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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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The loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line correlates with tp53 activity and oxidative stress in skin.(A–D) Test of tp53-dependency on the loss of NTR-hKikGR+ fluorescent signals by tp53 morpholino injection. (E) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in tp53 morphants treated with or without Met. (F–K) Test of oxidative status-dependency on the loss of NTR-hKikGR+ fluorescent signals by incubation with various concentrations of L-NAC as an anti-oxidant. (L) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in killer line embryos treated with Met and/or L-NAC. The cell number is presented as the mean±S.D. Different letters above the error bars indicate significant differences, as tested by one-way ANOVA with Tukey's pair-wise comparison method. L-NAC, N-acetyl-L-cysteine.
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pone-0020654-g006: The loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line correlates with tp53 activity and oxidative stress in skin.(A–D) Test of tp53-dependency on the loss of NTR-hKikGR+ fluorescent signals by tp53 morpholino injection. (E) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in tp53 morphants treated with or without Met. (F–K) Test of oxidative status-dependency on the loss of NTR-hKikGR+ fluorescent signals by incubation with various concentrations of L-NAC as an anti-oxidant. (L) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in killer line embryos treated with Met and/or L-NAC. The cell number is presented as the mean±S.D. Different letters above the error bars indicate significant differences, as tested by one-way ANOVA with Tukey's pair-wise comparison method. L-NAC, N-acetyl-L-cysteine.

Mentions: To investigate if NTR/Met-mediated skin apoptosis associates with tp53 activity and oxidative stress protein expression level of tp53 was knocked down by morpholino injection. Embryos were then incubated with 10 mM Met from 24 hpf onwards to trigger skin ablation. Compared to the Met-untreated killer line embryos (2426±693 mm−2, Figs. 6A, 6E) or tp53-MO injected killer line embryos (2324±627 mm−2, Figs. 6B, 6E), the loss of NTR-hKikGR+ cells in Met-treated killer line embryos (312±332 mm−2, Figs. 6C, 6E) was greatly attenuated by the blocking of tp53 expression (1956±743 mm−2, Figs. 6D, 6E). The impact of oxidative stress on skin cell death in Met-treated embryos was then assessed. Compared to Met-treated killer line embryos (226±186 mm−2, Fig. 6G, 6L), the loss of NTR-hKikGR+ cells significantly attenuated in a dose-dependent manner after incubation with the anti-oxidative agent N-acetyl-L-cysteine (L-NAC) from 10 to 100 µM to scavenge the oxidative stress (Figs. 6H–6K). These results indicated that elevated oxidative stress mediates the loss of NTR-hKikGR+ signals and skin cell death in Met-treated killer line embryos in a tp53-dependent manner.


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)

The loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line correlates with tp53 activity and oxidative stress in skin.(A–D) Test of tp53-dependency on the loss of NTR-hKikGR+ fluorescent signals by tp53 morpholino injection. (E) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in tp53 morphants treated with or without Met. (F–K) Test of oxidative status-dependency on the loss of NTR-hKikGR+ fluorescent signals by incubation with various concentrations of L-NAC as an anti-oxidant. (L) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in killer line embryos treated with Met and/or L-NAC. The cell number is presented as the mean±S.D. Different letters above the error bars indicate significant differences, as tested by one-way ANOVA with Tukey's pair-wise comparison method. L-NAC, N-acetyl-L-cysteine.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105106&req=5

pone-0020654-g006: The loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line correlates with tp53 activity and oxidative stress in skin.(A–D) Test of tp53-dependency on the loss of NTR-hKikGR+ fluorescent signals by tp53 morpholino injection. (E) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in tp53 morphants treated with or without Met. (F–K) Test of oxidative status-dependency on the loss of NTR-hKikGR+ fluorescent signals by incubation with various concentrations of L-NAC as an anti-oxidant. (L) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in killer line embryos treated with Met and/or L-NAC. The cell number is presented as the mean±S.D. Different letters above the error bars indicate significant differences, as tested by one-way ANOVA with Tukey's pair-wise comparison method. L-NAC, N-acetyl-L-cysteine.
Mentions: To investigate if NTR/Met-mediated skin apoptosis associates with tp53 activity and oxidative stress protein expression level of tp53 was knocked down by morpholino injection. Embryos were then incubated with 10 mM Met from 24 hpf onwards to trigger skin ablation. Compared to the Met-untreated killer line embryos (2426±693 mm−2, Figs. 6A, 6E) or tp53-MO injected killer line embryos (2324±627 mm−2, Figs. 6B, 6E), the loss of NTR-hKikGR+ cells in Met-treated killer line embryos (312±332 mm−2, Figs. 6C, 6E) was greatly attenuated by the blocking of tp53 expression (1956±743 mm−2, Figs. 6D, 6E). The impact of oxidative stress on skin cell death in Met-treated embryos was then assessed. Compared to Met-treated killer line embryos (226±186 mm−2, Fig. 6G, 6L), the loss of NTR-hKikGR+ cells significantly attenuated in a dose-dependent manner after incubation with the anti-oxidative agent N-acetyl-L-cysteine (L-NAC) from 10 to 100 µM to scavenge the oxidative stress (Figs. 6H–6K). These results indicated that elevated oxidative stress mediates the loss of NTR-hKikGR+ signals and skin cell death in Met-treated killer line embryos in a tp53-dependent manner.

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