Zebrafish have become a powerful new model organism for studies of vertebrate development. This is largely due to the optical clarity of zebrafish embryos and the advent of green fluorescent protein (GFP) transgenic technology. Combined, these features allow the characterization of organogenesis at the level of individual cells. Additionally, developmental genetic regulatory pathways have been found to be highly conserved between zebrafish and mammals. We first propose to exploit the advantages of this system to characterize the detailed normal morphogenesis of zebrafish teeth by the timelapse confocal microscopy of recently-generated GFP transgenic lines. We will use a Fli1:GFP line to specifically follow cells in the dental mesenchyme, a Shh:GFP line to trace the movements of the dental epithelium, and the histone H2A:GFP line to follow the nuclei of each cell in the tooth germ. We also propose to generate new transgenic zebrafish lines expressing GFP and DsRed driven by Fgf4 regulatory elements. We will use these lines to investigate the presence of a zebrafish homolog of the mammalian enamel knot, an organizing center within the mammalian tooth epithelium that expresses Fgf4. Lastly we propose to perturb the normal development of teeth with the FGF receptor inhibitor Su5402 and observe the effects on morphogenesis with the GFP lines mentioned. These studies will reveal cell behavior during normal and experimentally perturbed tooth morphogenesis at a level of detail never before described. ? ?
