Functional Consequences of Interactions between Pax9 and Msx1 Genes in Normal and Abnormal Tooth Development

Pax9 and Msx1 encode transcription factors that are known to be essential for the switch in odontogenic potential from the epithelium to the mesenchyme. Multiple lines of evidence suggest that these molecules play an important role in the maintenance of mesenchymal Bmp4 expression, which ultimately drives morphogenesis of the dental organ. Here we demonstrate that Pax9 is able to directly regulate Msx1 expression and interact with Msx1 at the protein level to enhance its ability to transactivate Msx1 and Bmp4 expression during tooth development. In addition, we tested how a missense mutation (T62C) in the paired domain of PAX9 that is responsible for human tooth agenesis (1Das P. Hai M. Elcock C. Leal S.M. Brown D.T. Brook A.H. Patel P.I. Am. J. Med. Genet. 2003; 118A: 35-42Crossref PubMed Scopus (103) Google Scholar) affects its functions. Our data indicate that although the mutant Pax9 protein (L21P) can bind to the Msx1 protein, it fails to transactivate the Msx1 and Bmp4 promoter, presumably because of its inability to bind cognate paired domain recognition sequences. In addition, synergistic transcriptional activation of the Bmp4 promoter was lost with coexpression of mutant Pax9 and wild-type Msx1. This suggests that Pax9 is critical for the regulation of Bmp4 expression through its paired domain rather than Msx1. Our findings demonstrate the partnership of Pax9 and Msx1 in a signaling pathway that involves Bmp4. Furthermore, the regulation of Bmp4 expression by the interaction of Pax9 with Msx1 at the level of transcription and through formation of a protein complex determines the fate of the transition from bud to cap stage during tooth development. Pax9 and Msx1 encode transcription factors that are known to be essential for the switch in odontogenic potential from the epithelium to the mesenchyme. Multiple lines of evidence suggest that these molecules play an important role in the maintenance of mesenchymal Bmp4 expression, which ultimately drives morphogenesis of the dental organ. Here we demonstrate that Pax9 is able to directly regulate Msx1 expression and interact with Msx1 at the protein level to enhance its ability to transactivate Msx1 and Bmp4 expression during tooth development. In addition, we tested how a missense mutation (T62C) in the paired domain of PAX9 that is responsible for human tooth agenesis (1Das P. Hai M. Elcock C. Leal S.M. Brown D.T. Brook A.H. Patel P.I. Am. J. Med. Genet. 2003; 118A: 35-42Crossref PubMed Scopus (103) Google Scholar) affects its functions. Our data indicate that although the mutant Pax9 protein (L21P) can bind to the Msx1 protein, it fails to transactivate the Msx1 and Bmp4 promoter, presumably because of its inability to bind cognate paired domain recognition sequences. In addition, synergistic transcriptional activation of the Bmp4 promoter was lost with coexpression of mutant Pax9 and wild-type Msx1. This suggests that Pax9 is critical for the regulation of Bmp4 expression through its paired domain rather than Msx1. Our findings demonstrate the partnership of Pax9 and Msx1 in a signaling pathway that involves Bmp4. Furthermore, the regulation of Bmp4 expression by the interaction of Pax9 with Msx1 at the level of transcription and through formation of a protein complex determines the fate of the transition from bud to cap stage during tooth development. The formation of mammalian dentition is a remarkable developmental process that provides a valuable model for studying genes that control three-dimensional patterning and morphogenesis. The genetic control of tooth development is underscored by the findings that mutations in genes that encode transcription factors involved in tooth signaling lead to tooth agenesis, a commonly inherited disorder in humans (hypodontia (MIM 106600)). Tooth agenesis occurs in syndromic and nonsyndromic forms and is classified as a genetically and clinically heterogeneous condition affecting various combinations of teeth (2Vastardis H. Am. J. Orthod. Dentofacial. Orthop. 2000; 117: 650-656Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 3Mostowska A. Kobielak A. Biedziak B. Trzeciak W.H. Eur. J. Oral Sci. 2003; 11: 272-276Crossref Scopus (69) Google Scholar, 4Vieira A.R. J. Dent. Res. 2003; 82: 162-165Crossref PubMed Scopus (96) Google Scholar). The presence of inter- and intra-familial variability as it relates to the number and types of missing teeth also suggests that common variants in genes that are involved in tooth signaling may influence the background responsible for the phenotype. To date, several reports have described the association of dominant mutations in MSX1 and PAX9, two transcription factors that are expressed in dental mesenchyme (1Das P. Hai M. Elcock C. Leal S.M. Brown D.T. Brook A.H. Patel P.I. Am. J. Med. Genet. 2003; 118A: 35-42Crossref PubMed Scopus (103) Google Scholar, 3Mostowska A. Kobielak A. Biedziak B. Trzeciak W.H. Eur. J. Oral Sci. 2003; 11: 272-276Crossref Scopus (69) Google Scholar, 4Vieira A.R. J. Dent. Res. 2003; 82: 162-165Crossref PubMed Scopus (96) Google Scholar, 5Vastardis H. Karimbux N. Guthua S.W. Seidman J.G. Seidman C.E. Nat. Genet. 1996; 13: 417-421Crossref PubMed Scopus (539) Google Scholar, 6Stockton D.W. Das P. Goldenberg M. D'Souza R.N. Patel P.I. Nat. Genet. 2000; 24: 18-19Crossref PubMed Scopus (393) Google Scholar, 7Nieminen P. Arte S. Tanner D. Paulin L. Alaluusua S. Thesleff I. Pirinen S. Euro. J. Hum. Gene. 2001; 9: 743-746Crossref PubMed Scopus (136) Google Scholar, 8Klein M.L. Nieminen P. Lammi L. Niebuhr E. Kreiborg S. J. Dent. Res. 2005; 84: 43-47Crossref PubMed Scopus (90) Google Scholar, 9Mostowska A. Kobielak A. Trzeciak W.H. Eur. J. Oral. Sci. 2003; 111: 365-370Crossref PubMed Scopus (80) Google Scholar, 10Vieira A.R. Meira R. Modesto A. Murray J.C. J. Dent. Res. 2004; 83: 723-727Crossref PubMed Scopus (132) Google Scholar, 11Frazier-Bowers S.A. Guo D.C. Cavender A. Xue L. Evans B. King T. Milewicz D. D'Souza R.N. J. Dent. Res. 2002; 81: 129-133Crossref PubMed Google Scholar). More recently, AXIN2, a Wnt-signaling receptor was identified as responsible for a nonsyndromic form of tooth agenesis (12Lammi L. Halonen K. Pirinen S. Thesleff I. Arte S. Nieminen P. Eur. J. Hum. Genet. 2003; 11: 866-871Crossref PubMed Scopus (89) Google Scholar, 13Lammi L. Arte S. Somer M. Jarvinen H. Lahermo P. Thesleff I. Pirinen S. Nieminen P. Am. J. Hum. Genet. 2004; 74: 1043-1050Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). When compared with a fairly mixed pattern of tooth agenesis seen in individuals with a nonsense mutation in AXIN2, the phenotypes reported in MSX1 and PAX9 affected families are more restricted to posterior dentition. Although mutations in MSX1 contribute to a pattern that mainly involves premolars and more rarely molars, PAX9 mutations show a strong association with molar agenesis and sometimes premolars. In family members with PAX9 mutations the congenital absence of premolars in addition to molars may reflect a secondary down-regulation in MSX1 expression. This implies that PAX9 shares an upstream genetic epistasis with MSX1. Studies of tooth development in mice also indicate a molecular relationship between Pax9 and Msx1. Both genes are coexpressed in dental mesenchyme and appear critical for tooth morphogenesis, because in Msx1 and Pax9 homozygous null mutants, tooth organs arrest at the bud stage (14Satokata I. Maas R. Nat. Genet. 1994; 6: 348-356Crossref PubMed Scopus (1047) Google Scholar, 15Peters H. Neubüser A. Balling R. Eur. J. Oral Sci. 1998; 106: 38-43Crossref PubMed Scopus (72) Google Scholar). However, there is little known about the interactions of Pax9 with Msx1 at the level of gene regulation and function. It is possible that Pax9 could interact directly with Msx1 or activate a regulator of Msx1. Alternatively, the molecular relationship shared by Pax9 and Msx1 could involve functional interactions on the post-transcriptional or protein level. Clues about a potential downstream effector gene of the Pax9-Msx1 pathway come from the observation that in Msx1 and Pax9 single homozygous mutant mice, Bmp4 expression is markedly reduced in dental mesenchyme (15Peters H. Neubüser A. Balling R. Eur. J. Oral Sci. 1998; 106: 38-43Crossref PubMed Scopus (72) Google Scholar, 16Chen Y. Bei M. Woo I. Satokata I. Maas R. Development. 1996; 122: 3035-3044Crossref PubMed Google Scholar). This suggests that both genes may be required for the modulation of Bmp4 in dental mesenchyme. As an effector molecule, Bmp4 is known to be involved in downstream signaling events that result in the induction of the enamel knot, a transient signaling center within dental epithelium (17Laurikkala J. Kassai Y. Pakkasjarvi L. Thesleff I. Itoh N. Dev. Biol. 2003; 264: 91-105Crossref PubMed Scopus (207) Google Scholar). Subsequent changes in the enamel organ result in the progress of cuspal morphogenesis. Initial molecular studies of the BMP4 promoter identified a region between –1100 and +45 as important for basal transcription (18Kawai S. Sugiura T. Bone. 2001; 29: 54-61Crossref PubMed Scopus (79) Google Scholar). Although a variant of the Bmp4 promoter has been shown to be a target for regulation by a number of transcription factors (16Chen Y. Bei M. Woo I. Satokata I. Maas R. Development. 1996; 122: 3035-3044Crossref PubMed Google Scholar, 19Bei M. Maas R. Development. 1998; 125: 4325-4333Crossref PubMed Google Scholar, 20Kettunen P. Thesleff I. Dev. Dyn. 1998; 211: 256-268Crossref PubMed Scopus (217) Google Scholar), there are no data available on the modulation of the Bmp4 promoter by either Pax9 or Msx1, or both. Here we report that Pax9 interacts with Msx1 at both the gene and protein levels and that the interaction enhances the ability of Pax9 to transactivate Msx1 and Bmp4 expression during tooth development. In addition, we tested how a missense mutation (T62C) in the paired domain of PAX9 that is responsible for human tooth agenesis (1Das P. Hai M. Elcock C. Leal S.M. Brown D.T. Brook A.H. Patel P.I. Am. J. Med. Genet. 2003; 118A: 35-42Crossref PubMed Scopus (103) Google Scholar) affects its functions. Our data indicate that although the mutant Pax9 protein (L21P) can bind to both wild-type Msx1 and Pax9 proteins, it fails to transactivate either the Msx1 or the Bmp4 promoter. Furthermore, synergistic transcriptional activation of the Bmp4 promoter was lost with coexpression of mutant Pax9 and wild-type Msx1. These data suggest that Pax9 is critical for the regulation of Bmp4 expression through its paired domain rather than Msx1. Together, these findings demonstrate a signaling pathway involving Pax9, Msx1, and Bmp4 that is critical for the progress of tooth morphogenesis from the bud to cap stage. Pax9-deficient Mice and in Situ Hybridization—The generation of Pax9 knock-out alleles in mice and the preparation of paraffin sections of mutant tooth organs have been described previously (15Peters H. Neubüser A. Balling R. Eur. J. Oral Sci. 1998; 106: 38-43Crossref PubMed Scopus (72) Google Scholar). Conditions for in situ hybridizations using a 35S-labeled riboprobe specific for murine Msx1 were followed as described earlier (21Peters H. Doll U. Niessing J. Dev. Dyn. 1995; 203: 1-16Crossref PubMed Scopus (57) Google Scholar). Plasmid Constructs and Site-directed Mutagenesis—The mammalian expression vector pCMV-Pax9 with c-Myc epitope tag was used as previously described (22Mensah J.K. Ogawa T. Kapadia H. Cavender A.C. D'Souza R.N. J. Biol. Chem. 2004; 279: 5924-5933Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Expression plasmids containing the cytomegalovirus promoter linked to the full coding sequence of Msx1 were constructed in pCMV-Tag2b (Stratagene, CA). A murine Msx1 cDNA clone comprising the full-length coding sequence region was kindly provided by Dr. John Rubenstein (University of California at San Francisco). The FLAG epitope is in frame with the amino terminus of Msx1. Three fragments of the Msx1 promoter (–3.5 kb/+106 bp, –1282/106 bp, and –165/+106 bp) containing the luciferase gene and the 5′ region of the Bmp4 promoter (–2372/+258 bp) within the luciferase vector were described previously (23Takahashi T. Guron C. Shetty S. Matsui H. Raghow R. J. Biol. Chem. 1997; 272: 22667-22678Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 24Shetty S. Takahashi T. Matsui H. Ayengar R. Raghow R. Biochem. J. 1999; 339: 751-758Crossref PubMed Scopus (41) Google Scholar, 25Zhang Z. Song Y. Zhao X. Zhang X. Fermin C. Chen Y. Development. 2002; 129: 4135-4146Crossref PubMed Google Scholar). To construct pCMV-L21PPax9, in vitro site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene). The specific primer sets used for the generation of mutant Pax9 cDNA were as follows: L21P, forward, 5′-CGGAAGGCCGCCGCCCCAACGCCAT-3′; and reverse, 5′-ATGGCGTTGGGCGGCGGCCTTCCG-3′. The mutated construct was sequenced entirely to confirm the point mutation. The Myc epitope was in frame with the amino terminus of mutant Pax9. Electrophoretic Mobility Shift Assay—Oligonucleotides corresponding to e5 and CD19-2(A-ins) were synthesized (Sigma/Genosys), and gel retardation assays were performed as previously described (22Mensah J.K. Ogawa T. Kapadia H. Cavender A.C. D'Souza R.N. J. Biol. Chem. 2004; 279: 5924-5933Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The oligonucleotide probes used are two previously established high affinity paired domain recognition sequences. The e5 sequence, which was originally derived from the Drosophila even skipped promoter for the Eve transcription factor (26Goulding M.D. Chalepakis G. Deutsch U. Erselius J.R. Gruss P. EMBO J. 1991; 10: 1135-1147Crossref PubMed Scopus (750) Google Scholar), is a known binding sequence for Pax9 (27Neubüser A. Koseki H. Balling R. Dev. Biol. 1995; 170: 701-716Crossref PubMed Scopus (240) Google Scholar). CD19-2(A-ins) is a classical paired domain recognition sequence (28Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; PubMed Scopus Google Scholar). e5 or CD19-2(A-ins) was with of from with the expression Pax9, or in The generation of the expression vector was previously described (22Mensah J.K. Ogawa T. Kapadia H. Cavender A.C. D'Souza R.N. J. Biol. Chem. 2004; 279: 5924-5933Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). at for of or was to the The was a gel as described previously (22Mensah J.K. Ogawa T. Kapadia H. Cavender A.C. D'Souza R.N. J. Biol. Chem. 2004; 279: 5924-5933Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). were from and as described previously (22Mensah J.K. Ogawa T. Kapadia H. Cavender A.C. D'Souza R.N. J. Biol. Chem. 2004; 279: 5924-5933Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). were with or and using to the the were in with and for on The were to of affinity gel and at The affinity were with and with and by with or using an kit of and of expression of was used as the control for were for and luciferase and using from and of the in demonstrate the in expression of wild-type and mutant Pax9, the were of were using were by the protein kit and of were by using and the kit were and with to with the c-Myc and with a was with an Pax9 for the Expression of Msx1 in during Tooth Pax9 is involved in the regulation of Msx1, the expression of Msx1 was in tooth of Pax9-deficient used no in expression of Msx1 was in the Pax9 mutant A and Msx1 expression reduced in the mesenchyme of Pax9 mutant molar organs and Although Msx1 were at in wild-type tooth no expression was in mutant tooth organs and Pax9 Msx1 in situ of Msx1 expression in Pax9-deficient dental mesenchyme that both genes within the signaling pathway and that Pax9 shares an upstream epistasis with Msx1. To Pax9 directly Msx1 expression, we performed assays using Msx1 promoter Three Msx1 promoter (23Takahashi T. Guron C. Shetty S. Matsui H. Raghow R. J. Biol. Chem. 1997; 272: 22667-22678Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 24Shetty S. Takahashi T. Matsui H. Ayengar R. Raghow R. Biochem. J. 1999; 339: 751-758Crossref PubMed Scopus (41) Google Scholar) with the luciferase gene were with Pax9 These either Pax9 or Msx1. The promoter of sequence upstream of the and upstream and downstream promoter It paired which are in the region within of the human and murine Msx1 promoter When Pax9 expression plasmids were a in gene was with of the Msx1 promoter of Pax9 and Msx1 on the Msx1 to the the potential and interactions of Pax9 and Msx1, we the can in we tested for a interaction between Pax9 and Msx1 by expressed in Our that both interact within tested the transcriptional of Pax9 and Msx1 are or using transient assays on the Msx1 promoter in of the Pax9 expression in activation of the of Msx1 expression that Msx1 can compared with the basal level. that of of Msx1 expression vector in activation of the construct However, the of Msx1 expression in a activation of the construct of Pax9 and Msx1 on the Bmp4 the of Pax9 and Msx1 on Bmp4 we performed assays using a Bmp4 promoter construct containing of sequence upstream of the tested the expression of Bmp4 is Pax9-Msx1 protein of the Pax9 expression in activation of the of Msx1 expression to regulate promoter. with Pax9 and Msx1 expression plasmids that the of Msx1 the of Pax9 the in a in activation from to that although the of Msx1 reduced transcriptional no changes were in Pax9 expression with levels of Msx1 This suggests that the of Msx1 the level of Pax9 to Msx1 to either Msx1 or Bmp4 Expression of in site-directed we a the and mutant from studies performed with c-Myc that protein is in mammalian and in the A and These data the to the mutant Pax9 protein could interact with wild-type Msx1. performed on combinations of wild-type and mutant Pax9 and Msx1 demonstrate that the protein interacts with the wild-type Msx1 to the as wild-type Pax9 protein To the functional of the paired domain mutation in PAX9, we the point mutation have on binding by the paired The mutant protein was to form a complex with either of the two cognate paired domain recognition tested transcriptional activation was by transient assays using a Msx1 promoter or Bmp4 promoter upstream of a luciferase of wild-type Pax9 was compared with the of wild-type Pax9 to activation of both Msx1 and Bmp4 However, no on these compared with basal levels of transcription A and of wild-type Pax9 and Msx1 in activation at the Bmp4 promoter, synergistic transcriptional activation was lost with of and Msx1 activation of the Msx1 promoter and the Bmp4 promoter. in transient with of wild-type Pax9 expression vector to a activation of the compared with as expression of the of expression vector to activate the promoter. with of wild-type Pax9 expression vector to a activation of the compared with activation at Bmp4 was reduced to by although of wild-type Pax9 and Msx1 activation to with coexpression of and Msx1. levels were by of in were to expression levels of The the of assays performed in the of available on the molecular required for tooth morphogenesis, there is little known about how the and of dental mesenchymal transcription factors Msx1 and Pax9 the progress of the tooth organ from the bud to cap stage of development. The down-regulation of Msx1 expression in Pax9-deficient mice and levels of Bmp4 expression in Pax9 and Msx1 homozygous null mice indicate that the genes within the signaling In these we the evidence in of a molecular relationship between Pax9 and Msx1 on the transcriptional level and interactions as report that Pax9 forms a complex with Msx1 and that the interaction enhances the ability of Pax9 to transactivate Msx1 and Bmp4 expression during tooth development. Our functional studies of the protein of a paired domain mutation demonstrate that although it with Msx1, the mutant protein is to activate both the Msx1 and Bmp4 Furthermore, we that coexpression of the Pax9 paired domain mutant and wild-type Msx1 in a of synergistic transcriptional activation of the Bmp4 promoter. These data suggest that the regulation of Bmp4 expression is by the paired domain of Pax9 rather than through its interactions with Msx1. Together, these findings demonstrate that the regulation of Bmp4 expression by the interaction of Pax9 with Msx1 determines the fate of the transition from bud stage to cap stage during tooth development Although mutations in MSX1 contribute to a that involves premolars and rarely molars, PAX9 mutations show a strong association with molar agenesis and sometimes premolars. In family members with PAX9 the congenital absence of premolars in addition to molars may reflect a secondary down-regulation in MSX1 expression. This is by two lines of in situ that although Msx1 expression is at the expression of Msx1 is at and This suggests that Pax9 is a transcriptional regulator of Msx1 at the of tooth that its presence is for tooth morphogenesis. of the Msx1 promoter several paired and with Pax9, a in promoter was seen with of the with the observation of down-regulation of Msx1 expression in Pax9-deficient mice, these findings suggest Pax9 shares an upstream genetic epistasis with Msx1 through the binding of Pax9 to the Msx1 promoter. data with expression of Pax9 and Msx1 during tooth development are of a molecular relationship between the two transcription Our of the formation of can interact on the protein level. Clues about the functional of interaction come from of a down-regulation of Bmp4 in Msx1 and Pax9 mutant mice (15Peters H. Neubüser A. Balling R. Eur. J. Oral Sci. 1998; 106: 38-43Crossref PubMed Scopus (72) Google Scholar, 16Chen Y. Bei M. Woo I. Satokata I. Maas R. Development. 1996; 122: 3035-3044Crossref PubMed Google Scholar) with studies that Bmp4 downstream of Msx1 to regulate tooth development. were able to show that although Pax9 can activate Msx1 and Bmp4 expression, Msx1 fails to the expression of either However, of a of Msx1 with Pax9 enhances the activation of Msx1 and Bmp4 of Pax9 and Msx1 This is to the modulation of Msx1 expression, on the stage of tooth development. we show a synergistic functional relationship between Pax9 and Msx1 in the regulation of Bmp4 expression, which is with the observation that of Bmp4 the tooth arrest shown in This is in to reports of interactions between Msx1 and transcription factors that result in of binding and transcriptional H. G. H. P. N. C. Biol. 1997; PubMed Scopus Google Scholar, J. C. 1998; PubMed Scopus Google Scholar). A possible of how occurs from a that Msx1 interacts with protein, to a that in H. R. C. 2004; PubMed Scopus Google Scholar). In the of tooth morphogenesis, it is possible that the presence of a transcriptional Pax9 can and for the transcription of a downstream target Bmp4. Bmp4 can signaling events that lead to of tooth development from the bud to cap stage. we that the synergistic regulation of Bmp4 expression by the interaction of Pax9 with Msx1 at both the transcriptional and protein level is essential at a critical stage of tooth morphogenesis. Although studies of human PAX9 mutations have the molecular that contribute to tooth agenesis are Our functional studies of a previously described mutation (L21P) in the of the paired domain (1Das P. Hai M. Elcock C. Leal S.M. Brown D.T. Brook A.H. Patel P.I. Am. J. Med. Genet. 2003; 118A: 35-42Crossref PubMed Scopus (103) Google Scholar) demonstrate transcriptional activation of the Msx1 and Bmp4 It is that the the and it from a we that the mutation of to interaction and the binding and transcription of a evidence has shown that the paired domain of and the of Msx1 are essential for the interaction between the two The of indicate that the mutation has no on its ability to interact with Msx1, that the of the paired domain is involved in it is that either the of the paired domain of Pax9 is critical for interaction or the mutation the of the paired domain to an that with Msx1. Although as is that interactions may also through the of Pax9, which is by the mutation D. G. B. Busslinger M. EMBO J. 2000; PubMed Scopus Google Scholar). also that synergistic transcriptional activation is reduced between and Msx1 at the Bmp4 promoter. with the observation that mutant Pax9 interacts with Msx1, suggest that in rather than are responsible for the of tooth agenesis for mutation. In data interactions between Pax9 and Msx1 on two The level is that of transcriptional The is that Pax9 and Msx1 interact as proteins, Furthermore, the of human data that the regulation of Bmp4 expression by the interaction of Pax9 with Msx1 through the paired domain of Pax9 determines the fate of the transition from the bud to cap stage of tooth development. Our studies the that Pax9 and Msx1 may control of important developmental by the expression of Bmp4 during tooth morphogenesis. data are important for of and tooth development. In addition, these studies valuable the of human tooth The and provided by and are as is the of