Global signaling reporters

We have developed a number of transgenic zebrafish reporter lines under the control of DNA elements responsive for specific cellular pathways involved in cell proliferation, stem cell maintenance and differentiation, tissue homeostasis and cancer (Wnt, BMP, Shh, Notch, TGFb, CREB/cAMP). These reporter lines function as biosensors allowing us to follow in vivo, both spatially and temporally, the activation and inhibition of these signalling pathways. We have validated the transgenic lines: 1) by observing their expression pattern in vivo; 2) by treating them with specific inhibitors and activators; 3) by focusing on pancreas during the postnatal development. The specificity of BMP, TGF beta and Wnt reporters was pharmacologically tested by using the appropriate inhibitors of each pathway and checking the alterations of the reporter transcript level by RNA in situ hybridization. The activation of the signalling pathways has initially been observed from 24 till 120 hours post fertilization (hpf) at the fluorescent microscope. For the Bone Morphogenic Protein (BMP) responsive (BRE) zebrafish line the main expression districts are vasculature, heart, muscle, maxillary and mandibular processes, pharyngeal arches, dorsal retina, otic vesicle, cloaca. For the TGF-beta reporter lines the expression is observed in the entire neural tube, particularly in the ventricular zone. To understand in which cells the TGF beta pathway is activated and what stage of differentiation/commitment, the reporter line has been crossed with GFAP, NeuroD and Ngn1 transgenic lines labeling ,in order, glia and radial glia, neuron precursors and mature neurons. Data seem to confirm the role of TGF beta in gliogenesis and differentiation of interneurons according to what found in literature. Transgenic lines specifically labelling pancreatic endocrine cells (Insulin, Glucagon and NeuroD transgenic lines), ductal cells (Nkx2.2a transgenic line) and exocrine cells (Ptf1a transgenic line) have been crossed with our reporter lines and the larvae were observed at the confocal microscope. During early larval stage the study of the pancreas has revealed the activation of the BMP pathway in this organ from 4 days post fertilization (dpf) till adult stage.