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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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Functional validation of potential apoptosis modulators in the living killer line embryos by crossing transgenic lines or transient plasmid DNA injection.(A–H) The NTR-hKikGR+ fluorescent signals in embryos derived from crossing of killer line with testing line carrying human constitutively active Akt1 (myrAkt1) (C, D), mouse constitutively active Stat3 (Stat3) (E, F), or HPV16 E6 (E6) transgenes (G, H). (I–N) NTR-hKikGR+ fluorescent signals in killer line embryos after injection with plasmids carrying myrAkt1 (I, J), Stat3 (K, L), or E6 transgenes (M, N). (O) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in embryos derived from stable line assay (black bars) or transient assay (red bars). The cell number is presented as the mean±S.D. Different letters above the error bars indicate significant differences as tested using one-way ANOVA with Tukey's pair-wise comparison method. Met, metrodinazole.
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pone-0020654-g008: Functional validation of potential apoptosis modulators in the living killer line embryos by crossing transgenic lines or transient plasmid DNA injection.(A–H) The NTR-hKikGR+ fluorescent signals in embryos derived from crossing of killer line with testing line carrying human constitutively active Akt1 (myrAkt1) (C, D), mouse constitutively active Stat3 (Stat3) (E, F), or HPV16 E6 (E6) transgenes (G, H). (I–N) NTR-hKikGR+ fluorescent signals in killer line embryos after injection with plasmids carrying myrAkt1 (I, J), Stat3 (K, L), or E6 transgenes (M, N). (O) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in embryos derived from stable line assay (black bars) or transient assay (red bars). The cell number is presented as the mean±S.D. Different letters above the error bars indicate significant differences as tested using one-way ANOVA with Tukey's pair-wise comparison method. Met, metrodinazole.

Mentions: The successful generation of a killer line which triggers apoptosis in the superficial skin layer provided the opportunity to test whether it is possible to validate potential apoptosis modulators in living zebrafish skin. Using rapid plasmid construction by Gateway recombination and germ line transmission by Tol2-mediated transgenic technology, three testing lines were created which overexpressed human constitutively active Akt1 (myrAkt1), mouse constitutively active Stat3 (Stat3), or HPV16 E6 (E6) in a superficial skin-specific manner. Previous studies documented the attenuation of UVB-induced apoptosis in skin culture cells or genetic modified mice by the three testing genes [50], [51], [52], [53]. Initially, the stable transmission and expression of the three testing transgenes in their corresponding testing lines were confirmed using PCR genotyping (Fig. S1A) and RT-PCR (Fig. S1B). Western blot analysis was performed on myrAkt1 transgenics (Fig. S1C) and real-time RT-PCR was performed on Stat3 and E6 transgenics (Figs. S1D–E) to validate the functionality of the exogenous transgenes. Detection of the activation of downstream targets confirmed that all three transgenes were functional. In myrAkt1 transgenics, the phosphorylation levels of downstream targets of GSK3α/β and p70S6K significantly elevated (Figs. S1C). The homozygotic killer line was then crossed with hemizygotic testing lines to generate double transgenics, which could be unambiguously identified based on their green skin and green heart appearance. Double transgenic embryos aged at 24 hpf were incubated with 10 mM Met to induce skin ablation and the number of NTR-hKikGR+ skin cells after ablation were calculated and statistically compared at 48 hpf by one-way ANOVA. In the untreated killer line (Fig. 8A) or untreated double lines derived from the crossing of the killer line and testing lines (Figs. 8C, 8E, 8G), the density of NTR-hKikGR+ cells maintained a consistent level of 2561±475 mm−2 (n = 134, Fig. 8O). Upon exposure to prodrug Met, the NTR-hKikGR+ cells in Met-treated killer line sharply declined to only 1% of the untreated control (32±55 mm−2, n = 69, Fig. 8B). However, in myrAkt1 (1104±409 mm−2, n = 16, Fig. 8D), Stat3 (1242±500 mm−2, n = 11, Fig. 8F) or E6 (1744±325 mm−2, n = 6, Fig. 8H) overexpression, the loss of NTR-hKikGR+ cells greatly attenuated when challenged with the NTR/Met ablation system.


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)

Functional validation of potential apoptosis modulators in the living killer line embryos by crossing transgenic lines or transient plasmid DNA injection.(A–H) The NTR-hKikGR+ fluorescent signals in embryos derived from crossing of killer line with testing line carrying human constitutively active Akt1 (myrAkt1) (C, D), mouse constitutively active Stat3 (Stat3) (E, F), or HPV16 E6 (E6) transgenes (G, H). (I–N) NTR-hKikGR+ fluorescent signals in killer line embryos after injection with plasmids carrying myrAkt1 (I, J), Stat3 (K, L), or E6 transgenes (M, N). (O) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in embryos derived from stable line assay (black bars) or transient assay (red bars). The cell number is presented as the mean±S.D. Different letters above the error bars indicate significant differences as tested using one-way ANOVA with Tukey's pair-wise comparison method. Met, metrodinazole.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020654-g008: Functional validation of potential apoptosis modulators in the living killer line embryos by crossing transgenic lines or transient plasmid DNA injection.(A–H) The NTR-hKikGR+ fluorescent signals in embryos derived from crossing of killer line with testing line carrying human constitutively active Akt1 (myrAkt1) (C, D), mouse constitutively active Stat3 (Stat3) (E, F), or HPV16 E6 (E6) transgenes (G, H). (I–N) NTR-hKikGR+ fluorescent signals in killer line embryos after injection with plasmids carrying myrAkt1 (I, J), Stat3 (K, L), or E6 transgenes (M, N). (O) Statistical comparison of the relative number of NTR-hKikGR+ fluorescent signals in embryos derived from stable line assay (black bars) or transient assay (red bars). The cell number is presented as the mean±S.D. Different letters above the error bars indicate significant differences as tested using one-way ANOVA with Tukey's pair-wise comparison method. Met, metrodinazole.
Mentions: The successful generation of a killer line which triggers apoptosis in the superficial skin layer provided the opportunity to test whether it is possible to validate potential apoptosis modulators in living zebrafish skin. Using rapid plasmid construction by Gateway recombination and germ line transmission by Tol2-mediated transgenic technology, three testing lines were created which overexpressed human constitutively active Akt1 (myrAkt1), mouse constitutively active Stat3 (Stat3), or HPV16 E6 (E6) in a superficial skin-specific manner. Previous studies documented the attenuation of UVB-induced apoptosis in skin culture cells or genetic modified mice by the three testing genes [50], [51], [52], [53]. Initially, the stable transmission and expression of the three testing transgenes in their corresponding testing lines were confirmed using PCR genotyping (Fig. S1A) and RT-PCR (Fig. S1B). Western blot analysis was performed on myrAkt1 transgenics (Fig. S1C) and real-time RT-PCR was performed on Stat3 and E6 transgenics (Figs. S1D–E) to validate the functionality of the exogenous transgenes. Detection of the activation of downstream targets confirmed that all three transgenes were functional. In myrAkt1 transgenics, the phosphorylation levels of downstream targets of GSK3α/β and p70S6K significantly elevated (Figs. S1C). The homozygotic killer line was then crossed with hemizygotic testing lines to generate double transgenics, which could be unambiguously identified based on their green skin and green heart appearance. Double transgenic embryos aged at 24 hpf were incubated with 10 mM Met to induce skin ablation and the number of NTR-hKikGR+ skin cells after ablation were calculated and statistically compared at 48 hpf by one-way ANOVA. In the untreated killer line (Fig. 8A) or untreated double lines derived from the crossing of the killer line and testing lines (Figs. 8C, 8E, 8G), the density of NTR-hKikGR+ cells maintained a consistent level of 2561±475 mm−2 (n = 134, Fig. 8O). Upon exposure to prodrug Met, the NTR-hKikGR+ cells in Met-treated killer line sharply declined to only 1% of the untreated control (32±55 mm−2, n = 69, Fig. 8B). However, in myrAkt1 (1104±409 mm−2, n = 16, Fig. 8D), Stat3 (1242±500 mm−2, n = 11, Fig. 8F) or E6 (1744±325 mm−2, n = 6, Fig. 8H) overexpression, the loss of NTR-hKikGR+ cells greatly attenuated when challenged with the NTR/Met ablation system.

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