OBJECTIVESAssessing the strength of integration among different regions of the modern human nasal complex is important for developing a more thorough understanding of the determinants of nasal morphology. Given the morphogenetic influence of cartilage on adjacent intramembranous growth sites, the interaction between chondrocranial- versus intramembranous-derived nasal structures may have a significant influence on patterns of nasal variation. The purpose of this study is to examine integration between the chondrocranial- and intramembranous-derived regions of the nasal complex.MATERIALS AND METHODSUsing computed tomograph (CT) scans, we collected three-dimensional coordinate landmark data from a static adult sample (n = 62). First, using centroid size, and the symmetric and asymmetric components of shape variation, we examined the strength of integration between landmarks representing chondrocranial-derived structures (e.g., ethmoid, external nasal cartilages) and landmarks representing intramembranous-derived structures (nasal floor, anterior nasal aperture, etc.). Second, given that the strength of integration is a relative measure, we compared integration between chondrocranial- and intramembranous-derived structures to the more modularized external and internal regions of the nasal complex.RESULTSThere was significant moderate morphological integration between chondrocranial- versus intramembranous-derived regions of the nasal complex. Moreover, integration between chondrocranial- versus intramembranous-derived structures was consistently stronger when compared to external versus internal regions for both the symmetric and asymmetric components of variation. Thus, more covariation within the nasal complex could be explained by the relationship between chondrocranial- and intramembranous-derived structures.CONCLUSIONSOur results suggest that the interaction between chondrocranial- and intramembranous-derived structures may be an important determinant in the patterning of nasal complex variation.

The mechanisms that regulate post-natal growth of the craniofacial complex and that ultimately determine the size and shape of our faces are not well understood. Hippo signaling is a general mechanism to control tissue growth and organ size, and although it is known that Hippo signaling functions in neural crest specification and patterning during embryogenesis and before birth, its specific role in postnatal craniofacial growth remains elusive. We have identified the transcription factor FoxO6 as an activator of Hippo signaling regulating neonatal growth of the face. During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation. Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology.

OBJECTIVES The long-term skeletal effects of Class II treatment in growing individuals using high-pull facebow headgear and fixed edgewise appliances have not been reported. The purpose of this study was to evaluate the long-term skeletal effects of treatment using high-pull headgear followed by fixed orthodontic appliances compared to an untreated control group. MATERIALS AND METHODS Changes in anteroposterior and vertical cephalometric measurements of 42 Class II subjects (n = 21, mean age = 10.7 years) before treatment, after headgear correction to Class I molar relationship, after treatment with fixed appliances, and after long-term retention (mean 4.1 years), were compared to similar changes in a matched control group (n = 21, mean age = 10.9 years) by multivariable linear regression models. RESULTS Compared to control, the study group displayed significant long-term horizontal restriction of A-point (SNA = -1.925°, P < .0001; FH-NA = -3.042°, P < .0001; linear measurement A-point to Vertical Reference = -3.859 mm, P < .0001) and reduction of the ANB angle (-1.767°, P < .0001), with no effect on mandibular horizontal growth or maxillary and mandibular vertical skeletal changes. A-point horizontal restriction and forward mandibular horizontal growth accompanied the study group correction to Class I molar, and these changes were stable long term. CONCLUSIONS One phase treatment for Class II malocclusion with high-pull headgear followed by fixed orthodontic appliances resulted in correction to Class I molar through restriction of horizontal maxillary growth with continued horizontal mandibular growth and vertical skeletal changes unaffected. The anteroposterior molar correction and skeletal effects of this treatment were stable long term.

s .................................................................................................................................................................. 26 Author/Abstract-Number Index ......................................................................................................................70 Iowa Section of AADR — Past Presidents .................................................................................................... 71 Acknowledgments ................................................................................................................................................ 72

INTRODUCTION:Children with high body mass index (BMI) values have been demonstrated to have precocious dental development. Research has largely focused on cross-sectional data sets, leaving an incomplete understanding of the longitudinal relationship between BMI and dental maturation. METHODS:We used a pure longitudinal growth series to examine the relationship between dental development and childhood BMI. Periapical radiographs from 77 children from the Iowa Growth Study were used to estimate dental development for those with high BMI values. RESULTS:We confirmed prior studies in finding that children with higher BMI values were more likely to have advanced dental development for their ages (P <0.001). BMI at age 4 years was predictive for the timing of dental development at age 12 (P = 0.052). The precocity of the rate of dental development accelerated across growth. Overall dental development scores also correlated with the age of dental eruption for the mandibular canines and first premolars (P <0.001). CONCLUSIONS:High BMI values at young ages predict advanced dental development at later times, suggesting a long-term effect of BMI on dental maturation and implying the need for earlier orthodontic interventions in obese children. These results corroborate those of previous studies, building further evidence that relatively early dental eruption is another consequence of childhood obesity.

ObjectivesMany studies over the past decade have employed morphological integration as a framework for understanding the interaction of the facial and basicranial skeleton in humans, the great apes, and our fossil lineage. Fewer studies, however, have examined morphological integration in the facial and basicranial portions of the skull in a true longitudinal sample. Most of these longitudinal studies have focused primarily on lateral cephalograms. While useful, these datasets only examine shape and morphological integration from the lateral aspect, limiting the types of questions that can be explored, primarily in the mediolateral axis. To rectify this gap in the data, we set out to study aspects of both symmetric and asymmetric shape variation in a longitudinal postero‐anterior (PA) celphalometric dataset in order to ascertain how patterns of morphological integration change in the mediolateral plane as a function of growth.MethodsPA cephalograms from a total of 55 individuals from the longitudinal Iowa Facial Growth Study were digitized using 13 coordinate landmarks across the mandible, face, and cranial base to capture patterns of integration and modularity. This data was collected at three time points per patient: age 4, age 11, and in adulthood. Data were submitted to MorphoJ for a Two‐Block Partial Least Squares (2‐B PLS) analysis of both the symmetric and asymmetric shape components in order to examine morphological integration between the face and cranial base.Results2‐B PLS results for the symmetric shape component show that integration between the face and cranial base decrease (becoming more modularized) from age 4 (RV=0.41, p<0.001), to age 11 (RV=0.25, p<0.001) before increasing again at the adult time point (RV=0.35, p<0.001). For the asymmetric component, however, levels of integration between the face and basicranium decrease consistently from age 4 (RV=0.59, p<0.001) to adult (RV=0.47, p<0.001). These results indicate that while levels of cranial integration tend to decrease throughout growth, this decrease is only consistent in the asymmetric shape component, potentially indicating that modularity of the face and cranial base has a strong mediolateral (side‐to‐side) component.ConclusionThese results show that while overall trajectories of facial/cranial base integration show a similar trend toward modularity as individuals grow, this pattern of modularity is only a linear when examining the asymmetric shape component. This aspect of shape has been largely overlooked in the integration literature to date and warrants further study.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Current tools for the inhibition of microRNA (miR) function are limited to modified antisense oligonucleotides, sponges, and decoy RNA molecules and none have been used to understand miR function during development. CRISPR/Cas mediated deletion of miR sequences within the genome requires multiple chromosomal deletions to remove all functional miR families due to the duplication of miR seed sequences and family members. We report a novel plasmid‐based miR inhibitor system (PMIS) that inhibits miR family members in cells and mice. The PMIS engineered optimal secondary structure, flanking sequences and specific antisense miR oligonucleotide sequence bind the miR in a stable complex to inhibit miR activity. In cells, one PMIS can effectively inhibit miR family members that share the same seed sequence. A complete family of miRs can be inhibited with a single plasmid. Different PMIS miR inhibitors can be linked together to knockdown multiple miRs expressed from different chromosomes. The PMIS shows no off‐target effects or toxicity and is highly specific for miRs sharing identical seed sequences. PMIS constructs associate with Dicer and Argonaute to form a stable miR inhibitor complex. Transgenic mice expressing PMIS‐miRs reveal different developmental processes affected by miRs. Genome‐wide analyses of PMIS transgenic mice and cells identified new miR regulated gene networks. Complete inhibition of the miR‐17‐92, miR‐106a‐363, miR‐106b‐25, and miR‐200 clusters reveals new mechanisms for bone regeneration and developmental defects for these miRs. These miRs also control inflammation through the direct targeting of pro‐inflammatory cytokines. We report a new tool to dissect the role of miRs in development without genome editing and as a potential new therapeutic reagent.Support or Funding InformationThis work was supported by grants from the State of Iowa Bio Venture group, College of Dentistry and Carver College of Medicine and NIH DE025328 to LH.

The role that noncoding regions of the genome play in the etiology of cleft palate is not well studied. A novel method of microRNA (miR) inhibition that allows for specific miR knockdown in vivo has been developed by our laboratory. To further understand the role of miRs in palatogenesis, we used a new mouse model to inhibit specific miRs within the miR-17-92 cluster. Transgenic mice expressing inhibitory complexes for miR-17 and miR-18 manifested a clefting phenotype that was distinct from that observed in mice carrying inhibitory complexes for miR-17, miR-18, miR-19, and miR-92. An in silico candidate gene analysis and bioinformatics review led us to identify TGFBR2 as a likely target of miR-17 and miR-19 family members. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that TGFBR1 and TGFBR2 expression levels were elevated in the palates of these miR transgenic embryos at embryonic day 15.5. RT-PCR data also showed that the expression of mature miRs from the miR-17-92 cluster was significantly decreased in the transgenic embryos. Decreased expression of TGFB pathway signaling ligands was also observed. Experiments in cells showed that inhibition of miR-17 and miR-18 was sufficient to induce increases in expression of TGFB receptors, while a concomitant decrease in TGFB signaling ligands was not observed. RT-PCR of mature miR-17-92 in cells demonstrated the selectivity and specificity of inhibitory complexes. While this study builds on previous studies that have implicated miR-17-92 in the regulation of important molecular components of the TGFB signaling pathway, it is likely that interactions remain to be elucidated between miR-17-92 and as-of-yet unidentified molecules important for the control of palatogenesis. The differential regulation of palatogenesis by members of the miR-17-92 cluster indicates that several gene combinations regulate palate elevation and extension during development.

Hippo signaling controls tissue growth and organ size but, its role in development of the craniofacial structures is unknown. We demonstrate that tissue specific Hippo signaling regulates growth of the face and is a crucial component in determining human facial characteristics. We have identified the transcriptional factor FoxO6 as an activator of Hippo signaling with specific expression in craniofacial tissues. FoxO6 loss‐of‐function mice undergo expansion of the face and skull, enlargement of the mandible and maxilla and lengthening of the incisors associated with increases in cell proliferation. FoxO6 activates Lats1 /2 expression, thereby increasing Yap phosphorylation to control Hippo signaling. A phenotype‐genotype correlation test identified significant associations (p<0.001) with three FOXO6 human single nucleotide polymorphisms in Caucasian adults with dento‐skeletal bite problems ranging from retrognathism to prognathism of both jaws. One mutation creates a new cFOS binding element in the FOXO6 5′flanking region that increases FOXO6 expression. This mutation is associated with bimaxillary retrusion and correlates with the role of FOXO6 and Hippo signaling. FoxO6 −/− mice also exhibited decreases in the expression of Shh and Runx2, suggesting that these factors are linked to Hippo signaling. Together, these results identify a FoxO6‐Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology. These data suggest that human FOXO6 mutations explain differences in the human face form.

INTRODUCTION:The aim of this in-vitro study was to evaluate the influence of cone-beam computed tomography scans on the diagnosis of chemically simulated external root resorption. METHODS:One hundred extracted anterior teeth were selected. Subsurface demineralization was induced on a limited area of the apical third of the root of 49 teeth. Each tooth was placed in an empty socket of a partially edentulous dry mandible. Cone-beam computed tomography images were obtained according to 3 protocols: (1) half scan, 0.40-mm voxel size; (2) full scan, 0.40-mm voxel size; and (3) full scan, 0.125-mm voxel size. Three observers evaluated the images. Sensitivity, specificity, accuracy, and area under the curve were compared with the Cochran Q and Mann-Whitney U tests. RESULTS:Protocol 3 had the highest sensitivity (81.63%), accuracy (80.67%), and area under the curve (0.807). There were statistically significant differences between protocol 3 and the other 2 protocols (P <0.001). The specificity of protocol 1 (84.97%) was greater than that of protocols 2 (69.93%) and 3 (79.74%); however, a statistically significant difference was found only between protocols 1 and 2 (P = 0.005). CONCLUSIONS:A more dedicated, high-resolution scan should be acquired when one intends to investigate the early stage of external root resorption during orthodontic treatment. However, this does not imply that all orthodontic patients should be subjected to high-dose cone-beam computed tomography scans.

A microRNA‐26b‐5p ( miR‐26b) over‐expression (OE) mouse was generated to understand the role of miR‐26b during embryonic development. The miR‐26b over expression mice have craniofacial defects including a lack of incisors, molars and hair. miR‐26b over‐expression mice have arrested early tooth development coincident with decreased epithelial progenitor cell proliferation. We demonstrate an inverse correlation between miR‐26b levels and Lef1 expression in the craniofacial region of these mice. miR‐26b targets Lef‐1 to modulate Lef‐1 transcriptional activity. Both cyclin D1 and c‐myc expression are decreased as well as other cell proliferation mechanisms. miR‐26b expression correlates with the transition of Lef‐1 expression in the dental epithelium. miR‐26b regulates all Lef‐1 isoforms and Wnt signaling, dependent on the Lef‐1 isoform expressed in specific dental tissues. Oral epithelial‐specific overexpression of Lef‐1 can rescue these specific tooth defects. This is the first demonstration of a mouse model for miRNA regulation that has tooth agenesis. miR‐26b regulation of Lef‐1 is essential for normal tooth and craniofacial development.

Prdm16 is involved in normal human and mouse craniofacial development. The cleft secondary palate 1 (csp1) ENU‐induced mutation displays cleft palate, shortening of the snout, and micrognathia on a congenic FVB/NJ strain background due to reduced Prdm16 expression, which mirrors the mechanism of Pierre Robin Sequence (PRS) in human. However, overt cleft palate is not evident on a congenic C57BL6/J background (csp1‐B6). Adult heterozygous csp1‐B6 mice have significantly shortened snouts, palates, and right‐side mandibles compared to wt littermates along with variable incidence of malocclusion and hydrocephalus. Homozygous mutant csp1‐B6 mice die shortly after birth for unknown reasons. Our aim is to quantitatively assess variation in bony facial morphology in newborn heterozygous (het) and homozygous mutant (mut) csp1‐B6 and contrast this with the wild type morphology.We compared the size, shape, and volume of the facial bones in newborn wt, het, and mut csp1‐B6 mice. Anatomical landmarks, bone surface area, and bone volume data were collected from three‐dimensional microcomputed tomography (microCT) images.Homozygous mutant newborn mice have significantly longer left‐sided premaxillae and maxillae, but shorter left‐side mandibles than the wt and het mice. The mut mice also showed significantly reduced left‐ and right‐side premaxillae heights versus wt and het mice and smaller individual facial bone volumes and surface areas. The wt and het newborn mice did not differ significantly from each other in any contrasts.Reduced Prdm16 expression in newborn homozygous mutant csp1‐B6 mice is correlated with overall, and asymmetric, micrognathia and undergrowth of the snout compared to the newborn wt and het mice. These findings complement our concurrent research on adult heterozygous csp1‐B6 mice, and will provide insight on the downstream effects of Prdm16 expression on craniofacial development and integration among the bones of the face.Support or Funding InformationNIH/NIDCR (R15DE023982)Midwestern University, College of Dental Medicine Illinois Intramural Research Grant

Gene trap mutagenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays to enable directional gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional gene trap cassettes, we developed a generic strategy for generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the Prdm16 transcription factor gene. We demonstrate that the gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other Prdm16 mutant mice. Consecutive breeding to Flpe and Emx1IREScre deleter mice spatially restricted Prdm16 loss to regions of the forebrain expressing the homeobox gene Emx1, demonstrating the utility of the technology for the analysis of tissue-specific gene functions.

The nasal septal cartilage is thought to be a key growth center that contributes to nasofacial skeletal development. Despite the developmental influence of the nasal septum however, humans often exhibit a high frequency of septal deviation suggesting discordance in the growth between the septum and surrounding nasofacial skeleton. While there are numerous etiological factors that contribute to septal deviation, the surrounding nasofacial skeleton may also act to constrain the septum, resulting in altered patterns of growth. That is, while the nasal septum has a direct morphogenetic influence on aspects of the nasofacial skeleton, other nasofacial skeletal components may restrict septal growth resulting in deviation. Detailing the developmental relationship between these structures is important not only for understanding the causal determinants of nasal septal deviation, but also for developing a broader understanding of the complex interaction between the facial skeleton and chondrocranium. We selected 66 non‐syndromic subjects from the University of Minnesota Orthodontic Clinic who ranged from 7 to 18 years in age and had an existing pretreatment cone‐beam computed tomography (CBCT) scan. Using CBCT data, we examined the developmental relationship between nasal septal deviation and the surrounding nasofacial skeleton. We measured septal deviation as a percentage of septal volume relative to a modeled non‐deviated septum. We then collected a series of coordinate landmark data in the region immediately surrounding the nasal septum in the midsagittal plane representing the nasofacial skeleton. First, we examined ontogenetic changes in the magnitude of nasal septal deviation relative to chronological age and nasofacial size. Next, using Procrustes‐based geometric morphometric techniques, we assessed the morphological relationship between nasal septal deviation and nasofacial skeletal shape. Our results indicate that variation in the magnitude of nasal septal deviation was established in our earliest age group and maintained throughout ontogeny. Moreover, nasal septal deviation was correlated with non‐allometric variation in nasofacial shape restricted to the region of the anterior sphenoid body. Ultimately, our results suggest that early developmental variation in midline basicranial components may act to alter or constrain patterns of nasal septal growth.