Chick limbs with mouse teeth: An effective in vivo culture system for tooth germ development and analysis

Mouse tooth germ development is currently studied by three main approaches: in wild-type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short-term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4-5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously.