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Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo.

Chen CF, Chu CY, Chen TH, Lee SJ, Shen CN, Hsiao CD - PLoS ONE (2011)

Bottom Line: Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis.In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling.The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.

ABSTRACT

Background: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish.

Methodology/principal findings: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling.

Conclusion/significance: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

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Skin ablation in Met-treated killer line specifically targeted the EVL and showed no bystander effect on the neighboring cells.Whole-mount immunostaining on killer line embryos aged at 48 hpf showing that the number of epidermal stem basal cells (A and B, stained with p63 antibody), NaRC (C and D, stained with Na,K-ATPase antibody), HRC (E and F, stained with H-ATPase antibody) and MC (G and H, stained with anterior gradient 2 antibody) did not significantly change between untreated (−Met) and treated (+Met) groups. (I–L) Statistical comparisons, using Student's t-test, of the relative cell numbers of p63+ cells, NaRC, HRC and MC between untreated (−Met) and Met-treated (+Met) groups. The cell number is presented as the mean±S.D. NaRC, Na-K-ATPase rich cell; HRC, H-ATPase rich cells; MC, mucous cells; Met, metrodinazole.
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pone-0020654-g007: Skin ablation in Met-treated killer line specifically targeted the EVL and showed no bystander effect on the neighboring cells.Whole-mount immunostaining on killer line embryos aged at 48 hpf showing that the number of epidermal stem basal cells (A and B, stained with p63 antibody), NaRC (C and D, stained with Na,K-ATPase antibody), HRC (E and F, stained with H-ATPase antibody) and MC (G and H, stained with anterior gradient 2 antibody) did not significantly change between untreated (−Met) and treated (+Met) groups. (I–L) Statistical comparisons, using Student's t-test, of the relative cell numbers of p63+ cells, NaRC, HRC and MC between untreated (−Met) and Met-treated (+Met) groups. The cell number is presented as the mean±S.D. NaRC, Na-K-ATPase rich cell; HRC, H-ATPase rich cells; MC, mucous cells; Met, metrodinazole.

Mentions: The bystander effect is a key component in tumor eradication using gene-directed enzyme prodrug therapy. Reportedly, NTR metabolizes another prototype NTR prodrug, CB1954 (5-aziridinyl-2, 4-dinitrobenzamide), to potent alkylating agents which can diffuse and kill non-NTR expressing neighboring cells by a bystander effect [48], [49]. In order to specifically ablate the superficial skin layer, the possible occurrence of the bystander effect was examined. Firstly, killer line embryos at 24 hpf were incubated with 10 mM Met and then fixed at 48 hpf to examine the integrity of other skin derived cells, such as basal epidermal cells/epidermal stem cells (Figs. 7A and 7B), Na,K-ATPase rich cells (which regulate ion homeostasis in zebrafish embryo skin, Figs. 7C and 7D), H-ATPase rich cells (which regulate acid-base and ion homeostasis in zebrafish embryo skin, Figs. 7E and 7F) and mucous cells (which secrete mucous in zebrafish embryo skin, Figs. 7G and 7H) by whole-mount antibody staining with respective antibodies as described in the Materials and Methods. The cell density for each cell type on the trunk region between untreated and Met-treated killer line embryos was quantified. The cell densities for p63+ cells (3402±421 mm−2 VS 3104±204 mm−2, P = 0.068, Fig. 7I), Na,K-ATPase rich cells (380±69 mm−2 VS 401±59 mm−2, P = 0.247, Fig. 7J), H-ATPase rich cells (328±112 mm−2 VS 316±113 mm−2, P = 0.822, Fig. 7K) or mucous cells (229±53 mm−2 VS 228±96 mm−2, P = 0.989, Fig. 7L) displayed no significant differences between killer line embryos treated without or with Met. These results clearly indicated that apoptosis specifically occurs in NTR-hKikGR-expressing cells and the possible bystander killing effect of NTR/Met-mediated skin ablation can be ignored.


Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo.

Chen CF, Chu CY, Chen TH, Lee SJ, Shen CN, Hsiao CD - PLoS ONE (2011)

Skin ablation in Met-treated killer line specifically targeted the EVL and showed no bystander effect on the neighboring cells.Whole-mount immunostaining on killer line embryos aged at 48 hpf showing that the number of epidermal stem basal cells (A and B, stained with p63 antibody), NaRC (C and D, stained with Na,K-ATPase antibody), HRC (E and F, stained with H-ATPase antibody) and MC (G and H, stained with anterior gradient 2 antibody) did not significantly change between untreated (−Met) and treated (+Met) groups. (I–L) Statistical comparisons, using Student's t-test, of the relative cell numbers of p63+ cells, NaRC, HRC and MC between untreated (−Met) and Met-treated (+Met) groups. The cell number is presented as the mean±S.D. NaRC, Na-K-ATPase rich cell; HRC, H-ATPase rich cells; MC, mucous cells; Met, metrodinazole.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105106&req=5

pone-0020654-g007: Skin ablation in Met-treated killer line specifically targeted the EVL and showed no bystander effect on the neighboring cells.Whole-mount immunostaining on killer line embryos aged at 48 hpf showing that the number of epidermal stem basal cells (A and B, stained with p63 antibody), NaRC (C and D, stained with Na,K-ATPase antibody), HRC (E and F, stained with H-ATPase antibody) and MC (G and H, stained with anterior gradient 2 antibody) did not significantly change between untreated (−Met) and treated (+Met) groups. (I–L) Statistical comparisons, using Student's t-test, of the relative cell numbers of p63+ cells, NaRC, HRC and MC between untreated (−Met) and Met-treated (+Met) groups. The cell number is presented as the mean±S.D. NaRC, Na-K-ATPase rich cell; HRC, H-ATPase rich cells; MC, mucous cells; Met, metrodinazole.
Mentions: The bystander effect is a key component in tumor eradication using gene-directed enzyme prodrug therapy. Reportedly, NTR metabolizes another prototype NTR prodrug, CB1954 (5-aziridinyl-2, 4-dinitrobenzamide), to potent alkylating agents which can diffuse and kill non-NTR expressing neighboring cells by a bystander effect [48], [49]. In order to specifically ablate the superficial skin layer, the possible occurrence of the bystander effect was examined. Firstly, killer line embryos at 24 hpf were incubated with 10 mM Met and then fixed at 48 hpf to examine the integrity of other skin derived cells, such as basal epidermal cells/epidermal stem cells (Figs. 7A and 7B), Na,K-ATPase rich cells (which regulate ion homeostasis in zebrafish embryo skin, Figs. 7C and 7D), H-ATPase rich cells (which regulate acid-base and ion homeostasis in zebrafish embryo skin, Figs. 7E and 7F) and mucous cells (which secrete mucous in zebrafish embryo skin, Figs. 7G and 7H) by whole-mount antibody staining with respective antibodies as described in the Materials and Methods. The cell density for each cell type on the trunk region between untreated and Met-treated killer line embryos was quantified. The cell densities for p63+ cells (3402±421 mm−2 VS 3104±204 mm−2, P = 0.068, Fig. 7I), Na,K-ATPase rich cells (380±69 mm−2 VS 401±59 mm−2, P = 0.247, Fig. 7J), H-ATPase rich cells (328±112 mm−2 VS 316±113 mm−2, P = 0.822, Fig. 7K) or mucous cells (229±53 mm−2 VS 228±96 mm−2, P = 0.989, Fig. 7L) displayed no significant differences between killer line embryos treated without or with Met. These results clearly indicated that apoptosis specifically occurs in NTR-hKikGR-expressing cells and the possible bystander killing effect of NTR/Met-mediated skin ablation can be ignored.

Bottom Line: Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis.In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling.The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.

ABSTRACT

Background: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish.

Methodology/principal findings: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling.

Conclusion/significance: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

Show MeSH
Related in: MedlinePlus