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The developing dentition is a paradigm for organogenesis, with morphogenesis and patterning (position, shape, and size), cell differentiation (enamel, dentin, cementum, bone), and cell function (mineralization, homeostasis, and regeneration) all having essential roles for its correct formation. Numerous genes are already known to function during the early stages of tooth development (Chai and Maxson, 2006) through both the genetic analysis of families presenting with a dental phenotype, such as PAX9 and third molar agenesis (Stockton et al., 2000), as well as direct investigation of the developing tooth. However, many of the genes involved in the latter stages of amelogenesis, root formation, and tooth eruption remain unknown (Chai and Maxson, 2006). Tooth development in non–human-like rodents is largely the same as that in humans (Peterkova et al., 2006). The mouse is, therefore, an excellent model system within which to investigate the molecular mechanisms of tooth development.
Tooth development involves the reciprocal interactive events of epithelial and mesenchymal tissues, which develop through the soft tissue phases: initiation, bud, cap, and bell stages (Chai and Maxson, 2006). This period is followed by the hard tissue formation stages of dentinogenesis and amelogenesis, or enamel formation. Tooth development involves the complex interplay of regulatory molecules that include transcription factors, growth factors, as well as cell surface and extracellular matrix molecules (Tucker and Sharpe, 2004). Many of these signaling molecules are also used recurrently in a variety of organs throughout vertebrate development and are remarkably well conserved throughout evolution (Peters and Balling, 1999; Pispa and Thesleff, 2003). Considerable insight into the identity and role of genes during the tooth development process has been gained from studies in both animal models and in humans (Thesleff and Nieminen, 1996; Stockton et al., 2000; Thesleff, 2003). There are currently more than 300 genes known to function during tooth development: 40 growth factors, 50 receptors, 105 signaling molecules, 57 transcription factors, 92 intracellular molecules, 114 extracellular molecules, and 81 plasma membrane proteins (genes may be categorized into more than one group; Thesleff, 2006). Despite many of these genes being used recurrently in the development of other organs, there are likely many additional genes that play a critical role in the tooth development process that are heretofore undiscovered.
To identify novel genes involved in tooth development for the purpose of furthering our knowledge of this developmental pathway and to provide a database of genes to aid future genetic dissection of dental phenotypes, we have investigated gene expression in the developing mouse molar tooth between postnatal days (P) 1 and P10. This period of tooth development primarily encompasses the amelogenesis stages where the enamel structures are formed, but also includes the early stages of root development (Chai and Maxson, 2006). Mouse molar teeth were chosen as their development is finite and, hence, more closely resembles that of human tooth development, unlike mouse incisor teeth, which grow continually. To this end, we carried out microarray gene expression, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and mRNA in situ hybridization analysis to identify genes that are specifically expressed in the developing tooth.
Microarray Analysis of Mouse Molar Tooth Gene Expression
To identify genes that function during mouse molar tooth development, the expression of the 34,000 mouse genes present on the Mouse Genome Expression 430 2.0 microarray (Affymetrix, Santa Clara, CA) were interrogated in a pool of mRNA from molar teeth extracted from Swiss Webster mouse pups between 1 and 10 days postnatal. These data were compared with a control set of 16 tissues obtained from the National Center for Biotechnology (NCBI) Gene Expression Omnibus (GEO) database (accession no. GSE1986), whose expression data were derived from tissue obtained from Swiss Webster mice between 2 and 4 weeks postnatal.
Analysis of the genes differentially expressed between the dental molar teeth (DMT) and control tissues (Fig. 1) found that the genes that exhibited increased expression in DMT were far more significant (10−9–10−23) than those that exhibited decreased expression (10−4–10−8). It was also apparent that more genes exhibited increased expression in the DMT over the controls (Fig. 1).
Analysis of the top 100 genes that exhibited decreased expression in the DMT (Table 1) found that none had been previously associated with tooth development. However, analysis of the top 100 genes that exhibited increased expression in the DMT found 29 that had been previously associated with tooth development (Table 2). Eight of these genes have also been associated with anomalies in tooth development. All five genes previously identified as causing amelogenesis imperfecta, a disease characterized by enamel defects (Aldred et al., 2003), when disrupted, are present within these 29 genes: amelogenin (Amelx; Lagerstrom et al., 1991), kallikrein 4 (Klk4; Hart et al., 2004), matrix metalloproteinase 20 (MMP20; enamelysin; Ozdemir et al., 2005), enamelin (Enam; Rajpar et al., 2001), and distal-less homeobox 3 (Dlx3; Price et al., 1999). Other genes present included ameloblastin (Ambn), a gene that when inactivated in the mouse presents with a similar enamel phenotype (Paine et al., 2003), as well as the dentin sialophosphoprotein (Dspp) gene associated with dentinogenesis imperfecta (Zhang et al., 2001) and the paired-like homeodomain transcription factor 2 (Pitx2) associated with Rieger Syndrome (Semina et al., 1996).
Table 1. Top 100 Genes Exhibiting Decreased Expression in the DMT Over Control Samples
Adj. P value
1.29 × 10−08
cytochrome c oxidase, subunit XVII assembly protein homolog (yeast)
solute carrier family 13 (sodium-dependent citrate transporter), member 5
1.03 × 10−09
1.10 × 10−09
growth factor receptor bound protein 10
1.10 × 10−09
insulin-like growth factor binding protein 5
1.12 × 10−09
After the removal of known tooth development genes, 56 (of 71) functionally characterized genes were identified as exhibiting increased expression in the DMT (Table 2) compared with 78 (of 100) exhibiting decreased expression (Table 1). Additionally, there were 14 and 23 additional unknown genes, respectively. Bioinformatics analysis of these unknown genes found that very few possessed a known domain within their sequence (data not shown) and that they were distributed between the different cellular compartments, making it difficult to assign a putative function.
Web-based gene ontology analysis of the top 100 genes exhibiting increased expression in the DMT found a greater than expected number of genes conferring compression resistance in an extracellular matrix and in integrin binding. There were also a greater than expected number of genes functioning in osteoblast differentiation, regulation of cell differentiation and signal transduction, skeletal development, tissue remodeling, anatomical structure formation, mesoderm development, biomineral formation, organ and appendage morphogenesis, all of which are processes that would be expected to be important during tooth development. Interestingly, similar analysis of those genes exhibiting decreased expression in the DMT found that a greater than expected number of genes were involved in the catalysis of redox reactions that use heme groups as a hydrogen or electron donors for the generation of a proton electrochemical gradient across a membrane and that are located in the organelle inner and outer membrane and in the organelle or mitochondrial envelope.
qRT-PCR Confirmation of Microarray Results
On the premise that genes involved in the amelogenesis and tooth development processes would most likely exhibit an increased expression in our DMT mRNA when compared with other control mRNA samples, which is strongly supported by our analysis above, we concentrated on the top 100 genes that were identified as exhibiting a significantly increased expression level in the DMT over the control samples for confirmation of their expression by qRT-PCR analysis. The 71 genes not previously associated with tooth development that were identified in the top 100 genes identified by microarray analysis to show increased expression in the DMT, as well as three control genes known to function in tooth development (Amelx, Enam, Shh), were chosen for qRT-PCR analysis. Of these 74, expression levels of 68 were found to be in agreement with the microarray data, of which six were unknown genes (data not shown). Based on bioinformatics and knowledge about pathways or functions of related genes, seven genes were selected for further analysis: Rspo4 (R-spondin family, member 4; Cristin-4), Papln (papilin, proteoglycan-like sulfated glycoprotein), Vrm (Vomeromodulin precursor), Amtn (Amelotin), Gja1 (gap junction protein, alpha 1; Connexin 43), Sp7 (Sp7 transcription factor; osterix), and Maf (avian musculoaponeurotic fibrosarcoma [v-maf] homolog), and the results of their qRT-PCR can be found in Table 3.
Table 3. qRT-PCR Results for Rspo4, Papln, Amtn, Gja1, Maf, Vrm, and Sp7, and the Amelx and Enam Controlsa
The ΔCt is the difference in cycle number between sample and negative data, and ΔΔCt is the Log2 fold difference between the DMT and either the Liver or Pool samples (ud = undetermined, nd = not determined). DMT, dental molar teeth; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.
Verification of Tooth Specific Expression of Genes by mRNA In Situ Hybridization
Seven genes were selected based on the microarray and qRT-PCR results, and the pattern of their expression in the developing incisor (Fig. 2) and molar (Fig. 3) tooth was analyzed in coronal sections of the heads of newborn mice using mRNA in situ hybridization. The results identified five genes that are expressed specifically in tooth tissues in the craniofacial region, including one expressed in the stratum intermedia (Gja1; Figs. 2A, 3A), two in the ameloblasts (Maf and Papln; Figs. 2B, 3B, and 2C, 3C, respectively), one only in incisor epithelia and the basal lamina of molar, but not incisor, ameloblasts (Amtn; Figs. 2D, 3D), and one in the dental papilla (Rspo4; Figs. 2E, 3E). Of the remaining two genes, Sp7 was found not to be expressed specifically in tooth tissues and the remaining gene (Vrm) was found to be expressed in the nasal serous gland, which is located close to, but distinct from, the developing tooth (data not shown).
Genetic dissection of the tooth development process over the past two decades has identified signaling molecules and factors that partner with them to allow precise development of the tooth at the appropriate developmental stage, with the correct pattern, shape, and size. The availability of the complete genomic sequence for many vertebrate species including rodents and humans as well as their representative transcriptomes, has provided a platform for large-scale interrogation of genes in specific organs. To identify genes heretofore undiscovered as critical players in the biogenesis of teeth, we have used microarray gene expression analysis of the developing mouse molar tooth between P1 and P10 to identify genes differentially expressed when compared with 16 control tissues. Our aim was to identify tooth-specific developmental genes. As many genes involved in developmental processes in the tooth are likely required during the development of other tissues, we wanted to minimize the inclusion of these more general developmental genes in our list of candidate tooth-specific genes. These control tissues (see the Experimental Procedures section) were chosen to provide a wide cross-section of tissues with which to compare DMT to ensure that the genes exhibiting the greatest differential expression, and by inference genes most likely to be tooth-specific, would be identified ahead of those genes that function more generally in tissue development.
Analysis of the top 100 genes showing increased expression in the DMT compared with control samples found that 29 had been previously associated with tooth development. However, analysis of the top 100 genes found to exhibit decreased expression in the DMT identified no genes previously associated with tooth development. This finding is striking, given that the repression of genes and signaling pathways during developmental processes is likely as important as their activation. These genes exhibiting decreased expression may, therefore, offer novel gene families that are involved in the regulation of tooth development, but further research is required to clarify their role, if any, in tooth development.
The high expression levels of 68 of 71 genes, which were identified as exhibiting a significantly increased expression level in the DMT and which had not been previously associated with tooth development, were confirmed by qRT-PCR analysis, which included six previously uncharacterized genes, indicating that the combination of microarray and qRT-PCR analyses is a good screening method to identify tissue/organ specific genes. Only five of seven genes analyzed by mRNA in situ hybridization were tooth-specific, indicating that microarray and qRT-PCR analyses may not be accurate enough to characterize particular tissue/organ specific genes. Our results suggest that the combination of microarray, qRT-PCR, and mRNA in situ hybridization is a better strategy to characterize the global tissue/organ specific expression pattern and to characterize accurately the specificity and expressing cell type of each target gene.
The expression of Maf and Amtn was found to be appreciably lower in the molar (Fig. 3B and D, respectively) compared with incisor (Fig. 2B and D, respectively) tooth by mRNA in situ hybridization. However, their expression had been found to be appreciable by microarray and qRT-PCR analysis of the molar tooth. The microarray and qRT-PCR analysis was performed on molar teeth from mouse pups between P1 and P10 and at P2, respectively, and the mRNA in situ hybridization was performed on tooth germs from P0. Their lower expression in the molar compared with incisor tooth germs at P0, but their appreciable expression at later time points in the molar tooth, would suggest that their expression is increasing from P0 to P10 in the molar tooth and that they are expressed earlier in incisor tooth development.
Amtn is reported to be an ameloblast-specific gene (Iwasaki et al., 2005; Moffatt et al., 2006; Trueb et al., 2007) whose expression is restricted to maturation-stage ameloblasts in developing murine molars and incisors (Iwasaki et al., 2005; Moffatt et al., 2006). Its greatest expression is found in the basal lamina of the ameloblasts in both the incisors and molars (Moffatt et al., 2006) from which it is secreted and is believed to play a role in cell adhesion during the latter stages of dental enamel formation (Iwasaki et al., 2005; Moffatt et al., 2006). However, we have found that Amtn is localized only in the incisor epithelia and the basal lamina of molar ameloblasts at P0 stage. Much of the previous research has been performed at later stages of tooth development (>P5), and our result would suggest that the localization of Amtn changes during tooth development. The temporospatial expression pattern of Amtn during tooth development needs further examination.
Gja1 has been previously reported to be expressed in the cells of the stratum intermedium and the inner enamel epithelium (Pinero et al., 1994; Kagayama et al., 1995) where it is distributed exclusively at the sites of contact between odontoblasts, suggesting a function in cell to cell signaling (Fried et al., 1996). Mutations in Gja1 have been found to cause oculodentodigital dysplasia and Hallermann–Streiff syndrome, conditions that include dental anomalies (Paznekas et al., 2003; Pizzuti et al., 2004). We have also found Gja1 to be expressed in the stratum intermedium, supporting the previous observations and putative function.
Rspo4 is a member of the R-spondin (Rspo) protein family that has purported essential activities in vertebrate development and their ligand-type activities overlap with those of the canonical Wnt ligands where both are capable of the activation of beta-catenin (Kim et al., 2006). However, there are likely independent receptor/signaling pathways for Rspo proteins that intersect those of Wnt at the level of beta-catenin (Kim et al., 2006). Both Wnt signaling (Sarkar and Sharpe, 1999) and beta-catenin (Obara et al., 2006) function during tooth development. However, the localization of beta-catenin (Obara et al., 2006) is different from that which we identified for Rspo4, suggesting that their interaction may have a role in cell–cell signaling between the dental papilla and the inner dental epithelium during tooth development. Plans are under way to inactivate this gene in the mouse and will lead to important insights on the role of this gene in various developmental pathways.
Papln is an extracellular matrix glycoprotein with an N-terminal region, called the papilin cassette, which shows similarity to the C-terminal region of matrix-associated metalloproteinases (Kramerova et al., 2000). This finding has led to a suggested role in influencing cell rearrangements and the modulation of metalloproteinases during organogenesis (Kramerova et al., 2000). There are many known metalloproteinases that function during tooth development, and the localized expression of Papln in the ameloblasts identified in this study would support this suggested role.
Maf contains a C-terminal conserved basic region leucine zipper (bZIP) domain, which likely mediates its dimerization and DNA binding property in its role as a transcription factor. c-Maf has been reported to be involved in lens development (Reza and Yasuda, 2004), the control of chondrocyte fate and differentiation (Lefebvre and Smits, 2005), and in the differentiation of TH2-type lymphocytes (Lavender et al., 2000; Agnello et al., 2003). We have found it to be expressed in the ameloblasts of the developing mouse incisor and molar tooth, which would suggest a putative role in the transcriptional regulation of genes specific to the ameloblast during amelogenesis.
Sp7 is a zinc-finger transcription factor and a member of the SP gene family, which is highly expressed in proliferating epithelial cells of hair follicles, limbs, and teeth (Nakamura et al., 2004), and it has also been reported to be ubiquitously expressed in adult tissues (Scohy et al., 2000). This is in agreement with the more general localization found by mRNA in situ hybridization in this study. During tooth development, Sp7 is expressed in proliferating dental epithelium and differentiated odontoblasts, and it has been proposed that it may function in the regulation of cell growth (Nakamura et al., 2004). Other members of the SP family, such as Sp4 and Sp6, are also known to be involved in tooth development (Supp et al., 1996; Nakamura et al., 2004).
Vrm is a novel glycoprotein originally identified in the lateral nasal gland where it is highly concentrated in the mucus of the vomeronasal organ, which has led to the hypothesis that vomeromodulin participates in perireceptor events that facilitate the process of pheromone access and detection as a chemosensory stimulus transporter in olfactory transduction (Khew-Goodall et al., 1991; Krishna et al., 1994). In this study, Vrm was identified by mRNA in situ hybridization to be located in the nasal serous gland that is located close to, but distinct from, the developing tooth.
We compared the developing tooth structure (P1–P10) against developed control tissues (2–4 weeks), an approach that may at first glance appear to have significant limitations. Given that microarray analysis is amenable to sequential experimentation, we reasoned that we could conduct the analysis with the DMT initially and compare the data obtained with those already available in the public domain, thereby enabling an economical experimental test. Our results demonstrate that, despite that the tissues being compared were not obtained from age-matched animals, this approach was indeed valid given that we identified a large number of known tooth development genes as most significantly differentially expressed between the DMT and control samples. Further validation of our approach was obtained by qRT-PCR analysis of these genes in RNA extracted from six control tissues obtained from P2 mice which supported the microarray data analysis for almost all genes. The final and most credible validation was obtained by mRNA in situ hybridization analysis of a subset of novel genes that confirmed the expression of five to seven genes as being specifically expressed in the tooth in the craniofacial region, and one in the nasal serous gland that is located close to the tooth. The presence of this latter gene in our analysis can possibly be explained by the structure within which it is expressed having been attached to the tooth structures extracted from the P1–P10 mouse pups and, therefore, its inclusion in the mRNA analyzed in both the microarray and qRT-PCR analysis. Future analysis would include the use of age-matched control tissues to allow for an improved comparison that may identify additional genes. The use of specific time points rather than the pool of time points used here may also allow for additional unknown and/or tooth-specific genes to be identified that may have been diluted in this analysis. Further analysis of other genes showing differential expression between the tooth and control samples but to a lesser degree than the 100 concentrated on here may also identify additional tooth development genes.
We have shown that microarray gene expression analysis of the developing tooth is a viable method for the identification of novel genes underlying this developmental process. Importantly, this approach has identified novel genes of unknown function, genes related to those known to have a role in tooth development, and genes known to be expressed in other tissues but not known to be expressed at significant levels in the developing tooth.
Isolation of Mouse Molar Tooth RNA
Molar teeth were manually extracted from 60 Swiss Webster mouse pups between 1 and 10 days postnatal (USC IACUC Protocol No. 7225). The teeth were pooled and ground in the presence of liquid nitrogen using a pestle and mortar, and the RNA were extracted using the RNeasy RNA purification kit (Qiagen, Valencia, CA) following the manufacturer's recommended protocol.
Microarray Analysis of Gene Expression in Mouse Molar Teeth
The mRNA was labeled and hybridized to the Affymetrix Mouse Genome Expression (MGE) 430 2.0 microarrays following the manufacturer's recommended protocols and reagents (Affymetrix, San Clara, CA). Microarrays hybridized with mouse molar tooth RNA were analyzed in quadruplicate and the raw microarray data, both CEL and data files, are available online at the NCBI-GEO (http://www.ncbi.nlm.nih.gov/geo/; Edgar et al., 2002) database, accession number GSE7164. The DMT microarray data was compared with a control set of 16 tissues (brain, CD4+ T-cells, eye, heart, kidney, liver, lung, lymph node, ovary, placenta, smooth muscle, spleen, stomach, submaxillary gland, testis, and thymus) whose data were obtained from the NCBI-GEO database (accession no. GSE1986), which were also obtained from Swiss Webster mice, but between 2 and 4 weeks postnatal. The comparison was performed using the Bioconductor open-source analysis program run within the R-script environment (Gentleman et al., 2004) and the limma package (Smyth, 2004) to identify genes exhibiting differential expression between DMT and control tissues. Briefly, raw microarray data was normalized using the log-additive robust-multichip-average (RMA) algorithm (Irizarry et al., 2003). The data from the four DMT microarray replicates were compared with that of the 16 control tissues by fitting a linear model to the expression data for each probe. The resulting coefficients for each probe were then subjected to a pair-wise comparison between the “DMT” vs. “control” data and differential expression identified by calculating moderated t statistics and log-odds of differential expression by empirical Bayes shrinkage of the gene-wise sample variances toward a common value (Smyth, 2004).
Analysis of Gene Ontology and Uncharacterized Genes
Six tissues (heart, lung, kidney, liver, spleen, and brain) were collected from 2-day-old Swiss Webster mice and were immersed in liquid nitrogen immediately after dissection. The RNAs were prepared according to the Trizol protocol (Invitrogen, Carlsbad, CA). Equal amounts of RNAs from each of these six tissues were pooled and first-strand cDNA was prepared using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Ten micrograms of RNA from either the pooled tissues or the molar tooth RNA from P1–P10 mice were mixed with 10 μl of 10 × buffer, 4 μl of 25 × dNTP, 10 μl of Random Primers, 5 μl of MultiScribe Reverse Transcriptase (50 U/μl) and H2O to 100 μl. The primers for qRT-PCR were designed with Primer Express version 3.1 (Applied Biosystems, Foster City, CA) based on the cDNA sequences deposited in the NCBI Genbank database (Supplementary Table S1, which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat). qRT-PCR reactions were prepared in triplicates with 1 μl of a 50 times dilution of first-strand cDNA mixed with 5 μl of 2 × ABsolute QPCR SYBR Green ROX mix (ABgene, Rochester, NY), 1 μl of 700 nM of the primer, and 3 μl of H2O. Reactions underwent thermal cycling as follows: 1 cycle of 95°C for 4 min, followed by 50 cycles of 95°C for 1 min, and 55°C for 1 min, in an ABI 7900HT real-time thermal cycler (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control to normalize for variation in the RNA concentrations.
mRNA In Situ Hybridization
Riboprobes for mRNA in situ hybridization were prepared using nested/half-nested PCR primers (Supplementary Table S2) designed with Primer3 (Rozen and Skaletsky, 2000) based on the cDNA sequences deposited in the NCBI GenBank database. The first PCR reaction was prepared using 1 μl of a 50 times dilution of the product from first-strand cDNA synthesis, 1 μl of 1.25 mM of dNTPs, 0.5 μl of 3.2 μM each primer, 2 μl of 10 × buffer, 1 unit of Taq DNA polymerase and H2O to 20 μl. The initial PCR reaction was performed as following: 1 cycle of 95°C for 5 min, followed by 10 cycles of 95°C for 45 sec, 65°C for 45 sec (reduced by 1 degree each subsequent cycle), and 72°C for 2 min, then 25 cycles of 95°C for 45 sec, 55°C for 45 sec, and 72°C for 2 min, and a final extension of 72°C for 8 min. The second PCR reaction consisted of 1 μl of a 10 times dilution of the initial PCR products, with the other PCR reagents and conditions as used for the initial PCR. One member of each pair of nested PCR primers included the Sp6 promoter sequence as a 5′ tag sequence so that the transcripts from the PCR products could be complemented with the sense strand of the gene. The nested PCR products were purified from a 1% agarose gel using the QIAquick Gel Extraction kit (Qiagen, Valencia, CA) according to the manufacturer's recommended protocol. The riboprobes were prepared with DIG RNA Labeling mix (Roche Applied Science, Indianapolis, IN) using Sp6 RNA polymerase according to the manufacturer's recommended protocol. The transcripts were examined by electrophoresis on a 1% agarose gel and diluted 2 to 20 times using water based on the intensity of bands.
Newborn mice were anesthetized using chloroform and killed immediately by decapitation. The heads were fixed in 4% paraformaldehyde at 4°C overnight, decalcified in 0.1 M ethylenediaminetetraacetic acid/2% paraformaldehyde/phosphate buffered saline for 1 week, embedded in paraffin, and sectioned. The 8-μm paraffin sections were rehydrated through successive incubations with xylene, ethanol at concentrations of 100%, 90%, 70%, and 50%, and PBS before treatment with 0.1 M of triethanolamine for 10 min and 0.1 M of triethanolamine with 0.25% of acetic anhydride for 10 min. Hybridization was performed in mRNA in situ hybridization solution (Dakocytomation, Carpinteria, CA) at 55°C. The sections were washed at 50°C in 2 × standard saline citrate (SSC)/50% formamide at 50°C for 1 hr and 1 × SSC/50% formamide at 50°C for 30 min, treated with 20 μg/ml of RNase A at 37°C for 30 min, and further washed with 2 × SSC at 55°C for 1 hr and 0.1 × SSC at 55°C for 2 hr. The sections were blocked with 10% lamb serum (Gibco; Invitrogen, Carlsbad, CA) at room temperature for 2 min and incubated with Anti-Digoxigenin-AP (Roche Applied Science; 1:1,000 dilution in 10% lamb serum) at 4°C overnight. The sections were treated with BCIP/NBT Liquid Substrate System (Sigma-Aldrich, St. Louis, MO) at room temperature overnight and counterstained with Methyl Green (Fluka; Sigma-Aldrich).
This research was supported by grant DE14102 (P.I.P.) from the National Institute of Dental and Craniofacial Research. M.L.S. is supported by DE06988. This research was conducted partly in a facility constructed with support from Research Facilities Improvement Program Grant No. C06 (RR10600-01, CA62528-01, RR14514-01) from the National Center for Research Resources, National Institutes of Health. Author contributions are as follows: T.J.P. conceived the study. P.I.P. and M.L.S. advised on strategy. M.L.S. harvested the mouse molar teeth. G.A.M. extracted the RNA. Y.H. conducted the microarray experiments under the supervision of R.M.-S. T.J.P. analyzed the microarray data. F.L. performed all RT-PCRs and qRT-PCRs, and in situ hybridizations (with guidance from S.O. in Y.C.'s laboratory on section preparation, and S.O. and P.B. in result interpretation). P.I.P. directed and provided funding for the studies.