Excess NF-κB induces ectopic odontogenesis in embryonic incisor epithelium.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKß, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkß). K5-Ikkß mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkß mice. The supernumerary incisors in K5-Ikkß mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

Three-dimensional analysis of the early development of the dentition.

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.

Three-dimensional analysis of molar development in the mouse from the cap to bell stage.

During four days of prenatal development in the mouse, the morphology of the first lower molar moves from the early cap to the bell stage. Five phenomena characterize this period: growth of the tooth germ; development of the cervical loop; histogenesis of the enamel organ; folding of the epithelial-mesenchymal junction associated with cusp formation; and change in cellular heterogeneity in the mesenchyme. All these processes are controlled by epithelial-mesenchymal interactions. These complex histo-morphogenetic events have been documented using histological sections and 3D reconstructions. When combined with functional tests in vitro, this approach allowed searching for possible relationships between simultaneous changes occurring in both the epithelial and ecto-mesenchymal compartments. Parallel changes that occur in the two tissues could result from different mechanisms, as illustrated by the increasing number of pre-odontoblasts and pre-ameloblasts during crown growth. Cell division was involved mainly in the ecto-mesenchyme, while proliferation and cell re-organization occurred in the inner dental epithelium. 3D reconstructions also raised still unsolved questions, such as the possible relationship between cusp size and spatial specification of cell kinetic parameters, changes in cell position within the inner dental epithelium, and tracing cell migration in the mesenchyme during development.

Evc regulates a symmetrical response to Shh signaling in molar development.

Tooth morphogenesis involves patterning through the activity of epithelial signaling centers that, among other molecules, secrete Sonic hedgehog (Shh). While it is known that Shh responding cells need intact primary cilia for signal transduction, the roles of individual cilia components for tooth morphogenesis are poorly understood. The clinical features of individuals with Ellis-van Creveld syndrome include various dental anomalies, and we show here that absence of the cilial protein Evc in mice causes various hypo- and hyperplasia defects during molar development. During first molar development, the response to Shh signaling is progressively lost in Evc-deficient embryos and, unexpectedly, the response consistently disappears in a buccal to lingual direction. The important role of Evc for establishing the buccal-lingual axis of the developing first molar is also supported by a displaced activity of the Wnt pathway in Evc mutants. The observed growth abnormalities eventually manifest in first molar microdontia, disruption of molar segmentation and symmetry, root fusions, and delayed differentiation. Analysis of our data indicates that both spatially and temporally disrupted activities of the Shh pathway are the primary cause for the variable dental anomalies seen in patients with Ellis-van Creveld syndrome or Weyers acrodental dysostosis.

Extension of orofacial cleft size and gestational bleeding in early pregnancy.

OBJECTIVE: The oronasal cavity in humans develops during embryonic day 30-60. There are three critical periods when this process can be affected, resulting in a specific type of orofacial cleft: cleft lip (CL), cleft palate (CP), or most serious, total cleft lip+palate (CLP). We assessed whether gestational bleeding during early pregnancy might act to produce a non-specific worsening of embryo status resulting in extension of the basic cleft type (CL or CP) into more serious CLP. STUDY DESIGN: In a group of the child patients with orofacial clefts, the cleft spectrum was correlated with first trimester gestational bleeding reported by the mother. Data were also related to the gender of patients, hereditary factors and additional malformations. RESULTS: Among 2524 mothers who gave birth to babies with an orofacial cleft in the Czech Republic during 1983-2009, 253 (10.0%) had gestational bleeding. Among the children with an orofacial cleft, 497 (19.7%) had an orofacial cleft among relatives and 297 (11.8%) exhibited an additional congenital malformation. In comparison with mothers without bleeding, there was significant increase of children with CLP (p < 0.01) at the expense of children with CP, whose number significantly decreased (p < 0.01) in the bleeding mothers. In the group of children with clefts among relatives we did not find any significant change associated with bleeding. The maternal bleeding was more frequent in children with additional malformations, but this difference was not significant (p = 0.112). CONCLUSION: We hypothesize that size/extent and therefore seriousness of orofacial cleft might increase as a consequence of hypoxia resulting from gestational bleeding.

A case of conjoined twin's cephalothoracopagus janiceps disymmetros.

Conjoined twins are rare variants of monozygotic twins, which result from an incomplete late division of the embryonic disk. Here we report the rarest case of conjoined twins - the male cephalothoracopagus janiceps disymmetros - born in prenatal week 30, from the archive of the Department of Teratology of the Institute of Experimental Medicine AS CR in Prague. The crown-rump length of each twin, 21cm, corresponded to prenatal week 22 in a normal gravidity. The head, chest and upper portion of the abdomen of the twins were fused. The anatomical features of these extremely rare conjoined twins and the observed external anomalies as a narrow nose with a single nostril, male hypoplastic genitalia, partially duplicated sella turcica, spina bifida and further abnormalities are described and documented.

The lack of isolated palatal clefts in Czech Gypsies.

Orofacial clefts are usually divided into three basic types: isolated cleft lip (CL), cleft lip and palate (CLP) and isolated cleft palate (CP). The incidence of specific cleft types in a population and their relative numbers show specific differences between ethnic groups and races. However, there are no available data about the incidence and relative numbers of orofacial cleft types (CL, CLP, CP) in the gypsy ethnic group. The aim of this study was to compare relative numbers of specific types of orofacial clefts between the Czech gypsy and non-gypsy populations. We conducted a retrospective epidemiological study using a set of all living patients with orofacial clefts born in the Czech Republic from 1964 until 2002. The cleft patients were subdivided into three groups: 5304 non-gypsy children, both parents of whom were non-gypsies (NN), 98 gypsy children, both parents of whom were gypsies (GG) and 18 children with one parent non-gypsy and one parent gypsy (NG). The relative number of isolated CP was 37.1% in NN children. However, the relative number of CP was significantly reduced to 5.1% (P < 0.01) in the GG group. Conversely, the relative number of CLP was higher (P < 0.01) in the GG group (62.2%) in comparison to the NN group (39.2%). The tendency to decrease in the relative number of CP and increase in the relative number of CLP was also apparent in the NG group, but not so well expressed. We hypothesize that the decrease in CP and increase in CLP and CL in gypsies might be caused by their genetic predis-position to CL. Since the CP originates later than CL during embryonic development, some CP arise in embryos with already existing CL giving rise to CLP. Consequently, the missing isolated CP might be hidden in the group of CLP patients postnatally.

The Prx1 homeobox gene is critical for molar tooth morphogenesis.

The paired-related homeobox genes, Prx1 and Prx2, encode transcription factors critical for orofacial development. Prx1(-/-)/Prx2(-/-) neonates have mandibular hypoplasia and malformed mandibular incisors. Although the mandibular incisor phenotype has been briefly described (ten Berge et al., 1998, 2001; Lu et al., 1999), very little is known about the role of Prx proteins during tooth morphogenesis. Since the posterior mandibular region was relatively normal, we examined molar tooth development in Prx1(-/-)/Prx2(-/-) embryos to determine whether the tooth malformation is primary to the loss of Prx protein or secondary to defects in surrounding tissues. Three-dimensional (3D) morphological reconstructions demonstrated that Prx1(-/-)/Prx2(-/-) embryos had molar malformations, including cuspal changes and ectopic epithelial projections. Although we demonstrate that Prx1 protein is expressed only mesenchymally, 3D reconstructions showed important morphological defects in epithelial tissues at the cap and bell stages. Analysis of these data suggests that the Prx homeoproteins are critical for mesenchymal-epithelial signaling during tooth morphogenesis.

Origin of the deciduous upper lateral incisor and its clinical aspects.

The upper lateral incisor in humans is often affected by dental anomalies that might be explained developmentally. To address this question, we investigated the origin of the deciduous upper lateral incisor (i2) in normal human embryos at prenatal weeks 6-8. We used serial frontal histological sections and computer-aided 3D reconstructions. At embryonic days 40-42, two thickenings of the dental epithelia in an "end-to-end" orientation were separated by a groove at the former fusion site of the medial nasal and maxillary processes. Later, these dental epithelia fused, forming a continuous dental lamina. At the fusion site, i2 started to develop. The fusion line was detectable on the i2 germ until the 8th prenatal week. The composite origin of the i2 may be associated with its developmental vulnerability. From a clinical aspect, a supernumerary i2 might be a form of cleft caused by a non-fusion of the dental epithelia.

Increased apoptosis during morphogenesis of the lower cheek teeth in tabby/EDA mice.

In wild-type (WT) mice, epithelial apoptosis is involved in reducing the embryonic tooth number and the mesial delimitation of the first molar. We investigated whether apoptosis could also be involved in the reduction of tooth number and the determination of anomalous tooth boundaries in tabby (Ta)/EDA mice. Using serial histological sections and computer-aided 3D reconstructions, we investigated epithelial apoptosis in the lower cheek dentition at embryonic days 14.5-17.5. In comparison with WT mice, apoptosis was increased mainly mesially in Ta dental epithelium from day 15.5. This apoptosis showed a similar mesio-distal extent in all 5 morphotypes (Ia,b,c and IIa,b) of Ta dentition and eliminated the first cheek tooth in morphotypes IIa,b. Apoptosis did not appear to play any causal role in positioning inter-dental gaps. Analysis of the present data suggests that the increased apoptosis in Ta mice is a consequence of impaired tooth development caused by a defect in segmentation of dental epithelium.

The supernumerary cheek tooth in tabby/EDA mice-a reminiscence of the premolar in mouse ancestors.

OBJECTIVE: A supernumerary cheek tooth occurs mesially to the first molar in tabby/EDA (Ta) mice affected by hypohidrotic ectodermal dysplasia. The supernumerary tooth (S) has been hypothetically homologized to the premolar, which has disappeared during mouse evolution. DESIGN: This hypothesis was tested using available morphological data on the lower cheek teeth in wild type (WT) and Ta mice. RESULTS: The presence of S is accompanied by a reduction in the mesial portion of the M(1) in mutant mice. 3D reconstructions suggest that the S in Ta homo/hemizygous embryos originates from a split off the mesial portion of the first molar (M(1)) cap. In WT embryos, two vestigial tooth primordia are transiently distinct in front of the M(1). The distal vestige has the form of a wide bud and participates during the development of the mesial portion of the M(1). This bud has been homologized with the vestigial primordium of the fourth premolar of mouse ancestors. The premolar disappearance coincided with a mesial lengthening of the M(1) during mouse evolution. The incorporation of the distal premolar vestige into the mesial part of the M(1) in WT embryos can be regarded as a repetition of the premolar disappearance during evolution. CONCLUSION: : Ontogenetic and phylogenetic data support that the S in Ta mice arises due to the segregation of the distal premolar vestige from the molar dentition and thus represents an evolutionary throwback (atavism).

[Effect of the Tabby mutation on the dentition of mice]. / Effets de la mutation Tabby sur la dentition chez la souris.

The X-linked hypohidrotic ectodermal dysplasia in man leads to dental defects and is homologous to the Tabby (Ta) mutation in mouse. We currently investigate the effects of the Ta mutation on odontogenesis. The incisor germ of Ta showed an abnormal size and shape, a change in the balance between prospective crown- and root-analogue tissues and retarded cytodifferentiation. Although the enamel organ in Ta incisors was smaller, a larger proportion of the dental papilla was covered by preameloblasts-ameloblasts. The independent development of the labial and lingual parts of the enamel organ in rodent lower incisor might reflect their heterogeneous origin, as demonstrated for the upper incisor. The mandibular cheek dentition in Ta mice exhibits large variations classified in five morphotypes, based on the tooth number, shape, size and position. In Ta embryos, the mesio-distal extent of the dental epithelium was similar to that in WT, but its segmentation was altered. These morphotypes could be explained by a tentative model suggesting that 1) the positions of tooth boundaries differ in Ta and WT molars and among the Ta morphotypes; 2) the tooth patterns are determined by the distal boundary of the most mesial tooth primordium while the distal teeth take advantage of the remaining dental epithelium; 3) one tooth primordium in Ta mice might derive from adjacent parts of two primordia in WT.

Tooth morphogenesis and pattern of odontoblast differentiation.

The terminal differentiation of odontoblasts is controlled by the inner dental epithelium (IDE) and occurs according to a tooth-specific pattern. It requires temporospatially regulated epigenetic signaling and the expression of specific competence. The patterning of cusp formation was compared with that of odontoblast differentiation in the first lower molar in mice. Histology, immunostaining, and three dimensional reconstructions were completed by experimental approaches in vitro. The mesenchyme controls the pattern of cusp formation. During the cap-bell transition in the molar, a subpopulation of nondividing IDE cells from the enamel knot (EK) undergo a tooth-specific segregation in as many subpopulations as cusps will form. Epithelial cell-basement membrane interactions seem to be involved in the segregation of EK cells. The timing and spatial pattern of the segregation of EK cells correlate with cusps formation. However, the temporal pattern of odontoblast terminal differentiation is different. This discrepancy might result from cusp-specific differences either in the timing of the initiation of odontoblast terminal differentiation and/or in cell proliferation kinetics.

Development of the vestigial tooth primordia as part of mouse odontogenesis.

The mouse functional dentition comprises one incisor separated from three molars by a toothless diastema in each dental quadrant. Between the incisor and molars, the embryonic tooth pattern also includes vestigial dental primordia, which undergo regression involving apoptosis in their epithelium. Apoptosis appears to play an important role in achieving the specific tooth pattern in the mouse. We documented similarities in the folding mechanism allowing the formation of the dental lamina in mice as well as in reptiles. While further budding on this dental lamina gives rise to many individual simple tooth primordia in crocodiles and lizards, budding morphogenesis of several simple tooth primordia appears to be integrated in the mouse, giving rise to enamel organs of a complex nature. The differentiation of a mammalian tooth germ during both ontogeny and phylogeny might thus include the concrescence (connation) of more primordia, putatively corresponding to simple teeth in mammalian ancestors.

Body weight in mouse embryos specifies staging of tooth development.

Great intra- and interlitter variation in morphological stages is known to exist among mouse embryos of the same strain at a similar chronological stage. With the aim of searching for an easily measurable parameter that correlates well with tooth development, the morpho- and histodifferentiation of teeth were compared in embryos classified according to the embryonic day (ED) specified by wet body weight. The embryos and fetuses were harvested at 12-h intervals from ED12.5 until birth, weighed, fixed, and processed histologically. The tooth age was determined according to the developmental progress of the first molar, evaluated based on morpho- and histodifferentiation criteria. The data documented a better correlation between tooth morpho-histodifferentiation with the age/weight staging than with only the embryonic day. The cyto-differentiation of odontoblasts exhibited a high interlitter variability and was similar within specimens of the same litter, regardless of their body weight differences.

Cell-cell and cell-matrix interactions during initial enamel organ histomorphogenesis in the mouse.

Relationships between cell-cell/cell-matrix interactions and enamel organ histomorphogenesis were examined by immunostaining and electron microscopy. During the cap-bell transition in the mouse molar, laminin-5 (LN5) disappeared from the basement membrane (BM) associated with the inner dental epithelium (IDE), and nondividing IDE cells from the enamel knot (EK) underwent a tooth-specific segregation in as many subpopulations as cusps develop. In the incisor, the basement membrane (BM) in contact with EK cells showed strong staining for LN5 and integrin alpha 6 beta 4. LN5 seems to provide stable adhesion, while its proteolytic processing might facilitate cell segregation. In both teeth, immunostaining for antigens associated with desmosomes or adherens junctions was similar for EK cells and neighboring IDE cells. Outside the EK, IDE cell-BM interactions changed locally during the initial molar cusp delimitation and on the labial part of the incisor cervical loop. Conversely, cell-cell junctions stabilized the anterior part of the incisor during completion of morphogenesis. Time and space regulation of cell-matrix and cell-cell interactions might thus play complementary roles in allowing plasticity during tooth morphogenesis and stabilization at later stages of epithelial histogenesis.

Postnatal lower jaw dentition in different phenotypes of tabby mice.

The tabby (Ta) syndrome in mouse is homologous to human anhidrotic ectodermal dysplasia, including defective development of hair, teeth, and glands. To complete the available data on the functional dentition in the Ta mice, we analyzed the mandibular cheek teeth in 261 postnatal specimens arranged in several phenotype/genotype groups: 51 Ta-hemizygous males, 56 Ta-homo/hemizygous females, 64 Ta-heterozygous females, and 40 and 50 wild-type control males and females, respectively. We evaluated tooth number, size, shape and eruption and compared these parameters in the different groups. In any individual group of Ta mice, there was variability mainly in the size and shape of the most mesial tooth and in the tooth patterns. The incidence of a reduction in tooth number in homozygous and hemizygous mice was dependent on the breeding scheme.

Different morphotypes of functional dentition in the lower molar region of tabby (EDA) mice.

OBJECTIVES: To sort and classify the highly variable lower molar dentition in tabby (Ta) mice postnatally. The Ta syndome is homologous to the anhidrotic (hypohidrotic) ectodermal dysplasia (EDA) in human and includes severe developmental defects of teeth, hair and sweat glands. DESIGN: Analysis of tooth shape and cusp pattern and measurement of the mesio-distal crown length. SETTING AND SAMPLE POPULATION: Institute of Experimental Medicine, Academy of Sciences, Prague. Fixed heads of 107 tabby (Ta) homozygous and hemizygous mice and 90 wild type mice aged from post-natal day 11 to adulthood, collected during 1995-2001. OUTCOME MEASURE: Identification of distinct morphotypes of Ta dentition. Reduced tooth length in Ta teeth and specific differences in tooth length between distinct morphotypes. RESULTS: The variable dentitions in the lower molar region of Ta mice were classified in two basic morphotypes I and II. The morphotype I was further subdivided into particular morphotypes Ia, Ib and Ic. Proportion of the basic morphotypes I and II was different in the offspring of heterozygous (84% and 12%) compared with homozygous + hemizygous (45% and 52%) mothers. The proportions of particular morphotypes within a basic morphotype were similar in both offspring groups. CONCLUSION: The identification of the distinct morphotypes made possible to classify the structural variability of the mandibular functional dentition in Ta mice.

Different morphotypes of the tabby (EDA) dentition in the mouse mandible result from a defect in the mesio-distal segmentation of dental epithelium.

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.