| Sign In to gain access to subscriptions and/or personal tools. |
Mesenchyme is Responsible for Tooth Suppression in the Mouse Lower Diastema
1 Key Laboratory for Oral Biomedical Engineering of the Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, Hubei, P.R. China 430079; and Correspondence: * corresponding author, zhichen{at}whu.edu.cn
Between the incisor and molars in each dental quadrant, mice have a toothless gap (diastema) that may contain vestigial tooth primordia. It is still not clear whether suppression of odontogenesis in the mouse lower diastema can be attributed to epithelium, mesenchyme, or both. Therefore, using recombination experiments with mouse tissues from E11.5 and E13.5 stages, we investigated whether the epithelium or mesenchyme is responsible for the suppression of odontogenesis. Five groups of recombinants were established and cultured under mouse kidney capsules. The results demonstrated that at E11.5, the lower diastemal epithelium and mesenchyme possessed odontogenic potential and competence, respectively; at E13.5, both the lower diastemal epithelium and mesenchyme had odontogenic competence, while the lower diastemal mesenchyme did not possess odontogenic potential. On the basis of comparison of the odontogenic capabilities between the lower diastemal and molar tooth primordia, we conclude that mesenchyme is responsible for tooth regression in the mouse lower diastema.
Key Words: Mouse diastema • odontogenic competence • odontogenic potential • recombination • tooth rudiments Abbreviations: E, embryonic day • Dia, diastema • Mol, molar • BA1, first branchial arch • BA2, second branchial arch • MS, mesial segment • R2, lower second rudimentary segment • M1, first lower molar
The basic mammalian dentition consists of 3 incisors, 1 canine, 4 premolars, and 3 molars in each quadrant (Peyer, 1968). The extant mouse functional dentition is highly reduced, consisting of 1 incisor, 3 molars, and a toothless diastema in each quadrant. However, vestigial tooth primordia have been found in the prospective edentulous diastema (Peterková et al., 1995, 1996; Keränen et al., 1999; Viriot et al., 2000; Yamamoto et al., 2005). The vestigial tooth rudiments represent a model of naturally aborted odontogenesis (Peterková et al., 1995, 2000). The developmental processes and fates of the lower diastemal rudimentary primordia are different from those of the molar germs. In the prospective lower diastema region, the oral epithelium thickens, and the density of the mesenchymal cells is similar to that of a normal molar at E11 (Yamamoto et al., 2005). After initiation, the lower diastemal rudimentary tooth germs develop well and attain characteristics of the bud stage as a maximum during E12.5–E13.5. However, condensation of the local mesenchymal cells is not observed beneath the mandibular diastemal buds. At E14, the lower diastemal buds regress via apoptosis, whereas the lower molar buds progress into an early cap stage (Viriot et al., 2000; Peterková et al., 2003, 2006; Yamamoto et al., 2005). To date, the reason for suppression of odontogenesis in the mouse diastema is ambiguous. According to some authors, suppression of odontogenesis might be determined by the diastemal epithelium (Keränen et al., 1999). In contrast, other authors hypothesized that the failure of the condensation of the mesenchymal cells might be the key to arresting further development of the mandibular diastemal bud (Yamamoto et al., 2005). Odontogenic potential represents the capability of a tissue to ’instruct’ the adjacent tissue to initiate tooth formation. Odontogenic competence represents the capability of a tissue to receive and respond to odontogenic signals and to support tooth development (Zhang et al., 2003, 2005; Song et al., 2006). Classic tissue recombination experiments have revealed the odontogenic potential and competence of molar epithelium and mesenchyme (Kollar and Baird, 1969, 1970a,b). Odontogenic potential resides in the molar epithelium prior to E12.5 and then shifts to the molar mesenchyme after E12.5, whereas the dental epithelium after E12.5 and the dental mesenchyme before E12.5 possess odontogenic competence (Kollar and Baird, 1969, 1970a,b; Mina and Kollar, 1987; Lumsden, 1988; Zhang et al., 2005; Song et al., 2006). Since teeth develop through a series of inductive and permissive epithelial-mesenchymal interactions, disparities in the odontogenic capabilities of the epithelium and mesenchyme between the lower diastema and molar, if they exist, could explain odontogenic suppression in the mouse lower diastema. In the present study, to determine whether tooth regression in the mouse mandibular diastema can be attributed to epithelium, mesenchyme, or both, we investigated, by heterotypic tissue recombinations, the odontogenic capacities of the epithelium and mesenchyme of the lower diastema at E11.5 and E13.5, and compared them with those of the lower molar.
Generation of Embryos The protocol for animal use was approved by the Animal Welfare Committee of the School and Hospital of Stomatology at Wuhan University. The ICR mice (Mus musculus) were mated overnight. Noon on the day on which vaginal plugs were detected was considered as E0.5.
Histological Staining of Lower Diastemal Rudiments
Lower Diastemal and Molar Transplants
Tissue Recombinations Enzymic digestion was continued long enough to ensure that epithelia were not contaminated by adherent mesenchymal cells as described previously (Lumsden, 1988). To detect any possible contamination between epithelium and mesenchyme, we constructed the tissue recombinations between E13.5 molar mesenchyme and E10.5 second branchial epithelium. In these recombinants, either epithelium or mesenchyme was from lacZ transgenic ROSA26 mice. β-galactosidase activity was present in the epithelium, but absent from the mesenchyme after X-gal staining in the recombinants with the lacZ transgenic epithelium and the wild-type mesenchyme (APPENDIX Fig. 2A). The recombinations with the wild-type epithelium and the lacZ transgenic mesenchyme showed β-galactosidase activity in the mesenchyme after X-gal staining (APPENDIX Fig. 2B). This showed that the epithelium and mesenchyme were clearly separated from each other (Cai et al., 2007; Cho et al., 2007).
To detect the odontogenic capabilities of the lower diastemal epithelium and mesenchyme at E11.5 and E13.5, we prepared 5 groups of tissue recombinations. The epithelial and mesenchymal components and objectives of each group are summarized in the Table
After subrenal culture for 2 or 3 wks, the subrenal grafts were harvested and processed with Azan dichromic staining for histological observation of tooth formation as described previously (Zhang et al., 2003).
Histological Observation of the Lower Diastemal Tooth Rudiments To investigate the developmental process of the mouse lower diastemal rudiments, we prepared and analyzed serial frontal sections of mouse embryos from E11.5 to E14.5. At E11.5, the epithelium of the lower diastemal region (Fig. 1A  ) became thickened, as did the lower molar epithelium (Fig. 1B). At E12.5, the diastemal epithelial folding gave rise to a budding lamina, bearing an accessory bud protruding from the lingual slope toward the surrounding mesenchyme (Fig. 1C), whereas the molar epithelium formed a dental lamina without any accessory bud (Fig. 1D). The mesenchyme underlying both the diastemal and molar epithelial lamina was slightly condensed (Figs. 1C, 1D). At E13.5, the diastemal bud (Fig. 1E), like a tooth germ at an early bud stage, was smaller than the molar bud (Fig. 1F), and the accessory lingual bud became smaller or even indistinguishable. Moreover, the diastemal mesenchyme (Fig. 1E) did not condense appropriately, as did the molar mesenchyme (Fig. 1F). At E14.5, the diastemal bud was reshaped into an epithelial lamina (Fig. 1G), while the molar germ developed to the early cap stage (Fig. 1H).
Development of the Lower Diastemal and Molar Transplants
The Odontogenic Capabilities of E11.5 Lower Diastemal Epithelium and Mesenchyme
To analyze the odontogenic potential of E11.5 lower diastemal epithelium, we established recombinants of group I. After subrenal culture for 2 wks, among the 8 experimental recombinants, 3 produced teeth with thin deposition of tooth hard tissues (Fig. 2A
To test the odontogenic competence of E11.5 lower diastemal mesenchyme, we prepared recombination tissues of group II. Four out of 9 experimental transplants produced teeth with 1 cusp or 2 shallow cusps on sections (Fig. 2C ), and all of the control transplants developed into teeth with more than 2 cusps on sections (n = 5/5, Fig. 2D). Thus, E11.5 diastemal mesenchyme possessed odontogenic competence like that of molar mesenchyme.
The Odontogenic Capabilities of E13.5 Lower Diastemal Epithelium and Mesenchyme
To detect the odontogenic potential of E13.5 lower diastemal mesenchyme, we constructed recombinants of group IV. All 9 experimental explants gave rise to bone and keratinizing cysts without dental structures (Fig. 3C ). The control transplants formed multicuspid molars (n = 7/8, Fig. 3D). Therefore, at E13.5, diastemal mesenchyme did not possess the inductive odontogenic potential that molar mesenchyme normally possessed.
To identify whether the E13.5 lower diastemal mesenchyme had an inhibitory effect on tooth development, we prepared recombinants of group V. Five of 9 experimental recombinants produced teeth with 1 cusp or 2 shallow cusps on sections (Fig. 3E
In this study, to determine whether odontogenic suppression in the mouse lower diastema can be attributed to epithelium and/or mesenchyme, we first observed the morphological characteristics of the mouse lower diastemal rudiments by serial frontal sections, and, second, identified the odontogenic capabilities of the lower diastemal epithelium and mesenchyme in 5 groups of tissue recombinations. Our results demonstrated that mesenchyme is responsible for tooth regression in the mouse lower diastema.
Early studies supported the absence of a diastemal tooth bud in the prospective mandibular diastema (Mina and Kollar, 1987; Peterková et al., 1995; Ture To our knowledge, this is the first report of the odontogenic capabilities of E11.5 lower diastemal epithelium and mesenchyme. In the present study, the odontogenic potential of E11.5 lower diastemal epithelium was detected in recombinants of group I. E10.5 second branchial mesenchyme, which is odontogenically competent and can be easily accessed (Mina and Kollar, 1987; Zhang et al., 2005), was used as undifferentiated neural-crest-derived non-dental mesenchyme for this group of tissue recombinants. Both experimental and control recombinations in group I were able to form teeth, demonstrating that E11.5 lower diastemal epithelium possessed odontogenic potential similar to that of E11.5 lower molar epithelium. The odontogenic competence of E11.5 lower diastemal mesenchyme was detected in recombinants of group II. E10.5 first branchial epithelium (from the future molar-forming sites) with odontogenic potential was used as the source of instructive stimulus (Song et al., 2006) in recombinants of group II. Both experimental and control transplants in group II yielded teeth, suggesting that E11.5 lower diastemal mesenchyme had odontogenic competence similar to that of E11.5 lower molar mesenchyme. Thus, at E11.5, the odontogenic capacities resided in both the lower diastemal epithelium and mesenchyme. Compared with E13.5 lower molar epithelium possessing odontogenic competence, and the odontogenic competence of E13.5 lower diastemal epithelium was identified in recombinations of group III. E13.5 lower molar mesenchymal components with odontogenic potential were used as the inductive components in these recombinants. Experimental recombinations in group III generated teeth, as did the controls. This confirmed a previous report, in which the recombinant tooth germ between the diastemal bud epithelium and the tooth bud mesenchyme formed a tooth (Yamamoto et al., 2005). Thus, E13.5 lower diastemal epithelium possessed odontogenic competence and did not have an inhibitory effect on tooth development. Subsequently, the odontogenic potential of E13.5 lower diastemal mesenchyme was detected in recombinations of group IV and compared with that of E13.5 molar mesenchyme. E10.5 second branchial epithelia with odontogenic competence were used as undifferentiated non-dental epithelial components (Song et al., 2006) in transplants of group IV. In contrast to the control explants of group IV, which were able to yield teeth, all of the experimental tissues in group IV failed to form dental structures. This was consistent with results of the previous study, in which the recombinant tooth germ between the diastemal bud mesenchyme and the tooth bud epithelium failed to form a tooth (Yamamoto et al., 2005). Hence, E13.5 lower diastemal mesenchyme did not possess inductive odontogenic potential, which was in contrast to the lower molar mesenchyme. Whether E13.5 diastemal mesenchyme had an inhibitory effect on tooth development was further determined in tissue recombinations of group V, in which E10.5 first branchial epithelium was also used as in group II. Experimental and control recombinants in group V were able to develop into teeth, demonstrating that E13.5 diastemal mesenchyme had odontogenic competence and had no inhibitory effect on tooth development. E11.5 lower diastemal epithelium and mesenchyme possessed odontogenic potential and competence, respectively. This may explain how diastemal rudiments can be initiated. At E13.5, both the diastemal epithelium and mesenchyme were odontogenically competent, while the diastemal mesenchyme lacked inductive odontogenic potential. Consequently, we postulated that E13.5 diastemal mesenchyme lacking the odontogenic potential was responsible for the regression of the mouse lower diastemal bud. However, we cannot rule out the possibility that the diastemal epithelium might play a partial role in lower diastemal tooth degeneration, due to the inhibitory effects of diastemal epithelium on the activity of Shh protein (Cobourne et al., 2004) and the lower success rate of experimental recombinations in group I in this study. The molecular mechanisms explaining the lack of inductive potential in E13.5 diastemal mesenchyme are still not clear. Over-expression of Eda or Edar or deficiency of Sprys might be sufficient for the odontogenic potential retrieval of E13.5 lower diastemal mesenchyme and further development of the lower diastemal buds into complete teeth, since extra diastemal teeth, an atavism of premolars in the ancestry of muroids, might occur in the lower jaws of the K14-Eda mice (Mustonen et al., 2003; Kangas et al., 2004), K14- Edar mice (Tucker et al., 2004), and Spry2-null and Spry4-null mice (Klein et al., 2006). For a better understanding of tooth suppression in the mouse diastema, further studies focused on expression patterns of developmental regulatory genes, both qualitatively and quantitatively, will be necessary.
We gratefully acknowledge Prof. Y.-P. Chen (The Ohio State University, Columbus, OH, USA) for his advice and critical reading of the manuscript. This study is supported by the grant from the National Natural Science Foundation of China to Z. Chen (No. 30572043) and by grants from the Fujian Provincial Department of Science and Technology to Y.-D. Zhang (Nos. 2002I006, C0320003).
 authors contributing equally to this work  A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/4/386/DC1. Received for publication March 18, 2007. Revision received November 30, 2007. Accepted for publication December 14, 2007.
Journal of Dental Research, Vol. 87, No. 4,
386-390 (2008) This article has been cited by other articles:
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
ková et al., 1996
