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Expression of neural cell-adhesion molecule mRNA during mouse molar tooth development
Archives of Oral Biology 47 (2002) 805–813
Expression of neural cell-adhesion molecule mRNA during mouse molar tooth development Nobuko Obara a,∗ , Yuko Suzuki a , Yasuko Nagai a , Hiromasa Nishiyama b , Itaru Mizoguchi b , Masako Takeda a b
a Department of Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Accepted 7 November 2002
Abstract This study employed in situ hybridisation using a probe recognising all isoforms of the molecule. Expression of the molecule in tooth germs started at embryonic day 13, when they were at the bud stage. Both inner cells of the epithelial bud and peripheral cells of the dental mesenchyme were positive. At the cap stage, positive cells were found in the inner part of the enamel organ but only in a limited area near the outer enamel epithelium. In the mesenchyme at the cap stage, expression was weak in the dental papilla and strong in the follicle. From the bell stage onward, epithelial cells in the enamel organ were negative except for the cells of the stratum intermedium, which were transiently positive at early and late bell stages. In the dental papilla, expression had mostly ceased during and after the bell stage, although transient expression was found in cuspal areas at the early bell stage. The dental follicle strongly expressed neural cell-adhesion molecule (NCAM) to the end of the experimental period, at post-natal day 4. In contrast to the first molar at its earliest stage of appearance, in which both the thickened epithelium and surrounding mesenchyme were negative for the expression of the molecule, the second molar appeared as a combination of extending epithelial thickenings and mesenchymal cells strongly positive for its expression. This study newly identifies the dental papilla and the stratum intermedium as NCAM-expressing sites. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: NCAM; Tooth germs; In situ hybridisation; Morphogenesis; Differentiation; Odontogenesis
1. Introduction Cell–cell adhesion is crucial for organogenesis. In developing organs, molecules that mediate specific cell–cell adhesion are expressed in a spatiotemporal manner and are thought to have a regulatory role in morphogenesis (Edelman, 1988; Edelman and Crossin, 1991; Takeichi, 1988, 1990, 1991). Among the various cell-adhesion molecules, cadherins, which mediate Ca2+ -dependent cell– cell adhesion, have been the best studied. In addition to Abbreviations: BCIP, 5-bromo-4-chloro-3-indol-phosphate; DIG, digoxigenin; NCAM, neural cell-adhesion molecule; NBT, tetrazolium chloride; PBS, phosphate-buffered saline; SSC, standard saline citrate buffer ∗ Corresponding author. Tel.: +81-1332-3-1236; fax: +81-1332-3-1236. E-mail address: [email protected] (N. Obara).
their basic function, i.e. the mediation of selective cell adhesion, they perform more specialised tasks including the establishment of cell-surface polarity and changes in gene expression as a consequence of adhesion (Marrs and Nelson, 1996). The expression of cadherin subclasses is developmentally regulated, indicating their correlation with a variety of morphogenetic events (Takeichi, 1988). Another well-characterised cell-adhesion molecule is neural cell-adhesion molecule (NCAM), which is also expressed in many developing organs and thus is thought to be involved in the regulation of morphogenesis (Crossin et al., 1985). NCAM belongs to the immunoglobulin superfamily and mediates Ca2+ -independent cell–cell adhesion by homophilic binding of proteins on neighbouring cells. Alternative RNA splicing and post-translational modification with polysialic acids generate many molecular forms with differences in molecular size and binding properties (Barbas et al., 1988; Owens et al., 1987; Santoni et al., 1989). In
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addition to mediating cell–cell adhesion, the binding of NCAM can affect intracellular signalling and thereby alter gene expression (Crossin and Krushel, 2000; Doherty and Walsh, 1994). This molecule is also known to participate in specific heterophilic interactions with extracellular matrix components (Cole et al., 1986; Cole and Akeson, 1989; Friedlander et al., 1994; Probstmeier et al., 1989). This complexity of molecular properties and functions raises the possibility that NCAM has multiple regulatory roles in developing organs, although the biological meaning of such complexity has not yet been fully established. Tooth development is an excellent model for studying organogenesis and involves several fundamental problems shared by other systems of organ development, e.g. neural crest-cell migration, epithelial–mesenchymal interactions, tissue patterning, morphogenesis and cytodifferentiation. In recent years, studies on tooth development have much advanced and now concern molecular mechanisms, in which growth factors, other signalling molecules and transcription factors are found to be involved in the above processes (Åberg et al., 1997; Jernvall and Thesleff, 2000; Thesleff and Sharpe, 1997; Tucker and Sharpe, 1999). There are reports of the expression patterns of several cell-adhesion molecules during tooth development (Chuong, 1990; Lüning et al., 1994, 1995; Fausser et al., 1998; Obara and Takeda, 1993a,b, 1997; Obara et al., 1998, 1999; Palacios et al., 1995; Terling et al., 1998), but less is known about these molecules than about other molecular factors in tooth development. In earlier studies, we localised NCAM polypeptides in tooth germs by using a monoclonal antibody and demonstrated a dynamic change in their expression pattern during tooth development (Obara and Takeda, 1993a,b, 1997). The spatiotemporally regulated expression of NCAM implied that it has a regulatory role in tooth development, but problems about how the expression of NCAM in tooth germs is regulated and its role in the particular sites where it is expressed remain to be resolved. As a basis for investigating these problems, we have now examined the expression pattern of NCAM during molar tooth development by means of in situ hybridisation using a probe that hybridises with a sequence included in all NCAM isoforms. Because we detected NCAM mRNA in the places where we had previously detected NCAM polypeptides, and also identified a few additional sites expressing NCAM transcripts, we also re-examined the newly identified NCAM-expressing sites by immunofluorescence to confirm the presence of NCAM polypeptides.
2. Materials and methods 2.1. Animals Embryos were obtained by mating male and female ddY mice. The ages of embryos were determined by the day of appearance of the vaginal plug (E0). The gestational period
of these animals is 19 days, and the day of birth was taken as day 0 of post-natal development (P0). 2.2. Tissue preparation To obtain embryos, pregnant females were killed by cervical dislocation. Tissues including the tooth germs or primordia were dissected from the embryos in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and fixed in the same solution overnight at 4 ◦ C. The post-natal mice used were anaesthetised with chloroform and perfused with the same fixative via the left ventricle. Their tissues were then dissected and fixed in the same way as for embryos. Fixed tissues were immersed in 20% sucrose in phosphate-buffered saline (PBS) for 2 h at 4 ◦ C, embedded in Tissue-Tek O.C.T. Compound (Miles Laboratories, Elkhart, Indiana), and frozen with Cryon Spray Freezer (Oken Shoji, Tokyo). Sections cut at 10 m thickness in a cryostat were picked up on silane-coated glass slides and air-dried for more than 30 min at room temperature. 2.3. In situ hybridisation Sections were immersed in absolute ethanol for 5 min and in 0.2N HCl for 20 min, and then washed twice in PBS for 5 min each. Next, the sections were treated with 2 g/ml of proteinase K (Takara Shuzo Co., Kyoto, Japan) at 37 ◦ C for 15 min, washed in PBS, and refixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 min. After having been washed twice in PBS and once in distilled water for 5 min each time, the sections were air-dried and hybridised. Hybridisation was performed at 45 ◦ C for 14–16 h with a digoxigenin (DIG)-labelled RNA probe (50 ng/ml) in a hybridisation solution containing 50% formamide, 0.3 M NaCl, 0.02 M Tris–HCl (pH 8.0), 1 mM EDTA, 10% dextran sulphate, one time in denhardt solution, 1 mg/ml yeast tRNA and 0.02 % sodium dodecyl sulphate. Hybridised sections were washed at 45 ◦ C in a solution containing 50% formamide and two times in standard saline citrate buffer (SSC; pH 7.0) for 60 min, and thereafter twice in two times in SSC for 5 min each time. They were then treated with 20 g/ml RNase (type II-A; Sigma, St. Louis, MO, USA)/two times in SSC at 37 ◦ C for 30 min, and washed at 45 ◦ C in 50% formamide/two times in SSC followed by 50% formamide/one time in SSC for 60 min each. After having been washed three times in PBS, the sections were incubated with 1% blocking reagent (Boeringer Manheim GmbH, Manheim, Germany) in maleic acid buffer (pH 7.5) for 60 min at room temperature. Subsequently, they were incubated overnight at 4 ◦ C with alkaline phosphatase-conjugated anti-DIG Fab fragments diluted 1:500 in PBS containing 1% bovine serum albumin. After three washes in Tris-buffered saline (0.1 M Tris–HCl, 0.15 M NaCl, pH 7.5), chromogenic reactions were carried out using tetrazolium chlo-
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ride (NBT)/5-bromo-4-chloro-3-indol-phosphate (BCIP; Boeringer Manheim).
expressing NCAM existed outside of the tooth primordia at this stage (Fig. 1a).
2.4. Hybridisation probes
3.2. Bud and cap stages
A complementary DNA corresponding to mouse NCAM mRNA at positions 397–1354 (Barthels et al., 1987) was prepared by reverse transcription–polymerase chain reaction from mouse brain total RNA, and subcloned into HindIII/ EcoRI sites of pT7/T3␣-18 (Life Technologies, Grand Island, NY, USA). DIG-labelled RNA probes were synthesised in the presence of DIG-UTP (DIG RNA labelling mix; Boeringer Manheim) by using T7 RNA polymerase and HindIII-linearised template (antisense probe) or T3 RNA polymerase and EcoRI-linearised template (sense probe). Probe length was reduced to 200 nucleotides by limited alkaline hydrolysis (Cox et al., 1984). The mRNA at positions 397–1354 corresponds to a part of exon 2, entire exons 3–7 and a part of exon 8 (Owens et al., 1987), and so the probe recognises mRNAs encoding the extracellular immunoglobulin-like domains that commonly exist in all isoforms of NCAM molecules (Santoni et al., 1989).
At E13, marginal cells of the epithelial tooth bud did not express NCAM but the inner cells did. The molecule was also expressed in mesenchyme surrounding the tooth bud, where expression was strong in the peripheral area and weak or almost negative in the inner area adjacent to the tip of the bud (Fig. 1b). In sections of the posterior region of E13 molar tooth germs, where the dental epithelium was shaped like an early bud or lamina at the posterior end, cells strongly expressing NCAM (Fig. 1c) surrounded the epithelium. In cap stage tooth germs, the expression of NCAM by epithelial cells had become more restricted to the dental lamina and the cells lining the outer enamel epithelium (Fig. 1d). Differentiating stellate reticulum and the enamel knot, as well as the inner and outer enamel epithelia, did not express NCAM. Both the dental papilla and the dental follicle at the cap stage expressed NCAM, but the strength of expression differed, being weak in the papilla and strong in the follicle (Fig. 1d). Through the bud and cap stages, cells expressing NCAM were also present in the mesenchyme outside of the tooth germs (Fig. 1b–f).
2.5. Indirect immunofluorescence staining Rat monoclonal antibody (IgG) to NCAM (H28.123; Hirn et al., 1981) was purchased from Immunotech S.A. (Marseille, France). Fluorescein isothiocyanate-conjugated, affinity-purified antibodies to rat IgG was purchased from Cappel, Organon Teknica (West Chester, PA) and used after absorption with purified mouse IgG (Zymed, San Francisco). Staining procedures were as previously described (Obara and Takeda, 1993a).
3. Results During all the experimental stages, in situ hybridisation using the sense sequence as a probe resulted in no reaction product by alkaline phosphatase as long as the hybridisation was performed under the same conditions as used in the experiments with the antisense probe (Fig. 2a). 3.1. Initiation stages The earliest morphological sign of tooth development became visible as epithelial thickenings on the day 11 of embryonic development (E11). At the site of tooth initiation, signals indicating NCAM mRNA expression were not detected in either the thickened epithelia or in the mesenchyme directly surrounding them. In embryos at E12 the dental epithelium had increased in thickness. NCAM transcripts were hardly detectable either in the mesenchyme adjacent to the developing dental epithelium or in the dental epithelium itself. However, mesenchymal cells weakly
3.3. Early stages of the second and third molars It was difficult in histological sections to define the initiation of the second and third molars as epithelial thickenings, which for first molars had appeared at E11. In fact, a distinct border between the first and the second molar could not be determined in embryos at stages earlier than E14. In E14 embryos, the epithelial bud of the second molar could be distinguished from the epithelial cap of the first molar. NCAM expression in the epithelial bud of the second molar was similar to that in the first molar, where only the inner epithelial cells expressed the molecule (Fig. 1e and f). However, the pattern of NCAM expression in the mesenchyme of the second molar tooth germs was slightly different from that of the first molars, especially at the early bud stage. The mesenchymal cells surrounding the tooth bud more strongly expressed NCAM than those in the first molar at the equivalent stage (Fig. 1e; compare with Fig. 1b). Particularly, the bud-shaped epithelium in the posterior part was in contact with mesenchymal cells strongly expressing NCAM (Fig. 1f). Whereas the sequential development of first and second molars begins within a short period, development of the third molar commences after a delay. Because of its position and smaller size, study of the initial stages of the third molar was more difficult than with the second molar, and its formation could be recognised in histological sections only after birth. Posterior to the second molar of P0 mice, there was a narrow space filled with NCAM-expressing mesenchymal cells. In P0 mice, an epithelial bud-like structure growing into the
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Fig. 1. Expression of NCAM during the early stages of tooth development. In situ hybridisation of frontal sections through areas bearing molar tooth germs. (a) Embryonic day (E) 12. Dental lamina (arrowheads) and surrounding mesenchyme are negative for NCAM expression. Mesenchymal cells apart from the tooth primordium weakly express the molecule. (b) Section through the middle parts of the tooth germs at E13. In the epithelial tooth buds (arrowheads), the peripheral cells are negative for NCAM, whereas the cells in the inner part are positive. In the dental mesenchyme, NCAM expression is stronger in the outer part (differentiating dental follicle), but very weak in the area surrounding the tip of the epithelial buds. (c) Section through the posterior ends of the tooth germs shown in (b). Mesenchymal cells surrounding the dental epithelia strongly express NCAM. (d) First molar tooth germs at E14. NCAM-expressing epithelial cells are localised beneath the outer enamel epithelium (arrows) and the inner area of the dental lamina. NCAM expression is weak in the dental papilla (P) and strong in the dental follicle (F). (e, f) Sections of a mandibular second molar tooth germ at E14 through middle (e) and posterior (f) parts. In the middle part, NCAM expression in the mesenchyme around the tip of the epithelial bud (∗) is weak, whereas the posterior part of the bud is surrounded by strongly NCAM-expressing cells. Note that NCAM expression is stronger than in the first molar tooth germs at the corresponding stage of tooth development (bar = 100 m).
space was apparent, and the budding epithelium expressed NCAM in its inner part (Fig. 2i). From the cap stage onward, the expression patterns of NCAM in the second (data not shown) and third (Fig. 2j) molars during the experimental period were fundamentally the same as in the first molar.
3.4. Bell and later stages At the early bell stage, NCAM was expressed in the mesenchyme of prospective cusp-forming areas, but not in the remaining part of the papilla. The molecule was expressed
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Fig. 2. Expression of NCAM in tooth germs at late embryonic and post-natal stages. (a) In situ hybridisation of a mandibular first molar tooth germ at embryonic day (E)16. Signals indicating NCAM transcripts are found in the stratum intermedium (SI), the dental follicle and the papillary mesenchyme in the cusp-forming areas (arrows). (b) Section of the tooth germ shown in (a), stained using anti-NCAM monoclonal antibody. Anti-NCAM immunoreactivity exists in the areas where NCAM transcripts are detected. Red blood cells with strong autofluorescence are found in both the dental papilla and the dental follicle. (c, d) In situ hybridisation of a mandibular tooth germ at E18, hybridised with antisense (c) and sense (d) probes. NCAM expression is strong in the dental follicle. Weak expression is found in the papillary mesenchyme close to the dental follicle (arrows). (e) In situ hybridisation of a mandibular first molar tooth germ at post-natal day (P) 0. NCAM expression is found in the dental follicle and in the stratum intermedium (arrows). (f) Higher magnification of the area surrounded by the square in (e). Only the stratum intermedium expresses NCAM. (g) Mandibular first molar tooth germ at P0 stained with anti-NCAM monoclonal antibody. Stratum intermedium exhibits weak NCAM immunoreactivity. (h) In situ hybridisation of a mandibular first molar tooth germ at P2. Expression of NCAM is found in the dental follicle and in the nearby papillary mesenchyme (arrows). (i) In situ hybridisation of a mandibular third molar at P1. Signals indicating NCAM transcripts are found in the epithelial bud (arrowheads). The surrounding mesenchyme is filled with NCAM-expressing cells. (j) In situ hybridisation of a mandibular third molar at P4. Strong expression of NCAM is found in the dental follicle, whereas the expression is weak in the dental papilla. Arrowheads indicate enamel organ. Sections were made in the anterior–posterior planes in (e)–(j). M2, mandibular second molar; M3, mandibular third molar; SR, stellate reticulum (bar = 100 m).
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by the epithelial cells in the stratum intermedium and in the narrow space between the outer and inner enamel epithelium of the lateral part of the enamel organ (Fig. 2a). During subsequent stages, NCAM expression in the dental papilla decreased; in tooth germs at late bell or later stages it became restricted to the basal part of the papilla, which is continuous with the dental follicle. In contrast, the follicle cells continued to express NCAM from the bud stage onward (Fig. 2c and h). Expression of NCAM in the stratum intermedium was temporary or regional in appearance. In the first molar tooth germs at E18 and P0 (and also in the second molars at equivalent stages of development), NCAM expression in the stratum intermedium was not uniform within a given germ (Fig. 2e). In tooth germs at more advanced stages, in which ameloblasts had become fully differentiated, the stratum intermedium no longer expressed NCAM (Fig. 2h). 3.5. Immunofluorescence detection In the course of this study, we found NCAM transcripts expressed in some positions that we had not recognised as NCAM expressing in our earlier immunofluorescence studies. So, we re-examined the presence of NCAM polypeptides in those positions by using some of the frozen sections prepared for in situ hybridisation and the same monoclonal antibody as before. Although non-specific fluorescence was observed in these sections, especially in red blood cells (Fig. 2b and g), all the areas in which we had detected NCAM polypeptides in our previous studies were NCAM-positive in these sections as well. In addition, the mesenchyme of the prospective cusp-forming areas at the early bell stage and some of the cells of the stratum intermedium during the bell stage exhibited NCAM expression (Fig. 2b and g). However, fluorescence indicating the presence of NCAM polypeptides was not clearly demonstrated in the dental papilla at the cap stage (data not shown), where NCAM transcripts had been demonstrated by in situ hybridisation.
4. Discussion 4.1. Evaluation of newly identified NCAM-expressing sites We confirm the presence of NCAM mRNA in developing molar tooth germs. NCAM transcripts were detected in both epithelial and mesenchymal cells. The time- and space-dependent expression pattern of NCAM mRNA was mostly consistent with that of the NCAM polypeptides we had reported from immunofluorescence studies (Obara and Takeda, 1993a,b). However, we detected hybridisation signals in three distinct positions that we had not previously described as NCAM-positive. The positions were the dental papilla at the cap stage, the papillary mesenchyme beneath the prospective cusps at the early bell stage, and some of the stratum intermedium in tooth germs during
the bell stage. Because adjacent sections treated under the same conditions but with the sense probe did not show such reaction products by alkaline phophatase, the signals did not seem to be caused by non-specific staining or by the activity of intrinsic enzymes. Then, we examined the presence of NCAM polypeptides by immunofluorescence using some of the frozen sections prepared for in situ hybridisation. In spite of the autofluorescence caused by the tissue preparation, NCAM polypeptides were apparently localised in two of the three positions mentioned above, i.e. the dental papilla beneath the prospective cusp at the early bell stage and in the stratum intermedium during the bell stage. This transient or regionally restricted expression might explain why we failed to detect these positions in our previous studies. On the other hand, the amount of NCAM in the dental papilla at the cap stage was very small, and the immunofluorescence difficult to distinguish from autofluorescence. Even in our previous immunofluorescence studies, the presence of NCAM in the dental papilla at the cap stage was ambiguous. As this faint fluorescence was hardly distinguishable from the autofluorescence found in control sections, we concluded that the dental papilla was negative. However, the present study did reveal weak but positive in situ hybridisation signals in the dental papilla at the cap stage, indicating a misinterpretation in our earlier studies. Immunohistochemical detection of proteins is often impaired totally or partially by numerous factors such as fixatives, embedding methods and staining procedures. A reduction in immunoreactivity may completely impair weak expression but have only a trivial effect on strong expression, which could explain the different appearances when NCAM mRNA and protein expression profiles were compared. 4.2. Expression of NCAM in the dental mesenchyme Recombination and cell labelling studies (Chai et al., 2000; Imai et al., 1996; Lumsden, 1988) have demonstrated the contribution of mammalian cranial neural crest cells to tooth development. These cells migrate into the first branchial arch and reside in the maxillary and mandibular processes before the morphological signs of tooth development appear (Chai et al., 2000; Imai et al., 1996; Osumi-Yamashita et al., 1994; Serbedzija et al., 1992; Tan and Morriss-Kay, 1986). In mouse embryos, the earliest known instructive potency for tooth induction resides in the oral epithelium at E9 (Lumsden, 1988; Mina and Kollar, 1987), and at that time the mesenchyme of the branchial arch is almost entirely occupied by cranial neural crest cells (Chai et al., 2000). In the following few days, the tooth-inductive potential shifts from the epithelium to the mesenchyme (Kollar and Baird, 1969; Lumsden, 1988; Mina and Kollar, 1987), while the neural crest cells become concentrated beneath the oral epithelium (Chai et al., 2000). In this and previous studies (Obara and Takeda, 1993a), we detected the earliest NCAM expression in the dental mesenchyme at the bud stage during first molar development. Therefore,
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it is unlikely that NCAM expression is involved in the cessation of the neural crest-cell migration or in their condensation beneath the epithelium. Even if specific cell–cell adhesion is required for these processes, molecules other than NCAM may be responsible. Otherwise, cell–matrix interaction could play a more significant part than cell–cell adhesion during these early stages. According to a study by Vainio et al. (1991), syndecan, a cell-surface matrix receptor, is expressed in the mesenchyme (and also in the epithelium) of the mandibular and maxillary processes as early as E10. At E11, this molecule is found together with tenascin, an extracellular matrix protein, in the dental mesenchyme under the thickened epithelium (Thesleff et al., 1996), where we could not detect any sign of NCAM expression. Syndecan and tenascin are induced in the mesenchyme by epithelial–mesenchymal interactions (Vainio et al., 1989) and are thought to play a part in the condensation of the dental mesenchymal cells (Thesleff et al., 1990, 1996). Consequently, these molecules seem to be responsible at least in a part for the behaviour of mesenchymal cells during the initial stages of tooth development. As mentioned earlier, NCAM expression in the dental mesenchyme obviously began after the initial epithelial– mesenchymal interactions and changed together with the mesenchymal differentiation. These facts imply that the expression of NCAM in the mesenchyme might be regulated directly or indirectly through epithelial–mesenchymal interactions. Studies on the control mechanisms of NCAM expression have revealed that the NCAM promoter contains binding sites for homeodomains (Edelman and Jones, 1998). Hence, it is conceivable that some of the transcription factors expressed during tooth development are involved in the regulation of NCAM expression in the dental mesenchymal cells. Among transcription factors known to be expressed during tooth development, Barx1 is a possible candidate, because it was found by virtue of its binding to the promoter of the NCAM gene and is expressed in molar mesenchyme (Tissier-Seta et al., 1995). However, the connections between Barx1 and NCAM gene expression in tooth germs should be further investigated in detail. In contrast with the dental papilla, the dental follicle constantly expressed NCAM from the time of its initial differentiation to P4, the oldest stage examined here. Once NCAM expression had commenced in the peripheral part of the dental mesenchyme, the periphery of the entire molar region was always occupied by the NCAM-expressing mesenchyme throughout the stages during which the second and third molars arose posterior to the previously formed teeth and developed. This continuous expression of NCAM in the dental follicle is in agreement with our earlier findings from immunofluorescence studies (Obara and Takeda, 1993a,b). The tissue in the molar region at stages later than E12 can generate all three molars if it is dissected and cultured in oculo, even though the second and third molars are not yet morphologically visible at the time of dissection (Lumsden, 1979). This means that additional immigration of neural
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crest cells is not necessarily required for the generation of these later formed teeth and that the mesenchymal cells already located in the region can provide all of the cells required for their development. If this is also the case in vivo, then when the dental lamina extends from the posterior end of the first molar epithelial cap, the mesenchymal cells might have to migrate along the epithelium as well as proliferate to increase their number. Similarly, before and during the time that the third molar develops posterior to the second, the mesenchymal cells competent for its formation would have to maintain their position around its primordium. Thus, NCAM expressed in the dental follicle could play a part in maintaining the dental mesenchyme as a mass. The strong expression around the posterior end of the extending dental epithelium also supports this idea. 4.3. Expression of NCAM in the enamel organ NCAM was also expressed in the epithelial components of the tooth germs. At the bud stage, expression was found in the inner cells of the enamel organ but not in the outer cells. In the enamel organ at the cap stage, expression was more restricted to just some of the inner cells; at the late bell stage, it appeared transiently in the stratum intermedium. On the other hand, cadherins and desmosomal proteins are more widely expressed in the enamel organ, indicating their involvement in both the maintenance and the regulation of its structural configuration (Fausser et al., 1998; Lüning et al., 1994; Obara et al., 1998; Palacios et al., 1995). Because the NCAM expression observed in the enamel organ overlaps with expression of E-cadherin and desmoglein, NCAM may cooperate with these molecules in the regulation of morphogenesis. An example of functional cooperation between NCAM and other adhesion molecules is found in NCAM knockout mice. In the pancreatic islets of NCAM-deficient mice, abnormal cell polarity accompanied by altered organisation of cadherins is observed, as well as impaired cell-type segregation, which suggests that cadherin-mediated adhesion and cell polarity are enhanced in the absence of proper NCAM function (Esni et al., 1999). If such a relation between NCAM and E-cadherin is applicable to the epithelial tooth bud, the expression of NCAM might support the morphological change in the epithelium by reducing cadherin-mediated adhesion and preventing polarisation of the cells in the inner part of the epithelial bud and cap. In contrast, the outer cells, which do not express NCAM, appear to keep their adhesion and polarity during the development of the enamel organ. The expression of NCAM in the stratum intermedium seemed complex. Signals indicating NCAM transcripts were detected in that stratum over a relatively wide range of developmental stages (from early to late bell), but neither in a continuous nor a uniform fashion (see Fig. 2a, c and e). The regional difference in expression is likely to be related to exact odontogenic stages (i.e. stages of ameloblast differentiation) at particular positions and thus probably with functional
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changes in the cells of the stratum intermedium. Because E-cadherin is expressed in the stratum intermedium (Fausser et al., 1998; Obara et al., 1998; Palacios et al., 1995), the expression of NCAM overlaps with that of E-cadherin in this case, too. Therefore, NCAM might also regulate functions in the stratum intermedium in concert with E-cadherin. The finding of the additional NCAM-expressing sites in both epithelial and mesenchymal components of the tooth germs suggests that the molecule might have more roles during tooth development than we had previously supposed.
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Edelman, G.M., Jones, F.S., 1998. Gene regulation of cell adhesion: a key step in neural morphogenesis. Brain Res. Rev. 26, 337–352. Esni, F., Täljedal, I.B., Perl, A.K., Cremer, H., Christofori, G., Semb, H., 1999. Neural cell adhesion molecule (N-CAM) is required for cell type segregation and normal ultrastructure in pancreatic islets. J. Cell Biol. 144, 325–337. Fausser, J.L., Schlepp, O., Aberdam, D., Meneguzzi, G., Ruch, J.V., Lesot, H., 1998. Localization of antigens associated with adherens junctions, desmosomes, and hemidesmosomes during murine molar morphogenesis. Differentiation 63, 1–11. Friedlander, D.R., Milev, P., Karthikeyan, L., Margolis, R.K., Margolis, R.U., Grumet, M., 1994. The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth, and inhibits neuronal adhesion and neurite outgrowth. J. Cell Biol. 125, 669–680. Hirn, M., Pieerres, M., Degostini-Bazin, H., Hirsch, M., Goridis, C., 1981. Monoclonal antibody against cell surface glycoprotein of neurons. Brain Res. 214, 433–439. Imai, H., Osumi-Yamashita, N., Ninomiya, Y., Eto, K., 1996. Contribution of early-emigrating midbrain crest cells to the dental mesenchyme of mandibular molar teeth in rat embryos. Dev. Biol. 176, 151–165. Jernvall, J., Thesleff, I., 2000. Reiterative signalling and patterning during mammalian tooth morphogenesis. Mechanisms Dev. 92, 19–29. Kollar, E.J., Baird, G.R., 1969. The influence of the dental papilla on the development of tooth shape in embryonic mouse tooth germs. J. Embryol. Exp. Morphol. 21, 131–148. Lumsden, A.G.S., 1979. Pattern formation in the molar dentition of the mouse. J. Biol. Buccale 7, 77–103. Lumsden, A.G.S., 1988. Spacial organisation of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germs. Development 103 (Suppl.), 155–169. Lüning, C., Rass, A., Rozell, B., Wroblewski, J., Öbrink, B., 1994. Expression of E-cadherin during craniofacial development. J. Craniofacial Genet. Dev. Biol. 14, 207–216. Lüning, C., Wroblewski, J., Öbrink, B., Hammarström, L., Rozell, B., 1995. Distribution of C-CAM in developing oral tissues. Anat. Embryol. 190, 251–261. Marrs, J., Nelson, J., 1996. Cadherin cell adhesion molecules in differentiation and embryogenesis. Int. Rev. Cytol. 165, 159–205. Mina, M., Kollar, E.J., 1987. The induction of odontogenesis in non-dental mesenchyme combined with early murine mandibular arch epithelium. Arch. Oral Biol. 32, 123–127. Obara, N., Takeda, M., 1993a. Expression of neural cell adhesion molecule (NCAM) during the first molar development in the mouse. Anat. Embryol. 187, 209–219. Obara, N., Takeda, M., 1993b. Expression of the neural cell adhesion molecule (NCAM) during second- and third-molar development in the mouse. Anat. Embryol. 188, 13–20. Obara, N., Takeda, M., 1997. Distribution of the neural cell adhesion molecule (NCAM) during pre- and post-natal development of mouse incisors. Anat. Embryol. 195, 193–202. Obara, N., Suzuki, Y., Nagai, Y., Takeda, M., 1998. Expression of E- and P-cadherin during tooth morphogenesis and cytodifferentiation of ameloblasts. Anat. Embryol. 197, 469–475. Obara, N., Suzuki, Y., Nagai, Y., Takeda, M., 1999. Immunofluorescence detection of cadherins in mouse tooth germs during root development. Arch. Oral Biol. 44, 415–421.
N. Obara et al. / Archives of Oral Biology 47 (2002) 805–813 Osumi-Yamashita, N., Ninomiya, Y., Doi, H., Eto, K., 1994. The contribution of both forebrain and midbrain crest cells to the mesenchyme in the frontonasal mass of mouse embryos. Dev. Biol. 164, 409–419. Owens, G.C., Edelman, G.M., Cunningham, B.A., 1987. Organisation of the neural cell adhesion molecule (N-CAM) gene: alternative exon usage as the basis for different membrane-associated domains. Proc. Natl. Acad. Sci. U.S.A. 84, 294–298. Palacios, J., Benito, N., Berpaquero, R., Pizarro, A., Cano, A., Gamallo, C., 1995. Differential spatiotemporal expression of E- and P-cadherin during mouse tooth development. Int. J. Dev. Biol. 39, 663–666. Probstmeier, R., Kuhn, K., Schachner, M., 1989. Binding properties of the neural cell adhesion molecule to different components of the extracellular matrix. J. Neurochem. 53, 1794–1801. Santoni, M.J., Barthels, D., Vooper, G., Boned, A., Goridis, C., Whille, W., 1989. Differential exon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain. EMBO J. 8, 385–392. Serbedzija, G.N., Bronner-Fraser, M., Fraser, S.E., 1992. Vital dye analysis of cranial neural crest cell migration in the mouse embryo. Development 116, 297–307. Takeichi, M., 1988. The cadherins: cell–cell adhesion molecules controlling animal morphogenesis. Development 102, 639– 655. Takeichi, M., 1990. Cadherins: a molecular family important in selective cell–cell adhesion. Annu. Rev. Biochem. 59, 237–252. Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451–1455.
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Tan, S.S., Morriss-Kay, G.M., 1986. Analysis of cranial neural crest cell migration and early fates in post-implantation rat chimaeras. J. Embryol. Exp. Morphol. 98, 21–58. Terling, C., Heymann, R., Rozell, B., Öbrink, B., Wroblewski, J., 1998. Dynamic expression of E-cadherin in ameloblasts and cementoblasts in mice. Eur. J. Oral Sci. 106 (Suppl.1), 137–142. Thesleff, I., Sharpe, P., 1997. Signalling networks regulating dental development. Mechanisms Dev. 67, 111–123. Thesleff, I., Vaahtokari, A., Vainio, S., 1990. Molecular changes during determination and differentiation of the dental mesenchymal cell lineage. J. Biol. Buccale 18, 179–188. Thesleff, I., Vaatokari, A., Vainio, S., Jowett, A., 1996. Molecular mechanisms of cell and tissue interactions during early tooth development. Anat. Record 245, 151–161. Tissier-Seta, J.P., Mucchielli, M.L., Mark, M., Mattei, M.G., Goridis, C., Brunet, J.F., 1995. Barx1, a new mouse homeodomain transcription factor expressed in craniofacial ectomesenchyme and the stomach. Mechanisms Dev. 51, 3–15. Tucker, A.S., Sharpe, P.T., 1999. Molecular genetics of tooth morphogenesis and patterning: the right shape in the right place. J. Dental Res. 78, 826–834. Vainio, S., Jalkanen, M., Thesleff, I., 1989. Syndecan and tenascin expression is induced by epithelial-mesenchymal interactions in embryonic tooth mesenchyme. J. Cell Biol. 108, 1945–1954. Vainio, S., Jalkanen, M., Vaahtokari, A., Sahlberg, C., Mali, M., Bernfield, M., Thesleff, I., 1991. Expression of syndecan gene is induced early, is transient, and correlates with changes in mesenchymal cell proliferation during tooth organogenesis. Dev. Biol. 147, 322–333.
Expression of neural cell-adhesion molecule mRNA during mouse molar tooth development Nobuko Obara a,∗ , Yuko Suzuki a , Yasuko Nagai a , Hiromasa Nishiyama b , Itaru Mizoguchi b , Masako Takeda a b
a Department of Anatomy, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Accepted 7 November 2002
Abstract This study employed in situ hybridisation using a probe recognising all isoforms of the molecule. Expression of the molecule in tooth germs started at embryonic day 13, when they were at the bud stage. Both inner cells of the epithelial bud and peripheral cells of the dental mesenchyme were positive. At the cap stage, positive cells were found in the inner part of the enamel organ but only in a limited area near the outer enamel epithelium. In the mesenchyme at the cap stage, expression was weak in the dental papilla and strong in the follicle. From the bell stage onward, epithelial cells in the enamel organ were negative except for the cells of the stratum intermedium, which were transiently positive at early and late bell stages. In the dental papilla, expression had mostly ceased during and after the bell stage, although transient expression was found in cuspal areas at the early bell stage. The dental follicle strongly expressed neural cell-adhesion molecule (NCAM) to the end of the experimental period, at post-natal day 4. In contrast to the first molar at its earliest stage of appearance, in which both the thickened epithelium and surrounding mesenchyme were negative for the expression of the molecule, the second molar appeared as a combination of extending epithelial thickenings and mesenchymal cells strongly positive for its expression. This study newly identifies the dental papilla and the stratum intermedium as NCAM-expressing sites. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: NCAM; Tooth germs; In situ hybridisation; Morphogenesis; Differentiation; Odontogenesis
1. Introduction Cell–cell adhesion is crucial for organogenesis. In developing organs, molecules that mediate specific cell–cell adhesion are expressed in a spatiotemporal manner and are thought to have a regulatory role in morphogenesis (Edelman, 1988; Edelman and Crossin, 1991; Takeichi, 1988, 1990, 1991). Among the various cell-adhesion molecules, cadherins, which mediate Ca2+ -dependent cell– cell adhesion, have been the best studied. In addition to Abbreviations: BCIP, 5-bromo-4-chloro-3-indol-phosphate; DIG, digoxigenin; NCAM, neural cell-adhesion molecule; NBT, tetrazolium chloride; PBS, phosphate-buffered saline; SSC, standard saline citrate buffer ∗ Corresponding author. Tel.: +81-1332-3-1236; fax: +81-1332-3-1236. E-mail address: [email protected] (N. Obara).
their basic function, i.e. the mediation of selective cell adhesion, they perform more specialised tasks including the establishment of cell-surface polarity and changes in gene expression as a consequence of adhesion (Marrs and Nelson, 1996). The expression of cadherin subclasses is developmentally regulated, indicating their correlation with a variety of morphogenetic events (Takeichi, 1988). Another well-characterised cell-adhesion molecule is neural cell-adhesion molecule (NCAM), which is also expressed in many developing organs and thus is thought to be involved in the regulation of morphogenesis (Crossin et al., 1985). NCAM belongs to the immunoglobulin superfamily and mediates Ca2+ -independent cell–cell adhesion by homophilic binding of proteins on neighbouring cells. Alternative RNA splicing and post-translational modification with polysialic acids generate many molecular forms with differences in molecular size and binding properties (Barbas et al., 1988; Owens et al., 1987; Santoni et al., 1989). In
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addition to mediating cell–cell adhesion, the binding of NCAM can affect intracellular signalling and thereby alter gene expression (Crossin and Krushel, 2000; Doherty and Walsh, 1994). This molecule is also known to participate in specific heterophilic interactions with extracellular matrix components (Cole et al., 1986; Cole and Akeson, 1989; Friedlander et al., 1994; Probstmeier et al., 1989). This complexity of molecular properties and functions raises the possibility that NCAM has multiple regulatory roles in developing organs, although the biological meaning of such complexity has not yet been fully established. Tooth development is an excellent model for studying organogenesis and involves several fundamental problems shared by other systems of organ development, e.g. neural crest-cell migration, epithelial–mesenchymal interactions, tissue patterning, morphogenesis and cytodifferentiation. In recent years, studies on tooth development have much advanced and now concern molecular mechanisms, in which growth factors, other signalling molecules and transcription factors are found to be involved in the above processes (Åberg et al., 1997; Jernvall and Thesleff, 2000; Thesleff and Sharpe, 1997; Tucker and Sharpe, 1999). There are reports of the expression patterns of several cell-adhesion molecules during tooth development (Chuong, 1990; Lüning et al., 1994, 1995; Fausser et al., 1998; Obara and Takeda, 1993a,b, 1997; Obara et al., 1998, 1999; Palacios et al., 1995; Terling et al., 1998), but less is known about these molecules than about other molecular factors in tooth development. In earlier studies, we localised NCAM polypeptides in tooth germs by using a monoclonal antibody and demonstrated a dynamic change in their expression pattern during tooth development (Obara and Takeda, 1993a,b, 1997). The spatiotemporally regulated expression of NCAM implied that it has a regulatory role in tooth development, but problems about how the expression of NCAM in tooth germs is regulated and its role in the particular sites where it is expressed remain to be resolved. As a basis for investigating these problems, we have now examined the expression pattern of NCAM during molar tooth development by means of in situ hybridisation using a probe that hybridises with a sequence included in all NCAM isoforms. Because we detected NCAM mRNA in the places where we had previously detected NCAM polypeptides, and also identified a few additional sites expressing NCAM transcripts, we also re-examined the newly identified NCAM-expressing sites by immunofluorescence to confirm the presence of NCAM polypeptides.
2. Materials and methods 2.1. Animals Embryos were obtained by mating male and female ddY mice. The ages of embryos were determined by the day of appearance of the vaginal plug (E0). The gestational period
of these animals is 19 days, and the day of birth was taken as day 0 of post-natal development (P0). 2.2. Tissue preparation To obtain embryos, pregnant females were killed by cervical dislocation. Tissues including the tooth germs or primordia were dissected from the embryos in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and fixed in the same solution overnight at 4 ◦ C. The post-natal mice used were anaesthetised with chloroform and perfused with the same fixative via the left ventricle. Their tissues were then dissected and fixed in the same way as for embryos. Fixed tissues were immersed in 20% sucrose in phosphate-buffered saline (PBS) for 2 h at 4 ◦ C, embedded in Tissue-Tek O.C.T. Compound (Miles Laboratories, Elkhart, Indiana), and frozen with Cryon Spray Freezer (Oken Shoji, Tokyo). Sections cut at 10 m thickness in a cryostat were picked up on silane-coated glass slides and air-dried for more than 30 min at room temperature. 2.3. In situ hybridisation Sections were immersed in absolute ethanol for 5 min and in 0.2N HCl for 20 min, and then washed twice in PBS for 5 min each. Next, the sections were treated with 2 g/ml of proteinase K (Takara Shuzo Co., Kyoto, Japan) at 37 ◦ C for 15 min, washed in PBS, and refixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 min. After having been washed twice in PBS and once in distilled water for 5 min each time, the sections were air-dried and hybridised. Hybridisation was performed at 45 ◦ C for 14–16 h with a digoxigenin (DIG)-labelled RNA probe (50 ng/ml) in a hybridisation solution containing 50% formamide, 0.3 M NaCl, 0.02 M Tris–HCl (pH 8.0), 1 mM EDTA, 10% dextran sulphate, one time in denhardt solution, 1 mg/ml yeast tRNA and 0.02 % sodium dodecyl sulphate. Hybridised sections were washed at 45 ◦ C in a solution containing 50% formamide and two times in standard saline citrate buffer (SSC; pH 7.0) for 60 min, and thereafter twice in two times in SSC for 5 min each time. They were then treated with 20 g/ml RNase (type II-A; Sigma, St. Louis, MO, USA)/two times in SSC at 37 ◦ C for 30 min, and washed at 45 ◦ C in 50% formamide/two times in SSC followed by 50% formamide/one time in SSC for 60 min each. After having been washed three times in PBS, the sections were incubated with 1% blocking reagent (Boeringer Manheim GmbH, Manheim, Germany) in maleic acid buffer (pH 7.5) for 60 min at room temperature. Subsequently, they were incubated overnight at 4 ◦ C with alkaline phosphatase-conjugated anti-DIG Fab fragments diluted 1:500 in PBS containing 1% bovine serum albumin. After three washes in Tris-buffered saline (0.1 M Tris–HCl, 0.15 M NaCl, pH 7.5), chromogenic reactions were carried out using tetrazolium chlo-
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ride (NBT)/5-bromo-4-chloro-3-indol-phosphate (BCIP; Boeringer Manheim).
expressing NCAM existed outside of the tooth primordia at this stage (Fig. 1a).
2.4. Hybridisation probes
3.2. Bud and cap stages
A complementary DNA corresponding to mouse NCAM mRNA at positions 397–1354 (Barthels et al., 1987) was prepared by reverse transcription–polymerase chain reaction from mouse brain total RNA, and subcloned into HindIII/ EcoRI sites of pT7/T3␣-18 (Life Technologies, Grand Island, NY, USA). DIG-labelled RNA probes were synthesised in the presence of DIG-UTP (DIG RNA labelling mix; Boeringer Manheim) by using T7 RNA polymerase and HindIII-linearised template (antisense probe) or T3 RNA polymerase and EcoRI-linearised template (sense probe). Probe length was reduced to 200 nucleotides by limited alkaline hydrolysis (Cox et al., 1984). The mRNA at positions 397–1354 corresponds to a part of exon 2, entire exons 3–7 and a part of exon 8 (Owens et al., 1987), and so the probe recognises mRNAs encoding the extracellular immunoglobulin-like domains that commonly exist in all isoforms of NCAM molecules (Santoni et al., 1989).
At E13, marginal cells of the epithelial tooth bud did not express NCAM but the inner cells did. The molecule was also expressed in mesenchyme surrounding the tooth bud, where expression was strong in the peripheral area and weak or almost negative in the inner area adjacent to the tip of the bud (Fig. 1b). In sections of the posterior region of E13 molar tooth germs, where the dental epithelium was shaped like an early bud or lamina at the posterior end, cells strongly expressing NCAM (Fig. 1c) surrounded the epithelium. In cap stage tooth germs, the expression of NCAM by epithelial cells had become more restricted to the dental lamina and the cells lining the outer enamel epithelium (Fig. 1d). Differentiating stellate reticulum and the enamel knot, as well as the inner and outer enamel epithelia, did not express NCAM. Both the dental papilla and the dental follicle at the cap stage expressed NCAM, but the strength of expression differed, being weak in the papilla and strong in the follicle (Fig. 1d). Through the bud and cap stages, cells expressing NCAM were also present in the mesenchyme outside of the tooth germs (Fig. 1b–f).
2.5. Indirect immunofluorescence staining Rat monoclonal antibody (IgG) to NCAM (H28.123; Hirn et al., 1981) was purchased from Immunotech S.A. (Marseille, France). Fluorescein isothiocyanate-conjugated, affinity-purified antibodies to rat IgG was purchased from Cappel, Organon Teknica (West Chester, PA) and used after absorption with purified mouse IgG (Zymed, San Francisco). Staining procedures were as previously described (Obara and Takeda, 1993a).
3. Results During all the experimental stages, in situ hybridisation using the sense sequence as a probe resulted in no reaction product by alkaline phosphatase as long as the hybridisation was performed under the same conditions as used in the experiments with the antisense probe (Fig. 2a). 3.1. Initiation stages The earliest morphological sign of tooth development became visible as epithelial thickenings on the day 11 of embryonic development (E11). At the site of tooth initiation, signals indicating NCAM mRNA expression were not detected in either the thickened epithelia or in the mesenchyme directly surrounding them. In embryos at E12 the dental epithelium had increased in thickness. NCAM transcripts were hardly detectable either in the mesenchyme adjacent to the developing dental epithelium or in the dental epithelium itself. However, mesenchymal cells weakly
3.3. Early stages of the second and third molars It was difficult in histological sections to define the initiation of the second and third molars as epithelial thickenings, which for first molars had appeared at E11. In fact, a distinct border between the first and the second molar could not be determined in embryos at stages earlier than E14. In E14 embryos, the epithelial bud of the second molar could be distinguished from the epithelial cap of the first molar. NCAM expression in the epithelial bud of the second molar was similar to that in the first molar, where only the inner epithelial cells expressed the molecule (Fig. 1e and f). However, the pattern of NCAM expression in the mesenchyme of the second molar tooth germs was slightly different from that of the first molars, especially at the early bud stage. The mesenchymal cells surrounding the tooth bud more strongly expressed NCAM than those in the first molar at the equivalent stage (Fig. 1e; compare with Fig. 1b). Particularly, the bud-shaped epithelium in the posterior part was in contact with mesenchymal cells strongly expressing NCAM (Fig. 1f). Whereas the sequential development of first and second molars begins within a short period, development of the third molar commences after a delay. Because of its position and smaller size, study of the initial stages of the third molar was more difficult than with the second molar, and its formation could be recognised in histological sections only after birth. Posterior to the second molar of P0 mice, there was a narrow space filled with NCAM-expressing mesenchymal cells. In P0 mice, an epithelial bud-like structure growing into the
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Fig. 1. Expression of NCAM during the early stages of tooth development. In situ hybridisation of frontal sections through areas bearing molar tooth germs. (a) Embryonic day (E) 12. Dental lamina (arrowheads) and surrounding mesenchyme are negative for NCAM expression. Mesenchymal cells apart from the tooth primordium weakly express the molecule. (b) Section through the middle parts of the tooth germs at E13. In the epithelial tooth buds (arrowheads), the peripheral cells are negative for NCAM, whereas the cells in the inner part are positive. In the dental mesenchyme, NCAM expression is stronger in the outer part (differentiating dental follicle), but very weak in the area surrounding the tip of the epithelial buds. (c) Section through the posterior ends of the tooth germs shown in (b). Mesenchymal cells surrounding the dental epithelia strongly express NCAM. (d) First molar tooth germs at E14. NCAM-expressing epithelial cells are localised beneath the outer enamel epithelium (arrows) and the inner area of the dental lamina. NCAM expression is weak in the dental papilla (P) and strong in the dental follicle (F). (e, f) Sections of a mandibular second molar tooth germ at E14 through middle (e) and posterior (f) parts. In the middle part, NCAM expression in the mesenchyme around the tip of the epithelial bud (∗) is weak, whereas the posterior part of the bud is surrounded by strongly NCAM-expressing cells. Note that NCAM expression is stronger than in the first molar tooth germs at the corresponding stage of tooth development (bar = 100 m).
space was apparent, and the budding epithelium expressed NCAM in its inner part (Fig. 2i). From the cap stage onward, the expression patterns of NCAM in the second (data not shown) and third (Fig. 2j) molars during the experimental period were fundamentally the same as in the first molar.
3.4. Bell and later stages At the early bell stage, NCAM was expressed in the mesenchyme of prospective cusp-forming areas, but not in the remaining part of the papilla. The molecule was expressed
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Fig. 2. Expression of NCAM in tooth germs at late embryonic and post-natal stages. (a) In situ hybridisation of a mandibular first molar tooth germ at embryonic day (E)16. Signals indicating NCAM transcripts are found in the stratum intermedium (SI), the dental follicle and the papillary mesenchyme in the cusp-forming areas (arrows). (b) Section of the tooth germ shown in (a), stained using anti-NCAM monoclonal antibody. Anti-NCAM immunoreactivity exists in the areas where NCAM transcripts are detected. Red blood cells with strong autofluorescence are found in both the dental papilla and the dental follicle. (c, d) In situ hybridisation of a mandibular tooth germ at E18, hybridised with antisense (c) and sense (d) probes. NCAM expression is strong in the dental follicle. Weak expression is found in the papillary mesenchyme close to the dental follicle (arrows). (e) In situ hybridisation of a mandibular first molar tooth germ at post-natal day (P) 0. NCAM expression is found in the dental follicle and in the stratum intermedium (arrows). (f) Higher magnification of the area surrounded by the square in (e). Only the stratum intermedium expresses NCAM. (g) Mandibular first molar tooth germ at P0 stained with anti-NCAM monoclonal antibody. Stratum intermedium exhibits weak NCAM immunoreactivity. (h) In situ hybridisation of a mandibular first molar tooth germ at P2. Expression of NCAM is found in the dental follicle and in the nearby papillary mesenchyme (arrows). (i) In situ hybridisation of a mandibular third molar at P1. Signals indicating NCAM transcripts are found in the epithelial bud (arrowheads). The surrounding mesenchyme is filled with NCAM-expressing cells. (j) In situ hybridisation of a mandibular third molar at P4. Strong expression of NCAM is found in the dental follicle, whereas the expression is weak in the dental papilla. Arrowheads indicate enamel organ. Sections were made in the anterior–posterior planes in (e)–(j). M2, mandibular second molar; M3, mandibular third molar; SR, stellate reticulum (bar = 100 m).
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by the epithelial cells in the stratum intermedium and in the narrow space between the outer and inner enamel epithelium of the lateral part of the enamel organ (Fig. 2a). During subsequent stages, NCAM expression in the dental papilla decreased; in tooth germs at late bell or later stages it became restricted to the basal part of the papilla, which is continuous with the dental follicle. In contrast, the follicle cells continued to express NCAM from the bud stage onward (Fig. 2c and h). Expression of NCAM in the stratum intermedium was temporary or regional in appearance. In the first molar tooth germs at E18 and P0 (and also in the second molars at equivalent stages of development), NCAM expression in the stratum intermedium was not uniform within a given germ (Fig. 2e). In tooth germs at more advanced stages, in which ameloblasts had become fully differentiated, the stratum intermedium no longer expressed NCAM (Fig. 2h). 3.5. Immunofluorescence detection In the course of this study, we found NCAM transcripts expressed in some positions that we had not recognised as NCAM expressing in our earlier immunofluorescence studies. So, we re-examined the presence of NCAM polypeptides in those positions by using some of the frozen sections prepared for in situ hybridisation and the same monoclonal antibody as before. Although non-specific fluorescence was observed in these sections, especially in red blood cells (Fig. 2b and g), all the areas in which we had detected NCAM polypeptides in our previous studies were NCAM-positive in these sections as well. In addition, the mesenchyme of the prospective cusp-forming areas at the early bell stage and some of the cells of the stratum intermedium during the bell stage exhibited NCAM expression (Fig. 2b and g). However, fluorescence indicating the presence of NCAM polypeptides was not clearly demonstrated in the dental papilla at the cap stage (data not shown), where NCAM transcripts had been demonstrated by in situ hybridisation.
4. Discussion 4.1. Evaluation of newly identified NCAM-expressing sites We confirm the presence of NCAM mRNA in developing molar tooth germs. NCAM transcripts were detected in both epithelial and mesenchymal cells. The time- and space-dependent expression pattern of NCAM mRNA was mostly consistent with that of the NCAM polypeptides we had reported from immunofluorescence studies (Obara and Takeda, 1993a,b). However, we detected hybridisation signals in three distinct positions that we had not previously described as NCAM-positive. The positions were the dental papilla at the cap stage, the papillary mesenchyme beneath the prospective cusps at the early bell stage, and some of the stratum intermedium in tooth germs during
the bell stage. Because adjacent sections treated under the same conditions but with the sense probe did not show such reaction products by alkaline phophatase, the signals did not seem to be caused by non-specific staining or by the activity of intrinsic enzymes. Then, we examined the presence of NCAM polypeptides by immunofluorescence using some of the frozen sections prepared for in situ hybridisation. In spite of the autofluorescence caused by the tissue preparation, NCAM polypeptides were apparently localised in two of the three positions mentioned above, i.e. the dental papilla beneath the prospective cusp at the early bell stage and in the stratum intermedium during the bell stage. This transient or regionally restricted expression might explain why we failed to detect these positions in our previous studies. On the other hand, the amount of NCAM in the dental papilla at the cap stage was very small, and the immunofluorescence difficult to distinguish from autofluorescence. Even in our previous immunofluorescence studies, the presence of NCAM in the dental papilla at the cap stage was ambiguous. As this faint fluorescence was hardly distinguishable from the autofluorescence found in control sections, we concluded that the dental papilla was negative. However, the present study did reveal weak but positive in situ hybridisation signals in the dental papilla at the cap stage, indicating a misinterpretation in our earlier studies. Immunohistochemical detection of proteins is often impaired totally or partially by numerous factors such as fixatives, embedding methods and staining procedures. A reduction in immunoreactivity may completely impair weak expression but have only a trivial effect on strong expression, which could explain the different appearances when NCAM mRNA and protein expression profiles were compared. 4.2. Expression of NCAM in the dental mesenchyme Recombination and cell labelling studies (Chai et al., 2000; Imai et al., 1996; Lumsden, 1988) have demonstrated the contribution of mammalian cranial neural crest cells to tooth development. These cells migrate into the first branchial arch and reside in the maxillary and mandibular processes before the morphological signs of tooth development appear (Chai et al., 2000; Imai et al., 1996; Osumi-Yamashita et al., 1994; Serbedzija et al., 1992; Tan and Morriss-Kay, 1986). In mouse embryos, the earliest known instructive potency for tooth induction resides in the oral epithelium at E9 (Lumsden, 1988; Mina and Kollar, 1987), and at that time the mesenchyme of the branchial arch is almost entirely occupied by cranial neural crest cells (Chai et al., 2000). In the following few days, the tooth-inductive potential shifts from the epithelium to the mesenchyme (Kollar and Baird, 1969; Lumsden, 1988; Mina and Kollar, 1987), while the neural crest cells become concentrated beneath the oral epithelium (Chai et al., 2000). In this and previous studies (Obara and Takeda, 1993a), we detected the earliest NCAM expression in the dental mesenchyme at the bud stage during first molar development. Therefore,
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it is unlikely that NCAM expression is involved in the cessation of the neural crest-cell migration or in their condensation beneath the epithelium. Even if specific cell–cell adhesion is required for these processes, molecules other than NCAM may be responsible. Otherwise, cell–matrix interaction could play a more significant part than cell–cell adhesion during these early stages. According to a study by Vainio et al. (1991), syndecan, a cell-surface matrix receptor, is expressed in the mesenchyme (and also in the epithelium) of the mandibular and maxillary processes as early as E10. At E11, this molecule is found together with tenascin, an extracellular matrix protein, in the dental mesenchyme under the thickened epithelium (Thesleff et al., 1996), where we could not detect any sign of NCAM expression. Syndecan and tenascin are induced in the mesenchyme by epithelial–mesenchymal interactions (Vainio et al., 1989) and are thought to play a part in the condensation of the dental mesenchymal cells (Thesleff et al., 1990, 1996). Consequently, these molecules seem to be responsible at least in a part for the behaviour of mesenchymal cells during the initial stages of tooth development. As mentioned earlier, NCAM expression in the dental mesenchyme obviously began after the initial epithelial– mesenchymal interactions and changed together with the mesenchymal differentiation. These facts imply that the expression of NCAM in the mesenchyme might be regulated directly or indirectly through epithelial–mesenchymal interactions. Studies on the control mechanisms of NCAM expression have revealed that the NCAM promoter contains binding sites for homeodomains (Edelman and Jones, 1998). Hence, it is conceivable that some of the transcription factors expressed during tooth development are involved in the regulation of NCAM expression in the dental mesenchymal cells. Among transcription factors known to be expressed during tooth development, Barx1 is a possible candidate, because it was found by virtue of its binding to the promoter of the NCAM gene and is expressed in molar mesenchyme (Tissier-Seta et al., 1995). However, the connections between Barx1 and NCAM gene expression in tooth germs should be further investigated in detail. In contrast with the dental papilla, the dental follicle constantly expressed NCAM from the time of its initial differentiation to P4, the oldest stage examined here. Once NCAM expression had commenced in the peripheral part of the dental mesenchyme, the periphery of the entire molar region was always occupied by the NCAM-expressing mesenchyme throughout the stages during which the second and third molars arose posterior to the previously formed teeth and developed. This continuous expression of NCAM in the dental follicle is in agreement with our earlier findings from immunofluorescence studies (Obara and Takeda, 1993a,b). The tissue in the molar region at stages later than E12 can generate all three molars if it is dissected and cultured in oculo, even though the second and third molars are not yet morphologically visible at the time of dissection (Lumsden, 1979). This means that additional immigration of neural
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crest cells is not necessarily required for the generation of these later formed teeth and that the mesenchymal cells already located in the region can provide all of the cells required for their development. If this is also the case in vivo, then when the dental lamina extends from the posterior end of the first molar epithelial cap, the mesenchymal cells might have to migrate along the epithelium as well as proliferate to increase their number. Similarly, before and during the time that the third molar develops posterior to the second, the mesenchymal cells competent for its formation would have to maintain their position around its primordium. Thus, NCAM expressed in the dental follicle could play a part in maintaining the dental mesenchyme as a mass. The strong expression around the posterior end of the extending dental epithelium also supports this idea. 4.3. Expression of NCAM in the enamel organ NCAM was also expressed in the epithelial components of the tooth germs. At the bud stage, expression was found in the inner cells of the enamel organ but not in the outer cells. In the enamel organ at the cap stage, expression was more restricted to just some of the inner cells; at the late bell stage, it appeared transiently in the stratum intermedium. On the other hand, cadherins and desmosomal proteins are more widely expressed in the enamel organ, indicating their involvement in both the maintenance and the regulation of its structural configuration (Fausser et al., 1998; Lüning et al., 1994; Obara et al., 1998; Palacios et al., 1995). Because the NCAM expression observed in the enamel organ overlaps with expression of E-cadherin and desmoglein, NCAM may cooperate with these molecules in the regulation of morphogenesis. An example of functional cooperation between NCAM and other adhesion molecules is found in NCAM knockout mice. In the pancreatic islets of NCAM-deficient mice, abnormal cell polarity accompanied by altered organisation of cadherins is observed, as well as impaired cell-type segregation, which suggests that cadherin-mediated adhesion and cell polarity are enhanced in the absence of proper NCAM function (Esni et al., 1999). If such a relation between NCAM and E-cadherin is applicable to the epithelial tooth bud, the expression of NCAM might support the morphological change in the epithelium by reducing cadherin-mediated adhesion and preventing polarisation of the cells in the inner part of the epithelial bud and cap. In contrast, the outer cells, which do not express NCAM, appear to keep their adhesion and polarity during the development of the enamel organ. The expression of NCAM in the stratum intermedium seemed complex. Signals indicating NCAM transcripts were detected in that stratum over a relatively wide range of developmental stages (from early to late bell), but neither in a continuous nor a uniform fashion (see Fig. 2a, c and e). The regional difference in expression is likely to be related to exact odontogenic stages (i.e. stages of ameloblast differentiation) at particular positions and thus probably with functional
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changes in the cells of the stratum intermedium. Because E-cadherin is expressed in the stratum intermedium (Fausser et al., 1998; Obara et al., 1998; Palacios et al., 1995), the expression of NCAM overlaps with that of E-cadherin in this case, too. Therefore, NCAM might also regulate functions in the stratum intermedium in concert with E-cadherin. The finding of the additional NCAM-expressing sites in both epithelial and mesenchymal components of the tooth germs suggests that the molecule might have more roles during tooth development than we had previously supposed.
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