Second permanent molars: embryological origin, development and eruption. Role of the RANK/RANKL/OPG pathway

Craniofacial skeleton formation, and more specifically alveolar bone development, follow a finely regulated controlled genetic program. A spatial and temporal combination of molecular signals determines the type and inter- and intra-maxillary position of each dental-alveolar unit. The volumetric growth and movement of tooth germs depend on a coordinated adaptive peripheral alveolar bone modeling process which is supported by signaling pathways between cells of the dental epithelium, dental follicle and alveolar bone. These signaling pathways involve transcription factors, proto-oncogenes and diffusible factors called “growth factors”. Transient impairment of these signals during time windows corresponding to the development of the various dental types could explain specific localized eruption deficits in a given dental type, as observed for second molars. Ongoing studies show that disturbances of bone resorption related to disruption of the RANKL/RANK/OPG signaling pathway affect tooth eruption and root morphogenesis, with a clear relationship between the time window of onset of disruption and the affected dental type.

Odontogenesis begins very early in embryonic development, with the establishment of the initial maxillary template. More precisely, a spatial combination of molecular signals determines the type and inter-and intramaxillary position of each dental-alveolar unit ( fig. 1B). In the mid-1990s, Paul Sharpe called this a "dental homeocode" 39,7,36 . According to this homeocode, the maxillary proximal-distal and oralaboral axes are established by the BMP (BMP4: Bone Morphogenetic Protein 4) and FGF (FGF8 and 9, Fibroblast Growth Factors 8 and 9) growth-factor family signaling pathways, expressing in the distal and proximal sectors, respectively 7 . FGF8 and 9 induce expression of homeoproteins LHX6 and 7 (Limhomeobox Gene), DLX1 and 2 (Distal-Less Homeobox Gene) and BARX1 (Bar Class Homeobox Gene) and mesenchymal markers of the proximal oral region 13,37 . In contrast, FGF8 and 9 suppress expression of GSC (Goosecoid Gene), an aboral mesenchyme marker. Expression of BMP4 in the distal oral ectoderm induces expression of MSX1 and 2 (Muscle Segment Homeobox Gene) in the incisor region and limits expression of BARX1 ion the proximal part of the maxilla where the molars form. If BMP4 signaling is inhibited, by Noggin for example, BARX1 is expressed in the region of the future incisors, which undergo "homeotic" transformation into molars 43,40 .
DLX family members are more widely involved in maxillary morphogenesis 8 . DLX-mutant mice show several skeletal defects. Expression of homeoproteins DLX-1 and -2 is restricted to cells of the maxillary proximal ectomesenchyme, where  20,40 . In the mandible, there is functional redundancy between DLX5 and DLX6 8 . These DLX homeoproteins are necessary for BARX1 expression in the odontogenic mesenchyme 43 , which otherwise loses its mandibular odontogenic potential, becoming chondrogenic, as is the case for superior molars in DLX-1 -2 double-knockout mice 37 . Following initiation (antenatal period), morphogenesis comprises a series of epithelial-mesenchymal interactions, morphologically consisting in invagination of the odontogenic epithelium of the underlying ectomesenchyme (developing from the cephalic neural crests), in successive stages known as bud, cap and bell ( fig. 1C 26 ).
In the cap stage (E14.5 in mice), two cellular layers form in the peripheral part of the dental epithelium: internal (IEE) and outer (OEE) enamel epithelium; The ectomesenchyme condenses adjacent to IEE to form the mesenchymal papilla and the cells of the follicular sac. Within the central IEE, the primary enamel knots begins to appear, in a signaling center expressing several transcription factors. At the bell stage (E16.5), the previous cellular developments increase. In the dental epithelium, four cellular layers can be distinguished: OEE, IEE, stellate reticulum (SR) and stratum intermedium (SI). Within the dental mesenchyme, two tissue regions can be distinguished: the mesenchymal papilla, facing the IEE, and the follicular sac, surrounding the system formed by the dental epithelium and the mesenchymal papilla Within the IEE, secondary enamel knots appear in the future cusp regions, underlying later morphogenesis 18 . They express and secrete the same factors as the primary knots, but are not involved in proliferation, rather controlling cell differentiation.
After the crown has been formed, root morphogenesis begins, with Hertwig's epithelial root sheath 49 , a double layer of epithelial cells which fuse at the extremities of the enamel organ once the crown is completely formed. On the classic theory of root formation, the Hertwig epithelial root sheath cells proliferate and migrate apically, separating the ecto-mesenchyme in two: radicular mesenchymal papilla, and follicle sac ( fig. 2A). The alveolar bone develops in coordination with the tooth. More precisely, growth in tooth volume, of both crown and root, and relative dental germ displacement during growth involve peripheral alveolar bone modeling 29,17,22 . Although dental growth has been shown not to depend on root formation 4 , the bone crypt still has to adapt to crown and root formation and bone modeling related to eruption 23,25,24 . This modeling, like the bone remodeling that has been clearly described in the axial and appendicular skeleton, involves resorption and apposition, and the emergence of site specificity. The two processes are known to be coupled, and are founded on the recruitment and activation of highly specialized cells: respectively, osteoclasts and osteoblasts.
Dental eruption is the process by which the tooth emerges from the bone sac after resorption of the overlying alveolar bone and reduction of the oral epithelium. The mechanism underlying the eruption pathway seems to be highly complex, depending not on the pressure of the tooth on the surrounding bone, but on programmed and coordinated local bone resorption, independently of root formation 41,4 . Eruption involves coupling and uncoupling between tooth and bone, via signaling pathways between dental epithelial cells, follicular sac and alveolar bone ( fig. 2B), some of which have been well described. They involve transcription factors, proto-oncogenes and growth factors 44,47 , and can be divided into two categories.
The first are those enabling timely recruitment, at the correct site, of effector cell precursors such as osteoclasts. The second are those inducing differentiation and modulating cell activation. In the first category, the main pathways initiating dental eruption are those triggering the arrival of mononuclear cells in the follicle. M-CSF and MCP-1 are the first two factors shown to be involved in mononuclear cell (osteoclast) recruitment in the dental follicle 42 . Interestingly,  3A). It is a soluble protein, structurally closer to the RANK protein, to which it can thus bind and block RANKL induction of osteoclast differentiation 41,43 .

Figure 2B Diagram of signaling pathways in coupling between dental epithelium cells, follicular sac and alveolar bone during alveolar bone modeling. M-CSF1: Macrophage
Expression of RANKL and OPG is governed by several hormones and cytokines that control bone resorption by direct action on the osteoblasts and stromal cell secreting them, and include parathyroid hormone, vitamin D, TNF (Tumor Necrosis Factor) alpha, interleukin 1, 6 and 11, M-CSF, prostaglandin E2 and EGF (Epidermal Growth Factor) 48 .
Most studies of the involvement of the RANKL/RANK/OPG triad confirmed its importance in alveolar bone modeling and remodeling and in maintaining periodontal ligament integrity 5,15 . They demonstrated that, under physiological conditions, RANKL and OPG are expressed in the follicle during eruption and in the  analysis showed that loss of PTHR1 function did not directly impact osteoclast activity but disturbed epithelial-mesenchymal interaction, impacting the teeth although occurring earlier than eruption 2 . Taken together, these findings show that dental and periodontal tissue signaling to the cells resorbing the bone is a key element in physiological dental eruption. Root formation, like eruption, requires bone lodge modeling coordinated in space and time with root lengthening and the formation of insertion tissue: cementum and periodontal ligament.
Hertwig's epithelial root sheath plays an important role in root lengthening by tissue interaction with the pulpar mesenchyme and dental follicle. Sheath cells express epithelial molecules such as cytokeratins, Ecadherin and ameloblastin, and also mesenchymal molecules such as BSP, vimentin and N-cadherin 1,10,11,33,50 . The interaction mechanisms between sheath epithelial cells and mesenchymal cells and the signaling pathways involved are as yet unknown. Interactions between epithelium and dental mesenchyme have been widely studied in early development 35 , but few studies have focused on communication between dental epithelial root cells and bone cells during later development and root growth, most being conducted in osteopetrosis, where root lengthening is strongly disordered 9 . Such root deformities are interpreted as dental lesions secondary to a mechanical problem of failed eruption pathway formation and alveolar bone pressure on the lengthening root 16 .
adult alveolodental ligament during periodontal homeostasis. RANKL activation of osteoclastogenesis is important for the formation of the eruption pathway and alveolar bone adaptation to growth in tooth volume. Overexpression of RANK in pre-osteoclasts accelerates molar eruption in mice 6 and, conversely, teeth fail to erupt in RANKL-knockout mice ( fig. 3B 12 ). In rats, Wise 46,47,48 showed reduced OPG expression at day 3 of life to be associated, in the alveolar bone, with increased RANKL expression and osteoclast production. In-vitro studies clearly established that OPG expression is inhibited by M-CSF and PTH-rP, promoting RANKL signaling 27 . It was therefore suggested that expression of M-CSF and PTH-rP in the dental follicle and stellate reticulum respectively lowers expression of OPG 22 , and increases RANKL expression and function and thus osteoclast differentiation.
The importance of PTH-rP for eruption was demonstrated in vivo: in PTH-rP knockout mice, eruption is totally interrupted 28,29 . PTH-rP was also shown to increase RANKL expression and reduce OPG expression in periodontal ligament cells 12 , inducing not only tooth displacement during eruption but also deciduous tooth root resorption 12  Recent studies by Berdal's team (Oral and Molecular Physiology Laboratory, UMRS 1138) showed that bone resorption disorder (deficient or excessive) impacts dental eruption and root morphogenesis ( fig. 3B) 5 . More precisely, resorption deficit (e.g., RANKL or RANKL-knockout mice) induces delayed eruption and shorter roots of greater diameter, whereas over-resorption (mice overexpressing RANK in pre-osteoclasts) greatly accelerates eruption and root formation, leading to roots of smaller diameter 6 . The implicated mechanisms are not yet fully understood, but it is clear that there is signaling from bone cells (osteoclasts and osteoblasts) toward dental and periodontal cells during dental lengthening and eruption. One explanation for inclusion specific to one type of tooth (2 nd molars or 2 nd premolars) is transiently impaired signaling between dental and bone cells during the time window of the specific tooth lengthening. Studies in mice, transiently blocking osteoclast activity at various time points, are underway, using a RANKL-blocking antibody; initial results suggest that very early blocking (days 1-5) inhibits eruption and root lengthening definitively in 1 st molars but transiently in 2 nd and 3 rd molars 21 . Later blocking (days 4-7) should induce maximal inhibition of 2 nd molars, with 1 st and 3 rd molars only transiently affected.