LPS-enhanced leukocyte migration assay in 4 dpf zebrafish larvae.

<p>All larvae (<i>nacre</i>) are four days post-fertilization (4 dpf), with anterior to the left, scale bar = 10 µm. <b>A</b>, tail of alive larva without tail cut; <b>B</b>, tail of alive larva with tail cut; <b>C–D</b>, whole-mount MPO staining in uncut tails of zebrafish larvae; <b>C</b>, without lipopolysaccharides (−LPS) and <b>D</b>, with lipopolysaccharides (+LPS); <b>E–F</b>, whole-mount MPO staining in cut tails of zebrafish larvae; <b>E,</b> without the inclusion of LPS; <b>F</b>, with the inclusion of LPS. Dark-spots (marked by arrows) represent the migrating leukocytes, which are semi-quantified in the region to the right of the dashed red arc.</p

Inhibitory effects of some natural products in various inflammatory targets.

<p>Inhibitory effects of some natural products in various inflammatory targets.</p

Anti-inflammatory activity of natural products.

a<p>Leukocyte migration is expressed as relative values (RLM).</p>***<p>p<0.001,</p>**<p>0.001*</p><p>0.01</p

Inhibition of the leukocyte migration by known anti-inflammatory drugs and non anti-inflammatory compounds.

<p>After exposing the larvae to mechanical and biological damages, anti-inflammatory drugs and non anti-inflammatory compounds were evaluated for their activity in the LPS-enhanced leukocyte migration assay. Migrating MPO-positive cells were counted in the tail tip and the results expressed as relative leukocyte migration (RLM) values. Results for NSAIDs (upper panel) and SAIDs (middle panel) show a significant inhibition of leukocytes migration in a concentration-dependent manner while non anti-inflammatory drugs (lower panel) indicate no significant effect on leukocyte migration. In each case, values are plotted as RLM (SEM, n = 3 replicates with 10 larvae each) and a value of 0.5 was established as a cut-off for anti-inflammatory activity. ***p<0.001, **0.001</p

Scoring of leukocyte migration in the transected tails of zebrafish larvae.

<p>All larvae (<i>nacre</i>) are four days post-fertilization (4 dpf), with anterior to the left, scale bar = 10 µm. Dark spots (marked by arrows) represent leukocytes migrating to the injured zone in the transected tails. Migrating leukocytes were counted in the region to the right of the dashed red arc. <b>A</b>, tail of an uncut larva; <b>B</b>, tail-cut with score 0; <b>C</b>, tail-cut with score 1; <b>D</b>, tail-cut with score 2; <b>E</b> tail-cut with score 3; <b>F,</b> tail-cut with score 4. <b>^a</b>Experimental values obtained for each larvae are normalized to a relative value expressed as relative leukocyte migration (RLM); +LPS: with inclusion of lipopolysaccharides. <b>^b</b>Percentage of anti-inflammatory activity is obtained as (1−RLM)x100.</p

Synergy between pCAME and dorsomorphin.

<p>(A) Wild type pulsed with 1% DMSO in Danieau’s solution; (B) 10 µM dorsomorphin pulse; (C) 70 µM pCAME pulse; (D) and (E) Combination of 10 µM dorsomorphin and 70 µM pCAME pulse. Black arrows denote ectopic tails. All AB embryos are at 48 hpf and pulsed for 1h at tailbud stage. A significant increase of the percentage of ectopic tail formation was observed as well as a more pronounced phenotype. </p

Analysis of tissue-specific marker expression in pCAME-induced ectopic tails.

<p>(A) <i>eve1</i>; (B) <i>ntl</i>; (C) <i>myoD</i>; (D) c<i>ol2a</i>; (E) <i>shh</i>; and (F) <i>cdx4</i> expression. All embryos were pulsed with 140 μM pCAME for 1h at tailbud stage and fixed at 30 hpf. Lateral views.</p

<i>jnk2</i> morpholino knockdown induces convergence extension defects.

<p>(A) and (B) 2 mM <i>jnk2</i> MO-injected fish A: 38 % B: 9 %; (C) Wild type; (D) 150 μM SP600125 pulsed for 1 h at tailbud stage. All embryos are at 48 hpf. Black arrow denotes duplicated tail. (E-H) Control MO injected fish; (E’-H’) <i>jnk2</i> MO injected fish; (E) <i>flh</i>; (F) <i>ntl</i>; (G) <i>myoD</i>; (H) <i>papc</i>.</p

Tissue marker analysis in SP600125-induced ectopic tails.

<p>(A) <i>eve1</i>; (B) <i>ntl</i>; (C) <i>myoD</i>; (D) c<i>ol2a</i>; (E) <i>shh</i>; and (F) c<i>dx4</i> expression. All embryos were pulsed with 140 μM pCAME for 1h at tailbud stage and fixed at 30 hpf. Lateral views.</p

pCAME induces convergence and extension defects.

<p>(A) and (B) <i>flh</i> expression; (C) and (D) <i>ntl</i>; (E) and (F) <i>papc</i>; (G) and (H) <i>myoD</i>. All embryos were treated at 2-4 cell stages with 14 or 28 μM pCAME and fixed at 1 somite stage. Dorsal view. Expression domains of compound-treated fish are altered as would be predicted for CE defects. Embryos have a broader and shorter notochord and impaired convergence and extension of the para-axial mesoderm and the somites.</p