Sodium nitrite caused defective development of zebrafish heart in a dose dependent way.

<p>(A–C) Zebrafish embryos exposed to 100 mg/l sodium nitrite from 10 hpf exhibited cardiac edema from slight phenotype (B) to severe phenotype (C) compared to control embryos with normal heart (A) at 108 hpf. (D) A scatter plot showing the edema index (EI) of embryos exposed with different concentration of sodium nitrite. EI is defined as b/a. Average ± standard errors of EIs were shown in lines. (E–G) Histological sections showing 100 mg/l sodium nitrite exposure caused defective structure of zebrafish heart at 108 hpf. Compared to control embryos with normal pericardial membrane, myocardium and both superior and inferior valve leaflets (E), 4/7 embryos exposed to the nitrite showed thinner pericardial membrane and myocardium, and only superior valve leaflet but no formation of inferior valve leaflet (F), whereas 3/7 of the treated embryos displayed thinner pericardial membrane and myocardium and no formation of either superior or inferior leaflets (G). a: the semi-diameter of ventricle; b: the semi-diameter of the pericardial cavity. Red star (*): pericardial membrane; Black star (*): myocardium; Black arrow: position of superior valve leaflet; Black arrowhead: position of inferior valve leaflet; A: atria; V: ventricle.</p

Inhibiting NO signaling in nitrite-exposed embryos partially rescued defective development of cardiac valve in zebrafish embryos.

<p>(A) The cGMP level was dramatically increased in the nitrite-exposed embryos at 24, 36, 48 and 76 hpf, respectively. **: P<0.01. The values of cGMP amount in all the control embryos were normalized to 1.0, respectively. The value of cGMP amount in the nitrite-exposed embryos was the fold of the control embryos at the same developmental stage. (B) A scatter plot showing the increased EIs in the nitrite-exposed embryos were significantly reduced by microinjecting ODQ (sGC inhibitor) into nitrite-exposed embryos. Different treatments of embryos were shown in X-axis. EI (shown in Y-axis) of each embryos was shown in the plot. Average ± standard errors of EIs were shown in lines. (C–E) Defective histological structures of heart caused by excessive nitrite were partially rescued by microinjection of ODQ into nitrite-exposed embryos. Embryos were observed at 108 hpf. Compared to control embryos with normal pericardial membrane, myocardium and both superior and inferior valve leaflets (C), 6/8 embryos exposed to the nitrite showed thinner myocardium and defective formation of either superior or inferior leaflets (D). ODQ microinjection resulted in 3/6 of the embryos developed both superior and inferior leaflets (E). (F–T) Microinjection of ODQ partially rescued the diminished expressions of valve progenitor makers in zebrafish embryos at 48 hpf. <i>nppa</i> is not expressed in the AVC (rectangular box) of control embryos (F) but was ectopically expressed in the AVC of nitrite-exposed embryos (K). Microinjection of ODQ into nitrite-exposed embryos prevented 7 of 15 embryos from expressing <i>nppa</i> in AVC (P). Compared to control embryos, nitrite exposure significantly decreased or abolished expressions of <i>bmp4</i> (G, L), <i>vcana</i> (H, M), <i>notch1b</i> (I, N) and <i>has2</i> (J, O) in AVC. However, microinjection of ODQ into nitrite-exposed embryos resumed expressions of <i>bmp4</i> (Q), <i>vcana</i> (R), <i>notch1b</i> (S) and <i>has2</i> (T) in about half embryos. Red star (*): pericardial membrane; Black arrow: position of superior valve leaflet; Black arrowhead: position of inferior valve leaflet; A: atria; V: ventricle.</p

Histological and molecular analyses revealing excessive nitrite affected zebrafish heart valve development directly.

<p>Nitrite-exposed embryos were treated with 100 mg/l sodium nitrite from 10 hpf. (A–F) Histological sections showing defective valve development caused by nitrite exposure occurred from 48 hpf. At 36 hpf, the embryos exposed to nitrite exhibited similar histological heart structure (B) to that of control zebrafish, comprising one layer of myocardium and one layer of endocardium (A). At 48 hpf, endocardial cells of control embryos exhibited cuboidal shape in AVC between two chambers (C) but the exposed embryos did not have cuboidal endocardial cells in AVC (D). At 76 hpf, invagination of endocardial cells into cardiac jelly in AVC in control embryos formed superior primitive valve leaflet consisting of multilayer of cells (E); however, no superior primitive valve leaflet (no multilayer cells in the superior part of AVC) was formed in nitrite-exposed embryos (F). (G–N) Cardiac looping in zebrafish embryos shown by the expression of <i>cmlc2</i> revealing abnormal cardiac looping caused by nitrite exposure occurred as early as 43 hpf. The expression pattern of <i>cmlc2</i> was not affected by nitrite exposure at 36 hpf (G–H) and 40 hpf (I–J), but slightly abnormal (shown in white dotted curve) at 43 hpf (K–L) and obviously abnormal (shown in white dotted curve) at 48 hpf (M–N). (O–X) Nitrite exposures altered the expressions of molecular makers of valve progenitors at 48 hpf. <i>nppa</i> was not expressed in the AVC (rectangular box) of control embryos (O) but was ectopically expressed in the AVC of nitrite-exposed embryos (P). Compared to control embryos, nitrite exposure significantly decreased or abolished expressions of <i>bmp4</i> (Q, R), <i>vcana</i> (S, T), <i>notch1b</i> (U, V) and <i>has2</i> (W, X) in AVC. The number shown in the lower right-hand corner was the number of embryos exhibiting the typical phenotype shown in the panel to the number of embryos totally observed. Black arrow: position of cuboidal endocardial cells; Black arrowhead: position of superior primitive leaflet; White arrow: endocardium; White arrowhead: myocardium.</p

A scatter plot showing that sodium nitrite affected zebrafish heart development starting from 36 hpf.

<p>Nitrite-exposed embryos were treated with 100 mg/l sodium nitrite for different time window shown in X-axis. EI (shown in Y-axis) of each embryos was measured and shown in the plot. Average ± standard errors of EIs were shown in lines.</p

RA restricts the primitive myelopoiesis mainly before 11 hpf.

<p>All embryos are positioned anterior left and lateral front. Embryos were treated with vehicle DMSO (A, H, O) or with 50 nM RA (B–G, I–N) from 3 to 5 (B, I), 5 to 7 (C, J), 7 to 9 (D, K), 9 to 11 (E, L), 11 to 13 (F, M) and 13 to 26 hpf (G, N), or with 250 nM RA form 10 to 11 (P), 11 to 13 (Q), 13 to 22 hpf (R), respectively. They were then examined for expressions of myeloid markers <i>lcp1</i> (A–G, O–R) and <i>mpx</i> (H–N) at 26 hpf (A–N) or 22 hpf (O–R) by whole mount <i>in situ</i> hybridization. The number shown in the lower left-hand corner of each panel is the number of embryos exhibiting the typical phenotype shown in the panel to the number of embryos totally observed. The typical embryos expressing <i>lcp1⁺</i> cells at 22 hpf were shown in O–R. The scatter plot (S) shows the number of <i>lcp1⁺</i> cells counted from each of the embryos at 22 hpf with different treatment (control; 10–11 hpf RA treatment; 11–13 hpf RA treatment and 13–22 hpf RA treatment).</p

Retinoic Acid Signaling Plays a Restrictive Role in Zebrafish Primitive Myelopoiesis

<div><p>Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers <em>scl</em>, <em>lmo2</em>, <em>gata2</em> and <em>etsrp</em> in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10–11 hpf should be due to ALPM patterning shift. Overexpressions of <em>scl</em> and <em>lmo2</em> partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10–11 hpf, suggesting RA acts upstream of <em>scl</em> to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing <em>gata4/6</em> whereas the abolished primitive myelopoiesis in <em>gata4/5/6</em> depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down <em>aldh1a2</em>, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing <em>gata4/6</em> inhibits <em>aldh1a2</em> expression whereas depleting <em>gata4/5/6</em> increases <em>aldh1a2</em> expression. The results reveal that RA signaling acts downstream of <em>gata4/5/6</em> to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either <em>cloche</em> embryos or <em>lycat</em> morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of <em>gata4/5/6</em>, upstream of, or parallel to, <em>cloche</em>, and upstream of <em>scl</em>.</p> </div

RA restricts the formation of anterior hemangioblasts in zebrafish embryos.

<p>All flat-mounted embryos are positioned anterior left and dorsal front. Embryos were treated with vehicle DMSO (A, D, G, J, M), 50 nM RA from 1–2-cell stage to 14 hpf (B, E, H, K, N) or 250 nM RA from 10 to 11 hpf (C, F, I, L, O), respectively. They were then examined for expressions of <i>pu.1</i> (A–C), <i>scl</i> (D–F), <i>lmo2</i> (G–I), <i>etsrp</i> (J–L), and <i>gata2</i> (M–O) in the rostral end of ALPM at 14 hpf by whole mount in <i>situ</i> hybridization. Expression of <i>myoD</i> in somites is used for staging. Bracket indicates the location of RBI. The number shown in the lower left-hand corner of each panel is the number of embryos exhibiting the typical phenotype shown in the panel to the number of embryos totally observed.</p

DEAB cannot rescue the defective primitive myelopoiesis in <i>cloche</i> or <i>lycat</i> knockdown embryos.

<p>All embryos are positioned anterior left and lateral front. Wild type siblings (A, G), <i>cloche</i> (B, H) and the embryos microinjected with <i>lycat</i>-MO at 1–2-cell stage (E, K) were treated with vehicle DMSO whereas <i>cloche</i> (C, I), <i>cloche</i> siblings (D, J) and <i>lycat</i>-MO knockdown (F, L) embryos were treated with 10 µM DEAB from 1–2-cell stage until 26 hpf. They were then examined for expressions of myeloid markers <i>lcp1</i> (A–F) and <i>mpx</i> (G–L) at 26 hpf by whole mount <i>in situ</i> hybridization. The number shown in the lower left-hand corner of each panel is the number of embryos exhibiting the typical phenotype shown in the panel to the number of embryos totally observed.</p

Overexpressions of <i>scl</i> and <i>lmo2</i> into RA-treated zebrafish embryos partially rescue the defective primitive myelopoiesis.

<p>Both flat-mounted embryos (A–H) and whole-mounted embryos (I–P) are positioned anterior left and dorsal front (A–H) or lateral front (I–P). Embryos were treated with vehicle DMSO (A, E, I, M), 250 nM RA during 10 to 11 hpf (B, F, J, N), or microinjected with <i>scl</i> and <i>lmo2</i> mRNA at 1–2-cell stage (C, G, K, O), or microinjected with <i>scl</i> and <i>lmo2</i> mRNA at 1–2-cell stage and then treated with 250 nM RA during 10 to 11 hpf (D, H, L, P), respectively. They were then examined for expressions of hemangioblast markers <i>etsrp</i> (A–D) and <i>gata2</i> (E–H) at 14 hpf, and myeloid markers <i>lcp1</i> (I–L) and <i>mpx</i> (M–P) at 24 hpf by whole mount in <i>situ</i> hybridization. Expression of <i>myoD</i> in somites was used for staging (A–H). Bracket indicates the location of RBI (A, B, E, F), and ALPM (C, D, G, H). The number shown in the lower left-hand corner of each panel is the number of embryos exhibiting the typical phenotype shown in the panel to the number of embryos totally observed.</p

<i>aldh1a2</i> is one of downstream target genes of <i>gata4/5/6</i> to affect zebrafish primitive myelopoiesis.

<p>Embryos are positioned animal pole top and ventral front (A–I), anterior top and dorsal front (J–O), and anterior left and lateral front (R–W). Embryos were microinjected with control MO (A, D, G, J, M, R, U), <i>gata5</i>-MO plus <i>gata6</i>-MO (B, E, H, K, N), <i>gata4</i> mRNA plus <i>gata6</i> mRNA (C, F, I, L, O), <i>gata5</i>-MO and <i>gata6</i>-MO plus control MO (S, V), <i>gata5</i>-MO and <i>gata6</i>-MO plus <i>aldh1a2</i> MO (T, W) at 1–2-cell stage, respectively. They were then examined for expressions of <i>aldh1a2</i> at 5 hpf (A–C), 7 hpf (D–F), 9 hpf (G–I), 11 hpf (J–L) and 13 hpf (M–O), <i>lcp1</i> (R–T) and <i>mpx</i> at 24 hpf (U–W) by whole mount <i>in situ</i> hybridization, respectively. qRT-PCR was performed to confirm the relative expression level changes of <i>aldh1a2</i> in <i>gata4/5/6</i> depleted embryos (P) or in <i>gata4/6</i> overexpressed embryos (Q) at 5, 7, 9, 11, 13 hpf, and those of <i>lcp1</i> and <i>mpx</i> at 24 hpf (X). The number shown in the lower left-hand corner of each panel (R–W) is the number of embryos exhibiting the typical phenotype shown in the panel to the number of embryos totally observed. *: P<0.05; **: P<0.01.</p