323 Introduction During development of the peripheral nervous system, growing axons navigate and establish connections to their developing target organs. Regulation of axon growth involves coordinated activity of diffusible and local contact-mediated attractive and repulsive guiding cues including members of for example the netrin, Slit, ephrin and semaphorin families (Dickson, 2002; Tessier-Lavigne and Goodman, 1996; Varela-Echavarria and Guthrie, 1997). Many of the guidance molecules show dynamic and restricted expression patterns in peripheral tissues and organs that correlate with regulation of axon growth. However, how the expression of guidance molecules is regulated during organ formation, and how axon navigation and patterning is coordinated spatiotemporally with organ formation has remained largely unknown. The developing tooth is a useful model in which to analyze the molecular mechanisms of organ formation. The teeth develop in the oral side of the maxillary and mandibular processes, and their formation is regulated by sequential and reciprocal interactions between the odontogenic epithelium and neural crest derived ectomesenchymal cells (Miletich and Sharpe, 2003; Thesleff, 2003). Signaling molecules have been shown to mediate inductive tissue interactions during odontogenesis. In particular, early oral epithelium- and oral epithelium-expressed signaling molecules regulate dental mesenchymal expression of signaling genes and transcription factors that are essential for tooth formation (Miletich and Sharpe, 2003; Thesleff, 2003). Trigeminal axon pathfinding and nerve fiber patterning, in particular in the murine lower first molar, takes place in a strictly spatiotemporally controlled manner and is tightly linked to tooth formation (Loes et al., 2002; Luukko et al., 1997b; Mohamed and Atkinson, 1983). During development, trigeminal nerve fibers navigate and establish their axonal projections to the developing tooth in a highly spatiotemporally controlled manner. By analyzing Sema3a and its receptor Npn1 knockout mouse embryos, we found that Sema3a regulates dental trigeminal axon navigation and patterning, as well as the timing of the first mandibular molar innervation, and that the effects of Sema3a appear to be mediated by Npn1 present in the axons. By performing tissue recombinant experiments and analyzing the effects of signaling molecules, we found that early oral and dental epithelia, which instruct tooth formation, and epithelial Wnt4 induce Sema3a expression in the presumptive dental mesenchyme before the arrival of the first dental nerve fibers. Later, at the bud stage, epithelial Wnt4 and Tgfβ1 regulate Sema3a expression in the dental mesenchyme. In addition, Wnt4 stimulates mesenchymal expression of Msx1 transcription factor, which is essential for tooth formation, and Tgfβ1 proliferation of the dental mesenchymal cells. Thus, epithelial-mesenchymal interactions control Sema3a expression and may coordinate axon navigation and patterning with tooth formation. Moreover, our results suggest that the odontogenic epithelium possesses the instructive information to control the formation of tooth nerve supply. Key words: Odontogenesis, Tissue interactions, Tooth, Axon growth, Mouse Summary Coordination of trigeminal axon navigation and patterning with tooth organ formation: epithelial-mesenchymal interactions, and epithelial Wnt4 and Tgfβ1 regulate semaphorin 3a expression in the dental mesenchyme Päivi Kettunen 1 , Sigbjørn Løes 1 , Tomasz Furmanek 1 , Karianne Fjeld 1 , Inger Hals Kvinnsland 1 , Oded Behar 2 , Takeshi Yagi 3 , Hajime Fujisawa 4 , Seppo Vainio 5 , Masahiko Taniguchi 6 and Keijo Luukko 1, * 1 Division of Anatomy and Cell Biology, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway 2 Department of Experimental Medicine and Cancer Research, The Hebrew University, Jerusalem, 91120, Israel 3 Laboratories of Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan 4 Group of Developmental Neurobiology, Division of Biological Science, Nagoya University Graduate School of Science, Chikusa-ku, Nagoya, 464-8602, Japan 5 Biocenter Oulu and Department of Biochemistry, Faculties of Science and Medicine, University of Oulu, 90014, Finland 6 Department of Biochemistry and Molecular Biology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan *Author for correspondence (e-mail: keijo.luukko@pki.uib.no) Accepted 20 October 2004 Development 132, 323-334 Published by The Company of Biologists 2005 doi:10.1242/dev.01541 Research article Development