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.

Sex differences and risk factors for bleeding in Alagille syndrome.

Spontaneous bleeds are a leading cause of death in the pediatric JAG1-related liver disease Alagille syndrome (ALGS). We asked whether there are sex differences in bleeding events in patients, whether Jag1Ndr/Ndr mice display bleeds or vascular defects, and whether discovered vascular pathology can be confirmed in patients non-invasively. We performed a systematic review of patients with ALGS and vascular events following PRISMA guidelines, in the context of patient sex, and found significantly more girls than boys reported with spontaneous intracranial hemorrhage. We investigated vascular development, homeostasis, and bleeding in Jag1Ndr/Ndr mice, using retina as a model. Jag1Ndr/Ndr mice displayed sporadic brain bleeds, a thin skull, tortuous blood vessels, sparse arterial smooth muscle cell coverage in multiple organs, which could be aggravated by hypertension, and sex-specific venous defects. Importantly, we demonstrated that retinographs from patients display similar characteristics with significantly increased vascular tortuosity. In conclusion, there are clinically important sex differences in vascular disease in ALGS, and retinography allows non-invasive vascular analysis in patients. Finally, Jag1Ndr/Ndr mice represent a new model for vascular compromise in ALGS.

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.

Standardization of mineral density maps of physiologic and pathologic biominerals in humans using cone-beam CT and micro-CT scanners.

OBJECTIVES: The lack of standardized X-ray imaging remains a challenge for comparative studies on spatial scans acquired from different clinic-specific X-ray scanners. The central objectives of this study are: 1) to delineate mineral density (MD) values, and 2) generate spatial MD maps of various physiologic and pathologic biominerals, and 3) propose a standardization protocol within the safe-operating zone of a CT scanner that underpins normalization of absorbed dose to shape and density of tissues. METHODS: A systematic approach to propose a standardization protocol for CT imaging in vivo included: 1) estimation of pathologic MD ranges by performing a comparative meta-analysis on 2009-2019 data from the PubMed database; 2) calibration of cone-beam CT (CBCT) and micro-CT scanners with phantoms of known mineral densities (0, 250, 500, 750 and 3000 mg/cc) and shapes (cylinders and polyhedrons); 3) scanning craniofacial bones (N = 5) and dental tissues (N = 5), and ectopic minerals from humans (N = 3 each, pulp, salivary gland, kidney and prostrate stones, and penile and vascular plaques); 4) underscoring the effect of shape-factor (surface area-to-volume ratio) on MD of biominerals. RESULTS: Higher MDs of physiologic and pathologic cortical bones (504-1009 mg/cc) compared to trabecular bone (82-212 mg/cc) were observed. An increase in shape-factor increased the CBCT error in MD measurement and revealed that the scanner resolution is dependent on the absorbed dose and shape-factor of detectable features. SIGNIFICANCE: CT scanners should be calibrated with phantoms containing segments of known shape-factors and mineral densities to identify safe-operating zones. The calibrated approach will narrow the gap between length-scale dependent measurements, and will permit spatiotemporal quantitative and reliable detection of pathologies.

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.

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.

IGF-1 TMJ injections enhance mandibular growth and bone quality in juvenile rats.

OBJECTIVES: Dentofacial orthopaedic treatment of mandibular hypoplasia has unpredictable skeletal outcomes. Although several biomodulators including insulin-like growth factor 1 (IGF-1) are known to contribute to chondrocyte proliferation, their efficacy in modulating mandibular growth has not been validated. The aim of this study was to determine the effect of locally delivered IGF-1 on mandibular growth and condylar bone quality/quantity in juvenile rats. SETTING AND SAMPLE POPULATION: Institutional vivarium using twenty-four 35-day-old male Sprague-Dawley rats. METHODS: PBS or 40 µg/kg (low-dose) IGF-1 or 80 µg/kg (high-dose) IGF-1 was injected bilaterally into the temporomandibular joints of the rats at weekly intervals for four weeks. Cephalometric and micro-computed tomography measurements were used to determine mandibular dimensions. Bone and tissue mineral density, volume fraction and mineral content were determined, and serum IGF-1 concentrations assayed. RESULTS: Intra-articular administration of high-dose IGF-1 contributed to a significant 6%-12% increase in mandibular body and condylar length compared to control and low-dose IGF-1-treated animals. Additionally, IGF-1 treatment resulted in a significant decrease in the angulation of the lower incisors to mandibular plane. Condylar bone volume, bone volume fraction, mineral content and mineral density were significantly increased with high-dose IGF-1 relative to control and low-dose IGF-1 groups. Serum IGF-1 levels were similar between all groups confirming limited systemic exposure to the locally administered IGF-1. CONCLUSION: Local administration of high-dose 80 µg/kg IGF-1 enhances mandibular growth and condylar bone quality and quantity in growing rats. The findings have implications for modulating mandibular growth and potentially enhancing condylar bone health and integrity.

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.

Comparative analysis of clinical characteristics, imaging and laboratory findings of different age groups with COVID-19.

Objective: This study aims to provide scientific basis for rapid screening and early diagnosis of the coronavirus disease 2019 (COVID-19) through analysing the clinical characteristics and early imaging/laboratory findings of the inpatients. Methods: Three hundred and three patients with laboratory-confirmed COVID-19 from the East Hospital of People's Hospital of Wuhan University (Wuhan, China) were selected and divided into four groups: youth (20-40 years, n = 64), middle-aged (41-60 years, n = 89), older (61-80 years, n = 118) and elderly (81-100 years, n = 32). The clinical characteristics and imaging/laboratory findings including chest computed tomography (CT), initial blood count, C-reactive protein [CRP]), procalcitonin (PCT) and serum total IgE were captured and analysed. Results: (1) The first symptoms of all age groups were primarily fever (76%), followed by cough (12%) and dyspnoea (5%). Beside fever, the most common initial symptom of elderly patients was fatigue (13%). (2) Fever was the most common clinical manifestation (80%), with moderate fever being the most common (40%), followed by low fever in patients above 40 years old and high fever in those under 40 years (35%). Cough was the second most common clinical manifestation and was most common (80%) in the middle-aged. Diarrhoea was more common in the middle-aged (21%) and the older (19%). Muscle ache was more common in the middle-aged (15%). Chest pain was more common in the youth (13%), and 13% of the youth had no symptoms. (3) The proportion of patients with comorbidities increased with age. (4) Seventy-one per cent of the patients had positive reverse transcription-polymerase chain reaction results and 29% had positive chest CT scans before admission to the hospital. (5) Lesions in all lobes of the lung were observed as the main chest CT findings (76%). (6) Decrease in lymphocytes and increase in monocytes were common in the patients over 40 years old but rare in the youth. Eosinophils (50%), red blood cells (39%) and haemoglobin (40%) decreased in all age groups. (7) The proportion of patients with CRP and PCT elevation increased with age. (8) Thirty-nine per cent of the patients had elevated IgE, with the highest proportion in the old (49%). Conclusion: The clinical characteristics and imaging/laboratory findings of COVID-19 patients vary in different age groups. Personalised criteria should be formulated according to different age groups in the early screening and diagnosis stage.

Downregulation of FGF Signaling by Spry4 Overexpression Leads to Shape Impairment, Enamel Irregularities, and Delayed Signaling Center Formation in the Mouse Molar.

FGF signaling plays a critical role in tooth development, and mutations in modulators of this pathway produce a number of striking phenotypes. However, many aspects of the role of the FGF pathway in regulating the morphological features and the mineral quality of the dentition remain unknown. Here, we used transgenic mice overexpressing the FGF negative feedback regulator Sprouty4 under the epithelial keratin 14 promoter (K14-Spry4) to achieve downregulation of signaling in the epithelium. This led to highly penetrant defects affecting both cusp morphology and the enamel layer. We characterized the phenotype of erupted molars, identified a developmental delay in K14-Spry4 transgenic embryos, and linked this with changes in the tooth developmental sequence. These data further delineate the role of FGF signaling in the development of the dentition and implicate the pathway in the regulation of tooth mineralization. © 2019 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

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.

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.

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.

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.

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.

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.

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.

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.

Craniofacial morphometric analysis of individuals with X-linked hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia (HED) is the most prevalent type of ectodermal dysplasia (ED). ED is an umbrella term for a group of syndromes characterized by missing or malformed ectodermal structures, including skin, hair, sweat glands, and teeth. The X-linked recessive (XL), autosomal recessive (AR), and autosomal dominant (AD) types of HED are caused by mutations in the genes encoding ectodysplasin (EDA1), EDA receptor (EDAR), or EDAR-associated death domain (EDARADD). Patients with HED have a distinctive facial appearance, yet a quantitative analysis of the HED craniofacial phenotype using advanced three-dimensional (3D) technologies has not been reported. In this study, we characterized craniofacial morphology in subjects with X-linked hypohidrotic ectodermal dysplasia (XLHED) by use of 3D imaging and geometric morphometrics (GM), a technique that uses defined landmarks to quantify size and shape in complex craniofacial morphologies. We found that the XLHED craniofacial phenotype differed significantly from controls. Patients had a smaller and shorter face with a proportionally longer chin and midface, prominent midfacial hypoplasia, a more protrusive chin and mandible, a narrower and more pointed nose, shorter philtrum, a narrower mouth, and a fuller and more rounded lower lip. Our findings refine the phenotype of XLHED and may be useful both for clinical diagnosis of XLHED and to extend understanding of the role of EDA in craniofacial development.