Cd

Fig. 3. Examples of the analysis of cultured explants. (A) Appearance of an explant in the stereomicroscope. The epithelium as well as the TGFP-1-releasing bead have induced a translucent zone in dental mesenchyme. (B) Localization of cell proliferation with BrdU incorporation under the epithelium as well as around an FGF-4-releasing bead. (C) Whole-mount in situ hybridization analysis of Msx-1 gene expression indicating induction by the epithelium in the mesenchyme. (D) Whole-mount immunohistochemical staining showing stimulation of tenascin expression in the mesenchyme around an FGF-4-releasing bead. (E) Section of an explant of dental mesenchyme and a TGF6-1-releasing bead (the filter has been detached during processing). (F) Dark-field illumination of the explant in E showing the induction of tenascin-C transcripts by in situ hybridization analysis. e, dental epithelium; m, dental mesenchyme; b, bead.

Fig. 3. Examples of the analysis of cultured explants. (A) Appearance of an explant in the stereomicroscope. The epithelium as well as the TGFP-1-releasing bead have induced a translucent zone in dental mesenchyme. (B) Localization of cell proliferation with BrdU incorporation under the epithelium as well as around an FGF-4-releasing bead. (C) Whole-mount in situ hybridization analysis of Msx-1 gene expression indicating induction by the epithelium in the mesenchyme. (D) Whole-mount immunohistochemical staining showing stimulation of tenascin expression in the mesenchyme around an FGF-4-releasing bead. (E) Section of an explant of dental mesenchyme and a TGF6-1-releasing bead (the filter has been detached during processing). (F) Dark-field illumination of the explant in E showing the induction of tenascin-C transcripts by in situ hybridization analysis. e, dental epithelium; m, dental mesenchyme; b, bead.

3. The dissection and culture techniques are basically similar when different organs or different developmental stages of tooth germs are studied. Separation of the epithelium and mesenchyme can be accomplished in young tissues, after enzyme treatment, even without dissection by briefly vortexing the tissues. On the other hand, more advanced tissues require a longer incubation in the enzyme solution (up to 10 min). The time needed for best separation depends also on the batches of enzymes, and therefore the optimal time must always be checked for new batches of enzyme and for different tissues.

4. Different supporting materials can be used for the cultured explants. Lens paper may be used for large tissue pieces. The supporting material must allow good diffusion of the medium to the tissue, and therefore Millipore filters of 100-|im thickness are not suitable. (The thickness of Nuclepore filters is approx 10 |im.) Different pore size Nuclepore filter may be used. Small pores (0.05-0.2 |im) allow better examination of the explants in the stereomicroscope using transmitted light, but the tissues tend to detach from these filters more readily during fixation and other treatments after culture. Therefore, larger pore sizes (up to 1 | m) may be preferable, depending on the experiment.

5. The Trowell-type organ culture can be used for a variety of other organ culture designs. One example is the transfilter culture, where the interacting tissues are cultured on opposite sides of the filter (4,9). The tissue to be grown below the filter is glued by heated 1% agar, after which the filter is turned upside down, and the other tissue is placed on top of the filter.

6. The composition of the optimal culture medium depends on the tissues. The medium in this protocol is good for a number of different organs at early stages of development, but during more advanced stages, different organs may have special requirements. Chemically defined media with varying compositions have been designed. For cultures of whole tooth germs, we routinely use chemically defined medium composed of D-MEM and F12 (Ham's Nutrient Mixture, Gibco-BRL) 1:1, supplemented with 50 |g/mL transferrin (Sigma, T-2252,10 mg/mL, 20-|L aliquots, stored at -20°C). For more advanced stages of tooth development, ascorbic acid is added at 150 |g/mL to allow deposition of dentin collagen (16). During prolonged culture, the medium should be changed at 2-3 d intervals.

7. Isolated epithelial tissue does not survive as well as the mesenchymal tissue, when cultured alone. The growth of the dental epithelium (as well as epithelium from other organs) is significantly improved by culture on extracellular matrix material. Collagen has been used, but by far the best results are obtained with the basement membrane matrix, Matrigel (Collaborative Biomedical Products, Bedford, MA, cat. no. 40234), which also promotes epithelial morphogenesis (17). Matrigel is kept on ice and pipeted on the filters. Dishes are transferred to the incubator for 30 min, allowing gelling of Matrigel. At room temperature, the tissue is placed on Matrigel. Covering of the tissue with a drop of Matrigel further improves epithelial growth.

8. Bromodeoxyuridine (BrdU) incorporation is commonly used for the analysis of cell proliferation (Fig. 3B). The explants are labeled by adding BrdU 0.5-3 h before fixation (we use cell proliferation kits from Amersham International, Little Chalfont, UK or Boehringer-Mannheim, Mannheim, Germany). After fixation in ice-cold methanol, the explants are washed in PBS and immunostained as whole mounts using antibodies against BrdU (12).

9. Usually, the tissues are analyzed after culture either as whole mounts (Fig. 3B-D), or they are paraffin-embedded and serially sectioned (Fig. 3E,F). For most purposes, they are fixed for 1 h in 4% paraformaldehyde in PBS (PFA) (after 5 min prefixation in ice-cold methanol). PFA should be fairly freshly made (not more than 7-d-old). The procedure used for whole-mount immunostaining is described in (ref. 11), and that for in situ hybridization in (ref. 12).

References

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2. Grobstein, C. (1953) Inductive epithelio-mesenchymal interaction in cultured organ rudiments of the mouse. Science 118, 52-55.

3. Saxen, I. (1973) Effects of hydrocortisone on the development in vitro of the secondary palate in two inbred strains of mice. Arch. Oral Biol. 18, 1469-1479.

4. Saxen, L., Lehtonen, E., Karkinen-Jaaskelainen, M., Nordling, S., and Wartiovaara, J. (1976) Morphogenetic tissue interactions: Mediation by transmissible signal substances or through cell contacts? Nature 259, 662,663.

5. Nogawa, H. and Takahashi, Y. (1991) Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium. Development 112, 855-861.

6. Nogawa, H. and Ito, T. (1995) Branching morphogenesis of embryonic mouse lung epithelium in mesenchyme-free culture. Development 121, 1015-1022.

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8. Thesleff, I., Vaahtokari, A., and Partanen, A. M. (1995) Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs. Int. J. Dev. Biol. 39, 35-50.

9. Thesleff, I., Lehtonen, E., Wartiovaara, J., and Saxen, L. (1977) Interference of tooth differentiation with interposed filters. Dev. Biol. 58, 197-203.

10. Partanen, A. M., Ekblom, P., and Thesleff, I. (1985) Epidermal growth factor inhibits tooth morphogenesis and differentiation. Dev. Biol. 111, 84-94.

11. Vainio, S. and Thesleff, I. (1992) Coordinated induction of cell proliferation and syndecan expression in dental mesenchyme by epithelium: evidence for diffusible signals. Dev. Dyn. 194, 105-117.

12. Vainio, S., Karavanova, I., Jowett, A., and Thesleff, I. (1993) Identification of BMP- 4 as a signal mediating secondary induction between epithelial and mesen-chymal tissues during early tooth development. Cell 75, 45-58.

13. Jernvall, J., Kettunen, P., Karavanova, I., Martin, L.B., and Thesleff, I. (1994) Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene. Int. J. Dev. Biol. 38, 463-469.

14. Vaahtokari, A., Aberg, T., and Thesleff, I. (1996) Apoptosis in the developing tooth: Association with an embryonic signalling center and suppression by EGF and FGF-4. Development 122, 121-129.

15. Mitsiadias, T., Muramatsu, T., Muramatsu, H., and Thesleff, I. (1995) Midkine (MK), a heparing-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the develping mouse tooth, and affects cell proliferation and morphogenesis. J. Cell Biol. 129, 267- 281.

16. Laine, M. and Thesleff, I. (1986) Development of mouse embryonic molars in vitro: An attempt to design defined culture conditions allowing mineralization. J. Biol. Buccale 14, 15-23.

17. Gittes, G., Galante, P. E., Hanahan, D., Rutter, W. J., and Debas, H. T. (1996) Lineage-specific moprhogenesis in the developing pancreas: role of mesecnhymal factors. Development 122, 439-447.

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