Current Understanding of Tooth Formation: Transfer - PDF

Australian Dental Journal The official journal of the Australian Dental Association Australian Dental Journal 2014; 59:(1 Suppl): 48–54...

Australian Dental Journal The official journal of the Australian Dental Association Australian Dental Journal 2014; 59:(1 Suppl): 48–54 doi: 10.1111/adj.12102 Current understanding of the process of tooth formation: transfer from the laboratory to the clinic I Thesleff* *Research Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Finland. ABSTRACT Teeth are typical examples of organs in which genes determine the progress of development from initiation to the final shape, size and structure, whereas environmental factors play a minor role. Advances in gene technology over the last three decades have led to powerful novel methods to explore the mechanisms of embryonic development. Today we know a few hundred genes that regulate tooth development, and mutations in dozens of these genes have been shown to cause aberrations in tooth development in mice and/or humans. The functions of an increasing number of genes in tooth development have been discovered using genetically modified mouse models. We are now beginning to understand the ‘programme’ underlying the process of tooth formation. Key components of the programme are signals mediating com- munication between cells and complex gene regulatory networks in which the signal pathways are integrated. Under- standing the mechanisms of tooth development at the level of genes, cells and molecules will lay the basis for new ways to prevent and treat dental defects and diseases. Over the last decade knowledge about dental stem cells has accumulated rapidly and novel stem cell technologies have been developed. Combining stem cell research with knowledge on the mechanisms of tooth development may open up novel possibilities for clinical tooth regeneration. Keywords: Hypodontia, signalling networks, tooth morphogenesis, tooth renewal. Abbreviations and acronyms: BMP = bone morphogenetic protein; FGF = fibroblast growth factor; Eda = ectodysplasin; HED = hypo- hidrotic ectodermal dysplasia; OODD = odonto-onycho-dermal dysplasia. complete teeth form when the tooth germs are trans- INTRODUCTION planted in adult mice, for instance in the anterior cham- The morphological aspects of tooth formation have ber of the eye or under the kidney capsule. They also been described in detail over the last 150 years in performed tissue recombination studies where they sepa- many animals, including humans. This work has been rated dental epithelium from underlying mesenchymal based on histological sections of embryonic teeth. All tissue at early developmental stages, and then recom- teeth form from oral epithelium and underlying mes- bined them with various tissues and transplanted the enchymal cells (embryonic stromal tissue). Their explants to adult mice. These experiments demonstrated development starts from the thickening of the epithe- that tooth development is regulated by communication lium which is followed by condensation of the mesen- between the two tissues. Today we know that there is a chymal cells. The tooth crown forms through stages chain of reciprocal epithelial-mesenchymal interactions defined by the shape of the epithelium (bud, cap, bell) which regulate tooth morphogenesis as well as the differ- followed by the development of the root (Fig. 1). entiation of tooth specific cell types, including odonto- Although dentitions vary greatly between different blasts, ameloblasts and cementoblasts. Later, tissue animals, the development of an individual tooth has culture techniques were developed allowing continuous remained similar throughout evolution. observation and experimental manipulation of tooth Experimental studies on the mechanisms regulating morphogenesis (as reviewed by Tummers and Thesleff1). tooth development started more than 50 years ago. In these studies, developmental biologists, or experimental The toolbox of genes and molecules regulating tooth embryologists as they were previously called, used the development mouse as the model animal, and dissected tooth germs from embryos at different stages of morphogenesis. They Research on tooth development at the level of genes observed that development proceeds normally and started between the late 1980s and early 1990s. It 48 © 2013 Australian Dental Association  The programme of tooth formation enamel epithelium dental enamel knot dentin enamel knot placode mesen- chyme root dental dental- ameloblasts pulp mesenchyme papilla bone odontoblasts dental lamina bud cap bell eruption initiation morphogenesis cell differentiation matrix secretion Fig. 1 The process of tooth development. The dental placode and enamel knots are signalling centres regulating tooth morphogenesis. then became possible to study the expression of genes families of signal molecules that are essential for cell in dental tissues during development. In situ hybrid- communication in all animals from flies to man as ization technology allowed the localization of mRNA well as in all different organs, including teeth. These (messenger RNA) indicating the transcription of a spe- are BMP (bone morphogenetic protein), FGF (fibro- cific gene in histological sections. Over the years, pat- blast growth factor), Hedgehog and Wnt and they terns of gene expression have been studied in also regulate tooth development during several steps developing teeth in numerous laboratories around the from initiation to root formation (Fig. 2). In addition, world. The patterns of some 300 genes can be viewed the toolbox contains mediators transmitting the signal in the ‘Bite-it’ database (http://bite-it.helsinki.fi). in the cell and numerous transcription factors regulat- Most gene expression data on teeth have come from ing gene expression in the nucleus. The transmission mice, but studies on tooth development in many dif- of the signals in the four families from cell surface to ferent animals including mammals, also humans, and nucleus is basically very similar to the ectodysplasin in addition several fish and reptile species indicate that (Eda) signal transduction shown in Fig. 4A. The dif- largely the same genes are involved in the regulation ferent types of genes and molecules operate in com- of tooth development across species. These genes plex gene regulatory networks (as reviewed by belong to the common toolbox of developmental reg- Tummers and Thesleff1). ulatory genes which has been conserved to an aston- It is noteworthy that no tooth-specific regulatory ishing extent during evolution. genes have been discovered, which is in line with the The signals mediating communication between cells remarkable conservation of the genes regulating devel- constitute one of the most important groups of mole- opment, and it is probable that such genes do not cules in the conserved toolbox. There are four major exist. This notion is of clinical importance in the oral ectoderm dental placode enamel knot pitx2 lef1, edar edar BMP BMP BMP FGF FGF FGF SHH SHH SHH WNT WNT WNT EDA BMP BMP BMP activin FGF FGF FGF WNT WNT dental mesenchyme condensed dental dental papilla msx1/msx2 gli2/gli3 mesenchyme lhx6/lhx7 dlx1/dlx2 msx1, pax9, runx2, lef1 Fig. 2 Teeth form from oral epithelium (green) and underlying mesenchyme (blue) and interactions between these tissues regulate development. The most important signal molecules mediating this communication are BMP (bone morphogenetic protein), WNT, SHH (sonic hedgehog) and FGF (fibroblast growth factor). They regulate the expression of important transcription factors indicated in boxes. Loss of function of many of these genes arrests the process of tooth development in genetically modified mice, and their mutations cause tooth agenesis in humans. © 2013 Australian Dental Association 49 I Thesleff tissue of the tooth germ which thereafter forms a bud. Shh Interestingly, the pattern of BMP4 expression suggested that this signal might mediate reciprocal communica- T tion between the epithelium and mesenchyme (Fig. 2). This was supported by experiments in explant cultures where BMP4 protein, when applied on isolated dental mesenchyme, induced the expression of the M transcription factor msx1 as well as its own expression. Functional evidence for the key role of msx1 was soon I gained by applying the then novel technology of genetic (a) modification in mice: knocking out msx1 function resulted in mice with no teeth.3 There was no Fgf4 expression of BMP4 in the dental mesenchyme of those mice and it could be concluded that msx1 is required for the mediation of the BMP4 signal in the mesenchyme.4 During the following years numerous studies apply- ing gene expression pattern analysis, ex vivo explants cultures, and in vivo mouse models gradually increased information on the roles of other signal molecules and many transcription factors. They pin- (b) pointed specific roles for individual genes and indi- cated that complex gene regulatory networks direct Shh the initiation and morphogenesis of teeth. The list of genetically modified mice exhibiting dental defects increased rapidly,5 and the more sophisticated trans- genic mouse technologies allowed the targeted or induced modification of genes, e.g. in a specific tissue and/or at a specific time. Transgenic and mutant mice continue to provide excellent tools for tooth develop- ment studies and add to information on the roles of M2 individual genes on specific developmental processes. M1 As a result of this research we are beginning to have (c) a better idea of the ‘programme’ of tooth development Fig. 3 The placodes and enamel knots are signalling centres producing (Fig. 2).1,6 The reciprocal and sequential interactions numerous signal molecules. (a). The placodes initiating the development of incisors (I) and molars (M) in the lower jaw of a mouse embryo are between dental mesenchyme and epithelium constitute visualized by the expression of Shh (in situ hybridization analysis). T, the core of the programme. The interactions are medi- tongue. (b). Histological section of mouse embryonic molar at the cap ated by the conserved signal molecules activating the stage shows the expression of FGF-4 in the primary enamel knot. (c). Occlusal view of developing mouse molars dissected from a 17-day-old expression of specific transcription factors, which in embryo. M1 is at the bell stage and M2 at the cap stage. The secondary turn regulate the expression of numerous other genes enamel knots marking future cusps in M1, and the primary enamel knot important for advancing morphogenesis and cell dif- in M2 are visualized by Shh expression. ferentiation in the developing tooth. If a necessary component of the programme is knocked out, tooth diagnosis and study of patients with dental aberra- development arrests. tions (the majority of which are genetic), because the Important players in the programme of tooth devel- gene mutation causing the dental defect may disturb opment are three sets of transient signalling centres in the development of other tissues and organs as well. dental epithelium. They appear just before key devel- opmental stages: first in dental placodes where they initiate the development of individual teeth, and next Functions of important genes and the programme of in the primary enamel knot forming at the tip of the tooth development tooth bud and initiating bud-to-cap stage transition One of the first genes localized in developing teeth was and tooth crown formation. The enamel knots subse- the signal BMP4.2 Its expression was observed at the quently induce the formation of the third set of signal- site of initiation of mouse teeth and, most interestingly, ling centres, the secondary enamel knots determining it first localized to the thickened oral epithelium and the sites of tooth cusps in molars (Fig. 1 and 3). The subsequently shifted to the underlying mesenchymal signalling centres produce locally more than a dozen 50 © 2013 Australian Dental Association  The programme of tooth formation different signals belonging to the four important sig- (a) Ectodysplasin nal families: BMP, FGF, Hh and Wnt.1 The function of enamel knots in regulation of tooth Edar crown patterning has been demonstrated in several genetically modified mouse lines. For example, knock- Edaradd ing out the signal molecule Shh (sonic hedgehog) results in the formation of numerous cusps and fusion of molars, whereas in the Fgf20 knockouts some IKKγ cytoplasm cusps are missing and the teeth are smaller than nor- mal.7,8 Another example is the knockout of Sostdc1 (also called ectodin and wise) which is an inhibitor of NFκB BMP and Wnt signalling and is expressed around the enamel knot. The first and second molars are fused and there are extra cusps.9 These phenotypes support nucleus the hypothesis that the patterning of the tooth crown and cusps is regulated by enamel knot signals by a transcription reaction diffusion mechanism where the interplay of NFκB activators and inhibitors determine the pattern. It is believed that fine-tuning of enamel knot signalling may account for the variation of mammalian cusp patterns during evolution.9 The genetic causes and pathogenesis of tooth agenesis (b) The first genes in which mutations were shown to cause tooth agenesis in humans were MSX1 and ecto- dysplasin (EDA), both reported in the same issue of Nature Genetics in 1996.10,11 Heterozygous loss of function of the MSX1 gene was discovered in one family as the cause of oligodontia (defined as more than six missing teeth excluding wisdom teeth), while mutations in EDA encoding a novel signal molecule, were identified as the cause of X-linked hypohidrotic ectodermal dysplasia (HED), a syndrome character- Fig. 4 Ectodysplasin (Eda) signalling is necessary for tooth formation. ized with severe tooth agenesis associated with other (a). The Eda pathway represents a typical signal pathway. The signal (Eda) binds to its cell surface receptor Edar mediating the signal to the symptoms including hair loss, dry mouth and inability cytoplasm. Edaradd and IKKgamma mediate the signal to activate the to sweat (Fig. 4). The MSX1 mutations, and soon transcription factor NFkappaB which moves to the nucleus and regulates thereafter PAX9 mutations causing oligodontia, were gene expression. (b). Severe tooth agenesis (oligodontia) caused by lack of Eda in XLHED (X-linked hypohidrotic ectodermal dysplasia). found using the candidate gene approach, since msx1 and pax9 knockout mice were known to lack all teeth.12 New genes associated with tooth agenesis in ectodermal dysplasias and identical tooth phenotypes transgenic mice and humans are being identified con- result from mutations in other components of the Eda stantly. Recently WNT10A has come up as the most signal pathway including the receptor EDAR and sig- common gene associated with human tooth agenesis. nal mediator EDARADD (Fig. 4). Interestingly, hyp- In one study, mutations were found in WNT10A in odontia patients with WNT10A mutations may also 56% of the cases of non-syndromic hypodontia.13 have defects in other ectodermal organs and represent Because the same genes are generally required for odonto-onycho-dermal dysplasia (OODD).14 development of many tissues, tooth agenesis is fre- All genes associated with hypodontia are obviously quently associated with congenital defects in other necessary for early tooth development, and the analy- organs, most often with ectodermal organs developing sis of their functions in mouse models has increased from the outer surface of the embryo. Such organs our understanding of the pathogenesis of tooth agene- include, in addition to teeth, hair and exocrine glands, sis and the genetic mechanisms of tooth formation. e.g. salivary glands. When two or more ectodermal Work on the functions of the Eda and Wnt pathways organs are affected, the conditions are called ectoder- provide examples of such approaches. mal dysplasias. X-linked HED, caused by mutations The functions of the Eda signal pathway have been, in the EDA gene, represents one of the most common and are currently, actively studied in many laboratories © 2013 Australian Dental Association 51 I Thesleff including our laboratory in Helsinki.15 The expression Initiation of new teeth from the dental lamina of the Eda receptor, Edar, was specifically localized in The capacity to generate new teeth has been reduced the placodes of teeth as well as in other derivatives of during evolution. Humans, like most other mammals, ectoderm indicating that Eda signalling affects the initi- can replace teeth once, while reptiles and snakes have ation of all ectodermal organs by direct effects on the the ability to replace teeth continuously. Because the placodes. The stimulation of Eda expression in trans- laboratory mouse, like other rodents, has completely genic mice induced the formation of extra teeth lost the capacity to replace teeth, there is very little (Fig. 5A and 5B) and mammary glands and stimulated information on the mechanisms of tooth replacement. the growth of hair and nails.16 However, work in other mammals, such as ferrets The Eda pathway is integrated with several other and some reptiles, has addressed the mechanisms of signal pathways. For example, expression of Eda is tooth replacement. It seems that all teeth are initiated stimulated by Wnt signals and regulates directly the from specific regions of the dental epithelium, called expression of Shh (sonic hedgehog), Wnt10b as well the dental lamina. The first teeth in all vertebrates are as Fgf20 in the placodes.8 The Eda pathway is unique initiated from the embryonic primary dental lamina, since it seems to be necessary, almost exclusively, for whereas all other teeth originate from the successional the formation of teeth and other ectodermal organs, dental lamina, associated with a previous tooth and unlike the other conserved signal pathways having forming as an extension from the primary dental lam- more widespread functions. Eda signalling is required ina. Teeth that form in succession include replacement for ectodermal organ development in all vertebrates teeth as well as primary teeth that develop serially, and, for example, fish scales fail to develop in its such as posterior molars. Both the primary and suc- absence. This indicates that the Eda pathway, like cessional dental laminae apparently contain stem cells the other key signal pathways, has been conserved in or progenitor cells which have the capacity to form evolution. teeth. Recently, the first marker gene was discovered The discovery of WNT10A mutations as the most for dental lamina cells.19 This gene, Sox2, is also common cause of oligodontia supports the current expressed in stem cells in several other tissues and is view, based on numerous experimental studies, that one of the factors used to reprogramme somatic cells Wnt signalling is of key importance for the initiation into iPS cells. It is also expressed in epithelial stem of tooth formation. For example, inhibition of Wnt cells fuelling the continuous growth of mouse incisors. signalling by overexpressing the Wnt inhibitor Dkk1 The maintenance, proliferation and differentiation of in transgenic mice prevents the formation of tooth the incisor stem cells are under active investigation in placodes and the initiation of teeth fails.17 Conversely, many laboratories around the world.20 the overactivation of Wnt signalling induces extensive The initiation of tooth development from the pri- formation of new teeth (Fig. 5C, and see below). mary dental lamina starts with the formation of the Thus, in light of the current evidence, Wnt may be dental placode and, as described above, many signal positioned highest in the hierarchy among the signals pathways, e.g. Wnt and Eda, as well as complex gene involved in tooth initiation. In addition, Wnt signal- regulatory networks, are involved in the process. ling like all other conserved pathways, regulates later Many of the same genes are expressed also when the events in tooth formation, e.g. shape development and replacement teeth are initiated from the successional cell differentiation.18 dental lamina in ferrets and reptiles.21,22 The most (a) (c) (b) Fig. 5 Stimulation of tooth initiation by excess Eda and Wnt signalling. (a). Three molars in a wild type mouse. (b). An extra premolar-like tooth has been induced in a mouse overexpressing Eda in oral epithelium. (c). Forced activation of Wnt signalling in oral epithelium (ß-cat ex3K14/+) results in con- tinuous initiation of new teeth. A tooth bud of a mutant mouse embryo was cultured ex vivo for 19 days. 52 © 2013 Australian Dental Association  The programme of tooth formation upstream signal pathway and the inducer of tooth ini- tion when they are building a tooth, and we actually tiation in mice appears to be the Wnt pathway. When know in great detail the other components of the this pathway is activated in the oral epithelium of programme underlying tooth development. We also transgenic mouse embryos (ß-catex3K14/+), dozens of know that when initiated, tooth development contin- teeth are generated, and they appear to develop in ues independently from the surrounding tissue. In succession: when one tooth bud of a transgenic mouse addition, there are genetic markers indicating stem- embryo is explanted in organ culture, new teeth are ness of progenitor cells, and much work is ongoing to initiated continuously during 19 days of culture elucidate the mechanisms of maintenance, prolifera- (Fig. 5C).23 This suggests that the capacity of continu- tion and differentiation of the dental stem cells. ous tooth formation, which was lost in the mouse Stem cells with abilities to generate differentiated during evolution, can be unlocked by increased Wnt dental tissues have been discovered in adult teeth, and signal activity in the epithelium. there is current research to characterize their proper- ties and functions.26 So far, it is not known whether these stem cells have the capacity to participate or Transfer of the laboratory findings to the clinic direct tooth morphogenesis. It is also noteworthy that Understanding the exact roles that individual genes no epithelial stem cells capable of producing dental play in tooth development may form the basis for epithelium have been found in adult teeth. Since teeth new ways to prevent and treat dental defects such as cannot be generated without epithelial cells, cells hypodontia. In addition, the mouse models generated exhibiting the capacity to generate dental epithelium for human dental aberrations will help to elucidate have to be found outside the teeth. Also, it may be their pathogenesis and the design of new treatments. challenging to use adult dental stem cells for regenera- There is already one potential treatment that may be tion purposes because their harvesting from a patient′s realized soon, namely, the prevention and cure of teeth may be problematic. Currently, reprogramming a X-linked HED, the ectodermal dysplasia syndrome patient′s other cells to become pluripotent stem cells caused by mutations in the EDA gene. The mouse (iPS cells) seems a more realistic approach. It may be model for this syndrome (Eda-/-) has similar pheno- possible in the future to programme the iPS cells to typic features as in human patients, including small or form dental epithelium and mesenchyme, but such missing teeth (Fig. 4B), lack of certain types of hair, protocols have not yet been discovered. Cells from and absence of sweat glands. The fact that Eda is a such dental cell lines might be used for tooth regenera- soluble signal molecule made it possible to test tion by following procedures based on the classical whether application of the Eda protein could compen- tissue recombination experiments demonstrating that sate for its lack in the mutant mice. Indeed, when the isolated tooth germ epithelium and mesenchyme can Eda protein was added in vitro to cultured Eda-/- form a tooth when recombined and transplanted.1 As embryonic skin explants it rescued the formation of proof of principle, it has been shown that teeth can the first hair follicles which otherwise fail to form in develop from dissociated cells derived from mouse the mutants.15 The effects of the Eda protein in vivo tooth germs.27 The cells were aggregated, and the epi- were more dramatic: prenatal or neonatal injections thelial and mesenchymal cells were combined and of recombinant Eda protein rescued the tooth, hair subsequently transplanted to the adult jaw where they and sweat gland phenotypes of the Eda mutant formed functional teeth. Obviously, more research on mice.24 Neonatal Eda protein injections had even tooth development and dental cell differentiation as more dramatic effects in dogs. The Eda-/- dogs have a well as on stem cell technologies is required before very severe tooth phenotype, characterized by absence the bioengineering of whole new teeth can become of most permanent premolars and incisors, and Eda feasible.28 protein rescued the development of all these teeth completely.25 Preparations have recently commenced DISCLOSURE in the USA for a clinical trial testing whether human X-linked HED can be prevented by neonatal injec- The author has no conflicts of interest to declare. tions of Eda protein with safety tests of the protein in affected adults. REFERENCES There are dreams that new teeth could be grown in the clinic to replace missing teeth in the future. This 1. Tummers M, Thesleff I. The importance of signal pathway modulation in all aspects of tooth development. J Exp Zool may become possible by novel cell-based technologies 2009;312B:309–319. combining current genetic and stem cell technologies 2. Vainio S, Karavanova I, Jowett A, Thesleff I. Identification of with accumulating knowledge on the mechanisms of BMP-4 as a signal mediating secondary induction between tooth morphogenesis. As described above, we already epithelial and mesenchymal tissues during early tooth develop- ment. Cell 1993;75:45–58. understand the language that cells use for communica- © 2013 Australian Dental Association 53 I Thesleff 3. Satokata I, Maas R. Msx1 deficient mice exhibit cleft palate 18. Liu F, Chu EY, Watt B, et al. Wnt/beta-catenin signaling directs and abnormalities of craniofacial and tooth development. Nat multiple stages of tooth morphogenesis. Dev Biol 2008;313: Genet 1994:348–356. 210–224. 4. Chen Y, Bei M, Woo I, Satokata I, Maas R. Msx1 controls 19. Juuri E, Jussila M, Seidel K, et al. Sox2 marks epithelial compe- inductive signaling in mammalian tooth morphogenesis. Devel- tence to generate teeth in mammals and reptiles. Development opment 1996;122:3035–3044. 2013;140:1424–1432. 5. Bei M. Molecular genetics of tooth development. Curr Opin 20. Juuri E, Saito K, Ahtiainen L, et al. Sox2+ stem cells contrib- Genet Dev 2009;19:504–510. ute to all epithelial lineages of the tooth via Sfrp5+ progeni- 6. O’Connell DJ, Ho JW, Mammoto T, et al. A Wnt-bmp feed- tors. Dev Cell 2012;23:317–328. back circuit controls intertissue signaling dynamics in tooth 21. J€arvinen E, Tummers M, Thesleff I. The role of the dental lam- organogenesis. Sci Signal 2012;5:ra4. ina in mammalian tooth replacement. J Exp Zool 2009;312B: 7. Dassule HR, Lewis P, Bei M, Maas R, McMahon AP. Sonic 281–291. hedgehog regulates growth and morphogenesis of the tooth. 22. Handrigan GR, Richman JM. A network of Wnt, hedgehog Development 2000;127:4775–4785. and BMP signaling pathways regulates tooth replacement in 8. H€a€ ar€ a O, Harjunmaa E, Lindfors P, et al. Ectodysplasin snakes. Dev Biol 2009;348:130–141. regulates activator-inhibitor balance in murine tooth develop- 23. J€arvinen E, Salazar-Ciudad I, Birchmeier W, Taketo MM, ment through Fgf20 signaling. Development 2012;139:3189– Jernvall J, Thesleff I. Continuous tooth generation in mouse is 3199. induced by activated epithelial Wnt/beta-catenin signaling. Proc 9. Jernvall J, Thesleff I. Tooth shape formation and tooth renewal: Natl Acad Sci USA 2006;103:18627–18632. evolving with the same signals. Development 2012;139:3487– 24. Gaide O, Schneider P. Permanent correction of an inherited 3497. ectodermal dysplasia with recombinant EDA. Nat Med 10. Kere J, Srivastava AK, Montonen O, et al. X-linked anhidrot- 2003;9:614–618. ic (hypohidrotic) ectodermal dysplasia is caused by mutation in 25. Casal ML, Lewis JR, Mauldin EA, et al. Significant correction a novel transmembrane protein. Nat Genet 1996;13:409–416. of disease after postnatal administration of recombinant ecto- 11. Vastardis H, Karimbux N, Guthua SW, Seidman JG, Seidman dysplasin A in canine X-linked ectodermal dysplasia. Am J CE. A human MSX1 homeodomain missense mutation causes Hum Genet 2007;81:1050–1056. selective tooth agenesis. Nat Genet 1996;13:417–421. 26. Han J, Menicanin D, Gronthos S, Bartold PM. Stem cells, tissue 12. Nieminen P. Genetic basis of tooth agenesis. J Exp Zool engineering and periodontal regeneration. Aust Dent J 2013; 2009;312B:320–342. doi: 10.1111/adj.12100 [Epub ahead of print]. 13. van den Boogaard MJ, Creton M, Bronkhorst Y, et al. Muta- 27. Oshima M, Mizuno M, Imamura A, et al. Functional tooth tions in Wnt10A are present in more than half of isolated regeneration using a bioengineered tooth unit as a mature organ hypodontia cases. J Med Genet 2012;49:327–331. replacement regenerative therapy. PLoS One 2011;6:e21531 14. Adaimy L, Chouery E, Megarbane H, et al. Mutation in 28. Lesot H, Hovorakova M, Peterka M, Peterkova R. Three- WNT10A is associated with an autosomal recessive ectodermal dimensional analysis of molar development in the mouse from dysplasia: the odonto-onycho-dermal dysplasia. Am J Hum the cap to bell stage. Aust Dent J 2013; doi: 10.1111/adj.12132 Genet 2007;81:821–828. [Epub ahead of print]. 15. Mikkola ML. Molecular aspects of hypohidrotic ectodermal dysplasia. Am J Med Genet A 2009;149A:2031–2036. Address for correspondence: 16. Mustonen T, Pispa J, Mikkola ML, et al. Stimulation of ecto- Professor Irma Thesleff dermal organ development by Ectodysplasin-A1. Dev Biol Institute of Biotechnology, POB 56 2003;259:123–136. 00014 University of Helsinki 17. Andl T, Reddy ST, Gaddapara T, Millar SE. WNT signals are required for the initiation of hair follicle development. Dev Cell Finland 2002;2:643–653. Email: [email protected] 54 © 2013 Australian Dental Association