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.

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.

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.

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.

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).

Contribution of 3-D computer-assisted reconstructions to the study of the initial steps of mouse odontogenesis.

The specific arrangement of mouse dentition in each dental quadrant (1-0-0-3) is supposed to result from the initiation of two independent dental laminae--one for the incisor and one for the three molars. In order to verify whether the adult mouse dental pattern really corresponds to the initial patterning, an analysis of development of the mouse antemolar part of the upper dental quadrant was performed in 10-13 day embryos using histological sections and computer-assisted 3-D reconstructions. Six primary dental laminae contributed to the formation of the upper incisor anlage, which is, therefore, a structure of multiple origin. In contrast to the lower diastema, where only a low epithelial band extended mesially from the first lower molar in 12-13 day embryos, in the upper diastema a dental lamina existed interconnecting transiently the incisor and molar anlagen and giving rise to 3 distinct epithelial rudiments. The rudiments exhibited growth retardation and regressed after reaching a maximum at the bud stage. Our results showed a discrepancy between the embryonic and adult dental patterns in the mouse upper jaw. The specific arrangement of the mouse dentition implied a reduction of the embryonic dental anlagen, which was achieved either by their integration into the one incisor primordium or regression in the prospective diastema. Odontogenesis in the mouse upper jaw provides a model of hypodontia of evolutionary origin, which can be employed in molecular studies of the control mechanisms of initiation, spatial organization and specific morphogenesis of teeth.

Multiple developmental origin of the upper incisor in mouse: histological and computer assisted 3-D-reconstruction studies.

Heads of 11-15-day-old mouse embryos were cut in frontal serial sections. Early development of the maxillary incisor was analyzed using series of thick (5 and 7 microns) and semi-thin (1 micron) frontal sections and computer assisted 3-D-reconstructions of the epithelial component. The enamel organ of the mouse maxillary incisor was found to be a complex structure of multiple origin, involving several epithelial anlagen--primary dental laminae--, which could hypothetically correspond to the 5 upper incisors of early mammals. The transitory existence of at once distinct and then fusing dental primordia could reflect heterochronic changes in ontogeny which might be related to phyletic trends.

Comparison of expression of the msx-1, msx-2, BMP-2 and BMP-4 genes in the mouse upper diastemal and molar tooth primordia.

The existence of transient putative tooth anlagen in the prospective mouse upper diastema region has been documented previously in morphological studies. By in situ hybridization we investigated the expression patterns of the msx-1, msx-2, BMP-2 and BMP-4 genes, supposed to regulate early tooth development, in day 10-14 mouse embryonic upper diastema and molar regions, using 49 series of frontal sections. On the basis of comparison of the temporo-spatial expression patterns in both diastemal and molar tooth primordia we conclude that each of the four genes was expressed at least for some period simultaneously and at a comparable developmental stage in the transient and persisting dental primordia. BMP-2 and BMP-4 expression was downregulated in the diastemal dental primordia during their regression starting at day 13. The temporo-spatial pattern of BMPs expression may be associated with the disappearance of diastemal rudiments. Contrary to the molar anlage, we did not detect msx-2 gene expression in the diastemal dental rudiments after the stage of epithelial thickening. The deficiency of the msx-2 gene products may play a role in the growth retardation of diastemal dental primordia resulting in their subsequent involution.

Apoptosis is involved in the disappearance of the diastemal dental primordia in mouse embryo.

Three transient dental primordia (D1, D2 and D3) exist in the upper diastema in mouse embryos and their regression is associated with the presence of cell death. In order to specify the type of cell death and its temporo-spatial distribution, staining with hematoxylin, supravital staining with Nile Blue, TUNEL method, electron microscopic analysis and computer assisted 3-D reconstructions were performed. These data demonstrated that apoptosis is involved in the disappearance of the diastemal dental rudiments. Apoptosis occurred first with prevalence in the buccal part of the epithelium of the diastemal dental primordia and extended later to the whole epithelium of the dental rudiments and the dental lamina interconnecting them with the incisor and molar epithelia. Cell death occurred only sporadically in the adjacent mesenchyme. The prospective upper diastema in mouse embryos may provide a model for studies of developmental determination of toothless areas in the jaw as well as a tool for analyses of regulatory mechanisms of programmed cell death in morphogenesis.

Mouse molar morphogenesis revisited by three dimensional reconstruction. I. Analysis of initial stages of the first upper molar development revealed two transient buds.

Early stages of tooth development in the maxillary cheek region in the mouse were investigated by combined analysis of histological sections, computer assisted 3D reconstructions and morphometry. In ED 12.5 embryos, 3D reconstructions revealed an accessory epithelial bud (R1) and a large bud (R2), which appeared as a single bud-shaped epithelium in frontal sections. This developmentally most advanced dental epithelium in the mouse embryonic maxilla until ED 13.5, generally considered as the bud of the first molar, regressed during later development. Meanwhile the bud and cap of the first upper molar originated more posteriorly, from ED 13.5. The regression of R1 and R2 was associated with epithelial apoptosis. Apoptotic cells and bodies were apparent on sections in the R1 epithelium from ED 12.5. The R2 epithelium maintained the large bud-shaped appearance on sections, representing the largest part of the dental epithelium in the maxillary cheek region until ED 14.0; apoptoses were detected there as late as from ED 13.5. During regression, the R2 rudiment was transformed into the medial and lateral epithelial ridges, posteriorly in continuity with the arising cap of the first molar. The reduced R1 epithelium seemed to contribute to the medial ridge. These results should be taken into consideration in the interpretation of early odontogenesis in the upper jaw in the mouse. The interesting problem of the identification of tooth homology of the rudiments should be elucidated by further comparative morphological and paleontological investigations.

Mouse molar morphogenesis revisited by three-dimensional reconstruction. II. Spatial distribution of mitoses and apoptosis in cap to bell staged first and second upper molar teeth.

Tooth morphogenesis is a complex multifactorial process in which differential mitotic activities and cell death play important roles. Upper first (m1) and second (m2) molars from mouse embryos were investigated from early cap to bell stage. m2 differed from m1 by delayed origin of the enamel grooves delimiting the protrusion of the cap bottom towards the dental papilla, and retardation of the enamel knot formation. The width of the m2 enamel organ was conspicuously smaller during cap formation and length remained smaller throughout the period of observation. Formation of the cap depression was comparable in m1 and m2, however margins delimiting the enamel organ cavity arose in m1 and m2 as mirror images. Attempts were made to correlate changes in the distribution of apoptotic cells and bodies and/or mitoses with morphogenesis. These cellular activities were recorded from histological sections and represented in space using computer-assisted three-dimensional reconstructions. Mitoses in the epithelial compartment were associated with the development of the cervical loop. In the mesenchyme of m1 at early bell stage, a postero-anterior increasing gradient of mitoses was observed which might be correlated with the anterior growth of the molar. Cells in the enamel knot demonstrated a high level of apoptosis, retarded in m2, but absolutely no division. Apoptotic processes were also involved in the anterior delimitation of the m1 epithelium. Apoptosis might correspond to the programmed destruction of cells whose function had to be suppressed or whose potential activity had to be avoided.

The presence of rudimentary odontogenic structures in the mouse embryonic mandible requires reinterpretation of developmental control of first lower molar histomorphogenesis.

In the mouse embryonic maxilla, rudimentary tooth primordia have been identified, which can be mistaken for the first upper molar. In order to determine whether such a situation might exist in the lower jaw as well, tooth development was investigated in the mouse mandibular cheek region during ED 12.5-15.0. A combination of histology, morphometry and computer-aided 3D reconstructions demonstrated the existence of rudimentary dental structures, whose gradual appearance and regression was associated with the segmental progress of odontogenesis along the mesio-distal axis of the jaw: 1) At ED 12.5, the mesial segment (MS) was the most prominent part of the dental epithelial invagination. It included an asymmetrically budding dental lamina. The MS, although generally mistaken for the lower first molar (M1, primordium, regressed and did not finally participate in M1 cap formation. 2) At ED 13.5, a wide dental bud (called segment R2) appeared distally to the MS. Although the R2 segment transiently represented the predominant part of the dental epithelium at ED13.5, it participated only in the formation of the mesial end of the M1 cap. 3) The top of the R2 segment at ED13.5 was not the precursor of the enamel knot (EK), contrary to what has been assumed. 4) The central segment of the M1 cap as well as the EK developed later and distally to the R2 segment. 5) Time-space specific apoptosis correlated with the retardation in growth of the R2 segment as well as with strong regressive changes in the epithelium situated mesially to it. These highlight the need to reinterpret current molecular data on early M1 development in the mouse in order to correlate the expression of signalling molecules with specific morphogenetic events in the appropriate antemolar or molar segments of the embryonic mandible.

Alterations in the incisor development in the Tabby mouse.

The X-linked tabby (Ta) syndrome in the mouse is homologous to the hypohidrotic ectodermal dysplasia (HED) in humans. As in humans with HED, Ta mice exhibit hypohidrosis, characteristic defects of hairs and tooth abnormalities. To analyze the effects of Ta mutation on lower incisor development, histology, morphometry and computer-aided 3D reconstructions were combined. We observed that Ta mutation had major consequences for incisor development leading to abnormal tooth size and shape, change in the balance between prospective crown- and root-analog tissues and retarded cytodifferentiations. The decrease in size of Ta incisor was observed at ED13.5 and mainly involved the width of the tooth bud. At ED14.5-15.5, the incisor appeared shorter and narrower in the Ta than in the wild type (WT). Growth alterations affected the diameter to a greater extent than the length of the Ta incisor. From ED14.5, changes in the shape interfered with the medio-lateral asymmetry and alterations in the posterior growth of the cervical loop led to a loss of the labio-lingual asymmetry until ED17.0. Although the enamel organ in Ta incisors was smaller than in the WT, a larger proportion of the dental papilla was covered by preameloblasts-ameloblasts. These changes apparently resulted from reduced development of the lingual part of the enamel organ and might be correlated with a possible heterogeneity in the development of the enamel organ, as demonstrated for upper incisors. Our observations suggest independent development of the labial and lingual parts of the cervical loop. Furthermore, it appeared that the consequences of Ta mutation could not be interpreted only as a delay in tooth development.

Morphogenesis of the lower incisor in the mouse from the bud to early bell stage.

The development of the lower incisor in the mouse was investigated from histological sections using computer-aided 3D reconstructions. At ED 13.0, the incisor was still at the bud stage. At ED 13.5, the initial cap was delimited by a short cervical loop, the development of which proceeded on the labial side, but was largely retarded on the medial side. This difference was maintained up to ED 15.0. From ED 16.0, the bell stage was achieved. Metaphases had a ubiquitous distribution both in the enamel organ and in the dental papilla from the bud to early bell stage. Apoptosis gradually increased in the mesenchyme posteriorly to the labial cervical loop from ED 13.5 to 14.0 and then disappeared; this apoptosis was not related to the posterior growth of the incisor. From ED 13.5, a high apoptotic activity was observed in the stalk. A focal area of apoptosis was observed at ED 13.5 in the enamel organ, approaching the epithelio-mesenchymal junction at the future tip of the incisor. There, the inner dental epithelium formed a bulbous protrusion towards dental papilla, reminiscent of the secondary enamel knot of mouse molars. This epithelial protrusion was still maintained at the bell stage. The enamel knot in the incisor demonstrated specific features, different from those characterizing the enamel knot in the molar: the concentric arrangement of epithelial cells was much less prominent and the occurrence of apoptosis was very transitory in the incisor at ED 13.5. The disappearance of the enamel knot despite a low apoptotic activity and the maintenance of the protrusion suggested a histological reorganization specific for rodent incisor.

Mouse molar morphogenesis revisited by three-dimensional reconstruction. III. Spatial distribution of mitoses and apoptoses up to bell-staged first lower molar teeth.

Computer-assisted 3D reconstructions were used to follow the development of the embryonic mouse first lower molar (M1). At ED 12.5, the thickening of the oral epithelium, which was thought to correspond to the molar dental lamina, regressed in its anterior part as a result of apoptosis. Only the posterior part later gave rise to molars. The transition to the cap stage entailed medial and lateral extensions of the dental epithelium. The growth and histo-morphogenesis of the enamel organ as well as cervical loop formation proceeded more rapidly in the anterior part of the M1 during the cap and early bell stages producing significant morphological differences along the antero-posterior axis. Apoptosis was temporarily intensive in the anterior part of the bud- and cap-shaped epithelium and thus pointed domains which do not participate in the formation of the final M1 enamel organ. In the well-formed cap, apoptoses displayed maximum concentration in the enamel knot (EK). No increase in the number of metaphases could be detected in the vicinity of the EK. Mitoses were distributed throughout the epithelial compartment until cap stage and then mainly concentrated in the inner dental epithelium at the early bell stage. At this later stage, either lateral views or thick virtual sections performed in the reconstruction demonstrated a clear cut distribution of mitoses and apoptoses in the enamel organ. At the early bell stage, mitoses in the mesenchyme demonstrated an increasing postero-anterior gradient.

Initial features of the inner dental epithelium histo-morphogenesis in the first lower molar in mouse.

First lower molar development in the mouse was investigated from the cap to early bell stage using histology, morphometry, TEM and 3D reconstructions. This period was characterized by the histogenesis of the enamel organ (EO), folding of the epithelio-mesenchymal junction and growth of the tooth. The histogenesis of the EO and appearance of the enamel knot (EK) were initiated at the early cap stage (ED14). From ED14 to ED15, the anterior and posterior extension of the EK was very prominent whilst the length of the enamel organ did not substantially change. The EK appeared as a dynamic and transitory histological structure including dying and replacement cells. At ED16, the folding of the IDE, which extended over the anterior two thirds of the molar, was the first sign of cuspidogenesis. It was accompanied by a local remodeling of the basement membrane (BM): IDE cells involved in this folding transitorily lost contact with the BM which formed a loop in the mesenchyme. During this period, the growth of the lower M1 along the antero-posterior axis was restricted to the posterior part of the molar. Histogenesis occurred in the whole EO, whilst initial cuspidogenesis was limited to the anterior part of the tooth. Distinct cell populations were thus involved in different contemporary processes leading to changes in the cell density in the mesenchyme, in the mitotic activity, in cell-shape, and cell-matrix interactions in the IDE, and remodeling of the BM where both epithelium and mesenchyme might participate.

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.

Crown morphology and pattern of odontoblast differentiation in lower molars of tabby mice.

The Tabby mutation leads to abnormal crown morphology in the developing molars. To identify cusps which were altered in number, size, and position in the first lower molars of mutant mice, we analyzed the patterning of odontoblast differentiation using morphological criteria on serial sections and 3D reconstructions. In wildtype mice, polarized and functional odontoblasts were first observed in the median L2 and B2 cusps, then in the distal cusps L3 and B3, and finally in L1, B1, and 4. In Tabby mice, terminal differentiation of odontoblasts was retarded by 24-36 hours compared with wild-type mice. Polarized odontoblasts first appeared in the most mesial part of the tooth and progressively extended distally. The mesial part of the M1 in Tabby fetuses may correspond to the L2, B2 area from wild-type mice. The ante-molar dental primordium observed in some samples would thus represent remnants of cusps L1 and B1.