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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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Administration of Met caused the killer line to lose the NTR-hKikGR+ fluorescent signals.(A–D) The ontogenic expression of NTR-hKikGR fusion protein in killer line embryos aged from 24 hpf to 96 hpf. (E–H) Consecutive incubation of killer line embryos with 10 mM Met, from 24 hpf to 96 hpf, caused the NTR-hKikGR+ fluorescent signals to gradually diminish by 48 hpf, totally disappear by 72 hpf, and show pericardial edema in Met-treated embryos by 96 hpf. (I–L) If Met was withdrawn and replaced with fresh fish water from 48 hpf onwards, the NTR-hKikGR+ fluorescent signals partially restored by 96 hpf. Scale bar = 100 µm in L (applies to A–L). The experimental design and work flow are illustrated at the top panel. Met, metrodinazole.
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pone-0020654-g003: Administration of Met caused the killer line to lose the NTR-hKikGR+ fluorescent signals.(A–D) The ontogenic expression of NTR-hKikGR fusion protein in killer line embryos aged from 24 hpf to 96 hpf. (E–H) Consecutive incubation of killer line embryos with 10 mM Met, from 24 hpf to 96 hpf, caused the NTR-hKikGR+ fluorescent signals to gradually diminish by 48 hpf, totally disappear by 72 hpf, and show pericardial edema in Met-treated embryos by 96 hpf. (I–L) If Met was withdrawn and replaced with fresh fish water from 48 hpf onwards, the NTR-hKikGR+ fluorescent signals partially restored by 96 hpf. Scale bar = 100 µm in L (applies to A–L). The experimental design and work flow are illustrated at the top panel. Met, metrodinazole.

Mentions: To validate whether the NTR/Met-based cell ablation system works properly in the killer line, killer line embryos were incubated with 10 mM Met from 24 to 48 hpf to ablate skin cells. Treatment was stopped by washing out Met after 48 hpf (Fig. 3, protocol indicated at upper panel). The NTR-hKikGR+ signals were monitored and photographed every 24 hours until 96 hpf. In the absence of Met (untreated), NTR-hKikGR+ fluorescent signals were robust throughout the entire experiment (Figs. 3A–3D). In the presence of Met, the NTR-hKikGR+ signals greatly diminished, and pericardial edema appeared at 96 hpf (Figs. 3E–3H). If Met was withdrawn after 48 hpf, the diminished NTR-hKikGR+ signals partially restored at 96 hpf (Figs. 3I–3L).


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)

Administration of Met caused the killer line to lose the NTR-hKikGR+ fluorescent signals.(A–D) The ontogenic expression of NTR-hKikGR fusion protein in killer line embryos aged from 24 hpf to 96 hpf. (E–H) Consecutive incubation of killer line embryos with 10 mM Met, from 24 hpf to 96 hpf, caused the NTR-hKikGR+ fluorescent signals to gradually diminish by 48 hpf, totally disappear by 72 hpf, and show pericardial edema in Met-treated embryos by 96 hpf. (I–L) If Met was withdrawn and replaced with fresh fish water from 48 hpf onwards, the NTR-hKikGR+ fluorescent signals partially restored by 96 hpf. Scale bar = 100 µm in L (applies to A–L). The experimental design and work flow are illustrated at the top panel. Met, metrodinazole.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105106&req=5

pone-0020654-g003: Administration of Met caused the killer line to lose the NTR-hKikGR+ fluorescent signals.(A–D) The ontogenic expression of NTR-hKikGR fusion protein in killer line embryos aged from 24 hpf to 96 hpf. (E–H) Consecutive incubation of killer line embryos with 10 mM Met, from 24 hpf to 96 hpf, caused the NTR-hKikGR+ fluorescent signals to gradually diminish by 48 hpf, totally disappear by 72 hpf, and show pericardial edema in Met-treated embryos by 96 hpf. (I–L) If Met was withdrawn and replaced with fresh fish water from 48 hpf onwards, the NTR-hKikGR+ fluorescent signals partially restored by 96 hpf. Scale bar = 100 µm in L (applies to A–L). The experimental design and work flow are illustrated at the top panel. Met, metrodinazole.
Mentions: To validate whether the NTR/Met-based cell ablation system works properly in the killer line, killer line embryos were incubated with 10 mM Met from 24 to 48 hpf to ablate skin cells. Treatment was stopped by washing out Met after 48 hpf (Fig. 3, protocol indicated at upper panel). The NTR-hKikGR+ signals were monitored and photographed every 24 hours until 96 hpf. In the absence of Met (untreated), NTR-hKikGR+ fluorescent signals were robust throughout the entire experiment (Figs. 3A–3D). In the presence of Met, the NTR-hKikGR+ signals greatly diminished, and pericardial edema appeared at 96 hpf (Figs. 3E–3H). If Met was withdrawn after 48 hpf, the diminished NTR-hKikGR+ signals partially restored at 96 hpf (Figs. 3I–3L).

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