Tooth development in mammals and other vertebrates is a complex multistage process, which has been studied for years (Lumsden, 1988; Ruch, 1995; Butler, 1995; Slavkin and Diekwisch, 1996; Jernvall and Thesleff, 2000). Tooth development initiates with the formation of a dental lamina. Dental lamina cells then invaginate into the underlying mesenchyme and give rise to the enamel organ. Epithelial cells in close contact with mesenchymal cells of the dental papilla constitute the inner dental epithelium, whereas those facing the surrounding mesenchymal cells constitute the outer dental epithelium. With further growth, inner and outer dental epithelia become separated by stellate reticulum; in addition, two to three cell layers of squamous epithelial cells adjacent to the inner dental epithelium become histologically recognizable and are defined as stratum intermedium. Further morphogenetic and cytodifferentiation events lead to the formation of epithelially derived ameloblasts and mesenchymally derived odontoblasts (Lumsden, 1988; Ruch, 1995; Slavkin and Diekwisch, 1996; Thesleff and Sharp, 1997).
As the above synopsis indicates, much has been learned about the overall morphogenetic and cytodifferentiation processes occurring during odontogenesis and about the important roles played by inner dental epithelium and dental mesenchyme (Kollar and Baird, 1969, 1970; Ruch et al., 1976; Thesleff and Hurmerinta, 1981; Mina and Kollar, 1987). For example, it is well established that these epithelial and mesenchymal populations establish critical reciprocal interactions early in odontogenesis (Kollar and Baird, 1970; Thesleff and Hurmerinta, 1981) and express several specific extracellular matrix molecules and transcription factors (Snead et al., 1988; Deutsch et al., 1991; Bashir et al., 1997). When interactions between inner dental epithelium and dental mesenchyme are experimentally interrupted, tooth germ development ceases and no cytodifferentiation events occur (Koch, 1967). On the other hand, there is far less information on stratum intermedium and stellate reticulum during tooth germ development, mainly because these populations are numerically small and consequently difficult to study (Wise and Fan, 1989; Nakamura et al., 1991). Yet, it is likely that these epithelially derived populations have critical but still undefined roles in odontogenesis, particularly given the morphologic fact that stratum intermedium is so intimately associated with developing ameloblasts (Nakamura et al., 1991). It has been shown that stratum intermedium cells produce high amounts of alkaline phosphatase, a finding interpreted to signify that stratum intermedium cells promote differentiation of ameloblasts (Wise and Fan, 1989; Gomez and Boyde, 1994). This idea was strengthened by the recent finding that, in Msx-2 null mice, stratum intermedium cells are not well formed and ameloblast function and enamel deposition are impaired (Maas and Bei, 1997). These findings raise important questions. If indeed stratum intermedium is important for ameloblast differentiation and function, how do these closely associated epithelially derived cell populations communicate? What signaling molecules are involved? And what roles do these signals play? Sonic hedgehog (Shh) is a signaling and morphogenetic factor involved in the development of several embryonic structures (Riddle et al., 1993; Echelard et al., 1993; Bumcrot et al., 1995; Porter et al., 1995, 1996; Chiang et al., 1996; Roessler et al., 1996). Shh influences behavior and fate of cells located within its range of diffusion by interaction with cell surface receptors such as Patched (Ptc), Patched2 (Ptch2), and Smoothened (Marigo et al., 1996; Stone et al., 1996; Motoyama et al., 1998; Chen and Struhl, 1998). Shh action has been studied in several systems. In the early limb, Shh is a key player in establishing the posterior–anterior axis of the limb (Riddle et al., 1993; Marti' et al., 1995; Drossopoulou et al., 2000). Studies on neural cells indicate that Shh is able to determine different classes of ventral neurons on the basis of concentration gradients (Ericson et al., 1997). Thus, Shh can act locally or at some distance from its site of production and its concentration seems to be important for action.
Recent studies from this and other laboratories have shown that Shh, Ptc, and Ptch2 are expressed during odontogenesis (Bitgood and McMahon, 1995; Vaahtokari et al., 1996; Koyama et al., 1996; Motoyama et al., 1998; Hardcastle et al., 1998). Shh transcripts were found in the dental lamina, enamel knot, and inner dental epithelium at different stages of murine odontogenesis (Bitgood and McMahon, 1995; Koyama et al., 1996; Vaahtokari et al., 1996). In addition, Ptch2 transcripts were detected in inner dental epithelium, largely overlapping the distribution of Shh transcripts, whereas Ptc transcripts were mainly found in dental mesenchyme. The data have indicated that Shh may act as an autocrine signal within the inner dental epithelial layer itself and as a paracrine signal between epithelial and mesenchymal layers (Motoyama et al., 1998; Hardcastle et al., 1998). These interesting studies, however, did not examine stratum intermedium in detail. Given its close proximity to inner dental epithelium, stratum intermedium could itself be a producer of Shh or a recipient of Shh action. To analyze this critical question and related issues, we decided to switch from the widely used mouse tooth germs to bovine tooth germs. We reasoned that by being much larger, bovine tooth germs may contain a more conspicuous stratum intermedium, greatly facilitating analysis of its numerically scarce cell population. Indeed, we found that stratum intermedium is readily apparent in bovine tooth germs, even at early stages. We show for the first time that this tissue is a Shh-producing structure and undergoes dramatic changes in organization, distribution, and phenotype during odontogenesis. The data lead us to propose that stratum intermedium is a previously underappreciated player in the progression of tooth germ development from cusp to cervix.