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Objectives The Patterning Cascade Model (PCM) provides an evolutionary developmental framework for exploring diversity in tooth crown form. According to the model, proximity of secondary enamel knots and tooth germ size track underlying developmental processes that dictate ultimate crown morphology (i.e., cusp number, accessory cusp presence/size). Previous research has shown the model to successfully predict variation in Carabelli's trait expression between antimeric and metameric pairs of human permanent molars. In this study, we quantify Carabelli's trait expression for metameres of the mixed dentition (dm2 and M1) and assess the PCM's potential for explaining differences in expression between the two elements. Materials and methods Crown dimensions, intercusp distances, and Carabelli's trait expression were collected from 49 subadults possessing observable dm2/M1 pairs. Wilcoxon signed-rank tests and paired t-tests were performed to assess whether metameres differ significantly in morphometric variables. We explored the relationships between relative intercusp distances (RICDs) and Carabelli's trait expression using proportional odds logistic regression. Results Intra-individual dm2/M1 pairs differed significantly in Carabelli's trait expression (p = 0.01), with dm2 exhibiting higher grades of expression more commonly despite its smaller crown size. Paired molars differed in only one statistically significant RICD: metacone-hypocone (p

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.

In mice, a toothless diastema separates the single incisor from the three molars in each dental quadrant. In the prospective diastema of the embryo, small rudimentary buds are found that are presumed to be rudiments of suppressed teeth. A supernumerary tooth occurs in the diastema of adult mice carrying mutations in either Spry2 or Spry4. In the case of Spry2 mutants, the origin of the supernumerary tooth involves the revitalization of a rudimentary tooth bud (called R2), whereas its origin in the Spry4 mutants is not known. In addition to R2, another rudimentary primordium (called MS) arises more anteriorly in the prospective diastema. We investigated the participation of both rudiments (MS and R2) in supernumerary tooth development in Spry2 and Spry4 mutants by comparing morphogenesis, proliferation, apoptosis, size and Shh expression in the dental epithelium of MS and R2 rudiments. Increased proliferation and decreased apoptosis were found in MS and R2 at embryonic day (ED) 12.5 and 13.5 in Spry2(-/-) embryos. Apoptosis was also decreased in both rudiments in Spry4(-/-) embryos, but the proliferation was lower (similar to WT mice), and supernumerary tooth development was accelerated, exhibiting a cap stage by ED13.5. Compared to Spry2(-/-) mice, a high number of Spry4(-/-) supernumerary tooth primordia degenerated after ED13.5, resulting in a low percentage of supernumerary teeth in adults. We propose that Sprouty genes were implicated during evolution in reduction of the cheek teeth in Muridae, and their deletion can reveal ancestral stages of murine dental evolution.

Background: Foxi3 is a member of the large forkhead box family of transcriptional regulators, which have a wide range of biological activities including manifold developmental processes. Heterozygous mutation in Foxi3 was identified in several hairless dog breeds characterized by sparse fur coat and missing teeth. A related phenotype called hypohidrotic ectodermal dysplasia (HED) is caused by mutations in the ectodysplasin (Eda) pathway genes. Results: Expression of Foxi3 was strictly confined to the epithelium in developing ectodermal appendages in mouse embryos, but no expression was detected in the epidermis. Foxi3 was expressed in teeth and hair follicles throughout embryogenesis, but in mammary glands only during the earliest stages of development. Foxi3 expression was decreased and increased in Eda loss- and gain-of-function embryos, respectively, and was highly induced by Eda protein in embryonic skin explants. Also activin A treatment up-regulated Foxi3 mRNA levels in vitro. Conclusions: Eda and activin A were identified as upstream regulators of Foxi3. Foxi3 is a likely transcriptional target of Eda in ectodermal appendage placodes suggesting that HED phenotype may in part be produced by compromised Foxi3 activity. In addition to hair and teeth, Foxi3 may have a role in nail, eye, and mammary, sweat, and salivary gland development. Developmental Dynamics 242:593–603, 2013. © 2013 Wiley Periodicals, Inc.

The patterning cascade model of tooth morphogenesis has emerged as a useful tool in explaining how tooth shape develops and how tooth evolution may occur. Enamel knots, specialized areas of dental epithelium where cusps initiate, act as signaling centers that direct the growth of surrounding tissues. For a new cusp to form, an enamel knot must form beyond the inhibition fields of other enamel knots. The model predicts that the number and size of cusps depends on the spacing between enamel knots, reflected in the spacing between cusps. Recently, work by our group demonstrated that the model predicted Carabelli trait expression in human first molars. Here we test whether differences in Carabelli trait expression along the molar row can also be predicted by the model. Crown areas and intercusp distances were measured from dental casts of 316 individuals with a digital microscope. Although absolute cusp spacing is similar in first and second molars, the smaller size and more triangular shape of second molars results in larger cusp spacing relative to size and, likely, less opportunity for the Carabelli trait to form. The presence and size of the hypocone (HY) and a range of small accessory cusps in a larger sample of 340 individuals were also found to covary with the Carabelli trait in a complex way. The results of this study lend further support to the view that the dentition develops, varies, and evolves as a single functional complex.

Dental patterns in vertebrates range from absence of teeth to multiple sets of teeth that are replaced throughout life. Despite this great variation, most of our understanding of tooth development is derived from studies on just a few model organisms. Here we introduce the reptile as an excellent model in which to study the molecular basis for early dental specification and, most importantly, for tooth replacement. We review recent snake studies that highlight the conserved role of Shh in marking the position of the odontogenic band. The distinctive molecular patterning of the dental lamina in the labial-lingual and oral-aboral axes is reviewed. We explain how these early signals help to specify the tooth-forming and non-tooth forming sides of the dental lamina as well as the presumptive successional lamina. Next, the simple architecture of the reptilian enamel organ is contrasted with the more complex, mammalian tooth bud and we discuss whether or not there is an enamel knot in reptilian teeth. The role of the successional lamina during tooth replacement in squamate reptiles is reviewed and we speculate on the possible formation of a vestigial, post-permanent dentition in mammals. In support of these ideas, we present data on agamid teeth in which development of a third generation is arrested. We suggest that in diphyodont mammals, similar mechanisms may be involved in reducing tooth replacement capacity. Finally, we review the location of label-retaining cells and suggest ways in which these putative dental epithelial stem cells contribute to continuous tooth replacement. genesis 49:247–260, 2011. © 2011 Wiley-Liss, Inc.

To understand developmental mechanisms of evolutionary change, we must first know how different morphologies form. The vast majority of our knowledge on the developmental genetics of tooth formation derives from studies in mice, which have relatively derived mammalian dentitions. The marsupial Monodelphis domestica has a more plesiomorphic heterodont dentition with incisors, canines, premolars, and molars on both the upper and the lower jaws, and a deciduous premolar. The complexity of the M. domestica dentition ranges from simple, unicusped incisors to conical, sharp canines to multicusped molars. We examine the development of the teeth in M. domestica, with a specific focus on the enamel knot, a signaling center in the embryonic tooth that controls shape. We show that the tooth germs of M. domestica express fibroblast growth factor (FGF) genes and Sprouty genes in a manner similar to wild-type mouse molar germs, but with a few key differences.

Pitrm1 is a zinc metalloendopeptidase that has been implicated in Alzheimer's disease and mitochondrial peptide degradation, but to date no major role in embryonic development has been documented. In a screen for genes regulated by hedgehog signaling in the mouse limb., we showed that expression of Pitrm1 is upregulated in response to loss of the Gli3 transcription factor. Here we confirm spatial changes in Pitrm1 expression in the Gli3 mutant mouse limb and examine Pitrm1 expression in Shh null and Ptch1 conditional deletion mouse mutants. In wild-type mice, Pitrm1 is expressed in a number of developing tissues known to be patterned by Sonic hedgehog, including the limbs, face, cortex, hippocampus, cerebellum, tectum, sub-mandibular gland, lung, genital tubercle, hair follicles, and the enamel knot of the teeth. Additionally, Pitrm1 is expressed in Pax3-expressing myoblast progenitors in the limb, the dermomyotome, and developing muscles of the face and torso. Developmental Dynarnics 238:3175-31.84, 2009. (C) 2009 Wiley-Liss, Inc.

SUMMARY A characteristic feature of mammalian dentition is the evolutionary reduction of tooth number and replacement. Because mice do not replace teeth, here we used Sorex araneus, the common shrew, as a model to investigate the loss of tooth replacement. Historically, shrews have been reported to initiate the development of several, milk or deciduous teeth but these soon become rudimentary and only the replacement teeth erupt. Shrews thus offer a living example of a derivedmammalianpattern where thedeciduous tooth development is being suppressed. Based on histological and gene expression analyses of serial sections, we suggest that S. araneus has discernible tooth replacement only in the premolar 4 (P4) position. Both generations of teeth express Shh in the enamel knot and in the inner enamel epithelium. Nevertheless, the deciduous P4 (dP4) is reduced in size during embryogenesis and is eventually lost without becoming functional. Analysis of growth shows that P4 replaces the dP4 in a ‘‘double-wedge’’ pattern indicative of competitive replacement where the suppression of the deciduous tooth coincides with the initiation of its replacement. Because activator–inhibitor mechanisms have been implicated in adjacent mouse molars and in transgenic mice with continuous tooth budding, we suggest that evolutionary suppression of deciduous teeth may involve early activation of replacement teeth, which in turn begin to suppress their deciduous predecessors.

Structured abstract Authors – Matalova E, Setkova J, Blackburn J, Misek I, Sharpe PT Introduction – Understanding of apoptotic mechanisms involved in tissue shaping is of particular interest because of possible targeted modulation of the development of organ structures such as teeth. Research of CD 95 mediated apoptosis has been focused particularly on cell death in the immune system and related disorders. However, CD 95 mediated apoptosis is also involved in embryogenesis of many organs as the kidney, the lung, the intestine and tissue networks such as the nervous system. Design – Narrative review. Results – This review briefly summarizes the current knowledge of CD 95 mediated apoptosis in embryogenesis with possible implication in tooth development. CD 95 receptor and CD 95 ligand are found at early stages of tooth development. The data suggest some positive correlations with dental apoptosis distribution, particularly in the primary enamel knot where apoptosis occurs during elimination of this structure. CD 95 deficient (lpr) adult mouse tooth phenotype, however, did not show any alterations in final tooth pattern and morphology. Conclusion – To date studies of apoptotic machinery during tooth development show spatial localization of many of the components together with precise and localized timing of cell death. There is still much to be learned about the regulation and importance of apoptosis in tooth development. Nevertheless, the involvement of apoptotic regulatory mechanisms interplaying with other molecules participates to the cellular cross-talk in developing tissues, which opens possible targeted modulations as suggested, e.g. for future molecular dentistry.

Signaling by Edar, a tumor necrosis factor receptor, is required for the development of ectodermal organs. Mutations in Edar or other molecules of the same signaling pathway cause ectodermal dysplasias in humans and mice. In these diseases, teeth are missing or malformed, and the development of hairs and several glands is hypoplastic. During tooth and hair development, Edar expression becomes patterned to ectodermal placodes and signaling centers. This localization has been suggested to be required for organogenesis. We have expressed Edar throughout the ectoderm using the keratin 14 promoter and show that this misexpression disrupts tooth patterning and differentiation. Tooth shape and cusp number are differentially affected, depending on the amount of transgene expression. In addition, tooth enamel formation is defective in a dose-dependent manner. We speculate that the tooth patterning defects are caused by ectopic Edar activity outside the signaling centers.

The gene expression and protein distribution of matrix metalloproteinase (MMP) -2, -9, membrane type-1 MMP (MT1-MMP), as well as of TIMP-1, -2, and -3 were analyzed during mouse molar development. Immunohistochemical data demonstrated that all the MMPs investigated were expressed in the dental epithelium and mesenchyme. In contrast, gene and protein expression analysis for TIMPs showed that they were differentially expressed. TIMP-1 was expressed in the dental epithelium and mesenchyme between E13 and E16 and was transiently up-regulated at E14, the cap stage. TIMP-1 expression was also detected in differentiating odontoblasts. TIMP-2 RNA transcripts were found in the peridental and dental mesenchyme, odontoblasts, and ameloblasts. Protein analysis revealed high expression on the lingual side of the dental epithelium and underlying mesenchyme together with transient expression in the enamel knot at E14 and expression in the gingival tissue and enamel matrix postnatally. TIMP-3 RNA transcripts were found in discrete regions of the dental epithelium, including at high levels in the cervical loop at E16. Expression was also detected in preodontoblasts at E16 and transiently during ameloblast differentiation. Analysis of the protein distribution revealed a lower level of TIMP-3 on the lingual side of the dental epithelium at E14. MT1-MMP was expressed in the dental mesenchyme between E13 and E16, at relatively high levels in the cervical loop at E14, and in the odontoblasts and ameloblasts. The distinct temporospatial distribution patterns of the TIMPs suggest that these inhibitors play several intrinsic roles during tooth development. Developmental Dynamics 228:105–112, 2003. © 2003 Wiley-Liss, Inc.

Structured Abstract Authors – Peterkova R., Kristenova P., Lesot H., Lisi S., Vonesch J-L., Gendrault J-L., Peterka M. Objectives –Prenatal identification of the different dentition morphotypes, which exist in the lower molar region of tabby (Ta) adult mice, and investigation of their origin. The mouse Ta syndrome and its counterpart anhidrotic (hypohidrotic) ectodermal dysplasia (EDA) in human are characterized by absence or hypoplasia of sweat glands, hair and teeth. Design –Analysis of tooth morphogenesis using serial histological sections and 3D computer aided reconstructions of the dental epithelium in the cheek region of the mandible. Setting and Sample Population –Institute of Experimental Medicine, Academy of Sciences, Prague. Heads of 75 Ta homozygous and hemizygous mice and 40 wild type (WT) control mice aged from embryonic day (ED) 14.0–20.5 (newborns), harvested during 1995–2001. Outcome Measure –Prenatal identification of five distinct morphotypes of Ta dentition on the basis of differences in tooth number, size, shape, position and developmental stage and of the morphology of the enamel knot in the most mesial tooth primordium. Results –The mesio-distal length of the dental epithelium was similar in the lower cheek region in Ta and WT mice. In Ta embryos, there was altered the mesio-distal segmentation of the dental epithelium giving rise to the individual tooth primordia. Prenatally, two basic morphotypes I and II and their particular subtypes (Ia, Ib, Ic, and IIa, IIb, respectively) of the developing dentition were identified from day 15.5. The incidence of the distinct morphotypes in the present sample did not differ from postnatal data. The proportion of the morphotype I and II was dependent on mother genotype. Conclusion –The different dentition morphotypes in Ta mice originate from a defect in the mesio-distal segmentation of the dental epithelium in mouse embryos. This defect presumably leads to variable positions of tooth boundaries that do not correspond to those of the WT molars. One tooth primordium of Ta mice might be derived from adjacent parts of two molar primordia in WT mice.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix. © 2001 Wiley-Liss, Inc.

To elucidate the roles of fibroblast growth factors (FGF) in tooth development, we have analyzed the expression patterns of fibroblast growth factor receptors (FGFR) in mouse teeth by in situ hybridization and studied the effects of FGF-2, -4, -8, and -9 on cell proliferation in vitro by local application with beads on isolated dental mesenchymes. mRNAs of FGFR-1, -2, and -3 were localized by probes specific for the alternative splice variants IIIb and IIIc. The expression patterns of FGFR1 -2, and -3 were completely different, and the two splicing variants of FGFR1 and 2 exhibited different expression domains. FGFR4 was not expressed in the developing teeth. The IIIb splice forms of FGFR1 and -2 were expressed in the dental epithelium during morphogenesis. The IIIc splice form of FGFR1 was expressed both in epithelium and mesenchyme whereas FGFR2 IIIc was confined to the mesenchymal cells of the dental follicle. Both splice forms of FGFR3 were expressed in dental papilla mesenchyme. None of the FGF-receptors was detected in the primary enamel knot, the putative signaling center regulating tooth morphogenesis. This may explain the fact that enamel knot cells do not proliferate, although they express intensely mitogenic FGFs. Beads releasing FGF-2, -4, -8, or -9 proteins stimulated cell proliferation in cultured dental mesenchymes. These data, together with our earlier data on FGF expression [Kettunen and Thesleff (1998): Dev Dyn 211:256-268] suggest that FGF-8 and -9 mediate epithelial-mesenchymal interactions during tooth initiation. During advancing morphogenesis FGF-3, -4, and -9 may act both on mesenchyme and epithelium. Finally, the intense expression of FGFR1 in odontoblasts and ameloblasts and FGFR2 IIIb in ameloblasts suggests that FGFs participate in regulation of their differentiation and/or secretory functions.

Bone morphogenetic proteins (BMP) are secretory signal molecules which have a variety of regulatory functions during morphogenesis and cell differentiation. Teeth are typical examples of vertebrate organs in which development is controlled by sequential and reciprocal signaling between the epithelium and mesenchyme. In addition, tooth development is characterized by formation of mineralized tissues: the bone-like dentin and cementum as well as epithelially derived enamel. We have performed a comparative in situ hybridization analysis of the expression of six different Bmps (Bmp-2 to Bmp-7) starting from initiation of tooth development to completion of crown morphogenesis when dentine and enamel matrices are being deposited. Bmps-2, -4, and -7 were frequently codistributed and showed marked associations with epithelial-mesenchymal interactions. Their expression shifted between the epithelium and mesenchyme starting from the stage of tooth initiation. They were subsequently expressed in the enamel knot, the putative signaling center regulating tooth shape. Their expression domains prior to and during the differentiation of the dentine-forming odontoblasts and enamel-forming ameloblasts was in line with functions in regulation of cell differentiation and/or secretory activities of the cells. The expression of Bmp-3 was confined to mesenchymal cells, in particular to the dental follicle cells which give rise to the cementoblasts, forming the hard tissue covering the roots of teeth. Bmp-5 was expressed only in the epithelial ameloblasts. It was upregulated as the cells started to polarize and intense expression continued in the secretory ameloblasts. Bmp-6 was expressed only weakly in the dental mesenchyme during bud and cap stages. Our results are in line with regulatory functions of Bmps at all stages of tooth morphogenesis. Bmps-2, -4, and -7 are conceivably parts of signaling networks regulating tooth initiation and shape development. They as well as Bmp-5 may be involved in the induction and formation of dentine and enamel, and Bmp-3 in the development of cementum. The remarkable overlaps in the expression domains of different Bmp genes may implicate functional redundancy and/or formation of active heterodimers between different BMPs. Dev. Dyn. 1997;210:383–396. © 1997 Wiley-Liss, Inc.

Tooth development involves reciprocal epithelial-mesenchymal interactions, polarized growth, mesenchyme condensation, and complex morphogenetic events. Because these processes bear similarities to those occurring in the developing limb, we asked whether morphogenetic signals found in the limb also occur in the developing tooth. We grafted mouse embryo tooth germs to the anterior margin of host chick embryo wing buds and determined whether the dental tissues had polarizing activity. Indeed, the grafts induced supernumerary digits. Activity of both molar and incisor tooth germs increased from bud to cap stages and was maximal at late bell stage in newborn. With further development the polarizing activity began to decrease, became undetectable in adult molar mesenchyme but persisted in incisor mesenchyme, correlating with the fact that incisors grow throughout postnatal life while molars do not. When different portions of neonatal incisors were assayed, a clear proximo-distal gradient of activity was apparent, with maximal activity restricted to the most proximal portion where undifferentiated mesenchyme and enamel organ reside. In situ hybridizations demonstrated that prior to induction of supernumerary digits, the tooth germ grafts induced expression in host tissue of Hoxd-12 and Hoxd-13. In addition, whole-mount in situ hybridizations and immunohistochemistry showed that developing tooth germs express Sonic hedgehog (Shh). Shh expression was first detected in bud stage tooth germs; at later stages Shh transcripts were prominent in enamel knot and differentiating ameloblasts at the cuspal region. We concluded that tooth germs possess polarizing activity and produce polarizing factors such as Shh. As in the limb, these factor(s) and activity probably play key roles in establishing polarity and regulating morphogenesis during early tooth development. Given its subsequent association with differentiating ameloblasts, Shh probably participates also in cytogenetic events during odontogenesis. © 1996 Wiley-Liss, Inc.

Teeth develop by a series of epithelial–mesenchymal interactions that govern not only where the tooth will form within the developing jaw, but also what type of tooth will develop. Before any sign of tooth development is observed, the patterning information regarding what type of tooth will form has already been determined. Thus an incisor forms in the distal jaw, whereas the molars form more proximally at the back of the jaw. Then, as the tooth develops, a signalling centre, known as the enamel knot, is set up at the centre of the tooth germ, which controls the shape (morphogenesis) of the tooth and the number of cusps which form. The genes involved in these patterning processes are starting to be elucidated and experiments involving knockout mice and misexpression studies have given insights into the molecular genetics of tooth morphogenesis. Key concepts: How a tooth develops in the embryo? How tooth identity is determined – i.e. to develop as a molar or incisor? How tooth shape is controlled – i.e. number of cusps? Keywords: tooth; development; shape; identity; enamel knot

SUMMARY The crown pattern of the tooth is essentially that of the surface of the dentine (dentine-enamel junction), modified by the deposition of enamel which may be uneven in thickness. The dentine-enamel junction preserves in the completed tooth the form of the membrana praeformativa, the basement membrane of the inner enamel epithelium of the enamel organ. Folding of this membrane creates the crown pattern. The inner enamel epithelium is subjected to pressure from both sides. On the basal side there is the rapidly growing mesenchyme of the dental papilla, and on the occlusal side there is the stellate reticulum, which swells by the accumulation of fluid. The stellate reticulum prevents distortion of the epithelium by growth of the papilla, and thus ensures that folding of the epithelium is due to its intrinsic growth pattern. This makes for more accurate control of the crown pattern, the details of which are of importance in the function of chewing. The enamel knot is a region of the inner enamel epithelium from which cells are contributed to the stellate reticulum. It represents the tip of the primary cusp. The enamel cord (‘enamel septum’) which consists of cells which are in process of transforming into stellate reticulum, has been confused with two other structures that develop later: a cleavage septum, preparatory to the formation of crown cementum, and an epithelial septum, found in marsupials and crocodilians. The epithelial nodules of monotremes are probably degenerate relics of an epithelial septum. The inner enamel epithelium is a diaphragm passing across the interior of the dental follicle, and folding to adapt its increased area to a confined space. The cement organ, which in some mammals develops from the follicle, probably plays no part in the deformation of the epithelium, but the follicle as a whole may be subject to compression by adjacent follicles. A cusp is a centre of precocious maturation of the cells of the inner enamel epithelium. Here growth ceases (perhaps after a transitory burst of mitosis) and eventually the hard tissues are deposited. The process starts at the tip of the cusp and extends basally, so that growth continues longest in the valleys, intensifying the crown relief. Dens in dente is due to retarded maturation of an area of the enamel epithelium. Throughout the development of the crown there is a marginal zona cingularis, where growth continues. The crown pattern depends upon the position and the stage of growth in which cusps are differentiated from the zona cingularis, by accelerated maturation of groups of cells. Cusps which appear late in development stand low on the crown and frequently form part of a cingulum. Changes in the timing of cusp formation play an important part in serial modifications of pattern, as well as in phylogeny. Ridges are probably produced by tensions set up in the epithelium by the relative movement of cusps, owing to unequal growth or to changes in the shape of the follicle. They form in areas where growth has slowed down but the apposition of hard tissues has not begun. The lobes of the basal outline of the tooth are produced by growth centres in the papilla, which cause evagination of the follicle. Each growth centre is supplied by a bundle of blood vessels. The roots form in relation to these blood vessels, and so reflect the organization of the papilla. Hertwig's epithelial sheath grows between the follicle and the base of the papilla, the direction of its growth being controlled by the surrounding tissues owing to the absence of stellate reticulum on the basal portion of the tooth. There is no constant relation between cusps and roots, although marginal cusps frequently arise in association with lobes of the basal outline. The crown pattern results from an interaction between the growth pattern of the inner enamel epithelium and that of the papilla, the latter controlling the shape of the margin which limits the folding epithelium.