Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis

Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify assoc...

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Autores principales:	Aina Higuchi, Eri Wakai, Tomoko Tada, Junko Koiwa, Yuka Adachi, Takashi Shiromizu, Hidemasa Goto, Toshio Tanaka, Yuhei Nishimura
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	liver apoptosis caspase Förster resonance energy transfer zebrafish in vivo fluorescence imaging Medicine R Pharmacy and materia medica RS1-441
Acceso en línea:	https://doaj.org/article/31971d8532744cd389bcecf1fb492e2b
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spelling	oai:doaj.org-article:31971d8532744cd389bcecf1fb492e2b2021-11-25T18:39:29ZGeneration of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis10.3390/ph141111171424-8247https://doaj.org/article/31971d8532744cd389bcecf1fb492e2b2021-10-01T00:00:00Zhttps://www.mdpi.com/1424-8247/14/11/1117https://doaj.org/toc/1424-8247Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify associated genes and potential drugs. In this study, we developed a transgenic zebrafish line that was suitable for the assessment of caspase 3 activity in the liver by using in vivo fluorescence imaging. The larvae of transgenic zebrafish dominantly expressed Casper3GR in the liver under control of the promoter of the phosphoenolpyruvate carboxykinase 1 gene. Casper3GR is composed of two fluorescent proteins, tagGFP and tagRFP, which are connected via a peptide linker that can be cleaved by activated caspase 3. Under tagGFP excitation conditions in zebrafish that were exposed to the well-characterized hepatotoxicant isoniazid, we detected increased and decreased fluorescence associated with tagGFP and tagRFP, respectively. This result suggests that isoniazid activates caspase 3 in the zebrafish liver, which digests the linker between tagGFP and tagRFP, resulting in a reduction in the Förster resonance energy transfer to tagRFP upon tagGFP excitation. We also detected isoniazid-induced inhibition of caspase 3 activity in zebrafish that were treated with the hepatoprotectants ursodeoxycholic acid and obeticholic acid. The transgenic zebrafish that were developed in this study could be a powerful tool for identifying both hepatotoxic and hepatoprotective drugs, as well as for analyzing the effects of the genes of interest to hepatic apoptosis.Aina HiguchiEri WakaiTomoko TadaJunko KoiwaYuka AdachiTakashi ShiromizuHidemasa GotoToshio TanakaYuhei NishimuraMDPI AGarticleliverapoptosiscaspaseFörster resonance energy transferzebrafishin vivo fluorescence imagingMedicineRPharmacy and materia medicaRS1-441ENPharmaceuticals, Vol 14, Iss 1117, p 1117 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	liver apoptosis caspase Förster resonance energy transfer zebrafish in vivo fluorescence imaging Medicine R Pharmacy and materia medica RS1-441
spellingShingle	liver apoptosis caspase Förster resonance energy transfer zebrafish in vivo fluorescence imaging Medicine R Pharmacy and materia medica RS1-441 Aina Higuchi Eri Wakai Tomoko Tada Junko Koiwa Yuka Adachi Takashi Shiromizu Hidemasa Goto Toshio Tanaka Yuhei Nishimura Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis
description	Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify associated genes and potential drugs. In this study, we developed a transgenic zebrafish line that was suitable for the assessment of caspase 3 activity in the liver by using in vivo fluorescence imaging. The larvae of transgenic zebrafish dominantly expressed Casper3GR in the liver under control of the promoter of the phosphoenolpyruvate carboxykinase 1 gene. Casper3GR is composed of two fluorescent proteins, tagGFP and tagRFP, which are connected via a peptide linker that can be cleaved by activated caspase 3. Under tagGFP excitation conditions in zebrafish that were exposed to the well-characterized hepatotoxicant isoniazid, we detected increased and decreased fluorescence associated with tagGFP and tagRFP, respectively. This result suggests that isoniazid activates caspase 3 in the zebrafish liver, which digests the linker between tagGFP and tagRFP, resulting in a reduction in the Förster resonance energy transfer to tagRFP upon tagGFP excitation. We also detected isoniazid-induced inhibition of caspase 3 activity in zebrafish that were treated with the hepatoprotectants ursodeoxycholic acid and obeticholic acid. The transgenic zebrafish that were developed in this study could be a powerful tool for identifying both hepatotoxic and hepatoprotective drugs, as well as for analyzing the effects of the genes of interest to hepatic apoptosis.
format	article
author	Aina Higuchi Eri Wakai Tomoko Tada Junko Koiwa Yuka Adachi Takashi Shiromizu Hidemasa Goto Toshio Tanaka Yuhei Nishimura
author_facet	Aina Higuchi Eri Wakai Tomoko Tada Junko Koiwa Yuka Adachi Takashi Shiromizu Hidemasa Goto Toshio Tanaka Yuhei Nishimura
author_sort	Aina Higuchi
title	Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis
title_short	Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis
title_full	Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis
title_fullStr	Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis
title_full_unstemmed	Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis
title_sort	generation of a transgenic zebrafish line for in vivo assessment of hepatic apoptosis
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/31971d8532744cd389bcecf1fb492e2b
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_version_	1718410825110126592

Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis

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