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Apoptosis distribution in the first molar tooth germ of the field vole (Microtus agrestis)
Tissue and Cell 36 (2004) 361–367
Apoptosis distribution in the first molar tooth germ of the field vole (Microtus agrestis) E. Matalova∗ , K. Witter, I. Misek Laboratory of Genetics and Embryology IAPG CAS, Institute of Animal Physiology and Genetics, Academy of Sciences, Veveri 97, 602 00 Brno, Czech Republic Received 18 February 2004; received in revised form 11 June 2004; accepted 17 June 2004
Abstract Apoptosis represents an important process in organ and tissue morphogenesis and remodeling during embryonic development. A role for apoptosis in shape formation of developing teeth has been suggested. The field vole is a useful model for comparative studies in odontogenesis, particularly because of its contrasting molar morphogenesis when compared to the mouse. However, little is known concerning apoptosis in tooth development of this species. Morphological (cellular and nuclear alterations) and biochemical (specific DNA breaks—TUNEL staining) characteristics of apoptotic cells were used to evaluate the temporal and spatial occurrence of apoptosis in epithelial and mesenchymal tissues of the developing first molar tooth germs of the field vole. Apoptotic cells were found in non-proliferating areas (identified previously) throughout bud to bell stages, particularly in the epithelium, however, scattered also in the mesenchyme. A high concentration of TUNEL positive cells was evident in primary enamel knots at late bud stage with increasing density of apoptotic cells until ED 16 when the primary enamel knot in the field vole disappears and mesenchyme becomes protruded in the middle axes of the bell forming two shallow areas with zig-zag located secondary enamel knots. Distribution of TUNEL positive cells corresponded with localisation of secondary enamel knots as shown using histological and 3D analysis. Apoptosis was shown to be involved in the first molar development of the field vole, however, exact mechanisms and roles of this process in tooth morphogenesis require further investigation. © 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Tooth development; DNA fragmentation; Field vole
1. Introduction Developing organisms are a complex of growing tissues and differentiating cells, that maintain a balance between cell division (proliferation) and programmed cell death (apoptosis). Apoptosis (Kerr et al., 1972) has been recognized as an essential component in animal development, in the construction, maintenance and repair of tissues as well as in removal of misplaced or damaged cells. Dysfunctions in the regulation or execution of apoptotic cell death are implicated in a wide range of developmental abnormalities and diseases (Meier et al., 2000; Ranger et al., 2001). ∗
Corresponding author. Tel.: +42 54 1562205; fax: +42 54 9211482. E-mail address: [email protected] (E. Matalova).
0040-8166/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2004.06.006
Apoptosis was originally defined as a distinct mode of cell death on the basis of the characteristic morphological features of the cells. More recently, internucleosomal DNA cleavage, membrane alterations, specific gene expression, and characteristic pathway activations have been used as markers of apoptosis. With increasing interest in apoptosis research, many assays have been developed to detect apoptosis in cell populations as well as at the individual cell level. However, exploitation of these methods in histological sections is often limited (Dubska et al., 2002). The TUNEL assay (Gavrieli et al., 1992) is acknowledged as the method of choice for the rapid identification and quantification of apoptotic cells on histological sections. Cell–cell signalling between epithelium and underlying mesenchyme underscores the embryonic development of
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teeth (reviewed in Thesleff and Sharpe, 1997; Tucker and Sharpe, 1999; Cobourne and Sharpe, 2003). During tooth formation these signals control key processes involved in odontogenesis—cell proliferation, migration, adhesion and cell death. In developing teeth, apoptosis occurs during all stages as shown in early morphogenesis (e.g. Peterkova et al., 1996; Vaahtokari et al., 1996; Jernvall et al., 1998), amelogenesis (e.g. Smith and Washawsky, 1977; Moe, 1979; Nishikawa and Sasaki, 1995; Kaneko et al., 1997), dentinogenesis (e.g. Bronckers et al., 1996; Vermelin et al., 1996; Franquin and Remusat, 1997) and during eruption (e.g. Schellens et al., 1982; ten Cate and Anderson, 1986). Thus, apoptosis seems to have a crucial role in morphogenetic processes involved in tooth development. Embryonic development of mammalian molar teeth involves the same general stages although the final tooth shape varies among species. The field vole represents a rodent species with hypselodont teeth with the same tooth formula as the mouse 1003/1003 (one incisor and three molars in each jaw quadrant), but the molar shape is quite different (Witter et al., 1996). Although field voles have been shown to be a suitable model for comparative studies in odontogenesis (Keranen et al., 1998, 1999; Jernvall et al., 2000), temporospatial localization of apoptotic cells in tooth development of the field vole has not yet been reported. Thus, knowledge of the distribution pattern of apoptotic cells, particularly versus proliferation, appears to be necessary in order to allow for further research into interspecies differences and general mechanisms engaged in tooth morphogenesis. In this study, apoptosis was identified from initiation of tooth buds to the bell stage of the developing field vole first molar by using morphological and biochemical criteria of apoptotic cell death.
2. Material and methods 2.1. Tissue sections Field vole (Microtus agrestis) embryos (two of each embryonic stage under study) from an embryonic collection (http://www.iach.cz/lge/sbirkae.htm), embryonic days (ED) 12–18, were fixed in 4% neutral formaldehyde in water (Bancroft and Cook, 1994). After paraffin processing 4 m serial tissue sections were prepared using a rotatory microtome and mounted on poly-l-lysine-coated glass slides. Every second section was used for TUNEL assay, all the others for hematoxylin–eosin (H&E) staining.
2.3. TUNEL POD assay—labeling Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (In Situ Cell Death Detection Kit, POD) was used for proving apoptosis in individual cells. The TUNEL working procedure was carried out following the producer’s directions (Roche Biochemicals, Germany). Endogenous peroxidase was blocked by incubation in 3% H2 O2 /10 min/RT before enzymatic labeling. During the TUNEL procedure samples were washed in phosphate-buffer saline (PBS, pH 7.4): 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2 PO4 , 8.1 mM Na2 HPO4 in distilled water. The fluorescent signal conversion using anti-fluorescence antibody conjugated with peroxidase and substrate color reaction applying chromogen DAB (3,3 -diaminobenzidine tetrahydrochloride; Sigma-Aldrich, Germany) were proceeded after enzymatic labeling. 2.4. TUNEL POD assay—controls Positive control of TUNEL POD labeling was prepared using DNase (Sigma-Aldrich, Germany) treatment. After pretreatment, histological sections were incubated with DNase (5 g/ml) at 37 ◦ C/humidified chamber/10 min to induce DNA strand breaks. Negative control was obtained by omitting terminal transferase from the labeling procedure (label solution only instead of TUNEL reaction mixture). As a positive control of correct fixation and staining of apoptotic cells, embryonic limb buds and adult small intestine were tested. 2.5. TUNEL POD assay—analysis Counterstaining with alcian blue (staining of intracellular matter) or light green (cytoplasma staining) were performed before microscopic analysis of apoptotic nuclei/bodies. Samples were photodocumented after dehydration and mounting in a permanent mounting medium. 2.6. Cell morphology For histological analysis tissue sections were stained with hematoxylin–eosin or alcian blue–hematoxylin–eosin to evaluate morphological changes characteristic of apoptosis.
3. Result evaluation 2.2. Pretreatments—cell permeabilisation The sections were deparaffinized and hydrated through a graded series of ethanol and distilled water and detergent permeabilisation was applied: 0.1% TRITON® X-100 in 0.1% sodium citrate/8 min/RT.
Field vole embryos were employed to localize apoptotic cells in both upper and lower developing first molars from initiation to bell stages, embryonic days 12–18. Positive and negative controls of the TUNEL reaction were prepared as described in Section 2 and as shown in Fig. 1.
E. Matalova et al. / Tissue and Cell 36 (2004) 361–367
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Fig. 1. Bud stage of first upper molar. Controls of TUNEL reaction—positive control (a) and negative control (b).
In our investigation, H&E and TUNEL staining were used to evaluate apoptotic cell death during tooth development. In the H&E sections, morphological criteria of apoptotic cell death as identified by condensed chromatin and formation of apoptotic bodies were applied. Positive TUNEL POD cells were characterized by brown or red-brown staining of the nuclei.
4. Results Early tooth development in the field vole follows a similar progression of well-characterized morphological stages from
initiation to bell structures as in other mammals (Luckett, 1993). At the initiation stage, TUNEL positive cells were scattered in the epithelial part of tooth germ from ED 12.5. During bud formation, apoptotic cells were concentrated in the middle axes of the prolonging bud and later appeared at the top of the tooth bud (Figs. 2 and 3). The concentration of apoptotic cells was particularly high in the anterior part of the tooth bud, where apoptotic cells were localized on the epithelium tip (Fig. 2c). This suggests apoptotic elimination of some expected antemolar rudimental structures (as described in the mouse—Tureckova et al., 1996). In the condensed mesenchyme around the epithelial thickening, no TUNEL positive cells were found at the bud stage. The localization
Fig. 2. Bud stage of the first upper molar, anterial part. Arrow showing TUNEL positive cells with brown nuclei (a) and apoptotic bodies in hematoxylin–eosin staining (b). Accumulation of apoptotic bodies is evident on the bud tip (c).
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Fig. 3. Bud formation of the first upper molar, posterial part. Arrow showing TUNEL positive cells with brown nuclei (a) and apoptotic bodies in hematoxylin–eosin staining (b).
Fig. 4. Late cup stage of first upper molar development. Arrows indicating the enamel knot structure where TUNEL positive cells (a) are clearly visible. Hematoxylin staining (b).
Fig. 5. Bell stage of first upper molar development. TUNEL positive cells were detected in area corresponding with localisation of secondary enamel knots. TUNEL labeling (a) and hematoxylin staining (b).
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Fig. 6. Schematic illustration of apoptotic cell distribution in first molar tooth development of the field vole (Microtus agrestis). Anterior (a) and posterior (b) parts of the bud, later cup (c) and bell (d) stages in the upper jaw.
of apoptotic cells was quite restricted at later developmental stages as the TUNEL positive area expanded along the middle axes of the tooth bud and in the advanced development of the tooth cap. At the cap stage, when the primary enamel knot is mature and the crown base starts its formation, a few TUNEL positive cells were observed also in the mesenchyme and remained throughout the bell stage. At ED 13.5–15.5, the primary enamel knot showed clear sign of apoptosis as evident after H&E as well as TUNEL staining (Fig. 4). Although the apoptotic cells were observed as soon as the enamel knot structure appears, the apoptotic area was significantly extended at later stages and finally most of the cells of the primary enamel knot were TUNEL positive. Secondary enamel knots in the field vole do not protrude into the mesenchyme and are less clear than in the mouse, however, some TUNEL positive cells were detected in this area at the bell stage, when cusps formation is initiated (Fig. 5). At the later bell stage, apoptotic cells were strongly present also in the stalk of the tooth primordium. Schematic apoptosis distribution is illustrated in Fig. 6. No remarkable differences were observed in distribution of apoptotic cells in lower and upper first molar tooth germs.
5. Discussion Apoptotic cell death has been recognized for several decades (Kerr et al., 1972) and the interest in this area keeps increasing. This fact reflects the importance of this process not only during embryogenesis and in tissue homeostasis but also in the contribution of apoptosis disorders to many human defects and diseases.
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As in other mammalian species (Peterkova et al., 1996; Shigemura et al., 1999), apoptosis was detected in the dental lamina of the field vole where it is suggested to play an important role in its reduction. Thus, apoptosis can contribute to prevention of the dental lamina persistence or even overgrowth, which may lead to cyst formation and tooth eruption delay as reported in human patients (Shear, 1994; Lombardi et al., 1995; Lo Muziol et al., 1999; Tsukamoto et al., 2001). Molars show species specific number of cusps, which must be shaped during the embryonic tissue remodeling. This can be achieved either by restricted proliferation or by elimination of cells by apoptosis as shown for example in limb development (reviewed in Zuzarte-Luis and Hurle, 2002). The balance between cell division and cell death seems to be crucial in all morphogenetic processes. In the mouse, apoptotic cells were demonstrated in non-dividing (Brd-U negative) areas (Lesot et al., 1996; Shigemura et al., 1999). Proliferation activity of epithelial and mesenchymal cells based on detection of proliferation cell nuclear antigen (PCNA) during molar development of the field vole has been reported previously (Matulova et al., 2002). Considered with the data from mouse embryos, our results show localization of apoptotic cells in non-proliferating areas also in the field vole. Despite the involvement of apoptosis in dental morphogenesis and possibly defects in craniofacial development (e.g. Clouthier et al., 2000; Beverdam et al., 2001), the roles and mechanisms of this process are poorly understood (Matalova et al., 2004). The distribution pattern of apoptotic cells has been described in some mammalian species, mainly in rodents (Vaahtokari et al., 1996; Lesot et al., 1996; Viriot et al., 1997; Shigemura et al., 1999; Bronckers et al., 2000; Sasaki et al., 2001). In the field vole, apoptotis pattern has not been reported yet. We observed a specific temporospatial distribution of apoptotic events during embryonic development of the molar teeth in this species. These characteristic patterns of apoptosis in different species suggest crucial roles in odontogenesis. Morphogenesis of dentition is based on reciprocal communication between epithelial cells and underlying mesenchymal cells (reviewed in Tucker and Sharpe, 1999; Cobourne and Sharpe, 2003). This process is mediated by factors employed in cell proliferation, adhesion and migration, in cell cycle regulation as well as in apoptotic inhibition and stimulation. The enamel knot has been described as a characteristic transitory structure with a specific packing of epithelial cells, decreased proliferation activity and accumulation of apoptotic activity. This structures has been identified as the areas with ceased or no proliferation in the mouse (Shigemura et al., 1999) and also in the field vole (Jernvall et al., 2000). Enamel knots are considered as signalling centres in tooth development (Jernvall et al., 1994), which are terminated by apoptotic cell death (Lesot et al., 1996; Keranen et al., 1998). Apoptosis in the signalling centres of enamel knots has been shown also in our study. Primary enamel knots in the field vole
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are less protruded towards mesenchyme than in the mouse. Secondary enamel knots in the field vole molars are shallower than in mouse and form a zig-zag pattern according to the molar shape (in the occlussal view). Apoptosis distribution corresponded with the zig-zag pattern where the apoptotic cells were alternately concentrated in the buccal and lingual depression of the tooth bell. The different shape of the entire tooth germ as well as of the enamel knots and thus occurrence of apoptosis may have an important impact on the final tooth crown morhogenesis, which must be species specific. The direct role of enamel knots and their apoptotic silencing has been shown in Tabby (Cermakova et al., 1998; Pispa et al., 1999; Lisi et al., 2001) and downless (Tucker et al., 2000) mutants where different shape and size of the enamel knots is linked to resultant cusps defects in the adult. As in the mouse, the field vole dentition consists of one incisor, toothless diastema and three molars in each jaw quadrant. In the diastema region of both species, vestigal dental primordia appear during embryonic development (Witter et al., 1996, Peterkova et al., 2002) and undergo their apoptotic fate at the bud stage (Tureckova et al., 1996; Peterkova et al., 2002). In the filed vole molar tooth germ, the apoptotic activity differed in the arterio-posterial axes, particularly at the bud stage (as shown in Figs. 2–3), suggesting apoptotic elimination of similar structures also in this species. This gradient of regressive processes may also play a role in positioning of the first molar cap. Apoptotis can be detected throughout embryonic tooth development in all animal models examined so far and thus seems to be highly conserved and involved in morphogenetic processes of tooth shaping and patterning. This morphogenetic mechanism seems to be involved also in molar tooth development in the field vole, as shown in our study. However, the exact pathways and roles of apoptotis in tooth development have not been understood so far (Matalova et al., 2004). A passive role for apoptosis has been shown in elimination of regressive tooth diastemal primordia where apoptosis can be considered more as a degenerative process than a process of creation. It remains to be clarified whether dental apoptosis has an active and essential role in enamel knot termination, modelling the final shape and governing the size and position of the teeth or if this process just removes silenced and unwanted cells. Further examination of apoptotic pathways and related molecules is therefore a challenge for future research.
Acknowledgement This work was supported by the Grant Agency of the Czech Republic (GACR-204/02/P112).
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Apoptosis distribution in the first molar tooth germ of the field vole (Microtus agrestis) E. Matalova∗ , K. Witter, I. Misek Laboratory of Genetics and Embryology IAPG CAS, Institute of Animal Physiology and Genetics, Academy of Sciences, Veveri 97, 602 00 Brno, Czech Republic Received 18 February 2004; received in revised form 11 June 2004; accepted 17 June 2004
Abstract Apoptosis represents an important process in organ and tissue morphogenesis and remodeling during embryonic development. A role for apoptosis in shape formation of developing teeth has been suggested. The field vole is a useful model for comparative studies in odontogenesis, particularly because of its contrasting molar morphogenesis when compared to the mouse. However, little is known concerning apoptosis in tooth development of this species. Morphological (cellular and nuclear alterations) and biochemical (specific DNA breaks—TUNEL staining) characteristics of apoptotic cells were used to evaluate the temporal and spatial occurrence of apoptosis in epithelial and mesenchymal tissues of the developing first molar tooth germs of the field vole. Apoptotic cells were found in non-proliferating areas (identified previously) throughout bud to bell stages, particularly in the epithelium, however, scattered also in the mesenchyme. A high concentration of TUNEL positive cells was evident in primary enamel knots at late bud stage with increasing density of apoptotic cells until ED 16 when the primary enamel knot in the field vole disappears and mesenchyme becomes protruded in the middle axes of the bell forming two shallow areas with zig-zag located secondary enamel knots. Distribution of TUNEL positive cells corresponded with localisation of secondary enamel knots as shown using histological and 3D analysis. Apoptosis was shown to be involved in the first molar development of the field vole, however, exact mechanisms and roles of this process in tooth morphogenesis require further investigation. © 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Tooth development; DNA fragmentation; Field vole
1. Introduction Developing organisms are a complex of growing tissues and differentiating cells, that maintain a balance between cell division (proliferation) and programmed cell death (apoptosis). Apoptosis (Kerr et al., 1972) has been recognized as an essential component in animal development, in the construction, maintenance and repair of tissues as well as in removal of misplaced or damaged cells. Dysfunctions in the regulation or execution of apoptotic cell death are implicated in a wide range of developmental abnormalities and diseases (Meier et al., 2000; Ranger et al., 2001). ∗
Corresponding author. Tel.: +42 54 1562205; fax: +42 54 9211482. E-mail address: [email protected] (E. Matalova).
0040-8166/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2004.06.006
Apoptosis was originally defined as a distinct mode of cell death on the basis of the characteristic morphological features of the cells. More recently, internucleosomal DNA cleavage, membrane alterations, specific gene expression, and characteristic pathway activations have been used as markers of apoptosis. With increasing interest in apoptosis research, many assays have been developed to detect apoptosis in cell populations as well as at the individual cell level. However, exploitation of these methods in histological sections is often limited (Dubska et al., 2002). The TUNEL assay (Gavrieli et al., 1992) is acknowledged as the method of choice for the rapid identification and quantification of apoptotic cells on histological sections. Cell–cell signalling between epithelium and underlying mesenchyme underscores the embryonic development of
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teeth (reviewed in Thesleff and Sharpe, 1997; Tucker and Sharpe, 1999; Cobourne and Sharpe, 2003). During tooth formation these signals control key processes involved in odontogenesis—cell proliferation, migration, adhesion and cell death. In developing teeth, apoptosis occurs during all stages as shown in early morphogenesis (e.g. Peterkova et al., 1996; Vaahtokari et al., 1996; Jernvall et al., 1998), amelogenesis (e.g. Smith and Washawsky, 1977; Moe, 1979; Nishikawa and Sasaki, 1995; Kaneko et al., 1997), dentinogenesis (e.g. Bronckers et al., 1996; Vermelin et al., 1996; Franquin and Remusat, 1997) and during eruption (e.g. Schellens et al., 1982; ten Cate and Anderson, 1986). Thus, apoptosis seems to have a crucial role in morphogenetic processes involved in tooth development. Embryonic development of mammalian molar teeth involves the same general stages although the final tooth shape varies among species. The field vole represents a rodent species with hypselodont teeth with the same tooth formula as the mouse 1003/1003 (one incisor and three molars in each jaw quadrant), but the molar shape is quite different (Witter et al., 1996). Although field voles have been shown to be a suitable model for comparative studies in odontogenesis (Keranen et al., 1998, 1999; Jernvall et al., 2000), temporospatial localization of apoptotic cells in tooth development of the field vole has not yet been reported. Thus, knowledge of the distribution pattern of apoptotic cells, particularly versus proliferation, appears to be necessary in order to allow for further research into interspecies differences and general mechanisms engaged in tooth morphogenesis. In this study, apoptosis was identified from initiation of tooth buds to the bell stage of the developing field vole first molar by using morphological and biochemical criteria of apoptotic cell death.
2. Material and methods 2.1. Tissue sections Field vole (Microtus agrestis) embryos (two of each embryonic stage under study) from an embryonic collection (http://www.iach.cz/lge/sbirkae.htm), embryonic days (ED) 12–18, were fixed in 4% neutral formaldehyde in water (Bancroft and Cook, 1994). After paraffin processing 4 m serial tissue sections were prepared using a rotatory microtome and mounted on poly-l-lysine-coated glass slides. Every second section was used for TUNEL assay, all the others for hematoxylin–eosin (H&E) staining.
2.3. TUNEL POD assay—labeling Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (In Situ Cell Death Detection Kit, POD) was used for proving apoptosis in individual cells. The TUNEL working procedure was carried out following the producer’s directions (Roche Biochemicals, Germany). Endogenous peroxidase was blocked by incubation in 3% H2 O2 /10 min/RT before enzymatic labeling. During the TUNEL procedure samples were washed in phosphate-buffer saline (PBS, pH 7.4): 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2 PO4 , 8.1 mM Na2 HPO4 in distilled water. The fluorescent signal conversion using anti-fluorescence antibody conjugated with peroxidase and substrate color reaction applying chromogen DAB (3,3 -diaminobenzidine tetrahydrochloride; Sigma-Aldrich, Germany) were proceeded after enzymatic labeling. 2.4. TUNEL POD assay—controls Positive control of TUNEL POD labeling was prepared using DNase (Sigma-Aldrich, Germany) treatment. After pretreatment, histological sections were incubated with DNase (5 g/ml) at 37 ◦ C/humidified chamber/10 min to induce DNA strand breaks. Negative control was obtained by omitting terminal transferase from the labeling procedure (label solution only instead of TUNEL reaction mixture). As a positive control of correct fixation and staining of apoptotic cells, embryonic limb buds and adult small intestine were tested. 2.5. TUNEL POD assay—analysis Counterstaining with alcian blue (staining of intracellular matter) or light green (cytoplasma staining) were performed before microscopic analysis of apoptotic nuclei/bodies. Samples were photodocumented after dehydration and mounting in a permanent mounting medium. 2.6. Cell morphology For histological analysis tissue sections were stained with hematoxylin–eosin or alcian blue–hematoxylin–eosin to evaluate morphological changes characteristic of apoptosis.
3. Result evaluation 2.2. Pretreatments—cell permeabilisation The sections were deparaffinized and hydrated through a graded series of ethanol and distilled water and detergent permeabilisation was applied: 0.1% TRITON® X-100 in 0.1% sodium citrate/8 min/RT.
Field vole embryos were employed to localize apoptotic cells in both upper and lower developing first molars from initiation to bell stages, embryonic days 12–18. Positive and negative controls of the TUNEL reaction were prepared as described in Section 2 and as shown in Fig. 1.
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Fig. 1. Bud stage of first upper molar. Controls of TUNEL reaction—positive control (a) and negative control (b).
In our investigation, H&E and TUNEL staining were used to evaluate apoptotic cell death during tooth development. In the H&E sections, morphological criteria of apoptotic cell death as identified by condensed chromatin and formation of apoptotic bodies were applied. Positive TUNEL POD cells were characterized by brown or red-brown staining of the nuclei.
4. Results Early tooth development in the field vole follows a similar progression of well-characterized morphological stages from
initiation to bell structures as in other mammals (Luckett, 1993). At the initiation stage, TUNEL positive cells were scattered in the epithelial part of tooth germ from ED 12.5. During bud formation, apoptotic cells were concentrated in the middle axes of the prolonging bud and later appeared at the top of the tooth bud (Figs. 2 and 3). The concentration of apoptotic cells was particularly high in the anterior part of the tooth bud, where apoptotic cells were localized on the epithelium tip (Fig. 2c). This suggests apoptotic elimination of some expected antemolar rudimental structures (as described in the mouse—Tureckova et al., 1996). In the condensed mesenchyme around the epithelial thickening, no TUNEL positive cells were found at the bud stage. The localization
Fig. 2. Bud stage of the first upper molar, anterial part. Arrow showing TUNEL positive cells with brown nuclei (a) and apoptotic bodies in hematoxylin–eosin staining (b). Accumulation of apoptotic bodies is evident on the bud tip (c).
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Fig. 3. Bud formation of the first upper molar, posterial part. Arrow showing TUNEL positive cells with brown nuclei (a) and apoptotic bodies in hematoxylin–eosin staining (b).
Fig. 4. Late cup stage of first upper molar development. Arrows indicating the enamel knot structure where TUNEL positive cells (a) are clearly visible. Hematoxylin staining (b).
Fig. 5. Bell stage of first upper molar development. TUNEL positive cells were detected in area corresponding with localisation of secondary enamel knots. TUNEL labeling (a) and hematoxylin staining (b).
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Fig. 6. Schematic illustration of apoptotic cell distribution in first molar tooth development of the field vole (Microtus agrestis). Anterior (a) and posterior (b) parts of the bud, later cup (c) and bell (d) stages in the upper jaw.
of apoptotic cells was quite restricted at later developmental stages as the TUNEL positive area expanded along the middle axes of the tooth bud and in the advanced development of the tooth cap. At the cap stage, when the primary enamel knot is mature and the crown base starts its formation, a few TUNEL positive cells were observed also in the mesenchyme and remained throughout the bell stage. At ED 13.5–15.5, the primary enamel knot showed clear sign of apoptosis as evident after H&E as well as TUNEL staining (Fig. 4). Although the apoptotic cells were observed as soon as the enamel knot structure appears, the apoptotic area was significantly extended at later stages and finally most of the cells of the primary enamel knot were TUNEL positive. Secondary enamel knots in the field vole do not protrude into the mesenchyme and are less clear than in the mouse, however, some TUNEL positive cells were detected in this area at the bell stage, when cusps formation is initiated (Fig. 5). At the later bell stage, apoptotic cells were strongly present also in the stalk of the tooth primordium. Schematic apoptosis distribution is illustrated in Fig. 6. No remarkable differences were observed in distribution of apoptotic cells in lower and upper first molar tooth germs.
5. Discussion Apoptotic cell death has been recognized for several decades (Kerr et al., 1972) and the interest in this area keeps increasing. This fact reflects the importance of this process not only during embryogenesis and in tissue homeostasis but also in the contribution of apoptosis disorders to many human defects and diseases.
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As in other mammalian species (Peterkova et al., 1996; Shigemura et al., 1999), apoptosis was detected in the dental lamina of the field vole where it is suggested to play an important role in its reduction. Thus, apoptosis can contribute to prevention of the dental lamina persistence or even overgrowth, which may lead to cyst formation and tooth eruption delay as reported in human patients (Shear, 1994; Lombardi et al., 1995; Lo Muziol et al., 1999; Tsukamoto et al., 2001). Molars show species specific number of cusps, which must be shaped during the embryonic tissue remodeling. This can be achieved either by restricted proliferation or by elimination of cells by apoptosis as shown for example in limb development (reviewed in Zuzarte-Luis and Hurle, 2002). The balance between cell division and cell death seems to be crucial in all morphogenetic processes. In the mouse, apoptotic cells were demonstrated in non-dividing (Brd-U negative) areas (Lesot et al., 1996; Shigemura et al., 1999). Proliferation activity of epithelial and mesenchymal cells based on detection of proliferation cell nuclear antigen (PCNA) during molar development of the field vole has been reported previously (Matulova et al., 2002). Considered with the data from mouse embryos, our results show localization of apoptotic cells in non-proliferating areas also in the field vole. Despite the involvement of apoptosis in dental morphogenesis and possibly defects in craniofacial development (e.g. Clouthier et al., 2000; Beverdam et al., 2001), the roles and mechanisms of this process are poorly understood (Matalova et al., 2004). The distribution pattern of apoptotic cells has been described in some mammalian species, mainly in rodents (Vaahtokari et al., 1996; Lesot et al., 1996; Viriot et al., 1997; Shigemura et al., 1999; Bronckers et al., 2000; Sasaki et al., 2001). In the field vole, apoptotis pattern has not been reported yet. We observed a specific temporospatial distribution of apoptotic events during embryonic development of the molar teeth in this species. These characteristic patterns of apoptosis in different species suggest crucial roles in odontogenesis. Morphogenesis of dentition is based on reciprocal communication between epithelial cells and underlying mesenchymal cells (reviewed in Tucker and Sharpe, 1999; Cobourne and Sharpe, 2003). This process is mediated by factors employed in cell proliferation, adhesion and migration, in cell cycle regulation as well as in apoptotic inhibition and stimulation. The enamel knot has been described as a characteristic transitory structure with a specific packing of epithelial cells, decreased proliferation activity and accumulation of apoptotic activity. This structures has been identified as the areas with ceased or no proliferation in the mouse (Shigemura et al., 1999) and also in the field vole (Jernvall et al., 2000). Enamel knots are considered as signalling centres in tooth development (Jernvall et al., 1994), which are terminated by apoptotic cell death (Lesot et al., 1996; Keranen et al., 1998). Apoptosis in the signalling centres of enamel knots has been shown also in our study. Primary enamel knots in the field vole
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are less protruded towards mesenchyme than in the mouse. Secondary enamel knots in the field vole molars are shallower than in mouse and form a zig-zag pattern according to the molar shape (in the occlussal view). Apoptosis distribution corresponded with the zig-zag pattern where the apoptotic cells were alternately concentrated in the buccal and lingual depression of the tooth bell. The different shape of the entire tooth germ as well as of the enamel knots and thus occurrence of apoptosis may have an important impact on the final tooth crown morhogenesis, which must be species specific. The direct role of enamel knots and their apoptotic silencing has been shown in Tabby (Cermakova et al., 1998; Pispa et al., 1999; Lisi et al., 2001) and downless (Tucker et al., 2000) mutants where different shape and size of the enamel knots is linked to resultant cusps defects in the adult. As in the mouse, the field vole dentition consists of one incisor, toothless diastema and three molars in each jaw quadrant. In the diastema region of both species, vestigal dental primordia appear during embryonic development (Witter et al., 1996, Peterkova et al., 2002) and undergo their apoptotic fate at the bud stage (Tureckova et al., 1996; Peterkova et al., 2002). In the filed vole molar tooth germ, the apoptotic activity differed in the arterio-posterial axes, particularly at the bud stage (as shown in Figs. 2–3), suggesting apoptotic elimination of similar structures also in this species. This gradient of regressive processes may also play a role in positioning of the first molar cap. Apoptotis can be detected throughout embryonic tooth development in all animal models examined so far and thus seems to be highly conserved and involved in morphogenetic processes of tooth shaping and patterning. This morphogenetic mechanism seems to be involved also in molar tooth development in the field vole, as shown in our study. However, the exact pathways and roles of apoptotis in tooth development have not been understood so far (Matalova et al., 2004). A passive role for apoptosis has been shown in elimination of regressive tooth diastemal primordia where apoptosis can be considered more as a degenerative process than a process of creation. It remains to be clarified whether dental apoptosis has an active and essential role in enamel knot termination, modelling the final shape and governing the size and position of the teeth or if this process just removes silenced and unwanted cells. Further examination of apoptotic pathways and related molecules is therefore a challenge for future research.
Acknowledgement This work was supported by the Grant Agency of the Czech Republic (GACR-204/02/P112).
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