set out to dissecting its mechanism.

Osmotic blood vessel permeabilization depends on Alox5a and Lta4h
The osmotic injury response of leukocytes is mediated by bioactive lipids of the eicosanoid pathway4. So, we wanted to test whether similar mechanisms control blood vessel permeabilization. To this end, we perturbed different branches of the eicosanoid cascade (Fig. 2a) through mutation and/or chemical antagonists: 12-lipoxygenase (ALOX12), 5-lipoxygenase (ALOX5), leukotriene A4 hydrolase (LTA4H), and cyclooxygenase30. ALOX12 (zebrafish ortholog: Alox12) is enriched in skin and catalyzes the conversion of AA into 12(S)-HETE, Hepoxilins and other epithelial lipid mediators31. ALOX5 (zebrafish ortholog: Alox5a) is enriched in leukocytes but also expressed in non-myeloid tissues4,32–35. Its downstream metabolite 5-KETE attracts zebrafish leukocytes to tail fin wounds in response to osmotic and oxidative stress4,6,26,27,36,37. LTA4H (zebrafish ortholog: Lta4h) mediates leukotriene synthesis downstream of ALOX5. Published in situ hybridization data show lta4h is expressed in leukocytes34,38. Cyclooxygenases are abundantly expressed in immune and non-immune cells and generate AA-derived prostaglandins, which also contribute to cardiovascular regulation39. They are clinically targeted by Non-steroidal Anti-Inflammatory Drugs (NSAIDs), such as diclofenac, aspirin and others.

The mutation of alox12 (alox12^mk218/mk218; Fig. 2a; Supplementary Fig. 2a, 2g-h, 2k) did not alter vessel- or wound-permeability (Fig. 2b, c). By contrast, alox5a36 or lta4h mutation (alox5a^mk211/mk211, lta4h^mk219/mk219; Supplementary Fig. 2i-j, 2l-m) suppressed dextran leakage from vesels after hypotonic shifting by ~55% or 52%, respectively (Fig. 2d–g; Supplementary Fig. 2c, 2e).

Pharmacological ALOX5 inhibition with licofelone (a cyclooxygenase/5-lipoxygenase inhibitor)40 or MK886 (a FLAP inhibitor)41, unlike inhibition with diclofenac42, resembled these effects (Fig. 2h, i; Supplementary Fig. 3a–c). None of the ALOX5 perturbations altered wound permeability/closure (Fig. 2d–i; Supplementary Fig. 2d, 2f), or gross vessel morphology/area (Supplementary Fig. 3d).

Direct bath supplementation with the ALOX5 substrate AA partially restored vessel leakage under isotonic conditions without affecting wound permeability (Supplementary Fig. 3f-I, 3k-l). Adenosine triphosphate (ATP), which is released by osmotic shock and promotes rapid epithelial wound closure5, strongly suppressed dextran leakage from wounds and modestly increased vessel leakage and dilation (Supplementary Fig. 3h-j, 3m). The ATP hydrolysis product adenosine (Ad) affected only vessel dilation, without altering vessel- or wound permeability.

Altogether, these experiments highlighted a key role for the Alox5a-Lta4h pathway in transducing osmotic vessel permeabilization. Given the pathway’s well-established involvement in inflammatory signaling by myeloid cells35,43–45, we wondered whether osmotic vessel permeabilization was mediated by immune cells.

Blood vessel permeabilization via Alox5a-Lta4h requires macrophages
At the larval stage, zebrafish do not possess functional adaptive immunity yet46. Neutrophils and macrophages, the two main types of larval leukocytes, rapidly respond to tail fin injury, infections, and osmotic shock1,4,26,27,37,47. This response can be imaged in vivo using fluorescent markers expressed under the control of the leukocyte-specific lyz or mpeg1.1 promoters, which light up neutrophils or macrophages, respectively (Supplementary Fig. 4a–b)48,49. Just as osmotic vessel permeabilization, osmotic leukocyte recruitment requires AA and Alox5a and is independent of Alox1236. Unlike osmotic vessel permeabilization, it initially does not depend on Lta4h activity4, but instead on the 5-Hydroxyeicosanoid dehydrogenase (5-HEDH) product 5-KETE and its receptor OXER1 (zebrafish ortholog: Hcar1-4). The OXER1 pathway constitutes a little studied, branch of the ALOX5 pathway37. Underlining that osmotic leukocyte recruitment and vessel permeabilization involve partially distinct mechanisms, dextran leakage from vessels was unaltered by hcar1-4 mutation (hcar1-4^mk214/mk214)26 (Supplementary Fig. 4c).

We used the lyz or mpeg1.1 promoters to specifically express nitroreductase (NTR) in neutrophils or macrophages. NTR converts the antibiotic metronidazole (Mtz) into a cytotoxic compound50,51. Ablating NTR-expressing leukocytes by bath supplementation of Mtz, allowed us to dissect neutrophil- and macrophage-contributions to osmotic vessel permeabilization (Fig. 3a-e; Supplementary Fig. 4d-i, 4j-m). Mtz treatment of lyz-NTR2.0 larvae abolished the fluorescent lyz marker signals and Sudan Black positivity in the tail fin, in line with complete neutrophil ablation (Fig. 3b, c; Supplementary Fig. 4j, 4k; Supplementary Movie 6). Yet, vessel leakage upon hypotonic shock remained unaltered (Fig. 3c; Figure S4d). In stark contrast, macrophage ablation reduced osmotic vessel permeability by ~54%, i.e., akin to Alox5a-Lta4h pathway perturbation (Fig. 3d, e; Supplementary Fig. 4f; Supplementary Movie 7). If vessel permeabilization via this pathway depends on macrophages, macrophage-deficient animals should be insensitive to ALOX5 inhibition, as observed (Fig. 3f, g; Supplementary Fig. 4h). None of the treatments affected rapid wound permeability/closure (Fig. 3b–g; Supplementary Fig. 4e, 4g, 4i), and macrophage ablation also did not alter early neutrophil recruitment to the wound (Supplementary Fig. 4l, 4m).

Part of the AA that drives Alox5a-dependent leukocyte migration is generated by cPla2 that adsorbs to the stretched INM of osmotically swollen cells4,6,52–54. Was cPla2 also fueling osmotic vessel permeabilization? More specifically, does nuclear membrane mechanotransduction via this enzyme allow macrophages to sense distant wounds in situ?

cPla2 mediates osmotic vessel permeabilization and wound sensing by macrophages
cPLA2 drives LTA4H-dependent eicosanoid production in peritoneal mouse macrophages55, Human56 (e.g., lung) macrophages show nuclear cPLA2, ALOX5 and LTA4H expression. Together with zebrafish single cell mRNA profiling data32,35,57–59, this underscore that the pathway is conserved in macrophages across phylae. The cPla2 mutants (pla2g4aa^mk220/mk220; Supplementary Fig. 5a-e) showed a ~59% reduction in osmotic vessel leakage with unaltered wound permeability (Fig. 4a, b).

To test whether and where macrophages osmotically activate cPla2 upon larval injury, we expressed fluorescently tagged cPla2 (cPla2-mKate2)4,6,13,20,21,52 using the mpeg1.1 promoter (Fig. 4c–f; Supplementary Fig. 5f). The animals were bathed in normal hypotonic (i.e., freshwater) or isotonic solution during the laser-wounding procedure. Under hypotonic but not isotonic bathing conditions, tail fin wounding triggered a pulse of cPla2-mKate2-INM adsorption, which passed through the field of view within ~10 s (Fig. 4g; Supplementary Fig. 5f; Supplementary Movie 8). Macrophages neighboring the wound (~20 µm) showed constitutive cPla2 binding to the INM. By contrast, macrophages at the vasculature (i.e., “perivascular”) showed highly reversible binding. Furthermore, none of the genetic cPla2-Alox5a-Lta4h larvae mutants altered gross vessel morphology/area, or baseline vessel permeability in non-wounded mutants compared to wt siblings (Supplementary Fig. 6a-c).

These observations argue that macrophages nearby the vasculature directly detect distant wounds via osmotic cPla2 activation. Two-photon resonance scanning microscopy corroborated these rapid dynamics at higher time-resolution (Fig. S7; Supplementary Movie 9). The respective translocation-wave propagates at ~50 µm/s through the tissue, i.e., consistent with interstitial sodium or chloride diffusion. Remarkably, endothelial cells at comparable wound distances did not show similar cPla2 responses (Supplementary Fig. S8a–c; Supplementary Movie 10), suggesting that the nuclei of perivascular macrophages are particularly sensitive to mechanical/osmotic stress (Fig. 4h).

Methods
Zebrafish Husbandry
Adult wild-type and mutant Casper74 Zebrafish (Danio rerio) strains and larvae were maintained28 and subjected to experiments according to the institutional compliance and approval of the animal ethics committee, the Institutional Animal Care and Use Committee (IACUC) and the Research Animal Resource Center (RARC) of the Memorial Sloan Kettering Cancer Center (MSKCC) (protocol no. 11-01-002). The adult fish are reared in either 2.8 or 6 L polycarbonate tanks at animal density 8 fish L−1, with a photoperiod of 14:10 light:dark cycles and maintained in salinity-conditioned system water surveillance, (pH= 7.53) sodium bicarbonate (Proline, SC12A), conductivity 700-850 μS cm−1 (Instant Ocean Sea Salt (#SS1-160P)) with TGP=99.4% (Total Gas Pressure) and DO=8.18 ppm (Dissolved Oxygen=8.155 mg L−1) at 28°C. The zebrafish are fed twice a day with live feed consisting of rotifers or artemia, followed by processed dry spirulina pellets (Zeigler). All anesthesia was conducted with 0.2 mg ml−1 3-aminobenzoic acid ethyl ester (Sigma, MS-222, E10521), (pH=7.0), buffered in 0.5 mg ml−1 anhydrous sodium phosphate dibasic (Fisher, BP332-500). The embryos were staged by dpf (days-post fertilization). Sex was indeterminate at 2.5–4 dpf and all experiments supporting findings of the manuscript were conducted at these larval stages. The animal embryos were collected from natural spawning and raised in standard hypotonic E3 containing 0.1% (w/v) methylene blue (Sigma-Aldrich, M9140) for first 24 hrs followed by E3 medium (5 mM NaCl (Sigma-Aldrich, S7653), 0.17 mM KCl (Sigma-Aldrich, P9333), 0.33 mM CaCl2 (Sigma-Aldrich, C5670), 0.33 mM MgSO4 (Sigma-Aldrich, M7506)) in 100 mm petri dishes (Fisher Scientific, FB0875713).

Transgenesis, plasmid construction and in vitro transcription
Fertilized Casper zebrafish embryos were collected and injected at the one-cell stage into the cytoplasm with a laser-pulled borosilicate glass microneedle (H=370, FIL=5, VEL=50, DEL=245, PUL=140; P-2000 Sutter Instrument CO.) and a Nanoject IITM microinjector (Drummond Scientific, Broomball, PA)75. Plasmids were assembled using Gateway multisite cloning kit using LR ClonaseTM (Invitrogen; C12537-023) and multicloning sites between the gateway att sites flanking cassette of p5E, pME and p3E plasmids that were recombined into Tol2kit76 destination vectors pDESTTol2CG2* or pDEST-crybb1 for generating following Q-system animals77: Tg(kdrl:Qf2; p5E-kdrl Addgene Catalog# 78687)78; Tg(mpeg1.1:Qf2; p5E-mpeg1.1, Addgene Catalog# 75023)79,80; Tg(lyz:Qf2; p5E-LyzC Addgene Catalog# 135200)79 or Tg(Quas:eGFP; pME-eGFP Tol2kit v1.2-383)76,80; Tg(5xQuas:GAP-tagYFP-P2A-NfsB_Vv)50 and Tg(Quas:cPla2-mKate2-P2A-eGFP-KDEL) or transgenic Casper zebrafish. The latter plasmid was constructed from Cytosolic Phospholipase A2 (cPla2, Ensembl: ENSDARG00000024546)1 where the open reading frame was amplified by PCR from a zebrafish cDNA clone (Open Biosystems, 9037889) using custom designed primers bearing unique palindromic overhangs, Bsu15I and EcoRI:(Fwd: 5’-AGTCatcgatGCCA-CAATGTCCAACATTATAG3’; (Rev: 5’-CAGTGTGGCTGTGGGAGCTGGAGgaattcGACT-3’). The PCR product was REdigested, gel-purified (Qiagen, 28106) ligated into pDONOR221 containing pME gateway compatible att sites and subsequently fused in-frame to mKate2 (Evrogen) as a C-terminus fluorescent tag with a 15 amino-acid GS-enriched linker6. The p3E entry vector was fused in-frame with self-cleaving P2A peptide in N-terminus of eGFP followed by endoplasmic reticulum localization signal KDEL and SV40 poly(A). The final transgenesis constructs were mixed and ~2.7 nL of 25 ng μL−1 of each plasmid co-injected with 25 ng μL−1 Tol2kit transposase mRNA, transcribed from NotI linearized pCS2FA-transposase plasmid with mMESSAGE mMACHINE SP6 reverse Transcription kit (Thermo Scientific, AM1340) in wt or Casper Larvae. Among the injected larvae, fluorescence-positive siblings in the heart (cmlc2:eGFP)76, lense (crybb1:mKate2)81 or chorion (he1:eCFP)50 were selected and raised in husbandry and backcrossed at sexual maturity to Casper fish. For establishing stable and constitutive tissue-specific fluorescent transgenic zebrafish lines, Qf2 founders were crossed with Quas animals and their progeny were identified through both transgenesis marker and indicated fluorescence in the designated tissue or cell type77.

Reagents, Isotonic reconstitution, and eicosanoid pathway Inhibition
For osmotic surveillance, a battery of iso-osmotic media was constituted by supplementing standard hypotonic E3 media (Π(osmolality)=10 mOsm) with the following osmolytes: 135 mM Na+·Cl- (Sigma-Aldrich, S7653); K+·Cl- (Sigma-Aldrich, P9333); K+·Br- (Sigma-Aldrich, P9881); Na+·Br- (Sigma-Aldrich, 220345); Cs+·Cl- (Sigma-Aldrich, C3011); Na+·C6H11O7- (TCI Chemicals, G0041); CH14NO+·Cl- (Fisher Scientific, 50213240) and 270 mM D-mannitol (Fisher Scientific, BP686), culminating in ISO_NaCl or ISO* (Π=280 mOsm) E3 media with the indicated reagents4. The final osmolality of solutions was approximated with ddH2O (18.2 MΩ·cm, Elga PURELAB®) calibrated handheld optical salinity refractometer (RHS-10ATC Cole-Parmer, EW-81150-31) ~1.0050:1.025-1.030 d20SGSalt (~8:35-40 ‰ PPT) for hypotonic (Π=10 mOsm) and all isotonic (Π=280 mOsm) E3 reagents respectively and used for Imaging and analysis. For reconstitution experiments the nucleotides, their derivatives were conducted by ISO_NaCl to ISO_NaCl shifting approach, where the latter was constituted with ISO_NaCl alone as vehicle or supplemented with 5 mM following nucleotide biomolecules: adenosine 5’-triphosphate disodium salt hydrate (ATP; Sigma-Aldrich, A26209) and adenosine (Ad; Sigma-Aldrich, A9251)5. The polyunsaturated arachidonic acid (20:4(5,8,11,14-all-cis-eicosatetranoic acid); Sigma-Aldrich, A3611) was administered in parallel to anesthetized and wounded 3 dpf Tg(kdrl:Qf2:Quas:eGFP) larvae at a final concentration of 5 μM in ISO_NaCl at 5 min during the imaging experiment. For eicosanoid pathway pharmacological inhibition experiments, Tg (kdrl:Qf2:Quas:eGFP) larvae were pre-incubated for 30-45 or 180 min in hypotonic (Π=10 mOsm) E3 supplemented with the following compounds: 50 μM Licofelone (Cayman Chemical Company, 100007692), 10 μM MK886 (Cayman Chemical Company, 10133) or 130 nM Diclofenac (Sigma-Aldrich, 1188800), respectively36. The imaging medium also contained the same concentration of the indicated inhibitors dissolved in hypotonic E3 (Π=10 mOsm) and administered 5 min after wounding. All water-insoluble biomolecules and inhibitors were dissolved at a maximal concentration of 1% dimethyl sulfoxide, DMSO (Millipore Sigma, 276855) hypotonic (Π=10 mOsm) or ISO_NaCl (Π=280 mOsm) E3 as a vehicle control.

Larvae preparation, microangiography, and tail fin wounding
Zebrafish larvae (2.5–4 dpf) were screened for transgenes, anesthetized, and oriented with their left lateral side facing up. They were positioned within slots imprinted from a custom-designed mold cast (NIH 3D Print Exchange, ID: 3DPX-021299) in 2% agarose (Fisher Scientific, BP160) dissolved in hypotonic (Π=10 mOsm) E3 medium. For microangiography, approximately 5 nL of 70 kDa dextran-tetramethylrhodamine (Fisher Scientific, D1818) or 500 kDa FITC-dextran (Fisher Scientific, D7136) dissolved in 1× phosphate-buffered saline (PBS; Sigma-Aldrich, 79382) at 1 mg/mL was microinjected into the Common Cardinal Vein (CCV) or the larval atrium, between the gill and pectoral fin. The injection was performed using a borosilicate microneedle (H=410, FIL=5, VEL=40, DEL=245, PUL=160; P-2000 Sutter Instrument Co.) and a microinjector (Drummond Scientific, Broomball, PA) under the guidance of a stereomicroscope (Discovery V.8, Zeiss).

Larvae with successful perfusion of fluorescent dextran dyes in the caudal arteries and veins were identified using a coaxial fluorescence dissection stereomicroscope (MVX10, Olympus) equipped with an MVPLAPO1X objective (NA 0.25, WD 65 mm, FN 22) and a mercury lamp (LM200B1-A, Prior Scientific) with dichroic mirror sets (MVX-RFA; 540/35 U-MGFPHQ/XL, 625/55 U-MRFPHQ/XL). For microangiography in non-casper background, dextran was injected in anesthetized AB larvae that were maintained in 0.2 mM N-phenylthiourea (PTU, Sigma-Aldrich, P7629) E3 to prevent pigment formation3. The injection site was accessed by making a small incision in the region between the gills and beneath the pectoral fin of 2.5-4 dpf larvae. Injections were performed percutaneously using a fine borosilicate microneedle (tip diameter ~10 µm), which gently penetrated the epidermis to reach the circulation. Successful entry into the circulation was confirmed by observing a slow retrograde flow column of red blood cells into the microneedle. Care was taken to avoid damage caused by dextran injection; larvae with such damage were excluded from further experiments. For the 500 kDa dextran, working solution is more viscous, the injection needle was slowly retracted from the circulation after ejection, rather than being removed directly, to prevent dye leakage (evisceration).

For tail fin imaging experiments, anesthetized larva were flat-mounted on their right side in a 35 mm glass-bottom microscopy imaging dish (MatTek Corporation, P12G-1.5-14F) and immersed in ~200 μL of 0.8% (w/v) low-melting agarose (Goldbio, A-204-100) dissolved in ISO_NaCl (Π=280 mOsm) E3 medium. For tail fin wounding, multiple larva were aligned in parallel, and the tips of the larval caudal fins were amputated at the posterior end of the notochord using a tungsten microblade needle (Fine Science Tools, 10318-14). Care was taken to avoid damaging the notochord or caudal vessels during the solidification of the low-melting agarose4,26. For non-wounded imaging experiments, the anesthetized zebrafish embryos were reared in borosilicate dish (Corning, 3160-60) and handled with manual pipette controller attached (Sigma-Aldrich, P7924) to borosillicate pasteur pipette (Corning, #7800; 7095D-9) to prevent any damage to the end of tail fin from larva handling during dextran injection and immobilization. After immobilization, the embedded live larvae were enveloped in 200 μL of ISO_NaCl (Π=280 mOsm) containing tricaine, to prevent desiccation. The immobilized larvae are allowed to rest at 28°C for approximately 15 min to restore cardiac function in the trunk. Following this, the tail fin region of the embedded larvae was carefully freed from the excess low-melting agarose using a tapered micro spatula (Fine Science Tools, 10089-11) and transferred to a pre-heated stage-top incubator (INU-TIZ-D35).

Spinning disk confocal microscopy
Tail fin amputation and non-wounded Imaging experiments were completed at 28°C with the heated imaging chamber (INUG2 KIW, TOKAI-HIT) and sub-stage heater (AirTherm ATX Invivo Scientific, WPI inc.), in an inverted Nikon Eclipse Ti microscope equipped with a CFI Apo LWD Lambda S-series 20X Objective lens (N.A.=0.9 ∞/0.11-0.23 WD=0.95 mm WI Objective, Nikon) or 63x oil objective lens (N.A.=1.4 ∞/ 0.11-0.23 WD=130 μm, CFI PLAN Apo Oil Objective, Nikon). The samples were excited with 488 and 561 nm diode laser (LUN-F100-240V~50/60Hz, Nikon Instruments inc.), where channel acquisition intensities/exposure times used in the manuscript were as follows: 40%/100 ms (488 nm, 30 mW or 0.03 VA) or 25%/100 ms (561 nm, 11.5 mW or 0.015 VA) laser power settings for illuminating either endothelium or innate immune cells (macrophages or neutrophils) and Dextran, respectively. For 63x imaging, 488 nm laser excitation was reduced to ~10%/100 ms to maintain same laser light density during excitation (up to 6.8 W cm−2). The fluorescence emission spectra were collected in 4D (XYZT) by automotive PCI hardware triggering (PXI-1033, National Instruments) with a Yokogawa CSU-W1 Spinning Disk unit (Nipkow disk pinhole=50 μm, 4000 rpm), incorporating a Photometrics Prime BSI Scientific CMOS (sCMOS) camera with analog Z-plane acquisition (MCL Nano-Drive NIDAQ Piezo Z, Nikon) and a motorized XY-stage (TI-S-ER, Nikon). The emission was collected for either green transgenic (Ex/Em: 488/525; Gain: Correlated Multi-Sampling) or red Dextran (Ex/Em: 561/605; Gain: High Dynamic Range) with a high-sensitive sCMOS camera using band-pass dichroic mirrors (Chroma Technology Corp., 89100bs) for filtering two separate fluorescence emission spectra (525/36 and 605/52, Chroma Technology Corp., 89000 Sedat Quad) placed in front of the detector for isolated detection of green and red fluorescence, respectively. The imaging plane (1331×1331 μm x 100 μm) was collected at 3 μm z step size to result with 35 slices and repeated with 30-second intervals per position for up to 60 min. Collectively, the live larvae were exposed to 5–6.81 W cm-2 and 2–3.1 W cm−2 laser power density for 488 and 561 nm, respectively. For all imaging experiments, the imaging dish was covered with a solution of interest at 270–300 s (t=10-11th frame) with a bolus of 10x agarose volume (~2 mL). The z-stack images of zebrafish larvae were captured using multiple scanning modes at 100 μm tissue depth in z-axis at a resolution of 2048×2048 pixels (16 bit) in the x and y plane, corresponding to 0.645 μm/pixel calibration with a voxel size of (0.65×0.65×3 μm) in xy and z, respectively. The high-resolution XYZ-T image files were pre-processed by (2×2) binning and acquired as (1024 × 1024 per z-plane) with corresponding 1.538 pixel μm-1 acquired image size (665.6×665.6×100 μm) through triggered excitation and emission collection for either red or green fluorescence in the NIS imaging software (NISElements, 3.22.14). Collectively, live unwounded larvae were exposed to 5 W cm−2 and 4 W cm−2 laser power density for 488 and 561 nm, respectively. The wounds were induced at ~1 min (t=5–7), with several successive laser pulses targeted at peripheral sentinel macrophages or nearby melanocytes for wounding in a single frame with 60 ms delay using microscope-mounted 435 nm ultraviolet Micropoint Laser (Andor), resulting in tail fin finfold ablation around the boundary of hematopoietic region in the trunk of the larvae.

Spinning disk confocal microscopy and laser Wounding
For laser wounding experiments, single intact and anesthetized 3 dpf Tg (mpeg1.1:Qf2; Quas:cPla2-mKate2-P2A-eGFP-KDEL) or 2-3 dpf Tg(kdrl:Qf2; Quas-cPla2-mKate2-P2A-eGFP-KDEL) were embedded in 60 mm plastic petri dish (Corning, 351007) and immobilized with ~200 μL 1% LM agarose ISO_NaCl (Π=280 mOsm) E3. Subsequently, the LM-agarose was enveloped with ~2-3 mL of hypotonic (Π=10 mOsm) or ISO_NaCl (Π=280 mOsm) E3 media for creating a submerging environment for the 25X Objective lens (N.A.=1.1 ∞/0-0.17 WD=2 μm Water Dipping Objective, Nikon) in Nikon Eclipse FN1 upright microscope. The samples were excited with 488 and 561 nm diode laser lines (Andor Revolution XD) and fluorescence channel excitation intensity/exposure was adjusted to 35%/80 ms (488 nm) and 30%/80 ms (561 nm) for illuminating macrophage endoplasmic reticulum (KDEL-eGFP) and cPla2-mKate2 in the caudal region of the larvae. The emission spectrum were excited in 4D (XYZT) by Smart Shutter controller (Sutter Instrument, LB10-B/IQ Lambda) triggered excitation carried by Andor Laser combiner (LC-501A, Andor Technology) housing Andor iXon3 897 thermoelectrically cooled EMCCD camera with analog Z-plane acquisition (STG-STEPPER-Piezo Focus, Ludl Electronic Products). The emission was collected with a Yokogawa CSU-X1 spinning disk unit (Nipkow disk pinhole=50 μm, 10,000 rpm) for green (Ex/Em: 488/525) or red (Ex/Em: 561/625) in electron-multiplying mode (Gain: 10 MHz at 14-bit; Multiplier 300, conversion 1.0X) and band-pass filters for green (525/40, Semrock., FF02-525/40-25) or red (617/73, Semrock., FF02-617/73-25) fluorescence emission spectra. The imaging plane (287X 287×50–80 µm) was collected at 1.5 μm z step size and repeated in no-delay intervals per position for up to 30 min, resulting with temporal step resolution of ~7–12 s per stack. The z-stacks were acquire data resolution of 512×512 pixels (14-bit), corresponding to 0.561 μm/pixel calibration with a voxel size (0.56×0.56×1.5 μm) in xy and z, in the NIS imaging software (NISElements, 3.22.14). Collectively, live unwounded larvae were exposed to 5 W cm−2 and 4 W cm−2 laser power density for 488 and 561 nm, respectively. The wounds were induced at ~1 min (t=5–7), with several successive laser pulses targeted at peripheral sentinel macrophages or nearby melanocytes for wounding in a single frame with 60 ms delay using microscope-mounted 435 nm ultraviolet Micropoint Laser (Andor), resulting in tail fin finfold ablation around the boundary of hematopoietic region in the trunk of the larvae.

Multiphoton imaging and FRAP-wounding
3 dpf intact larvae Tg(mpeg1.1:Qf2; Quas: cPla2-mKate2-P2A-eGFP-KDEL) were mounted in 35 mm glass-bottom microscopy imaging dish (MatTek Corporation, P12G-1.5-14F) and transferred to Ti2-E inverted Nikon microscope, housing AX-R multiphoton modality (AX-MPNDD) and stage top incubator set to 28°C (TOKAI-HIT, STX). The multiphoton images were excited through simultaneous absorption of 920 nm tuneable and 1045 nm fixed two photon lasers (mks, spectra-physics) for eGFP and mKate2, respectively. The excitation was adjusted to 920 nm (4.3%, Gain=45, Line Averaging=4X) and 1045 nm (5.3%, Gain=45, Line Averaging=4X) for eGFP and mKate2, respectively. The fluorescence emission was collected using 40X (Plan Apo lambda S 40XC, NA=1.25 ∞/0.13-0.21 WD=300 μm, Nikon) silicone objective with objective heating mantle (28°C) and detected with PMT GaAsP. The bidirectional images were acquired in resonant scanning mode. The imaging plane (147×90 ×15 µm) is collected at Nyquist optical resolution of 0.149 μm at 1.5 μm z-step size using NIDAQ piezo and repeated in no-delay intervals for up to 2 minutes, resulting with a temporal step resolution of 2.5 seconds per stack. The z-stacks were acquired at a pixel resolution of 1024×626 (14-bit), corresponding to 0.144 μm/pixel calibration with a voxel size (0.14×0.14×1.5 μm) in xy and z in the NIS imaging software (NISelements, 6.02.01). The wounds were induced at ~1 minute with a single high intensity 920 nm laser pulse (22%, Line Averaging=1X, Dwell time=1 s, area=20×20 µm) with photostimulation MPSTIM FRAP modality in Galvano scanner mode, resulting in abrupt epithelial tail fin fold laser ablation around the boundary of hematopoietic region in the trunk of the larvae. Furthermore, larvae were prepared similarly and also wounded in Leica stellaris 8 DIVE multiphoton microscope, equipped with two tunable IR excitation lasers: Mai Tai HP (690-1040 nm, continuous) and insight X3 (680-1300 nm, continuous). Within the Leica microsystem, tissues were wounded using the FRAP ab1 XYZT module in resnonant scanning mode with bidirectional scan direction, speed= 8,000 Hz and pixel dwell time 0.0319 µs. The images were acquired with HC PL IR APO 40x/1.10 WATER, with 2.25x zoom at Nyquist XY pixels size 0.11 μm pixel-1 resolution with a voxel size (0.129×0.129×1.5 μm) in xy and z, corresponding to 1184×1184 (14-bit) and stack depth of 15 microns. For dual color excitation, the MAITAI HP was tuned to 924 nm (MP1, 4.5%, power) for eGFP excitation and the insight X3 laser tuned to 1118 nm (MP2, 2.0% power) for mKate2. The wounding was performed using the FRAP module with a sequence of 4 pre-bleach iterations, followed by 6 bleach and 20 post-bleach iterations. For laser ablation, the bleaching intensity within ROI (area=20×20 µm) was set to 100% on MP2 (1118 nm) and 100% on MP1 (924 nm) in resonant scanning mode. We were not able to switch galvanic and resonant quickly within the same experiment with Leica stellaris 8 DIVE instead to induce efficient wounds at 8000 Hz scanning speed, WILL laser was also added at 100% and tuned to UV range with a total dwell time set to 2 s at mid-plane of the XYZT stack. Detection was performed using external spectral non-descanned hybrid detectors (NDD1 and NDD2) operating in photon-counting mode, with NDD1 configured for eGFP detection (480–527 nm) and NDD2 for mKate2 (620–732 nm). All imaging data were saved and processed using Leica LASX software (Leica LASX, 4.8.1.29271).

Denoise and 3D deconvolution
The 4D image stacks of tg(mpeg1.1:Qf2; Quas:cPla2-mKate2-P2A-eGFP-KDEL) were corrected by 3D deconvolution, where sample PSF was derived and computed automatically with depth-calibration and without image intensity subtraction or preprocessing in Nikon NIS elements (5.21.03, Build 1489). The deconvolution process was carried by default method using landweber algorithm for spinning-disc modality with 50 μm pinhole size with immersion refractive index 1.33 (water) for both 488 and 561 nm fluorescent light emission82. The deconvolved or MP image stacks are denoised using denoise.ai tool for improving signal-to-noise in Nikon NISelements (5.21.03).

Generation of zebrafish CRISPR mutants
To generate zebrafish mutants a CRISPR/Cas9 system with a single sgRNA (Alox12-sgRNA1) targeting zebrafish alox12 (ENSDARG00000069463) exon 7 is used. The ribonucleoprotein complex consisting of alox12-sgRNA1 and Cas9 recombinant protein is injected into the cytoplasm of one-cell stage fertilized zebrafish embryos. The injected F0 larvae were brought to adulthood and crossed with wild-type adults to produce F1 progeny. The alox12-sgRNA injected F1 zebrafish were grown to sexual maturity and their genomic DNA was isolated from their tail fins for genotyping. Tail fins were partially amputated, suspended in 250 μL of 0.05 M NaOH, incubated at 95°C for 10 min, cooled on ice for 10 min, then neutralized with 25 μL of 1 M Tris-HCl (pH 8), and vortexed. The DNA extracts were used as genomic templates for Polymerase Chain Reaction (PCR). 486 bp PCR products were digested with FastDigest BseLI (Thermo Fisher Scientific, FD1204) overnight at 37°C, producing two DNA fragments (310 and 176 bp). The 508 bp represents a mutant allele where the BseLI site has been disrupted by Cas9-induced mutation. The 508 bp band was isolated from the agarose gel and sequenced via Sanger, confirming a 22 bp insertion (Supplementary Fig. 2g-h, 2k). Subsequently, the F1 heterozygous adult zebrafish with 22 bp frameshift mutation were bred to homozygosity.

Two independent sgRNAs (lta4h-sgRNA-1 and lta4h-sgRNA-3) targeting zebrafish lta4h (ENSDARG00000006029) were used to establish a CRISPR-zebrafish line26. The Cas9-gRNA ribonucleoprotein complex (a combination of lta4h-sgRNA1 and lta4h-sgRNA3) was injected into the cytoplasm of one-cell-stage zebrafish embryos. After the injected F0 larvae matured (2-3 months post-fertilization), individual F0 adults were crossed with wild-type adults to produce F1 progeny. These F1 larvae were then grown to sexual maturity, and genomic DNA was isolated from their tail fins for genotyping. For lta4h-sgRNA1, PCR products were digested with FastDigest BseLI (Thermo fisher Scientific; FD1204), producing three DNA fragments (271, 140, 129 bp). For the lta4h-sgRNA3, PCR products were digested with FastDigest SalI (Thermo fisher Scientific; FD0644), producing three DNA fragments (271, 184, 85 bp). The 271 bp product represents a mutant allele where the BseLI or SalI site has been disrupted by Cas9-induced mutation. This 271 bp band was isolated from the agarose gel and sequenced via Sanger sequencing, confirming a 5 bp deletion (2, 2, 1 bp deletion) and a 7 bp insertion (Supplementary Fig. 2i-j, 2m). F1 heterozygous adult zebrafish with this frameshift mutation of interest were bred to achieve homozygosity.

A single guide was designed targeting zebrafish pla2g4aa (ENSDARG00000024546) for genetic disruption at exon 7. The Cas9-gRNA ribonucleoprotein complex consisting of pla2g4aa-sgRNA1 was injected into the cytoplasm of one-cell-stage fertilized embryos. The injected larvae were then brought to adulthood and the individual F0 animals were used to produce F1 progeny by backcrossing with wild-type Casper fish. The F1 larvae were grown to sexual maturity, genomic DNA was subsequently isolated from their tail fins and sequenced. The pla2g4aa^wt/mk220 DNA was PCR amplified, digested with FastDigest BseMI (Thermo fisher Scientific; FD1264) and then analysed by agarose gel electrophoresis for genotyping. The 447 bp PCR product from the wt pla2g4aa allele is cleaved into two smaller products upon overnight incubation with BseMI (281 and 166 bp) while the 437 bp PCR product of the mk220 allele is not cleaved by restriction digest into the smaller products. F1 heterozygous adult fish with the mk220 10 bp deletion (Supplementary Fig. 5a-b, 5e) were bred to achieve homozygosity. The Genomic exon-intron architectures (Supplementary Fig. 2g, 2i, 5a) were obtained from the Ensembl database for Danio rerio (GRz11 Genome assembly; GCA_000002035.4). The exon–intron schematics were downloaded and adapted (recoloured) for display visualization of exon targeting83.

Alox12 (Chr 7, alox12 Ensembl: ENSDARG00000069463) mk218 allele Exon 7-ins-22 bp
gRNA2: GGATCACTGGGCAGAAATACTGG
Fwd: 5’-CAAATGCATTGATGCAAAAAGT-’3
Rev: 5’-TGAGAAATAGCATTCATTTGCG-’3
Lta4h (Chr 4, lta4h Ensembl: ENSDARG00000006029) mk219 allele Exon 1-ins-7 bp-Δ5 bp
gRNA1: GAAAGTCGCCCTGACTGTGGAGG
gRNA3 CATGCCTGTCAAAGTCGACATGG
Fwd: 5’-TCAACCATGACTCCAGTTTCAG-’3
Rev: 5’-CAGTGCATTGGATCGTACTCAT-’3
cPla2 (Chr 2, pla2g4aa Ensembl: ENSDARG00000024546) mk220 allele Exon 7 Δ10 bp
gRNA1: GGAGGTTTTCGTGCAATGGTGGG
Fwd: 5’-CCATCCTCACCAAGAGAGGTAA-’3
Rev: 5’-ACTGCTTGAATTGACTGCAAAA-3’

Larval genotyping of established zebrafish CRISPR mutants
2.5, 3 or 4 dpf Zebrafish Larvae were recovered from the 35 mm imaging dish and subjected to 20 μL 0.05 M NaOH alkaline DNA extraction at 95°C and neutralization with 2 μL 1 M Tris-HCl (pH 7.4)26. The gRNA targeted exon was PCR amplified and subjected to restriction enzyme digestion followed by gel electrophoresis analysis. For imaging experiments where the genotype was unknown (in-cross of heterozygotes for pla2g4aa), blinding was inherent in the experimental design and the genotypes were matched to the assigned fish post hoc. For all genetic experiments supporting the findings of this paper use zebrafish larvae at age 2.5-4 dpf. The previously published and established zebrafish lines were genotyped using the following primers:
Alox5a (Chr 13, alox5a Ensembl: ENSDARG00000057273) mk211 allele Exon 7 Δ10 bp36
gRNA1: TGGGTGCCGCCAAGTACTGATGG
Fwd: 5’-GCTGTAATCCAGTGGTCATCAA-’3
Rev: 5’-TGATCTCACTGGAGACTGGAGA-’3
Hcar1-4 (Chr 12, Ensembl: ENSDARG00000087084) mk214 allele Exon 2 4 ins/Δ756 bp26
gRNA2: GGTAAAGGATCCTGAAGAAGCGG
gRNA4: GGCATGGAGACACACAATGAGGG
Fwd: 5’-TGCCTAAACATTTGTGTTCGTGT-3’
Rev: 5’-AGACTGCCGAATGTTGGTGT-3’

Chemogenetic depletion and Sudan Black staining
To perform macrophage-specific cell depletion, Tg(mpeg1.1:Qf2; 5x-Quas:GAP-tagYFP-P2A-NfsB_Vv) animals tagged with nitroreductase under the mpeg promoter were used. Tg(lyz:Qf2; 5xQuas:GAP-tagYFP-P2A-NfsB_Vv) was used as a genetic vehicle control. The 100 mM MTZ (Sigma-Aldrich, 1442009) stock solution was prepared in DMSO (Millipore Sigma, 276855) and protected from light. Embryos were dechorionated prior to metronidazole (MTZ) ablation using 1 mg/mL pronase (Roche, 165921). Starting at 2 dpf, the larvae were treated with 150 μM MTZ and 0.15% DMSO in hypotonic (Π=10 mOsm) E3 media or mock-treated with 0.15% DMSO in hypotonic E3 as a vehicle control for at least 24 hours. At 3 dpf, MTZ was temporarily removed and replaced with hypotonic (Π=10 mOsm) E3 media until the microangiography procedure and subsequent immobilization in ISO_NaCl (Π=280 mOsm) low-melting agarose for confocal imaging. During confocal imaging, larvae were shifted back to hypotonic (Π=10 mOsm) E3 media containing either 150 μM MTZ or 0.15% DMSO, depending on their respective treatment conditions. For pharmacological inhibition of eicosanoid pathway, macrophage depleted or DMSO treated larvae were both inhibited with 30-45 min 50 μM Licofelone dissolved in hypotonic (Π=10 mOsm) E3 and subjected to dextran microangiography and imaging. For Sudan Black staining, (Tg(mpeg1.1:Qf2; Quas:NTR2.0)) or (Tg(lyz:Qf2; Quas:NTR2.0)) larvae were used for MTZ or DMSO vehicle treatment regiment for 24 hrs, followed by wounding and fixed at 90 min post wounding using 4% paraformaldehyde (Fisher Scientific, BP531) in 1x PBS overnight at 4°C and then stained with Sudan-Black for 30 min. The larvae were rinsed three times in 70% ethanol (Decon Labs, 2405) and rehydrated with PBS-Tween-20 for 5 min. Prior to imaging, the larvae were depigmented (1% KOH, 1% H2O2) and washed for total of three times in PBST solution 5 min each and transferred for transmitted light imaging26.

Image processing and measurement of interstitial serum leakage and Dilation
Nikon ND2 Spinning disk confocal time-lapse stacks of wounded larvae were imported into Fiji (v1.54J Just ImageJ Package) using bio-formats plugin and z-projected (Maximum-Intensity Projection) with brightness and auto-contrast. To minimize the movement of the imaged plane, the 3D (XYTZ=MAX) was registered using 2D Linear Stack Alignment SIFT multichannel (PTBIOP plugin) tool and the dextran channel was set as reference on the imported composite image stacks (Scale Invariant Interest Point Detector) and rigid transformed (maximal alignment error: 50 px, inlier ratio: 0.05) for subsequent morphology or intensity analysis of endothelium or and dextran intensity and endothelial tail fin ratio. For genetic experiments, the registration was carried out using SIFT Linear Stack Alignment with default settings. For quantification of serum leakage, dextran intensity was traced per larva in a stratified manner in the extra-epidermal 20-50 μm wounded region (Ib(t)) and below the caudal arteries & veins (Iv(t)). Both Iv and Ib datasets were subtracted with background (empty area in field of view) intensity per time point for correcting the noise of sCMOS camera, except for 500 kDa imaging as the complete FOV does not capture extra epidermal dextran from the tail fin tissue wound in 2.5-4 dpf zebrafish larvae. For pharmacological experiments, quantifications were carried out with PTBIOP plugin. For ratio measurements of endothelium and tail fin, a custom script was developed in python, using scikit and numpy packages that allows filteration of small objects or cells in the epidermis and detection of single continuous binarized (=Otsu) endothelial signal and tail fin area based on 70 kDa dextran and calculated as ratio in FOV, the image was loaded and analyzed in napari. The quantifications were carried out manually to monitor the accuracy of defined registered regions in the tail fin using set rectangle tool with fixed dimensions for wound (50×50 pixels), vessel (300×100 pixels), respectively and quantified using plot z-axis profile function for all available time-frames for both 70 kDa and 500 kDa Dextran (ImageJ, fiji). For endothelial morphological parameter measurements and dilation, vertical line was drawn using line tool with fixed dimensions overlaying the split and registered endothelial fluorescence signal channel (length=50 pixels, with=30) and multi-kymograph function with default settings. The resulting kymograph was binarized (Threshold method=Otsu, Dark Background) to create an 8-bit image and area quantified using make line tool (width=30 pixels, length=121) and the diameter measured using the plot profile function.

To ensure comparability of results obtained across different experimental days and injection mixtures number of normalization methods were used. For systemic statistical comparisons, vessel dilation, vessel leakage and wound permeability were quantified using normalized formulations. Vessel dilation (D(t)=vessel diameter, Eq. 1), wound permeability (Ib(t)=wound leakage, Eq. 2), and vessel permeability (Iv(t)=vessel leakage, Eq. 3) were quantified by normalization to baseline (0–270 s) and expressed using the following mathematical formulations.

Dv_norm(t) = P Dv(t) / t=270s Dv(t) t=0s (1)

Vessel dilation (Eq. 1): Expressed as the relative change in vessel diameter over time, normalized to the cumulative baseline diameter measured during the first 270 s. This accounts for inter-experimental variation in baseline vessel size.

Ib_norm(t) = P Ib(t) / t=270s Ib(t) t=0s (2)

Wound leakage (Eq. 2): Reported as the relative wound intensity over time, normalized to the cumulative baseline wound intensity (0–270 s). This allows comparison of leakage dynamics independent of initial wound intensity values.

Iv_tot=norm = R t=3600s Iv(t) / P t=270s Iv(t) t=0s (3)

Total vessel leakage (Eq. 3): Calculated as the time-integrated vessel intensity over the full imaging window (0–3600 s), normalized to the cumulative baseline vessel intensity. This metric captures the overall leakage while correcting for baseline fluorescence variation.

The intensity measurements are reported as arbitrary values (arb. units). All imaging data was normalized to the first 10 time points per imaged larva using Python (Conda, v3.8.10 64-bit) and MATLAB (R2024a, v24.1.0.2537033, 64-bit) code and combined, except for leakage rate measurements. Leakage rates were derived from temporal derivatives of the intensity signals. The apparent wound leakage rate was defined as (Rb0 = dIb/dt) representing the rate of change in wound intensity over time. The vessel leakage rate (Rv0) was calculated from the corresponding vessel intensity dynamics.

Mathematically, if vessel intensity remains relatively stable or constant over the imaging period, then the area under the curve integration simply reflects the baseline signal scaled by the experiment duration (3600 s). Thus, Eq. (3) produces a normalized value of 3600, which can be interpreted as the expected hypothetical outcome in the absence of leakage. Deviations above this level indicate cumulative leakage relative to hypothetical baseline. Note that statistical analyses were performed without subtracting the fixed baseline integration value of 3600. The image acquisition and image analysis dimensions are set to be consistent between all samples, and these measurements are designated above in the microscopy and analysis section and independently represented. The 2D and 3D image analysis was performed with Anaconda distribution of Python (Python ≥3.8.10). Specifically, customized python scripts were developed using the Numpy (v.1.23.5)84, SciPy (v1.15.2)85 scikit-image (V0.19.1)86, matplotlib (v3.5)87, trackpy (v0.6.4)88,89, Allen Cell Structure Segmenter (v0.5.0)90 and napari (≥v0.4.10)91 libraries. GPU acceleration (when available) was provided by CuPy (v12.0)92.

500 kDa dextran onset of permeability quantification and kymograph area
Nikon ND2 Spinning disk confocal time-lapse stacks of wounded and non-wounded larvae were imported into Fiji (v1.54J Just ImageJ Package) using bio-formats plugin and z-projected (Maximum-Intensity Projection) with brightness and contrast-enhanced. The registration was carried out using SIFT Linear Stack Alignment with default settings. Similar to 70 kDa processing (Eq. 3). Furthermore, to trace the high-molecular weight dextran, the imaged stacks were loaded and analyzed for onset of leakage). The dextran channel was min-max normalized and projected for 1024×1024 pixels and collapsed 121 frames to form kymograph. The endothelial contour was segmented and overlayed with turbo look up table to correlate thresholded pixel position through time. The obtained mask is subsequently loaded for area analysis Iv (arb. units), where complete FOV area is 1.048578×106 pixel2 or 4.43 x 105 µm2. Unfortunately, we are not able to detect extra epidermal wound leakage with 500 kDa as it is absent outside tissue at baseline wound condition (Eq. 2). Iv statistical and comparative analysis was conducted similarly to 70 kDa dextran between matched controlled conditions.

Image-based measurement of cPla2 nuclear membrane binding in macrophages and endothelial cells
The 4D spinning disk confocal stacks were imported into napari (v0.4.10). Raw images were intensity-normalized and smoothed using a Gaussian filter, background fluorescence along the z-axis was subsequently subtracted using a rolling-ball algorithm. The cPla2-mKate2 channel was segmented in 3D using the watershed algorithm and middle frame is isolated based on the largest contour area and seeded for segmentation analysis. A standard particle tracking algorithm was employed to track the nuclei movement over time and the non-track objects were cleared. cPla2-mKate2 membrane binding was defined as the ratio between mKate2 intensity at nuclear rim and nucleoplasm at the middle frame. The obtained 3D tracks were directed to normalization and edge-preserving smooth function. The obtained tracks were quantified for nucleus volume geometry and protein binding over time and the discontinuous tracks were excluded from the analysis. All frames among different remaining track were unionized and cPla2-mKate2 binding ratio at subsequent time points are normalized to cPla2-mKate2 intensity in the 1st frame of the image stack.

Quantification of leukocyte recruitment in Sudan Black Staining
The still images of the Sudan-black stained larvae were processed in Fiji with z-project function (Maximum-Intensity Projection), duplicated and blurred (Gaussian, sigma=50 scaled). The background subtracted using image calculator function (“Subtract create 32-bit”, “”), resulting 32-bit floating image was median-filtered (radius=5) and quantified for number of leukocytes in the tail fin using find maxima tool (prominence= 1950 strict light) and counted. The counts were verified for accuracy by manual counting and analysed.

Time-lapse neutrophil and macrophage tracking quantification
For analyzing the migration patterns of neutrophils and macrophages to osmotic wounds, we developed a custom python script that relies on python 3.9.23, housing skimage, scipy numpy, napari and matplotlib plugins for analysis and segmentation of moving cells in (Tg(mpeg:Qf2; Quas:NTR2.0)) and (Tg(lyz:Qf2; Quas:NTR2.0)). In brief, approximately 10 parameters were defined within a single script to isolate the tracks of neutrophils and macrophages. The binarized cell channel is filtered per-frame and tracked for nearest-neighbor algorithm and to correct for ID assignment, centroid-tracking algorithm was implemented for fallback ID re-assignment of original tracks (“Ghost Tracks”). The obtained data was unionized for filtering redundant tracks, and filtered data is used to generate net object displacement for per-object and averaged across biologically independent experiments. For macrophage, the centroid-based tracking was not consistent due to amboeid movement. To isolate macrophage tracks, a hybrid of fallback and nearest neighbor algorithm that relies on hybrid of polygon and centroid-based tracking was used. Each object angular direction information was also extracted for rose polarity plots. The obtained angular data was extracted as angle_rad from biological independent experiments, averaged and plotted as rose plots for neutrophils and macrophages, respectively. Some of the files produced large amount of fragments due to centroid or polygon based tracking, those files that had excessive cell-cell interaction during the course of the imaging experiment, were not analyzed for tracks parameters.

Statistics and reproducibility
No statistical methods were used to predetermine sample size and the sample size is similar to those reported in previous publications4,36 in brief we always used sample sizes of 8 different animals or more in the study, for laser wounding 3 different animals or more. Unless otherwise indicated, the data analysis was performed blind to the condition of the experiments and each experiment was repeated at least twice with similar results. The specimens in the imaging experiments were randomly allocated to experimental groups without any bias or pre-selection. Investigators were not blinded to allocation except for genetic experiments with pla2g4aa where all the measurements were taken prior to the authors knowing the genotype status of the larvae. The data was sorted into groups post genotyping. Blinding was not performed for the sake of increasing experimental throughput. The calculations in Fiji and napari were completed before calculations in MATLAB and significance quantification in Prism. The statistical analysis was completed using GraphPad Prism 10 (version 10.2.3). Each dataset was tested for normal distribution (Shapiro-wilk or D’agostino-Pearson) test. Only if the data were normally distributed, a parametric method (unpaired two-tailed student t-test) was applied to the dataset. A non-parametric test (two-sided Mann-Whitney) was applied for non-normally distributed datasets. In case of multiple comparisons, one-way ANOVA or Welch one-way with Dunnett’s multiple comparison post-hoc test applied. Kruskal–Wallis testing, a nonparametric alternative to one-way ANOVA, was used due to non-normal data. For analysis with different variance in error distribution, Welch’s correction was used. Data from two groups were compared using two-tailed unpaired t-test. P<0.05 were considered significant. Data were represented as bar plots where one dot in a graph represents n=1 biologically independent larvae experiment (single larva). Dot plot annotated numbers represent the total n per group at the top and mean value in the bottom of the graph. In bar plots top and bottom edges indicate SD range and midline central marking is representative of mean values. The Data distributions errors are shaded and presented as 95% confidence intervals, unless otherwise indicated. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 and ns, not significant. For every pharmacological treatment, and control larvae were derived from the same spawn of embryos. For transgenic imaging experiments, larvae were derived from multiple independent clutch of embryos from the same day or more commonly independent clutch of embryos on multiple days. We did not include selective breeding approach for transgenic imaging epxeriments as these larvae derive from multiple parents per indicated date. Animals were never reused for different experiments and all statistical analyses were performed across biological replicates. Replication included variation in biological source conducted across several experimental days from independent clutch of embryos. Analysis was performed objectively without blinding.

The larvae were selected on the following criteria: normal morphology, a beating heart and circulating red blood cells. Data exclusion criteria were predefined. Animals were excluded if yolk and/or cardiac function did not restore after dextran injection experiments. The specimens were randomly chosen for pharmacological, chemogenetic genetic or transgenic imaging experiments without bias or pre-selection. Imaging datasets were excluded if affected by artificial fluorescence fluctuations (e.g., dust debris), incompletely acquired Z-stacks or experiments where tail fin tissue drift exceeded 300 pixels (~150 microns) in the XY plane or 50 pixels (~25 µm) in the Z-direction and were completely discarded, since no reliable data can be extracted. For x63 Oil objective imaging, the live zebrafish at 2.5-4 dpf do not have flat tail morphology and the depth of objective does not always reach the sample. Furthermore, Nikon’s Perfect Focus Function (PFS) was not able to focus an IR beam with the x63 objective, therefore most image data acquired drift upon shift in osmolyte or bathing medium, we have only retained data that do not show excessive XYZ drift in the imaging experiment. For each UV or IR-induced tissue wounding, directly damaged cells that were within the wound ROI ablation region were excluded from the analysis. No data was excluded based on distribution (no outlier exclusion). Details of statistical analyses used, exact P values, number of experiments and animals for all graphs are listed in figure or statistical source data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this article, Gelashvili and colleagues investigate the mecanims by which wounds are detected and responses are induced, using Zebrafish as a model system. They use fluorescent dextran in the blood ciruclation to measure leakage from the blood vessels and from the wound. Carefull quantification and use of the right controls shows that leakage from the blood vessels is induced by the osmotic shock accompanying the wound and requires Alox5a, suggesting a role for the cPLA2 pathway. The authors then show that immune cells, more specifically macrophages, are needed to induce leakage of dextran from blood vessels upon wounding, and that cPLA2 is translocated to the nuclear envelope in macrophages rapidly after a wound; they also show that cPLA2 mutant animals are defective in the induction of leakage by wounding. They propose a working model by which osmotic stress induced by the wound rapidly induces swelling of cells and nuclei in the tissue, leading to cPLA2 activation in macrophages, production of arachidonic acid by these cells, which promotes blood vesssel leakage via Alox5a. Overall the article is very clear, contains carefully executed experiments and controls, and provides a very nice demonstration of how the activation of cPLA2 in immune cells contributes to the response of tissues to wounds. I would thus recommend publication of the article. I have just a few minor concerns/questions.\n1) The authors write that they observe recruitement of cPLA2 to the INM of macrophages, refering to figure 4. While I understand that other articles demonstrate well this recruitement, it is not clear in figure 4. Maybe the authors mean enrichment at the nuclear periphery? This is quite clear from the quantifications, but it is hard to see how the INM can be distinguished here.\n2) In most perturbations, the amplitude of the reduction in dextran leakage appears quite small. The authors claim that the pathway they identified is 'required' to induce this leakage. Maybe they could rather say that it 'contributes'. It is not clear for me (I am not a specialist of this phenomenon nor these techniques), whether the reduction they observe would correspond to what is expected in the case of a total lack of leakage induction, or it is really just a partial effect, meaning that the cPLA2 pathway in macrophages is just one of the signals inducing this leakage. This should be clarified in the discussion of the results (but it could also be that I misunderstood something here).\n3) The authors showed several elements of their working model independantly of each other. If I understood correctly, they showed that macrophages contribute to the response, that cPLA2 also contributes (although the depletion is in the whole animal and not specifically cPLA2) and that they observe cPLA2 enrichment at the nuclear periphery in macrophages (but they did not seem to have stained other cell types). So they never test directly a role of cPLA2 in macrophages, and they also do not exclude a contribution of cPLA2 in other cells (like endothelial cells) - is cPLA2 also recruited to the nuclear periphery in endothelial cells, or in other cells of the tissue? Can they deplete cPLA2 in specific cell types? If more specific experiments are not feasible, I still think that their working model is not contradicted by their experiments, but they might want to tone down their conclusions and make it clear that there are other possible contributions to the induction of leakage upon wounding (like cPLA2 activation directly in other cell types, or indirectly, and the fact that there is no direct proof of the contribution of cPLA2 specifically in macrophages). Again, I apologize in case of misunderstanding on my side - this system is quite complexe and I might have missed important elements of proof for the working model proposed.", "reviewer_2": "In this work, Gelashvili et al., presents evidence that macrophages localizing to the perivascular space of embryonic zebrafish blood vessels trigger a quick response to wounding by regulating osmotic signals and vascular permeability. The work addresses an interesting and important aspect of wound healing and introduces a novel signaling pathways into the mechanism of tissue injury. The article is written is clear and detailed language, the data is of good quality and quantifications were carefully performed, however, there are several conclusions that would require further validation to support the author’s conclusions more directly, for example, a characterization describing the vascular phenotypes of the mutants described in this study under homeostasis would be a key point to interpret the results presented by the authors, as this would provide insights into the mutants vascular integrity, architecture and function. Some of my concerns and questions are described below:\nFigure 1\n- Is there also response to the wounds caused by intracardial injection? Even if this delivery is done under strict care, this procedure should still cause a wound on the site of puncture, therefore, a wound response may be seen at this site as well.\n- Dextrans tend to diffuse to tissues outside the vasculature – which is evident in all figures – the amount of diffusion may vary from fish to fish based on artifacts like the injury induced at the injection site, or the minimal differences in tail clipping sizes, the authors should show non-wounded animals as controls and include the values obtained in their quantifications. Alternative, they could use fluorescent qdots or nanobeads which stay in circulation for longer periods of time without diffusing to adjacent tissues and have been extensively used to label vascular structures in zebrafish. Any diffusion would be strictly at the wound site which could facilitate a more restricted quantification, but also help visualizing vascular phenotypes much more clearly.\n- It would be helpful to relabel this figure, assigning each panel a letter for easier reading, although this version is properly described, having single letters for each panel will make the manuscript easier to follow especially in figures with multiple panels.\nFigure 2\n- What is the vascular phenotype in the alox5a, alox12 and ita4h mutants? The few vessels showing in this figures hint of vessels having significant architectural differences (figure 2b,c and d), where they appear much thinner and less branched or developed, potentially explaining why some of these mutants (alox12 and Ita4h) showed reduced vessel leakage. Is the vasculature in these mutants leaky in general? Once again, showing an uninjured embryo’s vasculature labeled with qdots could provide insights into the vascular phenotypes of these mutants. This is also applicable to all the drugs quantified in Figure 2, where no embryo data is shown to support their vascular leakage observations.\nFigure 3\n- Based on references to zebrafish transcriptomics the author mention that alox5a and ita4h are expressed by immune cells – potentially macrophages and neutrophils, however they do not show a staining validating this, which makes their ablations informative but not conclusive on the specific role of these genes in vessel permeabilization – as many other factors coming from these immune cells could lead to their results. Similarly, expression of these genes by non-immune cells was not tested.\n- As the authors have already made alox5a and Itah4 mutants, they could repeat these experiments on these animals or test their hypothesis transplanting mutant macrophages/neutrophils into wild type animals and testing whether these mutant cells affect vascular permeability/leakage.\n- Here the authors mentions that the NTR/MTZ dependent ablation of neutrophils did not modulate vessel permeability, it would be good to see examples of these treated animals and the general distribution of their immune cells comparing inside and outside the vasculature.\n- It is also not clear how far are macrophages and neutrophils migrating in response to the wound and the change in osmotic solutions?\nFigure 4\n- Once again, showing the vascular phenotype of the cPla2 mutants would be helpful to get a sense of the vascular architecture and integrity.\n- The author mention ‘perivascular macrophages’ during their nuclear shape sensing analysis in cPla2 transgenics, and elaborate on this in the discussion, it would be helpful to characterize these cells and determine whether these are classic mpeg1+ macrophages localized outside vessels or actual perivascular immune cells with a specific cellular profile (e.g. mrc1/lyve1+)?\n- A general comment is that many of these experiments were performed on the casper mutants, do these mutants have any alterations in the way wound healing occurs, or immune cells react to wounding? the authors should either show this mutant background does not affect their results (compared to a WT) or reference studies pointing out to previous reports where this was addressed.", "reviewer_3": "The manuscript Gelashvili, et al, focuses on understanding how macrophages signal to respond to wound signals and initiate rapid vascular repair. Though the data is interesting, new insights since previously published work seem limited for the scope of Nature Communications. The imaging is carried out at fairly low resolution and while the responses to changes in osmotic stress are pertinent, the phenotypes are modest and definative links to mechanics and the proposed genetic pathway are lacking. The paper is overly technical to read, and somewhat hard to follow the logic of. I appreciate the immense amount of work presented; however, in this format, the paper is not well targeted for general readership."}, "author_response": "Response to Reviewers:\nWe would like to thank the reviewers for their interest and thorough assess-ment of our manuscript. Based on their critique we revised the manuscript providing a substantial number of new experiments and improved figure/data organization/presentation. We also largely revised the results and discussion section for better balance and focus and reduced technical terminology.\nThe revisions include:\n• Revised vessel leakage quantifications (baseline leakage now indicated and discussed) throughout the manuscript for easier estimation of perturbation effect sizes.\n• Inclusion of non-wounded animals into the analysis as requested.\n• New 500-kDa dextran assays, also at higher magnification (63x), in wounded and non-wounded animals along with an assessment of baseline vascular morphology/permeability as requested.\n• Figures streamlined and relabeled to improve logical flow; a single overview cartoon now integrates genetic, pharmacologic, and chemogenetic results. Reduced technical terminology as requested.\n• Cell-type specificity of cPla2 response: We now also test for osmotic cPla2 activation in the endothelium as requested.\n• We added additional OXER1/Hcar1-4 mutant experiments to further delineate the molecular mechanism of osmotic vessel permeabilization (i.e., distinguish it from osmotic surveillance by leukocytes).\nAltogether we believe that our extensive revisions significantly improved the overall strength, focus and accessibility of our findings.\nPlease find below a point-by-point discussion of all the comments.\nKind regards,\nP. Niethammer (for the authors.)\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this article, Gelashvili and colleagues investigate the mecanims by which wounds are detected and responses are induced, using Zebrafish as a model system. They use fluorescent dextran in the blood ciruclation to measure leakage from the blood vessels and from the wound. Carefull quantification and use of the right controls shows that leakage from the blood vessels is induced by the osmotic shock accompanying the wound and requires Alox5a, suggesting a role for the cPLA2 pathway. The authors then show that immune cells, more specifically macrophages, are needed to induce leakage of dextran from blood vessels upon wounding, and that cPLA2 is translocated to the nuclear envelope in macrophages rapidly after a wound; they also show that cPLA2 mutant animals are defective in the in-duction of leakage by wounding. They propose a working model by which osmotic stress induced by the wound rapidly induces swelling of cells and nuclei in the tissue, leading to cPLA2 activation in macrophages, produc-tion of arachidonic acid by these cells, which promotes blood vesssel leak-age via Alox5a. Overall the article is very clear, contains carefully executed experiments and controls, and provides a very nice demonstration of how the activation of cPLA2 in immune cells contributes to the response of tis-sues to wounds. I would thus recommend publication of the article. I have just a few minor concerns/questions.\n1) The authors write that they observe recruitment of cPLA2 to the INM of macrophages, referring to figure 4. While I understand that other articles demonstrate well this recruitment, it is not clear in figure 4. Maybe the au-thors mean enrichment at the nuclear periphery? This is quite clear from the quantifications, but it is hard to see how the INM can be distinguished here.\nResponse: Thank you for pointing this out. We now use contrast-adjusted single-plane confocal slice views instead of MIPs for the insets of Fig. 4c, 4e which makes the rim localization of cPla easier to discern in the still images.\n2) In most perturbations, the amplitude of the reduction in dextran leakage appears quite small. The authors claim that the pathway they identified is 'required' to induce this leakage. Maybe they could rather say that it 'con-tributes'. It is not clear for me (I am not a specialist of this phenomenon nor these techniques), whether the reduction they observe would correspond to what is expected in the case of a total lack of leakage induction, or it is re-ally just a partial effect, meaning that the cPLA2 pathway in macrophages is just one of the signals inducing this leakage. This should be clarified in the discussion of the results (but it could also be that I misunderstood something here).\nResponse: We agree and adjusted the presentation and discussion of our results for better judgement of effect size.\nIn the former version, our vessel leakage (Iv) plots only depicted the overall leakage without baseline correction, that did not easily allow to assess wound-induced-leakage, which is of main interest. Given the way we calcu-late Iv(tot/norm) as area under the curve of our normalized Iv plots, there is always a 3600 AU theoretical baseline included (now indicated by a dotted line as thoroughly explained in the revised results section). Only values above 3600 denote wound induced leakage, everything below is baseline leakage (which we also confirmed by the inclusion of unwounded animals). Using this baseline, osmotic inhibition of vessel permeabilization is ~90%, and all other effects around 60%. While these effects are still partial, they are nevertheless substantial.\nWe hope that our revised representation and discussion better highlights the importance of physically stimulated eicosanoids in rapid vessel permeabili-zation and the key role of macrophages in transducing these signals. We adjusted the discussion accordingly, and again apologize for the confusion.\n3) The authors showed several elements of their working model inde-pendently of each other. If I understood correctly, they showed that macro-phages contribute to the response, that cPLA2 also contributes (although the depletion is in the whole animal and not specifically cPLA2) and that they observe cPLA2 enrichment at the nuclear periphery in macrophages (but they did not seem to have stained other cell types). So they never test directly a role of cPLA2 in macrophages, and they also do not exclude a contribution of cPLA2 in other cells (like endothelial cells) - is cPLA2 also recruited to the nuclear periphery in endothelial cells, or in other cells of the tissue? Can they deplete cPLA2 in specific cell types? If more specific experiments are not feasible, I still think that their working model is not contradicted by their experiments, but they might want to tone down their conclusions and make it clear that there are other possible contributions to the induction of leakage upon wounding (like cPLA2 activation directly in other cell types, or indirectly, and the fact that there is no direct proof of the contribution of cPLA2 specifically in macrophages). Again, I apologize in case of misunderstanding on my side - this system is quite complex and I might have missed important elements of proof for the working model proposed.\nResponse: Thank you for your thoughtful comments. As suggested, we now studied cPla2 translocation in endothelial cells and found no significant nu-clear membrane translocation above background (Fig. S7a-c) showing that macrophages are more sensitive to osmotic signals than other cell types at similar distances from the wound.\nWe attempted macrophage transplantation experiments (as suggested by rev #2) to test whether introducing wt-macrophages into cPla2 pathway mu-tants would rescue the leakage defects. For such experiment to yield in-formative results, we need to be able to transfer a sufficient number of mac-rophages and ensure that they reach the perivascular tail fin region, which turned out to be very difficult in our hands.\nWe were able to isolate ~5×10⁵ viable fluorescent macrophages from >200 fish. However, this number was still far below the ~10⁷ cells typically required for robust transplantation experiments. The recipient embryos de-veloped severe edema, enlarged hearts, and swollen anterior vessels over-night, and we could not observe any of the transplanted macrophages in the perivascular space. Despite of our intense efforts, we were not able to turn larval macrophage transplantation into a robust and reproducible approach that we trust to report here.\nWe cannot exclude a cPla activation in other cells than macrophages\n2\nmay also contribute to arachidonic acid production and cPLA2-ALOX5-LTA4H dependent vessel permeabilization. In fact, we previously showed that cPla at the zebrafish wound margin, also by physical signals, just as in\n2\nmacrophages shown here 2,3. Thus, arachidonic acid could, in principle, dif-fuse from the wound margin to the perivascular macrophages to at least par-tially fuel their eicosanoid synthesis. But given that (i) macrophages are well-known to contain the full cPLA2-ALOX5-LTA4H pathway, (ii) activate cPla2 within seconds of osmotic shock, and (iii) are required for the sensitivity of vessel permeabilization to ALOX5 inhibitors, the balance of evidence suggests that macrophages are the most likely cells converting the physical wound signal (osmotic shock) into a vessel permeabilizing lipid. This conclu-sion does not exclude the possibility that other cell types contribute to this process. We tried to clarify this notion with a new overview scheme and bal-anced discussion.\nReviewer #2 (Remarks to the Author):\nIn this work, Gelashvili et al., presents evidence that macrophages localiz-ing to the perivascular space of embryonic zebrafish blood vessels trigger a quick response to wounding by regulating osmotic signals and vascular permeability. The work addresses an interesting and important aspect of wound healing and introduces a novel signaling pathways into the mecha-nism of tissue injury. The article is written is clear and detailed language, the data is of good quality and quantifications were carefully performed, however, there are several conclusions that would require further validation to support the author’s conclusions more directly, for example, a characteri-zation describing the vascular phenotypes of the mutants described in this study under homeostasis would be a key point to interpret the results pre-sented by the authors, as this would provide insights into the mutants vas-cular integrity, architecture and function. Some of my concerns and ques-tions are described below:\nFigure 1\n- Is there also response to the wounds caused by intracardial injection?\nResponse: This is a great question. We now analyzed vessel leakage in 70kDa dextran injected animals without tail fin wounding. Injected but non-wounded animals at HYPO show almost the same, or slightly less vessel leakage as wounded ISO, or ISO+ animals (Fig. 1d). These data suggest only minimal if any vessel leakage caused by the injection procedure.\n- Dextrans tend to diffuse to tissues outside the vasculature – which is evi-dent in all figures – the amount of diffusion may vary from fish to fish based on artifacts like the injury induced at the injection site, or the minimal differ-ences in tail clipping sizes, the authors should show non-wounded animals as controls and include the values obtained in their quantifications. Alterna-tive, they could use fluorescent qdots or nanobeads which stay in circula-tion for longer periods of time without diffusing to adjacent tissues and have been extensively used to label vascular structures in zebrafish. Any diffu-sion would be strictly at the wound site which could facilitate a more re-stricted quantification, but also help visualizing vascular phenotypes much more clearly.\nResponse: We agree and now included slow-diffusing 500 kDa dextran (in-stead of Q-dots) and non-wounded animals into the analysis as requested (Fig. S1j, S1l, S2g, S2h, S5f). Comparable to Q-dots, and unlike 70 kDa dex-tran, 500 kDa dextran remains nicely contained within the vasculature high-lighting its morphology. In injured animals, 500 kDa dextran leaks from the vessel regions nearby the amputation site (Fig. S1j-m), which we now quan-tified either by leakage intensity or thresholded leakage area at two different objective magnifications (20x: Fig. 1j-k; 63x: l-m). As with 70 kDa dextran, the leakage is largely abrogated by ISO bathing. Furthermore, our 500 kDa experiments in unwounded mutants (20x: Fig. S2g-h, S5f) did not indicate obvious differences in baseline vessel morphology or permeability.\n- It would be helpful to relabel this figure, assigning each panel a letter for easier reading, although this version is properly described, having single letters for each panel will make the manuscript easier to follow especially in figures with multiple panels.\nResponse: In the previous version the logic of our presentation was indeed a bit flawed, and we apologize for the confusion this may have caused.\nNamely, the representative images were not grouped with their respective quantification, which is not ideal, and might have been annoying. We re-vised all our main figures now for better readability, and less subpanel jumping.\nFigure 2\n- What is the vascular phenotype in the alox5a, alox12 and ita4h mutants?\nThe few vessels showing in this figures hint of vessels having significant ar-chitectural differences (figure 2b,c and d), where they appear much thinner and less branched or developed, potentially explaining why some of these mutants (alox12 and Ita4h) showed reduced vessel leakage.\nResponse: Vessel morphology is difficult to reliably assess using 70 kDa dextran, given its high baseline tissue leakage and fast diffusion. To better evaluate vascular baseline phenotypes, we thus now imaged 500 kDa dextran in non-wounded animals and observed no gross differences in ves-sel morphology and baseline permeability comparing alox5a, lta4h, or pla2g4aa mutants with their wild-type siblings (see 2nd response to Fig. 1 comment).\nIs the vasculature in these mutants leaky in general?\nResponse: Our new experiments indicate that mutants and wt siblings show no significant baseline differences in leakage (see above).\nOnce again, showing an uninjured embryo’s vasculature labeled with qdots could provide insights into the vascular phenotypes of these mutants. This is also applicable to all the drugs quantified in Figure 2, where no embryo data is shown to support their vascular leakage observations ->\nResponse: We completely agree and have addressed this concern. Our 500 kDa dextran experiments do not indicate gross differences in vascular mor-phology or permeability in unwounded animals (see above). For the pharma-cological treatments and vessel leakage, representative larva images are now provided for 70 kDa dextran for antagonist (Fig. 2h; Fig. S3a) and ago-nist (Fig. S3f, g). Licofelone-treatment had no gross effect on vessel mor-phology as judged by visual inspection of KDRL-eGFP images or calculation of the vessel/tail fin area ratio (Fig. S3d, S3e).\nFigure 3\n- Based on references to zebrafish transcriptomics the author mention that alox5a and ita4h are expressed by immune cells – potentially macrophages and neutrophils, however they do not show a staining validating this, which makes their ablations informative but not conclusive on the specific role of these genes in vessel permeabilization –\nResponse: This is correct. Unfortunately, there are no specific antibodies for these zebrafish proteins commercially available that would allow to con-duct reliable immunofluorescence or WB staining. Establishing new, reliable Abs for zebrafish proteins is generally desirable, but would constitute a con-siderable financial resources and time that are unfortunately beyond the scope of this study. However, mutually consistent zebrafish in situ hybridiza-tion and mRNAseq data supporting the leukocyte localization of these en-zymes in zebrafish are already available from multiple independent sources.\nWe are not sure that simply repeating those experiments will provide more insights.\nBased on all the available literature from mammals and zebrafish, the cPLA2-ALOX5-LTA4H is indeed highly conserved macrophages from fish to humans. In mammals, the biochemical activity of the cPLA2-ALOX5-LTA4H eicosanoid branch is typically assessed in isolated (e.g., peritoneal) macro-phages. We now provide a more thorough discussion and literature account of this work.\nas many other factors coming from these immune cells could lead to their results. Similarly, expression of these genes by non-immune cells was not tested.\nResponse: Our data show that (i) rapid vessel permeabilization is almost completely (~90%) mediated through osmotic cues (Fig. 1d), (ii) largely (~60%) mediated by the cPLA2-ALOX5-LTA4H pathway and macrophages (Fig. 2, 4; S7). They further show that (iii) macrophages are required for ves-sel regulation through this bioactive lipid pathway, and that (iv) the same pathway is directly activated by osmotic shock in macrophages. We think this balance of evidence argues that macrophages are main players in convert-ing physical wound signals into rapid vessel responses. Nevertheless –as the reviewer noted—we cannot exclude that other cells and pathways may contribute, too. Namely, ~40% of the vessel response is currently unac-counted by our genetic perturbation. We only know that neutrophils do not contribute to these unaccounted events. We revised our discussion and overview scheme (Fig. S7d) to better clarify these notions.\n- As the authors have already made alox5a and Itah4 mutants, they could repeat these experiments on these animals or test their hypothesis trans-planting mutant macrophages/neutrophils into wild type animals and testing whether these mutant cells affect vascular permeability/leakage.\nResponse: We agree that these would be great experiments and thus at-tempted to establish the outline transplantation approach. By FACS sorting, we were able to isolate ~5×10⁵ viable fluorescent macrophages from >200 fish. Unfortunately, this number was still below the ~10⁷ cells typically re-quired for robust transplantation experiments. The recipient embryos devel-oped severe edema, enlarged hearts, and swollen anterior vessels overnight, and we could not observe any of the transplanted macrophages in the perivascular space. Regrettably, and despite of our intense efforts, we were not able to turn larval macrophage transplantation into a robust and reproducible approach that we trust to report here.\n- Here the authors mentions that the NTR/MTZ dependent ablation of neu-trophils did not modulate vessel permeability, it would be good to see exam-ples of these treated animals and the general distribution of their immune cells comparing inside and outside the vasculature. –\nResponse: Thank you for the suggestion. To address the distribution im-mune cells of MTZ-treated animals, we provide examples of NTR2.0-lyz:MTZ ablation of neutrophils together with Sudan black staining post-wounding. Using our described protocol, neutrophils were eliminated within 24 h, as demonstrated by the absence of Sudan black–positive cells in every MTZ-treated embryo (Fig. S4l, S4m).\n- It is also not clear how far are macrophages and neutrophils migrating in response to the wound and the change in osmotic solutions?\nResponse: We now quantified the requested leukocyte migration patterns.\n(Fig. S4g, S4h). Based on the literature and these results, osmotic shock mobilizes leukocyte that are initially up to ~200 µm away from the wound8-11. In terms of directionality, neutrophils show a strong bias toward the wound, whereas macrophages display broader directional variability.\nFigure 4\n- Once again, showing the vascular phenotype of the cPla2 mutants would be helpful to get a sense of the vascular architecture and integrity.\nResponse: We now show the vascular phenotypes in age-matched siblings for both 70kDa (Fig. 4a) and 500kDa (Fig. S5f). We did not note reproducible differences.\n- The author mention ‘perivascular macrophages’ during their nuclear shape sensing analysis in cPla2 transgenics, and elaborate on this in the discussion, it would be helpful to characterize these cells and determine whether these are classic mpeg1+ macrophages localized outside vessels or actual perivascular immune cells with a specific cellular profile (e.g. mrc1/lyve1+)? -> both of these are membrane glycoproteins –\nResponse: We agree. What determines the increased osmotic sensitivity of these mpeg1+ cells nearby vessels is one of the most intriguing questions raised by our current work. The mpeg1 promoter we use here is perhaps the most well established and preferred macrophage reporter in the zebrafish field, so the 0-hypothesis is that these “perivascular” macrophages are just embryonic macrophages/monocytes neighboring blood vessels. Any other statement, e.g., the interesting reviewer hypothesis that these could be spe-cialized perivascular macrophages, would require an extraordinary amount of evidence, likely comprising an entirely new project.\nAt this time, we are not aware of any zebrafish antibodies (or cell-specific promoters) that would allow us to selectively stain and sort the perivascular mpeg1+ population for further mRNA profiling. This could answer the re-viewer’s question. Clearly, developing better tools to enable such approach (e.g., photoconversion and sorting of perivascular cells using robotic high throughput microscopy paired with machine learning) is a consequent next step transcending the scope of this focused osmotic vessel permeabilization study. We have revised our discussion to clarify that our usage of “perivascu-lar” does not allude to a specific differentiation state, although this possibility is not excluded and exciting.\n- A general comment is that many of these experiments were performed on the casper mutants, do these mutants have any alterations in the way wound healing occurs, or immune cells react to wounding? the authors should either show this mutant background does not affect their results (compared to a WT) or reference studies pointing out to previous reports where this was addressed.\nResponse: To experimentally address this comment, we extended our analyses from the casper background to AB animals and now provide new data with 70kDa dextran (Fig. S1g–i) showing that the casper and AB back-grounds respond similar with respect to wound-induced vessel leakage.\nReviewer #3 (Remarks to the Author):\nThe manuscript Gelashvili, et al, focuses on understanding how macro-phages signal to respond to wound signals and initiate rapid vascular re-pair. Though the data is interesting, new insights since previously published work seem limited for the scope of Nature Communications.\nResponse: We are saddened by the reviewer’s discouraging overall assess-ment of our work, though we admit that we could have done a much better job of highlighting its relevance in the first version. Thus, we have substan-tially revised our manuscript based on rev #1 & 2’s constructive critiques.\nWe respectfully disagree with this reviewer’s significance and novelty as-sessment. Neither we nor others have previously reported a role for macro-phages in osmotic vessel permeabilization and the cPLA2-ALOX5-LTA4H pathway, and nuclear membrane mechanotransduction in vivo.\nThe role of the nucleus as a mechanosensor and the contribution of nuclear mechanotransduction to physiological processes is still a largely uncharted field mostly studied in reconstituted cell culture environments, not in live an-imals – as we do here.\nTo clarify the broad relevance of our findings, we have completely revised our result and discussion sections.\nThe imaging is carried out at fairly low resolution\nResponse: We now include also a higher magnification experiment, along-side our standard 20x mag approach. Notably, 20x mag imaging allows to image larger tissue fields at higher temporal resolution than possible with 63x (Fig. S1j-m).\nand while the responses to changes in osmotic stress are pertinent, the phenotypes are modest and definative links to mechanics and the pro-posed genetic pathway are lacking.\nResponse: We admit that the previous presentation of our leakage (Iv) data might have caused confusion, as we did not explicitly distinguish baseline from wound-induced leakage. Indeed, this was a severe shortcoming that we now have addressed by indicating the leakage baseline in all applicable graphs. We now also experimentally determined baseline leakage in non-wounded animals. We estimate that wound-induced vessel permeabilization is (i) almost completely (~90%) caused by osmotic cues, (ii) and to ~ 60% via the cPLA2-ALOX5-LTA4H pathway and macrophages. We would not consider ~60% inhibition modest, although it is indeed partial. We have bal-anced our discussion to highlight this notion.\nWe regret that we might have contributed to this harsh reviewer judgement by not more explicitly discussing effect sizes in our earlier version, and due to unclear data presentation.\nThe paper is overly technical to read, and somewhat hard to follow the logic of. I appreciate the immense amount of work presented; however, in this format, the paper is not well targeted for general readership.\nResponse: We completely revised our results and discussion to reduce the use of technical language. The text should be now much more readable."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In their revised article, the authors have provided new data and analysis, including more controls and clarification of level of wound induced leakage, also being more subtle in their claims to leave open some alternatives when they did not provide perfect direct proofs - for example for the fact that the downstream products of the cPLA2 pathway are mostly coming from perivascular macrophages. They tried some direct transplantation experiments that failed, and as I am not a zebrafish expert, I unfortunately have no specific idea to suggest to go around this experiment to test their hypothesis more directly. I think that, despite this failure for a direct proof, their article provides very interesting and convincing data to support their working model, and that the way they now present their results and discuss them, is well adapted to the level of confidence they can have in their results. I would thus recommend publication without further revisions.", "reviewer_3": "The included revisions have decreased my initial concerns, and dramatically improved the manuscript", "reviewer_4": "I have been appointed to replace Reviewer #2, who are no longer available assess the authors’ response to their previous comments.\nAfter reviewing the revisions that the authors performed, I believe that the authors have in general satisfactorily addressed the Reviewer #2 comments.\nHowever, I would like to raise a few points:\n1. Given that the images of blood vessels are low in magnification and resolution, it is challenging to determine whether there is a vascular phenotype in the alox5a, alox12 and ita4h mutants; and between controls and after osmotic shock. Based on this resolution, there is no observed gross differences at the tissue scale, as the authors claim, although the ventral caudal vein of alox5a mutant appears more dilated than the wildtype embryo to me (Supplementary Fig. 2).\n2. I would have liked to see the effects of osmotic shock (after injury) on endothelial cell-cell junctions (morphology) and upon the loss of macrophages and/or perturbation of the cPLA2-ALOX5-LTA4H pathway. This will provide some insights into how blood vessel permeability is increased.\n3. The authors stressed the function of nuclear membrane mechanotransduction in transmitting wound signals to blood vessels and emphasized the importance of inner nuclear membrane (INM) tension in recruiting cPLA2 to the INM after osmotic shock. The evidence supporting this claim is rather weak in this manuscript. This is partly because it is not possible to see the shape of cell nuclei as well as the shape of the macrophages, thus making it difficult for non-macrophage experts to determine the subcellular localization of cPla2-mKate2. I would have appreciated a more detailed examination of cPla2 localization with, for example, a nuclear/nuclear membrane marker to clearly show that cPla2 redistributed from the nucleoplasm to the nuclear membrane. Along this line, why does cPla2-mKate2 distribution look different in Fig. 4e (more elongated, like the cell body) compared to Fig. 4c (more circular)? Does HYPO condition change the distribution of cPla2 or the shape of the nucleus?"}, "author_response": "> Thank you very much for lending your scientific perspective as it helped us\nstrengthen and improve the manuscript!\n> Thank you!\n> Thank you for the note! We think we have quantitatively addressed this\npoint in Fig. S1j-k and Fig. S6a-c\nWe agree. The Fig. S1j was full field of view, which we now cropped and\nenlarged for better visibility. To clarify this point, we now include a new sup-\nplemental figure with enlarged, native resolution (non-rasterized or kept at\n300ppi) insets (showing vessel morphology) for better visibility. From these\nenlarged, higher resolution images it should be clear that there are no overt\nmorphological differences in baseline vessel morphology between wild types\nand mutants.\nIn the new Fig. S6, we enlarged the quantitative panels showing that there\nis also no difference in basal vessel area (Iv , = morphological proxy) and\narea\nvessel permeability (Iv , functional proxy). The morphological differ-\ntot/norm\nences mentioned by the reviewer are most likely within the range of normal\nbiological variability.\n2. I would have liked to see the effects of osmotic shock (after injury) on\nendothelial cell-cell junctions (morphology) and upon the loss of macro-\nphages and/or perturbation of the cPLA2-ALOX5-LTA4H pathway. This will\nprovide some insights into how blood vessel permeability is increased.\n> Indeed, the next big question is how the blood vessel permeability is\nachieved, e.g., through chemical or physical interactions of the perivascular\nmacrophages with the blood vessels.\nCurrently, we do not have appropriate genetic marker tools to visualize en-\ndothelial–interstitial leakage routes in vivo at sufficient spatiotemporal reso-\nlution to make solid mechanistic statements. The leakage possibly involves\njunctional remodeling or trans-endothelial transport (e.g., caveolae). Care-\nfully testing these ideas, e.g., by high-speed/high-resolution intravital micros-\ncopy would be a new, dedicated project that is currently beyond our technical\ncapabilities.\n3. The authors stressed the function of nuclear membrane mechanotrans-\nduction in transmitting wound signals to blood vessels and emphasized the\nimportance of inner nuclear membrane (INM) tension in recruiting cPLA2 to\nthe INM after osmotic shock. The evidence supporting this claim is rather\nweak in this manuscript. This is partly because it is not possible to see the\nshape of cell nuclei as well as the shape of the macrophages, thus making it\ndifficult for non-macrophage experts to determine the subcellular localization\nof cPla2-mKate2. I would have appreciated a more detailed examination of\ncPla2 localization with, for example, a nuclear/nuclear membrane marker to\nclearly show that cPla2 redistributed from the nucleoplasm to the nuclear\nmembrane. Along this line, why does cPla2-mKate2 distribution look different\nin Fig. 4e (more elongated, like the cell body) compared to Fig. 4c (more\ncircular)? Does HYPO condition change the distribution of cPla2 or the shape\nof the nucleus?\n>Thank you for the comment. To clarify: The constitutive, nuclear localization\nof zebrafish cPla2 is well established (Enyedi et al., NCB, 2013; Enyedi et\nal., Cell, 2016). In addition, we provide with Fig. S5f a magnified representa-\ntive image of a perivascular macrophage with the ER marker (KDEL-eGFP,\nyellow). Since the ER and nuclear membrane are contiguous, this marker\noutlines the nucleoplasm. Before the translocation (left column), one can\nclearly see that cPla2 (magenta) is within the nucleoplasm, before it revers-\nibly translocates to the nuclear membrane (middle column) as marked by\nKDEL-eGFP (yellow). The same translocation (please note the change from\nhomogenous fluorescence distribution to rim-like fluorescence distribution)\nis also visible in the magnified insets of Fig. 4c and S7a,c), and quantified in\nthe respective profile plots (Fig. 4d; S7b-d). We have now added additional\nannotations to these rather complex figures to better guide the reader’s at-\ntention and avoid misunderstandings.\nHypotonic shock swells the nucleus (see Enyedi et al., Cell, 2016), but this\nmomentous volume increase upon shock is difficult to perceive or measure\nat our spatiotemporal resolution. The macrophage nuclei have complex,\nhighly variable shapes depending on how they are confined by the surround-\ning tissue at a given moment. The noted shape differences between 4c\n(hypo, round), and 4e (iso, elongated) most likely reflect biological variability.\nThis notion is corroborated by Fig. S5f (hypo, elongated) and S7 (hypo, more\nelongated; iso, more rounded), which show opposite shape trends then\nFig.4c/e."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have sufficiently addressed my comments as well as clarified my questions."}, "author_response": ""}]

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