Three-dimensional analysis of the early development of the dentition

Abstract

Tooth development has attracted the attention of researchers since the 19th century. It became obvious even then that morphogenesis could not fully be appreciated from two-dimensional histological sections. Therefore, methods of three-dimensional (3D) reconstructions were employed to visualize the surface morphology of developing structures and to help appreciate the complexity of early tooth morphogenesis. The present review surveys the data provided by computer-aided 3D analyses to update classical knowledge of early odontogenesis in the laboratory mouse and in humans. 3D reconstructions have demonstrated that odontogenesis in the early stages is a complex process which also includes the development of rudimentary odontogenic structures with different fates. Their developmental, evolutionary, and pathological aspects are discussed. The combination of in situ hybridization and 3D reconstruction have demonstrated the temporo-spatial dynamics of the signalling centres that reflect transient existence of rudimentary tooth primordia at loci where teeth were present in ancestors. The rudiments can rescue their suppressed development and revitalize, and then their subsequent autonomous development can give rise to oral pathologies. This shows that tooth-forming potential in mammals can be greater than that observed from their functional dentitions. From this perspective, the mouse rudimentary tooth primordia represent a natural model to test possibilities of tooth regeneration.

Keywords: 3D reconstruction; Tooth; development; human; mouse; odontogenesis.

Figure 1

Variability of teeth in vertebrates. (a) cartilaginous fish – shark; (b) an example of a bony fish; (c) amphibian – a frog (teeth are absent in the lower jaw); (d) reptile – turtle (teeth are absent); (e) reptile – python; (f) reptile – crocodile; (g) bird – goose (teeth are absent); (h) mammal – dolphin; (i) opossum; (j) hedgehog; (k) armadillo; (l) baboon; (m) porcupine; (n) deer (note the atavistic canine – c); (o) mandible of elephant (an example of horizontal replacement of teeth: the molars are permanently shifting anteriorly, where they are finally shed, while a new molar emerges posteriorly); (p) toothless ant-eater (tamanoir) (from the ref., with permission).

Figure 2

A schematic comparison of various tooth patterns in mammals. (a) The basic tooth pattern in mammals; (b, c, d) the tooth pattern in adult humans, squirrel and mouse, respectively; (e) mouse embryo. In the mouse embryonic upper jaw, the primordium of the functional incisor originates from joint development of several epithelial prominences (for details see Fig. 12). In the embryonic mandible, two to three epithelial prominences (bridges) come before the origin of the functional incisor primordium (for details see Fig. 17). I1, I2, I3 – the first, second and third incisor, respectively; C – canine; P1, P2, P3, P4 – the first, second, third and fourth premolar, respectively. M1, M2, M3 – the first, second and third molar, respectively. D1–D5, the small rudimental tooth primordia in the mouse diastema; MS, R1, R2 – the large rudimental tooth primordia in mouse diastema. Apoptosis accumulation – black.

Figure 3

A scheme presenting the ‘differentiation’ and ‘concrescence’ theories on the origin of a multicusped tooth in mammals. Tooth primordia of mammalian ancestors are represented by rings. (a, b) Green rings – tooth primordia involved in the evolution of a multicusped tooth in mammals. Dashed-line rings – tooth primordia that are not involved in the evolution of a multicusped mammalian tooth. (c, d) A hypothetical model of the regulation of tooth concrescence. (c) The individual tooth buds (rings) are induced and maintained by growth activators, while growth inhibitors regulate origin of inter-bud domains (zones where further budding is inhibited – inhibition zones). The final distance between bud primordia results from the interaction between the activating and inhibiting signals that co-express in the originating buds. (d) Tooth concrescence results from the deficient separation of tooth primordia due to deficient formation of the gaps (inhibition zones). The further joined development can be compared to that seen in Siamese twins (e).

Figure 4

Stages of tooth crown development in mammals. The shape of the dental epithelium is shown in schemes (a), frontal histological sections (b) and computer aided 3D reconstructions (c – left column) based on drawings of the epithelium on sections (c – right column). Dental epithelium – orange, dental mesenchyme – green. The first morphological sign of tooth formation is a thickening of the oral epithelium, which later forms a dental lamina. The dental lamina gives rise to epithelial tooth buds with surrounding condensed mesenchyme. Then the cervical loop (arrowhead) starts to grow at the cap stage, while a dental papilla fills in a cap activity. At the early bell stage, cusp formation is initiated, and the cervical loop progressively elongates. For later development see Lesot et al. in this supplement. (d) 3D reconstruction of an epithelial structure (ep) is based on the epithelium contours on sections. It makes visible the epithelium surface adjacent to the mesenchyme (mes). (e) Changes in the shape of dental epithelium along the antero-posterior jaw axes. Green – 3D reconstruction shows the mesenchymal aspect of the dental and adjacent oral epithelium in the cheek region of the mandible in a mouse embryo at embryonic day 14.5. M1 – the cap of the first lower molar. Bottom, frontal histological sections show the shape of the reconstructed dental epithelium at corresponding locations along the antero-posterior sequence: lamina – bud – cap – bud – lamina. White arrows point to the dental mound.

Figure 5

Distinction of early stages of odontogenesis on frontal sections in mouse embryos. The epithelial thickening differs from the adjacent oral epithelium by comprising a higher stratum of basal cells with prevalent orientation of the long axes of their nuclei perpendicular to the basement membrane. At the oral surface, one to two layers of flat cells are present, as elsewhere. The dental lamina is formed by folding of a thick stratum of columnar cells, and by the accumulation of smaller internal cells. Few layers of flat cells cover the oral surface. The angle included between the slope of the lamina and oral epithelium is larger than or equal to 90°. The tooth bud has a similar cell arrangement. At least one of the medial and lateral sides of the bud is vaulted, so that the angle between the bud and oral epithelium is smaller than 90°.

Figure 6

Three segments of the dentition in the mouse. I – incisor; M1, M2, M3 – the first, second and third molar, respectively. (a) A scheme of the skull of an adult mouse. The gnawing incisor and the 3 molars are separated by a toothless gap called a diastema in place of missing incisors, canine and premolars. (b) Scheme of the upper jaw dentition in an adult mouse. (c) The model of mouse odontogenesis provides an opportunity to investigate development of three different segments of the dentition, where different tooth primordia occur: a large incisor, prospective toothless diastema, and the three successively developing molars. The incisor is covered by enamel only on the anterior (labial) face (crown-like analogue), while cement covers the other side (root-like analogue). Due to the asymmetrical distribution of enamel, tooth abrasion is asymmetrical resulting in a sharp incisor margin. (d) Two parts of the mouse embryonic diastema according to the stage of odontogenesis arrest. Odontogenesis is blocked most strongly and/or early in the anterior part of the lower diastema (green), where it arrests at the epithelial thickening stage (dashed line). In the anterior part of the upper diastema (yellow), odontogenesis progresses to the stage of the dental lamina or small buds (D1–D5) before these structures are completely eliminated. The most advanced stage of tooth development is achieved in the posterior part of the upper or lower diastema (brown). Two large rudimentary buds develop there in each jaw (R1, R2 or MS, R2), before their development is stopped. The R1, R2 in the maxilla and MS in the mandible are transformed into epithelial ridges that are presumably later incorporated into the expanding enamel organ of the first molar (for a detailed review see the ref.14). The R2 bud in the mandible is incorporated in the M1 cap.

Figure 7

Determination of embryonic body weight and its correlation with tooth age in mouse embryos. (a) The whole mouse uterus is put in a Petri dish (Pd1) on a cold plate. Immediately after each individual embryo is dissected from the uterus, it is picked up at the waist, and its inferior part is gently touched several times on the bottom of the dry Petri dish (Pd2), to remove any excess of amniotic fluid on the body surface. (For the embryos after ED 14.4, a filter paper is put on the bottom of Pd2). Then the embryo is put on the Petri dish (Pd3) located on a balance set to zero. This weighing procedure takes just a few seconds. (b) The graph shows the distribution of the wet body weight in prenatal ICR mice during embryonic day (ED) 11.5–19.5. Each ring corresponds to one animal. (c) The correlation between body weight in milligrams (mg) and developmental stage (tooth age) of the first lower molar (M1) at ED 14.5. Contour drawings of dental epithelium on frontal sections document the central part of the M1 in different weight classes. The embryos have been randomly selected from each corresponding weight class. Note the step-by-step progress in tooth development according to increasing body weight. The margin of the enamel knot structure is indicated by a solid or dashed line. Black dots – apoptosis (modified according to the ref.49).

Figure 8

Different structures are called a ‘dental lamina’ in the literature. This term is used to refer to the structures indicated by an arrow: (a) early stage of tooth development; (b) the stalk of an enamel organ during later development; (c, d) the whole formation of the dental epithelium which is submerged into the mesenchyme and which bears developing tooth primordia in e.g. reptiles (c) or humans (d); (e) the proposed role of reorientation of mitotic spindles during origin of dental lamina: When the long axes of mitotic spindles are oriented in parallel to the basement membrane, the epithelium extends its surface. The long axes of mitotic spindles oriented perpendicular to the basement membrane result in an increase of epithelium thickness and dental lamina formation. (modified according to the ref.126) (f–i). Frontal sections of the maxilla in mouse embryos at ED 12.5. A small primordium in the anterior part of the upper diastema (f), the large posterior diastemal rudiment R1 at the dental lamina stage (h), R2 at an early bud stage (g), dental lamina formation in the molar area (i). The black arrow indicates the bulging of dental epithelium by the mejenchyme expanding medially to the forming dental anlage. Bar = 100 μm.

Figure 9

Developing dentition and oral vestibule in humans and their comparison with developing teeth in fish. (a) Embryological textbooks present two parallel U-shaped ridges in human embryos: DL – dental lamina (giving rise to the primary dentition) and VL – vestibular lamina or labio-gingival band (where the oral vestibule will form). (b-c) Summarization of data by 3D reconstructions showing that no continuous vestibular lamina exists. Instead, a set of discontinuous epithelial structures (ridges and bulges) transiently occurs externally to the dental epithelium. Red – dental epithelium. Yellow or blue – vestibular epithelium. c, m1, m2 – the deciduous canine, first and second molar, respectively. AC – the accessory cap-shaped structure (modified according to the refs25,44). (d) The schematic pattern of tooth rows (‘Zahnreihen’) in fishes. The empty rings and black spots indicate the older and younger teeth respectively, new teeth are formed at the posterior end of each Zahnreihen (modified according to the ref.161). (e) Dental and vestibular epithelia in an 8-week-old human embryonic maxilla in a 3D reconstruction viewed from the mesenchymal aspect. Note the reiterative fusions (white asterisks) between the dental epithelium and particular ridges of the vestibular epithelium. c, m1 – the deciduous canine and the first molar, respectively.

Figure 10

The tooth buds as swellings on a mound of dental epithelium. 3D reconstructions show. Mesenchyme-facing aspect of the dental and adjacent oral epithelium in the upper jaw in a 44–46 day old human embryo (a), and 13.5 day-old mouse embryo (b). The i1, i2, c and m1 – the swellings corresponding to the bud of the human deciduous upper first incisor, second incisor, canine and first molar, respectively. R1, R2 – the swellings corresponding to the rudimentary large buds in the posterior part of upper diastema. D – remnant of small diastemal buds in the anterior part of the upper diastema (compare to Fig. 16d). M1 – bud of the mouse upper first molar. (c, d) Frontal histological sections show the enamel knot (arrowhead) at the tip of the large diastemal bud R2 in mouse maxilla (c) and mandible (d) at ED13.5. A circle indicates the epithelium budding suggesting development of a successive tooth generation. Bar = 50 μm.

Figure 11

Incisor region of human embryos in 3D reconstructions. (a) A parallel regionalization of the dental and vestibular epithelium in the upper incisor region. On a mound of dental epithelium, there are distinct swellings corresponding to the first (i1) and second (i2) deciduous incisor at the bud stage. Externally to each swelling, a bulge of vestibular epithelium protrudes against the mesenchyme. (b) Differentiation of the incisor primordia and labial vestibular ridge in the lower incisor region. The bulges (1, 2) represent a common origin of the dental and vestibular epithelium. From the lingual and labial part of each bulge, the deciduous incisor and vestibular epithelium differentiate, respectively. The vestibular epithelium finally forms a labial vestibular ridge (LVR). c, m1 – the deciduous canine and the first molar. The middle line is dot-dashed.

Figure 12

The upper mouse incisor – phylogenetic and ontogenetic aspects. (a, b) Schemes show integration of several epithelial structures (black spots) during formation of the early bud of the functional mouse incisor. The morphology of these structures on sections is similar to the tooth placodes in reptiles, as described by Westergaard. (a) Dental epithelium at ED12.0. (b) Folding of the dental epithelial sheet forms a complex incisor primordium by an integrated (conjoint) development of original placodes at ED12.5. The arrows show the growth direction of the adjacent non-dental mesenchyme. Vertical or horizontal dashes indicate the layer of basal or superficial cells, respectively. (c) The placodes (white spots) give rise commonly to the incisor early bud in a 3D reconstruction at ED 13.5. (d) According to the generally accepted view, the number of incisors was progressively reduced during rodent evolution; the single incisor in rodents should correspond to the second incisor of placental mammals with unreduced incisor number (left scheme). Therefore, it is taken for granted that only one incisor develops in mice (middle scheme). However, embryological data document integrated development (concrescence) of 5–6 placodes that commonly give rise to the early bud of the upper incisor in mouse embryos; the most lateral placode takes its origin in the maxillary facial process (right scheme).,

Figure 13

Dental epithelium of the cheek region of mouse embryos. Dental epithelium in projection drawings of 3D reconstructions (green) and of frontal sections (orange) localized according to dashed lines in the appropriate 3D reconstruction. Note the sequential progress of growth of dental epithelium in a posterior direction. Consecutive appearance of two large rudimentary diastemal buds R1/MS and R2 is followed by the first molar (M1 – yellow field). The large rudimentary buds represent the most conspicuous structures in the cheek region before embryonic day (ED)14.0. Epithelial apoptosis – black dots. Black arrows suggest the final location of the rudimentary buds. (a) While the posteriorly situated M1 is still at the lamina stage, diastemal R1 and R2 buds reach maximum development at ED12.5 and ED 13.5 respectively, which is followed by growth arrest and apoptosis accumulation in the dental epithelium. Then the former buds become transformed into epithelial ridges fusing posteriorly with the first molar. (b) The anterior rudiment (MS) is the most conspicuous structure until ED12.5. Then it becomes affected by apoptosis and is transformed into the epithelial ridge. A large rudimentary bud R2 appears as a swelling in 3D at ED13.5. Apoptosis only transiently affects the tip of the R2, which becomes incorporated into the anterior end of the M1 cap. The growth of the upper and lower first molar is delayed compared with the diastemal rudiments. As the M1 cap develops, the third episodic concentration of cell death appears in its enamel knot (arrowhead) (modified according to the ref.84).

Figure 14

Three episodes of apoptosis in the dental epithelium in the mouse cheek region. Programmed cell death by apoptosis plays a part during formation of the mouse upper diastema and in the development of the cap of the first molar. The dashed line interconnects the most prominent tooth primordium (on a scheme of a jaw arch at each ED12.5, 13.5 and 14.5 with its projection drawing on a frontal section). The dashed arrow shows the final location of the structure in the 3D reconstruction (the dental epithelium in the cheek region of mouse embryos at ED 15.0). The disappearing or emerging tooth primordia are delineated by dotted or dashed lines, respectively. The rudimentary tooth primordia develop and then regress sequentially along the antero-posterior (bottom-up) direction. The third concentration of apoptosis appears in the enamel knot (arrowhead). (modified according to the ref.74).

Figure 15

A supernumerary cheek tooth in a mouse mandible. (a) Unilateral occurrence of a small supernumerary tooth (S) in the mandible of a Tabby heterozygous mouse. The presence of S is accompanied by a reduction of the anterior part of the adjacent first molar (M₁), when compared to the normal situation on the contra-lateral side of the same animal. (b) It has been hypothesized that the S is homologous to a premolar (P) lost during mouse evolution, and can be considered as an atavistic tooth.^{8, 65,148,} Occurrence of atavistic teeth at the place of suppressed teeth of remote ancestors is also known in other species of mammals (e.g. compare to Fig. 1n)., (c) 3D reconstructions of the dental and adjacent oral epithelium in the cheek region of the mandible of mutant Tabby (Ta), Spry2^-/-, Spry4^-/-, and wild type (WT) mouse fetuses at ED15.5. The mutant and the corresponding WT specimen have been coupled to exhibit not only a similar age (in ED) but also a similar body weight (in mg). Such detailed stage matching shows that the total length of dental epithelium is similar in the mutant and corresponding WT mouse. The graphic chimera (middle column) demonstrates the supernumerary tooth develops at the place corresponding to the anterior part of the M1 cap in WT embryo, where the large diastemal rudiment (R2) has been incorporated at an earlier stage (arrowhead).,,

Figure 16

Cap stage of tooth development. (a) Human embryo (prenatal week 8) in 3D reconstruction. The dashed-dotted line indicates the position of the section shown in the insert; c and m1, cap-staged upper deciduous canine and first molar; ac, accessory budding of the vestibular epithelium. (b) The cap of the upper or lower first molar cap (M or M₁ respectively) of the mouse at ED14.5. The arrow indicates the former posterior large diastemal rudiment in maxilla (R2) or mandible (R₂). (c) Lower first molar in a mouse embryo at ED14.5 on a frontal histological section. Histo-differentiation results in the appearance of the inner dental epithelium (IDE), outer dental epithelium (ODE), stellate reticulum (SR), dental sac (DS) and dental papilla (P). The arrow points to the enamel knot, the arrowheads to the enamel grooves; the double arrow indicates the stalk of the enamel organ. (d) The 3D reconstruction shows the lower molar germs in a mouse at ED 17.5. The first (M1), second (M2) and third (M3) molar are at the bell, cap and bud stages, respectively. Note the enamel organs of the molars are not separated by a low oral epithelium, but are attached on and interconnected by the mound of dental epithelium (arrowhead).

Figure 17

The tooth primordia in the prospective incisor region of wild type mice. Two generations of the Shh expression domains (a). Their sequential development is documented on the hybridized lower (b–d) and upper (h–j) jaws and corresponding 3D reconstructions (e–g and k–m). Two regions of the Shh expression located antero-posteriorly develop sequentially in each quadrant of the upper and lower jaws in mice. These domains reflect the sequential development of two generations of tooth primordia. The earlier-appearing anterior and more superficially located expression (green arrow) corresponds to the rudimentary prelacteal tooth development. The later-appearing, deeper and more posterior Shh expression domain (yellow arrow) is located in the central part of the germ of the functional incisor.,

Figure 18

The tooth primordia in a cheek region of the mouse embryonic mandible. Frontal histological sections show the three sequentially signalling structures: (a) the first diastemal rudiment (MS) at ED12.5; (b) the second diastemal rudiment (R2) at ED13.5; and (c) the first molar (M1) at ED14.5. Arrowheads point to the enamel knot of R2 and M1. (d–f) The three Shh expression domains are sequentially patterned along the antero-posterior axes in the cheek region of the mandible. The mandibles at ED12.7 (d), 13.3 (e) and 14.3 (f) have been hybridized with Shh anti-sense probe (left) and then sectioned frontally (right) to show the localization of the Shh expression domain in the dental epithelium using 3D reconstruction technique (middle). The Shh signalling centres (blue in MS, red in R2 and yellow in M1) correspond to the respective morphological structures (a, b, c). (g, h) A scheme compares the classical (g) and new (h) interpretations of the signalling structures during ED12.5–14.5. (g) According to the classical view, the characteristic structures in the cheek region of the mandible at respective days 12, 13 and 14 were generally assumed to correspond to various developmental stages of the M1 development (frame on the top). In these structures, similar signalling as well as concentration of apoptosis (black dots) have been found (compare to Fig. 14). (h) The 3D reconstructions have shown that the characteristic structures (a–c), in fact, are localized in different segments of the dental epithelium that appear sequentially in the posterior direction (arrow). The R2 becomes incorporated anteriorly into the M1 cap (grey area), as shown experimentally. A residuum of the MS might be implicated during the growth of the enamel organ at later stages (dashed line). The presence of the specific Shh expression domains along the antero-posterior jaw axes can be related to the transient manifestation of the tooth pattern in remote ancestors, where the premolars were still present (modified according to the refs.22,23).

Figure 19

A scheme summarizing the correlation between the signalling centres and developing teeth in a mandible of WT mice. Insert: mouse mandible at ED12.5 – whole mount Shh in situ hybridization. Rectangles – functional teeth; round spots – Shh expression domains. The yellow symbols indicate functional teeth; green, blue and red round symbols indicate the rudiments. Classical view: a single Shh expression domain is generally presented in each incisor and cheek area of the mandible during early development and interpreted as a signalling centre of the developing functional incisor or M1. New view: Recent studies have documented sequential appearance of several temporo-spatial distinct Shh expression domains along the antero-posterior jaw axes. The earlier-appearing domains correspond to rudimentary tooth primordia in both incisor and cheek regions. In the incisor region, Shh is expressed in two temporo-spatially distinct areas. The earlier, anterior and superficial Shh expression (green) reflects the development of rudimentary pre-lacteal teeth (pt). The later, posterior and deeper Shh expression (yellow domain) is located in the germ of the functional incisor (I). A similar situation is also found in the upper incisor region (compare to Fig. 17)., In the cheek region of mandible, three Shh signalling centres are sequentially patterned during early stages of development: the blue, red and yellow Shh domains indicate the signalling centre of the MS rudiment, R2 rudiment and M1, respectively (compare to Fig. 18). The adult M1 develops with the participation of the R2 diastemal rudiment (red rectangle); a minor contribution by the residuum of the rudiment MS (blue rectangle) is not excluded. The variable participation of the rudiments during M1 development has been proposed to result in variability of the anterior part of the adult M1. In the molar region, only the M1 is considered. That is why the region of the second and third molar is not presented in the scheme. These data supporting the existence of rudimentary structures are very helpful in searching for homologies of structures.