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

Abstract

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. Developmental Dynamics 226:149 -154, 2003.

Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture

“…This might reflect the accumulation of immunocompetent cells into a layer of odontoblasts (Jontell et al 1998;Shimizu et al 2000). Stem cells capable of forming dental tissues have been reported in transplants (Gronthos et al 2000(Gronthos et al , 2002Young et al 2002;Braut et al 2003;Koyama et al 2003). In cell culture, cells that can express both DSP and DPP genes specific for odontoblast differentiation have also been induced by overexpression of the DMP 1 gene (Narayanan et al 2001).…”

“…Furthermore, it has been suggested that the activity, function and morphology of cells under a three-dimensional microenvironment, including in vivo, are different from those in two dimensions (Cukierman et al 2001;Hotary et al 2003). Since these reports have emphasized that a three-dimensional microenvironment is needed in an in vitro culture system to provide conditions similar to those in vivo, several successful studies of tissue engineering, such as tooth formation, have been reported in in vivo culture systems in which grafts of dental tissues or cells are returned to a three-dimensional microenvironment, such as in transplants (Tucker et al 1998;Young et al 2002;Braut et al 2003;Koyama et al 2003). Therefore, as an in vitro model of cell culture to examine the origin of multi-potent adult progenitor cells/stem cells for regenerative odontoblasts, in a newly designed threedimensional cell culture system (Kikuchi et al 2002) we considered the possibility that the putative progenitor cells/stem cells in dental papillae may be able to differentiate into odontoblasts.…”

Hedgehog signaling is required at multiple stages of zebrafish tooth development

“…Experiments inhibiting or over-activating the hedgehog pathway in mouse embryos have demonstrated several hedgehog signaling requirements during tooth development. Examples include the inhibition of hedgehog signaling after the early epithelial-thickening stage arresting mouse tooth development [35], inhibition at the bud stage resulting in malformed teeth [35,36], and later inhibition at the bell stage affecting the timing of tooth growth [37]. These studies have revealed multiple effects of the pathway on tooth development, including in cell proliferation [38,39] and differentiation [40].…”

Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

“…The anti-Shh monoclonal antibody 5E1 (5E1) is a pathway antagonist that is widely used to study Hh signaling in both developmental biology (23)(24)(25)(26)(27)(28)(29) and cancer (3, 30 -32). 5E1 was generated with mouse hybridoma technology using the rat Shh N-terminal domain as the antigen (33).…”