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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

The krt4 promoter can target transgene expression in the superficial skin layer in zebrafish.(A) Plastic section at 2 µm thickness showing that zebrafish skin, at 5 days post-fertilization, consists of an outer enveloping layer (EVL) and inner basal epidermal layer (BEL). The basement membrane is highlighted by the dotted line. (B) Plastic section at 2 µm thickness showing that adult zebrafish (aged at 3 months) skin consists of three major layers, including the superficial cells (arrowhead), middle and basal cells (arrows), and mucous cells (asterisk). (C) BrdU incorporation experiment showing that most skin layers in adult zebrafish are mitotically active. The BrdU+ cells (brown signals) can be detected in most skin layers at adult stage. (D) CK5/6 and (E) p63 antibodies differentially label the superficial skin layer and the putative epidermal stem cells in adult zebrafish skin, respectively. For immunohistochemistry, the 5 µm thick paraffin sections were immunostained with antigen-specific antibodies and visualized with DAB coloring substrate (brown). To visualize the cell morphology, the slides were counterstained with hematoxylin (blue). (F) Whole-mount immunostaining of p63 (red) on Tg(krt4:nlsEGFP)cy34 (green) embryonic yolk aged at 24 hpf, showing that the krt4 promoter targets the outermost EVL. The relative position of the captured image is highlighted at the upper right corner. (G–I) Immunohistochemistry of GFP (brown) on paraffin sections derived from Tg(krt4:nlsEGFP)cy34 aged at 3 months. The positive signals (brown) show that the krt4 promoter targets the superficial layer in the skin (G), esophagus (H) and gill (I) at adult stage.
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pone-0020654-g001: The krt4 promoter can target transgene expression in the superficial skin layer in zebrafish.(A) Plastic section at 2 µm thickness showing that zebrafish skin, at 5 days post-fertilization, consists of an outer enveloping layer (EVL) and inner basal epidermal layer (BEL). The basement membrane is highlighted by the dotted line. (B) Plastic section at 2 µm thickness showing that adult zebrafish (aged at 3 months) skin consists of three major layers, including the superficial cells (arrowhead), middle and basal cells (arrows), and mucous cells (asterisk). (C) BrdU incorporation experiment showing that most skin layers in adult zebrafish are mitotically active. The BrdU+ cells (brown signals) can be detected in most skin layers at adult stage. (D) CK5/6 and (E) p63 antibodies differentially label the superficial skin layer and the putative epidermal stem cells in adult zebrafish skin, respectively. For immunohistochemistry, the 5 µm thick paraffin sections were immunostained with antigen-specific antibodies and visualized with DAB coloring substrate (brown). To visualize the cell morphology, the slides were counterstained with hematoxylin (blue). (F) Whole-mount immunostaining of p63 (red) on Tg(krt4:nlsEGFP)cy34 (green) embryonic yolk aged at 24 hpf, showing that the krt4 promoter targets the outermost EVL. The relative position of the captured image is highlighted at the upper right corner. (G–I) Immunohistochemistry of GFP (brown) on paraffin sections derived from Tg(krt4:nlsEGFP)cy34 aged at 3 months. The positive signals (brown) show that the krt4 promoter targets the superficial layer in the skin (G), esophagus (H) and gill (I) at adult stage.

Mentions: Prior to performing skin ablation, the basic architecture of zebrafish skin was characterized. Consistent with previous studies [43], [44], zebrafish larvae skin consisted of EVL and BEL (Fig. 1A). The sagittal section of fully adult zebrafish skin (age greater than 3 months) was also analyzed. Results showed that the thickness of the skin varies in different positions. The epithelium surrounding the head and jaw was thicker than in other regions (data not shown). In contrast, the epithelium covering the scales was much thinner and usually organized into three cell layers. At higher magnification, cell nuclei of the upper superficial layer were flat and elongated (Fig. 1B, arrow head) while cell nuclei of the middle and basal skin layer appeared round and much larger than those in the superficial layer (Fig. 1B, arrows). High glycoprotein content and positive Periodic Acid Schiff (PAS) staining (data not shown) characterized larger cells, oval in shape and with peripheral cell nuclei, as mucous cells (Fig. 1B, asterisk). Cell morphology suggested the upper skin layer might be the differentiated layer while the middle and basal layers might form the undifferentiated layer. To test this hypothesis, BrdU incorporation experiment was performed to investigate cell proliferation activity in different skin layers of adult zebrafish. Result showed that BrdU+ skin cells widely distributed in the middle and basal layers, while the upper superficial layer displayed very few BrdU+ signals in most cases (Fig. 1C). This result is consistent with the findings obtained from tritiated thymidine injection [2], showing that the middle and basal skin layers have strong cell division/proliferation potential in adult zebrafish. To further clarify the skin differentiation pattern at the molecular level, immunohistochemistry was performed on adult skin to label the putative differentiated skin cells (using CK5/6 antibody) and epidermal stem cells (using p63 antibody). CK5/6 antibody staining strongly labeled the keratinocytes of the superficial skin layer (Fig. 1D) while p63 antibody staining strongly labeled most of the cell nuclei in the basal and middle skin layers (Fig. 1E). The BrdU, CK5/6 and p63 immunoreactive patterns suggested that the superficial skin layer is the differentiated layer while the middle and basal layers are the undifferentiated stem cell layers in 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)

The krt4 promoter can target transgene expression in the superficial skin layer in zebrafish.(A) Plastic section at 2 µm thickness showing that zebrafish skin, at 5 days post-fertilization, consists of an outer enveloping layer (EVL) and inner basal epidermal layer (BEL). The basement membrane is highlighted by the dotted line. (B) Plastic section at 2 µm thickness showing that adult zebrafish (aged at 3 months) skin consists of three major layers, including the superficial cells (arrowhead), middle and basal cells (arrows), and mucous cells (asterisk). (C) BrdU incorporation experiment showing that most skin layers in adult zebrafish are mitotically active. The BrdU+ cells (brown signals) can be detected in most skin layers at adult stage. (D) CK5/6 and (E) p63 antibodies differentially label the superficial skin layer and the putative epidermal stem cells in adult zebrafish skin, respectively. For immunohistochemistry, the 5 µm thick paraffin sections were immunostained with antigen-specific antibodies and visualized with DAB coloring substrate (brown). To visualize the cell morphology, the slides were counterstained with hematoxylin (blue). (F) Whole-mount immunostaining of p63 (red) on Tg(krt4:nlsEGFP)cy34 (green) embryonic yolk aged at 24 hpf, showing that the krt4 promoter targets the outermost EVL. The relative position of the captured image is highlighted at the upper right corner. (G–I) Immunohistochemistry of GFP (brown) on paraffin sections derived from Tg(krt4:nlsEGFP)cy34 aged at 3 months. The positive signals (brown) show that the krt4 promoter targets the superficial layer in the skin (G), esophagus (H) and gill (I) at adult stage.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020654-g001: The krt4 promoter can target transgene expression in the superficial skin layer in zebrafish.(A) Plastic section at 2 µm thickness showing that zebrafish skin, at 5 days post-fertilization, consists of an outer enveloping layer (EVL) and inner basal epidermal layer (BEL). The basement membrane is highlighted by the dotted line. (B) Plastic section at 2 µm thickness showing that adult zebrafish (aged at 3 months) skin consists of three major layers, including the superficial cells (arrowhead), middle and basal cells (arrows), and mucous cells (asterisk). (C) BrdU incorporation experiment showing that most skin layers in adult zebrafish are mitotically active. The BrdU+ cells (brown signals) can be detected in most skin layers at adult stage. (D) CK5/6 and (E) p63 antibodies differentially label the superficial skin layer and the putative epidermal stem cells in adult zebrafish skin, respectively. For immunohistochemistry, the 5 µm thick paraffin sections were immunostained with antigen-specific antibodies and visualized with DAB coloring substrate (brown). To visualize the cell morphology, the slides were counterstained with hematoxylin (blue). (F) Whole-mount immunostaining of p63 (red) on Tg(krt4:nlsEGFP)cy34 (green) embryonic yolk aged at 24 hpf, showing that the krt4 promoter targets the outermost EVL. The relative position of the captured image is highlighted at the upper right corner. (G–I) Immunohistochemistry of GFP (brown) on paraffin sections derived from Tg(krt4:nlsEGFP)cy34 aged at 3 months. The positive signals (brown) show that the krt4 promoter targets the superficial layer in the skin (G), esophagus (H) and gill (I) at adult stage.
Mentions: Prior to performing skin ablation, the basic architecture of zebrafish skin was characterized. Consistent with previous studies [43], [44], zebrafish larvae skin consisted of EVL and BEL (Fig. 1A). The sagittal section of fully adult zebrafish skin (age greater than 3 months) was also analyzed. Results showed that the thickness of the skin varies in different positions. The epithelium surrounding the head and jaw was thicker than in other regions (data not shown). In contrast, the epithelium covering the scales was much thinner and usually organized into three cell layers. At higher magnification, cell nuclei of the upper superficial layer were flat and elongated (Fig. 1B, arrow head) while cell nuclei of the middle and basal skin layer appeared round and much larger than those in the superficial layer (Fig. 1B, arrows). High glycoprotein content and positive Periodic Acid Schiff (PAS) staining (data not shown) characterized larger cells, oval in shape and with peripheral cell nuclei, as mucous cells (Fig. 1B, asterisk). Cell morphology suggested the upper skin layer might be the differentiated layer while the middle and basal layers might form the undifferentiated layer. To test this hypothesis, BrdU incorporation experiment was performed to investigate cell proliferation activity in different skin layers of adult zebrafish. Result showed that BrdU+ skin cells widely distributed in the middle and basal layers, while the upper superficial layer displayed very few BrdU+ signals in most cases (Fig. 1C). This result is consistent with the findings obtained from tritiated thymidine injection [2], showing that the middle and basal skin layers have strong cell division/proliferation potential in adult zebrafish. To further clarify the skin differentiation pattern at the molecular level, immunohistochemistry was performed on adult skin to label the putative differentiated skin cells (using CK5/6 antibody) and epidermal stem cells (using p63 antibody). CK5/6 antibody staining strongly labeled the keratinocytes of the superficial skin layer (Fig. 1D) while p63 antibody staining strongly labeled most of the cell nuclei in the basal and middle skin layers (Fig. 1E). The BrdU, CK5/6 and p63 immunoreactive patterns suggested that the superficial skin layer is the differentiated layer while the middle and basal layers are the undifferentiated stem cell layers in 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