- Email: [email protected]
Dental epithelial histo-morphogenesis in the mouse: positional information versus cell history
Archives of Oral Biology (2005) 50, 131—136
www.intl.elsevierhealth.com/journals/arob
Dental epithelial histo-morphogenesis in the mouse: positional information versus cell history Bing Hua,b, Amal Nadiria, Sabine Bopp-Kuchlera, ´ Lesota,* Fabienne Perrin-Schmittc, Songlin Wangb, Herve a
INSERM U595, Faculty of Medicine, 11, rue Humann, 67085 Strasbourg Cedex, France Faculty of Stomatology, Capital University of Medical Sciences, Beijing, PR China c LGME, CNRS-ULP UMR 7104, INSERM U596, Faculty of Medicine, Strasbourg, France b
Accepted 17 September 2004
KEYWORDS Enamel organ; Epithelial histogenesis; Positional information; Enamel knot; Odontogenesis
Summary Reciprocal epithelial—mesenchymal interactions control odontogenesis and the cap stage tooth germ mesenchyme specifies crown morphogenesis. The aim of this work was to determine whether this mesenchyme could also control epithelial histogenesis. Dental mesenchyme and enamel organ were dissociated from mouse first lower molars at E14. At this early cap stage, the enamel organ consists of four cell types forming the inner dental epithelium (IDE), primary enamel knot (PEK), outer dental epithelium (ODE) and the stellate reticulum (SR). Pelleted trypsin-dissociated single dental epithelial cells, which had lost all positional information, were reassociated to either dental mesenchyme or dissociated mesenchymal cells and cultured in vitro. Although with different timings, teeth developed in both types of experiments showing a characteristic dental epithelial histogenesis, cusp formation, and the differentiation of functional odontoblasts and ameloblasts. The rapid progression of the initial steps of histogenesis suggested that the cell history was not memorized. The dental mesenchyme, as well as dissociated mesenchymal cells, induced the formation of a PEK indicating that no specific organisation in the mesenchyme is required for this step. However, the proportion of well-formed multicusped teeth was much higher when intact mesenchyme was used instead of dissociated mesenchymal cells. The mesenchymal cell dissociation had consequences for the functionality of the newly-formed PEK. # 2004 Elsevier Ltd. All rights reserved.
Abbreviations: BrdU, 5-bromo-2-deoxyuridine; IDE, inner dental epithelium; ODE, outer dental epithelium; PEK, primary enamel knot; SR, stellate reticulum * Corresponding author. Tel.: +33 3 90 24 33 78/31 11; fax: +33 3 90 24 35 64. E-mail address: [email protected] (H. Lesot).
Introduction Reciprocal epithelial—mesenchymal interactions control all steps of odontogenesis. Crown morpho-
0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.09.007
132
B. Hu et al.
genesis (i.e. cusp number, size and position) is specific for each molar in the upper and lower jaws. Tissue reassociation experiments have shown that crown morphogenesis is controlled by the dental mesenchyme.1,2 Epithelial histogenesis is already initiated at the bud stage since two cell types can be distinguished morphologically. Cells in contact with the basement membrane are elongated, while internal epithelial cells are small and round. Positional information might thus be involved in specifying the cell shape and cell fate as illustrated for other models.3—6 At the early cap stage, epithelial histogenesis becomes much more complex when the inner dental epithelium (IDE) and outer dental epithelium (ODE), the stellate reticulum (SR) and the primary enamel knot (PEK) become distinct. The molecular mechanisms, which control dental epithelial histogenesis, involve cell—cell and cell—matrix interactions as well as diffusible signaling molecules.7—15 The aim of the present work was to determine whether the mesenchyme could control epithelial histogenesis. For this purpose, the dental mesenchyme from the cap stage (E14) was reassociated with pelleted single dental epithelial cells, which had lost all positional information, and cultured in vitro. In complementary experiments, dissociated single dental mesenchymal cells were pelleted and reassociated with epithelial cells to test whether the potentialities of the mesenchyme required any specific tissue organisation.
Figure 1
Materials and methods ICR mice were mated overnight and the detection of the vaginal plug was determined as Embryonic day (E)0. The first lower molars (n = 2420) were dissected from embryos at E14 under a stereomicroscope (Leica MZ9).
Tissue dissociation The dental epithelium and mesenchyme were dissociated by using 1% trypsin in Hanks’ at 4 8C.2 After separation of the two tissues, the dental epithelium, and sometimes mesenchyme, were dissociated into cells by sequentially using fine needle and filtered through 70 mm nylon filters. Epithelial cells were then pelleted by centrifugation at 9000 g, the pellets were cut into fragments, reassociated with either intact mesenchyme or pelleted mesenchymal cells and cultured (Fig. 1).
Cultures The reassociations were cultured for up to 12 days on a semi-solid medium, which consisted of DMEM/ F-12 (Gibco) containing 20% fetal bovine serum (CAMBREX), and supplemented with ascorbic acid (0.18 mg/ml, Merck), L-glutamine (2 mM, Gibco), penicilline/streptomycine (50 units/ml, Gibco) and agar (0.36%, Sigma). Cultures were performed at 37 8C in a humidified atmosphere of 5% CO2. The medium was changed every 2 days. In this work, 234
Schematic representation of the experimental procedures.
Epithelial cell positional information
133
Figure 2 In vitro development of reassociations from first lower molars at ED14: (A—R) reassociations between dental mesenchyme and dissociated epithelial cells; (S—AJ) reassociations between dissociated dental mesenchymal cells and epithelial cells. The reassociations were cultured for 12 h (A, J, S, AB); 24 h (B, K, T, AC); 2 days (C, L, U, AD); 3 days (D, M, V, AE); 4 days (E, N, W, AF); 6 days (F, O, X, AG); 8 days (G, P, Y, AH); 10 days (H, Q, Z, AI) and 12 days (I, R, AA, AJ). AM: ameloblast; BM: basement membrane; D: Dentin; DE: dental epithelium; DM: dental mesenchyme; DP: dental papilla; IDE: inner dental epithelium; OD: odontoblast. ODE: outer dental epithelium; PEK: primary enamel knot; SEK: secondary enamel knot; SI: stratum intermedium; SR: stellate reticulum. Bar = 40 mm.
134
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reassociations were performed between mesenchyme and dissociated epithelial cells and 56 between epithelial and mesenchymal cells.
differentiation, cell—cell reassociations were also retarded compared to tissue—cell reassociations.
Histology
Discussion
All samples were fixed in Bouin—Hollande, embedded in paraffin, and serial sections (5 mm) were stained with Mallory’s stain.
When epithelial cells were dissociated, the enamel organ already comprised four distinct cell populations (IDE, PEK, ODE and SR). Their specific fate may be determined by positional information,3—5 which can be specified by differential cell—cell interactions,8,9,15,17 cell—matrix interactions7,10—12,14,18 and signaling molecules.13,19,20 After trypsin treatment, the breakdown of the basement membrane and cell-surface molecules resulted in the loss of positional information. For each experiment, tissues from about 60 teeth were used. The dissociated epithelial cells were mixed together and pelleted by centrifugation. In a first set of experiments, fragments of the pellet were reassociated to dental mesenchyme and cultured. During the first 12 h in culture, an important cell death, probably a consequence of trypsin treatment, took place while surviving cells deposited a new basement membrane at the epithelial—mesenchymal junction. After 24 h, most of the dead cells had disappeared and the epithelial cells in contact with the newly deposited basement membrane started to elongate, a first sign of histogenesis. This was similar to that observed at the bud stage in vivo.8 Cell elongation was found to be slightly faster when using tissues from E13 instead of E14 (data not shown). Although with different timings, the same was observed when epithelial cells were cultured in contact with either a dental mesenchyme or dental mesenchymal cells. The cap stage including the formation of a PEK was achieved after 2 days when epithelial cells were reassociated to a dental mesenchyme, instead of 3 days when they were reassociated with mesenchymal cells. The PEK was characterized by (1) its specific cell arrangement, (2) the absence of BrdU incorporation, (3) the expression of Shh as observed by in situ hybridisation, (4) a positive staining for WNT5a and Frizzled receptor and the absence of WNT10b, as observed in cap stage molars.20 This PEK was functional since cusps formed in both types of experiments. The two types of reassociations led to teeth with functional odontoblasts and polarized ameloblasts. Epithelial cells showed a remarkable plasticity. They rapidly adjusted to their new environment and restored the histological characteristics of IDE, ODE, PEK and SR cells. During the first 12 h, different processes might take place: epithelial cell selection since cell death was important, segregation21 possibly mediated by cell migration as reported in the
Results Mouse first lower molars were trypsin-dissociated in order to separate the mesenchyme from the epithelium at E14, when the PEK was formed and expressed transcripts for signaling molecules.16 Reassociations between the dental mesenchyme and dissociated single epithelial cells were cultured in vitro (Fig. 1). Complementary experiments were also performed by reassociating pelleted dissociated mesenchymal cells and pelleted dissociated epithelial cells (Fig. 1). In both experimental groups, the epithelial histo-morphogenesis was successfully achieved. The epithelial—mesenchymal junction was restored after 12 h in tissue—cell reassociations despite an initial high frequency of cell death, especially in the internal epithelial cells (Fig. 2A and J). During the first 12 h, the deposition of a new basement membrane progressed more slowly in cell—cell reassociations (compare Fig. 2S and AB with Fig. 2A and J). Tissue—cell reassociation reached the bud stage after 24 h (Fig. 2B and K), instead of 48 h for cell—cell reassociations (Fig. 2U and AD). At this stage, the epithelial cells in contact with the basement membrane started to elongate. A 24 h delay was observed for cell—cell reassociations to reach the early cap stage (Fig. 2V and AE) when compared to tissue—cell reassociations (Fig. 2C and L). A clear epithelial histogenesis, with a stellate reticulum intercalated between the inner and outer dental epithelium was achieved after 3 days for tissue—cell reassociations (Fig. 2D and M) instead of 4 days for cell—cell reassociations (Fig. 2W and AF). After 4 days, the PEK were still visible in cell— cell reassociations (Fig. 2AF) while secondary enamel knots (SEKs) were formed in tissue—cell reassociations (Fig. 2E and N). Cusp formation occurred in both types of reassociations, but again with a delay in cell—cell reassociations (compare Fig. 2F and O with Fig. 2Y and AH). Odontoblast differentiation was initiated at the tip of the cusps and progressed, forming gradients in both types of cultures (Fig. 2G, P, Z and AI) and then ameloblast differentiation was initiated with the same pattern (Fig. 2H, Q, I, R, AA and AJ). In terms of cell
Epithelial cell positional information dental papilla during reparative processes22 or cell reprogramming. A detailed study of the reexpression of surface molecules will have to be performed during the initial stages of the culture to follow cadherins,8,15,23 integrins,7,14 and changes in the basement membrane composition during the bud to cap transition, as investigated during tooth development in vivo. The fast progression in the initial steps of epithelial histogenesis in the reassociations suggested that the cell history4 was not memorized. New positional information was acquired at the bud stage, since epithelial cells adapted their morphology to being either in contact with a basement membrane or not, and then changing again at the cap stage when being localized in the region of either the IDE or ODE. This latter change is controlled by the mesenchyme as shown by heterotopic tissue reassociation experiments,24 suggesting that regional specificities might exist in the mesenchyme (dental versus peridental). It was shown that the changes in the basement membrane composition during the bud to cap transition, depending on whether it is reassociated to the ODE or IDE, might be a consequence of MMP and TIMP expression in the mesenchyme.11 The dental mesenchyme, as well as mesenchymal cells from E14, induced the formation of a PEK indicating that no specific organisation in the mesenchyme is required for this step. Furthermore, mesenchyme from E13 had the same potentialities to support tooth development (not shown) showing that a specification by signaling molecules from the PEK is not required. However, the proportion of multicusped teeth was much higher when epithelial cells were reassociated with intact mesenchymes instead of mesenchymal cells, suggesting that the dissociation of the mesenchyme had consequences on the functionality of the PEK. Further investigation at the molecular level will have to be performed to compare the PEK, which forms in the two types of reassociations.
Acknowledgement The authors wish to thank Mr. A. Ackermann for histology.
References 1. Kollar EJ, Baird GR. Tissue interactions in embryonic mouse tooth germs. II. The inductive role of the dental papilla. J Embryol Exp Morphol 1970;24:173—86.
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2. Schmitt R, Lesot H, Vonesch JL, Ruch JV. Mouse odontogenesis in vitro: the cap-stage mesenchyme controls individual molar crown morphogenesis. Int J Dev Biol 1999;43:255—60. 3. Hata RI. Where am I? How a cell recognizes its positional information during morphogenesis Cell Biol Int 1996;20:59— 65. 4. Wolpert L. The progress zone model for specifying positional information. Int J Dev Biol 2002;46:869—70. 5. Rudel D, Sommer RJ. The evolution of developmental mechanisms. Dev Biol 2003;264:15—37. 6. Salinas PC. The morphogen sonic hedgehog collaborates with netrin-1 to guide axons in the spinal cord. Trends Neurosci 2003;26:641—3. 7. Salmivirta K, Gullberg D, Hirsch E, Altruda F, Ekblom P. Integrin subunit expression associated with epithelial— mesenchymal interactions during murine tooth development. Dev Dyn 1996;205:104—13. 8. Obara N, Suzuki Y, Nagai Y, Takeda M. Expression of E- and Pcadherin during tooth morphogenesis and cytodifferentiation of ameloblasts. Anat Embryol Berl 1998;197:469—75. 9. Fausser JL, Schlepp O, Aberdam D, Meneguzzi G, Ruch JV, Lesot H. Localization of antigens associated with adherens junctions, desmosomes, and hemidesmosomes during murine molar morphogenesis. Differentiation 1998;63:1—11. 10. Yoshiba K, Yoshiba N, Aberdam D, Meneguzzi G, PerrinSchmitt F, Stoetzel C, et al. Expression and localization of laminin-5 subunits during mouse tooth development. Dev Dyn 1998;211:164—76. 11. Yoshiba N, Yoshiba K, Stoetzel C, Perrin-Schmitt F, Cam Y, Ruch JV, et al. Temporo-spatial gene expression and protein localization of matrix metalloproteinases and their inhibitors during mouse molar tooth development. Dev Dyn 2003;228:105—12. 12. Nagai N, Nakano K, Sado Y, Naito I, Gunduz M, Tsujigiwa H, et al. Localization of type IV collagen a1 to a6 chains in basement membrane during mouse molar germ development. Int J Dev Biol 2001;45:827—31. 13. Mustonen T, Tummers M, Mikami T, Itoh N, Zhang N, Gridley T, et al. Lunatic fringe, FGF, and BMP regulate the Notch pathway during epithelial morphogenesis of teeth. Dev Biol 2002;248:281—93. 14. Lesot H, Kieffer-Combeau S, Fausser JL, Meyer JM, PerrinSchmitt F, Peterkova R, et al. Cell—cell and cell—matrix interactions during initial enamel organ histomorphogenesis in the mouse. Connect Tissue Res 2002;43:191—200. 15. Obara N, Lesot H. Subcellular localiztion of b-catenin and cadherin expression in the cap stage enamel organ of the mouse molar. Histochem Cell Biol 2004;121:351—8. 16. Thesleff I. Epithelial—mesenchymal signalling regulating tooth morphogenesis. J Cell Sci 2003;116:1647—8. 17. Braga VM. Cell—cell adhesion and signalling. Curr Opin Cell Biol 2002;14:546—56. 18. Ingber DE. Mechanosensation through integrins: cells act locally but think globally. Proc Natl Acad Sci USA 2003;100:1472—4. 19. Osterfield M, Kirschner MW, Flanagan JG. Graded positional information: interpretation for both fate and guidance. Cell 2003;113:425—8. 20. Nadiri A, Kuchler-Bopp S, Haı¨kel Y, Lesot H. Immunolocalization of BMP-2/-4, FGF-4 and WNT10b in the developing mouse first lower molar. J Histochem Cytochem 2004;52:103—12. 21. Duguay D, Foty RA, Steinberg MS. Cadherin-mediated cell adhesion and tissue segregation: qualitative and quantitative determinants. Dev Biol 2003;253:309—23. 22. Melin M, Joffre-Romeas A, Farges JC, Couble ML, Magloire H, Bleicher F. Effects of TGFbeta1 on dental pulp cells in cultured human tooth slices. J Dent Res 2000;79:1689—96.
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23. Hirai Y, Nose A, Kobayashi S, Takeichi M. Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. I. Lung epithelial morphogenesis. Development 1989;105:263—70.
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24. Olive M, Ruch JV. Does the basement membrane control the mitotic activity of the inner dental epithelium of the embryonic mouse first lower molar? Dev Biol 1982;93: 301—7.
www.intl.elsevierhealth.com/journals/arob
Dental epithelial histo-morphogenesis in the mouse: positional information versus cell history Bing Hua,b, Amal Nadiria, Sabine Bopp-Kuchlera, ´ Lesota,* Fabienne Perrin-Schmittc, Songlin Wangb, Herve a
INSERM U595, Faculty of Medicine, 11, rue Humann, 67085 Strasbourg Cedex, France Faculty of Stomatology, Capital University of Medical Sciences, Beijing, PR China c LGME, CNRS-ULP UMR 7104, INSERM U596, Faculty of Medicine, Strasbourg, France b
Accepted 17 September 2004
KEYWORDS Enamel organ; Epithelial histogenesis; Positional information; Enamel knot; Odontogenesis
Summary Reciprocal epithelial—mesenchymal interactions control odontogenesis and the cap stage tooth germ mesenchyme specifies crown morphogenesis. The aim of this work was to determine whether this mesenchyme could also control epithelial histogenesis. Dental mesenchyme and enamel organ were dissociated from mouse first lower molars at E14. At this early cap stage, the enamel organ consists of four cell types forming the inner dental epithelium (IDE), primary enamel knot (PEK), outer dental epithelium (ODE) and the stellate reticulum (SR). Pelleted trypsin-dissociated single dental epithelial cells, which had lost all positional information, were reassociated to either dental mesenchyme or dissociated mesenchymal cells and cultured in vitro. Although with different timings, teeth developed in both types of experiments showing a characteristic dental epithelial histogenesis, cusp formation, and the differentiation of functional odontoblasts and ameloblasts. The rapid progression of the initial steps of histogenesis suggested that the cell history was not memorized. The dental mesenchyme, as well as dissociated mesenchymal cells, induced the formation of a PEK indicating that no specific organisation in the mesenchyme is required for this step. However, the proportion of well-formed multicusped teeth was much higher when intact mesenchyme was used instead of dissociated mesenchymal cells. The mesenchymal cell dissociation had consequences for the functionality of the newly-formed PEK. # 2004 Elsevier Ltd. All rights reserved.
Abbreviations: BrdU, 5-bromo-2-deoxyuridine; IDE, inner dental epithelium; ODE, outer dental epithelium; PEK, primary enamel knot; SR, stellate reticulum * Corresponding author. Tel.: +33 3 90 24 33 78/31 11; fax: +33 3 90 24 35 64. E-mail address: [email protected] (H. Lesot).
Introduction Reciprocal epithelial—mesenchymal interactions control all steps of odontogenesis. Crown morpho-
0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.09.007
132
B. Hu et al.
genesis (i.e. cusp number, size and position) is specific for each molar in the upper and lower jaws. Tissue reassociation experiments have shown that crown morphogenesis is controlled by the dental mesenchyme.1,2 Epithelial histogenesis is already initiated at the bud stage since two cell types can be distinguished morphologically. Cells in contact with the basement membrane are elongated, while internal epithelial cells are small and round. Positional information might thus be involved in specifying the cell shape and cell fate as illustrated for other models.3—6 At the early cap stage, epithelial histogenesis becomes much more complex when the inner dental epithelium (IDE) and outer dental epithelium (ODE), the stellate reticulum (SR) and the primary enamel knot (PEK) become distinct. The molecular mechanisms, which control dental epithelial histogenesis, involve cell—cell and cell—matrix interactions as well as diffusible signaling molecules.7—15 The aim of the present work was to determine whether the mesenchyme could control epithelial histogenesis. For this purpose, the dental mesenchyme from the cap stage (E14) was reassociated with pelleted single dental epithelial cells, which had lost all positional information, and cultured in vitro. In complementary experiments, dissociated single dental mesenchymal cells were pelleted and reassociated with epithelial cells to test whether the potentialities of the mesenchyme required any specific tissue organisation.
Figure 1
Materials and methods ICR mice were mated overnight and the detection of the vaginal plug was determined as Embryonic day (E)0. The first lower molars (n = 2420) were dissected from embryos at E14 under a stereomicroscope (Leica MZ9).
Tissue dissociation The dental epithelium and mesenchyme were dissociated by using 1% trypsin in Hanks’ at 4 8C.2 After separation of the two tissues, the dental epithelium, and sometimes mesenchyme, were dissociated into cells by sequentially using fine needle and filtered through 70 mm nylon filters. Epithelial cells were then pelleted by centrifugation at 9000 g, the pellets were cut into fragments, reassociated with either intact mesenchyme or pelleted mesenchymal cells and cultured (Fig. 1).
Cultures The reassociations were cultured for up to 12 days on a semi-solid medium, which consisted of DMEM/ F-12 (Gibco) containing 20% fetal bovine serum (CAMBREX), and supplemented with ascorbic acid (0.18 mg/ml, Merck), L-glutamine (2 mM, Gibco), penicilline/streptomycine (50 units/ml, Gibco) and agar (0.36%, Sigma). Cultures were performed at 37 8C in a humidified atmosphere of 5% CO2. The medium was changed every 2 days. In this work, 234
Schematic representation of the experimental procedures.
Epithelial cell positional information
133
Figure 2 In vitro development of reassociations from first lower molars at ED14: (A—R) reassociations between dental mesenchyme and dissociated epithelial cells; (S—AJ) reassociations between dissociated dental mesenchymal cells and epithelial cells. The reassociations were cultured for 12 h (A, J, S, AB); 24 h (B, K, T, AC); 2 days (C, L, U, AD); 3 days (D, M, V, AE); 4 days (E, N, W, AF); 6 days (F, O, X, AG); 8 days (G, P, Y, AH); 10 days (H, Q, Z, AI) and 12 days (I, R, AA, AJ). AM: ameloblast; BM: basement membrane; D: Dentin; DE: dental epithelium; DM: dental mesenchyme; DP: dental papilla; IDE: inner dental epithelium; OD: odontoblast. ODE: outer dental epithelium; PEK: primary enamel knot; SEK: secondary enamel knot; SI: stratum intermedium; SR: stellate reticulum. Bar = 40 mm.
134
B. Hu et al.
reassociations were performed between mesenchyme and dissociated epithelial cells and 56 between epithelial and mesenchymal cells.
differentiation, cell—cell reassociations were also retarded compared to tissue—cell reassociations.
Histology
Discussion
All samples were fixed in Bouin—Hollande, embedded in paraffin, and serial sections (5 mm) were stained with Mallory’s stain.
When epithelial cells were dissociated, the enamel organ already comprised four distinct cell populations (IDE, PEK, ODE and SR). Their specific fate may be determined by positional information,3—5 which can be specified by differential cell—cell interactions,8,9,15,17 cell—matrix interactions7,10—12,14,18 and signaling molecules.13,19,20 After trypsin treatment, the breakdown of the basement membrane and cell-surface molecules resulted in the loss of positional information. For each experiment, tissues from about 60 teeth were used. The dissociated epithelial cells were mixed together and pelleted by centrifugation. In a first set of experiments, fragments of the pellet were reassociated to dental mesenchyme and cultured. During the first 12 h in culture, an important cell death, probably a consequence of trypsin treatment, took place while surviving cells deposited a new basement membrane at the epithelial—mesenchymal junction. After 24 h, most of the dead cells had disappeared and the epithelial cells in contact with the newly deposited basement membrane started to elongate, a first sign of histogenesis. This was similar to that observed at the bud stage in vivo.8 Cell elongation was found to be slightly faster when using tissues from E13 instead of E14 (data not shown). Although with different timings, the same was observed when epithelial cells were cultured in contact with either a dental mesenchyme or dental mesenchymal cells. The cap stage including the formation of a PEK was achieved after 2 days when epithelial cells were reassociated to a dental mesenchyme, instead of 3 days when they were reassociated with mesenchymal cells. The PEK was characterized by (1) its specific cell arrangement, (2) the absence of BrdU incorporation, (3) the expression of Shh as observed by in situ hybridisation, (4) a positive staining for WNT5a and Frizzled receptor and the absence of WNT10b, as observed in cap stage molars.20 This PEK was functional since cusps formed in both types of experiments. The two types of reassociations led to teeth with functional odontoblasts and polarized ameloblasts. Epithelial cells showed a remarkable plasticity. They rapidly adjusted to their new environment and restored the histological characteristics of IDE, ODE, PEK and SR cells. During the first 12 h, different processes might take place: epithelial cell selection since cell death was important, segregation21 possibly mediated by cell migration as reported in the
Results Mouse first lower molars were trypsin-dissociated in order to separate the mesenchyme from the epithelium at E14, when the PEK was formed and expressed transcripts for signaling molecules.16 Reassociations between the dental mesenchyme and dissociated single epithelial cells were cultured in vitro (Fig. 1). Complementary experiments were also performed by reassociating pelleted dissociated mesenchymal cells and pelleted dissociated epithelial cells (Fig. 1). In both experimental groups, the epithelial histo-morphogenesis was successfully achieved. The epithelial—mesenchymal junction was restored after 12 h in tissue—cell reassociations despite an initial high frequency of cell death, especially in the internal epithelial cells (Fig. 2A and J). During the first 12 h, the deposition of a new basement membrane progressed more slowly in cell—cell reassociations (compare Fig. 2S and AB with Fig. 2A and J). Tissue—cell reassociation reached the bud stage after 24 h (Fig. 2B and K), instead of 48 h for cell—cell reassociations (Fig. 2U and AD). At this stage, the epithelial cells in contact with the basement membrane started to elongate. A 24 h delay was observed for cell—cell reassociations to reach the early cap stage (Fig. 2V and AE) when compared to tissue—cell reassociations (Fig. 2C and L). A clear epithelial histogenesis, with a stellate reticulum intercalated between the inner and outer dental epithelium was achieved after 3 days for tissue—cell reassociations (Fig. 2D and M) instead of 4 days for cell—cell reassociations (Fig. 2W and AF). After 4 days, the PEK were still visible in cell— cell reassociations (Fig. 2AF) while secondary enamel knots (SEKs) were formed in tissue—cell reassociations (Fig. 2E and N). Cusp formation occurred in both types of reassociations, but again with a delay in cell—cell reassociations (compare Fig. 2F and O with Fig. 2Y and AH). Odontoblast differentiation was initiated at the tip of the cusps and progressed, forming gradients in both types of cultures (Fig. 2G, P, Z and AI) and then ameloblast differentiation was initiated with the same pattern (Fig. 2H, Q, I, R, AA and AJ). In terms of cell
Epithelial cell positional information dental papilla during reparative processes22 or cell reprogramming. A detailed study of the reexpression of surface molecules will have to be performed during the initial stages of the culture to follow cadherins,8,15,23 integrins,7,14 and changes in the basement membrane composition during the bud to cap transition, as investigated during tooth development in vivo. The fast progression in the initial steps of epithelial histogenesis in the reassociations suggested that the cell history4 was not memorized. New positional information was acquired at the bud stage, since epithelial cells adapted their morphology to being either in contact with a basement membrane or not, and then changing again at the cap stage when being localized in the region of either the IDE or ODE. This latter change is controlled by the mesenchyme as shown by heterotopic tissue reassociation experiments,24 suggesting that regional specificities might exist in the mesenchyme (dental versus peridental). It was shown that the changes in the basement membrane composition during the bud to cap transition, depending on whether it is reassociated to the ODE or IDE, might be a consequence of MMP and TIMP expression in the mesenchyme.11 The dental mesenchyme, as well as mesenchymal cells from E14, induced the formation of a PEK indicating that no specific organisation in the mesenchyme is required for this step. Furthermore, mesenchyme from E13 had the same potentialities to support tooth development (not shown) showing that a specification by signaling molecules from the PEK is not required. However, the proportion of multicusped teeth was much higher when epithelial cells were reassociated with intact mesenchymes instead of mesenchymal cells, suggesting that the dissociation of the mesenchyme had consequences on the functionality of the PEK. Further investigation at the molecular level will have to be performed to compare the PEK, which forms in the two types of reassociations.
Acknowledgement The authors wish to thank Mr. A. Ackermann for histology.
References 1. Kollar EJ, Baird GR. Tissue interactions in embryonic mouse tooth germs. II. The inductive role of the dental papilla. J Embryol Exp Morphol 1970;24:173—86.
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