Genetics of taste buds development using zebrafish – ZASTE

Taste is in charge of evaluating the nutritious content of food and preventing the ingestion of toxic substances. Sweet taste helps us choose high energy food whereas bitter taste protects us agains ingesting toxic substances. Taste sense organs are located in the pharyngeal cavity (tongue in mice and man) and composed of taste buds that each harbors three main cell types, the taste sensory receptor cells, the presynaptic cells and supportive cells. Mammalian taste buds are assembled further into papillae. Whereas the mechanism through which tastants are perceived and interpreted by the nervous system is the focus of intense research, relatively little is known about the mechanism allowing taste organs development. Yet, taste is altered in a number of human diseases or following treament. Taste also participates in the enjoyment of the meal and is thus a important factor in the development of major health issues such as obesity. Papillae formation has been analyzed in mice. Wnt signalling induces rostral tongue papillae, whereas Shh and EGF signalling represse papillae formation. Using zebrafish as a model system, we have shown that the pharyngeal cavity develops a network of taste buds, derived from endoderm. We have also shown that FGF signalling is critical within the pharyngeal epithelium for posterior but not for rostral (lip) taste buds development. In addition, we have preliminary evidence that Notch signalling is important for controlling cell type formation. However the exact mechanisms leading to the build up of taste organs and, in particular, the generation of the different cell types has not been analysed in detail. To adress this issue, we propose the following project which aims at using zebrafish to decipher the cellular events involved in the build up of the taste buds, as well as identifying key molecular processes sustaining these cellular events. To achieve the first goal, being able to follow on live tissues the development of the tastes buds from the pharyngeal epithelium is a major prerequisite which is fulfilled by the use of the optically clear zebrafish larva in which these structures are directly accessible. During the last years, we have developped a number of tools to adress this issue including a number of markers labelling either the presynaptic cell (expression of serotonin and tph1b), taste receptor cell (calb 2) as well as microRNA family miR-200, which labels a subset of taste bud cells to be defined. Interestingly, FGF8, is expressed before taste bud specification in isolated cells within the pharyngeal epithelium. Quite strikingly we have also characterized a transgenic fluorescent zebrafish line whose expression is controlled by elements of the FGF8 enhancer and localised to isolated cells within the differentiating taste buds. Altogether, these results suggests that FGF8 may represent a critical signal to allow patterning within the taste bud itself. This project is thus organised into three different tasks. The first task involves the development of new tools and refinement of previously identified tools. In particular, new tools will include the development of fluorescent transgenic lines recapitulating the expression of early and possibly late markers to be able to follow live development of taste organs. Refinement will include a better definition of the type of territories/cell types defined by these markers and transgenic lines. The second task, based on the use of markers and on transgenic lines aims at deciphering the cellular aspects of taste bud morphogenesis. This approach will involve, on the one hand, the in vivo time lapse analysis of morphogenesis. Since the FGF8 fluorescent transgenic line exhibits expression in single discrete cells, we will study morphogenesis using this expression as an anchor and define the fate of the FGF8 cell as well as the one of its closest neighbours. Then, we will study the function of specific cells of the epithelium by physically (laser) or chemically ablating them. The third task aims at defining the molecular interactions controlling taste bud build up. On the one hand, we will further define by again an loss of function strategies, using Morpholino oligonucleotides/DNA injections/electroporation the function of FGF and Notch signalling in the generation of the different cell types, but also define the epistatic relationship between the different signalling pathways. Last, we will attempt to define new genes controlling taste bud development by carrying out a genetic screen based on two different assays, an immunohistological assay and a feeding behaviour assay for larvae insensitive to the repulsive effect of bitter compounds.