8 research outputs found
Over-expression of <i>bmp2b</i> specifically localized in the CHT area at 79 hpf upon ginger or 10-G exposure in normal and in anemic zebrafish embryos.
<p>Whole-mount in situ hybridization of <i>bmp2b.</i> (A–C, left) Normal non-anemic control embryos or embryos treated with ginger/10-G. (D–F, right) Anemic control embryos or anemic embryos treated with ginger/10-G. Anemic groups were treated with 0.5 µM PHZ from 33 to 48 hpf. Embryos express <i>bmp2b</i> in the CHT region (arrows) following exposure to ginger (B, E) or 10-G (C, F). (G) A table shows the percentage of embryos with <i>bmp2b</i> expression in the CHT area at 79 hpf. Scale bars = 420 µm.</p
Ginger/10-G treatment after gastrulation promotes <i>bmp2b/7a</i> in the developing caudal hematopoietic tissue.
<p>(A–B) Zebrafish embryos were treated with ginger (5 µg/ml) or 10-G (2 µg/ml) from 10 to 48 hpf, followed by whole-mount in situ hybridization of <i>bmp2b</i> (A) and <i>bmp7a</i> (B). Both <i>bmp2b</i> and <i>bmp7a</i> were up-regulated locally in the CHT (and underlying fin) upon ginger or 10-G exposure (whereas they are not expressed in the CHT of control embryos at 48 hpf). Scale bars = 700 µm.</p
Ginger extract and its purified phenolic compounds promote <i>Tg(gata1:dsRed)</i> fluorescence and <i>gata1</i> mRNA expression.
<p>(A) Bright field (top left) and <i>Tg(gata1:dsRed)</i> fluorescence of zebrafish embryos at about 22 hpf, before the onset of circulation (anterior to the left). Exposure to ginger extract or its compounds 8-gingerol (8-G), 10-gingerol (10-G), 8-shogaol (8-S) and 10-shogaol (10-S) promoted <i>Tg(gata1:dsRed)</i> fluorescent erythroid cell development in the ICM and PBI (arrows), as compared to control embryos. N = 35 embryos per group. In this panel, we show an embryo treated with a lower concentration of 6-S (2 µg/ml) as this compound was toxic at higher doses. Scale bar = 400 µm. (B) Whole-mount in situ hybridization of ginger or 10-G treated embryos (8 hpf to 21 hpf exposure) revealed increased expression of <i>gata1</i> transcript at 22 hpf. N = 50 embryos per group. Scale bar = 350 µm. (C) At 48 hpf, control embryos at the top; ginger or 10-G treated embryos at the bottom. Scale bar = 500 µm. Fluorescent erythrocytes circulating in the axial vasculature (arrows) and in the pericardial space (arrow heads).</p
Ginger/10-G treatment during gastrulation promotes <i>bmp2b/7a</i> and Bmp target gene expression in zebrafish embryos.
<p>(A) Treatment of late gastrulae with ginger at 15 or 20 µg/ml induces the <i>mercedes</i> mutant-like phenotype (partial duplication of the tail fin) at 1 dpf in 8% or 10% of the treated embryos, respectively. Thus, the zebrafish embryos exposed to ginger extract mimic the phenotype of the <i>ogon</i> mutant, which has a mutation in <i>sizzled</i>, a <i>bmp</i> suppressor gene, at 1 dpf. (B) <i>bmp7a</i> expression was strongly increased and extended to the entire blastoderm at 60% epiboly, following short-term exposure to ginger (5 µg/ml) or 10-G (1 µg/ml) from sphere (4 hpf) to 60% epiboly (7 hpf) stages. (C) Up-regulation and extension of the expression domain were observed for <i>bmp2b</i> at 60% epiboly. (D–E) Accordingly, BMP target genes were up-regulated after ginger/10G treatment from the sphere stage (4 hpf) to 7 hpf, as illustrated by enhanced <i>eve1</i> extended towards the dorsal side (arrow heads), a ventral mesoderm marker (D), and <i>gata2,</i> a non-neural ectoderm marker (E), in zebrafish embryos at 60% epiboly. Pictures on left panels show gastrulae, dorsal side to the right (B–E) and statistics tables (right panels) are representative of three independent experiments. N = number of embryos per group. Scale bars = 250 µm.</p
Over-expression of <i>bmp7a</i> specifically localized in the CHT region at 79 hpf upon ginger or 10-G exposure in normal and in anemic zebrafish embryos.
<p>Whole-mount in situ hybridization of <i>bmp7a</i>. (A–C, left) Normal non-anemic control embryos or embryos treated with ginger/10-G. (D–F, right) Anemic control embryos or anemic embryos treated with ginger/10-G. Anemic group were treated with 0.5 µM PHZ from 33 to 48 hpf. Embryos express <i>bmp7a</i> in the CHT area (arrows) following exposure to ginger (B, E) or 10-G (C, F). (G) A table shows the percentage of embryos with <i>bmp7a</i> expression in the CHT region at 79 hpf. Scale bars = 420 µm.</p
Ginger/10-G treatment increases hematopoietic progenitor markers expression.
<p>Zebrafish embryos were treated with ginger or 10-G from 9 to 21 hpf. (A) <i>Tg(gata1:dsRed)</i> for erythrocyte and <i>Tg(flk1:GFP)</i> for blood vessels, double-fluorescent overlay pictures of embryos at 22 hpf after exposure to ginger or 10-G. Hypertrophy of the PBI vascular plexus in <i>Tg(flk1:GFP)</i> after ginger or 10-G treatment, with <i>Tg(gata1:dsRed)</i> red fluorescent erythrocytes accumulated inside the honeycomb-like vasculature (arrows). Scale bars = 500 µm. Whole-mount in situ hybridization of <i>c-myb</i> (B) and <i>scl</i> (C) in zebrafish embryos at 22 hpf. Both hematopoietic progenitor markers were up-regulated in primitive hematopoietic tissues (ICM+PBI) upon ginger (arrow head) or 10-G (arrow) exposure. Scale bar = 350 µm.</p
Ginger or 10-G exposure promotes erythrocyte recovery from anemia via a Bmp/Smad signal-dependent mechanism.
<p>Bmp/Smad inhibition abolishes the hematopoiesis promoting effect of ginger and 10-G. (A–B) The effect of ginger on hematopoiesis was quantitated in zebrafish embryos after phenylhydrazine (PHZ) induced acute hemolytic anemia, followed by extensive washes and treatments with ginger extract (A) or 10-G (B) with or without dorsomorphin (DMP; 0.1 µM). Ginger and 10-G promote hematopoietic recovery in PHZ treated embryos. Videos of circulating erythrocytes were analyzed and erythrocyte numbers for “PHZ+ginger” and “PHZ+ginger+DMP” assays were calculated and normalized with blood flow (velocity) using the PHZ control value as a reference. Tables summarize the results of one representative experiment. Experiments were repeated 3 times. n = number of embryos analyzed per group. <i>p</i> values were determined by using the Student’s t-test. (C) Regions of erythropoiesis promoted by ginger and 10-G are indicated on cartoons of zebrafish embryos at 22 hpf (primitive wave; before circulation), and at 5–6 dpf during the definitive wave of hematopoiesis. DMP-mediated inhibition of Bmp/Smad signal refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone.0039327.s005" target="_blank">Figures S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone.0039327.s006" target="_blank">S6</a>, 6 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone.0039327.s007" target="_blank">S7</a> data. DMH1-mediated inhibition of Bmp/Smad signaling refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone.0039327.s005" target="_blank">Figures S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone.0039327.s006" target="_blank">S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone.0039327.s008" target="_blank">S8</a> data. During the primitive wave of hematopoiesis, expression of <i>gata1</i> and <i>Tg(gata1:dsRed)</i> were increased in the ICM and PBI, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone-0039327-g001" target="_blank">Figure 1</a>, and the hematopoietic progenitor markers <i>cmyb</i> and <i>scl</i> were up-regulated in the same hematopoietic tissues, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone-0039327-g002" target="_blank">Figure 2</a>. During the definitive wave, <i>Tg(gata1:dsRed)</i> circulating cells were promoted at 5/6 dpf upon ginger/10-G exposure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone-0039327-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone-0039327-g006" target="_blank">6</a>), and the hematopoietic progenitor markers <i>cmyb</i>, <i>scl</i> and <i>lmo2</i> were up-regulated in the CHT/hemogenic endothelium at 6 dpf (<i>cmyb, </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone-0039327-g007" target="_blank">Figure 7</a>) or in the CHT only at 5 dpf (<i>scl</i> and <i>lmo2, </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039327#pone.0039327.s009" target="_blank">Figure S9</a>).</p
Effect of ginger and 10-G treatments on <i>c-myb</i> expression in zebrafish embryos at 6 dpf.
<p>(A–C) Ginger (B) or 10-G (C) treatment from 2 dpf to 6 dpf promotes <i>cmyb</i> expression in the CHT (black arrows) and the hemogenic endothelium (white arrows) along the ventral wall of the dorsal aorta (AGM equivalent) in the trunk and tail regions of normal embryos. (D–F) In phenylhydrazine-induced anemic embryos, ginger (E) or 10-G (F) treatment similarly promotes <i>cmyb</i> expression both in the CHT (black arrows) and the hemogenic endothelium (white arrows). (G–H) Graphical representation of the percentage of embryos showing <i>cmyb</i> expression in the CHT (G) and in the hemogenic endothelium (H). CTRL: control; PHZ: phenylhydrazine; n = number of embryos. Scale bar = 500 µm.</p