Tooth development or odontogenesis is governed by an array of Epithelial–Mesenchymal interactions formed by a cascade of complex, predetermined, interdependent communications between adjacent layers of the epithelium ectomesenchyme, mediated by multiple signalling pathways resulting in growth differentiation of embryonic cells. Teeth are appendages that develop from epithelial cells of the mucosa lining the stomodeum or the primitive oral cavity, along with the ectomesenchyme cells, which originate from the cranial neural crest cells. A cascade of interactions between the two layers, the epithelium and underlying mesenchyme, occurs sequentially and reciprocally under the influence of multiple cell-signalling pathways for tooth formation. These pathways affected by genetic modulation determine the patterning of dentition. ^,

Growth factors are signalling molecules that help adjacent cells communicate through specific cell membrane receptors. They act intercellularly between embryonic cells and stimulate cellular differentiation and proliferation. Families of growth factors involved in tooth development are EGF (Epidermal growth factor), FGF (Fibroblast growth factor), TGF (Transforming growth factor) and its receptors, EDAR (Ectodysplasin A receptor), BMP (Bone morphogenetic proteins, member of TGF β family) and PDGF (Platelet-derived growth factor).

Various signalling pathways via transcription factors act on specific promoter/enhancer regions of the DNA to regulate target gene expression. Gene expression is the activation of a gene that produces polypeptide/protein that further activates or deactivates other genes with the help of transcription factors (growth factors). Mutation in genes involved in major signalling pathways results in genetic defects. More than 300 genes have been reported to be actively involved in the development of teeth.

Determination of specific tooth types at their correct position in the jaws is referred to as patterning. Patterning is a spatial temporal event comprising the regional development of incisors, canines, premolars and molars, which occur at different ages and involve the process of induction competence differentiation. It occurs around embryonic day E10 in the mouse, ^, which is equivalent to four weeks of human IUL .

The dental epithelium is the point of origin of signals for the number and positioning of tooth primordial. The epithelium shows the expression of Fgf8, which is critical in establishing the oral-aboral axis. Pitx2 appears in the stomodeum, subsequently localised in dental placodes, whereas Pax9 may be responsible for instructing the mesenchyme to respond to epithelial signals regulation of signalling molecules produced by it. Once the signals initiating tooth development have conferred the ability for tooth development to the ectomesenchyme, the dental papillary cells maintain it. ^, Fig. 14.1 shows the pathways determining tooth type.

Pathway involved in regulating determination of tooth type in murine dentition.

Green colour denotes expression in dental epithelium and yellow denotes expression in dental mesenchyme. Bmp4 and Fgf8 are expressed in murine epithelium of future incisor and molar, respectively. A positive feedback loop is created by Bmp4 with islet 1 as it induces Msx1 expression in the region of presumptive incisal ectomesenchyme, repressing Pitx1 and Barx1 expression in molar ectomesenchyme. Fgf8 induces expression of Pitx1 and Barx1 in ectomesenchyme of future molar. Isl1: LIM homeodomain protein islet1.

A particular set of homeobox genes present within the ectomesenchyme, the odontogenic homeobox code, directs the final morphology of the tooth primordium. It includes the LIM-homeobox domain genes transcription factors LHX6 and 7. ^,

Other genes involved in tooth development, like Otlx2, Dlx2, Msx1, Msx2 and Lef1, are expressed at the same time. Dlx1, Dlx2 and Barx1 genes are involved specifically in the development of molar teeth.

Expression of several genes in the ecto-mesenchyme marks the site of tooth germ initiation, including Pax9 and Activin-A. Fgf8 induces Pax9, which is, in turn, inhibited by Bmp2 and 4. Fgf8, Bmp2 and Bmp4 were expressed in non-overlapping areas. Tumour necrosis factor (Tnf), Fgf, Bmp, Sonic hedgehog (Shh) and Wnt pathways are involved in signalling pathways of organogenesis on the 9th to 11th embryonic days to initiate tooth epithelium. Ectodysplastin, a signalling molecule belonging to the Tnf family, mediates signalling between the ectodermal components in tooth germs. ^, Transient signalling centres appear in the epithelium during key morphogenetic stages, first appearing in dental placodes during the budding of epithelium and subsequently during bud-to-cap transition.

At embryonic day E11.5 in the mouse embryo and 37 days in humans in utero, tooth morphogenesis begins as localised thickenings of the oral epithelium within the dental placodes. At around the 6th week of human embryogenesis, they form dental lamina, followed by invagination of the actively proliferating dental epithelium into the underlying mesenchyme. The ectoderm produces paracrine signals during this stage, which mediates cell communication, initiating tooth development.

The bud stage is represented by the first epithelial incursion into the ectomesenchyme of the jaw between E12.5 and E13.5 in mice, 55 to 56 days of human embryonic development. BCL11B transcription factor is essential to the timing of epithelial proliferation by down-regulation of epithelial BMP-4. Shh affects epithelial cell proliferation to produce a tooth bud. BMP4 acts as a paracrine molecule that induces and maintains the gene expression of Shh and Bmp2 at the bud stage. Msx1, Pdgfa, Pax9, Lef1, Inhba, Runx2 contribute to the transition from bud to cap stage. Enamel knot formation involving various pathways occurs at this transition stage. ^,

The cap-shaped ectoderm surrounding the papilla at E14 is called an enamel organ. The appearance of a transitory yet important structure in the dental epithelium, the primary enamel knot (PEK), controls the formation of cusps. Multi-cuspid teeth show an impression of secondary enamel knots (SEK), which appear where inner epithelium infolding occurs, displacing the stellate reticulum and giving it a shape resembling a bell. The bell stage for primary teeth is marked at 14 weeks. The height of the cusps has been seen to correspond to the timing of the appearance of SEKs.

The key signalling molecule for EK formation is Bmp4. In teeth destined to be multi-cuspid, Eda expression around PEK is stimulated by Bmp2, Bmp4 and Bmp7, establishing the locations of SEK. Wnt/β- catenin signalling is an important signalling pathway required for the maintenance of the EKs, thus tooth shape determination. ^{, ,}

By the end of E16, EKs show apoptosis balanced by cell proliferation co-occurring in the dental epithelium. This proliferation involves factors like FGF4, FGF9, HGF, Sp6, Tβ4, YAP and EGF. ^,

Odontoblasts differentiate from mesenchymal cells adjacent to the inner enamel epithelium in the dental papilla, laying down pre-dentin. Twist 1 expression is seen at this time at the interface. Meanwhile, at around 18 weeks, differentiation of cells of the inner enamel epithelium adjacent to the freshly layered dentin differentiate into enamel-forming cells called ameloblasts. They secrete the enamel matrix, which ultimately mineralises. At the same time, root formation begins with the formation of root dentin, cementum and periodontal ligaments, which hold the tooth in its socket after the eruption ( Fig. 14.2 , Table 14.1–14.2 ). ^{, , ,}

Signalling and transcription factors mediating tooth development.

(A) At initiation, signals from epithelium activate a set of transcription factors (Wnt, Fgf8, Shh, Bmp, Eda, Pitx2) in mesenchyme leading to mesenchymal condensation (MC) and formation of dental placode (DP). Bmp4 is one of the first genes to be expressed at the site of presumptive dental epithelium. Shh regulates proliferation of dental epithelium (DE) cells to produce tooth bud. (B) Epithelial Bmp induces expression of Bmp4 in mesenchyme, correlating with shift in odontogenic potential to dental mesenchyme (DM) at bud stage. Eda, Edar, Pitx2 expressed in DE. Runx2, Dlx1,2, Fgf3,10 are also expressed in DM. (C) Primary enamel knot (PEK) appears in DE. Lef1 regulates expression of Fgf4 in the epithelium, which in turn induces Runx2 expression in the DM through Msx1. Runx2 further induces expression of Fgf3 regulating expression of Shh in EK. The Wnt cannonical pathway functions through β-catenin acting on Bmp4 mediated by TCF/Lef. (D and D1) EK is fully functional and expresses Shh and Fgf4 that regulate dental papilla formation in adjacent mesenchyme and proliferation of DE to form cervical loops (CL). Fgf3 and Runx2 show remarkable expression in mesenchyme induced by FGF signals from DE. Mesenchymal Bmp4 targets p21, resulting in exiting of EK by inducing apoptosis. Other factors are also expressed. (E and E1) Early bell stage shows slit-1 expression which initiates proliferation within SEK and Fgf4 which promotes proliferation of adjacent DE and DM cells. Bmp4; Wnt5a,6,10a expression is also seen. Tgf β/Bmp signals odontoblast induction. After which they signal back to DE for ameloblast induction through Bmp2,4, Tgf β1. Shh from DE supports ameloblast differentiation along with Tgf β1, Wnt3, Eda and follistatin. Ameloblasts express Sp6 & Msx2.