Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstnnju6) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstnnju7), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstnnju6/+ and 14 mstnnju7/+ yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish

Excessive nitrite affects zebrafish valvulogenesis through yielding too much NO signaling.

Sodium nitrite, a common food additive, exists widely not only in the environment but also in our body. Excessive nitrite causes toxicological effects on human health; however, whether it affects vertebrate heart valve development remains unknown. In vertebrates, developmental defects of cardiac valves usually lead to congenital heart disease. To understand the toxic effects of nitrite on valvulogenesis, we exposed zebrafish embryos with different concentrations of sodium nitrite. Our results showed that sodium nitrite caused developmental defects of zebrafish heart dose dependently. It affected zebrafish heart development starting from 36 hpf (hour post fertilization) when heart initiates looping process. Comprehensive analysis on the embryos at 24 hpf and 48 hpf showed that excessive nitrite did not affect blood circulation, vascular network, myocardium and endocardium development. But development of endocardial cells in atrioventricular canal (AVC) of the embryos at 48 hpf was disrupted by too much nitrite, leading to defective formation of primitive valve leaflets at 76 hpf. Consistently, excessive nitrite diminished expressions of valve progenitor markers including bmp4, has2, vcana and notch1b at 48 hpf. Furthermore, 3', 5'-cyclic guanosine monophosphate (cGMP), downstream of nitric oxide (NO) signaling, was increased its level significantly in the embryos exposed with excessive nitrite and microinjection of soluble guanylate cyclase inhibitor ODQ (1H-[1], [2], [4]Oxadiazolo[4,3-a] quinoxalin-1-one), an antagonist of NO signaling, into nitrite-exposed embryos could partly rescue the cardiac valve malformation. Taken together, our results show that excessive nitrite affects early valve leaflet formation by producing too much NO signaling

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

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

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

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 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

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

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