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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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Related in: MedlinePlus

Evaluation of skin ablation by NTR/Met ablation system in adult killer line.The lateral (A) and ventral (B) views of fluorescent appearance of NTR-hKikGR fusion protein in the killer line at adult stage. Some regions like gill operculum (A, B), oral cavity (C), scale (D) and tail fin (E), which are rich in epithelial structures, showed robust fluorescent signals (heighted by arrows). (F–G) Test of the possibility of performing skin ablation in the adult killer line. Treatment of the killer line with 2.5 mM Met for three consecutive days resulted in greatly compromised skin integrity and some detached skin debris (NTR-hKikGR+) in the fish tank. (H–S) Histological assessment of skin integrity in wild-types or killer lines treated with or without 2.5 mM Met. Paraffin sections stained with hematoxylin and eosin showing the serial morphological changes in the regions of superficial skin (H–K), esophagus (L–O) and gill epithelium (P–S). Included for comparison, the normal epidermal histology in superficial skin (H), esophagus (L) and gill (P) before performing skin ablation. To highlight the position of mucous cells, PAS staining (blue color) in paraffin sections derived from skin (H–K) and esophagus (L–O) shown in lower right corners. (T–W) Detection of cell apoptosis in the damaged skin by activated caspase 3 antibody staining on paraffin sections (brown signals). The killer line adults were first incubated with 2.5 mM Met solution for three consecutive days to execute cell ablation and then paraffin sections, at 5 µm intervals, were cut for histological assay or immunohistochemistry. Met, metrodinazole; PAS, Periodic Acid Schiff.
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pone-0020654-g005: Evaluation of skin ablation by NTR/Met ablation system in adult killer line.The lateral (A) and ventral (B) views of fluorescent appearance of NTR-hKikGR fusion protein in the killer line at adult stage. Some regions like gill operculum (A, B), oral cavity (C), scale (D) and tail fin (E), which are rich in epithelial structures, showed robust fluorescent signals (heighted by arrows). (F–G) Test of the possibility of performing skin ablation in the adult killer line. Treatment of the killer line with 2.5 mM Met for three consecutive days resulted in greatly compromised skin integrity and some detached skin debris (NTR-hKikGR+) in the fish tank. (H–S) Histological assessment of skin integrity in wild-types or killer lines treated with or without 2.5 mM Met. Paraffin sections stained with hematoxylin and eosin showing the serial morphological changes in the regions of superficial skin (H–K), esophagus (L–O) and gill epithelium (P–S). Included for comparison, the normal epidermal histology in superficial skin (H), esophagus (L) and gill (P) before performing skin ablation. To highlight the position of mucous cells, PAS staining (blue color) in paraffin sections derived from skin (H–K) and esophagus (L–O) shown in lower right corners. (T–W) Detection of cell apoptosis in the damaged skin by activated caspase 3 antibody staining on paraffin sections (brown signals). The killer line adults were first incubated with 2.5 mM Met solution for three consecutive days to execute cell ablation and then paraffin sections, at 5 µm intervals, were cut for histological assay or immunohistochemistry. Met, metrodinazole; PAS, Periodic Acid Schiff.

Mentions: In addition to embryos, skin ablation in adult zebrafish was also investigated. At adult stage, the killer line continued to express strong NTR-hKikGR+ signals specifically in the skin tissues. Some regions like the gill operculum (Figs. 5A and 5B), oral cavity (Fig. 5C), scales (Fig. 5D) and tail fin fold (Fig. 5E), which are rich in epithelial folding, displayed robust NTR-hKikGR+ fluorescent signals (indicated by arrows). Compared to embryos, the adult killer line displayed higher efficacy of NTR/Met-ablation system. Administration of Met concentration higher than 2.5 mM was too stressful and, therefore, lethal. Compared to the untreated group (Fig. 5F), some NTR-hKikGR+ skin cells detached from the living fish (Fig. 5G) in the killer line after continuous exposure to 2.5 mM Met for three consecutive days. The fish also presented behavioral and physiological evidences of hypoxic exposure, such as difficulty in maintaining upright posture and vertical swimming balance, bleeding, and reduced swimming mobility (Movie S1). To examine the integrity of skin before and after Met-incubation, paraffin sectioning was performed on adult killer lines treated without or with Met. Consistent with findings in embryos, the superficial skin (Fig. 5K), esophagus (Fig. 5O) and gill epithelium (Fig. 5S) of Met-treated killer lines presented significant injuries. In skin and esophagus, the disruption of superficial skin led to the release of cellular content stored in the underlying mucous cells. PAS staining characterized this material. In gill epithelium, formation of pseudobranchia-like structures, characterized by fusion between secondary lamellae, provided evidence of disruption to the epilthelium (Fig. 5S). In contrast, wild-types treated without (Figs. 5H, 5L, 5P) or with (Figs. 5I, 5M, 5Q) Met, or untreated killer lines (Figs. 5J, 5N, 5R) displayed no evidence of skin injuries. Activated caspase 3 immunostaining revealed robust apoptotic activity in the Met-treated killer line (Fig. 5W). In contrast, Met-untreated (Fig. 5T), treated wild-types (Fig. 5U), and Met-untreated killer lines (Fig. 5V) demonstrated minimal activated caspase 3 activity. These results clearly demonstrated that disruption to skin integrity in Met-treated killer line is mediated by apoptotic cell death, and also highlighted the specificity and low leakage of the NTR/Met-skin ablation system when applied in living zebrafish.


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)

Evaluation of skin ablation by NTR/Met ablation system in adult killer line.The lateral (A) and ventral (B) views of fluorescent appearance of NTR-hKikGR fusion protein in the killer line at adult stage. Some regions like gill operculum (A, B), oral cavity (C), scale (D) and tail fin (E), which are rich in epithelial structures, showed robust fluorescent signals (heighted by arrows). (F–G) Test of the possibility of performing skin ablation in the adult killer line. Treatment of the killer line with 2.5 mM Met for three consecutive days resulted in greatly compromised skin integrity and some detached skin debris (NTR-hKikGR+) in the fish tank. (H–S) Histological assessment of skin integrity in wild-types or killer lines treated with or without 2.5 mM Met. Paraffin sections stained with hematoxylin and eosin showing the serial morphological changes in the regions of superficial skin (H–K), esophagus (L–O) and gill epithelium (P–S). Included for comparison, the normal epidermal histology in superficial skin (H), esophagus (L) and gill (P) before performing skin ablation. To highlight the position of mucous cells, PAS staining (blue color) in paraffin sections derived from skin (H–K) and esophagus (L–O) shown in lower right corners. (T–W) Detection of cell apoptosis in the damaged skin by activated caspase 3 antibody staining on paraffin sections (brown signals). The killer line adults were first incubated with 2.5 mM Met solution for three consecutive days to execute cell ablation and then paraffin sections, at 5 µm intervals, were cut for histological assay or immunohistochemistry. Met, metrodinazole; PAS, Periodic Acid Schiff.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020654-g005: Evaluation of skin ablation by NTR/Met ablation system in adult killer line.The lateral (A) and ventral (B) views of fluorescent appearance of NTR-hKikGR fusion protein in the killer line at adult stage. Some regions like gill operculum (A, B), oral cavity (C), scale (D) and tail fin (E), which are rich in epithelial structures, showed robust fluorescent signals (heighted by arrows). (F–G) Test of the possibility of performing skin ablation in the adult killer line. Treatment of the killer line with 2.5 mM Met for three consecutive days resulted in greatly compromised skin integrity and some detached skin debris (NTR-hKikGR+) in the fish tank. (H–S) Histological assessment of skin integrity in wild-types or killer lines treated with or without 2.5 mM Met. Paraffin sections stained with hematoxylin and eosin showing the serial morphological changes in the regions of superficial skin (H–K), esophagus (L–O) and gill epithelium (P–S). Included for comparison, the normal epidermal histology in superficial skin (H), esophagus (L) and gill (P) before performing skin ablation. To highlight the position of mucous cells, PAS staining (blue color) in paraffin sections derived from skin (H–K) and esophagus (L–O) shown in lower right corners. (T–W) Detection of cell apoptosis in the damaged skin by activated caspase 3 antibody staining on paraffin sections (brown signals). The killer line adults were first incubated with 2.5 mM Met solution for three consecutive days to execute cell ablation and then paraffin sections, at 5 µm intervals, were cut for histological assay or immunohistochemistry. Met, metrodinazole; PAS, Periodic Acid Schiff.
Mentions: In addition to embryos, skin ablation in adult zebrafish was also investigated. At adult stage, the killer line continued to express strong NTR-hKikGR+ signals specifically in the skin tissues. Some regions like the gill operculum (Figs. 5A and 5B), oral cavity (Fig. 5C), scales (Fig. 5D) and tail fin fold (Fig. 5E), which are rich in epithelial folding, displayed robust NTR-hKikGR+ fluorescent signals (indicated by arrows). Compared to embryos, the adult killer line displayed higher efficacy of NTR/Met-ablation system. Administration of Met concentration higher than 2.5 mM was too stressful and, therefore, lethal. Compared to the untreated group (Fig. 5F), some NTR-hKikGR+ skin cells detached from the living fish (Fig. 5G) in the killer line after continuous exposure to 2.5 mM Met for three consecutive days. The fish also presented behavioral and physiological evidences of hypoxic exposure, such as difficulty in maintaining upright posture and vertical swimming balance, bleeding, and reduced swimming mobility (Movie S1). To examine the integrity of skin before and after Met-incubation, paraffin sectioning was performed on adult killer lines treated without or with Met. Consistent with findings in embryos, the superficial skin (Fig. 5K), esophagus (Fig. 5O) and gill epithelium (Fig. 5S) of Met-treated killer lines presented significant injuries. In skin and esophagus, the disruption of superficial skin led to the release of cellular content stored in the underlying mucous cells. PAS staining characterized this material. In gill epithelium, formation of pseudobranchia-like structures, characterized by fusion between secondary lamellae, provided evidence of disruption to the epilthelium (Fig. 5S). In contrast, wild-types treated without (Figs. 5H, 5L, 5P) or with (Figs. 5I, 5M, 5Q) Met, or untreated killer lines (Figs. 5J, 5N, 5R) displayed no evidence of skin injuries. Activated caspase 3 immunostaining revealed robust apoptotic activity in the Met-treated killer line (Fig. 5W). In contrast, Met-untreated (Fig. 5T), treated wild-types (Fig. 5U), and Met-untreated killer lines (Fig. 5V) demonstrated minimal activated caspase 3 activity. These results clearly demonstrated that disruption to skin integrity in Met-treated killer line is mediated by apoptotic cell death, and also highlighted the specificity and low leakage of the NTR/Met-skin ablation system when applied in living zebrafish.

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