Limits...
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.

Show MeSH

Related in: MedlinePlus

Establishment of Tg(krt4:NTR-hKikGR)cy17 killer line.(A) The work flow to conditionally ablate zebrafish skin using NTR/Met-mediated system. The superficial skin-specific krt4 promoter controls NTR-hKikGR fusion protein. Tol2 transposon elements flank the whole transgene cassette and enhance the germ-line transmission rate. Dimerization of NTR-hKikGR transfers electrons from NADH/NADPH to Met prodrug. Activated Met crosslinks DNA and specifically triggers apoptotic death in skin. (B) The killer line carrying the krt4:NTR-hKikGR transgene was crossed with several testing lines which overexpress human constitutively active myrAkt1 (myrAkt1), mouse constitutively active Stat3 (Stat3), or HPV16 E6 (E6) genes. The double transgenics were then subjected to Met incubation to assay the potential function of apoptosis modulators. (C–F) The ontogenic expression pattern of NTR-hKikGR fusion protein in killer line aged from 24 to 96 hpf. (G) The living fluorescent signals detected in double transgenics from the crossing of the killer line and Tg(krt4:nlsEGFP)cy34 aged at 72 hpf show that the NTR-hKikGR+ signals (yellow) aggregated adjacently to the nlsEGFP+ (green) skin nucleus. (H) Model to illustrate the spatial distribution of NTR-hKikGR fusion protein in zebrafish embryo skin. (I) The native hKikGR protein displays cytoplasmic distribution pattern in the skeletal muscle of Tg(mylz2:hKikGR). The relative position of the captured image is highlighted at the upper right corner. (J) The NTR-hKikGR fusion protein aggregated in skeletal muscle of Tg(mylz2:NTR-hKikGR). EVL, enveloping layer; BEL, basal epidermal layer; Met, metrodinazole.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105106&req=5

pone-0020654-g002: Establishment of Tg(krt4:NTR-hKikGR)cy17 killer line.(A) The work flow to conditionally ablate zebrafish skin using NTR/Met-mediated system. The superficial skin-specific krt4 promoter controls NTR-hKikGR fusion protein. Tol2 transposon elements flank the whole transgene cassette and enhance the germ-line transmission rate. Dimerization of NTR-hKikGR transfers electrons from NADH/NADPH to Met prodrug. Activated Met crosslinks DNA and specifically triggers apoptotic death in skin. (B) The killer line carrying the krt4:NTR-hKikGR transgene was crossed with several testing lines which overexpress human constitutively active myrAkt1 (myrAkt1), mouse constitutively active Stat3 (Stat3), or HPV16 E6 (E6) genes. The double transgenics were then subjected to Met incubation to assay the potential function of apoptosis modulators. (C–F) The ontogenic expression pattern of NTR-hKikGR fusion protein in killer line aged from 24 to 96 hpf. (G) The living fluorescent signals detected in double transgenics from the crossing of the killer line and Tg(krt4:nlsEGFP)cy34 aged at 72 hpf show that the NTR-hKikGR+ signals (yellow) aggregated adjacently to the nlsEGFP+ (green) skin nucleus. (H) Model to illustrate the spatial distribution of NTR-hKikGR fusion protein in zebrafish embryo skin. (I) The native hKikGR protein displays cytoplasmic distribution pattern in the skeletal muscle of Tg(mylz2:hKikGR). The relative position of the captured image is highlighted at the upper right corner. (J) The NTR-hKikGR fusion protein aggregated in skeletal muscle of Tg(mylz2:NTR-hKikGR). EVL, enveloping layer; BEL, basal epidermal layer; Met, metrodinazole.

Mentions: To enhance conditional targeted skin ablation in zebrafish, the mutated version of NTR (T41Q/N71S/F124T), the most sensitive version of NTR tested in vitro, was utilized [30]. In order to facilitate the transgenic line screening and monitor the dynamic expression of NTR in real-time, in-frame fused NTR with hKikGR photoconvertiable fluorescent protein at the C-terminus (hereafter, recognized as NTR-hKikGR) was used. The design of the transgenic cassette, mechanism of NTR/Met ablation system, and strategy for screening of apoptosis modulators using the killer/testing line binary system are illustrated in Figs. 2A and 2B. 15 independent lines out of 92 putative founders carrying the krt4:NTR-hKikGR transgene were identified (germ-line transmission rate = 16%). Among all transgenic lines identified, line number 17 (Tg(krt4:NTR-hKikGR)cy17, killer line) displayed the strongest fluorescent signal and exhibited normal skin development, with no evidence of cell toxicity in the absence of prodrug treatment. It was, therefore, selected to generate the homozygotic line for further skin ablation studies. In the killer line embryos, detection of the NTR-hKikGR+ signals first occurred at the 5-somite stage (data not shown). NTR-hKikGR+ signals in the skin gradually up-regulated and finally displayed robust expression in the pharyngeal arch epithelium (Figs. 2C–2F). The fluorescent signal of NTR-hKikGR fusion protein did not evenly distribute in the cytoplasm but aggregated into distinct small spots. These fluorescent dots seemed to localize in specific compartments of skin cells. To clarify the subcellular identity of NTR-hKikGR+ signals, the killer line was crossed with Tg(krt4:nlsEGFP)cy34, whose skin nucleus was highlighted with nlsEGFP fluorescent signals. Results showed that the NTR-hKikGR+ signals were located adjacent to the nuclei of EVL in the compound transgenics (Fig. 2G and also summarized in Fig. 2H). This unique localization of NTR-hKikGR fusion protein is not specific to the skin compartment, since NTR-hKikGR fusion protein demonstrated a similar dotted distribution pattern when driven by a muscle-specific mlyz2 promoter (Fig. 2J).


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)

Establishment of Tg(krt4:NTR-hKikGR)cy17 killer line.(A) The work flow to conditionally ablate zebrafish skin using NTR/Met-mediated system. The superficial skin-specific krt4 promoter controls NTR-hKikGR fusion protein. Tol2 transposon elements flank the whole transgene cassette and enhance the germ-line transmission rate. Dimerization of NTR-hKikGR transfers electrons from NADH/NADPH to Met prodrug. Activated Met crosslinks DNA and specifically triggers apoptotic death in skin. (B) The killer line carrying the krt4:NTR-hKikGR transgene was crossed with several testing lines which overexpress human constitutively active myrAkt1 (myrAkt1), mouse constitutively active Stat3 (Stat3), or HPV16 E6 (E6) genes. The double transgenics were then subjected to Met incubation to assay the potential function of apoptosis modulators. (C–F) The ontogenic expression pattern of NTR-hKikGR fusion protein in killer line aged from 24 to 96 hpf. (G) The living fluorescent signals detected in double transgenics from the crossing of the killer line and Tg(krt4:nlsEGFP)cy34 aged at 72 hpf show that the NTR-hKikGR+ signals (yellow) aggregated adjacently to the nlsEGFP+ (green) skin nucleus. (H) Model to illustrate the spatial distribution of NTR-hKikGR fusion protein in zebrafish embryo skin. (I) The native hKikGR protein displays cytoplasmic distribution pattern in the skeletal muscle of Tg(mylz2:hKikGR). The relative position of the captured image is highlighted at the upper right corner. (J) The NTR-hKikGR fusion protein aggregated in skeletal muscle of Tg(mylz2:NTR-hKikGR). EVL, enveloping layer; BEL, basal epidermal layer; Met, metrodinazole.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020654-g002: Establishment of Tg(krt4:NTR-hKikGR)cy17 killer line.(A) The work flow to conditionally ablate zebrafish skin using NTR/Met-mediated system. The superficial skin-specific krt4 promoter controls NTR-hKikGR fusion protein. Tol2 transposon elements flank the whole transgene cassette and enhance the germ-line transmission rate. Dimerization of NTR-hKikGR transfers electrons from NADH/NADPH to Met prodrug. Activated Met crosslinks DNA and specifically triggers apoptotic death in skin. (B) The killer line carrying the krt4:NTR-hKikGR transgene was crossed with several testing lines which overexpress human constitutively active myrAkt1 (myrAkt1), mouse constitutively active Stat3 (Stat3), or HPV16 E6 (E6) genes. The double transgenics were then subjected to Met incubation to assay the potential function of apoptosis modulators. (C–F) The ontogenic expression pattern of NTR-hKikGR fusion protein in killer line aged from 24 to 96 hpf. (G) The living fluorescent signals detected in double transgenics from the crossing of the killer line and Tg(krt4:nlsEGFP)cy34 aged at 72 hpf show that the NTR-hKikGR+ signals (yellow) aggregated adjacently to the nlsEGFP+ (green) skin nucleus. (H) Model to illustrate the spatial distribution of NTR-hKikGR fusion protein in zebrafish embryo skin. (I) The native hKikGR protein displays cytoplasmic distribution pattern in the skeletal muscle of Tg(mylz2:hKikGR). The relative position of the captured image is highlighted at the upper right corner. (J) The NTR-hKikGR fusion protein aggregated in skeletal muscle of Tg(mylz2:NTR-hKikGR). EVL, enveloping layer; BEL, basal epidermal layer; Met, metrodinazole.
Mentions: To enhance conditional targeted skin ablation in zebrafish, the mutated version of NTR (T41Q/N71S/F124T), the most sensitive version of NTR tested in vitro, was utilized [30]. In order to facilitate the transgenic line screening and monitor the dynamic expression of NTR in real-time, in-frame fused NTR with hKikGR photoconvertiable fluorescent protein at the C-terminus (hereafter, recognized as NTR-hKikGR) was used. The design of the transgenic cassette, mechanism of NTR/Met ablation system, and strategy for screening of apoptosis modulators using the killer/testing line binary system are illustrated in Figs. 2A and 2B. 15 independent lines out of 92 putative founders carrying the krt4:NTR-hKikGR transgene were identified (germ-line transmission rate = 16%). Among all transgenic lines identified, line number 17 (Tg(krt4:NTR-hKikGR)cy17, killer line) displayed the strongest fluorescent signal and exhibited normal skin development, with no evidence of cell toxicity in the absence of prodrug treatment. It was, therefore, selected to generate the homozygotic line for further skin ablation studies. In the killer line embryos, detection of the NTR-hKikGR+ signals first occurred at the 5-somite stage (data not shown). NTR-hKikGR+ signals in the skin gradually up-regulated and finally displayed robust expression in the pharyngeal arch epithelium (Figs. 2C–2F). The fluorescent signal of NTR-hKikGR fusion protein did not evenly distribute in the cytoplasm but aggregated into distinct small spots. These fluorescent dots seemed to localize in specific compartments of skin cells. To clarify the subcellular identity of NTR-hKikGR+ signals, the killer line was crossed with Tg(krt4:nlsEGFP)cy34, whose skin nucleus was highlighted with nlsEGFP fluorescent signals. Results showed that the NTR-hKikGR+ signals were located adjacent to the nuclei of EVL in the compound transgenics (Fig. 2G and also summarized in Fig. 2H). This unique localization of NTR-hKikGR fusion protein is not specific to the skin compartment, since NTR-hKikGR fusion protein demonstrated a similar dotted distribution pattern when driven by a muscle-specific mlyz2 promoter (Fig. 2J).

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