The availability of new tools to study the activation of specific cells signaling regulating cell survival and death, proliferation and differentiation are the new frontiers for understanding in-vivo cell development, tissue homeostasis and tumor growth. Here we describe the generation and validation of a transgenic reporter line to study the hypoxia signaling pathway regulated by Hypoxia-inducible factor1 (Hif1) transcription factor, the central component of oxygen sensing in the cell. Hif1 activity is deregulated in many human cancers, especially those that are usually resistant to antiangiogenic drugs and to radiotherapy. For these reasons, understanding the role of the hypoxia signaling pathway in-vivo, could be a big step in the study the molecular mechanisms of solid tumors. To investigate Hif1 activity we developed two novel zebrafish transgenic lines named Tg(4xhre.tata:GFP) and Tg(4xhre.tata:mCherry), expressing GFP or mCherry under the control of a synthetic promoter containing Hif1 DNA binding sites. Independent founders expressing the transgene were identified and raised to adulthood. GFP or mCherry expression was stable in subsequent generations without changes in the expression pattern. At first, we observed that Tg(4xhre) zebrafish transgenic lines are responsive to changes in environmental oxygen levels and to chemical compounds mimicking hypoxic condition. Then, we monitored Hif1 activation during development, observing the expression of the transgene in retina, heart, eye lens, notochord and muscle cells. GFP or mCherry signals were present also in different areas of the head, such as the cerebral vascular system and the craniofacial skeleton. Under physiological condition the activity of Hif1 was also maintained in adult zebrafish, namely in the kidney, ovary, heart, eye lens and cerebral vascular system. In conclusion, we demonstrated that Tg(4xhre) lines are a reliable and sensitive biosensor to monitor Hif1 activity in vivo and to study at the whole organism level the hypoxia signaling pathway both in physiological and pathological conditions such as tumor angiogenesis and chronic inflammation.
Generation and characterization of a novel zebrafish transgenic line reporting the activity of Hypoxia-inducible factor1 (Hif1) the central component of hypoxia sensing in the cell
VETTORI, ANDREA;ASTONE, MATTEO;M. Schiavone;MILANETTO, MARTINA;MORO, ENRICO;TISO, NATASCIA;ARGENTON, FRANCESCO
2013
Abstract
The availability of new tools to study the activation of specific cells signaling regulating cell survival and death, proliferation and differentiation are the new frontiers for understanding in-vivo cell development, tissue homeostasis and tumor growth. Here we describe the generation and validation of a transgenic reporter line to study the hypoxia signaling pathway regulated by Hypoxia-inducible factor1 (Hif1) transcription factor, the central component of oxygen sensing in the cell. Hif1 activity is deregulated in many human cancers, especially those that are usually resistant to antiangiogenic drugs and to radiotherapy. For these reasons, understanding the role of the hypoxia signaling pathway in-vivo, could be a big step in the study the molecular mechanisms of solid tumors. To investigate Hif1 activity we developed two novel zebrafish transgenic lines named Tg(4xhre.tata:GFP) and Tg(4xhre.tata:mCherry), expressing GFP or mCherry under the control of a synthetic promoter containing Hif1 DNA binding sites. Independent founders expressing the transgene were identified and raised to adulthood. GFP or mCherry expression was stable in subsequent generations without changes in the expression pattern. At first, we observed that Tg(4xhre) zebrafish transgenic lines are responsive to changes in environmental oxygen levels and to chemical compounds mimicking hypoxic condition. Then, we monitored Hif1 activation during development, observing the expression of the transgene in retina, heart, eye lens, notochord and muscle cells. GFP or mCherry signals were present also in different areas of the head, such as the cerebral vascular system and the craniofacial skeleton. Under physiological condition the activity of Hif1 was also maintained in adult zebrafish, namely in the kidney, ovary, heart, eye lens and cerebral vascular system. In conclusion, we demonstrated that Tg(4xhre) lines are a reliable and sensitive biosensor to monitor Hif1 activity in vivo and to study at the whole organism level the hypoxia signaling pathway both in physiological and pathological conditions such as tumor angiogenesis and chronic inflammation.
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