This study aimed to evaluate the disturbances in normal coronal suture development resulting in craniosynostosis, a congenital disorder in which the calvarial sutures close prematurely. Craniosynostosis syndromes can be caused by mutations in the genes encoding for the fibroblast growth factor receptors (FGFRs) 1, 2, and 3. These gain-of-function mutations cause the transcribed receptor to be constitutively activated. To mimic this genetic defect, fibroblast growth factor (FGF) 2 or 4 was administered near the developing coronal suture in normal mouse embryos through ex utero surgery. The effect on apoptosis and bone differentiation, as collagen type I expression and mineralization, within the FGF-exposed coronal suture was investigated through (immuno)histochemical staining. An increase in the number of apoptotic cells together with ectopic collagen type I expression within the suture and accelerated mineralization followed FGF application. Macroscopically, this presented as a synostotic coronal suture. These results suggest that both apoptosis and differentiation are two processes that are simultaneously implicated in synostosis of the coronal suture in case of a FGFR-related craniosynostosis.
Oculo-auriculo-vertebral (OAV) spectrum or Goldenhar syndrome is a complex and heterogeneous condition characterized by hemifacial microsomia (unilateral ear abnormalities and ipsilateral mandibular hypoplasia) as well as vertebral anomalies and epibulbar dermoids or lipodermoids. Although most cases of OAV spectrum are sporadic, both autosomal recessive and autosomal dominant inheritance have been reported. Furthermore, the association of OAV spectrum with different types of chromosomal abnormalities has been described. We present a premature newborn delivered after 36 weeks of gestation, whose birth weight was 2,100 g, birth length 43 cm and head circumference 32.5 cm. OAV spectrum with associated axial skeleton anomalies, eventration of the right hemidiaphragm, accessory spleen, unlobulated right lung, agenesis of right kidney, right ovary and right uterine horn, and partial agenesis of corpus callosum were found. She was the second child of unrelated parents, who have a healthy boy. Both parents refused chromosomal analysis of their peripheral blood. Trypsin G-banding and C-banding chromosomal analysis of the patient's peripheral blood revealed a pericentric inversion of chromosome 9 with break points at p11 and q13. This may be a coincidental or causal finding.
The secondary palate formation in mouse has been associated with the period of fast growth of the mandible from embryonic days (ED) 13.0 to 16.0. During that time, the incisors and first molars develop from the bud to the bell stage. We investigated the position and growth of the tooth during prenatal elongation of the lower and upper jaws, and searched for the developmental stage when alignment of opposing teeth was achieved. Computer-aided 3D representations allowed us to represent the position of incisors and molars in the embryonic head from ED 13.5 to 18.0 on the basis of data obtained from histological sections. The atlas-hypophysis connection exhibited minimum change in length and orientation during the prenatal period, and thus was used as a reference line. The length of the teeth was calculated from 3D data. The upper first and second molars were longer than the lower ones. When viewed from the upper side, the upper and lower molar primordia were parallel from ED 13.5 to 15.0. During this period, the upper molars had a more lateral position than the lower ones. This situation was maintained in the anterior extremity of the first molars at later stages, while the posterior part of the upper and lower molar epithelia reached opposition in the medio-lateral direction from ED 16.0. The lower incisors exhibited an apparently backward position when compared to the upper incisors at earlier stages. However, the distance between the prospective anterior tips of the opposing incisors gradually decreased. The part of Meckel's cartilage associated with the lower dental quadrant elongated more than 3-fold from ED 13.5 to 18.0, and the lower jaw grew faster than the upper one. This difference resulted from the fast growth of the lower diastema from ED 14.0 to 18.0. The different growth speeds of the upper and lower jaws did not change the relative antero-posterior adjustment of the upper and lower molars, but contributed to achieving the opposition of the gnawing ends of the incisors.
Cleidocranial dysplasia (CCD), which is caused by mutations of the core binding factor alpha 1 (CBFA1)/runt-related gene 2 (Runx2), is an autosomal, dominantly inherited disorder of high penetrance affecting skeletal ossification and tooth development. Recently, we found a novel frameshift mutation 383-T-insertion (S128F) in exon 3 in the CBFA1 gene of a Japanese classic CCD patient. We describe our detailed investigation of the patient with CCD associated with the CBFA1 mutation. The patient showed the characteristic expression of CCD, such as dysplasia of the clavicles, patent fontanelles, short stature, impacted supernumerary teeth, and delayed eruption of the permanent teeth. In addition to these characteristics, orthopantomography delayed ossification of the mandibular symphysis and a three-dimensional computed tomograph (3D-CT) analysis showed hypoplasia of the zygomatic arch. Furthermore, the acellular cementum of an impacted supernumerary tooth was absent in this patient. Thus, the CBFA1 mutation was critical for the pathogenesis of CCD in this patient.
The molecular biology of the homeobox genes MSX1 and MSX2 is reviewed. In a selective type of tooth agenesis, an MSX1 G --> C transversion results in a missense mutation Arg31Pro. The phenotype is due to haploinsufficiency. Boston-type craniosynostosis involves an MSX2 C --> A transversion, resulting in a missense mutation Pro7His. Three different mutations on MSX2 cause parietal foramina by haploinsufficiency. These mutations, which result in decreased parietal ossification, are in marked contrast to the gain-of-function mutation for Boston-type craniosynostosis, which results in increased sutural ossification.
The objective of this study was to investigate bone mineral density (BMD) in the growing human mandible by dual-energy X-ray absorptiometry, comparing densitometric data with the nature of the dentition and hence, indirectly, with variation of mechanical stresses. Postero-anterior scans were performed on 58 dried mandibles (between 15 months and 14 years) by a Hologic QDR 1000 X-ray densitometer with "Ultra-Hi-Resolution" software (Hologic Inc., Waltham, MA). The analyses were performed on a polygonal area corresponding to the infero-median region of the mandibular body, and divided into three sections. The sample was divided into two groups according to the appearance of the first permanent molar. A high correlation is detectable between BMD values and age in the group with decidual dentition, while BMD values are significantly less correlated with age in the group with mixed dentition. There are no significant differences between the BMD values of males and females.
The development of functional teeth in the mouse has been widely used as a model to study general mechanisms of organogenesis. Compared with other mammals, in which three incisors, one canine, four premolars, and three molars may occur even in each dental quadrant, the mouse functional dentition is strongly reduced. It comprises only one incisor separated from three molars by a toothless gap diastema - at the location of the missing teeth. However, mouse embryos also develop transient vestigial dental primordia between the incisor and molar germs in both the upper and lower jaws. These rudimental structures regress, and epithelial apoptosis is involved in this process. The existence of the vestigial dental structures allowed a better assessment of the periodicity in the mouse dentition, which extends opportunities for the interpretation of molecular data on tooth development. We compared the dentition development with tentative models of budding morphogenesis in other epithelial appendages lungs and feathers. We suggested how developmental control by signaling molecules, including bone morphogenetic protein (Bmp), sonic hedgehog (Shh), and fibroblast growth factor (Fgf), can be similarly involved during budding morphogenesis of dentition and other epithelial appendages, We propose that epithelial apoptosis plays an important role in achieving specific features of dentition, whose development involves both budding and its more complex variant branching. The failure of segregation of the originating buds supports the participation of the concrescence of several tooth primordia in the evolutionary differentiation of mammalian teeth.
The purpose of the present study was to elucidate the relationship between fusions in the primary dentition and the occurrence of agenesis in the succedaneous permanent dentition in a Danish child population and to elucidate this relationship from the recently described normal embryological development of the anterior parts of the human maxilla and mandible. The material included radiographs, either as intraoral film or as orthopantomograms from a total of 19 primary dentitions with a total of 21 fusions. Radiographs of the permanent dentition in the fusion regions were available for all 19 dentitions. Of 21 fusions, a total of 20 were in the mandible and one in the maxilla. In 15 cases, the fusions were between primary incisors and in six cases between lateral incisors and canines. Agenesis of a permanent lateral incisor always occurred when there had been fusion of a primary lateral incisor and a canine in the primary dentition. When fusion had been between primary incisors, there was only agenesis of an incisor in the permanent dentition in a few cases. The degree of fusion between the involved teeth was not related to the occurrence of agenesis. It is suggested that the intra-jaw differences are related to the recently reported prenatal developmental patterns of the alveoli of the incisors and canines. Moreover, it is suggested that neural crest developmental field differences between the developing maxilla and mandible may explain the inter-jaw differences in phenotypic abnormalities.
Secalonic acid D (SAD), a cleft palate-inducing mycotoxin, reduces palatal cyclic AMP (cAMP) levels. cAMP relays its signals via the transcription factors (TF) such as cAMP response element (CRE) binding protein (CREB), CRE modulator (CREM) and activator transcription factor-1 (ATF-1) to CRE-containing genes. These studies tested the hypothesis that these TF are present and functional in the developing palate and that SAD alters their expression and function along with that of the CRE-containing gene, proliferating cell nuclear antigen (PCNA). Electrophoretic mobility shift assays (EMSA), using nuclear extracts of control and SAD-treated developing palate tissues and 32P-labeled CRE revealed the formation of a DNA-protein complex. Supershift/ablation assays with TF antibodies showed the presence of CREB, CREM and another unidentified TF but not ATF-1 in the complex. Western analyses of the DNA-protein complex from preparative EMSA revealed increased binding of CREB to CRE in direct correlation with increase in phospho-CREB (pCREB) in the controls. Exposure to SAD significantly reduced CREB binding throughout palate development. This was in correlation with reductions in pCREB levels on gestational day (GD) 13 and 14 palates. On GD 12, however, SAD dramatically increased CREB phosphorylation. The ontogeny of palatal CREB and CREM (several isoforms) expression remained unchanged in controls whereas SAD increased that of the repressor isoform of CREM. The expression of PCNA was inhibited by SAD on GD 12. These results show that the cAMP signaling pathway is functional in the palate and that SAD alters CREB phosphorylation and inhibits its binding to CRE. leading to altered expression of genes involved in cell proliferation, an event critical for normal palate development.
In a previous study, we observed the strong expression of a stress protein of the HSP100/Clp family (HSP110) in apoptotic mesectodermal cells during early mouse facial development.In the present study, we describe the strong expression of the same HSP110 in mesectodermal cells undergoing apoptosis after all-trans retinoic acid (RA) administration.We used a teratological model known to increase cell deaths mainly in the first and second branchial arches during mammalian cephalogenesis: the treatment of E9 mouse embryos with all-trans RA, which results in craniofacial malformations comparable to those that characterize mandibulofacial dysostosis in man. Pregnant NMRI mice were treated with 60 mg/kg body weight of all-trans RA, given orally on day 9 of gestation; embryos were taken 4, 12 or 24 hr after RA administration.The apoptotic pattern of RA-induced cell deaths was confirmed using the dUTP biotin nick-end labeling (TUNEL) method and transmission electron microscopy (TEM).HSP110 expression was detected using an immunohistochemical approach.The increase in the number of TUNEL-positive cells and HSP110-positive cells after all-trans RA administration was quantified in the first branchial arch using a computerized method.Twelve hours after RA administration, the increase in the number of HSP110-positive cells is greater than the increase in the number of TUNEL-positive cells. Twenty-four hours after RA administration, only TUNEL-positive cells remain strong in number.We suggest that HSP110 expression could represent a biochemical event of apoptotic cell death induced by RA, associated with early stages of the apoptotic process.In order to find out if HSP110 expression resulted from neosynthesis, we performed in situ hybridization, which demonstrated that the expression of HSP110 occurred at the level of mRNA.
Moire topography was used to quantitatively determine the shape of the palatal vault in 57 Spanish patients with Down syndrome (DS) (38 males and 19 females: age range 18-36 years) and in 100 normal controls (N) (76 males and 24 females; age range 20-29 years).The topographic image for each palatal vault was analyzed and approximately 40 sets of coordinates (x, y, z) were recorded. Other parameters, including length, width and maximum height, were recorded from the palate vault images.No appreciable sexual dimorphism in palate dimensions was observed in DS subjects versus the situation in N subjects. Globally, the average N dimensions were significantly greater than those in the DS patients (P < 0.005). A special palatal morphology was found to be associated with DS, with different ratios for the three dimensions (mean factor 0.88 for length, 0.81 for width and 0.73 for height), versus the healthy palatal vaults. It is concluded that palatal morphology in DS fits an elliptic paraboloid.On the other hand, no lineal correlation was observed between height, width and length in the DS and N groups. The scatter plots of bivariate data exhibited a shapeless morphology. The lineal correlation coefficients ranged from 0.008 to 0.33 for the DS and N groups.
An immunohistochemical study analyzing the pattern of distribution of some intermediate filament proteins, keratin and vimentin and, one adhesion molecule, cadherin in different stages of developing secondary palate in two strains of mice with different H-2 backgrounds was undertaken to investigate differences between a strain that is susceptible to glucocorticoid-induced cleft palate (A/Sn) and one that is resistant to glucocorticoid-induced cleft palate (C57/BL). The heads of embryos were processed by standard immunohistochemistry with antipancytokeratin (KAE1), antikeratins 18 (K18) and 19 (K19), antivimentin, and anti E-cadherin antibodies. Immunostaining with KAE1 antibody showed differences between the strains. The reaction was stronger in the medial edge epithelia of palatal processes in the A/Sn strain at all stages of palatogenesis. The C57/BL strain showed a weak immunostain to KAE1. Antivimentin antibody stained the mesenchymal cells of palatal processes and K18 and K19 showed no reaction in either strain of mice. Anti E-cadherin antibody was detected in the medial palatal epithelium of both strains of mice and in all stages of palate development. No differences were observed in E-cadherin and vimentin immunostain in palatal epithelium between the strains. The different expression of some cytokeratins in the embryonic palatal epithelium suggests that these intermediate filament proteins may be involved in different susceptibility to glucocorticoid-induced cleft palate in the mouse. The decreased immunoreaction of cytokeratins observed in the resistant strain would facilitate the disappearance of this molecule during the transformation from an epithelial to a mesenchymal phenotype that takes place during the development of the palate. These results may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformation in the embryonic palate.
The distribution of type I and II collagen synthesis in the temporomandibular joint (TMJ) area of 1- to 28-day-old rats was studied after hybridization with probes to pro alpha1(I) and pro alpha1(II) collagen mRNA, and stain intensity through the various cartilaginous zones of the mandibular condyle and other areas of TMJ was assessed. The pro alpha(I) collagen mRNA was detected in the perichondrium/periosteum, in the fibrous and undifferentiated cell layers of the mandibular condyle, in the articular disc, and in all bone structures and muscles. The pro alpha1(II) collagen mRNA was found in the condylar cartilage and the articular fossa. Intensity in the condyle was highest in the chondroblastic layer and decreased towards the lower hypertrophic layer. In the condylar cartilage of the 21- to 28-day-old rats the chondroblastic cell zone was relatively narrow compared with the younger animals, whereas the reverse seems to be the case in the cartilage of the articular fossa. Changes in the pro alpha1(II) collagen mRNA were observed in the osseochondral junction area of the primary spongiosa, in that at the age of 5 days intense staining was found, whereas no staining was observed by 14 days. In the mineralizing zone, however, the majority of osteoblastic cells gave a positive signal with the pro alpha1(I) collagen probe. In conclusion, type II collagen synthesis of the mandibular condyle is restricted to its upper area. This differs from the long bone epiphyseal plate, where this type of collagen is produced virtually throughout the cartilage. Type II collagen synthesis of the fossal cartilage seems to increase as a function of age.
The aim of this study was to analyze the CBFA1 gene in a phenotypically variable family with autosomal dominant cleidocranial dysplasia (CCD). Five members of a family with CCD were characterized clinically. X-rays and photographs of the two clinically affected family members were taken. The genotype of all five affected family members was determined with the use of single strand conformation polymorphism (SSCP) and direct sequencing. A point-mutation in exon 2 (R148G) was detected in a patient with the full-blown clinical phenotype. His son, demonstrating the same mutation, showed only the dental CCD characteristics. No mutation could be found in the three clinically healthy family members. To conclude, a missense mutation in the CBFA1 gene was detected in a family with variably expressed CCD syndrome. A detailed clinical examination is necessary to detect minimally affected gene mutation carriers.
We report a case of postnatal onset short stature and a distinctive pitted enamel hypoplasia in a 19-year-old woman. Growth hormone deficiency and other endocrine deficiencies were excluded. Additional observations of similar cases might outline a newly recognized syndrome.
Molecular aspects of bone formation and bone growth are discussed together with selected genetic disorders of the involved genes. Topics covered include: collagenopathies and osteogenesis imperfecta; core binding factor transcription factors and cleidocranial dysplasia; bone morphogenetic proteins and fibrodysplasia ossificans progressiva; transforming growth factor beta, suture closure, and craniosynostosis; Indian hedgehog, parathyroid hormone-related protein together with its receptor, and Jansen metaphyseal chondrodysplasia.
Glucosyltransferase from oral bacteria Streptococcus mutans is the most significant virulent factor in causing dental caries. The enzyme has two subsites. The binding specificity of divalent metal ions to glucosyl or fructosyl subsite was examined using multiple inhibition kinetics. The interaction factor "alpha" identifies whether the two subsites are exclusive or non-exclusive.
The formation of the midbrain region depends mainly on the activity of a signalling center located in the isthmus region, on the border between the prospective mesencephalon and metencephalon. FGF-8 has been proposed as a signalling molecule responsible for this specification because of its expression pattern and its ability to elicit duplication of the midbrain region when expressed ectopically in the neuroepithelium. Here we present evidence that members of the FGF family of growth factors when released in the cephalic mesenchyme are able to extend the expression of the mesencephalic marker En-2 to both the anterior and the posterior regions of its original landmark. This alteration in the expression pattern of En-2 is not accompanied by a significant alteration in the later development of the midbrain-cerebellar anlage, although the eye development is severely altered. Members of the bone morphogenetic protein family ectopically released from the mesenchyme down-regulate the expression of En-2 and also have an effect on the development of the eye. These results demonstrate that growth factor molecules produced in the mesenchyme (vertical signalling) participate in the correct establishment of the antero-posterior patterning of the cephalic nervous system during development.
Skeletogenesis and chondrogenesis result from a sequence of events involving epithelial-mesenchymal interaction, condensation, and differentiation. Types of bone and cartilage formation include: (1) intramembranous ossification, (2) endochondral ossification, (3) combined endochondral and intramembranous ossification, (4) heterotopic bone and cartilage formation, and (5) secondary cartilage formation. Pathologic conditions with bone and cartilage include: (1) benign and malignant tumors and (2) reactive osseous and cartilaginous metaplasia.
