Epiprofin/Sp6 regulates Wnt-BMP signaling and the establishment of ...

Cell Tissue Res (2012) 350:95–107 DOI 10.1007/s00441-012-1459-8

REGULAR ARTICLE

Epiprofin/Sp6 regulates Wnt-BMP signaling and the establishment of cellular junctions during the bell stage of tooth development Gaskon Ibarretxe & Maitane Aurrekoetxea & Olatz Crende & Iker Badiola & Lucia Jimenez-Rojo & Takashi Nakamura & Yoshihiko Yamada & Fernando Unda

Received: 14 March 2012 / Accepted: 24 May 2012 / Published online: 7 August 2012 # Springer-Verlag 2012

Abstract Epiprofin/Specificity Protein 6 (Epfn) is a Krüppel-like family (KLF) transcription factor that is critically involved in tooth morphogenesis and dental cell differentiation. However, its mechanism of action is still not fully understood. We have employed both loss-of-function and gain-of-function approaches to address the role of Epfn in the formation of cell junctions in dental cells and in the regulation of junction-associated signal transduction pathways. We have evaluated the expression of junction proteins in bell-stage incisor and molar tooth sections from Epfn(−/−) This work was supported by research projects from the University of the Basque Country (UPV/EHU; GIU09/70) and Unidades de Formación e Investigación (UFI11/44), by Basque Government project grant SAIOTEK SPE11UN051, and by projects from the University of Zurich to L.J. M.A. received PhD fellowships from UPV/EHU and the Jesús Gangoiti Barrera Foundation. G. Ibarretxe : M. Aurrekoetxea : O. Crende : I. Badiola : F. Unda (*) Cell Biology & Histology Department, Faculty of Medicine and Dentistry, University of the Basque Country (UPV/EHU), 48940 Bizkaia, Spain e-mail: [email protected]

mice and in dental pulp MDPC-23 cells overexpressing Epfn. In Epfn(−/−) mice, a dramatic reduction occurs in the expression of tight junction and adherens junction proteins and of the adherens-junction-associated β-catenin protein, a major effector of canonical Wnt signaling. Loss of cell junctions and βcatenin in Epfn(−/−) mice is correlated with a clear decrease in bone morphogenetic protein 4 (BMP-4) expression, a decrease in nestin in the tooth mesenchyme, altered cell proliferation, and failure of ameloblast cell differentiation. Overexpression of Epfn in MDPC-23 cells results in an increased cellular accumulation of β-catenin protein, indicative of upregulation of canonical Wnt signaling. Together, these results suggest that Epfn enhances canonical Wnt/β-catenin signaling in the developing dental pulp mesenchyme, a condition that promotes the activity of other downstream signaling pathways, such as BMP, which are fundamental for cellular induction and ameloblast differentiation. These altered signaling events might underlie some of the most prominent dental defects observed in Epfn(−/−) mice, such as the absence of ameloblasts and enamel, and might throw light on developmental malformations of the tooth, including hyperdontia.

L. Jimenez-Rojo Institute of Oral Biology, ZZM, Faculty of Medicine, University of Zurich, Plattenstrasse 11, 8032 Zurich, Switzerland

Keywords Tooth development . Epiprofin/Sp6 . Cell junction . Wnt/β-catenin . MDPC-23 . Mouse Epfn(+/−); Epfn(−/−)

T. Nakamura Department of Oral Health and Development Sciences, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan

Introduction

Y. Yamada Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda MD 20892, USA

Epiprofin/Specificity Protein 6 (Epfn) is a member of the Krüppel-like family (KLF) of transcription factors that regulate gene expression during development. Epfn has been shown to play a crucial role during the morphogenesis of ectodermal organs. Thus, Epfn(−/−) mice present serious developmental malformations in hair follicles, limbs, lungs, and teeth

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(Hertveldt et al. 2008; Nakamura et al. 2008; Talamillo et al. 2010). Concerning teeth development, this process critically depends on epithelial-mesenchymal interactions, whereby communication by secreted paracrine signals is established to direct dental morphogenesis and the polarization and differentiation of the cells producing hard dental tissue, namely, first, the odontoblasts, which produce dentin, and then, the ameloblasts, which produce enamel. The dental phenotype of Epfn (−/−) animals is striking; they present severe hyperdontia of incisor and molar teeth, together with a distinct loss of ameloblasts and enamel tissue. Odontoblast function is also affected by the loss of Epfn, which results in defective polarization of these cells and the formation of brittle dentin (Nakamura et al. 2008; Jimenez-Rojo et al. 2010a). The molecular mechanisms of these alterations have not yet been fully characterized, although evidence suggests that the absence of Epfn leads to the disruption of important cell signaling pathways during development, such as those involving follistatin/bone morphogenetic protein (BMP; Ruspita et al. 2008), sonic hedgehog (Shh), or Wnt/β-catenin (Jimenez-Rojo et al. 2010a). Tight and adherens junctions are prime regulators of cell polarization and differentiation. During development, the ameloblasts and odontoblasts that produce hard dental tissue acquire a rod-like elongated shape, polarize, and attain lengths of over 100 μm at which point they terminally differentiate. The formation of cell junctions depends upon paracrine signals that are secreted by interacting epithelial preameloblast and mesenchymal preodontoblast cells, which are placed facing each other in close proximity (Thesleff and Tummers 2009). Junctions develop in a temporally coincident manner in both cell types. During this natural differentiation process, proliferation-inducing nuclear coactivators are permanently recruited to the plasma membrane and thus inactivated. Tight and adherens junctions bind a large number of cytoplasmic proteins, some of which are translocatable nuclear coactivators linking cell adhesion and gene expression and even cell division (Balda and Matter 2009; Harris and Tepass 2010). We have shown elsewhere that tight-junction-associated claudin-1 and ZONA-B immunoreactivity is largely reduced in Epfn(−/−) mutant teeth (Jimenez-Rojo et al. 2010b). This is important because impaired cell junction formation might be one responsible mechanism for ameloblast and odontoblast differentiation defects in the absence of Epfn. Another type of nuclear coactivator that binds to cell junctions is βcatenin, which can be sequestered and inactivated by cadherin-dependent adherens junctions or alternatively can be accumulated in the cytoplasm and eventually translocated to the nucleus to upregulate the transcription of specific Wnt signaling genes. The impaired degradation and intracellular accumulation of β-catenin is a major consequence of the activation of the canonical Wnt/β-catenin signaling pathway (Tian et al. 2011).

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At the bell stage of tooth development, which in the mouse occurs between embryonic day (E) 16 and E18, ameloblast and odontoblast lineage cells are known to undergo intensive differentiation. Terminal differentiation of ameloblasts and odontoblasts is highly dependent on the secretion of intercellular paracrine signaling molecules, such as BMP. Adequate mesenchymal BMP-4 signaling is known to be essential for the differentiation of epithelial ameloblasts, as perturbation of BMP signaling leads to impaired amelogenesis (Wang et al. 2004). In this regard, canonical Wnt/β-catenin signaling in the dental papilla mesenchyme has recently been reported to be a fundamental event in tooth development, since it is a necessary upstream signal for Bmp-4 expression (Chen et al. 2009; Fujimori et al. 2010). Our recent results support the idea that this coupled Wnt-BMP signaling system operates during the bell stage of tooth development, since pharmacological Wnt activation in tooth organotypic cultures at this stage also activates Bmp-4 expression (Aurrekoetxea et al. 2012). In the present work, we have addressed the role of Epfn in the formation of cell junctions during the dental cell predifferentiation (bell) stages of the development of mouse molar and incisor teeth, between E16 and E18. At the bell stage of tooth development, Epfn expression is known to be restricted to ameloblast- and odontoblast-lineage cells (Nakamura et al. 2004). Loss of Epfn affects the establishment of tight and adherens junctions in differentiating preameloblasts and preodontoblasts/odontoblasts. This is correlated with a substantial downregulation of dental mesenchymal Wnt/β-catenin and BMP signaling in Epfn incisor and molars, as well as with a failure of ameloblast differentiation. Conversely, the overexpression of Epfn in dental pulp MDPC-23 cells induces the accumulation and nuclear distribution of cellular β-catenin, which is associated with enhanced canonical Wnt/β-catenin signaling.

Materials and methods Breeding of Epfn(−/−) mice and tissue sectioning Control Epfn(+/−) and defective Epfn(−/−) mice were generated as described elsewhere (Nakamura et al. 2008). Mouse heads were dissected out, fixed, and cryoprotected with 4% paraformaldehyde+30% sucrose in phosphate-buffered saline (PBS) overnight, embedded, and frozen in Tissue-Tek OCT compound (Sakura). Frontal head sections (15 μM) were cut by using a cryostat and stored at −80 °C until use. Histology and immunostaining Mouse incisor and molar tooth sections were stained with hematoxylin/eosin or were processed for immunohistochemistry with primary rabbit polyclonal antibodies against ZO-1 (617300; Invitrogen), pan-cadherin and β-catenin (ab6529, ab6302; Abcam),

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BMP-4 (ab39973, Abcam; AB1049, Millipore), nestin (ab5968, Abcam), and histone-3P (H3P; 06–570, Upstate). Antisera dilutions, fixation, blocking, and incubation conditions were as recommended by the manufacturer in each case. Fluorescent labeling was achieved by incubating the sections with Alexa 488 or Alexa 594 goat-anti rabbit secondary antibodies (A11008, A11012; Invitrogen) diluted 1:200 in PBS+3% bovine serum albumin for 2 h at room temperature. Cell culture, transfection, and pharmacological activation of Wnt signaling We cultured MDPC-23 cells, which were derived from mouse dental pulp and of between 60–70 total culture passages, in standard DMEM medium (Gibco) supplemented with 20% fetal bovine serum (FBS), L-glutamine, and antibiotics. We transfected cells with the green fluorescent protein (GFP)-containing Epfn-GFP plasmid DNA ppurotGFP constructs or with the equivalent nuclear localization signal (NLS)-containing (nuclear-targeted) NLS-GFP control constructs (custom designed by Innoprot), by using TransIT-LT1 Reagent (Mirus Bio), following the instructions of the manufacturer. Cells were fixed for 48 h after transfection and processed for immunocytochemistry as before. Pharmacological activation of Wnt signaling was performed by incubating cells with the glycogen synthase kinase-3 (GSK-3) inhibitor BIO ((2′Z,3′E)-6-bromoindirubin-3′-oxime; Calbiochem) or with its inactive chemical analog methyl-BIO (MetBIO) at 10 μM for 24 h. Conventional and quantitative reverse transcription polymerase chain reaction Total RNA from E19 mouse embryos and MDPC23 cells was extracted with the RNeasy Mini kit (Qiagen), according to the manufacturer's instructions. Total RNA was used to generate first-strand complementary DNA (cDNA) with the Multiscribe reverse transcriptase (Applied Biosystems). Subsequently, the cDNA was used as a template for amplification and detection of Epfn expression by conventional reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qPCR) by utilizing intron-spanning primers and optimized reaction conditions. Each sample was normalized with RPL-13 as an internal control. Normal PCR was performed by using 40 ng total cDNA in a 10-μl reaction volume (AmpliTaq Gold 5 U/μl, Applied Biosystems). Aliquots of each PCR were run on a 2% agarose gel, and DNA bands were stained with ethidium bromide. qPCR was performed in triplicate by using 40 ng total cDNA in a 10-μl reaction volume (SYBR Green PCR Reagent kit, Applied Biosystems) and run on an ABI/PRISM 7900 Sequence Detector System (Applied Biosystems). Data were expressed as the 2-ΔCt relative quantification; 2-ΔCt is the difference between the threshold cycle of a selected gene and a reference gene for the same sample. For each sample, real-time PCR was performed in triplicate, with replicas

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having Ct differences of less than 0.3 cycles being admitted as good data; the standard deviation (SD) was calculated from these replicates. In vitro tooth culture The first lower molars of E14.5 embryos were dissected by microsurgery. Teeth were placed over tissue culture membranes (0.4 μm diameter, Millicell PICMORG50, Millipore) and maintained in vitro for 6 days in DMEM/F12 medium supplemented with 20% FBS, Lglutamine, ascorbic acid, and antibiotics, in the presence of BIO or control Met-BIO at 20 μM. A higher concentration of BIO was employed in tooth organotypic cultures because of the lower penetration of the drug under these conditions, as based on previous results (Aurrekoetxea et al. 2012). Image acquisition and analysis Microscopy slides were analyzed by using microscopy equipment at the SGIKER researchsupport unit in the Basque Country University (UPV/EHU). Confocal Olympus FV500 and Leica LCS SP2 AOBS microscopes were used. Alternatively, cell samples were also observed under a conventional epifluorescence microscope. Image acquisition was performed by using identical conditions for different samples. Quantification of the labeling in images was performed by using Image J software. Labeling quantification was always normalized to cell surface area. For H3P immunoreactivity quantification, the numbers of positive (mitotic) cells where assessed by counting. Data handling and statistical tests were performed by using Excel and SPSS software.

Results and discussion Reduced expression of cell junction protein markers and βcatenin in the enamel organ, preameloblasts, preodontoblasts, and dental papilla of Epfn(−/−) mice We investigated whether alterations in ameloblast and odontoblast differentiation in Epfn(−/−) mice were caused by the defective formation of cellular junctions and the consequent disruption of their associated cell signaling networks. We employed frozen sections of bell-stage E16.5 to E17.5 control Epfn(+/−) and mutant Epfn(−/−) mouse embryos and visualized developing incisor and molar teeth. Histological assessment of incisor and molar development in Epfn(−/−) mice revealed profound morphogenetic anomalies, as previously reported (Nakamura et al. 2008). At E16.5-E17.5, bell-stage Epfn(−/−) incisor and molar sections exhibited impaired polarization of the preameloblastic inner enamel epithelium, indicative of disrupted amelogenesis (Fig. 1a–h). The mutant incisor displayed a highly aberrant enamel organ with multiple dental papilla spaces and no distinguishable labial amelogenic and lingual non-amelogenic sides (Fig. 1b, f). In Epfn(−/−) teeth, cells at the boundary between the enamel organ and the dental papilla

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retained a rounded shape indicative of a lack of differentiation, in contrast to the well-defined, rod-shaped and palisading preameloblasts and preodontoblasts found in the corresponding molar ridges and incisor labial areas of the Epfn(+/−) mouse (Fig. 1e, g versus f, h). We next compared the formation of tight and adherens junctions in bell-stage teeth of Epfn(+/−) and Epfn(−/−) mice. We chose ZO-1 (zonula occludens-1) and pan-cadherin as immunohistochemical markers of tight and adherens junctions, respectively. Tight and adherens junctions recruit important nuclear transcription cofactors and mediators of cell division, such as ZONA-B, CDK-4, or β-catenin. Immunohistochemistry of Epfn(+/−) tooth sections revealed four main areas showing the enrichment of tight and adherens junctions in the labial side of the incisor: the stratum intermedium, the apical and basal sides of the preameloblastic epithelium, and the apical side of preodontoblastic neighboring cells (preodontoblast/odontoblast cells). In contrast, a clear loss of ZO-1 and pan-cadherin labeling was found in the mutant Epfn(−/−) incisor (Fig. 1i, m versus j, n; Fig. 2a, e versus b, f). ZO-1 labeling, which was clearly present over the stratum intermedium and inner enamel epithelium of Epfn(+/−) molars, was virtually absent in Epfn(−/−) teeth (Fig. 1k, o versus l, p). Of note, the ZO-1 labeling detected in tight junctions between endothelial vascular cells in the dental papilla, dental follicle, and other mesenchymal areas was found to be unaltered in mutant Epfn(−/−) teeth. Adherens junctions recruit β-catenin protein, which is a fundamental effector of canonical Wnt signaling. In previous studies, we have reported that canonical Wnt signaling is altered during early dental development in Epfn(−/−) mutants, from E11.5 to E14.5 (Nakamura et al. 2008; Jimenez-Rojo et al. 2010a). Thus, we wondered whether the loss of cadherin proteins at the late-bell stages of tooth development would eventually lead to overactivation of the canonical Wnt/β-catenin pathway because of the impaired sequestration and release of β-catenin into the cytoplasm and, in this way, contribute to the impaired ameloblast and odontoblast differentiation in Epfn(−/ −) mice. The pattern of β-catenin immunostaining in labial side Epfn(+/−) incisors was similar to that mentioned for pancadherin, although β-catenin immunoreactivity was distributed more prominently than pan-cadherin in stratum intermedium cells and in the apical side of preameloblasts (Fig. 2c, g versus a, e). In contrast, Epfn(−/−) incisors exhibited substantially decreased pan-cadherin and β-catenin levels in the enamel organ and, particularly, in the dental papilla mesenchyme (Fig. 2a, e versus b, f; Fig. 2c, g versus d, h). Dental-pulp-derived MDPC-23 cell lines do not express Epfn under basal conditions Since we found that the expression of cell adhesion complexes and their associated transcriptional mediators such as βcatenin was reduced and sometimes even lost in developing

Cell Tissue Res (2012) 350:95–107 Fig. 1 Alterations in morphogenesis and tight junction formation in„ incisor and molar teeth of Epfn(−/−) mice. a–h Hematoxylin/eosinstained sections of bell-stage (E16.5) incisor and molar teeth from (a, c) Epfn(+/−) and (b, d) Epfn(−/−) mice. Boxed areas in a–d are shown at higher magnification in e–h. Epfn(+/−) incisors display a round dental papilla (dp) space surrounded by a thin enamel organ (eo) that exhibits clearly distinguishable labial amelogenic (Lab) and lingual non-amelogenic (Lin) sides. Polarizing preameloblasts (am) and preodontoblasts/odontoblasts (od) are found facing each other on the incisor labial amelogenic side. In Epfn(+/−) molars, the differentiation of preameloblast and preodontoblast cells is slightly delayed at this stage compared with the incisor, although preameloblasts are also evident in the dental ridges (see arrow). In Epfn(−/−) mice, incisor histomorphology is highly aberrant, with multiple dental papilla spaces and no apparent signs of preameloblast and preodontoblast polarization and differentiation. Epfn(−/−) molars also present a dysmorphic underdeveloped enamel organ at this stage (c, d, g, h) and no signs of preameloblast and preodontoblast differentiation. i–p Confocal merged images of ZO-1 (zonula occludens-1) immunohistochemistry (green) and 4,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue). Boxed areas in i–l are shown at higher magnification in m–p. Epfn(+/ −) incisors show ZO-1 enrichment in palisading preameloblast and preodontoblast/odontoblast cells facing each other on the labial side (e, i). This labeling is virtually lost in Epfn(−/−) incisors (f, j). Epfn(+/ −) molars show ZO-1 labeling in preameloblasts (g, k); this is largely diminished in Epfn(−/−) molars (h, l). Strong ZO-1 immunofluorescent areas in the dental papilla correspond to developing blood vessels (si stratum intermedium, ii preameloblast apical side, iii preameloblast basal side). Bars 50 μm (a–d, i–l), 25 μm (e–h, m–p)

teeth in the absence of Epfn, we decided to examine whether Epfn was directly involved in cell junction formation by using an Epfn overexpression model. We chose a system of dentalpulp-derived MDPC-23 cells, which can be induced to differentiate into an odontoblastic phenotype under specific conditions (Hanks et al. 1998). Thus, MDPC-23 cells can be considered to be a model of in vivo bell-stage dental papilla cells. Assessment of the basal levels of Epfn expression in MDPC-23 cells by conventional and qRT-PCR revealed the absence of Epfn under basal conditions. In contrast, E19 latebell molar teeth, which were used as a positive control, expressed Epfn (Fig. 3). Epfn overexpression induces accumulation of β-catenin in MDPC-23 cells Epfn expression in the dental papilla of bell-stage teeth is restricted to odontoblast and subodontoblast cell layers (Nakamura et al. 2004). We wanted to investigate whether forced overexpression of Epfn would affect the formation of cell junctions in MDPC-23 cells. To do so, we transiently transfected MDPC-23 cells with Epfn-GFP and control NLS-GFP transgenes, the latter including an NLS for a better comparison of the introduced recombinant proteins. The GFP products of both transgenes prominently accumulated in the nucleus by 48 h post-transfection. The nuclear location of the Epfn-GFP protein was consistent with Epfn being a transcription factor (Fig. 4a). The extent of successful transfection of MDPC-23

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cells with Epfn-GFP was low, even when using different transfection reagents and conditions (always less than 1%, assessed by cell counting; data not shown). In contrast, the number of active recombinant protein-expressing (GFP-fluorescent) MDPC-23 cells was much higher on using control NLS-GFP plasmids, with the rate of successful GFP expression (green fluorescent cells) being about 25-fold higher with respect to Epfn-GFP (assessed by cell counting; data not

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shown). This striking difference in NLS versus Epfn recombinant GFP protein accumulation after 48 h is probably attributable to the extremely rapid proteasomal degradation of the Epfn protein in dental cell types. Indeed, the half-life of Epfn protein in ameloblast cell lines has recently been reported to be a mere 40 min (Utami et al. 2011). We evaluated changes in ZO-1, pan-cadherin, and βcatenin levels by quantitative immunocytochemistry in

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MDPC-23 cells. Under our culture conditions, MDPC-23 cells did not exhibit ZO-1 immunoreactivity or labeling with other tight junction protein markers (data not shown). Thus, we focused our examination on adherens junction proteins. Quantification of the intensity of pan-cadherin immunostaining did not reveal significant differences in adherens junction formation between Epfn-transfected versus control non-transfected cells (normalized mean±SEM: 111±10% versus 100±8%; P00.37, Student’s t-test; Fig. 4b). Therefore, adherens junction formation in MDPC-23 cells was not affected by Epfn overexpression. However, β-catenin staining revealed consistent differences. Thus, Epfn-GFP-transfected MDPC-23 cells exhibited a 62±13% (mean±SEM) increase in β-catenin labeling, with respect to control nontransfected or NLS-GFP-transfected cells (Fig. 5). Similarly, β-catenin protein in Epfn-GFP-transfected MDPC-23 cells was differently distributed, adopting a more widespread localization, including extension to nuclear areas, in contrast with non-transfected cells in which β-catenin immunoreactivity was segregated from the nucleus (Fig. 4c). Thus, βcatenin immunoreactivity increased in Epfn-overexpressing

MDPC-23 cells, whereas we could not observe any change of pan-cadherin immunoreactivity under the same conditions. These results suggest that the accummulation of βcatenin in Epfn-transfected MDPC-23 cells is not dependent on adherens junction formation.

Fig. 2 Loss of adherens junction proteins in Epfn(−/−) mouse teeth. Immunohistochemical detection of pan-cadherin and β-catenin in E17.5 bell-stage incisor teeth. Confocal merged images of pancadherin (green in a, b, e, f), β-catenin (green in c, d, g, h), and DAPI nuclear counterstain (blue). Boxed areas in a–d are shown at higher magnification in e–h. Pan-cadherin is largely concentrated in preameloblasts (am) and preodontoblasts/odontoblasts (od) on the labial side of Epfn(+/−) incisors (a, e). This signal is virtually lost in Epfn(−/−) incisors (b, f). In (+/−) teeth, β-catenin is also located predominantly in

preameloblast and preodontoblast/odontoblast cells. However, its expression domain is wider than that of pan-cadherin and includes the rest of the enamel organ (eo) on the labial side (Lab) of the incisor. βcatenin expression is weaker and homogeneously distributed in the enamel organ of Epfn(−/−) teeth and appears to be absent in the dental papilla mesenchyme (Lin lingual non-amelogenic sides, si stratum intermedium, ii preameloblast apical side, iii preameloblast basal side, dp dental papilla). Bars 50 μm (a–d), 25 μm (e–h)

Accumulation of β-catenin is reproduced by incubation with the GSK-3 inhibitor BIO, in MDPC-23 cells and tooth organ cultures In order to corroborate these findings regarding β-catenin, we incubated MDPC-23 cells for 24 h with 10 μM BIO, which is known to activate Wnt/β-catenin signaling (thereby acting as a positive control) or its inactive structural analog Met-BIO. In MDPC-23 cells, the intensity of βcatenin immunostaining almost tripled in cells incubated in the presence of BIO for 24 h, with respect to non-incubated cells (Figs. 5, 6a versus b). Cells incubated with Met-BIO for 24 h showed no significant increase in β-catenin immunoreactivity compared with controls (Fig. 5). β-catenin expression did not increase further in Epfn-transfected MDPC-

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23 cells treated with BIO, compared with cells treated with BIO alone (Fig. 5). We also tested pharmacologically induced Wnt activation in whole tooth organ cultures. Teeth were dissected at E14.5 (cap stage) and maintained for 6 days in vitro in the presence or absence of BIO. Control samples progressed from cap to bell stage over that period. Samples incubated with BIO showed intense β-catenin labeling with respect to controls, in addition to profound morphogenetic anomalies (Fig. 6c versus d). Therefore, incubation with BIO dramatically increased β-catenin immunoreactivity in both MDPC-23 cells and in tooth organotypic cultures. Epfn overexpression did not further enhance β-catenin levels in BIO-treated MDPC-23 cells. BMP signaling markers are downregulated in bell-stage Epfn(−/−) incisor and molar teeth Canonical Wnt/β-catenin signaling in the tooth mesenchyme is intimately related with the production of BMPs to control multiple aspects of tooth development, such as tooth morphogenesis, tooth number, and dental cell differentiation. Recently, canonical Wnt signaling in the dental papilla mesenchyme has been reported to be a fundamental event in tooth development, i.e., as a necessary upstream signal for Bmp-4 expression (Chen et al. 2009; Fujimori et al. 2010). Similar coupled Wnt-BMP signaling systems have been found to regulate the morphogenesis of other related ectodermal organs (Shu et al. 2005; Hill et al. 2006). Conditional inactivation of β-catenin in the tooth mesenchyme can lead to either dental development arrest (Chen et al. 2009) or hyperdontia (Fujimori et al. 2010), depending on the transgenic mouse strain. The arising

Fig. 3 Dental-pulp-derived MDPC-23 cells do not express Epfn under basal conditions. a Epfn expression analysis by conventional reverse transcription with polymerase chain reaction (RT-PCR) amplification. PCR products derived from RPL13 (149 bp) and Epfn (101 bp) primer sets were separated by using a 2% agarose gel in TRIS-acetate-EDTA buffer (M DNA ladder marker, lane 1 E19 molar teeth sample as a positive control, lane 2 MDPC-23 cell line). The Epfn amplification

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hyperdontia has been attributed to a downregulation of BMP-4 (Fujimori et al. 2010). Interestingly, a similar type of hyperdontia can be induced by applying BMP inhibitors, such as Noggin, in early-stage (E10-E12) organotypic tooth cultures. This effect is dependent on the Noggin concentration and on the developmental stage of the tooth (Munne et al. 2010). BMP signals at later stages of tooth development are fundamental for ameloblast and odontoblast terminal differentiation. BMPs are, in turn, regulated by inhibitors, such as follistatin, to control amelogenesis, as also determined in experiments involving the use of genetically modified mice (Thesleff and Tummers 2009; Wang et al. 2004). The absence of amelogenesis in Epfn(−/−) mice has been proposed to be attributable to the repression of follistatin, eventually leading to follistatin protein accumulation and hence to a local disruption of BMP signals. This mechanism is supported by Epfn overexpression studies in dental epithelial cell lines in which Epfn downregulates follistatin expression (Ruspita et al. 2008). The same group has recently shown that Epfn upregulates the expression of ameloblast markers in Epfn overexpressing C9 cells (Utami et al. 2011), partially confirming some of our earlier results in preameloblastic SF2 cells (Nakamura et al. 2008). Without excluding these possibilities, another critical regulatory switch of amelogenesis by Epfn may rely on its role as an activator of Wnt/β-catenin signaling in dental mesenchymal preodontoblastic cells. In order to evaluate this possibility, we assessed BMP-4 protein levels by immunohistochemistry in incisor and molar tooth sections from Epfn(−/−) mice. BMP-4 expression was found to be noticeably reduced in both incisor and molar tooth sections,

product is absent in the MDPC-23 lane (arrow). b Epfn expression analysis by quantitative RT-PCR (qPCR). The expression level was calculated as the 2-ΔCt index and is represented as normalized values with respect to RPL13 gene expression. Data represent mean±SD values from triplicate samples of E19 embryos (E19) and MDPC-23 cells (MDPC23)

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Fig. 4 Overexpression of recombinant Epfn increases accumulation of β-catenin, but not of cadherin proteins, in MDPC-23 cells. a Nuclear localization of the recombinant Epfn-GFP protein in transfected MDPC-23 cells, as revealed by confocal merged images of the DAPI nuclear stain (blue) and Epfn-GFP (green). b Epfn overexpression does not affect cadherin immunoreactivity in MDPC-23 cells. Confocal merged images of Epfn-GFP (green) and pan-cadherin (red). c Epfn overexpression increases the accumulation of β-catenin in transfected MDPC-23 cells. Confocal merged images of Epfn-GFP (green) and βcatenin (red). β-catenin accumulates and adopts a more widespread distribution in Epfn-GFP-labeled cells (arrow; compare with the adjacent nontransfected cell). MDPC-23 cells were examined at 48 h after transfection. Bar 25 μm

particularly in dental papilla mesenchymal areas (Fig. 7a, c versus b, d). Consistent with the decrease in BMP-4, we found that the expression of the intermediate filament nestin, which is a BMP-4 downstream target (About et al. 2000), was also remarkably downregulated in Epfn(−/−) incisor and molar teeth (Fig. 7e, g versus f, h). Therefore, coupled Wnt-BMP signaling events might be operating in the dental papilla mesenchyme during the bell stage of dental development to direct the differentiation of ameloblasts and odontoblasts. The existence of this combined Wnt-BMP signaling is supported by our own previous results showing that BIO applications in tooth organ cultures, under similar conditions to those described in this study, increased BMP-4 expression in bell-stage cultured molars (Aurrekoetxea et al. 2012). Both β-catenin and BMP signaling markers are downregulated in the dental papilla of bell-stage incisor and molar Epfn(−/−) teeth, a result supporting a role for Epfn in the regulation of this coupled Wnt-BMP signaling system. Since BMP signaling appeared to be enhanced in BIOtreated teeth and inhibited in Epfn(−/−) teeth, and since the overexpression of Epfn seemed to increase β-catenin protein

accumulation, we next wanted to address whether the alterations in BMP-4 and nestin expression would also be reproduced in Epfn-overexpressing MDPC-23 cells. MDPC-23 cells under basal conditions express both nestin and BMP-4 (Fig. 8). However, none of these markers presented altered levels of expression, as assessed by immunoreactivity quantification, when we compared Epfn-overexpressing MDPC-23 cells with control non-transfected cells (normalized mean± SEM immunolabeling for nestin: 95.8±10.4% versus 100 ±6%; for Bmp-4: 107±5% versus 100±4%). This failure to induce an increase in BMP production by Epfn overexpression probably occurs because MDPC-23 cells constitute a highly limited model of odontogenesis, whereby other molecular players, including paracrine signals secreted by epithelial cells of the enamel organ, are necessary to induce BMP-4 expression in the tooth mesenchyme (Thesleff and Tummers 2009). Cell proliferation is affected in bell-stage Epfn(−/−) incisor and molar teeth Finally, we measured a 70±10% (mean±SEM) reduction in the number of histone-3P (H3P)-labeled cells in the dental

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papilla mesenchyme of E17.5 molar teeth from Epfn(−/−) mice with respect to Epfn(+/−) mice (Fig. 7k versus l). Canonical Wnt/β-catenin signaling appears to be an important enhancer of dental papilla cell proliferation at the cap to bell transition (Aurrekoetxea et al. 2012). Accordingly, the loss of mitotic (H3P-positive; H3P+) cells would reflect impaired Wnt/β-catenin signaling in Epfn(−/−) molars and a delay in organ growth. The effect of Epfn as a promoter of cell proliferation has previously been reported in non-dental COS-7 cells (Nakamura et al. 2004). However, in E17.5 bell-stage Epfn(−/−) incisor sections, we curiously found an increase in H3P+proliferating cells (Fig. 7i versius j). Indeed, H3P+cells were virtually absent in Epfn(+/−) incisors, which again might be explained by the advanced stage of cytodifferentiation in the incisors at E17.5 with respect to molar teeth (Fig. 7i versus k). Advanced cell differentiation correlates with the exit from the cell cycle and therefore a reduction in the number of H3P+ cells. Moreover, we have previously reported that tooth development appears to be retarded in Epfn(−/−) mice with respect to wild-types (Jimenez-Rojo et al. 2010a). The canonical Wnt/β-catenin pathway sustains cell proliferation at early (bud, cap, and early bell) stages of tooth development (Liu and Millar 2010). Therefore, reduced Wnt signaling would be accompanied by a reduction in cell proliferation leading to delayed organ growth, which is a consistent dental phenotype found in Epfn(−/−) mice (Nakamura et al. 2008). This delay in dental development in the absence

Fig. 5 Quantification of β-catenin immunoreactivity in MDPC-23 cells under various experimental conditions. Bar chart showing means+SEM of normalized β-catenin-labeling intensity after 48 h in MDPC-23 cells under basal non-transfection conditions (control), transfection with NLS-GFP or with Epfn-GFP, or non-transfected and treated with Met-BIO (Met-(2′Z,3′E)-6-bromoindirubin-3′-oxime) or BIO (10 μM) for 24 h, or finally BIO-treated Epfn-GFP-expressing cells. A total of 15–30 cells were analyzed for each experimental condition. Confocal image acquisition was performed under strictly similar conditions. Total β-catenin labeling was normalized to cell area for quantification. β-catenin protein significantly accumulates in Epfntransfected MDPC-23 cells and in transfected and non-transfected MDPC-23 cells exposed to BIO for 24 h before fixation (Tukey´s test; two way analysis of variance. **P