Altering maternal calcium and phosphorus dietary intake induces persistent sex-specific changes in the dentition of the offspring.

BACKGROUND: The maternal diet is essential to offspring development, but the specific effects on tooth morphology are still unknown. The aim of this study was to evaluate the effects of altering maternal calcium (Ca) and phosphorus (P) supplementation during gestation and lactation on offspring dentition. METHODS: Pregnant mice were fed an experimental diet containing a threefold increase in Ca and a threefold decrease in P compared to the standard mouse chow diet at embryonic Day 0.5 (E0.5). Offspring mice were maintained on standard or experimental diets from post-natal Day 0 to weaning, then fed control diets until 6 weeks of age. Six-week-old offspring heads were collected and scanned using micro-computed tomography. Dental morphometrics of offspring maxillary and mandibular first and third molars (n = 5-6 per diet/per sex) were determined. A two-way ANOVA test was employed to verify the existence of any significant differences between groups. The significance level was set at P < .05. RESULTS: A two-way ANOVA revealed a statistically significant interaction between the effects of diet and sex on the upper and lower dentition. Moreover, experimental diet-fed female offspring exhibited smaller molars with shorter mesiodistal width and larger pulp chambers relative to controls, while experimental diet-fed male offspring possessed larger molars with wider mesiodistal width and smaller pulp chambers. CONCLUSION: Our findings reveal that altering the maternal and offspring dietary Ca:P ratio during gestation, lactation and weaning led to significant, sex-specific changes in the offspring dentition. The differences in dentition appeared to be correlated with the sex-specific changes in the craniofacial skeleton.

Altering osteoclast numbers using CTSK models in utero affects mice offspring craniofacial morphology.

BACKGROUND: Bone remodelling during development and growth is important for craniofacial integrity of offspring. The aim of this study was to investigate the changes in offspring adult skull morphology when the osteoclasts number was altered in utero, using three-dimensional (3D) geometric morphometric analysis (GMA). MATERIALS AND METHODS: We altered osteoclasts number in utero via two approaches. First, we generated heterozygous CtskCre ;DTAfl/+ (diphtheria toxin A) mice. Second, we altered Ctsk expression in vivo by injecting pregnant wild-type dams at embryonic day (E) 12.5 with in vivo siRNA specific for Ctsk. Mice were collected at 6 weeks and analysed using geometric morphometric analysis via computed tomography, histomorphometry and gene expression analysis. RESULTS: Altering osteoclasts number in utero affected the offspring adult skull morphology. Decreased Ctsk and osteoclast numbers were associated with a decrease in cranial vault height and an increase in mandibular body length. Changes in size and shape were observed with an increased number of osteoclasts in CtskCre ;DTAfl/+ mice, including an increase in cranial vault height, as well as a shortening of mandibular body length and ramus height. CONCLUSION: The findings of this study suggest that modulation of osteoclast numbers during pre- and post-natal development may be a previously unknown factor in the aetiology of skeletal malocclusions. An improved understanding of the factors affecting bone homeostasis during development and growth may help in the development of future therapies that would target the early intervention of skeletal malocclusion.

Alterations in subgingival microbiota during full-fixed appliance orthodontic treatment-A prospective study.

BACKGROUND: Full-fixed appliance orthodontic treatment (commonly called braces) increases plaque accumulation and the risk of gingivitis and periodontitis. However, little consensus exists on changes to subgingival microbiota and specific periodontopathogens during treatment with braces. Prior studies have been hampered by selection biases due to dependence on culture conditions, candidate-based PCR and shallow sequencing methods. OBJECTIVE: The objective was to provide the first longitudinal, culture-free and deep-sequence profiling of subgingival bacteria in subjects during early stages of full-fixed orthodontic treatment. METHODS: We performed 16S rRNA next-generation sequencing (NGS) on 168 subgingival samples collected at 4 distinct mandibular tooth sites per subject before (0 weeks) and during (6 and 12 weeks) orthodontic intervention in 9 experimental and 5 control subjects not undergoing treatment. RESULTS: Overall, we noted that orthodontic intervention led to increased microbial richness, accompanied by an increased incidence of localized gingivitis/mild periodontitis in subjects requiring orthodontic treatment compared to controls, as well as significant baseline variations in subgingival microbiomes in all subjects. Moreover, we confirmed individual- and site-dependent microbiome variability (in particular, the lingual site harboured higher microbiome diversity than buccal sites) that orthodontic bands may lead to more prolonged shifts in microbial changes compared to brackets, and evidence of adaptive enrichment of consensus bacteria with orthodontic intervention (12 novel, consensus bacterial species were identified). CONCLUSION: Our study, along with evolving global profiling methods and data analyses, builds a strong foundation for further analyses of subgingival microbiomes during full-fixed orthodontic treatment.

Altering calcium and phosphorus supplementation in pregnancy and lactation affects offspring craniofacial morphology in a sex-specific pattern.

INTRODUCTION: The effects on offspring craniofacial bone morphology and accretion because of altered maternal exposure to dietary components such as calcium (Ca) and phosphorus (P) are unclear. The objective of this study was to investigate the changes in offspring skull morphology and tissue mineral density (TMD), including sex-specific changes, with exposure to a maternal diet high in Ca-to-P levels during gestation and lactation in mice. METHODS: Time-mated FVB wild-type mice were fed a normal or experimental diet during gestation until weaning. The experimental diet contained a 3-fold increase in Ca and a 3-fold decrease in P (Ca:P molar ratio, 10.5) compared with normal mouse chow (Ca:P molar ratio, 1.5). The heads of 6-week-old control and experimental offspring mice were collected and scanned using microcomputed tomography. Three-dimensional geometric morphometric analysis was performed to analyze changes in craniofacial morphology. TMD measurements were also analyzed. RESULTS: We observed subtle changes and no significant differences between offspring control and experimental skulls when we compared all samples. However, when we separated skulls by sex, we discovered significant differences in craniofacial morphology and TMD. Experimental female offspring possessed skulls that were smaller, narrower transversely, taller vertically, and decreased in TMD. Experimental male offspring possessed skulls that were larger, wider transversely, shorter vertically, and increased in TMD. CONCLUSIONS: Maternal exposure to diet and increased Ca:P molar ratio during gestation and lactation led to significant, sex-specific morphologic and TMD changes in 6-week-old mouse skulls.

FGF signaling refines Wnt gradients to regulate the patterning of taste papillae.

The patterning of repeated structures is a major theme in developmental biology, and the inter-relationship between spacing and size of such structures is an unresolved issue. Fungiform papillae are repeated epithelial structures that house taste buds on the anterior tongue. Here, we report that FGF signaling is a crucial regulator of fungiform papillae development. We found that mesenchymal FGF10 controls the size of the papillary area, while overall patterning remains unchanged. Our results show that FGF signaling negatively affects the extent of canonical Wnt signaling, which is the main activation pathway during fungiform papillae development; however, this effect does not occur at the level of gene transcription. Rather, our experimental data, together with computational modeling, indicate that FGF10 modulates the range of Wnt effects, likely via induction of Sostdc1 expression. We suggest that modification of the reach of Wnt signaling could be due to local changes in morphogen diffusion, representing a novel mechanism in this tissue context, and we propose that this phenomenon might be involved in a broader array of mammalian developmental processes.

Microanatomical changes and biomolecular expression at the PDL-entheses during experimental tooth movement.

The novel aspect of this study was to contextualize the co-localization of biomolecular expression in widened and narrowed periodontal ligament (PDL)-space within a mechanically activated periodontal complex. The PDL is unique as it is the only ligament with both innervation and vascularization. Maxillary molars in 6-week-old male C57BL/6 mice (N = 5) were experimentally translated for 2 weeks using an elastic spacer. Contralateral teeth were used as controls. Mechanical testing of the periodontal complex of a mouse in situ and imaging using X-ray micro-computed tomography (micro-XCT) illustrated deformations within blood vessels (BV) of the PDL. PDL-bone and PDL-cementum entheses at the widened and narrowed PDL-spaces following experimental tooth movement (ETM) illustrated osterix (OSX), bone sialoprotein (BSP), cluster of differentiation 146 (CD146), and protein gene product 9.5 (PGP9.5), indicating active remodeling at these sites. PGP9.5 positive nerve bundles (NBs) were co-localized with multinucleated cells (MCs), Howship's resorption lacunae, and CD146 positive BVs. Association between nerves and MC was complemented by visualizing the proximity of osmium tetroxide stained NBs with the ultrastructure of MCs by performing scanning transmission electron microscopy. Spatial association of NB with BV, and NB with MC, provided insights into the plausible co-activation of NBs to initiate osteoclastic activity. Resorption of mineral occurred as an attempt to restore PDL-space of the load-bearing complex, specifically at the PDL-entheses. Mapping of anatomy-specific structural elements and their association with regenerative molecules by correlating light and electron micrographs provided insights into the use of these extracellular matrix molecules as plausible targets for pharmacological interventions related to tooth movement. Within the realm of tissue regeneration, modulation of load can reverse naturally occurring mineral formation to experimentally induced resorption, and naturally occurring mineral resorption to experimentally induced formation at the enthesial sites to permit tooth translation.

Altering calcium and phosphorus levels in utero affects adult mouse mandibular morphology.

OBJECTIVES: The purpose of our study was to determine morphological changes and bone mineral density (BMD) differences in the adult mandible of offspring exposed to high calcium, low phosphorus diets in utero until weaning age. MATERIALS AND METHODS: Time-mated FVB wild-type mice were fed normal or experimental diet during gestation and until weaning of offspring. Experimental diet contained 3-fold increase in calcium and 3-fold decrease in phosphorus compared to normal diet. Adult mandibles of offspring exposed to experimental diet were sacrificed and heads scanned using micro-computed tomography. Three-dimensional 3D geometric morphometric analysis GMA was utilized to detect morphological changes to the mandible including the condyle. RESULTS: Experimental females showed the greatest morphological differences including shortened mandibular ramus width and height, shortened mandibular body length and height, a wider but shortened condylar neck and a wider condylar head in the lateral-medial direction. Experimental male mandibles trended towards increased mandibular body height and length, opposite the changes observed in experimental female mandibles, whereas condyles were similar to that observed in experimental females. Bone mineral density (BMD) was lowered in experimental females. CONCLUSION: Increased calcium and decreased phosphorus levels led to a retrognathic mandible associated with lowered BMD in experimental females, whereas experimental showed partly opposite effects. Further studies are required to understand the mechanism underlying diet- and gender-specific differences in mandibular morphology.

The Role of Epithelial Stat3 in Amelogenesis during Mouse Incisor Renewal.

The aim of this study was to evaluate the role of epithelial signal transducer and activator of transcription 3 (STAT3) in mouse incisor amelogenesis. Since Stat3 is expressed in the epithelial component of developing and adult mouse teeth, we generated and analyzed Krt14Cre/+;Stat3fl/fl mutant mice in which Stat3 was inactivated in epithelia including ameloblast progenitors and ameloblasts, the cells responsible for enamel formation. Histological analysis showed little enamel matrix in mutant incisors compared to controls. Delayed incisor enamel mineralization was demonstrated using micro-computed X-ray tomography analysis and was supported by an increase in the pre-expression distance of enamel-enriched proteins such as amelogenin, ameloblastin, and kallikrein-4. Lastly, scanning electron microscopy analysis showed little enamel mineralization in mutant incisors underneath the mesial root of the 1st molar; however, the micro-architecture of enamel mineralization was similar in the erupted portion of control and mutant incisors. Taken together, our findings demonstrate for the first time that the absence of epithelial Stat3 in mice leads to delayed incisor amelogenesis.

Neural crest-mediated bone resorption is a determinant of species-specific jaw length.

Precise control of jaw length during development is crucial for proper form and function. Previously we have shown that in birds, neural crest mesenchyme (NCM) confers species-specific size and shape to the beak by regulating molecular and histological programs for the induction and deposition of cartilage and bone. Here we reveal that a hitherto unrecognized but similarly essential mechanism for establishing jaw length is the ability of NCM to mediate bone resorption. Osteoclasts are considered the predominant cells that resorb bone, although osteocytes have also been shown to participate in this process. In adults, bone resorption is tightly coupled to bone deposition as a means to maintain skeletal homeostasis. Yet, the role and regulation of bone resorption during growth of the embryonic skeleton have remained relatively unexplored. We compare jaw development in short-beaked quail versus long-billed duck and find that quail have substantially higher levels of enzymes expressed by bone-resorbing cells including tartrate-resistant acid phosphatase (TRAP), Matrix metalloproteinase 13 (Mmp13), and Mmp9. Then, we transplant NCM destined to form the jaw skeleton from quail to duck and generate chimeras in which osteocytes arise from quail donor NCM and osteoclasts come exclusively from the duck host. Chimeras develop quail-like jaw skeletons coincident with dramatically elevated expression of TRAP, Mmp13, and Mmp9. To test for a link between bone resorption and jaw length, we block resorption using a bisphosphonate, osteoprotegerin protein, or an MMP13 inhibitor, and this significantly lengthens the jaw. Conversely, activating resorption with RANKL protein shortens the jaw. Finally, we find that higher resorption in quail presages their relatively lower adult jaw bone mineral density (BMD) and that BMD is also NCM-mediated. Thus, our experiments suggest that NCM not only controls bone resorption by its own derivatives but also modulates the activity of mesoderm-derived osteoclasts, and in so doing enlists bone resorption as a key patterning mechanism underlying the functional morphology and evolution of the jaw.

Abnormal Ras signaling in Costello syndrome (CS) negatively regulates enamel formation.

RASopathies are syndromes caused by gain-of-function mutations in the Ras signaling pathway. One of these conditions, Costello syndrome (CS), is typically caused by an activating de novo germline mutation in HRAS and is characterized by a wide range of cardiac, musculoskeletal, dermatological and developmental abnormalities. We report that a majority of individuals with CS have hypo-mineralization of enamel, the outer covering of teeth, and that similar defects are present in a CS mouse model. Comprehensive analysis of the mouse model revealed that ameloblasts, the cells that generate enamel, lacked polarity, and the ameloblast progenitor cells were hyperproliferative. Ras signals through two main effector cascades, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. To determine through which pathway Ras affects enamel formation, inhibitors targeting either PI3K or MEK 1 and 2 (MEK 1/2), kinases in the MAPK pathway, were utilized. MEK1/2 inhibition rescued the hypo-mineralized enamel, normalized the ameloblast polarity defect and restored normal progenitor cell proliferation. In contrast, PI3K inhibition only corrected the progenitor cell proliferation phenotype. We demonstrate for the first time the central role of Ras signaling in enamel formation in CS individuals and present the mouse incisor as a model system to dissect the roles of the Ras effector pathways in vivo.

Evolution of a developmental mechanism: Species-specific regulation of the cell cycle and the timing of events during craniofacial osteogenesis.

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.

FGF signaling regulates the number of posterior taste papillae by controlling progenitor field size.

The sense of taste is fundamental to our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Sensory taste buds are housed in papillae that develop from epithelial placodes. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found in the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP). Despite the great variation in the number of CVPs in mammals, its importance in taste function, and its status as the largest of the taste papillae, very little is known about the development of this structure. Here, we report that a balance between Sprouty (Spry) genes and Fgf10, which respectively antagonize and activate receptor tyrosine kinase (RTK) signaling, regulates the number of CVPs. Deletion of Spry2 alone resulted in duplication of the CVP as a result of an increase in the size of the placode progenitor field, and Spry1(-/-);Spry2(-/-) embryos had multiple CVPs, demonstrating the redundancy of Sprouty genes in regulating the progenitor field size. By contrast, deletion of Fgf10 led to absence of the CVP, identifying FGF10 as the first inductive, mesenchyme-derived factor for taste papillae. Our results provide the first demonstration of the role of epithelial-mesenchymal FGF signaling in taste papilla development, indicate that regulation of the progenitor field size by FGF signaling is a critical determinant of papilla number, and suggest that the great variation in CVP number among mammalian species may be linked to levels of signaling by the FGF pathway.

PERP regulates enamel formation via effects on cell-cell adhesion and gene expression.

Little is known about the role of cell-cell adhesion in the development of mineralized tissues. Here we report that PERP, a tetraspan membrane protein essential for epithelial integrity, regulates enamel formation. PERP is necessary for proper cell attachment and gene expression during tooth development, and its expression is controlled by P63, a master regulator of stratified epithelial development. During enamel formation, PERP is localized to the interface between the enamel-producing ameloblasts and the stratum intermedium (SI), a layer of cells subjacent to the ameloblasts. Perp-null mice display dramatic enamel defects, which are caused, in part, by the detachment of ameloblasts from the SI. Microarray analysis comparing gene expression in teeth of wild-type and Perp-null mice identified several differentially expressed genes during enamel formation. Analysis of these genes in ameloblast-derived LS8 cells upon knockdown of PERP confirmed the role for PERP in the regulation of gene expression. Together, our data show that PERP is necessary for the integrity of the ameloblast-SI interface and that a lack of Perp causes downregulation of genes that are required for proper enamel formation.

Characterization of X-linked hypohidrotic ectodermal dysplasia (XL-HED) hair and sweat gland phenotypes using phototrichogram analysis and live confocal imaging.

Hypohidrotic ectodermal dysplasia (HED) is the most common type of ectodermal dysplasia (ED), which encompasses a large group of syndromes that share several phenotypic features such as missing or malformed ectodermal structures, including skin, hair, sweat glands, and teeth. X-linked hypohidrotic ectodermal dysplasia (XL-HED) is associated with mutations in ectodysplasin (EDA1). Hypohidrosis due to hypoplastic sweat glands and thin, sparse hair are phenotypic features that significantly affect the daily lives of XL-HED individuals and therefore require systematic analysis. We sought to determine the quality of life of individuals with XL-HED and to quantify sweat duct and hair phenotypes using confocal imaging, pilocarpine iontophoresis, and phototrichogram analysis. Using these highly sensitive and non-invasive techniques, we demonstrated that 11/12 XL-HED individuals presented with a complete absence of sweat ducts and that none produced sweat. We determined that the thin hair phenotype observed in XL-HED was due to multiple factors, such as fewer terminal hairs with decreased thickness and slower growth rate, as well as fewer follicular units and fewer hairs per unit. The precise characterization of XL-HED phenotypes using sensitive and non-invasive techniques presented in our study will improve upon larger genotype-phenotype studies and the assessment of future therapies in XL-HED.

Unilateral Loss of Maxillary Molars in Young Mice Leads to Bilateral Condylar Adaptation and Degenerative Disease.

The adaptive response of the mandible and temporomandibular joint (TMJ) to altered occlusion in juvenile patients is presently unclear. To address this question, we established a mouse model in which all molars were extracted from the maxillary right quadrant in prepubertal, 3-week-old mice and analyzed morphological, tissue, cellular, and molecular changes in the mandible and condyle 3 weeks later. Unilateral loss of maxillary molars led to significant, robust, bilateral changes, primarily in condylar morphology, including anteroposterior narrowing of the condylar head and neck and increased convexity at the condylar surface, as determined by geometric morphometric analysis. Furthermore, both condyles in experimental mice exhibited a degenerative phenotype, which included decreased bone volume and increased mineral density near the condylar head surface compared to control mice. Changes in condylar morphology and mineralized tissue composition were associated with alterations in the cellular architecture of the mandibular condylar cartilage, including increased expression of markers for mature (Col2a1) and hypertrophic (Col10a1) chondrocytes, suggesting a shift toward differentiating chondrocytes. Our results show significant bilateral condylar morphological changes, alterations in tissue composition, cellular organization, and molecular expression, as well as degenerative disease, in response to the unilateral loss of teeth. Our study provides a relatively simple, tractable mouse tooth extraction system that will be of utility in uncovering the cellular and molecular mechanisms of condylar and mandibular adaptation in response to altered occlusion. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Effects of Multi-Generational Soft Diet Consumption on Mouse Craniofacial Morphology.

Variations in craniofacial morphology may arise as a result of adaptation to different environmental factors such as soft diet (SD), which lessens functional masticatory load. Prior studies have shown that changes in the masticatory muscle function associated with a switch to short-term SD led to changes in craniofacial morphology and alveolar bone architecture. However, the long-term effects of SD and the associated adaptive changes in craniofacial shape are unclear. Our novel study set out to profile prospective skull changes in mice fed with SDs over multiple generations using three-dimensional (3D) geometric morphometric analysis (GMA). Our results revealed that short-term SD consumption led to a significant decrease in craniofacial size, along with numerous shape changes. Long-term SD consumption over 15 continuous generations was not associated with changes in craniofacial size; however, shape analysis revealed mice with shortened crania and mandibles in the anteroposterior dimension, as well as relative widening in the transverse dimension compared to the average shape of all mice analyzed in our study. Moreover, changes in shape and size associated with different functional loads appeared to be independent - shape changes persisted after diets were switched for one generation, whereas size decreased after one generation and then returned to baseline size. Our study is the first to study the role of prolonged, multi-generational SD consumption in the determination of craniofacial size and shape.

Isl1 Controls Patterning and Mineralization of Enamel in the Continuously Renewing Mouse Incisor.

Rodents are characterized by continuously renewing incisors whose growth is fueled by epithelial and mesenchymal stem cells housed in the proximal compartments of the tooth. The epithelial stem cells reside in structures known as the labial (toward the lip) and lingual (toward the tongue) cervical loops (laCL and liCL, respectively). An important feature of the rodent incisor is that enamel, the outer, highly mineralized layer, is asymmetrically distributed, because it is normally generated by the laCL but not the liCL. Here, we show that epithelial-specific deletion of the transcription factor Islet1 (Isl1) is sufficient to drive formation of ectopic enamel by the liCL stem cells, and also that it leads to production of altered enamel on the labial surface. Molecular analyses of developing and adult incisors revealed that epithelial deletion of Isl1 affected multiple, major pathways: Bmp (bone morphogenetic protein), Hh (hedgehog), Fgf (fibroblast growth factor), and Notch signaling were upregulated and associated with liCL-generated ectopic enamel; on the labial side, upregulation of Bmp and Fgf signaling, and downregulation of Shh were associated with premature enamel formation. Transcriptome profiling studies identified a suite of differentially regulated genes in developing Isl1 mutant incisors. Our studies demonstrate that ISL1 plays a central role in proper patterning of stem cell-derived enamel in the incisor and indicate that this factor is an important upstream regulator of signaling pathways during tooth development and renewal. © 2017 American Society for Bone and Mineral Research.

Inhibition of Notch Signaling During Mouse Incisor Renewal Leads to Enamel Defects.

The continuously growing rodent incisor is an emerging model for the study of renewal of mineralized tissues by adult stem cells. Although the Bmp, Fgf, Shh, and Wnt pathways have been studied in this organ previously, relatively little is known about the role of Notch signaling during incisor renewal. Notch signaling components are expressed in enamel-forming ameloblasts and the underlying stratum intermedium (SI), which suggested distinct roles in incisor renewal and enamel mineralization. Here, we injected adult mice with inhibitory antibodies against several components of the Notch pathway. This blockade led to defects in the interaction between ameloblasts and the SI cells, which ultimately affected enamel formation. Furthermore, Notch signaling inhibition led to the downregulation of desmosome-specific proteins such as PERP and desmoplakin, consistent with the importance of desmosomes in the integrity of ameloblast-SI attachment and enamel formation. Together, our data demonstrate that Notch signaling is critical for proper enamel formation during incisor renewal, in part by regulating desmosome-specific components, and that the mouse incisor provides a model system to dissect Jag-Notch signaling mechanisms in the context of mineralized tissue renewal.

Spontaneous emergence of overgrown molar teeth in a colony of Prairie voles (Microtus ochrogaster).

Continuously growing incisors are common to all rodents, which include the Microtus genus of voles. However, unlike many rodents, voles also possess continuously growing molars. Here, we report spontaneous molar defects in a population of Prairie voles (Microtus ochrogaster). We identified bilateral protuberances on the ventral surface of the mandible in several voles in our colony. In some cases, the protuberances broke through the cortical bone. The mandibular molars became exposed and infected, and the maxillary molars entered the cranial vault. Visualisation upon soft tissue removal and microcomputed tomography (microCT) analyses confirmed that the protuberances were caused by the overgrowth of the apical ends of the molar teeth. We speculate that the unrestricted growth of the molars was due to the misregulation of the molar dental stem cell niche. Further study of this molar phenotype may yield additional insight into stem cell regulation and the evolution and development of continuously growing teeth.

Characterization of the 5'-flanking region of the rat AJ18 gene.

Krüppel-associated box (KRAB) domains are present in one-third of all C(2)H(2) zinc finger containing proteins, making the KRAB/C(2)H(2) proteins one of the largest known families of putative transcription repressors. AJ18 has been identified as a novel KRAB/C(2)H(2) gene that is involved in the differentiation of osteogenic cells. To study the regulation of expression of the AJ18 gene, the 5'-flanking region of the AJ18 gene was obtained by screening a rat genomic library. This region was sequenced, and the transcription start site mapped by primer extension. The AJ18 gene consists of at least four exons, the first exon coding for an unusually long 2.3 kb 5'-UTR region. A putative internal ribosome entry site, immediately upstream of the translation initiation site, is indicated from the complementarity of a 12 nucleotide sequence with a region in the rat 18S rRNA. Chimeric constructs encompassing the region surrounding the transcription start site (-77-+171), as well as constructs with additional 1.9 kb upstream from this region revealed strong transcriptional activity when ligated to a luciferase reporter gene and tested in transient transfection assays. This activity was lost on deletion of the 5'-flanking region to -77. In addition, transcriptional activity was progressively lost with the inclusion of downstream sequences extending into the 5'-UTR. Several known response elements for proteins such as Runx2, NFkappaB, Smads, Sp1, and Ets1 are retained within the conserved sequences of rat and mouse AJ18, which was retrieved from mouse genomic libraries. Interestingly, the transcriptional activity was approximately 100-fold higher in the osteocarcinoma cell line ROS 2.8/17 compared to the fibroblast-like C3H10T1/2. Notably, this is the first gene promoter from the large KRAB/C(2)H(2) zinc finger family of proteins to be identified and characterized.