Disturbed tooth germ development in the absence of MINT in the cultured mouse mandibular explants

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1 Disturbed tooth germ development in the absence of MINT in the cultured mouse mandibular explants Molecular Biology Reports An International Journal on Molecular and Cellular Biology ISSN Volume 38 Number 2 Mol Biol Rep (2010) 38: DOI / s

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3 Mol Biol Rep (2011) 38: DOI /s Disturbed tooth germ development in the absence of MINT in the cultured mouse mandibular explants Ming-Hui Zhu Wen-Bo Dong Guang-Ying Dong Ping Zhang Yong-Jin Chen Bu-Ling Wu Hua Han Received: 4 November 2009 / Accepted: 30 March 2010 / Published online: 15 April 2010 Ó Springer Science+Business Media B.V Abstract The Msx2-interacting nuclear target protein (MINT) is a nuclear matrix protein that regulates the development of many tissues. However, little is known regarding the role of MINT in tooth development. In this study, we prepared polyclonal antibodies against MINT, and found that that MINT was expressed in different cells at each stage of tooth germ development by immunohistochemistry. The role of MINT in tooth development was further illustrated by the misshapen and severely hypoplastic tooth organ in the cultured mandibular explants of MINT deficient mice. From the initiation to cap stage, the differences between mutants and wild-type molars were more and more distinguished histologically. In the MINT-deficient mandibular explants, the tooth germ was reduced in the overall size and lacked enamel knot, with abnormal dental lamina and collapsed stellate reticulum. Furthermore, the BrdU incorporation experiment showed that the proliferation activity was significantly reduced in MINT-deficient dental epithelium. Our results suggest that MINT plays an important role in tooth development, in particular, epithelial morphogenesis. Keywords MINT Tooth development Epithelial-mesenchymal interactions Organ culture Cell proliferation Ming-Hui Zhu, Wen-Bo Dong, Guang-Ying Dong and Ping Zhang contributed equally to this work. M.-H. Zhu W.-B. Dong G.-Y. Dong Y.-J. Chen B.-L. Wu Department of General and Emergency Dentistry, College of Stomatology, Xian, China M.-H. Zhu P. Zhang H. Han (&) Department of Medical Genetics and Developmental Biology, Fourth Military Medical University, Chang-Le Xi Street #17, Xian , China huahan@fmmu.edu.cn Introduction Tooth development is accomplished by the interactions between the odontogenic epithelium and the neural crestderived mesenchyme [1]. The earliest morphological sign of tooth formation is the dental lamina which forms a thickening oral epithelium at the site of the future tooth row. Dental placode, which buds to the underlying mesenchyme, forms along the dental lamina in the embryonic day 10.5 (E10.5) mouse. At the bud stage (E12.5), the epithelium signals to the mesenchyme and induces it to acquire odontogenic potential [2]. Classical tissue recombination experiments have also shown that the inductive capacity for mouse tooth formation reside in the epithelium until embryonic day 12.5 (E12.5). The induced mesenchyme is capable to instruct a non-dental epithelium permitting it to participate in tooth formation [3]. Signals from the mesenchyme are required for further tooth morphogenesis [4, 5]. During subsequent morphogenesis the cap stage (E14.5) follows, signals from the mesenchyme induce the epithelium to fold and grow to encompass the mesenchymal dental papilla. When the epithelial tooth bud has reached its full size, it invaginates and folds at its tip. At the same time, the enamel knot which is a transient structure of condensed epithelial cells, forms at the tip of the folding bud. The size and shape of the tooth crown are regulated by the enamel knot. During the bell stage (E18.5), the final size and shape of the tooth crown are fully established as the cells at the epithelial-mesenchymal interface differentiate into ameloblasts and odontoblasts, and secrete the mineralizing matrices of enamel and dentin, respectively. The same signals are responsible for the morphogenetic stages of tooth development from initiation through bell stage [2].

4 778 Mol Biol Rep (2011) 38: The main proteins involved in this signaling process include members of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), sonic hedgehog (Shh), and wingless (Wnt) family molecules [6, 7]. The evolutionarily conserved Notch signaling pathway is also essential for tooth morphogenesis [8], which is tightly regulated by several modifiers. Among them, MINT (Msx2-interacting nuclear target protein) interacts with RBP-J (recombination signal binding protein-jj), the critical transcription factor downstream to the Notch receptors, and represses the RBP-J-mediated transactivation by competing with the Notch intracellular domain(nic) for binding to RBP-J and recruiting co-repressors, thus acting as co-suppressor of Notch pathway[9, 10]. MINT was originally cloned as an interacting molecule of Msx2, a homeodomain transcription repressor involved in the craniofacial skeletal and neural development [11]. As a DNA and RNA binding nuclear matrix protein, MINT is highly homologous to Drosophila Spen, a transcription factor regulating the head development in concert with Hox homeodomain proteins [12, 13]. Similar to Spen proteins, MINT is characterized by a conserved domain structure, which is composed of three repeated RNA recognition motifs (RRMs) near the N terminus and a conserved SPOC (Spen paralog and ortholog C-terminal domain) domain at the C terminus [14]. MINT may self-interact in vitro and in vivo through its C-terminus, and the integrity of the SPOC domain is indispensable to self-interaction of MINT [15]. Being a scaffold protein, MINT potentially exerts both positive and negative regulatory actions by organizing transcriptional complexes in the nuclear matrix. For example, MINT competes with NIC for binding to a DNA binding protein RBP-J and suppresses the transactivation activity of Notch signaling [16]. MINT is highly expressed in the lymphoid tissue, central nervous system, the cardiac tissue, and the calvarial osteoblasts [9]. Kuroda et al. showed that targeted disruption of MINT gene leads to embryonic lethality at E14.5, with a developmental retardation of multiple organs [9]. However, as a widely expressed protein, possible function of MINT in many organs remains elusive. Emerging evidence suggested MINT may play a vital role in tooth morphogenesis. MINT could interact with transcription factor Runx2/Cbfa1 and Msx2 [17], which play an important role in the signaling networks regulating tooth development [18, 19]. In osteoblasts, MINT enhances the transcription of OC, a key molecular regulating tooth development [20, 21], by synergistically acting with Runx2/Cbfa1 in the presence of activated FGFR2 [17]. Furthermore, osteoblasts and odontoblasts share several molecular characteristics [20 23]. We hypothesized that MINT may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses in tooth morphogenesis. To gain a better understanding of the function of MINT during tooth morphogenesis, we prepared a polyclonal antibody (pab) against MINT and analyzed its temporal and spatial expression from early to late phases of odontogenesis. We further analyzed tooth germ development in MINT-deficient mandibular explants cultured, which is a classic experimental system and widely utilized for studying the molecular mechanisms underlying epithelial-mesenchymal interactions in vitro [24, 25]. Furthermore, we employed BrdU incorporation analysis to investigate the pattern of cell proliferation in the MINT-deficient tooth germ in mandibular explants. Our results suggested that MINT played an important role in tooth development, in particular, epithelial morphogenesis. Materials and methods Animals MINT knockout mice were described previously [9]. Heterozygous mice were intercrossed to obtain homozygous embryos, which were genotyped by PCR using genomic DNA from extra-embryonic membranes as described [9]. Mice were maintained in a C57BL/6 background. Embryonic age was determined by set mating, and the noon of the plug day was taken as E0.5. All animal experiments were approved by the Animal Experiment Administration Committee of Fourth Military Medical University. Preparation of polyclonal antibodies against MINT A fragment of MINT was amplified by PCR and was subcloned. The fragment was inserted into pet32 (a) (Novagen, Darmstadt, Germany) to construct pet-32a- MINT. The expression of the His-tagged MINT protein fused with Trx was induced with IPTG. The recombinant protein was purified using Ni?? -NTA columns (Novagen, Darmstadt, Germany), according to the protocols provided by the manufacturer. The His-MINT-Trx fusion protein was further purified after 12% SDS-PAGE, and the gel fragment containing the fusion protein (39.2 kda) was used as an antigen to immunize rabbits for the production of polyclonal antibodies. The specificity and the titer of the anti-mint polyclonal antibody were determined by Western blotting and enzyme-linked immunosorbent assay (ELISA) using standard protocols. In vitro mandible organ culture Pregnant mice of E10.5 were killed. The first branchial arch explants were dissected from embryos and were

5 Mol Biol Rep (2011) 38: cultured under the conditions of 100% humidity in an atmosphere containing 5% CO 2 95% air at 37 C in a modified Trowell s system [24, 25] for a period of up to 12 days. The explants were cultured in the BGJb medium (Invitrogen, Carlsbad, CA) containing 100 lg/ml ascorbic acid (Sigma, St. Louis, MI). The culture medium was changed every 2 days. Histology Tissues were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), ph7.4, for 12 h at 4 C, and were embedded in paraffin wax. Serial Sections 4 lm thickness were cut in the anterior-posterior direction of the mandible, and were then stained with hematoxylin. For the embryos of later stages and neonates, tissues were decalcified for 7 days with ethylene diamietetracetic acid (EDTA) before further process. For immunohistochemistry, the sections were deparaffinized, and were treated with 0.3% H 2 O 2 in phosphate-buffered saline (PBS), ph 7.4 for 30 min at room temperature, followed by the pre-treatment with the normal rabbit serum for 1 h at room temperature. The samples were incubated with the primary antisera (1:100) for 16 h at room temperature, washed, and were then incubated with the biotinylated secondary antibody for 30 min. The immune complexes were visualized by the incubation with 3, 3 0 -diaminobenzidine hydrochloride (Sigma, St. Louis, MI) for 5 min. MINT. For this purpose, the amino acid fragment of MINT was obtained by PCR, with MINT cdna as a template (Fig. 1a). The PCR product was cloned, sequenced, and inserted in frame into pet-32a to construct pet-32a-mint, which was expressed in E. coli as a His-tagged fusion protein with Trx (Fig. 1b). The expression of the His-tagged MINT protein fused with Trx was induced with IPTG. The fusion protein was purified using Ni?? -NTA columns, followed by SDS-PAGE analysis (Fig. 1c). The gel fragment containing the MINT fusion (39.2 kda) was recovered, and was directly used as an antigen to immunize rabbits. Polyclonal antibody against MINT specifically recognized the His-MINT fusion protein, as shown by Western blotting, demonstrating that it was effectively produced (Fig. 1d). The antibodies were then purified and titered by ELISA using standard protocols for later use. Expression of MINT in murine tooth germ We then employed the MINT-specific polyclonal antibody to investigate the expression pattern of MINT during early murine tooth development. BrdU incorporation assay The 5-bromo-2 0 -deoxy-uridine (BrdU, 0.5 mg/ml, Sigma) solution was added to the medium of the organ cultures, which were incubated further for 5 h before the end of the culture. Samples were then fixed and sectioned as described above. The sections were stained with the anti-brdu antibody (Sigma, St. Louis, MI) by immunohistochemistry, as described above. To quantify proliferating cells, the BrdU-labeled cells and the total number of cells within the enamel organ epithelium or within the adjacent dental mesenchyme of a tooth germ were counted from five randomly selected sections per mandibular explant. Data were expressed as mean ± SD. The significance of difference was evaluated using Student s t-test in SPSS Version P \ 0.05 was considered to be statistically significant. Results Preparation of polyclonal antibodies against MINT To examine the expression of MINT during murine tooth development, we first tried to prepare antibodies against Fig. 1 The production of polyclonal antibodies against MINT. a The construction of pet32a-mint. Lane 1, the amplified MINT fragment, as indicated by the arrow (511 bp). Lane 2, pet32a-mint digested with EcoRI and BamHI. M, DNA marker ranging from 100 bp to 2,000 bp. b SDS-PAGE of the His-MINT fusion protein (39.2 kda). Lane 1, uninduced, lanes 2 6, induced with IPTG for 1, 2, 3, 4, and 5 h, respectively. M, molecular weight marker. c Purification of the His-MINT fusion protein. Lane 1, total protein, lane 2, the supernatant of the bacterial lysates, lane 3, the purified His-MINT protein. M, molecular weight marker. The arrow indicates 39.2 kda. d Western blot. Bacterial lysates containing His tag (lane 1) or His- MINT (lane 2 8) were separated by SDS-PAGE, blotted, and probed with pre-immune rabbit serum (lane 1, 2, 1:1000), monoclonal anti- His tag antibody (lane 3, 1:500), or polyclonal anti-mint (1:50, 1:200, 1:500, 1:1000, 1:5000, for lane 4 8, respectively. M, molecular weight marker. The arrow indicates 39.2 kda

6 780 Mol Biol Rep (2011) 38: At the bud stage (E12.5, Fig. 2a), the dental epithelium, which consists of an outer layer of basal cells surrounding inner loose epithelial tissue [26], invaginate into the underlying mesenchyme and to form epithelial buds. MINT was weakly detected in the dental epithelial cells (Fig. 2a, arrow). Condensed dental mesenchymal cells around the bud also showed faint and diffuse immunopositivities for MINT (Fig. 2a, arrowhead). At the cap stage (E15.5, Fig. 2b), the epithelial bud has undergone folding morphogenesis and the basal epithelial cells of the bud have contributed to the inner enamel epithelium (IEE) facing the forming dental papilla mesenchyme, to the outer enamel epithelium (OEE) facing the dental follicle cells, and to the stellate reticulum in the middle of IEE and OEE, which separate the tooth germ from the surrounding jaw mesenchyme [27]. MINT was strongly expressed in most of the epithelial cells of the enamel organ, especially in the enamel knots. In addition, the surrounding mesenchymal cells, including those in the dental papilla cells facing IEE showed discontinuous and faint immunopositivities for MINT (Fig. 2b, arrow). During the early bell stage (E17.5, Fig. 2c), the characteristic cusp pattern of the enamel organ starts to be formed, and mesenchymal tissues are condensed and enclosed by the enamel organ as definite forms of the dental papilla. Odontoblasts differentiate at the main cusp tips and their differentiation progresses towards the basal region of each cusp in a specific time space pattern [21]. As tooth germ development progressed, MINT staining intensity in the IEE/preameloblasts (Fig. 2c, arrow) decreased. At the same time, strongly positive reactions were visualized in the odontoblasts (Fig. 2c, arrowhead). In addition, immunopositivities for MINT was also seen in the stellate reticulum and stratum intermedium as well as in mesenchymal cells in the dental papilla facing the odontoblasts. At the postnatal differentiation stage (P3, Fig. 2d), the tooth germ begins to assume the crown shape of molar teeth. Cusps can be clearly recognized, and the dental papilla space is proportionally enlarged. Alignments of tall columnar ameloblasts and odontoblasts in their functional state are obvious. At this stage, MINT expression was absent from the ameloblasts, the narrowed stellate reticulum and the immature dental pulp, whereas MINT expression was enhanced in the odontoblasts (Fig. 2d, arrow) and was moderate in predentin (Fig. 2d, arrowhead) with the development of the enamel organ (Fig. 2d). At the postnatal root developmental stage (P7, Fig. 2e and f), the outline of a tooth crown is almost defined and tooth roots start to be elongated in the molar tooth germ [28]. With the apical elongation of the cervical loop of the enamel organ, the stellate reticulum starts to diminution, and both enamel and dentin matrices start to be calcified cuspids. During this stage, on one hand, MINT was heterogeneously expressed in the odontoblasts, with a stronger staining intensity at the cusp tips (Fig. 2e, arrow) and a weaker intensity in the pit and fissure regions. On the other hand, MINT expression was preserved in the dentin (Fig. 2e, arrowhead). Moreover, the positive staining was also found in ERS (Fig. 2f, arrow). The expression pattern described above strongly suggested MINT might play a role in early murine tooth development. Abnormal early tooth germ development in the MINT mutant embryos Fig. 2 Immunohistochemistry for the expression of MINT. Sections were prepared from embryos of E12.5 (a, bud stage, 9200), E15.5 (b, cap stage, 9100), E17.5 (c, early bell stage, 9100), or neonates of P3 (d, late bell stage, 9100) and P7 (e, f, crown hard tissue development stage, 1009), and were immunohistochemically stained with the anti- MINT polyclonal antibodies. OEE, outer enamel epithelium; IEE, inner enamel epithelium; sr, stellate reticulum; oe, oral epithelium; dpc, dental papilla cells; dfc, dental follicle cells; mc, mesenchymal cells; bm, basement membrane; ab, ameloblasts; em, enamel matrix; ERS, epithelial root sheath; od, odontoblasts; pd, predentins; ek, enamel knot To address the issue whether MINT plays a role in early murine tooth development, we employed in vitro mandible culture system to observe early tooth development in the absence of MINT, since MINT deficiency leads to embryonic lethality. The mandibles were collected from the E10.5 MINT?/? and MINT -/- embryos and were cultured in vitro. Morphological examinations were chronologically performed from the 2nd to 12th day during the culture.

7 Mol Biol Rep (2011) 38: On the 2nd day of the cultivation (Fig. 3a and b), the initial invagination of the dental placode underlying mesenchymal tissue and epithelial thickening where molars were supposed to form occurred [1], which indicates the morphological initiation of tooth development. In MINT -/- mandibles, the initial invagination of the dental placode occurred, but the superficial layer of MINT -/- thickened epithelium was defected (Fig. 3b, arrow) and the overall size of epithelial thickening was smaller compared with that of MINT?/?. On the 7th day of the cultivation (Fig. 3c and d), the thickening epithelium became an epithelial bud, invaginating toward the underlying mesenchymal tissue and formed tooth bud both in MINT?/? and MINT -/- mandibles cultured. In MINT?/? mandibles cultured, the tooth bud was composed of 3 6 layers of epithelial cells and extended into the mesenchyme with its end bulge. Mesenchymal cells around the tooth bud condensed (Fig. 3c, arrowhead). In the MINT -/- mandibles cultured, however, the dental epithelium was poorly organized and the dental lamina was defect (Fig. 3d, arrow). Furthermore, mesenchymal cells were also affected and hardly condensed (Fig. 3d, arrowhead). On the 10th day of the cultivation (Fig. 3e and f), the tooth bud transforms into the cap stage and enamel organ forms. In MINT?/? tooth germ, the enamel organ was well-defined in structure and morphological features. The Fig. 3 Hematoxylin staining. The wild-type (a, c, e) and MINTdeficient (b, d, f) mandibles were cultured in vitro for 2 (a, b, 9400), 7(c, d, 9200) and 10 (e, f, 9200) days, sectioned, and were stained dental epithelium differentiated into the enamel organ, in which the IEE and stellate reticulum could be distinguished clearly. And the enamel knot was clearly detected (Fig. 3e, arrowhead). In MINT -/- tooth germ, epithelial tooth buds seemed to fold, but did not form a distinctive cap-like structure surrounding the mesenchymal cells. The enamel organ had an irregular contour. The abnormal morphology of MINT -/- enamel organ was witnessed by smaller size, apparently atrophied stellate reticulum (Fig. 3f asterisk), malformed dental lamina (Fig. 3f, arrow), absence of formed enamel knot (Fig. 3f, arrowhead), shallowly folded IEE, and reduced mesenchymal cells enclosed by IEE. Reduced cell proliferation in the cultured MINT deficient tooth germs To elucidate the mechanism of abnormal structures in MINT-deficient tooth germs, we examined cell proliferation in the cultured mandibles by BrdU incorporation. As a result of cell count, only 9% of total counted cells within enamel organ epithelium were labeled with BrdU in MINT -/- mandibles cultured for 4 days. MINT -/- mandibles cultured for 7 and 10 days also showed a significant reduced number of BrdU-labeled cells (8 and 9%, respectively) within enamel organ epithelium (Fig. 4). In the tooth germs in the cultured explants for 4 and 7 days, although BrdU-positive cells in MINT -/- tooth germs exhibited a distribution similar to that in MINT?/?, a reduction of proliferative cells in dental epithelium was observed in MINT -/- tooth organ (Fig. 4a d). After cultured for 10 days, BrdU-positive cells were observed in the dental papilla and in the IEE and OEE in wild type. Interestingly, in the dental epithelium, the number of proliferating cells in MINT -/- was comparable with that in MINT?/?, but their distribution differed. In MINT?/? dental epithelium, a zone of increased cell proliferation corresponding to the prospective cervical loop was observed in the cap-stage tooth germ (Fig. 4e arrow). This cell population was not seen in MINT -/- dental epithelium (Fig. 4f arrow) and BrdU-positive cells were dispersedly distributed in the dental papilla and in the IEE and OEE. Quantitation of the percentage of BrdU labeled cells showed that the ablation of MINT significantly reduced proliferation within enamel organ epithelium (P \ 0.05) (Fig. 4g). These results suggested that MINT might have a role in the folding of the inner enamel epithelium by regulating cell proliferation. In addition, the MINT-dependent proliferation of IEE may be important for the architecture of the prospective cusps, thus providing a potential explanation for the cusp possible shape abnormalities in MINTdeficient teeth.

8 782 Mol Biol Rep (2011) 38: Fig. 4 Altered cell proliferation in the MINT-deficient tooth germs. BrdU was added to the medium of the organ cultures and was incubated for 5 h further. The samples were fixed, sectioned, and were stained with the anti-brdu antibody by immunohistochemistry. (a d) Labeling of the cultured explants for 4 (a, b) and 7 (c, d) days. Although BrdU-positive cells in MINT -/- tooth germs exhibited a distribution similar to that in MINT?/?, a reduction of proliferative cells in dental epithelium was observed in the MINT -/- tooth germs. (e, f) Labeling of the 10th day cultivation. In MINT?/? dental epithelium (e), a zone of increased cell proliferation corresponding to the prospective cervical loop was observed in the cap-stage tooth germ (arrow). This cell population was not seen in MINT -/- dental epithelium and BrdU-positive cells were dispersedly distributed in the dental papilla and in the IEE and OEE (f). (g) Quantification of the percentage of BrdU? cells in tooth germ cultured in vitro. Data represent means ± SD. *, P \ Discussion To clarify the role of MINT in regulating tooth morphogenesis, we firstly prepared polyclonal antibodies against murine MINT, and then examined the expression pattern of it throughout tooth germ development with immunohistochemistry. MINT featured a temporo-spatial localization pattern in the epithelium and mesenchyme during odontogenesis, especially in epithelium-derived tissues, which was very similar to the expression pattern of Msx2, a nuclear protein interacting with MINT [17], indicating these two proteins may share similar functions in vivo. We then analyzed the tooth phenotypes in MINT-deficient mice, using in vitro cultured mandibles, and found that the differences between mutants and wild-type molars were distinguishable from the initiation of the cap stage, although MINT-deficient molars developed beyond the bud stage. Tooth organogenesis is a multi-step process, regulated by reciprocal and sequential interactions between the epithelium and mesenchyme. The reiterated appearance of transient signaling centers in the epithelium during key morphogenetic steps is a characteristic feature of tooth development. As the first signaling center, the dental placode expresses at least Bmp2, Bmp4, Fgf8, Shh, Wnt10b, Msx2, Lef1, and p21 [29, 30] and components of Notch pathway including Notch 1, Notch 2 and Notch 3 [2, 31]. We hypothesized that MINT, the co-suppressor of Notch receptors (Notch 1 4), may regulate the dental placode formation by manipulating the activity of Notch signaling pathway. In consistent with our hypothesis, after 2 days cultivation in vitro, the MINT mutant dental placode was not invaginated as deeply as the wild type. BrdU incorporation also showed reduced proliferating cells in the invaginated epithelium. These results clearly suggested the involvement of MINT in the initiation of tooth development. Immunohistochemical analysis showed that MINT was positively expressed in the dental lamina, stellate reticulum and enamel knot during the cap stage. Accordingly, after 4 and 7 days cultivation, different from normal process of the bud to cap transition, which is characterized by formation of the cervical loop and histogenesis of the dental epithelium when the IEE and OEE are separated by the stellate reticulum [27], MINT mutant molar exhibited impaired dental lamina, leading to strikingly aberrant tooth germ development at the cap stage. In addition, a much compromised mesenchymal condensation was visualized around the tooth bud. After 10 days cultivation, the MINTdeficient tooth germ showed the impaired cap stage,

9 Mol Biol Rep (2011) 38: evidenced by abnormal dental lamina, shallow folded IEE, collapsed stellate reticulum and absent enamel knot. These results indicated MINT was apparently required for the rapid proliferation and folding of the enamel organ epithelium, perhaps by affecting the expression of mesenchymal genes involved in the formation or degradation of basement membrane or other extracellular matrix components, as the lack of a stable epithelial mesenchymal interface has been shown to affect proper tooth morphogenesis [32]. We also found tooth germ development proceeded abnormally through the bud to cap stages in MINT-deficient embryos. During the bud to cap stage transition, the size of normal enamel organ significantly increased. The enlargement of tooth enamel organ results from both a histological reorganization of the IEE and cell proliferation in the IEE. However, MINT-deficient enamel organ displayed a smaller size. Thus, BrdU incorporation analysis was employed to determine whether the smaller enamel organ and shallowly folded IEE in MINT-deficient tooth germ resulted from defects in epithelial cell proliferationin in MINT-deficient tooth germs. Notably, proliferating cells in the IEE of MINT-deficient tooth germs were much reduced and distributed with no regular pattern. Particularly, MINT-deficient tooth germs showed no accumulation of proliferating cells at the location of the future cervical loop where IEE and OEE join. Reduced proliferation in the mutant dental epithelium conceivably leaded to the failure of cervical loop formation. These results indicated that MINT probably regulated the proliferation and folding of the dental epithelium during the bud and cap stage. Notably, MINT-deficient tooth germs exhibited morphological defects in the stellate reticulum, which is the main bulk of the enamel organ and provides large intercellular spaces to the developing tooth crown [33]. Stellate reticulum has been thought to act as a spacer device to provide mechanical protection for the developing tooth cusps or to define their spatial growth, or it might participate in nutritional recruitment for the stellate cells from the outlying vascular circulation. Harada has reported that the stellate reticulum has been identified as a source of stem cells in the developing tooth organ [34]. From the bud to cap stage transition, stellate reticulum as well as other two cell types IEE and OEE are differentiated from the enamel organ, which is regulated by Notch signaling. Notch1-3, Jagged1, Lunatic fringe (L-Fng) and Hes1 were all expressed in stellate reticulum cells [28, 35]. In MINTdeficient tooth germs the collapsed stellate reticulum suggested that MINT may be involved in the differentiation of enamel organ through modulating Notch signaling or MINT may regulate extracellular matrix assembly in the stellate reticulum, including the establishment of a proteoglycan matrix that has been shown to be an essential part of the stellate reticulum [36]. Interestingly, in MINT-deficient tooth germs, enamel knot failed to form. The enamel knot is developed from epithelial cells at the tip of the tooth bud and has been fully differentiated by the cap stage. Expressing at least ten different signals belonging to the FGF, BMP, Hh, and Wnt families, enamel knot is believed to be the second signaling center during the tooth organogenesis. The function of the enamel knot is tightly related to epithelial-mesenchymal interactions, whose formation is regulated by signals from the mesenchyme [37]. Among those signals, Notch signaling is involved in demarcating the boundary between the enamel knot and the remainder of the epithelial compartment [35, 38]. Hes1, a downstream target gene of Notch signaling, was intensely positive in the condensed mesenchyme at early bud stage and was mostly negative in the enamel knot at cap stage [34]. As a transcriptional suppressor of Hes1, MINT possessed a contrary expression pattern to that of Hes1. Thus in MINT-deficient tooth germs, the signaling pathway between epithelium and mesenchyme was probably disturbed, resulting in the absence of enamel knot and shallowly folded dental epithelium. In this study, with the polyclonal antibodies against MINT prepared by ourselves, we found that MINT was expressed in different cells at each stage of tooth germ development. The misshapen and severely hypoplastic tooth organ in the cultured mandibular explants of MINT deficient mice illustrated a significant role of MINT in tooth development. From the initiation to cap stage, the differences between mutants and wild-type molars were more and more distinguished histologically. In the MINTdeficient mandibular explants, the tooth germ displayed reduction in overall size, disappearing enamel knot, abnormal dental lamina and collapsed stellate reticulum. Moreover, the proliferation activity was significantly reduced in MINT-deficient dental epithelium revealed by BrdU incorporation experiment. Our results suggest that MINT plays an important role in tooth development, in particular, epithelial morphogenesis. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China ( , , ). References 1. Tucker A, Sharpe P (2004) The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet 5(7): Thesleff I (2003) Epithelial-mesenchymal signalling regulating tooth morphogenesis. J Cell Sci 116(Pt9): Takahashi C, Yoshida H, Komine A, Nakao K, Tsuji T, Tomooka Y (2009) Newly established cell lines from mouse oral

10 784 Mol Biol Rep (2011) 38: epithelium regenerate teeth when combined with dental mesenchyme. In Vitro Cell Dev Biol Anim. doi: /s Salazar-Ciudad I, Jernvall J (2002) A gene network model accounting for development and evolution of mammalian teeth. Proc Natl Acad Sci USA 99(12): Bei M (2009) Molecular genetics of tooth development. Curr Opin Genet Dev 19(5): Thesleff I, Mikkola M (2002) The role of growth factors in tooth development. Int Rev Cytol 217: Tummers M, Thesleff I (2009) The importance of signal pathway modulation in all aspects of tooth development. J Exp Zool Mol Dev Evol 312B(4): Mitsiadis TA, Regaudiat L, Gridley T (2005) Role of the Notch signalling pathway in tooth morphogenesis. Arch Oral Biol 50(2): Kuroda K, Han H, Tani S, Tanigaki K, Tun T, Furukawa T, Taniguchi Y, Kurooka H, Hamada Y, Toyokuni S, Honjo T (2003) Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. Immunity 18(2): Oswald F, Kostezka U, Astrahantseff K, Bourteele S, Dillinger K, Zechner U, Ludwig L, Wilda M, Hameister H, Knöchel W, Liptay S, Schmid RM (2002) SHARP is a novel component of the Notch/RBP-J kappa signalling pathway. EMBO J 21(20): Newberry EP, Latifi T, Towler DA (1999) The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates the rat osteocalcin promoter. Biochemistry 38(33): Kuang B, Wu SC, Shin Y, Luo L, Kolodziej P (2000) Split ends encodes large nuclear proteins that regulate neuronal cell fate and axon extension in the Drosophila embryo. Development 127(7): Chung N, Jee BK, Chae SW, Jeon YW, Lee KH, Rha HK (2009) HOX gene analysis of endothelial cell differentiation in human bone marrow-derived mesenchymal stem cells. Mol Biol Rep 36(2): Ariyoshi M, Schwabe JW (2003) A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling. Genes Dev 17(15): Li J, Li J, Yang X, Qin H, Zhou P, Liang Y, Han H (2005) The C terminus of MINT forms homodimers and abrogates MINTmediated transcriptional repression. Biochim Biophys Acta 1729(1): Tsuji M, Shinkura R, Kuroda K, Yabe D, Honjo T (2007) Msx2- interacting nuclear target protein (Mint) deficiency reveals negative regulation of early thymocyte differentiation by Notch/ RBP-J signaling. Proc Natl Acad Sci USA 104(5): SierraOL,ChengSL,LoewyAP,Charlton-KachigianN,TowlerDA (2004) MINT, The Msx2 interacting nuclear matrix target, enhances Runx2-dependent activation of the Osteocalcin fibroblast growth factorresponseelement.jbiolchem279(31): Bidder M, Latifi T, Towler DA (1998) Reciprocal temporospatial patterns of Msx2 and Osteocalcin gene expression during murine odontogenesis. J Bone Miner Res 13(4): Xiao G, Jiang D, Ge C, Zhao Z, Lai Y, Boules H, Phimphilai M, Yang X, Karsenty G, Franceschi RT (2005) Cooperative interactions between activating transcription factor 4 and Runx2/ Cbfa1 stimulate osteoblast-specific osteocalcin gene expression. J Biol Chem 280(35): Stein GS, Lian JB, van Wijnen AJ, Stein JL (1997) The osteocalcin gene: a model for multiple parameters of skeletal-specific transcriptional control. Mol Biol Rep 24(3): Ruch JV, Lesot H, Bègue-Kirn C (1995) Odontoblast differentiation. Int J Dev Biol 39(1): Harris SE, Harris MA (2001) Gene expression profiling in osteoblast biology: bioinformatic tools. Mol Biol Rep 28(3): Ryu YM, Hah YS, Park BW, Kim DR, Roh GS, Kim JR, Kim UK, Rho GJ, Maeng GH, Byun JH (2010) Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold. Mol Biol Rep. doi: /s Yamada M, Bringas P Jr, Grodin M, MacDougall M, Cummings E, Grimmett J, Weliky B, Slavkin HC (1980) Chemically-defined organ culture of embryonic mouse tooth organs: morphogenesis, dentinogenesis and amelogenesis. J Biol Buccale 8(2): Chai Y, Bringas P Jr, Shuler C, Devaney E, Grosschedl R, Slavkin HC (1998) A mouse mandibular culture model permits the study of neural crest cell migration and tooth development. Int J Dev Biol 42(1): Thesleff I, Nieminen P (1996) Tooth morphogenesis and cell differentiation. Curr Opin Cell Biol 8(6): Thesleff I, Vaahtokari A, Partanen AM (1995) Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs. Int J Dev Biol 39(1): Thomas HF, Kollar EJ (1989) Differentiation of odontoblasts in grafted recombinants of murine epithelial root sheath and dental mesenchyme. Arch Oral Biol 34(1): Dassule HR, McMahon AP (1998) Analysis of epithelial-mesenchymal interactions in the initial morphogenesis of the mammalian tooth. Dev Biol 202(2): Tompkins K (2006) Molecular mechanisms of cytodifferentiation in mammalian tooth development. Connect Tissue Res 47(3): Mitsiadis TA, Lardelli M, Lendahl U, Thesleff I (1995) Expression of Notch 1, 2 and 3 is regulated by epithelial-mesenchymal interactions and retinoic acid in the developing mouse tooth and associated with determination of ameloblast cell fate. J Cell Biol 130(2): Diekwisch TG, Luan X, McIntosh JE (2002) CP27 localization in the dental lamina basement membrane and in the stellate reticulum of developing teeth. J Histochem Cytochem 50(4): Kallenbach E (1980) Access of horseradish peroxidase (HRP) to the extracellular spaces of the maturation zone of the rat incisor enamel organ. Tissue Cell 12(1): Harada H, Kettunen P, Jung HS, Mustonen T, Wang YA, Thesleff I (1999) Localization of putative stem cells in dental epithelium and their association with notch and FGF signaling. J Cell Biol 147(1): Mustonen T, Tümmers M, Mikami T, Itoh N, Zhang N, Gridley T, Thesleff I (2002) Lunatic fringe, FGF, and BMP regulate the notch pathway during epithelial morphogenesis of teeth. Dev Biol 248(2): Sasaki T, Goldberg M, Takuma S, Garant PR (1990) Cell biology of tooth enamel formation: functional electron microscopic monographs. Monogr Oral Sci 14: Vaahtokari A, Aberg T, Jernvall J, Keränen S, Thesleff I (1996) The enamel knot as a signaling center in the developing mouse tooth. Mech Dev 54(1): Borkosky SS, Nagatsuka H, Orita Y, Tsujigiwa H, Yoshinobu J, Gunduz M, Rodriguez AP, Missana LR, Nishizaki K, Nagai N (2008) Sequential expressions of Notch1, Jagged2 and Math1 in molar tooth germ of mouse. Biocell 32(3):