BMP Signaling Pathway in Dentin Development and Diseases

BMP signaling plays an important role in dentin development. BMPs and antagonists regulate odontoblast differentiation and downstream gene expression via canonical Smad and non-canonical Smad signaling pathways. The interaction of BMPs with their receptors leads to the formation of complexes and the transduction of signals to the canonical Smad signaling pathway (for example, BMP ligands, receptors, and Smads) and the non-canonical Smad signaling pathway (for example, MAPKs, p38, Erk, JNK, and PI3K/Akt) to regulate dental mesenchymal stem cell/progenitor proliferation and differentiation during dentin development and homeostasis. Both the canonical Smad and non-canonical Smad signaling pathways converge at transcription factors, such as Dlx3, Osx, Runx2, and others, to promote the differentiation of dental pulp mesenchymal cells into odontoblasts and downregulated gene expressions, such as those of DSPP and DMP1. Dysregulated BMP signaling causes a number of tooth disorders in humans. Mutation or knockout of BMP signaling-associated genes in mice results in dentin defects which enable a better understanding of the BMP signaling networks underlying odontoblast differentiation and dentin formation. This review summarizes the recent advances in our understanding of BMP signaling in odontoblast differentiation and dentin formation. It includes discussion of the expression of BMPs, their receptors, and the implicated downstream genes during dentinogenesis. In addition, the structures of BMPs, BMP receptors, antagonists, and dysregulation of BMP signaling pathways associated with dentin defects are described.


Introduction
The tooth is an amazing sensory organ. Teeth perform basic physiological functions in our daily life, playing essential roles in mastication and speech. The tooth is composed of enamel, dental pulp, dentin, and periodontium, consisting of cementum, the periodontal ligament (PDL), alveolar bone, and gingival tissue ( Figure 1a) [1,2]. Tooth development and formation are characterized as resulting from sequential and reciprocal interactions between dental oral epithelial and ectomesenchymal cells. The mouse is widely used as an animal model for studying tooth development and signaling pathways. At embryonic day 10 (E10) of mouse tooth development, oral epithelial cells invade, interact with mesenchymal cells, and initiate the mesenchyme directing the process. At E11.5, a local thickening of the dental epithelium becomes evident as the presumptive dental epithelial cells elongate along the apical-basal axis and cell shape changes from cuboidal to columnar, forming the dental placode (the lamina stage) (Figure 1b). At E12.5, the thickened dental epithelial cells derived from the ectoderm of the first branchial arch proliferate and invaginate into the subjacent ectomesenchyme of the neural crest and continue until E13.5, forming a definite tooth bud (the bud stage). The tooth bud is organized into three major parts, including the enamel (dental) organ, the dental papilla, and the dental follicle. Subsequently, the Subsequently, the epithelial bud progresses to the cap stage at E14.5 (the cap stage) and goes through specific folding, with enamel knot formation in the dental epithelium to control the patterning of tooth cusps. A small group of ectomesenchymal cells below the dental epithelium proliferate and produce extracellular substances, resulting in an aggregation of these cells termed the dental papilla. On the other hand, ectomesenchymal cells surrounding the enamel organ become dense and form what is known as the dental follicle. Eventually, the epithelial cells from the enamel organ differentiate into ameloblasts, which produce enamel, while mesenchymal cells from the dental papilla differentiate into odontoblasts and produce dentin and dental pulp. The dental follicle produces all the supporting structures of the tooth, such as cementum, alveolar bone, and PDL. When the dental epithelial and mesenchymal cells proceed to histodifferentiation and morpho-differentiation and the enamel organ forms a bell-shaped morphology at E16.5 (the bell stage), these cells begin to differentiate into ameloblasts and odontoblasts, respectively [3]. During the late bell stage of tooth development, odontoblasts synthesize and produce dentin extracellular matrix (ECM), including collagen and non-collagenous proteins (NCPs) required for dentin formation. The crown of the tooth is covered with enamel, while the root is covered with cementum and periodontal ligament. The cementoenamel junction is located between the enamel and root. The root is surrounded by the alveolar bone through periodontal ligaments. The dentin surrounds the dental pulp. Nerves, lymph, and blood vessels enter the dental pulp from the apical foramen of the tooth and provide nutrition and innervation to odontoblasts and dental The crown of the tooth is covered with enamel, while the root is covered with cementum and periodontal ligament. The cementoenamel junction is located between the enamel and root. The root is surrounded by the alveolar bone through periodontal ligaments. The dentin surrounds the dental pulp. Nerves, lymph, and blood vessels enter the dental pulp from the apical foramen of the tooth and provide nutrition and innervation to odontoblasts and dental pulp. (b) Teeth form from the surface epithelium and underlying mesenchyme, and their development

BMP-2
At the initiation stage of mouse tooth development (E10-12), the dental lamin formed as an epithelial clustering, distinguishing the dental epithelium and mesenchy Bmp-2 gene transcription is seen in those areas of dental lamina where buds have star to form [5,[25][26][27]. At the bud stage (E12-13), Bmp-2 expression is detectable in the de epithelium and mesenchyme throughout the budding period. At the cap stage, Bm expression is prominent at E14 and mainly localized at the epithelial enamel knot dur the late cap stage. Bmp-2 expression then expands to the neighboring inner dental epi lium, where the secondary enamel knots will be formed, and its signal can be seen in d tal mesenchyme using in situ hybridization and immunohistochemistry analyses. At bell stage, in situ hybridization and immunostaining showed that Bmp-2 expressio detected in the dental mesenchymal cells [28,29]. Later, Bmp-2 expression spreads to dental papilla and is intense in the pre-odontoblasts. In postnatal days (PNs), Bmpcontinually expressed in odontoblasts and ameloblasts and is detected in the dental pilla as well as adjacent tissues, including the dental follicle, PDL cells, cementoena junction, Hertwig's epithelial root sheath (HERS), and osteoblasts in alveolar bo At the initiation stage of mouse tooth development (E10-12), the dental lamina is formed as an epithelial clustering, distinguishing the dental epithelium and mesenchyme. Bmp-2 gene transcription is seen in those areas of dental lamina where buds have started to form [5,[25][26][27]. At the bud stage (E12-13), Bmp-2 expression is detectable in the dental epithelium and mesenchyme throughout the budding period. At the cap stage, Bmp-2 expression is prominent at E14 and mainly localized at the epithelial enamel knot during the late cap stage. Bmp-2 expression then expands to the neighboring inner dental epithelium, where the secondary enamel knots will be formed, and its signal can be seen in dental mesenchyme using in situ hybridization and immunohistochemistry analyses. At the bell stage, in situ hybridization and immunostaining showed that Bmp-2 expression is detected in the dental mesenchymal cells [28,29]. Later, Bmp-2 expression spreads to the dental papilla and is intense in the pre-odontoblasts. In postnatal days (PNs), Bmp-2 is continually expressed in odontoblasts and ameloblasts and is detected in the dental papilla as well as adjacent tissues, including the dental follicle, PDL cells, cementoenamel junction, Hertwig's epithelial root sheath (HERS), and osteoblasts in alveolar bones analyzed by in situ hybridization and immunohistochemistry assays ( Figure 3 and Table 1) [30,31]. Gao et al. reported that in miniature pigs Bmp-2 was expressed in both the enamel organ and in the dental mesenchyme at E40 and E50 and was expressed mainly in pre-odontoblasts at E60 by in situ hybridization and immunohistochemistry assays [29]. In the human fetus, BMP-2 expression was detected in the enamel organ and dental papilla, with a slightly stronger intensive signal in the inner epithelium of the incisor and molar in the developing human primary tooth germ at the cap stage (12th embryo week). At the bell stage (14th week of fetal development), BMP-2 expression pattern was similar to that at the cap stage in the incisor but was mainly restricted to the inner enamel epithelium in the molars, as determined by in situ hybridization assay [28]. analyzed by in situ hybridization and immunohistochemistry assays ( Figure 3 and Table  1) [30,31]. Gao et al. reported that in miniature pigs Bmp-2 was expressed in both the enamel organ and in the dental mesenchyme at E40 and E50 and was expressed mainly in pre-odontoblasts at E60 by in situ hybridization and immunohistochemistry assays [29].
In the human fetus, BMP-2 expression was detected in the enamel organ and dental papilla, with a slightly stronger intensive signal in the inner epithelium of the incisor and molar in the developing human primary tooth germ at the cap stage (12th embryo week). At the bell stage (14th week of fetal development), BMP-2 expression pattern was similar to that at the cap stage in the incisor but was mainly restricted to the inner enamel epithelium in the molars, as determined by in situ hybridization assay [28]. . Bmp-2 expression during mouse tooth development. At the cap stage (E15) of mouse tooth development, Bmp-2 mRNA was highly expressed in the dental epithelium and moderately detectable in dental mesenchymal cells, osteogenetic mesenchyme, and Meckel's cartilage. At the early bell stage (E16), the Bmp-2 transcript was present in ameloblasts, odontoblasts, dental pulp cells, dental follicles, and mesenchyme in the alveolar bone. At postnatal days ranging from 1 to 14, an in situ hybridization signal was apparent in odontoblasts, dental pulp cells, and osteoblasts in the alveolar bone and was weakly detected in ameloblasts. The figure is adapted with permission from Figure 3. Bmp-2 expression during mouse tooth development. At the cap stage (E15) of mouse tooth development, Bmp-2 mRNA was highly expressed in the dental epithelium and moderately detectable in dental mesenchymal cells, osteogenetic mesenchyme, and Meckel's cartilage. At the early bell stage (E16), the Bmp-2 transcript was present in ameloblasts, odontoblasts, dental pulp cells, dental follicles, and mesenchyme in the alveolar bone. At postnatal days ranging from 1 to 14, an in situ hybridization signal was apparent in odontoblasts, dental pulp cells, and osteoblasts in the alveolar bone and was weakly detected in ameloblasts. The figure is adapted with permission from Chen et al., 2008, ref. [5]. ab, alveolar bone; am, ameloblasts; mc, Meckel's cartilage; D, dentin; de, dental epithelium; df, dental follicle; dm, dental mesenchyme; dp, dental papilla; E, enamel; E, embryonic day; od, odontoblasts.

BMP-3
At the initiation stage of tooth development, there is no Bmp-3 expression in tooth tissue, but Bmp-3 transcripts are visible in the lingual aspect of the dental lamina at the bud stage. At the cap stage, expression of Bmp-3 is detected in the dental papilla mesenchyme underlying the enamel knot and mesial part of the dental mesenchyme. Bmp-3 expression is also seen in the dental follicle mesenchyme and its intense expression is observed in the lingual side of the outer dental epithelium. Bmp-3 expression is strongly detected in osteoblasts of alveolar bones as well as in peripheral chondrocytes of Meckel's cartilage. At the bell stage and PNs, expression of Bmp-3 is apparent in odontoblasts, but its expression gradually decreases with the advancement of odontoblast differentiation. Bmp-3 expression is observed in odontoblasts, dental follicles, PDL cells, cementoblasts, tooth root-lining cells, and osteoblasts on the active bone-forming surface [25,30,31,[40][41][42]. BMP-3 expression was found to be evenly distributed in human incisors and molars at the cap and bell stages (12th and 14th weeks of fetal development, respectively) by in situ hybridization assay [28].

BMP-4
At the tooth initiation stage, the Bmp-4 transcript is detected in the oral epithelium and dental mesenchyme and continues to the dental epithelium and the condensed dental mesenchyme around the epithelial bud [25]. At the bud stage, Bmp-4 is detected in both the dental epithelium and mesenchyme with similar intensities [25,26]. At the cap stage, Bmp-4 expression is present in the epithelium and primary enamel knot cells distant from the basement membrane in the dental papilla mesenchyme and dental sac. At the bell stage, expression of Bmp-4 becomes prominent in the dental papilla, pre-odontoblasts, inner enamel epithelium, outer enamel epithelium, and stellate reticulum, but is absent in the dental epithelium with the removal of the enamel knot. Bmp-4 expression is weak in the central cells of the dental papilla [26,28,29]. During PNs, Bmp-4 expression is observed in the odontoblasts, ameloblasts, and osteoblasts in alveolar bone [30]. In miniature pigs, in situ hybridization and immunostaining analyses showed that Bmp-4 was expressed mainly in the epithelium, with some weak expression in the mesenchyme at E40. At E50, Bmp-4 expression was stronger in the mesenchyme and weaker in the epithelium. Bmp-4 expression was mainly detected in the mesenchyme at E60 [29]. In human tooth germs, BMP-4 expression was visible in the inner enamel epithelium and dental papilla, with slightly weaker expression in outer enamel epithelium in the human primary incisor at the cap stage (12th embryo week), whereas BMP-4 expression was intense in the molar, with a higher level in the inner enamel epithelium and dental mesenchyme underneath it and a lower level in the remaining dental tissues as determined by anti-BMP-4 mRNA probe. By the bell stage (14th week of the fetus), BMP-4 transcription was more prominent located in the inner enamel epithelium and dental papilla than in the outer enamel epithelium and the stellate reticulum of the incisor and the molar. Immunohistochemistry further confirmed the BMP-4 expression patterns in the developing human tooth germs [28].
BMP-4 plays an important role in the signaling interactions between tooth bud epithelium and mesenchyme during tooth morphogenesis [43]. BMP-4 mutation in humans was associated with different types of dental anomalies, including tooth agenesis [43,44]. Bmp-4 mutation influences storage or transport of the mature Bmp-4 protein in the ECM of the developing teeth and significantly decreased levels of phosphorylated Smad-1/5/8 in the cells. Homozygous Bmp-4 null mice die between E6.5 and E9.5 and show little or no mesodermal differentiation [45]. Thus, Bmp-4 cKO mice (Collα1-Cre;Bmp-4fx/fx) were generated and exhibited dentin deficiency at the late stages of mouse tooth development [46]. In Bmp-4 mutant mice, dentin thickness was reduced and root eruption was delayed, while dental pulp chamber volume was increased. Odontoblastic cell differentiation was impaired in Bmp-4 cKO mice, and Bmp-4 deletion led to decreased expression of osteocalcin, Dmp1, Col1α1, Dlx5, Osx, and phosphorylated Smad1/5/8 in the mutant odontoblasts [43].
Bmp-2 and Bmp-4 double cKO mice (DcKO, Dmp1-Cre; Bmp-2 fx/fx/Bmp-4 fx/fx) were generated [47]. The Bmp-2/Bmp-4 DcKO mice showed reduced dentin thickness and enlarged dental pulp cavities compared to the wild-type mice. Dentin in Bmp-2/Bmp-4 DcKO mice was characterized by small, disorganized dentin fibers and a wide predentin layer; decreased expression of bone sialoprotein (Bsp), Dmp1, and Dspp genes was also observed. This indicates that the loss of Bmp signaling alters gene expression in odontoblasts and dentin and leads to improper odontoblast differentiation and odontogenesis.

BMP-5
Expression of Bmp-5 is not detected at the initiation, bud, and cap stages of mouse tooth development [25]. However, Bmp-5 transcripts are present in the differentiating pre-ameloblasts during the late bell stage [25]. On PNs, Bmp-5 expression is evident in ameloblasts during amelogenesis and is seen in chondrocytes and osteoblasts [48][49][50]. Mutations of BMP-5 gene are associated with short ears in humans and mice [51,52]. Additionally, genetic variants of BMP-5 are associated with susceptibility to osteoarthritis (OA) [53,54]. Bmp-5 is required for neural crest progenitor cell survival and proliferation through the Smad-Msxb and Mek-Erk-Id3 signaling pathways, respectively [55]. Although BMP-5 is expressed in dental cells during tooth development, the roles of BMP-5 in tooth formation have not been elucidated in detail.

BMP-6
During the initiation stage of tooth development (E10-E12), the Bmp-6 transcript is not detectable, but at the bud and cap stages Bmp-6 expression is weakly observed in dental mesenchyme, dental lamina, and enamel-organ epithelial cells [25,56]. During the bell stage, Bmp-6 protein reaction in developing odontoblasts and dental papilla can be found to be positive using anti-Bmp-6 immunostaining. Bmp-6 immunoreactivity is weakly present in the outer enamel epithelium and stratum intermedium but is not visible by developing ameloblasts. The dental lamina continues to react positively and the dental follicle negatively. When dentin matrix production begins, polarized odontoblasts appear strongly Bmp-6-immunoreactive. Positive staining with Bmp-6 antibody is also observed in the dental pulp. Expression of Bmp-6 persists in secretory odontoblasts during the period of dentin formation, whereas enamel organ epithelial, secretory ameloblasts, and dental follicles react negatively [56]. On PNs, Bmp-6 protein is present in mesenchymal cells in the freshly formed periodontium and in the layer of cementoblasts along with the root, the HERS, as well as in the osteoblasts lining the alveolar bones [57].
Mutations of BMP-6 in humans and mice induce massive iron overload [58][59][60]. Despite their severe iron overload, Bmp-6 null mice are viable and fertile. Hepatocytes of Bmp-6 mice express low levels of phosphorylated Smad1/5/8/ proteins and hepcidin synthesis is significantly reduced. This indicates that Bmp-6 is critical for iron homeostasis. Additionally, histomorphometry of the proximal tibial metaphysis exhibited a defect in bone formation immediately adjacent to the growth plate in Bmp-6 KO mice compared to wild-type mice following estrogen treatment, and growth plate functions were decreased in Bmp-6 mutant mice after estrogen treatment, causing an impaired cancellous bone. This suggests that Bmp-6 also plays a physiological function in maintaining growth plate function [61,62]. Cleves et al. found that Bmp-6 is expressed in epithelial and mesenchymal cells during tooth development in sticklebacks and is required for viability, growth, and tooth patterning [63]. Homozygous deletion of the Bmp-6 gene in the fish resulted in reductions in both tooth number and tooth plate area at the early juvenile stage compared to wild-type or heterozygous fish. In addition, heterozygous mutations of Bmp-6 revealed a reduction in tooth number and tooth plate area in the late juvenile and adult stages compared to wild-type fish at the same ages. Gene expression of TGF-β and Smad signaling in Bmp-6 null fish was found to be decreased compared to wild-type fish [63].

BMP-7
At the initiation stage of mouse tooth development (E10-E12), Bmp-7 transcription is visible throughout the oral epithelium at the time of the formation of the dental lamina; however, in other parts of the epithelium covering the facial processes, Bmp-7 mRNA is weakly expressed [25,64]. At the bud stage, Bmp-7 expression is localized at the early bud stage in the tip of the bud, and its expression patterns are seen in these areas of the dental lamina where it starts to form a bud. At the cap stage, the anti-Bmp-7 probe is apparent and restricted to the epithelial enamel knot. As the enamel knot begins to disappear during the late cap stage (E15), Bmp-7 expression spreads to the neighboring inner dental epithelium and its expression appears to localize to sites where the secondary enamel knots will be formed [25,30,65]. At the bell stage, the Bmp-7 transcript continues throughout the inner enamel epithelium to the preameloblasts. Its expression is detectable in the dental papilla and the preodontoblasts when they start to secrete predentin matrix (E-19). On PNs, during deposition of dentin and enamel, the expression patterns of Bmp-7 appear in the dental papilla, odontoblasts, and in functional ameloblasts [25]. During root development, Bmp-7 staining is positive in PDL cells, dental follicles, tooth root-lining cells, chondrocytes, osteoblasts, osteocytes, and osteoclasts in alveolar bones [31,40,41,65]. In miniature pigs, Bmp-7 gene expression patterns in developmental teeth were similar to Bmp-2 as determined by anti-Bmp-7 probe and immunohistochemistry assays. However, Bmp-7 expression was also present in the inner enamel epithelium at E60 [29].
In human fetuses of 12th (equal to the cap stage of mouse tissues) and 14th (equal to the bell stage of mouse tissue) week gestations, BMP-7 expression was weakly detected in both the incisors and the molars at the cap and bell stages of the human tooth germs. BMP-7 expression patterns were rather consistent in the dental epithelium and mesenchyme at the cap and bell stages and were relatively higher in the inner enamel epithelium than in the dental mesenchyme and inner enamel epithelium [28]. However, BMP-7 expression was detected in both the dental epithelium and mesenchyme of human fetuses in the 12 th week of gestation, in contrast to mouse tooth germs at the cap stage [25,28,29,64,65].
Mutations of BMP-7 in humans and mice are associated with anomalies in several organs, including the kidneys, eyes, skeleton, and genitourinary ducts [66][67][68]. Genetic variants of BMP-7 are suspected to be implicated in molar-incisor hypomineralization (MIH) [69]. Global homozygous Bmp-7-deficient mice die shortly after birth because of poor kidney development. Histochemistry analysis demonstrated that mutant embryos during organ development reveal failure of metanephric mesenchymal cell differentiation, resulting in a virtual absence of glomeruli in newborn kidneys. Additionally, Bmp-7 null mice display eye deficiencies that appear to originate during lens induction. Bmp-7 mutant mice exhibit minor defects in the skeleton [70,71]. Heterozygous Bmp-7 mice exhibited significantly increased tooth sizes in adult incisors compared to wild-type adult mice. Cell proliferation in the cervical loop at the bell stage (E15) of heterozygous Bmp-7 mice was significantly higher than in wild-type mice of the same age [72]. Bmp-7 cKO mice (Wnt1-Cre:Bmp-7 fx/fx) were generated and mutant molars were short and broad both mesiodistally and buccolingually and displayed extra cusps on the first upper and lower molars. Multiple morphotypes of the 'extra cusp' morphology were present in both the upper and lower first molars in the null mice [73]. In addition, Bmp-7 cKO mice showed interruption in the initial onset of mineralization and a lack of dentin/enamel formation at birth and delayed tooth eruption. Expression of Dspp and amelogenin genes in odontoblasts and ameloblasts were decreased in Bmp-7 cKO mice. Bmp-7 contributes to the regulation of initiation of tooth mineralization via a complex epithelial-mesenchymal Bmp/Wnt signaling pathway [74].
2.1.7. Activin-like Kinase-1 (ALK1, ACVRL1, or TSR-1) and ALK2 (ACVR1, ActR-IA) At the initiation stage of mouse tooth development (E-12), Alk-2 mRNA transcripts are not present in the tooth primordium of mouse embryos but can be seen in other tissues, such as the brain [75]. At the bud stage (E13-13.5), Alk-1 and Alk-2 proteins are detected in the dental epithelium. The expression of Alk-1 and Alk-2 proteins is apparent in the basal layer of the dental epithelial bud. Both proteins are weakly present in the dental mesenchyme [76,77]. Alk-2 mRNAs are expressed in dental pulp cells in adult rat, bovine, and human teeth, but the Alk-1 gene is only detected in rat dental pulp cells [78][79][80][81]. Additionally, the Alk-2 protein is detected in HERS, the dental follicle, osteoblasts of alveolar bone, PDL cells, and tooth root-lining cells [31,82]. An in vitro study showed that the expression level of Alk-2 is increased with PDL cell differentiation [79,82]. Mutations of ALK1 cause type 2 hereditary hemorrhagic telangiectasia (HHT2), a devastating disorder that leads to arteriovenous malformations [83][84][85]. However, mutations of ALK-1 related to tooth development and formation have not been described so far.
Alk-2 cKO mice (Osx-Cre; Alk-2 fx/fx) exhibited dentin defects in molars and osteodentin formation in incisors. Alk-2 cKO mice displayed striking tooth phenotypes characterized by enlarged dental pulp cavities, wide predentin, and thin dentin, as well as delayed odontoblast differentiation in molars compared to wild-type mice. Expression of Dspp, Dmp1, and Osx in odontoblasts of Alk-2 null mice was reduced, whereas Bsp expression was increased in the mutant odontoblastic cells, leading to an alteration of cell fate from odontoblasts to osteoblasts. In addition, the expressions of the WNT antagonists Dkk1 and Sost were downregulated, but β-catenin was upregulated in Alk-2 cKO incisors. This indicates that Alk-2 plays an important role in dentinogenesis [86]. Furthermore, mutations of the ALK-2 gene in humans and mice cause a rare genetic disorder known as fibrodysplasia ossificans progressiva (FOP) characterized by progressive heterotopic ossification (HO), where especially muscles, tendons, and ligaments are converted into bone. The mutations of ALK-2 enhance the BMP-Smad and BMP-p38 signaling pathways [87][88][89].
At the bud stage of mouse tooth development, Alk-3 peptides are detected in the dental epithelium but are mainly localized to the tip and stalk and oral epithelium. At the cap stage, immunostaining of Alk-3 is present in the dental epithelium and the stalk as well as the upper part of the enamel organ. Its signal is also seen in dental mesenchyme. At the late cap stage (E15), Alk-3 expression is localized in the cells of the primary enamel knot distant from the basement membrane, in the stellate reticulum, and in the outer dental epithelium. Additionally, at this stage, the staining of this protein appears in the dental mesenchyme. At the bell stage, Alk-3 protein is present in the stratum intermedium, the stalk, the inner dental epithelium, and ameloblasts. The immunohistochemistry of Alk-3 is positive in the dental papilla and odontoblasts [26,81]. On PNs, besides ameloblasts and odontoblasts, Alk-3 protein expression is observed in HERS, the dental follicle, osteoblasts in alveolar bones, PDL cells, and tooth root-lining cells [31,79,82]. Expression of Alk-3 is increased with PDL cell differentiation [31,82]. Alk-3 mRNA transcription is detected in dental pulp cells from adult rat, bovine, and human teeth and is increased with dental pulp cell differentiation [78][79][80].
Global null mutation of the Alk-3 gene causes embryonic lethality in mice and animals die at E9.5. Homozygous deleted mice with morphological defects are detected at E7.5. No mesoderm forms in the mutant embryos, suggesting that Alk-3 is required for the inductive events, leading to the formation of mesoderm during gastrulation [89]. Alk-3 cKO mice (K14-Cre;Alk-3 fx/fx; Wnt1-Cre;Alk-3 F/-) showed an arrest of tooth development at the bud/early cap stages; the tooth buds were significantly smaller and less well developed in mutant embryos than in controls. Defective tooth development is accompanied by the downregulation of BMP-responsive genes, including Bmp-4, Msx1, and Pax9, in the mesenchyme at the bud stage and reduced cell proliferation levels in the dental mesenchyme [90,91]. In Osx-Cre;Alk-3 fx/fx mice, loss of Alk-3 in odontoblasts led to impaired dentin formation and short molar roots at PN 21. Alk-3 cKO mutants exhibited thin dentin, reduced dentin matrix production, and decreased expression of Osx, Dspp, phosphorylated Smad1/5/9, and phosphorylated p38 as compared to controls [92]. Transgenic mice expressing a constitutively active form of Alk-3 (caAlk-3; Wnt1-Cre; pMescaAlk-3) were generated, and all binary transgenic mice of caAlk-3 died shortly after birth [93]. The transgenic molars showed less differentiated odontoblasts and ameloblasts and dentin deposition was lacking in molars at PN 0. The number of phosphorylated Smad1/5/8-positive cells was significantly increased in the dental mesenchyme of the molar germ at E13.5 in the transgenic mice. The expression of odontogenesis, amelogenesis differentiation markers, Dspp, and amelogenesis were remarkably reduced within odontoblasts and ameloblasts in PN 0 molars in the transgenic mice. Overall, this indicates that the accurate balance of Alk-3 expression levels is essential for tooth development and formation.

ALK-5 (TGFβR1 or NDSTN)
At the bud stage of mouse tooth development, the dental lamina is invaginated into the condensed mesenchyme, and immunostaining of Alk-5 is detected in the dental epithelium and dental mesenchyme of the tooth bud [94,95]. At the cap stage, Alk-5 mRNA transcription is seen in the dental epithelial and mesenchymal cells and tooth germ mesenchymal cells (TGMCs) from the lower first molar tooth germs at E15.5 [94,96]. Expression of Alk-5 protein is observed in undifferentiated mesenchyme cells around Meckel's cartilage through E12 to E16 of mouse embryogenesis [94]. At the late bell stage (E18.5), Alk-5 protein is clearly evident in the inner dental epithelium layer and weakly stained in dental mesenchymal cells [97]. During PNs, the expression level of Alk-5 was elevated in secretory ameloblasts and odontoblasts in developing mouse teeth and immortalized mouse ameloblast-lineage cells [97]. Alk-5 mRNA transcription is observed in dental pulp cells and odontoblasts from rat, bovine, and human teeth [79,80,98,99]. The expression level of Alk-5 increased with dental pulp cell differentiation [79,98,99].

ALK-6 (BMPR-IB)
At the bud stage of tooth development, Alk-6 is present in the dental epithelial and mesenchymal cells surrounding the epithelial bud [26,81]. In the mesenchyme, the staining is intense on the buccal side. At the cap stage, expression of Alk-6 in the epithelium is more prominent in the inner dental enamel than in the outer dental enamel, and the inner cells of the primary enamel knot are intensely stained. The relative amount of Alk-6 expression is slightly decreased from E14 to E17 but is increased at the bell stage when cell differentiation occurs. At the bell stage, the expression of Alk-6 appears strong in ameloblasts and odontoblasts. ALK-6 expression patterns at the cap and bell stages of human tooth development are similar to those in mice. ALK-6 is present in both the wholeenamel organ and the dental mesenchyme, with the strongest expression levels in the inner dental enamel at the cap and bell stages [28,97]. During PNs, ALK-6 is strongly apparent in secretory ameloblasts and odontoblasts, as well as dental pulp cells from human, rat, and bovine molars [78,80]. With tooth root development, expression of ALK-6 is positive in HERS, tooth root-lining cells, the dental follicle, PDL cells, and osteoblasts in alveolar bones [31,82,100].
Homozygous Alk-6 null mice are viable and fertile. However, in Alk-6-deficient mice, proliferation of prechondrogenic cells and chondrocyte differentiation in the phalangeal region were significantly reduced. In adult null mice, the proximal interphalangeal joint was absent, the phalanges were replaced by a single rudimentary element, and the metacarpals and metatarsals were reduced, but the lengths of the radius, ulna, and tibia were not affected [101]. Recently, it has been reported that Alk-6 KO resulted in osteopenia in 8-week-old male mice, and the phenotype was transient and gender-specific. The decreased bone mass was not related to either the changes in osteoblastic bone formation activity or osteoclastic bone resorption activity in vivo. Thus, Alk-6 plays a unique role in maintaining bone mass and transducing BMP signaling [102]. In Alk-3 cKO mice, tooth development was arrested at the bud/early cap stages and the tooth buds were significantly smaller and less well developed in mutant embryos than in controls. Expression of Bmp-4, Msx1, and Pax9 at the bud stage and of Dspp and amelogenin at PN 0 was reduced in the mesenchyme; reduced cell proliferation levels in the dental mesenchyme were also observed [90,91]. However, when Alk-3 cKO mice (Wnt1-Cre;Alk-3F/-) were crossed with a conditional transgenic line containing a constitutively active form (with Gln203 to Asp change) of Alk-6 (named caAlk-6) to generate mice lacking Alk-3 but expressing caAlk-6 in the neural crest cells (Wnt1-Cre;Alk-3F/-; caIb), most Wnt1-Cre;Alk-3F/-;caIb embryos died mid-gestation. caAlk-6 only partially substitutes for the loss of Alk-3 in the cranial neural crest cells to regulate tooth development; it is unable to fully replace Alk-3 function, as evidenced by delayed odontoblast and ameloblast differentiation as well as a reduction in Dspp and amelogenin gene expression within odontoblasts and ameloblasts at PN 0 [91]. In addition, in immortalized ameloblast-lineage cells, Alk-6 and odontogenic ameloblast-associated protein (ODAM) display similar expression patterns, and Alk-6 physiologically interacts with ODAM, resulting in an increase in ODAM protein phosphorylation mediated by BMP-2. The phosphorylated ODAM induces MAPK signaling cascades, inducing ameloblast differentiation and enamel mineralization [103].

Type II Bmp Receptor (Bmpr-II)
Immunohistochemistry showed that BmpR-II protein is present in the dental epithelium and in the dental mesenchyme at the bud stage (E13) in mouse tooth development. BmpR-II is apparent in the internal part of the bud, in the stalk, and in the oral epithelium.
At the cap stage (E14), BmpR-II is mainly localized in the mesenchyme, but weak staining is present in the oral epithelium by RT-PCR and immunoblotting assays. At the late cap stage (E15), BmpR-II is detected in the mesenchyme and in the cells of the primary enamel knot near the stratum intermedium. BmpR-II immunostaining is intense in the stalk and in the oral epithelium. BmpR-II protein is also stained in the outer dental epithelia and in the cells of the stellate reticulum, close to these outer dental epithelia. At the bell stage, expression of BmpR-II is seen in the dental papilla, odontoblasts, and ameloblasts as well as the stratum intermedium [26,81]. BmpR-II is also expressed in the dental pulp and preodontoblasts in rat, bovine, and human teeth [78][79][80]. Furthermore, during mouse root development from PNs 5 to 23, the study demonstrated that expression of BmpR-II was detected in odontoblasts, ameloblasts, HERS, dental follicles, osteoblasts in alveolar bone, PDL cells, and tooth root-lining cells [31,100].

Structure of BMPs and Their Partners
Cells such as odontoblasts and osteoblasts produce BMPs, which are secreted into the ECM. All BMPs, like other TGF-β family members, initially act as a single chain containing a larger amino-terminal prodomain and a smaller carboxy-terminal mature signaling domain [104][105][106]. TGF-β ligands are initially synthesized as large dimers containing a secretion signal peptide in the amino-terminal element termed the prodomain and a cystineknot mature domain in the carboxy-terminal domain [107]. TGF-β-secreted precursors are attached to a prodomain called a latent associated peptide (LAP), which regulates the folding of the prodomain and prevents interaction with the receptor at the cell membrane [108]. By contrast, BMPs do not display such latency [106] and are generally catalyzed by cellular serine endoproteases to liberate the mature BMP polypeptides before secretion [109]. Recently, studies have shown that although BMP processing is predominantly localized intracellularly, it might also occur at the plasma membrane or ECM [105]. The active signaling molecules act as homodimers, and the mature BMP monomers contain seven cysteines, six of which form intramolecular disulfide bonds. The remaining seventh cysteine residue is involved in the dimerization with another BMP monomer through a covalent disulfide bond, resulting in a biologically active dimeric ligand for BMP receptor activation [21,110]. BMPs are dimeric molecules that display sites for N-and O-glycosylation, which increase the stability and half-life of the protein in the body as well as determine the specificity of receptor coupling [111][112][113]. In addition, heterodimers in particular experimental settings in vitro and in vivo have been shown to be more potent than the corresponding homodimers (i.e., BMP-2/-5; BMP-2/-6; BMP-2/-7) [114][115][116]. For instance, BMP-7 has been shown to function in development largely as a heterodimer with BMP-2 and BMP-4 [117]. The BMP-2/BMP-7, BMP-4/BMP-7, and BMP-2/BMP-6 heterodimers have increased signaling activity compared with homodimers [22,[118][119][120][121][122][123]. This increased bioactivity has been attributed to a relatively higher affinity for type I receptors of BMP-2/BMP-4 and a relatively higher affinity for type II receptors of BMP-6/BMP-7, creating a hybrid signaling molecule with higher total receptor affinity [118].
Classically portrayed as a hand, each chain consists of two sets of anti-parallel βstrands forming finger-like extensions that protrude from a central stabilizing "wrist" α-helix. The structure is stabilized by a conserved cystine knot formed by three internal disulfide bonds, similar to other growth factors [22] (Figure 4). Crystal structures of BMP homodimers revealed that the core structures of BMP dimers consist of a "cystine knot" structure and that the overall structure of BMPs has a "wrist and knuckle" or "two bananas" shape [124][125][126][127][128]. Studies of the crystal structures of BMPs have also identified possible binding epitopes of BMPs to specific receptors and antagonists [126][127][128][129][130][131][132][133][134] (Figure 5).

Runt-Related Transcription Factor 2 (Runx2)
Runx2 (also designated AML-3, PEBP2αA, CBFA1, Osf2) is a critical transcriptional regulator of odontoblast and osteoblast differentiation. The functions of Runx2 in bone and tooth development are dependent on gene expression at given stages during dentinogenesis and osteogenesis [135][136][137][138][139][140][141]. Runx2 has a unique expression pattern in dental mesenchymal cells from the bud to early bell stages during active epithelial morphogenesis. At the initiation stage of tooth development (E11), an in situ hybridization assay showed that there is no Runx2 expression in any craniofacial tissues. However, at the bud stage (E12-E13), the Runx2 transcript is intense in the mesenchymal condensates of forming bones and teeth in the developing maxillary and mandibular arches [142][143][144]. At the cap stage (E14), the expression of Runx2 appears prominent in the dental papilla, dental follicle, and mesenchyme in osteogenic zones. However, the Runx2 mRNA transcript is barely seen in the dental epithelium. At the bell stage (E16), the Runx2 transcript is expressed in ameloblasts, odontoblasts, dental pulp cells, dental follicles, and differentiating osteogenic mesenchyme. At the late bell stage (E18), Runx2 expression is dramatically downregulated in the ameloblasts, odontoblasts, and dental pulp cells, except for cells near the mesenchyme within the alveolar bones in the developing incisors and molars. Its signal is apparent within osteoblasts in differentiating alveolar bones ( Figure 6). On PN 1, Runx2 mRNA is highly expressed within osteoblasts in alveolar bones and dental follicles, which give rise to cementoblasts and PDL cells, but its signal is weakly expressed in ameloblasts, odontoblasts, and dental pulp cells in the developing incisors and molars. On PNs 5 to 14, Runx2 expression patterns are similar to those on PN 1. Notably, Runx2 expression continues to PDL cells, cells within the cementoenamel junction, and roots during tooth root development. Following the eruption of the first molar at PN 28, expression of Runx2 is present in the PDL, cementoblasts, cementocytes, and osteoblastic cells in the alveolar bones [145][146][147]. Therefore, the developmental profiles of Runx2 expression in odontoblasts and osteoblasts appear to differ greatly, suggesting that this gene is differentially regulated in these cells and plays different biological roles in dentinogenesis and osteogenesis [148,149]. Heterozygous mutations of the Runx2 gene cause an autosomal dominant human disease termed cleidocranial dysplasia (CCD; OMIM119600). CCD patients exhibit supernumerary teeth, delayed eruption, malformed roots, and absence of cellular cementum [137,140,150]. The expression of Runx2 was upregulated within odontoblasts at the early stages of mouse tooth development but downregulated in odontoblasts at the later stages of tooth development [143,148]. This indicates that Runx2 plays a dual role in dentinogenesis and tooth-related gene expression. Runx2 upregulated Dspp expression in pre-odontoblastic cells but downregulated Dspp transcription in matured odontoblastic cells [148,151]. Overexpression of Runx2 in mice resulted in dentin defects in the late stages of tooth development and downregulated Dspp gene expression in odontoblasts [152]. This indicates that, unlike bone, the biological functions of Runx2 during dentinogenesis are performed in a spatial-temporal manner. Dlx5 competes with Msx2 and binds to homeodomain response motifs in the Runx2 promoter and Dlx5 significantly upregulates Runx2 transcription, whereas Msx2 suppresses Runx2 expression [153]. Smads are able to interact with Runx2 synergically to promote gene expression [154]. In addition, Smad ubiquitin regulatory factor 1 (Smurf1) interacts with the proline-tyrosine (PY) domain in the COOH-terminus of the Runx2 protein and induces Runx2 protein degradation [155]. Conditional Smad4 KO mice (Dmp1-Cre;Smad4fx/fx) led to dentin defects, with wide predentin, thin dentin, disorganized dentin tubules, and decreased expression of Bsp, Collα1, Dmp1, and Dspp genes exhibited [156]. Canonical Smad signaling enhanced dental mesenchymal cell differentiation and Dspp expression via the action of Dlx5, Msx2, Osx, Runx2, Smads, and other compounds [92,[157][158][159][160].

Osterix (Osx or Sp7)
Osx is a zinc finger-containing transcriptional factor distinctly expressed in all developing bones and teeth [143,161]. It is important for odontoblast and osteoblast differentiation. At the bud stage (E12) of mouse tooth development, the antisense Osx probe is intense in the mesenchymal condensates of forming bones and teeth in the developing maxillary and mandibular arches. At the cap stage (E14), Osx mRNA is expressed in mesenchymal cells in the alveolar bone, dental papilla, and dental follicle, while Osx expression is barely detected in the dental epithelium. At the bell stage (E16-E18), Osx transcripts are present in differentiating osteogenic mesenchyme, ameloblasts, odontoblasts, and dental pulp. On PNs 1 to 14, Osx expression remains abundant in odontoblasts, dental pulp cells, and osteoblasts in alveolar bone, but its expression is weakly detected in secretory ameloblasts. Furthermore, Osx expression is also seen in PDL cells, cementoenamel junction, and HERS [143,146,[162][163][164] (Figure 6). During dentin regeneration and orthodontic tooth movement, expression levels of Osx are increased in odontoblasts, osteoblasts, and cementoblasts in experimental groups compared to control groups [147,165,166]. From this, it is implied that Osx participates in dentin regeneration and root formation.
Mutations of the Osx gene in humans and mice are associated with defects in both teeth and bones [161,[167][168][169][170][171]. In Osx null cells, tooth roots were short with a cuboidlike odontoblast layer containing few dentin tubules [168]. Further studies showed that Osx induces dental mesenchymal differentiation and mineralization and expression of Alp, Col1, Dmp1, and Dspp genes [37,143,172], while the expression of Dspp, Dmp1, and Collα1 was also significantly declined in Osx cKO mice, which may contribute to the resultant promotion of cell proliferation and differentiation [168,173,174]. Further experiments demonstrated that Osx directly binds to the Dspp gene promoter and that it upregulated their transcriptions in dental papilla mesenchymal cells [37,175]. The effect of Osx on gene expression is regulated by either BMP-Smad-Runx2-dependent or BMP-Smad-Runx2-independent signaling pathways [174,176,177]. BMP-mediated Dlx5, Msx2, and Runx2 facilitated their downstream gene expression via Osx [178,179]. Tao et al. reported that Klf4 promotes odontoblast differentiation and Dmp1 expression through Osx [180]. Furthermore, Nfic induced expression of Dmp1 and Dspp genes, as well as promoted odontogenesis via regulating Osx expression [168,174,181].

Distal-Less (Dlx) Homeobox Gene 3 (Dlx3)
Dlx3, one of the Dlx family members, is required for epithelium, hair, bone, tooth, and placental development [182,183]. At the tooth initiation stage (E9.5-E10.5), Dlx3 expression is visualized in the mesenchyme of the first branchial arch and the mandibular epithelium in the most mesial region of the mandibular arch [184,185]. At the bud stage (E12.5-E13.5), Dlx3 is expressed in the dental lamina of lower incisors and lower molars, and the labial enamel epithelium of upper molars. Dlx3 is also detected in mesenchyme adjacent to the dental lamina [183,[185][186][187]. After the dental lamina forms, dental epithelial cells invaginate into the dental mesenchyme to form an epithelial bud and subsequently develop into the cap-and bell-stage-shaped tooth germs. The spatial-temporal expression patterns of Dlx3 are highly evident in the dental epithelium, pre-ameloblasts, ameloblasts, dental follicles, and weakly evident in dental papilla and pre-odontoblasts through the cap to bell stages [183,[185][186][187][188]. Dlx3 is expressed in the dental follicle mesenchyme, which is involved in the development of PDL and the cementum. After birth, Dlx3 continues to show strong expression in ameloblasts and weak expression in odontoblasts. In addition to the expression of Dlx3 in ameloblasts and odontoblasts during dental morphogenesis and histodifferentiation, Dlx3 expression is also present in chondrocytes and osteoblasts [183,189] (Figure 7).

Msh (Muscle Segment Homeobox) Drosophila Homolog 1 (Msx1) and Msx2
Msx1 (also designated homeobox 7 (Hox7)) is involved in tooth development and formation. Through tooth development, Msx1 transcripts are highly restricted to the dental mesenchyme [30,[200][201][202]. At the tooth initiation stage (E11), expression of Msx1 is broadly localized to the mesenchyme surrounding the thickened epithelium in the maxillary processes and the mesenchyme at the tip of the mandibular arch [200]. By E12, expression of Msx1 is restricted to the mesenchyme immediately surrounding the developing tooth germs in the maxillary and mandibular processes. Msx1 expression in the mesenchyme of the dental papilla and dental follicle is maximal at the cap stage of mouse tooth development and progressively declines in the bell stage prior to the differentiation of odontoblasts and ameloblasts [200]. Through the cap stage, the Msx1 transcript is intense in the dental papilla and dental follicle and osteoblasts in future alveolar bones. However, the labeling of Msx1 is not visible in the enamel organ and other epithelia in all tooth germs [200]. At the bell stage, Msx1 expression continues in the dental papillae and dental follicle as well as osteoblasts, but its signal decreases in intensity in the dental papilla, though it is still higher than in the oral mesenchyme surrounding the tooth germ [201]. During root development, Msx1 expression is detected in the dental pulp and weakly seen within osteoblasts in the alveolar bone and PDL cells [30].  At new birth (NB) and PNs 1 and 5, expression of Dlx3 can be detected in ameloblasts, odontoblasts, dental papilla, and dental follicles, as well as in the mesenchyme in the alveolar bone. At PN 1, Dlx3 expression is more intense in ameloblasts, odontoblasts, and dental papilla than in developing alveolar bone mesenchymal cells. (f,f'). Tooth tissues were immunostained with anti-Dlx3 antibody, showing that the Dlx3 expression is apparent in ameloblasts, dental pulp cells, and odontoblasts at PN 2 of mouse tooth development. (f') shows higher a magnification of the box in (f). The tissue sections were stained with anti-IgG serum as the negative control. (g') shows a higher magnification of the box in (g). ab, alveolar bone; am, ameloblasts; con, control; D, dentin; de, dental epithelium; dm, dental mesenchyme; df, dental follicle; dp, dental papilla; E, enamel; E, embryonic day; mc, Meckel's cartilage; od, odontoblasts; PN, postnatal day. The data are unpublished.
MSXs are homeobox genes and constitute a large family of transcription factors. Mutations of MSX genes result in cleft lip, cleft palate, and tooth agenesis. Patients with MSX1 mutations exhibited teeth with oligodontia [203,204]. Mutations of MSX1 in humans and mice cause tooth deficiency [203,[205][206][207]. BMP-Smad signals induced tooth development via MSX1. MSX1 induced human DPSC proliferation and differentiation as well as expression of BSP and DSPP genes in human DPSCs [208,209]. However, Feng et al. reported that MSX1 promotes proliferation and prevents the differentiation of dental mesenchymal cells by the inhibition of BMP-2 and BMP-4 expression and downregulation of DSPP, DMP1, Runx2, OC, and Collα1 gene expression [210,211]. Of note, an atypical canonical BMP signaling pathway in a Smad1/5/8-dependent, but Smad4-independent manner in the dental mesenchyme during odontogenesis upregulated Msx1 expression via binding of pSmad1/5/8 proteins in Msx1 gene promoter [212,213].
At the tooth initiation stage of mouse tooth development (E9-E11), Msx2 expression is present in the neural crest-derived mesenchyme of branchial arches and maxillary and mandibular processes. Msx2 transcript in the oral epithelium is first evident at the sites of the dental placodes [214][215][216][217]. By E10.5, Msx2 mRNA is not detectable in the epithelium, but by Ell.5 Msx2 expression is visible in dental epithelial cells, immediately above its previous area of the mesenchymal expression. At the bud stage, in situ hybridization anti-Msx2 probe is evident in external enamel epithelium and the mesenchymal cells of the dental papilla and dental follicle immediately surrounding the invaginating dental epithelium. At the cap stage, Msx2 transcript is visible in the dense epithelium of the enamel knot adjacent to the internal enamel epithelium and septum and associated external enamel epithelium. Additionally, its signal is apparent in the layer of dental papilla mesenchyme cells and dental follicles. When the transitory enamel knot, septum, and navel disappear at the bell stage, Msx2 is expressed throughout the internal enamel epithelium, the external enamel epithelium, ameloblasts, and the cervical loop. Low levels of Msx2 expression are also evident in the cellular condensations of the stellate reticulum in the cervical loops. Expression of Msx2 also appears in the dental papilla mesenchyme, pre-odontoblasts, and odontoblasts as well as dental follicle mesenchyme [214][215][216][217]. In PNs, Msx2 expression is detected in the dental papillae cells, odontoblasts, and ameloblasts, but barely in osteoblasts in the alveolar bone. Msx2 expression is also seen in the cervical loop and in the stratum intermedium [218]. During mouse root development, Msx2 is expressed in HERS besides dental pulp and dental follicles, the adult periodontium, inner enamel epithelium, and outer enamel epithelium [219,220].
MSX2 gene mutation in humans caused Boston-type craniosynostosis characterized by the premature fusion of skull bones together with certain orofacial bone abnormalities [221,222]. Contrarily, haploinsufficiency of MSX2 causes midline cranial defects reflected in the occurrence of wide-open fontanels in the skull vault [223]. In Msx2 null mice, tooth root eruption was delayed, dentin deposition was affected, and dental pulp chambers were larger in size than in wild-type mice. The odontoblast row and odontoblastic layer were highly disorganized, and abnormal polarization ended with cellular inclusion within the pre-dentin and dentin matrix in Msx2 mutant mice. Different layers were visible in the dentin thickness of the mutant mice [224]. Deletion of Msx2 in mice reduced the expression of Alp, Col1, Dspp, Oc, Runx2, and Bmp-4 genes in odontoblasts [224,225]. Msx2 competed with Dlx5 in homeodomain response elements (ATTA) in the Dspp regulatory region and repressed Dspp gene transcription in C2C12 cells. By contrast, Dlx5 upregulated Dspp expression [157,226]. On the other hand, Msx2 interacts with CCAAT/enhancerbinding protein (C/EBP) and binds to its binding site(s) in the mouse amelogenin gene promoter, repressing amelogenin transcription [227]. MSX2 and BMP-4 have mutuality between one another [217,228,229], and BMP induces Msx2 expression through Smad signaling [228,[230][231][232]. In addition, BMP-2-mediated Msx2 stimulated Osx gene expression in Runx2-deficienct mesenchymal cells [179].

Paired Box 9 (Pax9)
Pax9 is a transcription factor that plays an important role in the mechanisms of ontogeny in all classes of vertebrates. At the initiation stage of mouse tooth development, Pax9 transcript is low in prospective incisor mesenchyme and positive prospective molars as well as mesenchyme in maxillary and mandibular arches [218,233,234]. At the cap stage (E14.5), Pax9 expression is only detected in mesenchymal cells in incisors and molars but is visible in both dental epithelium and mesenchyme in the incisors and premolars in human 12-week-old embryos, which expression pattern is different from that in mice [235]. At the bell stage (E16.5), the Pax9 signal is apparent in the mesenchyme of all teeth [234].
Pax9 plays a role in the development of human dentition. Mutations of Pax9 in humans cause hypodontia, including abnormal incisor and canine shapes and missing premolars and permanent molars [233,[236][237][238]. Pax9 is one of the most widely studied genes participating in odontogenesis [235,239,240]. Tooth development is arrested at the bud stage in Pax9-deficient mice and tooth germs are small in Pax9 null mice compared to controls, indicating that Pax9 is required for tooth development at or before this stage [233,234,241]. The arrested tooth development in Pax9 null mice was partially rescued by BMP-4, but Pax9 also upregulated BMP-4 and Msx1 gene expression [241][242][243][244]. Overexpression of Pax9 enhanced the expression of Dmp1 and Msx1 in the induced pluripotent stem cells (iPSCs) derived from neural crest-like cells (iNCLCs), but BMP-4 also upregulated the expression of Dmp1, Dspp, and Msx1 genes in iNCLCs. Co-expression of Pax9 and BMP-4 synergistically promoted the expression of Dmp1 and Pax9 genes in iNCLCs [245]. BMP-Smad signaling controls odontogenesis and Dspp expression via Pax9 [246].

Dentin Matrix Protein 1 (DMP1)
DMP1 is a non-collagenous protein originally identified in dentin and belongs to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs), which are essential for the proper development and formation of hard tissues, such as teeth and bones [247,248]. In mouse tooth development, at the bud stage (E13), no signal for Dmp1 is detectable within either tooth ectoderm or mesenchyme, but Dmp1 transcripts are visible in the first mouse mandibular molar at E13 and E14 by RT-qPCR assay [249,250]. At the cap stage (E15), Dmp1 mRNA is intense in newly differentiated osteoblasts within the forming alveolar bone, while a weak signal of Dmp1 is seen in the enamel organ and dental papilla. At the bell stage, Dmp1 transcription is evident in the inner dental epithelium of the enamel organ and newly differentiated or young odontoblasts at the mesiobuccal cusp tip of the first mouse molar. On PNs, an antisense Dmp1 riboprobe is accumulated in polarized odontoblasts and faintly hybridized to preameloblasts at PN 1. Dmp1 mRNA is notably present in osteoblasts (Figure 8). At PN 6, Dmp1 mRNA appears clearly in differentiated odontoblasts, dentinal tubules, and predentin, and is significantly downregulated in mature odontoblasts as well as upregulated in ameloblasts at the maturated stage of amelogenesis. At the same stage, osteoblasts in the alveolar bone maintain high levels of Dmp1 mRNA expression. In erupted molars at PN 26, Dmp1 is expressed in odontoblasts, while a more intense signal is observed in osteoblasts and osteocytes of inter-radicular bone [249][250][251][252][253]. During root development, Dmp1 mRNA is detected in cementoblasts and cementocytes in the cementum, besides odontoblasts, dentinal tubules, and osteoblasts [254].
amelogenesis. At the same stage, osteoblasts in the alveolar bone maintain high leve Dmp1 mRNA expression. In erupted molars at PN 26, Dmp1 is expressed in odontobla while a more intense signal is observed in osteoblasts and osteocytes of inter-radic bone [249][250][251][252][253]. During root development, Dmp1 mRNA is detected in cementoblasts cementocytes in the cementum, besides odontoblasts, dentinal tubules, and osteobl [254]. By in situ hybridization as Dmp1 mRNA transcription was clearly detected in odontoblasts near the dental crown and ro the mouse tooth and osteoblasts in alveolar bones by PN 1, while by PN 5, Dmp1 expression downregulated in odontoblasts and only appeared in odontoblasts located at the dental root was highly intense in the alveolar bone. By PN 10-14, Dmp1 transcription was weakly visib odontoblasts, but still highly expressed in osteoblasts in alveolar bones. By PN 1, Dspp transc were evident in odontoblasts, dental papilla, and pre-ameloblasts in the mouse mandibular mo By PN 5, Dspp mRNA was strongly expressed in odontoblasts and weakly seen in ameloblasts Figure 8. Dmp1 and Dspp expression patterns in developing teeth. By in situ hybridization assay, Dmp1 mRNA transcription was clearly detected in odontoblasts near the dental crown and root of the mouse tooth and osteoblasts in alveolar bones by PN 1, while by PN 5, Dmp1 expression was downregulated in odontoblasts and only appeared in odontoblasts located at the dental root but was highly intense in the alveolar bone. By PN 10-14, Dmp1 transcription was weakly visible in odontoblasts, but still highly expressed in osteoblasts in alveolar bones. By PN 1, Dspp transcripts were evident in odontoblasts, dental papilla, and pre-ameloblasts in the mouse mandibular molars. By PN 5, Dspp mRNA was strongly expressed in odontoblasts and weakly seen in ameloblasts and dental pulp cells. From PN 10 to 14, Dspp mRNA was mainly expressed in odontoblasts and weakly in dental pulp cells. Dspp expression was barely detected in osteoblasts in alveolar bone from PN 1 to PN 14 during mouse tooth development. ab, alveolar bone; am, ameloblasts; D, dentin; df, dental follicle; dp, dental papilla; E, enamel; od, odontoblasts; mc, Meckel's cartilage; pAm, pre-ameloblasts; pd, dental pulp; PN, postnatal day. The photos are partially adapted with permission from Chen et al., 2009, ref. [143]. DMP1 mutations in humans and animals are associated with tooth and bone deficiency [255][256][257][258][259]. Patients with DMP1 mutations displayed normal tooth growth and shape, but their deciduous and permanent teeth exhibited enlarged pulp cavities, thin dentin and enamel, as well as the absence of lower third molars and hypomineralization compared to healthy controls [258]. In Dmp1 null mice, as analyzed from PN 3 to 1 year of age, tooth morphology is similar to dentinogenesis imperfecta type III (DGI-III) in humans. Teeth in the mutant mice displayed enlarged dental pulp chambers, wide predentin, thin dentin and altered dentin matrix structure, and hypomineralization. Expression of Dspp, one of the important non-collagenous proteins in dentin, was decreased in odontoblasts, whereas biglycan, one of the small leucine-rich proteoglycan family members, was increased in odontoblasts of the null mice compared to the controls [255].

Dentin Sialophosphoprotein (DSPP)
Both DMP1 and DSPP are phosphorylated matrix proteins [247,248], but DSPP has distinct expression patterns and is highly distributed within pre-ameloblasts and odontoblasts during tooth development. At the bud stage (E13) of molar development, an in situ hybridization assay showed that no notable signal of both Dmp1 and Dspp was detected in either the ectodermal or mesenchymal components of the mouse tooth organ and surrounding tissues. However, while the Dmp1 transcript was amplified from the first mandibular molar by RT-qPCR analysis, there was no Dspp signal [143,249]. At the cap stage (E15), a high level of Dmp1 mRNA appears in newly differentiated osteoblasts in the alveolar bone, whereas no signal of Dspp is evident in osteoblasts within the same serial section. At the bell stage (E16-E17), Dspp transcription is first visible in young and mature odontoblasts and pre-ameloblasts and is weakly seen in dental pulp cells [143,249,260,261]. On PN 1, a high level of Dspp expression is mainly restricted to the tooth organ and progresses along a developmental gradient in odontoblasts, dental pulp cells, and preameloblasts [262,263] ( Figure 8). By PN 6 and later stages of tooth formation, Dspp expression retains in odontoblasts through all phases of primary dentinogenesis, and transient Dspp transcripts are present in early secretory ameloblasts at the growing end of the molar organ [264]. Besides Dspp expression in ameloblasts, odontoblasts, dental pulp cells, and dentin matrix, low expression levels of Dspp are found in osteoblasts, osteocytes, cementoblasts, PDL cells, and non-mineralized tissues [251,[265][266][267][268].
DSPP mutations in humans are associated with dentinogenesis imperfecta type II (DGI-II, OMIM 125490) and DGI-III (OMIM 125500), as well as dentin dysplasia type II (DD-II, OMIM 125420) and DD-I (MIM 125400) [269][270][271][272][273]. These hereditary dentin diseases are the most common dentin genetic diseases. DGI-II is characterized by bulbous crown shape, opalescent discolored dentition, and pulpal calcification, as well as impaired odontoblast cell differentiation and delayed conversion of predentine to dentine. DGI-III was originally regarded as a Brandywine isolate [274], along with a severe form of DGI-II with multiple dental pulp exposures and shell-like teeth. In DD-I, teeth are normal in shape and form as well as consistent in deciduous and permanent dentitions. In some cases, teeth may show a slightly amber discoloration. However, the roots are short, and pulp obliteration causes a crescent-shaped pulpal remnant in the permanent dentition and a total pulpal obliteration in the deciduous dentition. Teeth in DD-II patients are similar to the deciduous dentition in DGI-II, but tooth discoloration is minimal and dental pulp chambers are thistle-tube-shaped with pulp stones in the permanent dentition.
Homozygous null mice (Dspp−/−) analyzed from 2-3 months after birth showed tooth deficiencies similar to those seen in patients suffering from DGI and DD, with enlarged pulp chambers, a wide predentine zone, thin dentin, dentin hypomineralization, and dental pulp exposure. Expression of biglycan and decorin in PNs 6, 15 and 1-year-old teeth in mutant mice was increased compared to wild-type mice [275].

BMP Signaling during Tooth Development
The BMP ligands, as dimers, bind to two distinct receptors, type I serine/threonine kinase and type II receptors, which are necessary for signal transduction [276][277][278][279]. The serine/threonine kinase domains of type II receptors are constitutively active and phosphorylate Gly-Ser (GS) domains in the type I receptors upon ligand binding, leading to the activation of type I receptor kinases (Figure 9). BMPs bind to three distinct type II receptors, for instance, BMP type II receptor (BmpR-II), activin type II receptor (ActR-IIA or AcvR-2A), and activin type II B receptors (ActR-IIB or AcvR-2B), while BMPs bind to the type I receptors, ALK-1/-2/-3/-6 [105,[279][280][281][282]. The specificities of BMP binding to type receptors depend on the identities of the interacting type II receptors and cell types [283] (Figure 2). BMPs display a different affinity for BMP type I receptors: BMP-2 and BMP-4 bind to ALK-3 and ALK-6 [22,132,284] (Figures 2 and 5), while BMP-6 and BMP-7 bind strongly to ALK-2 and weakly to ALK-6 [285]. BMP-14 preferentially binds to ALK-6 [286]. BMP-9 and BMP-10 bind to ALK-1 and ALK-2 [126,287] (Figure 2). Some BMP type I receptors are shared by certain members of the TGF-β family members. TGF-β binds to ALK-5 (TGFβRI) as well as ALK-1 [282]. In contrast to other BMP family members, BMP-3 and BMP-11, as well as BMP-15 and GDF-8, have been described to activate TGF-β and activin type I receptors ALK-4, ALK-5, and ALK-7, respectively (Figure 2), leading to activation of Smad-2 and Smad-3 [22,288,289] (Figure 2). Thus, BMP-3, BMP-11 and BMP-15 appear to activate signals similar to TGF-β/activins, but distinct from those induced by other BMPs [22]. BMP-3 has been shown to bind to ActR-IIB, which transduces ActR-IIB-Smad-2/-3 signaling to suppress the other BMPs [290,291]. BMP signaling regulates downstream gene expressions through either Smad-dependent or Smad-independent pathways (Figure 9). Following heterodimerization, type I receptors are phosphorylated by type II receptors and subsequently activate the receptor-regulated Smad-1/5/8 through phosphorylation [292,293]. The phosphorylated Smad-1/5/8 heterodimers form a complex with the only common mediator Smad-4 (Co-Smad-4). Following nuclear translocation of the Smad-1/5/8/Co-Smad-4 complex, BMP target gene expression is induced (Figure 10) [292]. On the other hand, BMPs also activate Smad-independent signaling pathways, such as mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs), p38MAPKs, c-Jun amino-terminal kinase (JNK), phosphoinositol-3 kinase (PI3K), v-Akt murine thymoma viral oncogene (Akt)/PKB (protein kinase-B), and small GTPases (Figure 11) [294]. Thus, these Smad-independent pathways cooperate with Smad-dependent pathways to regulate various cellular responses. Expression patterns of BMP ligands and BMP receptor types I and II, including BMP-2/-3/-4/-5/-6/-7, ALK-2/-3/-5/-6, and BMPR-II, are observed in tooth tissues at the given stages during tooth development [25,26,30,31,64,75,94]. . BMP ligands, receptors, and interacting receptors. BMP signal transduction involves a number of ligands, type I and type II serine/threonine kinase receptors, and coreceptors, which regulate the activation of intracellular mediators in interactions with extracellular stimuli. BMPs are classified into several subfamilies according to sequence homology. The BMP signaling pathways. Canonical Smad signaling executes its function through Smad proteins. BMPs interact with the type I and type II receptors to form a heterotetrameric complex. Complex formation and ligand binding can be potentiated by a coreceptor, that is, endoglin, betaglycan, or repulsive guidance molecule (RGM). Upon phosphorylation by the type II receptor, the type I receptor recruits and phosphorylates pathway-specific R Smads (Smad-1, Smad-5 and Smad-8, and Smad-2 and Smad-3), which can form trimers with Smad-4 and translocate to the nucleus. Smads have intrinsic DNA-binding activity and can regulate gene expression by recruitment of chromatin-remodeling machinery and integration with tissue-specific transcription factors. Non-canonical Smad signaling, through phosphorylated TAK (pTAK). Ligand binding induces the formation of the receptor complex. BmpR-I is linked to the TAK1/TAB1 complex through the X-linked inhibitor of apoptosis (XIAP) to phosphorylate and activate the downstream mitogen-activated protein kinases (MAPKs), including p38, Figure 9. BMP ligands, receptors, and interacting receptors. BMP signal transduction involves a number of ligands, type I and type II serine/threonine kinase receptors, and coreceptors, which regulate the activation of intracellular mediators in interactions with extracellular stimuli. BMPs are classified into several subfamilies according to sequence homology. The BMP signaling pathways. Canonical Smad signaling executes its function through Smad proteins. BMPs interact with the type I and type II receptors to form a heterotetrameric complex. Complex formation and ligand binding can be potentiated by a coreceptor, that is, endoglin, betaglycan, or repulsive guidance molecule (RGM). Upon phosphorylation by the type II receptor, the type I receptor recruits and phosphorylates pathway-specific R Smads (Smad-1, Smad-5 and Smad-8, and Smad-2 and Smad-3), which can form trimers with Smad-4 and translocate to the nucleus. Smads have intrinsic DNA-binding activity and can regulate gene expression by recruitment of chromatin-remodeling machinery and integration with tissue-specific transcription factors. Non-canonical Smad signaling, through phosphorylated TAK (pTAK). Ligand binding induces the formation of the receptor complex. BmpR-I is linked to the TAK1/TAB1 complex through the X-linked inhibitor of apoptosis (XIAP) to phosphorylate and activate the downstream mitogen-activated protein kinases (MAPKs), including p38, ERK1/2, and JNK. Consequently, activation of these kinases leads to their translocation into the nucleus where they phosphorylate and activate and regulate the transcription of downstream target genes. Canonical Smad signaling is intracellularly inhibited by inhibitory Smads, that is, Smad-6 and/or Smad-7, and E3 ubiquitin ligases, such as Smurf1 or Smurf2, whose expression provides the cell with a negative feedback mechanism. The pathway can be antagonized by many mechanisms,   Ligand binding induces the formation of hetero-tetrameric receptor complexes con sisting of the type I and type II receptors [24,133]. Tight regulation of signaling intensit duration, specificity, and diversity is further achieved by the interaction of this recepto complex with co-receptors, e.g., endoglin, betaglycan, repulsive guidance molecule Ligand binding induces the formation of hetero-tetrameric receptor complexes consisting of the type I and type II receptors [24,133]. Tight regulation of signaling intensity, duration, specificity, and diversity is further achieved by the interaction of this receptor complex with co-receptors, e.g., endoglin, betaglycan, repulsive guidance molecules (RGMs, including RGMa, RGMb or DRAGON, and RGMc or Hemojuvelin), neogenin, TrkC, ROR2, MuSK, neuropilins, BAMBI, or integrins [295][296][297]. Co-receptors do not produce a signal directly, but rather promote ligand docking on the cell surface, regulating specificity and likely controlling signaling kinetics [297,298]. Recent reviews have highlighted aspects of co-receptor biology and have been linked to BMP signaling modulation [297].

Negative Regulation of BMP Signaling in Odontoblast Differentiation and Dentinogenesis
BMP signaling is under elaborate regulation to maintain accurate dentin and tissue development and formation. BMP signaling is negatively controlled by several mechanisms, including ligand antagonists, transcriptional repressors, intracellular regulation, and epigenetic factors. Common antagonists targeting BMPs, such as chordin [334], follistatin [335], gremlin [336], and noggin [337,338], have been reported ( Figure 12). Noggin, a BMP antagonist, competitively binds BMPs to prevent their binding to receptors, and negatively regulates BMP activities during odontogenesis and osteogenesis. Mutations of noggin in humans are associated with stapes ankylosis, with broad thumbs and toes, hyperopia, and skeletal anomalies, as well as mandibular micrognathia (OMIM 186500) [339][340][341][342]. With noggin deletion, mice died at birth with severe malformed skeletons [343,344]. Skeletal noggin overexpression in mice resulted in osteopenia and retarded bone formation [345,346]. Transgenic mice with noggin gene overexpression showed dentin defects, an increase in dental pulp chamber, a decrease in dentin volume, impaired odontoblastic differentiation, and reduced tooth-related gene expression, including pSmad1/5/8 and Dspp [47,337,338]. Yuan et al. reported that noggin impaired the non-canonical Smad signal of p38 and Erk kinases and Wnt/β-catenin of Pitx2 expression, inhibiting dental cell proliferation and fate as well as tooth development [347]. In addition, Chen et al. found that BMP-2 activated Dspp gene transcription in mouse odontoblastic cells through the NY-F transcriptional factor, but noggin was able to block this activity [5]. Noggin suppressed BMP-2-mediated phosphorylated Smad1/5/8 expression and BMP-2-mediated Akt signaling, inhibiting the expression of ALP, BSP, OCN, OPN, Osx, and Runx2 genes in dental cells [348]. Furthermore, noggin gene expression in osteoblastic cells was induced by BMP-2, -4, and -6 proteins and acted as a negative feedback loop to regulate BMP activity [349].
Gremlin is another BMP antagonist for preventing BMP signals. It is required for normal limb patterns and morphology [336]. In gremlin null mice, the expression of Msx1 and Msx2 was high, suggesting that gremlin plays an essential role in BMP signaling during limb development [336]. GREMLIN 2 (GREM2) mutations in humans are associated with dental anomalies, showing isolated tooth agenesis, microdontia, short tooth roots, and taurodontism [350]. Grem2 null mice displayed incisor wear and reduced incisors, as well as dentin layers that were narrow and irregular in appearance and did not completely encircle the dental pulp [351]. Overexpression of the gremlin gene in mice caused developmental defects in teeth. Teeth in the gremlin transgenic mice exhibited tooth fragility and attrition of the tips of the incisors as a result of periodic trimming in an effort to prevent malocclusion and malnutrition, as well as an enlarged pulp chamber with ectopic calcification and thinner dentin and enamel compared with the wild-type controls. In addition, the transgenic mice of gremlin displayed enlarged dental chambers and lacked odontoblast polarization and columnar shape as well as wide pre-dentin and thin dentin, indicating the retarded maturation of odontoblasts and dentin mineralization [352]. Overexpression of GREM1 and GREM2 inhibited bone/tooth-related gene expression of BSP, DMP1, DSPP, OCN, and OPN, as well as transcription factors Osx and Runx2, repressing cell differentiation and biomineralization. By contrast, the knock-down of GREM1 and GREM2 promoted the above gene expression, enhancing ALP activity and mineralization. Furthermore, GREM1 repressed mRNA expression levels of BMP-2, BMP-6, and BMP-7 [353,354]. Guan et al. reported that Grem1 is related to the progress of periodontitis and enhanced activity of NF-κB and interleukin-1β via MAPK signaling and Smad3-β-catenin signaling pathways [355]. GREM2 expression was strong in the bone specimens of osteoporosis patients compared to people without osteoporosis, and human bone marrow stem cells (hBMSCs) mixed with Matrigel containing siRNA GREM2 were transplanted into defect areas of femoral bones in nude mice. siRNA GREM2 robustly promoted new bone formation compared to control groups [353]. Chordin (CHD) belongs to a family of proteins that share a cysteine-rich pro-collagen repeat (or chordin-like cysteine-rich repeat (CR)), which is also found in various extracellular matrix proteins [356,357]. Without exception, the homology between CHD family members, including chordin-like 1 (CHDL1) and chordin-like 2 (CHL2), lies within their CRs. The CRs within CHD and CHDLs are responsible for BMP binding, specifically and  Chordin (CHD) belongs to a family of proteins that share a cysteine-rich pro-collagen repeat (or chordin-like cysteine-rich repeat (CR)), which is also found in various extracellular matrix proteins [356,357]. Without exception, the homology between CHD family members, including chordin-like 1 (CHDL1) and chordin-like 2 (CHL2), lies within their CRs. The CRs within CHD and CHDLs are responsible for BMP binding, specifically and , inhibition. Wnt, wingless type MMTV integration site family; SBE, Smad binding element; BMP, bone morphogenetic protein; MAPK, mitogen-activated protein kinase; p38, p38 mitogen-activated protein kinases; ERK, mitogenactivated protein kinase; TF, transcriptional factor; Akt, v-Akt murine thymoma viral oncogene/PKB (protein kinase B). Chordin (CHD) belongs to a family of proteins that share a cysteine-rich pro-collagen repeat (or chordin-like cysteine-rich repeat (CR)), which is also found in various extracellular matrix proteins [356,357]. Without exception, the homology between CHD family members, including chordin-like 1 (CHDL1) and chordin-like 2 (CHL2), lies within their CRs. The CRs within CHD and CHDLs are responsible for BMP binding, specifically and tightly to BMP-4 [358][359][360][361]. As inhibitors, the interaction of CHD or CHDLs with BMPs prevents BMPs acting on their signaling pathways. Chd, Chdl1, and Chdl2 are expressed in mesenchymal cells [361][362][363]. Overexpression of Chd delayed chondrocyte differentiation and maturation in developing skeletal and cartilage elements [361,363]. Expression of Chd, Chdl1 and Chdl2 genes was detected in mesenchymal cells in the pharynx during early mandibular outgrowth and later in the mandibular process [360][361][362]. Some homozygous mutant Chd mice survived, while others died of cardio-respiratory failure at birth, showing pharyngeal malformations similar to DiGeorge/velocardiofacial (DGS/VCFS) syndromes in humans characterized by cleft palate, facial dysmorphism with low setting of the external ear, small jaw, deafness, and cardiac defects [364,365]. Mice mutant for Chd exhibited mild mandibular defects and craniofacial anomalies characterized by a reduced distance between eye and pinna [362]. CHDL1 mutations in humans are associated with X-linked megalocornea characterized by an increased corneal diameter and deep anterior chamber evident at birth with later onset of mosaic corneal degeneration, arcus juvenilis, and presenile cataracts (MIM 300350) [366,367]. Additionally, Exotic BMP-4 represses Fgf8 and Pax9 transcription and induces Chd expression in mandibular explants in vitro [362]. Chd inhibited BMP-4 expression and osteoblast-like cell (MC3T3-E1) differentiation and mineralization via downregulation of the expression of phosphorylated Smad1/5/8, Alp, Bsp, and Ocn genes [368]. Conversely, Liu et al. reported that knockdown of CHDL1 did not affect hBMSC proliferation but inhibited BMP-4-dependent osteogenic differentiation, showing decreased mRNA expression levels of osteogenic markers, such as ALP, Collα1, OPN, pSmad1/5/8, and Runx2, and reduced mineralization in hBMSCs. Moreover, hBM-SCs containing overexpressing pLVX-CHDL1 were mixed with Matrigel and transplanted into defect regions of femoral bones in nude mice. The pLVX-CHDL1 significantly induced new bone formation compared to the controls and Si-CHDL1 mice [369]. These results support the hypothesis that CHD and CHL1 play different biological functions in osteogenesis and that these functions may be dependent on different cell types.

Therapeutic Implications of BMPs for Endodontic Regeneration
Tooth decay is the most prevalent non-communicable disease [370]. The World Health Organization (WHO) has defined early childhood caries as a worldwide problem, with a prevalence between 60% and 90%. In addition, more than 90% of all adults have experienced this disease [371][372][373][374][375]. When left untreated, dental caries can lead to disease of the dental pulp with sequent pulpal infection, necrosis, and loss of tooth vitality and possibly function, as well as eventual tooth loss. However, approximately 50% of cases require revision within 5-10 years after restorative treatment [376]. Additionally, any traditional artificial restorative materials might fail due to inappropriate physical, biocompatible, and mechanical properties [372,377,378]. The material pulls away from the cavity wall and microleakage can occur between the cavity and dental material. Microleakage may cause bacterial invasion, resulting in recurrent caries [377,378]. Therefore, despite several advances in dental restorative materials, it is required for novel therapeutic restorative approaches in dentistry to maintain a healthy dentition. Regenerative endodontic treatment (RET) is defined as 'biologically based procedures designed to replace damaged tooth structures, including dentin and root structures as well as the pulp-dentine complex' damaged by infection, trauma, or developmental anomalies with respect to immature permanent teeth with necrotic pulp [379]. In general, RET involves three principles of bioengineering: stem cells (SCs), biomaterials, and growth factors [380]. Dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs) and from the apical papilla (SCAPs) were isolated and these SCs were able to proliferate and differentiate into odontoblast-like cells, forming dentin structures in the given niches [381][382][383]. Recent preclinical and clinical studies have demonstrated that exogenous DPSCs were transplanted into injured teeth in patients, and these SCs regenerated dentin formation and provided evidence of neuro-vascularization [384,385]. SC proliferation and differentiation are regulated by intrinsic and extrinsic signaling pathways. Growth factors and advanced biomaterials provide a physicochemical and biological three-dimensional niche for cell attachment, proliferation, differentiation, and neo-tissue genesis.
Previously, Nakashima used rhBMP-2 and rhBMP-4 capped with an inactive dentin matrix, respectively, and the mixed compounds were transplanted into amputated pulps in dogs. At two months, tubular dentin was generated in the lower part and osteo-dentin in the upper part of the amputated pulps in the rhBMP-2 or -4, with the inactive dentin matrix groups compared to the inactive dentin matrix groups alone, indicating that rhBMP-2 and rhBMP-4 are able to induce the differentiation of adult dental pulp cells into odontoblastic cells, forming dentin structures [386,404,405]. Human SCAPs with nanofibrous microspheres (NF-MSs) as carriers in combination with controlled release of rhBMP-2 were implanted into subcutaneous regions in nude mice. rhBMP-2 induced differentiation of SCAPs into odontoblast-like cells and DSPP gene expression and osteodentin formation [393]. BMP-2 promotes dental mesenchymal cell differentiation and dentin formation through DSPP signaling [5,157]. DSP and domains of DSP peptides are able to induce dental mesenchymal cells into odontoblasts and mineralization as well as vascularization [406][407][408][409]. However, recently, side effects of BMP-2 usage have been reported with increasing frequency, causing safety concerns, including concerns about ectopic bone formation, osteoclast-mediated bone resorption, postoperative inflammation, and even tumorigenesis [410]. Thus, BMP-2 and other BMPs in the RET for precision medicine need to be further investigated in the future.

Conclusions
In this review, we have summarized recent achievements in research into BMP signaling pathways during odontogenesis. Despite new insights gained during the past decades, many questions still need to be answered regarding genetic, cellular, and molecular understanding of the biological functions of BMPs. Downstream signaling pathways of BMPs and BMP receptors are much more complex in tooth development and formation, especially odontogenesis. Little is known about the target genes of divergent BMP signaling. Genetic analyses in humans and knockout mouse models obviously demonstrate that BMPs and their target genes play critical roles in communication between ameloblasts and odontoblasts and spatial-temporal dental mesenchymal cell proliferation and differentiation, as well as biomineralization during dentin development and formation, besides other signaling pathways. Further analyses of these cell and tissue morphologies should be conducted and the molecular signals should be explored further for a better understanding of the mechanisms underlying each step in the cellular processes that occur during dentinogenesis.
BMP signaling is essential to and has diverse roles in odontogenesis. Intensive studies in human genetics reveal that mutation of BMPs, BMP receptors, antagonists, and their downstream genes, such as transcription factors, Runx2, Osx, Dlx3, MSX1, MSX2, and PAX9, as well as collagen and non-collagen proteins, including DSPP and DMP1, lead to dentin anomalies. Conditional gene knockout of BMPs and their signaling molecules in mice results in their developing similar phenotypes to those observed in human beings. In vitro and in vivo studies further dissect the atlas of the BMP signaling pathways of physiology and pathogenesis during dentin development and formation. Knowledge obtained from the analyses of BMP signaling in dentinogenesis should also shed light on understanding the molecular mechanisms of these human diseases and provide potential clues for treating dentin defects.

Conflicts of Interest:
The authors declare no conflict of interest.