Generation and characterization of a novel transgenic line reporting the activity Hif1alpha, the central component of hypoxia sensing in the cell.

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 Hif1alpha, the central component of oxygen sensing in the cell. Hif1alpha activity is deregulated in many human cancers, especially those that are highly hypoxic. For these reasons, understanding the hypoxia signaling pathway in vivo could be a big step in the study the molecular mechanisms of solid tumors. To investigate Hif1alpha activity we developed two novel zebrafish transgenic lines named Tg(4xHRE:GFP) and Tg(4xHRE:nlsmCherry), expressing GFP or mCherry under the control of a synthetic promoter containing Hif1alpha DNA binding sites. 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. A strong Hif1alpha activation was observed in embryos from early development stages. We detect Hif1alpha activity in several tissues of developing embryos; particularly evident districts are retina, heart, eye lens, notochord and muscle cells. GFP or mCherry signals were present in different areas of the head, such as the cerebral vascular system and the craniofacial skeleton. Under physiological condition the activity of Hif1alpha was also maintained in adult fish, namely in the kidney, ovary, heart, eye lens, cerebral vascular system and the swim bladder. In conclusion, we demonstrated that Tg(4xHRE) lines are a reliable and sensitive biosensor to monitor Hif1alpha activity in vivo and to study at the whole organism level the hypoxia signaling pathway both in physiological and pathological conditions.