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Notch in the Dental Epithelial Stem Cells


  1. Ectodermal Organs

Organogenesis of appendages derived from the ectoderm leads to the differentiation of diverse well-specialized organs including the salivary glands, hair follicle, mammary glands, and teeth. Irrespective of their shape differences, the level of specialization, number and function, the various ectodermal organs undergo continuous development and renewal throughout life. They also exhibit a reciprocal but continuous interaction between mesenchymal and the epithelia while eliciting common molecular and morphological characteristics in the course of embryonic development. According to a stem cell fate determination review by Jiménez-Rojo, Granchi, Graf and Mitsiadis, diseases occurring during pregnancy, such as ectodermal dysplasia are known to affect ectodermal organs at the same time, an indication pointing the same origin of these organs from a common multipotent precursor originating from the ectoderm layer during embryonic development (Jiménez-Rojo, Granchi, Graf and Mitsiadis 2). It has been established that the precursor ectodermal stem cells may follow diverse differentiation paths and ultimately develop to distinct tissue-specific cells harboring various cell lineages, which further grow into diverse organs. Various signaling pathways leading to specification of different common stem cell precursors have been documented including the bone morphogenesis protein, Wnt, fibroblast growth factor, and Notch signaling pathways.

Studies have shown that the various organs arising from the ectoderm, particularly the teeth, hair follicle, sweat glands, salivary glands and mammary glands have a common origin occurring between the mesenchyme and epithelium. As noted above, various signal regulars of ectodermal organogenesis have been isolated, for instance, the transforming growth factor beta (TGF-β), the regulators belonging to the hedgehog family, and Wnt (Dell'Accio et al. 3). The definite manner in which ectodermal organogenesis occurs, as well as its timing, depends on the invagination process involving epithelial cells. With respect to teeth development, FGF-9 is one of the mediating factors regulating the activation of tooth germ development (Mitsiadis et al. 3025). The activity of FGF-9 increases the rate of invagination of the epithelium, a factor only expressed in the epithelium. Besides, the FGF-10 factor is expressed stimulating cellular proliferation in the epithelium making the tooth germs larger. As Jiménez-Rojo et al., established, the oral ectoderm and neural crest form the ectoderm arising from the mesenchyme from which mammalian teeth develop. The epithelial elements with stem cells commanding continuous teeth growth constitute suprabasal layer and stellate reticular tissue of the surface ectoderm.

As it occurs in tooth development, other ectodermal appendages go though analogous stem cell developmental mechanisms, which entail a close but regulated communication and exchange between the mesenchyme and epithelium (Pispa and Thesleff 195). Additionly, the appendages share general characteristics morphologically in the course of early ectodermal organogenesis. Such common features include epithelial thickening to form the placode of the appendages. Secondly, the formed placode forms an invagination into the mesenchyme, and through guided proliferation results in the formation of a bud as the surrounding mesenchyme begins condensing (Jiménez-Rojo et al. 2). The formed epithelial bud proliferates and grows into the adjacent mesenchyme with a final acquisition of the organ-specific configuration during morphogenesis into the skin ectoderm-derived organs, including the hair follicle and mammary gland, or the oral ectoderm-derived organs including the salivary gland and teeth.

2.0 Tooth Overview

Teeth are diminutive, calcified organs, white in color fixed on the jaw or mouth cavity of vertebrates. Functionally, teeth are used for food breakdown, hunting prey or for defense, and are indispensable in speech modulation. A tooth is composed of different cellular tissues that undergo differentiation into the final structure. In essence, it is not composed of a bony structure but a combination of diverse tissues eliciting varying solidity and densities (McCollum and Sharpe 153). Every tooth is made of different parts including the enamel, which is the hardest part is the body. It forms the white outer surface of a tooth and is made of calcified phosphates. It is also composed of dentin, which is a layer of living cells underlying the enamel. The living cells of dentin secret calcification factors into the enamel. The pulp is another component of a tooth. It is a soft inner lining of teeth into which blood vasculature and innervations are interspersed. The cementum, a lining of connective tissues, anchors the root of a tooth into the jawbone and gums firmly (Tummers and Thesleff 1054). Finally, there are periodontal ligaments, which are additional connective tissues that facilitate in holding teeth firmly into the jaw. While the crown of a tooth projects above the gums, he root descends into the jaw below the gum line. During organogenesis and morphogenesis of teeth, the cellular tissues that differentiate into teeth are components of the embryonic ectoderm.

3.0 Tooth Development

Studies in odontogenesis resolve the complexity of the developmental processes elaborating on the formation of teeth from embryonic germ line cells, their growth, and finally eruption into the oral cavity. Under healthy conditions, all parts of teeth are anticipated to develop and grow during their respective fetal development stages. During prenatal development, primary teeth grow between the sixth and eighth week, while permanent teeth development starts from the 20th week. In case teeth development does not commence within the identified times, hypodontia and/or anodontia may result due to a partial or total failure of teeth development (Blanpain, Horsley and Fuchs 445). Studies have established the necessity of factors in tissues within the primary pharyngeal arch, which takes a crucial role in initiating teeth development.

The enamel of teeth, the hardest tissue in the human body, takes a crucial role in the health and functioning of teeth. The differences exhibited within patterns and structures of teeth define the evolutionary patterns among different species with respect to their diets. For instance, the dentition in a mouse is composed of a single mono- cuspid incisor teeth in addition to three multi-cuspid molars situated on both sides of each jaw. Developmental stages of teeth occur in similar fashion as other ectodermal organs, wherein a group of molecular regulators and a crosstalk between the mesenchyme and epithelium mediate the dental patterning and growth (Mitsiadis et al. 276).

Odontogenesis has been shown to involve tooth germ cells, which entails cellular aggregation with an eventual tooth formation. As indicated above, tooth germ cells are derivatives of the embryonic ectoderm of the crosswalk between the ectomesenchyme and the initial pharyngeal arc (Mitsiadis and Smith 177). Essentially, tooth development is a coalition of a series of processes entailing mesenchymal-epithelial interactions with cells derived from the neural crest ectomesenchyme and oral epithelium differentiating into the tooth with is associated mineralized structures. Ectomesenchymal cells of the neural crest give rise to dental pulp, its follicle together with odontoblasts that are involved in the synthesis of dentin matrix (Mitsiadis and Graf 200). On the other hand, cells arising from the oral epithelium develop into ameloblasts that are implicated in the synthesis of the enamel (Bluteau et al. 2).

3.1 Development of Mineralized Tissue

3.1.1 Dentinogenesis

Among teeth constituents, dentin is the largest mineralized tissue. It is regarded as a connective tissue, wherein the extracellular matrix has undergone a series of modifications to give rise to a mineral phase, which enable the dentin tissue to function fully in teeth. Dentinogenesis entails a series of different processes, including cellular interaction and differentiation, the formation of organic matrices, and subsequent synthesis of mineralized crystals within an extracellular matrix (Mitsiadis et al. 199). Dentin synthesis begins from odontoblasts, specialized cells that are derived from the outer surface of dental pulps, and commences in the late bell stages of teeth development. The differentiation of cells of the dental papilla results in the formation of odontoblasts. On the outset, ectodermal-mesenchymal interactions initiate the differentiation and morphogenesis processes entailed in odontogenesis (Bluteau et al. 4). Matrix interactions together with reciprocal cells are important in initiating terminal differentiation, particularly of ameloblasts and odontoblasts. In a critical review on dentinogenesis by Linde, is was established that early dentinogenic odontoblasts actively participate in the synthesis, as well as secretion and reabsorption, of extracellular matrix constituents. While the above synthesis takes place within cell bodies, endocytotic and exocytotic processes are part of the cellular activities. The product of cell processes is a complex structure whose main trunk diameter measures between 0.5 and 1 pm. The trunk, most often, bifurcates into two or more consistently thin branchings laterally (Linde 690). The synthesis of non-mineralized dentin matrix is mediated by a series of processes, including secretion of collagen and non-collagen materials by the newly differentiated odontoblasts. The collagen formed is packaged into bundles grouped into large collagen fiber bundles globular configuration.

Prior to mineral formation, the presence of voluminous amounts of amorphous plaques composed of osmiophilic substances is identified at the dentin-enamel junction (Linde 691). These plaques are reabsorbed in later stages in the course of tooth formation. The initial stage of dentinogenesis gives rise to a mantle dentin that lacks dentinal tubules, but only canaliculi, which are thin (Mitsiadis et al. 199). In some cases, globular structures that are mineralized may be seen occurring in groups or in isolation, embedded within interglobular dentin networks in the mantle dentin crown (Linde 690). As opposed to the formation of mantle dentin, which occurs on ground substances within the dental papilla, circumpulpal dentin formation occurs through the synthesis of an organic predentin and ensued by mineralization along the mineralization border. The synthesis of circumpulpal dentin is controlled by odontoblast secretory activity.

Predentin forms in sound teeth at the proximal aspect exhibiting a constant thickness, as executed by cell bodies of odontoblasts while communicating through their distal junction complex connections (Linde 693). During predentin formation, exocytosis of collagen take place near cell bodies and is attached to fibrils where intermolecular and intramolecular cross-links form for stabilization (Linde 694). The process of mineralization of the collagenous cross-links takes place at the mineralization front. Root dentinogenesis commences during morphogenesis of dental crown and subsequent onset of tooth eruption. Hertwig's epithelial root sheath begins growing at the cervical loop in the enamel within the inner and outer enamel epithelia junction (Mitsiadis et al. 200). The sheath is composed of two layers, outer and inner epithelia layers that set off odontoblast differentiation at the border of dental papilla. Differentiated odontoblasts become polarized, and commence secreting predentin at the root that give rise to root dentin in the long run (Linde 694). Root predentin is narrower that in the crown.

3.1.2 Amelogenesis

The process of amelogenesis begins in the course of advanced bell stage of odontogenesis, and subsequent to dentinogenesis. Reciprocal induction defines dentinogenesis and amelogenesis considering the relation requiring dentin formation to occur first for amelogenesis to proceed, and that ameloblasts must be present for dentinogenesis to occur. Signals are sent from odontoblasts that are newly differentiated to the epithelium of inner enamel inducing further differentiation of the epithelial cells into secretory ameloblasts that are active. The formation of enamel is a three-step process involving the induction stage, secretory stage, and maturation stage. The pre-secretory stage is characterized by morphodifferentiation into the shape of the dental crown as defined by the bell stage. The newly formed predentin is the initiator of differentiation phase, followed by elongation of inner enamel epithelial cells into preameloblasts. The transformation of these cells shifts their polarity, accompanied by their elongation and further development and polarization into postmitotic secretory ameloblasts.

The secondary stage of amelogenesis is characterized by ameloblasts that have assumed the attributes of polarized columnar cells. Enamel proteins within the rough endoplasmic reticulum of the ameloblasts are secreted into the surrounding inducing the synthesis of enamel matrix. Alkaline phosphatase contributes to partial mineralization of the formed matrix. After the formation of the first layer, ameloblasts recede from the dentinoenamel interface, to enable the growth of Tome’s processes in ameloblasts that are communicating with dentinoenamel junction. The Tome’s processes develop at an angle facilitating the presence of crystalline orientation differences as they deposit enamel matrix crystals. Amelogenesis continues into the adjoining cells leading to the formation of a wall or pit that encapsulates the Tomes’ processes, and associated deposits of enamel matrix crystals within the wall or dental pits. The matrix deposited in the pits gives rise to enamel rods while that in the walls become interod enamel.

The final stage of amelogenesis, maturation stage, entails transport of substances used in the process by ameloblasts. In this regard, studies show the transition of ameloblasts from secretory cells to transport entities. The proteins engaged in the mineralization process include ameloblastins, amelogenins, tuftelins enamelins. Calcium ions used in the process is derived from the enamel organs through active transport, or passive extracellular transport mechanism (Mitsiadis et al. 180). Whereas enamel is being secreted, part of mineralization is accomplished using calcium ion deposition by the amelogenins. Some studies also suggest the involvement of tuftelins in regulating the ion deposition. The porosity of the forming enamel decreases with mineralization, a process that occurs only once. Enamelins and tuftelins are lost in the enamel and are responsible for tufts in the enamel. Termination of amelogenesis indicates the end of enamel synthesis, as opposed to dentinogenesis, which occurs continuously in one’s lifetime (Blanpain et al. 455).

3.2 Molecular Bases of Tooth Development

Odontogenesis begin from a continued reciprocal but sequential interaction involving epithelium and neural crest-derived mesenchyme. Tooth formation entails specially coordinated series of morphogenetic and molecular processes defining the type of signals deliberated to regulate cell fates and subsequent patterning (Mitsiadis and Graf 199). Mesenchymal cells migrate from the neural tube dorsum and consequently assume various cell fetes leading to the development of different structures with distinct morphological shapes and functions, including teeth, bone, hair follicle and mammary glands (Yoshizaki et al. 3; Yildirim 55). In order to generate cells of such diversity, various cellular and molecular interactions limit cells to differentiate into a specific cell phenotype.

Through genetic marking of cell fates and use of lineage tracers for dental progenitor cell microinjection, it has been established that molecular and cell-to-cell interactions regulate the progress of odontogenetic processes by controlling the involved cellular processes including cellular proliferation, differentiation and its termination, apoptosis, morphogenesis through cytoskeletal modifications, and synthesis of extracellular matrix. Similar signals are relayed cyclically throughout different odontogenetic stages. Various factors are produced in the early stages of tooth organogenesis, including bone morphogenetic proteins (BMPs), Wnt molecules, and fibroblast growth factors (FGFs), which may manipulate lineage commitment by instructing precursor cells to choose a specific fate outplaying others in response to a particular signal. The factors also manipulate lineage commitment by assisting in choosing a particular fate randomly and controlling their proliferation and survival.

A primary transcription factor, Pitx2, which is bicoid-related, defines the fate of dental epithelium early in the course of embryonic development. In the late stages of embryonic development, the expression of Pitx2 becomes limited primarily on the epithelium of teeth germ line. In addition, the dentition pattern is defined as early as before the elicitation of tooth development. Various genetic pathways controlling teeth development run concurrently to influence the growth of different teeth categories such as the molars, premolars, canines, and incisors. This intuition is anchored on the cell population within the cranial neural crest, which are pre-patterned, contributing to the development of specific teeth morphology.

The magnitude of signals, defining the shape and number of teeth, arising from the oral epithelium determines the fate and arrangement of responding cells. Several molecular factors, including elements within the transforming growth factor-b (TGF-β) group, Wnt factor, and hedgehog (Hh) category have been identified functioning as morphogens (Charron and Tessier-Lavigne 120). Notable is the permissive influence of FGF on teeth development. The control signaling factors regulating various stages of odontogenesis act independently or through cellular and molecular interactions. The Midkine (MK), BMP2 and BMP4, for instance, control epithelial–mesenchymal interactions (Bluteau et al. 2008).

On the other hand, FGF family, including FGF3, FGF4, and FGF8 to FGF10, are implicated in the regulation of cell proliferation and a direct control of expression of target genes. The Wnt3, Wnt7β, Wnt10α, and Wnt10β mediate proliferation of cells, their relocation and differentiation at some stages in the course of teeth synthesis initiation and morphogenesis. A study by Khna et al. confirmed the involvement of sonic hedgehog in the regulation of teeth initiation and morphogenesis (Khan et al. 240).

Among the identified molecular factors, FGF8 and BMP4 are critical for early epithelial initiation and play indispensable roles in activating the control genes within the mesenchyme (Mitsiadis, 25). The role of these two signals has been exhibited in rodents by their influence in teeth patterning. In this regard, FGF8 controls the shape f molars while BMP4 controls the shape of incisors (Mitsiadis and Smith, 178). Specific gene complements, including Msx1 and Msx2, are produced in the mesenchyme of developing incisors. Additionally, the mesenchyme of developing molars expresses Barx1, Dlx1 and Dlx2 gene complements. The specific gene complements of the above-mentioned transcription factors activate teeth germ into the molar or incisor configuration.

Considering the limited expression realm of the molecular signaling factors and the genes arising from the cranial neural crest mesenchyme of the mandible and maxilla, the production of Msx1 and Msx2 in the mesenchyme requires stimulation by the expression of BMP4 in the oral epithelium. Msx1 takes a crucial role in the course of incisor morphogenesis and provides the spatial details required for dental patterning (Mitsiadis and Smith 180). Conversely, FGF8 sends positive signals to the mesenchyme to express Dlx2 and Barx1, which are necessary for molar morphogenesis (Cobourne and Mitsiadis 252). It is argued that Barx1 is important in defining the neural crest-derived mesenchymal cell identity in the mandible and maxilla. Together with Pax9 and Sox2 expressed in the mesenchyme underlying the epithelial pits, transcription factors are indispensable in regulating the transcriptional program required for dental morphogenesis (Cobourne and Mitsiadis 258; Juuri et al. 319).

4.0 Mouse Tooth as a Model to Study Development and Regeneration

A mouse tooth is used as an in vitro model for studying odontogenesis in higher vertebrates, especially in humans. In the elicitation of the functions of Notch1 and calcium ions in the regulation of oral epithelium differentiation under the influence of Mediator (Med)1, cervical loop-derived oral epithelial (CLDE) cells from the embryonic incisors of a mouse are isolated and cultured until self renewal and immortalization (Yoshizaki et al., 5). Such studies have established that majority of CLDE cells express Sox2, a stem cell marker, which is a potent transcription factor for the growth of incisors (Juuri et al. 318). A mouse tooth model is ideal for such studies considering the continuous growth of their incisors, making it possible to enumerate on stem cell regulation, differentiation, and renewal. Stem cells in the oral epithelium are restricted within the incisors’ proximal end towards the labial cervical loop. The mouse teeth allow restriction of cells containing the transcription factors Sox2 to the labial cervical loop in the course of teeth morphogenesis leading to the renewal of ameloblasts that synthesis enamel. Moreover, it has been possible to enumerate the presence of Sfrp5, a Wnt inhibitor that is present in newly formed stem cells expressing Sox2 transcripts (Juuri et al. 318).

The entire odontogenetic process has been demonstrated in a mouse to understand various developmental stages and the modulator factors regulating various stages. The initial indicators of the onset of odontogenesis in mouse have been shown to entail the thickening of dental epithelium at day ten of embryonic development. The thickening is accompanied by the invagination of the underlying cranial neural crest-derived mesenchyme to gradually produce teeth buds between embryonic days 12 and 13 (Mitsiadis and Graf 199). Further, the oral epithelium continues growing and develops to a dental cap by the 14th or 15th embryonic day followed by the formation of a dental bell configuration by the 16th to 18th day. During the late dental bell stage, cells of the mesenchyme give rise to a dental follicle and subsequently the formation of a dental pulp. The pulp cells embedded in the oral epithelium give rise to odontoblasts on differentiation, while epithelial cells embedded in the dental pulp grow into ameloblasts (Mitsiadis and Graf 199). The odontoblasts then coalesce to form a layer that is in close communication with the dentin odontoblastic processes form distally penetrating into the dentin while participating in the secretion of dentin minerals and matrix. Mouse teeth model studies have established that the contents of the dentin matrix include 90% collagen proteins, blended with non-collagenous proteins. Dentinogenesis is an important process that has been shown through a mouse model, its development and renewal potential throughout life.

4.1 Mouse Epithelial-Mesenchymal Stem Cells

Pluripotency of the embryonic epithelial-mesenchymal stem cells is the differentiating capacity of the cells into various cell lineages. In addition, embryonic epithelial-mesenchymal stem cells have the competence to undergo self-renewal, which entails a lifelong ability to proliferate without differentiating (Boyer et al. 948). Recent studies on the mouse epithelial-mesenchymal stem cells have established the upregulation of expression of genes, which control self-renewal through core transcription factors, particularly Sox2, Oct4, and Nanog. These transcription factors are also involved in repressing the genes that regulate cellular differentiation in a mouse model (Bernstein et al. 315). The transcription repressor, Rest, according to a recent study, expressed abundantly in the mouse epithelial-mesenchymal stem cells, is a prime target of the Sox2-Oct3-Oct4-Nanog transcription control network, but is not a regulatory requirement in the preservation of pluripotency of the epithelial-mesenchymal stem cells (Yamada et al. 13). The transcription repressor is known to enhance cellular differentiation through suppression of self-renewal genes.

Signaling networks, particularly Stat3, leukemia inhibitory factor, Smad/BMP, MAPK, Calcineurin-NFAT, and Ras pathways have been shown to control molecular switch between differentiation and self-renewal proliferation in mouse epithelial-mesenchymal stem cells. In this regard, reports indicate that Zap70, for instance, regulates the modulation of the equilibrium between MAPK/Ras pathway and leukemia inhibitory factor-Stat3 pathway in mouse epithelial-mesenchymal stem cells in order to maintain their differentiation capacity and pluripotency (Cha and Park 4244; Koster et al. 42). Epigenetic processes, in addition to signaling pathways coupled to transcription factors, especially through chromatin remodeling and DNA methylation are indispensable in the determination of cell fate in mouse epithelial-mesenchymal stem cells between differentiation and self-renewal (Kalluri and Neilson 1776).

Epithelial cells give rise to complete tissue layers and structures since their lateral membranes are in close communication with intercellular adhesion complexes, especially the gap junctions, tight junctions and adherens junctions. Moreover, the cranial neural crest-derived mesenchymal cells are capable of relocating on their own into the extracellular matrix since they lack polarity and intracellular junctions (Kalluri and Neilson 1782). Mouse epithelial-mesenchymal stem cells are capable of eliciting epithelial-mesenchymal transition, which is a process entailing epithelial cell phenotypic transformation into mesenchymal cells. Transcription factor signaling via calcineurin-NFAT signaling has been enumerated to support EMT in the course of epithelial stem cell switch during differentiation into lineage precursors. It is the ability of mouse epithelial-mesenchymal stem cells to engage a variety of transcription factors to bind to EMT regulator gene promoters to facilitate lineage specification (Ahn et al. 2).

5.0 Organ Renewal and Tissue Specification

An important characterization of stem cells is their pluripotency capacity to undergo self-renewal through lop-sided proliferation to remodel stem cells and differentiated cells with more limited progeny that have restricted potential for proliferation and differentiation. During embryonic development, stem cells are localized in a specific cellular niche identified as their microenvironment. In tooth development, the stem cell niche is within the dental pulp. The potency of cell renewal and differentiation is controlled by stem cell niche (Mitsiadis et al. 275). The specific microenvironment of stem cell niche in tooth specification and renewal define the way in which stem cell populations in the dental pulp carry on tissue maintenance, dental self-renewal, and teeth regeneration. In order to function properly, stem cells require transcription factors and signaling molecules for regulation by eliciting different levels of control to induce cellular proliferation, and/or differentiation. Following dental injury, odontoblasts undergo apoptosis, a process that stimulates and activates dental pulp stem cells to undergo proliferation, stem cell transitioning, and differentiation into morphologically alike odontoblast cells. These newly formed cells have the capacity to repair dental dentin (Neunzehn et al. 25).

Regulation of stem cells within the dental pulp microenvironment is indispensable in dental regeneration. The activation of stem cells in the dental pulp niche initiates an intricate process that entails a sequence of epithelial-mesenchymal interactions, leading to the derivation of diverse mineralized tooth structures. In essence, ameloblasts, which are responsible for the synthesis of enamel arise from cells of epithelial origin while cranial neural crest-derived mesenchymal cells develop into dental pulp spheres, dental follicle, and odontoblasts, which synthesize dentin matrix (Abe et al. 2; Borena et al. 1302). Identification of dental pulp stem cells, including their isolation, may require enumeration of stem cell surface markers using FACS-based assays or magnetic bead–based assays that enrich stem cells utilizing their expression of surface markers (Nemeth and Karpati 3). In addition, side population analysis has been used to make a distinction between dental pulp stem cells and their progenitors from the surrounding somatic cells based on their excretory capacity of eliminating alien molecules extracellular to the cells (Nemeth and Karpati 3).

6.0 Notch Signaling Overview

The concept of the Notch signaling pathway has been highly studied to understand the mechanisms underlying the highly preserved cell-signaling pathway during cell proliferation, differentiation and renewal. Higher vertebrates elicit four distinct types of Notch receptors, which include Notch1, Notch2, Notch3, and Notch4 (Mitsiadis et al. 407). The structure of the Notch signaling system is such that it is described as a single-pass transmembrane receptor protein. The 3D structure of this protein is a hetero-oligomer that is made of a long extracellular component that exhibits a non-covalent interaction that is calcium-dependent. Additionally, it has a diminutive component of Notch protein that is made of a small intracellular facet, and small extracellular facet functioning as a singular transmembrane pass. Notch signaling is a component of regulatory pathways that stimulates and supports stem cell proliferation for neurogenesis to occur. Notch signaling is inhibited by Numb signaling to allow for neural differentiation.

A Notch protein, which is a transmembrane protein, spans a stem cell membrane leaving some portions intracellularly and extracellularly. Ligands bind to the Notch's extracellular domain leading to the induction of a proteolytic cut of the intracellular Notch domain, which is carried into the nucleus to induce alterations in gene expression (Brou et al. 208). The Notch receptors are usually activated by cell-to-cell communication such that the extracellular domains of the transmembrane proteins that are in express contact constitute the ligand proteins that attach to the Notch receptors. Following ligand binding onto Notch receptors, cells organize themselves into groups so that the expression of a particular phenotypic train in one cell results in a switch-off in other cells in the group via intracellular Notch signaling. As a result, cellular coalitions interact leading to the synthesis of functional multicellular structures. It has been established that the Notch cascade entail the Notch protein, Notch ligand, and intracellular proteins facilitating the transportation of intracellular Notch component into the nucleus (Lardelli et al. 177). Studies have isolated the formation of complexes between the intracellular Notch domain, and MAML1 and RBPJ in the process of activating target gene transcription.

6.1 Notch Signaling in the Ectodermal Organs

Notch signaling is a regulatory system defining cell fate and organogenesis regulation. It is necessary in embryonic development to regulate proliferation and differentiation of stem cells. As a single-pass transmembrane protein, Notch forms a surface receptor directed to transmembrane ligands including Delta1, Delta3 and Delta4, Jagged1, and Jagged2 (Shutter et al. 1313; Yoneya et al., 27). The extracellular Notch domains harbor tandem repeats of EGF and membrane proximal repeats rich in cysteine. The intracellular Notch component is made of six cdc10/ankyrin tandem repeats that play a major role in protein-to-protein interactions. Notch proteins harbor a domain rich in glutamine, in addition to a PEST sequence that is important for downstream signal transduction (Harada et al. 107).

During ectodermal organogenesis, the activation of Notch signaling through the attachment of its ligands to extracellular domains results in the cleavage of the entire Notch at least three times prior to the generation of an intracellular peptide for signal transduction to activate the downstream receptors. Initially, a furin-like protease localized in the Golgi complex configures embryonic Notch protein forming heterodimers followed by cleavage process to facilitate surface expression of the Notch signaling system (Mustonen et al. 281). The intramembrane cleavage is accomplished by presenilins, an aspartyl protease that is active as a macromolecular complex mediating the activity of γ-secretase enzyme within cell membranes (Li, 198; Selkoe and Kopan, 565). Intracellular Notch components that are soluble translocate into the nucleus to combine with RBPJ and begin functioning as a transcription factor.

O-fucosylation occurring within the EGF tandem repeats control the activation and ligand binding of Notch signaling (Lei et al. 6412). The EGF domain modifies Notch signaling through the addition of fucose to threonine or serine amino acid residues. The enzyme O-fucosyltransferase 1 mediates O-fucosylation of Notch signaling leading to its upregulation while downregulating the expression of the transferase hinders Notch domain-ligand binding, as evidenced in Drosophila species (Okajima et al. 42340). In mouse, FNGS modulate the activation of Notch system by modifying the O-linked fucose moieties (Shao et al. 7775). In this regard, the Notch-ligand interactions rely on the type of O-fucose glycans present.

6.2 Notch Signaling in the Tooth

During odontogenesis, it has been shown that cells in front of anterior enamel epithelium experience intense Notch1 mRNA signal. It has been argued that these cells arise from the stem cell pool and proliferate more rapidly to commit to ameloblast cell fate leading to the synthesis of inner enamel epithelium. Similarly, Notch2 is expressed on the outer epithelium of enamel together with the stellate reticulum that is underlying (Lane et al. 795). In contrast, Notch3 is not expressed within the odontogenetic processes. In tooth repair and regeneration, Notch signaling is stopped in one of the two progeny cells, while it is stimulated in one, to facilitate differentiation of the cells into two different types of cells. While Notch signaling is important in cell fate determination, there are no detectable Notch receptors on adult dental pulp. Notch signaling is activated after a dental injury to facilitate renewal (Ornitz and Itoh 3006). Moreover, although Notch activity is linked to differentiation inhibition in adult mice, the inhibition of Notch signaling facilitates cellular commitment to the ameloblast cell fate, with the commencement of differentiation and synthesis of enamel. Stem cells in the dental pulp eliciting Notch1 signaling activation that is not inhibited may remain in the stellate reticulum within primitive cellular compartments in the development of incisors.

In the development of mouse molar at embryonic day 13 to 14, the bud stage transitions to cap stage characterized by the development of mature enamel knot (Mustonen et al. 123). During this stage, Notch1 and Notch2 transcription factors are detectable during embryonic day 13 within the oral epithelium excluding the basal layer (Lin and Kopan 343). The two Notch genes are highly expressed at mRNA transcription in the buccal line of stellate reticulum. On further development, Notch 1 domain is highly expressed on the buccal side than the lingual side of the molar. In all cases, their expression is in the regulation of cell fate for apposite teeth development.