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Keywords:

  • microRNA;
  • Dicer;
  • tooth development;
  • Wnt1Cre;
  • ShhCre;
  • epithelial–mesenchymal interaction

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Background: Tooth development is known to be mediated by the cross-talk between signaling pathways, including Shh, Fgf, Bmp, and Wnt. MicroRNAs (miRNAs) are 19- to 25-nt noncoding small single-stranded RNAs that negatively regulate gene expression by binding target mRNAs, which is believed to be important for the fine-tuning signaling pathways in development. To investigate the role of miRNAs in tooth development, we examined mice with either mesenchymal (Wnt1Cre/Dicerfl/fl) or epithelial (ShhCre/Dicerfl/fl) conditional deletion of Dicer, which is essential for miRNA processing. Results: By using a CD1 genetic background for Wnt1Cre/Dicerfl/fl, we were able to examine tooth development, because the mutants retained mandible and maxilla primordia. Wnt1Cre/Dicerfl/fl mice showed an arrest or absence of teeth development, which varied in frequency between incisors and molars. Extra incisor tooth formation was found in ShhCre/Dicerfl/fl mice, whereas molars showed no significant anomalies. Microarray and in situ hybridization analysis identified several miRNAs that showed differential expression between incisors and molars. Conclusion: In tooth development, miRNAs thus play different roles in epithelium and mesenchyme, and in incisors and molars. Developmental Dynamics 241:1465–1472, 2012. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

MicroRNAs (miRNAs) are 19- to 25-nt noncoding small single-stranded RNAs that negatively regulate gene expression by binding targets in mRNAs. miRNAs bind to 3′UTR regions of target mRNAs in a sequence-specific manner to impair their translation and stability. To date, more than 400 miRNAs have been identified in humans, but it is predicted that the true figure may be between 500 and 1,000 (Griffiths-Jones, 2004; Bentwich et al., 2005; Berezikov et al., 2005, 2006; Xie et al., 2005). The miRNAs are synthesized by a multiple-step process where initially, miRNAs are transcribed as long primary transcripts by RNA polymerase II. The first transcript (pri-RNAs) folds into a characteristic hairpin, that is cleaved by the nuclear RNase III-like enzyme Drosha in a complex with DGCR8 to release 60-nt stem-loop precursors (pre-miRNAs) that are exported into cytoplasm. The cytoplasmic pre-miRNAs undergo final cleavage by Dicer, another RNA III-like enzyme, to produce mature miRNAs that assemble into an RNA-induced silencing complex (RISC).

MiRNAs are evolutionarily highly conserved in eukaryotic organisms including fish and mammals. Dicer null mutation leads to early embryonic lethality due to the loss of the inner cell mass of the blastocyst, suggesting that miRNAs are essential molecules in development (Bernstein et al., 2003). Conditional deletion of Dicer shows that Dicer plays diverse roles in the development of many tissue such as heart, lung, hair, skin and limb (Hornstein et al., 2005; Harfe et al., 2005; Andl et al., 2006; Yi et al., 2006; Harris et al., 2006; Chen et al., 2008).

Teeth develop through epithelial–mesenchymal interactions, controlled by intricate cross-talk between signaling pathways such as Shh, Fgf, Bmp, and Wnt (Pispa and Thesleff, 2003; Tucker and Sharpe, 2004). The fine-tuning of these signaling pathways is known to be crucial for the interactions to govern precise organogenesis.

Cranial neural crest cells migrate ventrolaterally during craniofacial development to contribute extensively to the formation of many structures of the head and neck. Previous studies have reported that mice with mesenchymal conditional Dicer mutation (Wnt1Cre/Dicerfl/fl) have major abnormalities including no mandible, tongue or maxillae, that are thus too severe to examine the role of miRNAs in tooth development (Cao et al., 2010). We show here on a CD1 genetic background, Wnt1Cre/Dicerfl/fl mice exhibit milder craniofacial phenotypes that enabled us to examine tooth development. In addition to mesenchymal conditional deletion, we also examined tooth development in mice with epithelial conditional Dicer mutation using ShhCre (ShhCre/Dicerfl/fl) because Shh is known to be expressed in tooth epithelium from early stages (Hardcastle et al., 1998; Dassule et al., 2000; Gritli-Linde et al., 2002). Supernumerary incisor teeth were found in ShhCre/Dicerfl/fl mice, whereas Wnt1Cre/Dicerfl/fl mice showed an arrest or absence of tooth development. The miRNAs thus play critical and complex roles in regulating tooth development.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Teeth in Wnt1Cre/Dicerfl/fl Mice

Most of the tooth mesenchymal cells are known to be Wnt1 expressing (Fig. 1A; Chai et al., 2000). Previous studies have reported that mice with conditional deletion of Dicer using Wnt1Cre (Wnt1Cre/Dicerfl/fl) have no mandible, tongue, or maxillae, phenotypes that are too severe to permit investigating of the role of miRNAs in the development of orofacial ectodermal organs (Cao et al., 2010). We however found that mandible, tongue, and maxillae were all present in Wnt1Cre/Dicerfl/fl mice on a CD1 genetic background at embryonic day (E) 18.5, although they were smaller than those of wild-type mice. CD1 background mutant mice thus provided a source to investigate the role of miRNAs in tooth development. Because Dicer is an essential molecule for miRNA processing, conditional deletion of Dicer using Wnt1Cre results in an absence of miRNAs from most of tooth mesenchymal cells, which was confirmed by the lack of miR203 expression (Fig. 1C).

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Figure 1. LacZ in Wnt1Cre/R26R mice and miR203 expression in Wnt1Cre/Dicerfl/fl mice. A: LacZ positive cells in mandibular incisor tooth germs in Wnt1Cre/R26R mice. B,C: In situ hybridization on frontal sections showing expression of miR-203 in wild-type (B) and Wnt1Cre/Dicerfl/fl mice (C) at E13.5.

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In Wnt1Cre/Dicerfl/fl mice, maxillary molars had slightly abnormal shapes, that showed prolonged epithelial projections in the inner enamel epithelium (Fig. 2B). Maxillary incisors also showed subtle abnormal shapes. The mesiolateral axis of the lingual side of mutant incisors was wider than that of the labial side, whereas the labial side of incisor teeth were wider in wild-type (Fig. 2D). The length of mutant incisors in the labiolingual axis was shorter than those of wild-types (Fig. 2D). Mutant maxillary molars were frequently observed at cap or bud stage at E18.5, whereas wild-type molar tooth germs reached to bell stages. An arrest of tooth development was also observed in maxillary incisor of Wnt1Cre/Dicerfl/fl mice. Mutant maxillary incisor tooth germs were often completely absent, whereas all mutant maxillary molar tooth germs were present (Fig. 2I).

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Figure 2. Teeth in Wnt1Cre/Dicerfl/fl mice. Frontal sections showing maxillary molar (A,B), maxillary incisor (C,D), mandibular incisor (E,F,H) and mandibular molar (G, H) of wild-type (A,C,E,G) and Wnt1Cre/Dicerfl/f (B,D,F,H). Arrow indicating protrusion (B). H: Arrow and arrowhead indicating incisor and molar, respectively. I: Frequency of tooth abnormalities in mutants.

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Unlike maxillary teeth, no tooth germs beyond the cap stage at E18.5 were observed in the mandibles (Fig. 2H,I). No epithelial buds were often observed in mandibles of Wnt1Cre/Dicerfl/fl mice. Mandibular incisor tooth germs were localized alongside the tongue in Wnt1Cre/Dicerfl/fl mice, suggesting abnormal tooth position (Fig. 2F,H).

To identify molecular changes that might be the cause of arrest or absence tooth development, we examined key tooth related molecules. Pax9 is known to be essential molecule for tooth development, because Pax9 mutants show an arrest of tooth development at the bud stage (Peters et al., 1998). Pax9 expression was found in both incisor and molar presumptive regions of mutants, although epithelial buds could not be seen in mutant mandibles (Fig. 3A,B). Bmp signaling is also known to be an important pathway in tooth development (Andl et al., 2004). A marker for activation of Bmp signaling, phosphorylated Smads 1, 5, and 8 (p-Smad1/5/8) positive cells were lost in mutant mandibular tooth germs that were arrested at cap stage, whereas they showed no significant changes in mutant maxillary tooth germs that reached to bell stage at E18.5 (Fig. 3D,F). Canonical Wnt signaling plays multiple roles in tooth development (Andl et al., 2002). The canonical Wnt target gene, Axin2 expression was lost in presumptive mandibular tooth regions that showed arrest of tooth development, whereas no significant changes of Axin2 expression were observed in mutant maxillary tooth germs that reached the cap stage at E15.5 (Fig. 3H). Wnt and Bmp signaling were thus down-regulated in mutant tooth germs that were arrested in their development.

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Figure 3. Molecular changes in tooth development of Wnt1Cre/Dicerfl/fl mice. A: Pax9 expression in presumptive mandibular incisor (arrow) and molar (arrowhead) region at E13.5. B: Light field image of A. C–F: Immunohistochemistly for p-Smad1/5/8 in maxillary incisors (C,D) and mandibular incisors (E,F) of wild-type (C,E) and Wnt1Cre/Dicerfl/f (D,F) at E17.5. F: arrowhead indicating mutant mandibular incisor. (G, H) Arrow and arrowhead indicating presumptive lower and upper molar, respectively at E15.5. A,G,H: Radioactive in situ hybridization on frontal sections showing Pax9 (A) and Axin2 (G,H).

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Teeth in ShhCre/Dicerfl/fl Mice

Shh is expressed in tooth epithelium from early stages, which was confirmed by LacZ staining in ShhCre/R26R mice (Fig. 4A,B). To investigate the role of epithelial miRNAs in tooth development, we crossed ShhCre mice with Dicerfl/fl mice (ShhCre/Dicerfl/fl). ShhCre/Dicerfl/fl mice showed supernumerary upper incisors, whereas there were no significant abnormalities in molars or lower incisors (Fig. 4D,F; data not shown). Extra incisor formation has been previously shown to be caused by alteration of apoptosis and cell proliferation (Munne et al., 2009; Charles et al., 2011). No significant changes in apoptosis or cell proliferation could be detected in upper incisors of ShhCre/Dicerfl/fl mice at E14.5 (Supp. Fig. S1, which is available online). Shh is known to be expressed in ameloblasts and is an essential molecule for ameloblast differentiation (Dassule et al., 2000; Gritli-Linde et al., 2002). Differentiation of odontoblasts is also known to rely on the interaction with ameloblasts in wild-type. To investigate whether the differentiation of ameloblasts and odontoblasts was impaired in mutants, we examined Amelogenin, Ameloblastin, and Dspp in ShhCre/Dicerfl/fl mice. Amelogenin, Ameloblastin, or Dspp however showed no significant abnormal expression in mutant tooth (Fig. 4H,J,L).

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Figure 4. Teeth in ShhCre/Dicerfl/fl mice. A: Shh expression in maxillary incisors (arrow) and molars (arrowhead) at E10.5 (in situ hybridization). B: LacZ positive cells in incisor tooth germs in ShhCre/R26R mice (arrow). (D) Frontal sections showing extra maxillary incisor (arrow in D) in ShhCre/Dicerfl/fl at E18.5. Molars in wild-type (D) and ShhCre/Dicerfl/fl (F) at E18.5. G–L: In situ hybridization of Amelogenin (G,H), Ameloblastin (I,J), and Dspp (K,L) in wild-type (G,I,K) and ShhCre/Dicerfl/f mice (H,J,L) at E18.5.

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Microarray Analysis and Expression Patterns of Selected miRNAs

Both Wnt1Cre/Dicerfl/fl and ShhCre/Dicerfl/fl mice showed different tooth phenotypes between incisors and molars. To identify differentially expression of miRNAs between incisors and molars, miRNA microarray analysis was performed between incisor and molar regions of E13.5 mandible primordia. To profile miRNA expression in tooth development at early stages, miRNA microarrays was also screened to compare miRNA expression between E10.5, E11.5, and E13.5 mandible primordia. The heat map showed a specific miRNA profile at each stage (Supp. Fig. S2). Only few miRNAs were found to be significantly changed (>4-fold) between E10.5 and E11.5 mandible primordia, E10.5 and E13.5, and E11.5 and E13.5. Most of miRNAs showing significant changes (>4-fold) were down-regulated in E10.5 or E11.5 mandible primordia (data not shown). Seventeen miRNAs were identified to be significantly down- or up-regulated between incisor and molar regions at E13.5 (Table 1; >4-fold). Based on our microarray results, 10 miRNAs (miR23b, miR98, miR148, miR193, miR203, miR205, miR218, miR363, miR378, and miR27560) were selected and their expression were examined in tooth development by in situ hybridization.

Table 1. Summary of miRNAs Up- or Down-regulated in E13 Incisor Vs E13 Molara
mmu-Incisor (E13.5)Molar (13.5)
  • a

    −, 4-fold down-regulated; 0 +, 4-fold up-regulated.

  • *

    Expressed from the opposite arm of the precursor.

miR-1+
miR-124+
miR-133a+
miR-133b+
miR-135b+
miR-138+
miR-140+
miR-193+
miR-193b+
miR-23b+
miR-203+
miR-205+
miR-206+
miR-363+
miR-7a+
miR-9+
miR-9*+

Microarray results showed that miR23b was up-regulated in incisor regions compared with molar regions at E13.5 (Table 1). MiR23b was found to be strongly expressed in both epithelium and mesenchyme of incisors, whereas it showed weak expression in the outer epithelium of molars at E13.5 (Fig. 5A,B). Weak miR23b expression was observed in enamel knots and outer enamel epithelium at E14.5 (Fig. 5C). Microarray results showed that miR27560 was up-regulated in mandibles at E11.5 compared with incisor or molar regions at E13.5. MiR27560 was expressed ubiquitously in presumptive molar regions at E11.5, whereas it showed restricted expression in molar tooth germs at E13.5 (Fig. 5D,E). Strong miR27560 expression was observed in the cervical loop, whereas enamel knots showed no expression at E14.5 (Fig. 5F). Microarray results showed that miR193 was up-regulated in incisor regions compared with molar regions at E13.5 (Table 1). MiR193 was strongly expressed in both epithelium and mesenchyme of incisors and showed weak expression in outer epithelium and mesenchyme of molars at E13.5 (Fig. 5G,H). At E14.5, miR193 expression was found in incisors, whereas it could not be detected in molars (Fig. 5I, data not shown). Microarray results showed that miR203 and miR363 were up-regulated and miR205 was down-regulated in molar tooth regions compared with incisor regions. In situ hybridization analysis however failed to show any significant differences in expression between incisors and molars. MiR203 was expressed in both epithelium and mesenchyme of both incisors and molars at E13.5 (Fig. 5J,K). At E14.5, miR203 showed restricted expression in outer enamel epithelium and the cervical loop except enamel knots (Fig. 5L). MiR205 showed restricted expression in epithelium of both incisors and molars from E10.5 (Fig. 5M–O). Weak miR363 expression was observed in epithelium of both incisors and molars (Fig. 5P,Q). Microarray results showed that miR98, miR218, miR378, and miR148a were significant up-regulated in both incisor and molar regions at E13.5 compared with mandible primordia at E10.5 or E11.5. In situ hybridization analysis, however, failed to show significant differences in expression between them. MiR98 expression was observed in outer epithelium of tooth buds and mesenchyme at E13.5, and showed restricted expression in the buccal cervical loops at E14.5 (Fig. 5R,S). MiR378, miR148a, and miR218 were expressed in outer epithelium and mesenchyme at E13.5, whereas expression were observed in outer epithelium and the cervical loops, except enamel knots at E14.5 (Fig. 5T–X).

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Figure 5. The expression of miRNAs in tooth development. In situ hybridization on frontal sections showing expression of miR-23b (A–C), miR-27569 (D–F), miR-193 (G–I), miR-203 (J–L), miR-205 (M–O), miR-363 (P,Q), miR-98 (R,S), miR-378 (T,U), miR-148a (V,W), and miR-218 (X) in wild-type at E10.5 (M), E11.5 (D), E12.5 (N–P), E13.5 (A,B,E,G,H,J,K,R,T,V,X) and E14.5 (C,F,I,L,Q,S,U,W).

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The expression of miRNAs we examined is summarized in Tables 2 and 3.

Table 2. Summary of Expression of miRNAs in Tooth Development at Embryonic Day 13.5a
 Incisor (E13.5)Molar (13.5)
mmu-Basal epitheliumStellate reticulumMesenchymeBasal epitheliumStellate reticulumMesenchyme
  • a

    +, low; ++, medium; and +++, high level of expression. The level of expression was assessed of each probe separately.

miR-23b+++++++
miR-98+++++++++
miR-148a++++++++++
miR-193+++++++++++
miR-203++++++++++++++
miR-205++++++
miR-218++++++++++++
miR-363++++
miR-378++++++++++
miR-27560+++++++++++++
Table 3. Summary of Expression of miRNAs in Molar Tooth Development at Embryonic Day 14.5a
mmu-Inner epitheliumOuter epitheliumCervical loopEnamel knotStellate reticulumDental follicle
  • a

    +, low; ++, medium; and +++, high level of expression. The level of expression was assessed of each probe separately.

miR-23b++++
miR-98+++
miR-148a+++++++++
miR-193++++++
miR-203++++++++++
miR-205++++
miR-218+++++
miR-363+++
miR-378++++++
miR-27560+++++++++++

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Epithelial deletion of Dicer using ShhCre resulted in extra tooth formation, a phenotype also seen in mice with epithelial mutation of Dicer using Pitx2Cre (Cao et al., 2010). Both Shh and Pitx2 are expressed from E10.5, whereas K14 starts to be expressed from around E12 (St Amand et al., 2000; Dassule et al., 2000). In rodents, several rudimentary tooth epithelial buds are known to be present in the incisor region at early stages of tooth development. These vestigial buds are believed to merge to form incisor tooth germs or are removed by apoptosis (Peterkova et al., 2000, 2009). It is possible that in the absence of miRNAs, bud merger, or apoptotic activity is inhibited, which can rescue the development of vestigial tooth germs. In Wnt1Cre/Dicerfl/f mice, upper molars had abnormal shapes similar to those in mice with mutation of Caspase-3 (Matalova et al., 2006). It has been shown that miRNAs in neural crest-derived cells are involved in apoptotic activity in jaw, brain, nerve, tongue, and palate development (Zehir et al., 2010; Sheehy et al., 2010; Huang et al., 2010; Nie et al., 2011). It is thus possible that mesenchymal miRNAs inhibit gene expression that reduces apoptosis in tooth development. Loss of miRNAs thus leads to increase in these inhibitory factors, resulting in reduced apoptotic activity in tooth development.

In Wnt1Cre/Dicerfl/f mice, incisor tooth germs were found to abnormally locate alongside the tongue. The anterior mandible has been shown to be shortened in the anterior–posterior axis in Wnt1Cre/Dicerfl/f mice and we also found expansion of the posterior part confirmed by expression of the posterior marker, Barx1 (Sheehy et al., 2010; data not shown). Mandible development is thus altered in the anterior–posterior axis. The abnormal position of incisors is thus likely to be caused by the abnormal mandible growth. Although extra tooth germs were never observed in mutant jaws, we could not exclude the possibility that the abnormal positioned tooth was an extra tooth, while the incisor was absence.

In Wnt1Cre/Dicerfl/f mice, Wnt and Bmp signaling was down-regulated in mutant tooth germs that were arrested in their development. This raised the possibility that inhibitory factors for Bmp and Wnt signaling were up-regulated by loss of miRNAs. MiR-203 was expressed in tooth mesenchyme, and one of its targets is the Bmp antagonist, Wise (Ectodin, Sostdc1, USAG-1) and Bambi that are expressed in tooth mesenchyme (Laurikkala et al., 2003; Pummila et al., 2007). MiR-7a, miR-23b, miR-193, and miR-218 expression were observed in tooth mesenchyme, and one of their targets is the Bmp antagonist, Gremlin1 that is also expressed in tooth mesenchyme (data not shown; Pummila et al., 2007). MiR-7a, miR-98, and miR-203 were expressed in tooth mesenchyme, and one of their targets is the Wnt antagonist, Dkk3 that is expressed in tooth mesenchyme (data not shown; Fjeld et al., 2005). It is thus possible that the lack of miRNAs leads to the up-regulation of these antagonists, resulting in reduced Bmp and Wnt signaling in tooth development.

Fine-tuning of molecular mechanisms is known to be crucial in tooth development because single gene mutation often leads to either increasing or reducing number of tooth, and hypomorphic mutation has been shown to lead to multiple tooth anomalies (Peterkova et al., 2005; Ohazama et al., 2008; Charles et al., 2011). The miRNAs are known to be fine-tuners of many signaling pathways in multiple organs (Shyu et al., 2008; Guo et al., 2010; O'Neill et al., 2011). Variation of tooth phenotypes seen in Wnt1Cre/Dicerfl/f mice is likely to be caused by perturbation of the fine-tuning of molecular pathways.

Wnt1Cre/Dicerfl/f mice showed a lack of incisors, whereas extra incisors were observed in ShhCre/Dicerfl/f mice. The role of epithelial miRNAs is thus opposite from those of mesenchymal miRNAs. Correct epithelial–mesenchymal interaction is essential for tooth development, which requires miRNAs in upper incisor development. Unlike upper incisors, ShhCre/Dicerfl/f mice showed no significant anomalies in lower incisors and molars, whereas Wnt1Cre/Dicerfl/f mice exhibited abnormalities in both these types of tooth. The role of miRNAs is thus different between tooth types.

MiRNAs were found to show restricted expression in tooth germs including the primary enamel knots, inner enamel epithelium, and dental papillae. Teeth are known to develop through interaction between these components of tooth germs. The miRNAs are thus likely to be involved in these interactions.

In ShhCre/Dicerfl/fl mice, no significant changes of morphology or expression of ameloblast differentiation marker, Amelogenin or Ameloblastin could be detected in molars or lower incisors at birth. Development of molars or lower incisors was thus not impaired until the early bell stage in ShhCre/Dicerfl/fl mice. K14 is also expressed in tooth epithelium including ameloblasts and mice with epithelial deletion of Dicer using K14Cre show only minor enamel defects (Michon et al., 2010). It is possibility that enamel defects would be present in ShhCre/Dicerfl/fl mice if they survived after birth.

Dicer mutant mice showed tooth phenotypes that were different between incisors and molars. ShhCre/Dicerfl/fl mice also showed different tooth phenotypes from those in Wnt1Cre/Dicerfl/fl mice. Microarray and in situ hybridization analysis identified several miRNAs that showed differentially expression between incisors and molars. In tooth development, miRNAs thus play multiple roles in epithelium and mesenchyme, and in incisors and molars.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Production and Analysis of Transgenic Mice

Dicerfl/fl, Wnt1Cre, R26R, and ShhCre mice were produced as described by Harfe et al. (2005), Danielian et al. (1998), Soriano (1999), and Joksimovic et al. (2009), respectively. CD1 genetic background Wnt1Cre/Dicerfl/fl mice were generated by six generation of backcrossing to inbred CD1 mice.

Micro-array Analysis of miRNAs

Total RNA including miRNA was extracted from whole mandible primordia of E10.5 or E11.5 and incisor or molar regions of E13.5 mandible primordia using the miRNeasy Mini kit as described by the manufacturer (Qiagen). RNA quality and concentration was monitored using a 2100 Bioanalyzer (Agilent Technologies). Microarray including labeling, hybridization, and analysis of the data were carried out by Exicon A/S (miRCURY LNA Array v.9.2.).

In Situ Hybridization

Mouse heads were fixed in 4% paraformaldehyde, wax embedded, and serially sectioned at 7 μm. Sections were split over 5–10 slides and prepared for in situ hybridization.

For detecting mRNAs, radioactive in situ hybridization with [35S]UTP-labeled riboprobes were carried out as described previously (Ohazama et al., 2008). Decalcification using 0.5 M EDTA (pH 7.6) was performed after fixation of E16 and newborn mice.

For detecting miRNAs, locked nucleic acid probes labeled with digoxigenin (DIG) were purchased from Exicon A/S. After deparaffinization, the slides were pretreated with 5 mg/ml proteinase K and 0.25% (vol/vol) acetic anhydride. Hybridization was carried out overnight in a humidified chamber. After hybridization, tissues were washed in high-stringency conditions and preblocked in antibody blocking solution, then incubated with preabsorbed antibody overnight. DIG-labeled antisense riboprobes were detected with alkaline phosphatase-coupled anti-DIG antibodies using NBT and BCIP as the color substrates in NMT solution.

Immunohistochemistry

After deparaffinization, sections were treated by proteinase K and then incubated with antibody to Phosphorylated Smad1, Smad5, and Smad8 (p-Smad1/5/8; Cell signaling Technology), active-Caspase3 (Promega), or Ki67 (Vector). As a negative control, normal rabbit serum were used instead of primary antibody. Tyramide signal amplification system was performed (Parkin Elmer Life Science) for detecting the anti-p-Smad1/5/8 or active-Caspase3 antibody. Alexa488 (Molecular probe) were used for detecting the anti-Ki67 antibody. Pictures were taken with same exposure between control wild-type and mutant mice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Alex Huhn for technical supports. Y.O.-K. is supported by Nihon University. K.K. is supported by JSPS International Program for Young Researcher Overseas Visits.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_23828_sm_SuppFig1.tif406KSupp. Fig. S1. Cell proliferation and apoptosis in ShhCre/Dicer fl/fl mice. Immunohistochemistry of Ki67 (A,B) and active-Caspase3 (C,D) in upper incisors in ShhCre/Dicer fl/fl (B,D) and wild-type (A,C) at E14.5 (C,D). Tooth epithelium is outlined by red dots.
DVDY_23828_sm_SuppFig2.tif595KSupp. Fig. S2. Differentially expressed miRNAs. The heat map showed differentially expressed 2.5-fold.

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