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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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Apoptotic cell death mediates the loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line.krt4 promoter suppression does not mediate loss of NTR-hKikGR+ fluorescence in Met-treated killer line because downregulation of nlsEGFP+ fluorescence signal intensity (A and B) or nlsEGFP+ cells (C) in Tg(krt4:nlsEGFP)cy34 did not occur with or without Met treatment. (D–F) Western blot analysis showed that apoptotic cell death mediates the loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line embryos because the relative expression levels of NTR-hKikGR fusion protein (D), pan-cytokeratin (E) and pro-caspase 3 (F) greatly reduced in Met-treated embryos. Note that the relative intensities of the cleavaged caspase 3-immunoreactive signals greatly increased in the Met-treated killer line (F). (G–H) Plastic sections at 2 µm thickness show the greatly compromised superficial skin integrity in Met-treated killer line embryos (arrow). Whole-mount TUNEL assay demonstrated significantly enhanced cell death signals in Met treated killer line embryos (J) compared to the untreated group (I). The cell number is presented as the mean±S.D. Student's t-test was used to make statistical comparisons between untreated (−Met) and Met-treated (+Met) killer lines. Met, metrodinazole.
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pone-0020654-g004: Apoptotic cell death mediates the loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line.krt4 promoter suppression does not mediate loss of NTR-hKikGR+ fluorescence in Met-treated killer line because downregulation of nlsEGFP+ fluorescence signal intensity (A and B) or nlsEGFP+ cells (C) in Tg(krt4:nlsEGFP)cy34 did not occur with or without Met treatment. (D–F) Western blot analysis showed that apoptotic cell death mediates the loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line embryos because the relative expression levels of NTR-hKikGR fusion protein (D), pan-cytokeratin (E) and pro-caspase 3 (F) greatly reduced in Met-treated embryos. Note that the relative intensities of the cleavaged caspase 3-immunoreactive signals greatly increased in the Met-treated killer line (F). (G–H) Plastic sections at 2 µm thickness show the greatly compromised superficial skin integrity in Met-treated killer line embryos (arrow). Whole-mount TUNEL assay demonstrated significantly enhanced cell death signals in Met treated killer line embryos (J) compared to the untreated group (I). The cell number is presented as the mean±S.D. Student's t-test was used to make statistical comparisons between untreated (−Met) and Met-treated (+Met) killer lines. Met, metrodinazole.

Mentions: This study hypothesized the following reasons for the loss of NTR-hKikGR+ signals: (1) activated Met suppressed the krt4 promoter and blocked the expression of NTR-hKikGR+ fusion protein; (2) activated Met caused redistribution of NTR-hKikGR+ fusion protein back to the cytoplasm compartment causing it to lose its aggregated, high fluorescent nature; (3) activated Met triggered cell apoptosis in skin. The lack of significant difference in nlsEGFP+ cells between untreated (2365±124 mm−2, n = 22) and Met-treated Tg(krt4:nlsEGFP)cy34 (2372±177 mm−2, n = 17) ruled out the hypothesis that the same krt4 promoter also drives the nlsEGFP reporter gene (Figs. 4A–4C). Western blot analysis showed that the relative amount of NTR-hKikGR fusion protein greatly reduced after Met treatment, indicating that the redistribution of NTR-hKikGR+ proteins was also not feasible (Fig. 4D). The loss of pan-cytokeratin provided further evidence of the death of skin cells triggered by activated Met (Fig. 4E). To examine whether Met-induced cell death occurred via cell apoptosis, the expression of an apoptosis downstream effector of caspase 3 was analyzed by Western blot. Results showed a significant reduction in pro-caspase 3 signals and a sharp increase in activated caspase 3 signals in Met-treated killer lines (Fig. 4F). Histological examination demonstrated strong apoptotic skin death in Met-treated killer line embryos. Compared to the untreated groups (Figs. 4G and 4I), the integrity of the NTR-hKikGR-expressing EVL greatly reduced after exposure of the killer line embryos to short term 10 mM Met incubation (Fig. 4H). To detect the apoptotic event in situ, killer line embryos aged at 24 hpf were treated with Met and TUNEL assay was performed on embryos at 48 hpf. Results showed that TUNEL+ cells in Met-treated killer line embryos (3548±341 mm−2, n = 18, Fig. 4J) have a much higher cell densities than in untreated killer line embryos (1098±668 mm−2, n = 19, p<0.001, Fig. 4I). This result supported the theory that apoptosis mediates loss of NTR-hKikGR+ signal in Met-treated killer line and this can be used as a real marker to report skin apoptosis in vivo.


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)

Apoptotic cell death mediates the loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line.krt4 promoter suppression does not mediate loss of NTR-hKikGR+ fluorescence in Met-treated killer line because downregulation of nlsEGFP+ fluorescence signal intensity (A and B) or nlsEGFP+ cells (C) in Tg(krt4:nlsEGFP)cy34 did not occur with or without Met treatment. (D–F) Western blot analysis showed that apoptotic cell death mediates the loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line embryos because the relative expression levels of NTR-hKikGR fusion protein (D), pan-cytokeratin (E) and pro-caspase 3 (F) greatly reduced in Met-treated embryos. Note that the relative intensities of the cleavaged caspase 3-immunoreactive signals greatly increased in the Met-treated killer line (F). (G–H) Plastic sections at 2 µm thickness show the greatly compromised superficial skin integrity in Met-treated killer line embryos (arrow). Whole-mount TUNEL assay demonstrated significantly enhanced cell death signals in Met treated killer line embryos (J) compared to the untreated group (I). The cell number is presented as the mean±S.D. Student's t-test was used to make statistical comparisons between untreated (−Met) and Met-treated (+Met) killer lines. Met, metrodinazole.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020654-g004: Apoptotic cell death mediates the loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line.krt4 promoter suppression does not mediate loss of NTR-hKikGR+ fluorescence in Met-treated killer line because downregulation of nlsEGFP+ fluorescence signal intensity (A and B) or nlsEGFP+ cells (C) in Tg(krt4:nlsEGFP)cy34 did not occur with or without Met treatment. (D–F) Western blot analysis showed that apoptotic cell death mediates the loss of NTR-hKikGR+ fluorescent signals in Met-treated killer line embryos because the relative expression levels of NTR-hKikGR fusion protein (D), pan-cytokeratin (E) and pro-caspase 3 (F) greatly reduced in Met-treated embryos. Note that the relative intensities of the cleavaged caspase 3-immunoreactive signals greatly increased in the Met-treated killer line (F). (G–H) Plastic sections at 2 µm thickness show the greatly compromised superficial skin integrity in Met-treated killer line embryos (arrow). Whole-mount TUNEL assay demonstrated significantly enhanced cell death signals in Met treated killer line embryos (J) compared to the untreated group (I). The cell number is presented as the mean±S.D. Student's t-test was used to make statistical comparisons between untreated (−Met) and Met-treated (+Met) killer lines. Met, metrodinazole.
Mentions: This study hypothesized the following reasons for the loss of NTR-hKikGR+ signals: (1) activated Met suppressed the krt4 promoter and blocked the expression of NTR-hKikGR+ fusion protein; (2) activated Met caused redistribution of NTR-hKikGR+ fusion protein back to the cytoplasm compartment causing it to lose its aggregated, high fluorescent nature; (3) activated Met triggered cell apoptosis in skin. The lack of significant difference in nlsEGFP+ cells between untreated (2365±124 mm−2, n = 22) and Met-treated Tg(krt4:nlsEGFP)cy34 (2372±177 mm−2, n = 17) ruled out the hypothesis that the same krt4 promoter also drives the nlsEGFP reporter gene (Figs. 4A–4C). Western blot analysis showed that the relative amount of NTR-hKikGR fusion protein greatly reduced after Met treatment, indicating that the redistribution of NTR-hKikGR+ proteins was also not feasible (Fig. 4D). The loss of pan-cytokeratin provided further evidence of the death of skin cells triggered by activated Met (Fig. 4E). To examine whether Met-induced cell death occurred via cell apoptosis, the expression of an apoptosis downstream effector of caspase 3 was analyzed by Western blot. Results showed a significant reduction in pro-caspase 3 signals and a sharp increase in activated caspase 3 signals in Met-treated killer lines (Fig. 4F). Histological examination demonstrated strong apoptotic skin death in Met-treated killer line embryos. Compared to the untreated groups (Figs. 4G and 4I), the integrity of the NTR-hKikGR-expressing EVL greatly reduced after exposure of the killer line embryos to short term 10 mM Met incubation (Fig. 4H). To detect the apoptotic event in situ, killer line embryos aged at 24 hpf were treated with Met and TUNEL assay was performed on embryos at 48 hpf. Results showed that TUNEL+ cells in Met-treated killer line embryos (3548±341 mm−2, n = 18, Fig. 4J) have a much higher cell densities than in untreated killer line embryos (1098±668 mm−2, n = 19, p<0.001, Fig. 4I). This result supported the theory that apoptosis mediates loss of NTR-hKikGR+ signal in Met-treated killer line and this can be used as a real marker to report skin apoptosis in vivo.

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