Connective Tissue Research, 43: 120–128, 2002Copyright c© 2002 Taylor and Francis0300-8207/02 $12.00+ .00DOI: 10.1080/03008200290000745

Development of the Vestigial Tooth Primordia as Partof Mouse Odontogenesis

R. Peterkova,1 M. Peterka,1 L. Viriot, 2 and H. Lesot31Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vıdenska 1083,142 20 Prague 4, Czech Republic2CNRS UMR 6046–Laboratoire de Geobiologie, Biochronologie et Paleontologie Humaine, Faculte SFA,Universite de Poitiers, 40, avenue du Recteur Pineau, 86022 Poitiers Cedex, France3INSERM U424–Faculte de Medecine, 11, rue Humann, 67085 Strasbourg Cedex, France

The mouse functional dentition comprises one incisor separatedfrom three molars by a toothless diastema in each dental quadrant.Between the incisor and molars, the embryonic tooth pattern alsoincludes vestigial dental primordia, which undergo regression in-volving apoptosis in their epithelium. Apoptosis appears to play animportant role in achieving the specific tooth pattern in the mouse.We documented similarities in the folding mechanism allowing theformation of the dental lamina in mice as well as in reptiles. Whilefurther budding on this dental lamina gives rise to many individualsimple tooth primordia in crocodiles and lizards, budding morpho-genesis of several simple tooth primordia appears to be integratedin the mouse, giving rise to enamel organs of a complex nature. Thedifferentiation of a mammalian tooth germ during both ontogenyand phylogeny might thus include the concrescence (connation) ofmore primordia, putatively corresponding to simple teeth in mam-malian ancestors.

Keywords Dentition, Epithelium, Budding/Branching, Mouse,Evolution

INTRODUCTIONThe development of the mouse incisor and molar are clas-

sical models in odontogenesis studies. Data have accumulatedenhancing understanding of the control mechanisms of tooth de-velopment and helping to elucidate general principles of organo-genesis [1–6].

Incisor and molar teeth, separated by a toothless diastema,compose the reduced functional dentition of the mouse (com-pare Figure 1, A and B). However, vestigial (rudimentary) toothprimordia are transiently present in the mouse antemolar region

Received 19 June 2001; accepted 2 January 2002.Address correspondence to Renata Peterkov´a, Institute of Ex-

perimental Medicine, Academy of Sciences of the Czech Republic,Vıdenska 1083, 142 20 Prague 4, Czech Republic. E-mail: repete@biomed.cas.cz

during early stages [7–12] (Figure 1, C and D). The largest vesti-gial buds are the most conspicuous structures distally to incisorsin the upper and lower jaw until ED 13.5 [9, 12] (Figure 2).The vestigial tooth primordia do not complete their individualdevelopment and suggest an attempt to repeat the ancestral sit-uation where many much more teeth existed [13]. Taking intoaccount the vestigial primordia allows for a better assessmentof the conservation of an ancestral pattern in mouse dentition(Figure 1, C and D) and for a reevaluation of the interpretationsof the molecular signaling that regulates odontogenesis [13].

Existence of vestigial tooth primordia has also been reportedin other mammals and has been interpreted as an intermedi-ary stage in the loss of functional teeth during evolution [14,15]. Modification of an older genetic control, as well as hete-rochronic changes (i.e., changes in timing) in gene expressionduring evolution, can lead to a modification or extinction of an-cestral structures, or to the initiation of new structures allowingfunctional adaptation [16]. Phylogenetic changes in the dentitionassociated with its functional adaptation raise questions aboutthe evolution of developmental decisions and the mechanismsof their realization [17–19]. In this article we seek (a) to reviewdata on the occurrence of the vestigial structures during mouseodontogenesis, (b) to complete them by new observations ondental lamina formation in the mouse and (c) to discuss the pu-tative implications of the vestigial structures from ontogeneticand phylogenetic aspects.

STAGES OF TOOTH DEVELOPMENTThe teeth in mice pass classical developmental stages named

according to the shape of the dental epithelium on frontal sec-tions (Figure 3). However, three-dimensional (3D) reconstruc-tions revealed that the terms “bud,” “cap,” and “bell” do notexpress the real spatial shape of the dental epithelium at therespective stages (Figure 3), and document the existence of a

120

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009

VESTIGIAL PRIMORDIA AND ODONTOGENESIS 121

Figure 1. Tooth pattern in one jaw quadrant: (A) presumed ancestral dentalformula in mammals [50]; (B) adult mouse; (C, D) mouse embryonic upperand lower jaw, respectively. The regressing antemolar structures affected byapoptosis are in black. D1−5, small vestigial tooth primordia, which finallydisappear in the mesial part of the upper diastema. Dashed line, the dentalepithelial thickening transiently present in the mesial part of the lower diastema.R1, R2, and MS, large vestigial tooth buds in the distal part of the embryonicdiastema. R2, the wide bud integrated to the mesial end of the first lower molar.The dashed arrow shows the generally assumed correspondence of the upperincisor in mouse to the middle incisor in other mammals. The full arrows indicatethe integration of six placodes during the upper incisor formation (the most distalprimordium is later affected by apoptosis). Dashed rectangles suggest that atleast three primordia might connate during the origin of the mouse lower incisor(Peterkova, unpublished data).

mesio-distal gradient during development of the dental epithe-lium in the cheek region [9, 12, 20, 21].

Folding of Dental Epithelium as a Mechanism of DentalLamina Formation

The first morphological sign of odontogenesis is representedby epithelial thickening and a condensation of the adjacent mes-enchyme, which compose the odontogenic zone [22]— “la zonedentaire pr´esomptive” [23]. Although epithelial cell prolifera-tion plays an important role during the later stages of mouseodontogenesis [24], the origin of a dental lamina or bud doesnot involve a local increase in epithelial cell proliferation, butrather a specific orientation of mitotic spindles perpendicular tothe basement membrane [25]. The dental lamina/bud has been

Figure 2. The most conspicuous dental structures are only shown in a schemeof the upper and lower jaw quadrants of ED 12.5 and 13.5 mouse embryos.These patterns are similar to the expression patterns of several genes that havebeen documented in the dental epithelium (e.g., in refs. 2 and 35.) Oval, incisor;R1, R2, MS, and R2, large vestigial tooth buds.

suggested to result from the infolding of dental epithelium intothe mesenchyme. In the meantime, surface epithelial cells slideto fill in the forming groove [8, 23] (see Figures 5–7). We as-sume that the surface (peridermal) cells (see Figure 5C) maybe involved in the origin of the stellate reticulum of the toothenamel organ (see Figures 5–7).

The existence of an infolding of the sheet of dental epithe-lium during odontogenesis has been reported in sharks, amphib-ians and reptiles [26, 27]. During the infolding, a crease of oralmucous membrane actively grows towards the oral cavity tooverlap the prospective dental epithelium from the lingual side(Figure 4). Bolk [26] introduced the termoperculumfor thiscrease of oral mucous membrane and presented a tentative modelof operculum development, similar during both phylogeny andontogeny in reptiles (Figure 4).

The infolding of the dental epithelium in the mouse does notseem to result from an active sinking of the dental epitheliuminto the mesenchyme, but rather from a swelling of the adjacentnon-dental mesenchyme towards the oral cavity [8, 22]. In thisrespect, the crease of the lingually adjacent oral mucous mem-brane in the mouse [22] (see Figures 6–8) can be compared tothe operculum in reptiles (Figure 4). Such a mechanism of pas-sive “invagination” of the dental epithelium due to the activeexpansion of the nondental mesenchyme in the oral direction isin agreement with previous observations [29].

VESTIGIAL TOOTH PRIMORDIA IN MOUSE

Incisor RegionThe mouse incisor is a complex structure of multiple origins

(Figure 1, C and D). In the upper incisor region, the epitheliumthickens in a large area. It gives rise to six structures [8], withsimilar morphology on frontal sections to placodes of simpleteeth documented in lizards [28]. This area is folded into themesenchyme to form a composite primordium (Figure 5, A andB) known as the incisor dental lamina. The integrated develop-ment of several connate tooth primordia (Figure 1C) can also bedetected during later stages [8] (Figure 5, D and F). Participa-tion of the original primordia seems to differ along the antero-posterior axis of the developing incisor (Figure 5, D and F).This has been related to the difference between the incisor labial(enamel covered) and lingual (enamel free) side [8], originatingpredominantly from the respective antero-medial and postero-lateral parts of the complex incisor bud (compare to Figure 5D).

A preliminary study suggested that at least three connate pri-mordia might be implicated in the development of the mouselower incisor during early stages (Peterkov´a, unpublished data)(Figure 1D). Later on, three components participate in theformation of the cervical loop of the lower incisor enamelorgan [30].

At later stages, an abortive vestigial incisor forms at the bot-tom of the bell-shaped enamel organ of the mouse functionalincisor [14] (Figure 5E).

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009

122 R. PETERKOVA ET AL.

Figure 3. Stages of the M1 formation in ICR mouse embryo at ED 12.5 (A–C), ED 13.5 (D, E), ED 14.0 (F, G), 14.5 (H, I), and 16.0 (J, K). The M1 is documentedin projection drawing of a frontal section at the mid-level of the tooth (B, D, F, H, J) and respective 3D computer-aided reconstruction of the dental epitheliumsituated in the cheek region (C, E, G, I, K). (A) The epithelial thickening (not shown in 3-D). (B) The dental lamina exhibits on its lingual slope (C) accessorybuddings (white spots). Bud (D) and early cap (F) stages correspond to single (E) or double (G) long cylindrical shapes in space, respectively. At the cap (H)and bell (J) stages, the dental epithelium in 3D reconstructions is reminiscent of a “canoe” (I) or a “boat” (K), respectively. MS, the regressing antemolar mesialsegment, R2, a wide bud at the mesial end of M1; pEK–level of the prospective primary enamel knot.

Diastema RegionIn rodents, the diastema is a characteristic toothless gap be-

tween the incisor and cheek teeth (Figure 1B). In the uppermouse embryonic diastema (Figure 1C), five small “D” primor-dia are transiently present in its mesial part located in the lip re-gion [10, 11] and two large “R” buds originate in its distal part lo-cated in the cheek region [9]. In the lower diastema (Figure 1D),

Figure 4. (A–D) Stages of dental lamina formation in reptiles. Dentalepithelium—dotted area (A); blue and orange, the basal and superficial epithelialcell layer, respectively. The operculum (black arrow) forms at the lingual aspectof the dental lamina. The teeth are assumed to develop on the buccal aspect of thedental lamina (D) arising as a duplicature of the oral epithelial lining. Modifiedaccording to Bolk [26] and Edmund [27].

there are no small “D” primordia—only a thin lamina transientlydevelops and disappears at ED 12.5 [13]. More distally (in thecheek region), the lower dental epithelium is more abundant. Itsmesial part gives rise to two vestigial structures: a mesial seg-ment (MS) and the wide R2 bud [12] (Figure 3E). In contrastto the R2 bud, whose epithelium becomes incorporated into themesial end of the first lower molar (M1) cap (Figure 1D), theregressing R1, R2, and MS (Figure 1, C and D) are reshaped intoepithelial ridges [13]. The ridges are first situated mesially fromthe first molar and become incorporated into its enamel organ,when it expands mesially at later stages (see Figure 10C). Thelower number of vestigial dental primordia in the mandible com-pared to the maxilla (Figure 1, C and D) is in agreement withthe always more advanced reduction of mandibular ante-molarteeth in rodent functional dentition [15].

Similar to the upper incisor (Figure 5, A and B), the largevestigial tooth buds (“R” and MS) originate from the folding ofa sheet of dental epithelium toward the mesenchyme. The for-mation of the operculum is involved in this process (Figures 6B,7B, and 7C). A distinct placode/bud could be detected on thelingual slope of the infolding of the dental epithelium (Figures6B, 7B, and 7C). The MS and “R” primordia might correspondto a remnant of premolars lost during muroid evolution [13].

In contrast, the “D” primordia in the upper jaw maximallyreach the shape of a small placode/bud arising from the epitheliallayer, which remains in contact with the oral cavity. Formationof the operculum is not completed there (Figures 6A, 7A, and 8).In this respect, the “D” rudiments are similar to the first formedabortive teeth in crocodiles and lizards. Such “placoid teeth” inthe reptiles are laid down on the surface of the jaw and become

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009

VESTIGIAL PRIMORDIA AND ODONTOGENESIS 123

Figure 5. Upper mouse incisor region. (A, B) Simplified schemes show inte-gration of several incisor placodes (black spots) during dental lamina formation.(A) Dental epithelium at ED 12.0. (B) Folding of the dental epithelial sheetforms a complex dental lamina at ED 12.5. The arrows show the growth direc-tion of the swelling of the adjacent nondental mesenchyme. Vertical or horizontaldashes indicate the layer of basal or peridermal cells, respectively. (C) Electronmicroscopic view of the incisor dental epithelium at ED 12.0 documents the dif-ference in morphology and arrangement between the basal (Ba) and peridermal(Pe) cells. Bar, 5µm. (D) Connate buddings (white spots) compose the incisorbud in a 3D reconstruction at ED 13.5. (E) A semithin frontal section shows(white arrow) the vestigial incisor [14] at the anterior slope of the functionalincisor at ED 16.0. Bar, 100µm. (F) A semithin frontal section shows two con-nate caps (black arrows) at ED 15.0. The two black spots label the distal endsof two more distant components, after their participation in the anterior part ofthe incisor. Bar, 100µm.

aborted, similar to the vestigial incisor in mouse (Figure 5E);the functional dentition is produced by a dental lamina, infoldedduring operculum formation [26, 27] (Figure 4). A failure in theoperculum formation (Figures 6A and 7A) might play a role inthe abortion of the “D” primordia in mouse.

Molar RegionSimilar to the incisor, the molar dental lamina in mouse also

arises by a folding of the dental epithelial sheet into the mes-enchyme (Figure 7D). The molar epithelial primordium is com-

Figure 6. Frontal histological sections of the maxillary diastema in a mouseembryo at ED 12.5. (A) The initial stage of formation of a small “D” primordium.(B) The R2 antemolar primordium. The double arrowhead points to the diastemaldental lamina (A) or to its distal extension corresponding to the lingual half ofthe R2 primordium (B). The single arrowhead indicates the vestibular laminalining the lip furrow (A) and its distal extension involved in the buccal half ofthe R2 (B). In contrast to the “D” primordium, the operculum formation (blackarrow), associated with a folding of the epithelial sheet and the influx of surfacecells in the rising groove, are clearly apparent in place of the R2 primordium.Bar, 100µm.

plex and integrates more buddings [13]. This complexity furtherincreases during the bud–bell stages. The bell-shaped enamelorgan has been suggested to result from the rapidly ensuingbudding (branching) morphogenesis. During this process, thedevelopment of more buds and bud generations (putatively cor-responding to the primordia of simple ancestral teeth) can beintegrated to ultimately give rise to the bell-shaped enamel or-gan [13]—see also further discussion.

COMMON ORIGIN OF TEETH, PALATAL RIDGES, ANDTHE ORAL VESTIBULE

The dental lamina, vestibular lamina and palatal ridges aredevelopmentally related in the mouse [22]. Initially, the epithe-lial thickening (odontogenic epithelial zone, OEZ) covers thewhole oral surface of the maxilla in ED 11.5 mouse embryos(Figure 8). In ED 12.0–13.0 mouse embryos, a segmentation ofthe OEZ along the bucco-lingual direction breaks it up into thepalatal ridges region, dental lamina, and vestibular (lip furrow)lamina [22] (Figures 7A and 8). While the dental and lip furrow

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009

124 R. PETERKOVA ET AL.

Figure 7. The epithelium on the oral surface of maxilla on projection draw-ings of frontal histological sections (bar, 100µm) with a detail of the dentalepithelium on semi-thin sections (bar, 50µm) in ED 12.5 mouse embryos. (A–D) The respective level of the primordia D3, R1, R2, and the first upper molar(M1). The oral vestibule originates in the lip area from the vestibular lamina(VL) lining the lip furrow (empty triangle). The VL (single arrowhead) anddental lamina (double arrowhead) are separated in the mesial diastema (A), butfuse distally (B) to form the dental primordia located in the cheek region (C, D).There, the oral vestibule arises from the cheek furrow (black triangle). Opercu-lum formation (black arrow) is associated with the influx of surface cells in therising folding of the epithelial sheet. The operculum is absent or only poorlyformed (dashed arrow) in the region of “D” primordia (A) and not yet completedin the molar area (D).

laminae are clearly separated (segregated) in the lip region, bothlaminae jointly give rise to the large dental primordia in thecheek region of the maxilla [22] (Figure 7, B, C, and D).

A segmentation of the rugal and dental epithelium along themesio-distal axis of maxilla gives rise to individual palatal ridgeanlagen [31] and teeth/cusp primordia [13], which occur peri-odically along the mesio-distal jaw axis. There is a correlationbetween the mesio-distal periodicity of the palatal ridges andteeth/cusps in some mammals [22]. Such a correlation is bestexpressed in armadillo [32], but can also be traced in, for exam-ple, macaco, dog, or cat.

Palatal ridges and oral vestibule appear to be related to thedentition having a common precursor both in ontogeny (odon-togenic zone) and phylogeny (a maxillary area bearing teethin non-mammalian vertebrates) [22]. This can be supported bythe synchronous occurrence of apoptosis in the epithelium of

Figure 8. The epithelium on the oral surface of maxilla in the mouse di-astemal region at ED 11.5–13.0 on projection drawings of frontal sections lo-cated mesially from the mouth corner. The odontogenic epithelial zone (OEZ)and its derivatives palatal ruga, diastemal dental lamina (DL) and vestibularlamina (VL) lining the lip furrow are in gray. The OEZ is segmented by mes-enchymal bulging (empty arrow) or swelling towards oral cavity. The latterprocess is associated to the operculum formation (black arrow), which remainsuncompleted. A star indicates the margin of the palatal shelf. Bar, 100µm.

the palatal ridges, oral vestibule, and dentition in the mouseembryonic maxilla [33].Msx1, Msx2, andBmp4are expressedsynchronously in the dental and vestibular regions, whileBmp2has been detected only in the vestibular mesenchyme in mouseembryos during early stages of development [34]. In both thepalatal ridges and dental epithelium, there has been documentedthe expression ofShhat day 12–13 [35] andBmp4at day 14(Peterkova, unpublished data). All these genes are involved inthe regulation of odontogenesis [6].

APOPTOSISDevelopmentally programmed cell death (apoptosis) is

involved in the elimination of unwanted cells [36].Glucksmann [37] distinguished (a) “phylogenetic” cell deathparticipating in the loss of vestigial structures, (b) “morpho-genetic” cell death involved in the specific shaping of structures,and (c) “histogenetic” cell death associated with cell differen-tiation. Apoptosis appears to play all three roles during mouseodontogenesis.

Dying epithelial cells accumulate during regression of themost lateral part of the composite upper incisor bud [8] and

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009

VESTIGIAL PRIMORDIA AND ODONTOGENESIS 125

in the “D” primordia [33]. Similar to the segmental growth ofthe dental epithelium in the cheek region, apoptosis appearsin three distinct waves [13] affecting sequentially (a) the largevestigial antemolar primordia (R1, MS) at ED 12.5 [9, 12],(b) the wide antemolar buds (R2 and R2) at ED 13.5 [9, 12],and (c) the primary enamel knot (pEK) of the first molar at day14 [20, 38], (compare with Figure 3E). In the R2 buds, apopto-sis concentrates in the transient enamel knot-like structure at thebud tip (see Figure 10B). The secondary enamel knot (sEK) [6]is affected by apoptosis at later stages [39]. The apoptosis alsooccur during the disappearance of the stalk of the enamel organ[38] and ameloblast differentiation [40].

BUDDING MORPHOGENESIS OF MOUSE DENTITIONA gradient model has been proposed to explain how differ-

ing concentrations of regulatory molecules (morphogens) canprovide cell positional information in a coordinate system [41].Reaction-diffusion (physicochemical) processes produce a spa-tial variation in activator and inhibitor morphogens (signals)produced from the same source (for review see ref. 3). Thesemechanisms may be involved in the patterning of epithelial ap-pendages [42].

The above principles have been applied in tentative modelsfor feather and lung development [43, 44]. Feathers and lung areperiodically organized epithelial appendages [42] that developfrom simple buds by budding morphogenesis [44]. The feather orlung bud is induced and maintained by activators (e.g., Fgf, Shh),while inhibitors (e.g., Bmp-2, Bmp-4) regulate the interbud do-mains [42–44] (Figure 9A). After experimental downregulationof the inhibition effect, the interbud domains are reduced andthe feather primordia fuse [43, 45] (Figure 9B).

Weiss et al. have assumed that the positional informationincluding the reaction-diffusion mechanisms can also act dur-ing different steps of odontogenesis [3]. We have supported thisassumption [3] by suggesting how activators (Fgf, Shh) andinhibitors (Bmps) may act during budding morphogenesis of

Figure 9. A tentative model of bud patterning. (A) The development of aplacode/bud (ring) is induced and maintained by activators, while inhibitorsregulate the interbud domains. Both the activating and inhibiting signals coex-press in the placode/bud, and their effect at a specific locus is influenced byreaction–diffusion processes [43, 44]. (B) After an experimental downregula-tion of inhibition, the interbud domains are reduced and the bud primordia fuse[43, 45].

dentition in mouse embryos [13]. There, the fundamental mesio-distal periodicity of tooth elements in the dentition is better rep-resented than in the adult mouse (Figure 1), due to the existenceof the vestigial tooth primordia.

Epithelial Thickening StageDifferent molecules have been reported to determine tooth

position and type during the initial stage of mouse odontogen-esis [1–6]. Similar to what occurs in feather and lung budding[43, 44], experimental approaches have demonstrated that Bmpinhibits the effects of Fgf during initial odontogenesis in themouse mandible [4]. There, Bmp2 and Bmp4 antagonize thePax9-inducing activity of Fgf8 in the tooth mesenchyme. Thisinhibitory effect by BMPs results in the absence ofPax9 ex-pression and in the suppression of tooth formation in the lowerdiastema [4], where only an epithelial thickening (Figure 3A) istransiently present mesially (Figure 1D). In the maxilla,BMP-4 [34, 35] as well asPax9 [35] expression is present in theprospective place of “D” vestiges. However, thePax9expressionis weaker than in the incisor region [35]. This downregulationof Pax9has been explained by the stronger expression ofBmp4thanFgf8, and related to the regression of the “D” primordia[35]. In contrast,BMP4does not express in the chick mandible,where the odontogenic epithelial thickening is located. This fail-ure ofBmp-4expression may be associated with early arrest ofodontogenesis in birds [46]. A different regulation of tooth ar-rest may be expected in the mesial part of the mouse upper di-astema (where the “D” vestiges arrest at the placode/bud stage;Figure 7A) and in the distal part of the diastema in both jaws(where the R1, R2 and MS vestiges cease their development atthe well-formed bud stage). A comparison of molecular con-trols in the different regions of the upper and lower diastema inmouse (Figure 1, C and D) and in the chick odontogenic zonecould help to detect similarities/differences in the odontogene-sis arrest in these natural models of hypodontia of evolutionaryorigin.

Tooth Bud StageAt the bud stage, the molar epithelium appears as a long

cylinder (Figure 3E). Such a structure has been assumed to re-sult from fusion and further unique development of more toothplacodes along the mesio-distal jaw axis [13].

During early odontogenesis in the cheek region of the mand-ible, several genes are coexpressed in specific areas. These areashave been indicated as the “early signaling center” and the “pEKsignaling center,” and are assumed to regulate the M1 develop-ment [6]. We have correlated the published data on gene expres-sion with those on the morphology of the dental epithelium andproposed that the colocalized signaling might indicate the po-sition of distinct mesio-distal segments in the dental epithelium[13]. It appears that the colocalized gene expression (signalingcenters) may occur sequentially in time and space—in three dis-tinct structures along the mesio-distal jaw axis: R1, R2, and the

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009

126 R. PETERKOVA ET AL.

pEK of the first molar in the maxilla, and similarly the MS, R2,and the pEK of M1 in the mandible [11–13] (compare to Fig-ure 3E). The reiterative signaling during this period [6] mightthus be associated with the progression of budding of the dentalepithelium along the mesio-distal jaw axis [13].

The R2 bud appears to participate in the formation of themesial part (L1 and B1 cusps) of M1 in mouse [12] (Figure 1D).This bud has been hypothetically related to a vestigial

Figure 10. Examples of budding morphogenesis of the dental epithelium. (A) A long projection (red spot) of the R2 vestigial primordium at ED 13.5 is similarto the dental lamina of successive tooth generation. (B) The enamel knotlike structure (dashed ring) at the top of the wide R2 bud at ED 13.5. (C) The arrowindicates the location where the mesial extension of the M1 enamel organ overwhelms the antemolar epithelial ridge at ED 16.0. (D) The frontal section of M1 atED 15.5 and its contour drawing suggest a branching of the enamel organ related to the differentiation of two concrescent caps joined at the enamel septum [26](dashed line). The pEK (single arrowhead) seems to split off the sEK (double arrowhead) during cap-bell transition. This supports the previous observations aboutcontinuity between the pEK and sEK in this area [39, 53]. (E, F) Frontal sections of the upper first molar during cap–bell transition and bell stage, respectively,document the putative developmental relationship between the budding/branching (arrow) of the lingual cervical loop (E) and the location (spot) ofthe prospectivelingual row of cusps (F). Bars, 50µm.

primordium of the most distal premolar, incorporated duringmesial elongation of the M1 in the course of muroid evolution[47]. The presence of a small “supernumerary” tooth in front ofthe lower cheek teeth in the tabby mice might suggest a defectin incorporation of a vestigial dental primordium (R2) into thefirst molar germ [48], as similarly proposed in the upper jaw [7].This situation in tabby mice would thus approach the ancestralstate in muroids.

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009

VESTIGIAL PRIMORDIA AND ODONTOGENESIS 127

Budding Model of Tooth Enamel Organ DevelopmentWe have proposed a model of enamel organ development

based on the rapidly ensuing budding (branching) of the dentalepithelium [13]. The growth and delimitation of this buddingmay be regulated by activators (e.g., Fgfs, Shh) and inhibitors(e.g., Bmps). A reduction in the inhibition effect might resultin the reduction or even absence of interbud domains and in thefusion of several buds, as similarly described in feather morpho-genesis [43, 45] (compare to Figure 9).

In accordance with general principles of budding morpho-genesis [44], the enamel knot signaling [6] can be addressed tothe tip of a tooth bud, whose further growth has to be stopped.The growth arrest is associated with branching at the bud tipleading to cap formation [13]. The onset of apoptosis in theenamel knot epithelium has been explained by a local predom-inance of inhibiting signals (e.g., BMP), as similarly suggestedfor the regulation of apoptosis in the vestigial antemolar struc-tures [13]. The reiterative signaling by the pEK and sEK in theM1 [6] might be related to tips of buds belonging to differentgenerations of budding, including the M1 bud before ED 14.5and the accessory buds protruding towards papilla from ED 15.5(e.g., Figure 10D). Similarly, a reiterative signaling regulatesgenerations of budding/branching in the lung [49].

CONCLUSIONIn mouse, the existence of small placodes/buds (e.g.,

Figures 5A and 6A), as well as the formation of the operculumduring dental epithelium infolding (Figures 6–8), can be con-sidered as a repetition of features that occur in nonmammalianvertebrates. The present data suggest that a specific sector ofthe operculated dental lamina, giving rise to several generationsof individual simple teeth at several positions along the mesio-distal jaw axis in reptiles (Figure 4), might participate in theformation of one complex tooth primordium in the mouse. Con-sequently, a mouse functional tooth germ integrates the develop-ment of several connate structures [13] (e.g., Figures 3E, 5B, 5D,5F, and 10D). The connation (concrescence) putatively resultsfrom failed/reduced inhibition to delineate a gap between indi-vidual structures (compare to Figure 9B). The connate buds/capsmight reflect developmental repetition of simple dental primor-dia of mammalian ancestors [13].

As reviewed by Peyer [50], according to the “concrescencetheory,” multicusped teeth in mammals evolved by concrescenceof several simple tooth primordia inherited from their ances-tors. The alternative “differentiation theory” proposes that mul-ticusped teeth in mammals evolved by differentiation from uni-form teeth in the ancestor [50]. The budding model of dentitiondevelopment suggests that the “differentiation” of the postna-tal tooth shape in mammalian ancestors might include gradualmesio-distal and bucco-lingual “concrescence” (connation) ofsimple tooth primordia during prenatal development in the re-spective animals [13].

Although the developmental mechanism of concrescence oftooth primordia has been disputed by many odontologists in

mammals, a fusion of several simple teeth to form tooth plates,as well as heterodonty, are documented in some fishes [18]. Afunctional junction of several simple teeth formed a single com-posite unit (a “dental battery”) in some fossil reptiles [27]. Theperiodic pattern and its regulation by inhibition zones in primi-tive teeth [51], in reptilian teeth [52], in the mouse molar toothcusps [3, 6], and in feathers [43] exhibit a similarity (compareto Figure 9A). This supports the hypothesis that a decrease inthe efficiency of the inhibition zone (Figure 9B) might lead tofusion of several dental elements to form more complex teeth innonmammalian vertebrates [51] as well as in mammals.

ACKNOWLEDGMENTSWe thank Dr. M. Titlbach for electron microscopic pictures

and kind help with the preparation of semithin sections, andJ. Dutt for his critical reading. The technical assistance of B.Rokytova is gratefully acknowledged. We thank Dr. Z. Likovsk´yand M. Praˇzanova for their help in the preparation of micropho-tographs. Supported by the Grant Agency of the Academy ofSciences CR (grant A-7039901) and by the Ministry of Educa-tion, Youth, and Sports (Cost B-8.10) of the Czech Republic.

REFERENCES[1] Maas, R., and Bei, M. (1997). The genetic control of early tooth develop-

ment.Crit. Rev. Oral Biol. Med.8:4–39.[2] Dassule, H.R., and McMahon, A.P. (1998). Analysis of epithelial–

mesenchymal interactions in the initial morphogenesis of the mammaliantooth.Dev. Biol.202:215–227.

[3] Weiss, K.M., Stock, D.W., and Zhao, Z. (1998). Dynamic interactions andthe evolutionary genetics of dental patterning.Crit. Rev. Oral Biol. Med.9:369–398.

[4] Peters, H., and Balling, R. (1999). Teeth—Where and how to make them.Trends Genet.15:59–65.

[5] Tucker, A.S., and Sharpe, P.T. (1999). Molecular genetics of tooth mor-phogenesis and patterning: The right shape in the right place.J. Dent. Res.78:826–834.

[6] Jernvall, J., and Thesleff, I. (2000). Reiterative signaling and patterningduring mammalian tooth morphogenesis.Mech. Dev.92:19–29.

[7] Peterkova, R. (1983). Dental lamina develops even within the mouse di-astema.J. Craniofac. Genet. Dev. Biol.3:133–142.

[8] Peterkova, R., Peterka, M., Vonesch, J.-L., and Ruch, J.-V. (1993). Multi-ple developmental origin of the upper incisor in mouse: Histological andcomputer assisted 3-D-reconstruction studies.Int. J. Dev. Biol.37:581–588.

[9] Peterkova, R., Lesot, H., Vonesch, J.-L., Peterka, M., and Ruch, J.-V.(1996). Mouse molar morphogenesis revisited by three dimensional recon-struction: I. Analysis of initial stages of the first upper molar developmentrevealed two transient buds.Int. J. Dev. Biol.40:1009–1016.

[10] Lesot, H., Peterkov´a, R., Vonesch, J.-L., Viriot, L., Tureˇckova, J., Peterka,M., and Ruch, J.-V. (1998). Early stages of tooth morphogenesis in mouseanalyzed by 3D reconstructions.Eur. J. Oral Sci.106:64–70.

[11] Peterkova, R., Peterka, M., Vonesch, J.-L., Tureˇckova, J., Viriot, L., Ruch,J.-V., and Lesot, H. (1998). Correlation between apoptosis distribution andBMP-2 and BMP-4 expression in vestigial tooth primordia in mice.Eur.J. Oral Sci.106:667–670.

[12] Viriot, L., Lesot, H., Vonesch, J.-L., Ruch, J.-V., Peterka, M., andPeterkova, R. (2000). The presence of rudimentary odontogenic struc-tures in the mouse embryonic mandible requires reinterpretation of de-velopmental control of first lower molar histomorphogenesis.Int. J. Dev.Biol. 44:233–240.

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009

128 R. PETERKOVA ET AL.

[13] Peterkova, R., Peterka, M., Viriot, L., and Lesot, H. (2000). Dentitiondevelopment and budding morphogenesis.J. Craniofac. Genet. Dev. Biol.20:158–172.

[14] Moss-Salentijn, L. (1978). “Vestigial teeth in rabbit, rat and mouse; theirrelationship to the problem of lacteal dentitions.” In:Development, Func-tion and Evolution of Teeth, P.M. Butler and K.A. Joysey (eds.), pp. 13–29.(Academic Press, London).

[15] Luckett, W.P. (1985).“Superordinal and intraordinal affinities of rodents:Developmental evidence from the dentition and placentation.” In:Evolu-tionary Relationships Among Rodents, W.P. Luckett and J.-L. Hartenberger(eds.), pp 227–276. (Plenum Press, New York.).

[16] Maderson, P.F.A. (1975). Embryonic tissue interactions as the basis formorphological change in evolution.Am. Zool.15:315–327.

[17] Smith, M.M., and Hall, B.K. (1990). Development and evolutionary ori-gins of vertebrate skeletogenic and odontogenic tissues.Biol. Rev.65:277–373.

[18] Huysseune, A., and Sire, J.-Y. (1998). Evolution of patterns and processesin teeth and tooth-related tissues in non-mammalian vertebrates.Eur. J.Oral Sci.106(Suppl. 1), 437–481.

[19] Jernvall, J., and Jung, H.S. (2000). Genotype, phenotype and developmen-tal biology of molar tooth characters.Yearbook Phys. Anthropol.43:171–190.

[20] Lesot, H., Vonesch, J.-L., Peterka, M., Tureˇckova, J., Peterkov´a, R., andRuch, J.-V. (1996). Mouse molar morphogenesis revisited by three dimen-sional reconstruction: II. Spatial distribution of mitoses and apoptosis incap to bell stages first and second molar teeth.Int. J. Dev. Biol.40:1017–1031.

[21] Lesot, H., Peterkov´a, R., Schmitt, R., Meyer, J.-M., Viriot, L., Vonesch,J.-L., Senger, B., Peterka, M., and Ruch, J.-V. (1999). Initial features ofthe inner dental epithelium histo-morphogenesis in the first lower molar.Int. J. Dev. Biol.43:245–254.

[22] Peterkova, R. (1985). The common developmental origin and phylogeneticaspects of teeth, rugae palatinae, and fornix vestibuli oris in the mouse.J.Craniofac. Genet. Dev. Biol.5:89–104.

[23] Pourtois, M. (1961). Contribution a l’´etude des bourgeons dentaires dela souris: I. P´eriodes d’´ınduction et de morphodiff´erentiation.Arch. Biol.(Liege)72:17–95.

[24] Lehmann, R., and Slavkin, H.C. (1984). Identification of inner and outercell proliferation centers during fetal tooth morphogenesis.J. Craniofac.Genet. Dev. Biol.4:47–57.

[25] Ruch, J.-V. (1984). “Tooth morphogenesis and differentiation.” In:Dentinand Dentinogenesis, A. Linde (ed.), pp. 47–79. (CRC Press, Boca Raton,FL).

[26] Bolk, L. (1922). Odontological essays. On the relation between reptilianand mammalian teeth.J. Anat.56:107–136.

[27] Edmund, A.G. (1960). Tooth replacement phenomena in the lower verte-brates.R. Ont. Mus. Life Sci.52:1–190.

[28] Westergaard, B. (1986). “The pattern of embryonic tooth initiation in rep-tiles.” In Teeth Revisited, D.E. Russel, J.-P. Santoro, and D. Sigogneau-Russel (eds.), pp. 55–63. (M´em. Mus. Natn. Hist. Nat., Paris, S´erie C, 53,Paris).

[29] Moss-Salentijn, L. (1982). “Morphological aspects of the growth behaviorof the early dental lamina in the cat and rat.” In:Teeth: Form, Functionand Evolution, B. Kurten (ed.), pp. 7–20. (Columbia University Press,New York).

[30] Kieffer, S., Peterkov´a, R., Vonesch, J.-L., Ruch, J.-V., Peterka, M., andLesot, H. (1999). Morphogenesis of the lower incisor in the mouse frombud to early bell stage.Int. J. Dev. Biol.43:531–539.

[31] Peterkova, R., Klepacek, I., and Peterka, M. (1987). Prenatal developmentof rugae palatinae in mice: Scanning electron microscope and histologicalstudies.J. Craniofac. Genet. Dev. Biol.7:169–189.

[32] Linton, R.G. (1905). On the morphology of the mammalian palatal rugae.Vet. J.12:220–252.

[33] Tureckova, J., Lesot, H., Vonesch, J.-L., Peterka, M., Peterkov´a, R., andRuch, J.-V. (1996). Apoptosis is involved in disappearance of the di-astemal dental primordia in mouse embryo.Int. J. Dev. Biol.40:483–489.

[34] Tureckova, J., Sahlberg, C.,Aberg, T., Ruch, J.-V., Thesleff, I., andPeterkova, R. (1995). Comparison of expression of the msx-1, msx-2,BMP-2 and BMP-4 genes in the mouse upper diastemal and molar toothprimordia.Int. J. Dev. Biol.39:459–468.

[35] Keranen, S.V.E., Kettunen, P.,Aberg, T., Thesleff, I., and Jernvall, J.(1999). Gene expression patterns associated with suppression of odonto-genesis in mouse and vole diastema regions.Dev. Genes. Evol.209:495–506.

[36] Jacobson, M.D., Weil, M., and Raff, M.C. (1996). Programmed cell deathin animal development.Cell 88:347–354.

[37] Glucksmann, A. (1951). Cell deaths in normal vertebrate ontogeny.Biol.Rev.26:59–86.

[38] Vaahtokari, A.,Aberg, T., and Thesleff, I. (1996). Apoptosis in the develop-ing tooth: Association with an embryonic signaling center and suppressionby EGF and FGF-4.Development122:121–129.

[39] Shigemura, N., Kiyoshima, T., Kobayashi, I., Matsuo, K., and Yamaza,H. (1999). The distribution of BrdU- and TUNEL-positive cells dur-ing odontogenesis in mouse lower first molar.Histochem. J.31:367–377.

[40] Bronckers, A.L.J.J., Lyaruu, D.M., Goei, W., Litz, M., Luo, G., Karsenty,G., Woltgens, J.H.M., and D’Souza, R.N. (1996). Nuclear DNA frag-mentation during postnatal tooth development of mouse and hamster andduring dentin repair in rat.Eur. J. Oral Sci.104:102–111.

[41] Wolpert, L. (1978). Pattern formation in biological development.Sci. Am.239:154–164.

[42] Chuong, C.M., and Noveen, A. (1999). Phenotypic determination of ep-ithelial appendages: Genes, developmental pathways, and evolution.J.Invest. Dermatol. Symp. Proc.4:307–311.

[43] Jung, H.S., Francis-West, P.H., Widelitz, R.B., Jiang, T.X., Ting-Berreth,S., Tickle, C., Wolpert, L., and Chuong, C.M. (1998). Local inhibitoryaction of BMPs and their relationships with activators in feather formation:Implications for periodic patterning.Dev. Biol.196:11–23.

[44] Hogan, B.L.M. (1999). Morphogenesis.Cell 6:225–233.[45] Noramly, S., and Morgan, B.A. (1998). BMPs mediate lateral inhibition

at successive stages in feather tract development.Development125:3775–3787.

[46] Chen, Y.P., Zhang, Y., Jiang, T.-X., Barlow, A.J., Amand, T.R.S., Hu,Y., Heanley, S., Francis-West, P., Chuong, C.M., and Maas, R. (2000).Conservation of early odontogenic signaling pathways in aves.Proc. Natl.Acad. Sci. USA97:10044–10049.

[47] Viriot, L., Peterkova, R., Peterka, M., and Lesot, H. (2002). Evolutionaryimplications of the occurrence of two vestigial tooth germs during earlyodontogenesis in the mouse lower jaw. This issue.

[48] Kristenova-Cermakova, P., Peterka, M., Lisi, S., Lesot, H., and Peterkov´a,R. (2002). Postnatal lower jaw dentition in different phenotypes of tabbymice. This issue.

[49] Metzger, R.J., and Krasnow, M.A. (1999). Genetic control of branchingmorphogenesis.Science284:1635–1639.

[50] Peyer, B. (1968).Comparative Odontology, R. Zangler (ed.). (Universityof Chicago Press, Chicago).

[51] Reif, W.E. (1982). Evolution of dermal skeleton and dentition in verte-brates: The odontode regulation theory.Evol. Biol.15:287–368.

[52] Osborn, J.W. (1978). “Morphogenetic gradients: Fields versus clones.”In: Development, Function and Evolution of Teeth, P.M. Butler and K.A.Joysey (eds.), pp. 171–201. (Academic Press, London).

[53] Coin, R., Lesot, H., Vonesch, J.-L., Haikel, Y., and Ruch, J.-V. (1999).Aspects of cell proliferation kinetics of the dental epithelium during mousemolar and incisor morphogenesis: A reappraisal of the role of the enamelknot area.Int. J. Dev. Biol.43:261–267.

Downloaded By: [CDL Journals Account] At: 17:44 16 April 2009