Ellis et al., 2016 - Experimental Dissection of Metalloproteinase Inhibition-Mediated and Toxic Effects of Phenanthroline on Zebrafish Development. International Journal of Molecular Sciences   17(9) Full text @ Int. J. Mol. Sci.

Fig. 2

PhN and PhE both disrupt normal development, but embryos are more sensitive to PhN. Embryos were treated with either PhN or PhE at various concentrations from 24 to 48 hours post-fertilization (hpf), and assessed (blinded) for gross morphological defects visually. (A) A normal embryo from the vehicle control group. Embryos treated with PhN representing individuals scored as: (B) "Normal"; (C) "Mild"; and (E) "Severe" phenotypes (pericardial and yolk-sac edema indicated with arrowheads); (D) Summary of results from scoring 44 embryos exposed to vehicle only, 69 exposed to 5 µM PhN, 63 exposed to 10 µM PhN, 62 exposed to 25 µM PhN, and 88 exposed to 40 µM PhN. Embryos exposed to PhE exhibiting: (F) "Normal"; (G) "Mild"; and (H) "Severe" phenotypes; (I) Summary of results from scoring of 60 embryos exposed to vehicle only, 48 exposed to 5 µM PhE, 47 exposed to 10 µM PhE, 51 exposed to 25 µM PhE, and 40 exposed to 40 µM PhE. In addition to tail curvature, necrosis, pericardial and yolk-sac edema seen in embryos exposed to either PhE or PhN, embryos exposed to PhN exhibited loss of pigmentation, craniofacial abnormalities, and absence of intersomitic circulation. Scale bar = 200 µm.

Fig. 3

PhN perturbs development of neural crest-derived pigment cells. (A) A 48 hpf Tg(mitfa:eGFP) embryo exposed from 24 hpf to vehicle control, exhibiting normal distribution of GFP-expressing presumptive pigment cells. At high magnification (A′), the stellate morphology of these cells is apparent as they invade the overlying epidermis; (B) A 48 hpf Tg(mitfa:eGFP) embryo exposed from 24 hpf to 10 µM PhN, in which presumptive pigment cells in the head and anterior trunk have successfully emigrated from the neural tube and become distributed relatively normally, but in which presumptive pigment cells in the posterior tail have failed to emigrate from the neural tube (arrowhead). At high magnification (B′), these cells have a rounded amoeboid appearance. Boxes indicate regions magnified. Scale bar = 200 µm.

Fig. 4

Pigment cells fail to invade epidermis in the presence of PhN. (A) A single focal plane through the head epidermis of a Tg(mitfa:eGFP) embryo exposed from 18 to 24 hpf to 10 µM PhE and stained with anti-laminin (magenta) and anti-GFP (green) showing presumptive pigment cells that have crossed the basal lamina and successfully invaded the epidermis (arrowheads); (B) A comparable single focal plane through the head epidermis of a Tg(mitfa:eGFP) embryo exposed from 18 to 24 hpf to 10 µM PhN, showing presumptive pigment cells (arrowheads) having failed to invade across the basal lamina of the epidermis; (C) A slightly lower magnification view of a Tg(mitfa:eGFP) embryo exposed from 18 to 24 hpf to 10 µM PhN, showing several presumptive pigment cells trapped below the epidermis. Dotted lines indicate the basal lamina. Scale bars are 20 µm.

Fig. 5

PhN disrupts craniofacial neural crest. (A) Epifluorescence micrograph of the head of a 48 hpf Tg(sox10a:eGFP) embryo treated from 24 hpf with 10 µM PhE showing normal development of the craniofacial neural crest (arrowheads indicate the otic vesicle and jaw cartilage). Embryos treated with: (B) 5; (C) 10; (D) 25; and (E) 40 µM PhN, exhibiting progressively more severe defects in these structures. Arrowhead in (E) indicates presumptive pigment cells trapped below the epidermis. Scale bars are 200 µm.

Fig. 6

PhE does not disrupt angiogenesis. Forty-eight hpf Tg(fli1:eGFP) embryos treated from 24 hpf with: (A) vehicle alone; and (B) 10 µM; (C) 25 µM; or (D) 40 µM PhE exhibiting normal trunk vasculature.

Fig. 7

PhN disrupts angiogenesis. Forty-eight hpf Tg(fli1:eGFP) embryos treated from 24 to 48 hpf with either: (A) vehicle alone; or various concentrations of PhN (B-D), illustrating: (B) "Normal"; (C) "Mild"; and (D) "Severe" disruption of angiogenesis. The DLAV is indicated with an arrowhead in (B,C); and examples of disrupted angiogenesis are highlighted with asterisks; (E) Summary of blind scoring of 40 embryos exposed to vehicle alone, 96 embryos exposed to 5 µM PhN, 102 embryos exposed to 10 µM PhN, 91 embryos exposed to 25 µM PhN, and 77 embryos exposed to 40 µM PhN.

Fig. 8

Morpholino knock-down of the aryl hydrocarbon receptor rescues some but not all effects of PhN treatment. Tg(fli1:eGFP) embryos were injected with morpholinos demonstrated to interfere with the expression of AhR2, thereby protecting them from PAH-toxicity, or control morpholinos, and were then exposed to 10 µM PhN from 24 to 48 hpf. (A) Control morphants exhibit typical PAH toxicity effects (tail curvature, necrosis, pericardial and yolk-sac edema), as well as disrupted angiogenesis, loss of pigmentation, and craniofacial defects; (B) Ahr2 morphants exhibit reduced pericardial and yolk-sac edema, but still exhibit loss of pigmentation, craniofacial defects; and (C) disrupted angiogenesis.

Acknowledgments:
ZFIN wishes to thank the journal International Journal of Molecular Sciences for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Int. J. Mol. Sci.