Generation of a zebrafish reporter line for in vivo visualization of cAMP signaling activation

cAMP, the classical second messenger discovered more than 50 years ago, has more recently considered as a relevant signal in a number of human pathologies, ranging from heart failure, cancer, metabolic, endocrine, respiratory and neuromuscular diseases. In the last decade, the generation and use of fluorescent probes has allowed visualization and quantification of cAMP signaling at a pharmacological, spatial and temporal level, adding significant contributions to the understanding of this pathway. However, these studies have been mainly performed by in vitro systems, thus losing information at the whole animal level. By exploiting a Gateway/Tol2-based transgenic strategy, our group has recently generated a zebrafish-based cAMP signaling reporter system. For this purpose, six cAMP responsive elements have been multimerized upstream of a minimal promoter, to drive the expression of GFP or mCherry fluorescent reporters in response to cAMP signaling activation. Starting from founder fish with multiple insertions, stable reporter lines have been obtained, with Mendelian inheritance of the transgene; a maternal contribution has been also observed. Both green and red reporters appear to consistently label the same anatomical regions, throughout the main steps of zebrafish embryonic and larval development. Positive areas include skin, lens epithelium, olfactory pits, proctodeum, heart, fast muscle fibers, cranial cartilage, Mauthner cells, thyroid, liver, vessels and blood cells. Our current analyses are evaluating cAMP reporter activation during regeneration and adult stages. To functionally validate our reporter, a pharmacological approach has been adopted, using a set of compounds with known agonistic or antagonistic effect on cAMP signaling. Preliminary data show in vivo variation of reporter fluorescence according to the expected activity of each drug. These results point to our transgenic lines as bona fide reporters of cAMP signaling activation, indicating their suitability as in vivo systems for large scale screening of small molecules with therapeutic effects on cAMP–linked human diseases.