INTRODUCTION By taking advantage of reporter fish lines generated in the frame of the ZF‐HEALTH project, Partner 12 has started a methodical application of these biosensors to analyze signaling pathways activation during zebrafish organ formation. As a pilot study, we have focused our attention on the zebrafish eye, taking into consideration a series of key properties of this organ, among which: ‐ early formation during embryonic development; ‐ optical accessibility due to its superficial position and relatively large size compared to other embryonic structures; ‐ high evolutionary conservation of its components, both in the anterior and posterior segments (e.g. lens structure; retinal cell types); ‐ available information on some signaling mechanisms, derived from previous zebrafish studies or different model systems. METHODOLOGY As a first step, we have selected four key developmental time points for our analysis: early somitogenesis (optic lobe evagination, lens induction); 1 dpf (lens and retina both detectable); 2 dpf (retinal cell type formation); 3 dpf (retinal layer formation, visual function). For each time points, we have performed both in vivo and post mortem confocal documentation of reporter activity, by imaging fluorescent protein distribution or reporter messenger localization (either alone or in combination with ocular markers). FUTURE PERSPECTIVES We are currently completing this general picture by analyzing more advanced developmental stages, as well as including two additional pathway reporters, Stat3 and Glucocorticoid Receptor, more recently generated. To evaluate roles and epistatic relationships of signaling pathways activated in the same ocular district, we will perform reporter inter-crosses and functional activation/inactivation of each involved molecular cascade.
Application of zebrafish reporter lines for eye tissues analysis
TISO, NATASCIA;R. Dahm;MORO, ENRICO;VETTORI, ANDREA;ARGENTON, FRANCESCO
2012
Abstract
INTRODUCTION By taking advantage of reporter fish lines generated in the frame of the ZF‐HEALTH project, Partner 12 has started a methodical application of these biosensors to analyze signaling pathways activation during zebrafish organ formation. As a pilot study, we have focused our attention on the zebrafish eye, taking into consideration a series of key properties of this organ, among which: ‐ early formation during embryonic development; ‐ optical accessibility due to its superficial position and relatively large size compared to other embryonic structures; ‐ high evolutionary conservation of its components, both in the anterior and posterior segments (e.g. lens structure; retinal cell types); ‐ available information on some signaling mechanisms, derived from previous zebrafish studies or different model systems. METHODOLOGY As a first step, we have selected four key developmental time points for our analysis: early somitogenesis (optic lobe evagination, lens induction); 1 dpf (lens and retina both detectable); 2 dpf (retinal cell type formation); 3 dpf (retinal layer formation, visual function). For each time points, we have performed both in vivo and post mortem confocal documentation of reporter activity, by imaging fluorescent protein distribution or reporter messenger localization (either alone or in combination with ocular markers). FUTURE PERSPECTIVES We are currently completing this general picture by analyzing more advanced developmental stages, as well as including two additional pathway reporters, Stat3 and Glucocorticoid Receptor, more recently generated. To evaluate roles and epistatic relationships of signaling pathways activated in the same ocular district, we will perform reporter inter-crosses and functional activation/inactivation of each involved molecular cascade.
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