Quantitative Morphologic Analysis of Cranial Vault in Twist1+/- Mice: Implications in Craniosynostosis.

BACKGROUND: The haploinsufficiency in the TWIST1 gene encoding a basic helix-loop-helix transcription factor is a cause of one of the craniosynostosis syndromes, Saethre-Chotzen syndrome. Patients with craniosynostosis usually require operative release of affected sutures, which makes it difficult to observe the long-term consequence of suture fusion on craniofacial growth. METHODS: In this study, we performed quantitative analysis of morphologic changes of the skull in Twist1 heterozygously-deleted mice (Twist1+/-) with micro-computed tomographic images. RESULTS: In Twist1+/- mice, fusion of the coronal suture began before postnatal day 14 and progressed until postnatal day 56, during which morphologic changes occurred. The growth of the skull was not achieved by a constant increase in the measured distances in wild type mice; some distances in the top-basal axis were decreased during the observation period. In the Twist1+/- mouse, growth in the top-basal axis was accelerated and that of the frontal cranium was reduced. In the unicoronal suture fusion mouse, the length of the zygomatic arch of affected side was shorter in the Twist1+/- mouse. In one postnatal day 56 Twist1+/- mouse with bilateral coronal suture fusion, asymmetric zygomatic arch length was identified. CONCLUSION: The authors'results suggest that measuring the length of the left and right zygomatic arches may be useful for early diagnosis of coronal suture fusion and for estimation of the timing of synostosis, and that more detailed study on the growth pattern of the normal and the synostosed skull could provide prediction of the risk of resynostosis. CLINICAL RELEVANCE STATEMENT: The data from this study can be useful to better understand the cranial growth pattern in patients with craniosynostosis.

Temporal Progression of Craniofacial Dysmorphology in Unilateral Coronal Synostosis: A Mechanistic Hypothesis.

AIM: This study chronicles skull base and face development in nonsyndromic unilateral coronal synostosis (UCS) during infancy, to characterize the mechanistic progression of facial dysmorphology. METHODS: Computed tomography scans from 51 subjects were reviewed (26 UCS, 25 controls) and data were reconstructed. Patients were stratified into 5 age groups. A series of measurements were taken from the reconstructions. RESULTS: All patients had a unilaterally fused coronal suture at the time of analysis. Asymmetry of the sphenoid wings was present across all age groups. The sphenoid wing ipsilateral to the fused suture consistently had a more acute angle from the midline. At 19 days of age, ipsilateral nasal root and cribriform plate deviation are noted, as well as increased contralateral zygoma antero-posterior length. Patients younger than 2 months also had elongated posterior cranial bases. At 2 to 3 months of age, the cranial base widens in the anterior portion of the middle cranial fossa with an increased ipsilateral pterion to sella distance. The most delayed change observed was the increase in contralateral orbital rim angle at 7 to 12 months of age compared to normal. CONCLUSION: After suture fusion, sphenoid wing changes are among the earliest restructural malformations to take place. This suggests that the cascade of dysmorphology in UCS originates in the cranial vault, then progresses to the skull base, and lastly to the facial structures. Ipsilateral orbital changes are early facial changes in UCS that begin before 2 months of age. This is then followed by changes in the contralateral face later in development.

Zygomaticomaxillary suture maturation: A predictor of maxillary protraction? Part I - A classification method.

OBJECTIVE: The aim of this study was to present a method of classifying the maturational level of the zygomaticomaxillary sutures (ZMSs). METHODS: Cone-beam CT (CBCT) images from 74 subjects (5.6-58.4 years) were examined to define the radiographic stages of ZMS maturation. Five stages of maturation of the ZMS were identified and defined: Stage A-uniform high-density sutural line, with no or little interdigitation; Stage B-scalloped appearance of the high-density sutural line; Stage C-two parallel, scalloped, high-density lines, separated in some areas by small low-density spaces; Stage D-fusion in the inferior portion of the suture; and Stage E-complete fusion. Intra- and inter-examiner agreements were evaluated by weighted kappa tests. RESULTS: The intra- and inter-examiners reproducibility values demonstrated substantial to almost perfect agreement. No fusion of ZMSs was observed in patients up to 10 years of age. From 10 to 15 years, all maturational stages were identified. After 15 years of age, the majority of patients showed fusion of ZMSs. CONCLUSIONS: The classification of ZMS maturation using CBCT is a reliable method that allows the assessment of the morphology of the ZMSs in the individual patient.

Zygomatic bone shape in intentional cranial deformations: a model for the study of the interactions between skull growth and facial morphology.

Intentional cranial deformations (ICD) were obtained by exerting external mechanical constraints on the skull vault during the first years of life to permanently modify head shape. The repercussions of ICD on the face are not well described in the midfacial region. Here we assessed the shape of the zygomatic bone in different types of ICDs. We considered 14 non-deformed skulls, 19 skulls with antero-posterior deformation, nine skulls with circumferential deformation and seven skulls with Toulouse deformation. The shape of the zygomatic bone was assessed using a statistical shape model after mesh registration. Euclidian distances between mean models and Mahalanobis distances after canonical variate analysis were computed. Classification accuracy was computed using a cross-validation approach. Different ICDs cause specific zygomatic shape modifications corresponding to different degrees of retrusion but the shape of the zygomatic bone alone is not a sufficient parameter for classifying populations into ICD groups defined by deformation types. We illustrate the fact that external mechanical constraints on the skull vault influence midfacial growth. ICDs are a model for the study of the influence of epigenetic factors on craniofacial growth and can help to understand the facial effects of congenital skull malformations such as single or multi-suture synostoses, or of external orthopedic devices such as helmets used to correct deformational plagiocephaly.

The Periosteum of the Zygomatic Arch: Vascularization and Growth.

In addition to conveying the forces of attaching muscles and ligaments to the zygomatic and temporal bones, the arch periosteum is responsible for lateral apposition and medial resorption during the growth period. In this contribution, we describe the vasculature of the zygomatic arch in young pigs (Sus scrofa dom.) in order to understand the relationship of osseous and periosteal vessels to each other, to surrounding tissues, and to patterns of modeling. Subjects 2-6 weeks of age were perfused with vascular fill; some also received the vital bone label calcein. Whole mounts were prepared of the decalcified bony arch and of its lateral periosteum. Undecalcified arches were plastic-embedded and thick-sectioned. Additional observations on cell replication were made using material from a previous study. The osseous and periosteal vascular supplies were largely independent, joined only by a fine network at the tissue interface. Osseous vessels entered the medial side of the arch through clusters of nutrient foramina. The intraosseous branching pattern resembled the direction of appositional growth, which in turn describes the disposition of bony trabeculae in older pigs. In contrast, vessels arrived at the periosteum via muscles and ligaments and thus its perfusion may partially depend on functional activity. The open weave of periosteal vessels bore little similarity to bone architecture, especially for the temporal bone, but the appositional lateral periosteum showed indications of angiogenesis, whereas the thinner, resorptive periosteum on the medial side featured composite, possibly fusing vessels at the bone surface. Anat Rec, 299:1661-1670, 2016. © 2016 Wiley Periodicals, Inc.

Skeletal effects of the alteration of masseter muscle function.

AIM: To investigate the effects of muscle denervation and the introduction of the ß2-adrenoceptor agonist, formoterol, on the relationship between muscles and underlying skeletal growth. METHOD: Thirty-one (4-week-old) male Sprague-Dawley rats were assigned to four groups: Surgical Sham; Denervated; Denervated + ß2-agonist; and ß2-agonist only. The Surgical Sham group had the left masseteric nerve exposed but not sectioned. Both of the denervated groups had the left masseteric nerve exposed and sectioned. The groups receiving the ß2-agonist had formoterol directly injected into the left masseter muscle every three days for eight weeks. Sixteen angular and linear skeletal measurements were assessed in the overall craniofacial region and the mandible via standardised digital radiography in three views: lateral head, submento-vertex and right and left disarticulated hemi-mandibles. RESULTS: The findings indicated that, following surgical denervation of the masseter muscle, there were significant changes in the muscle and in the subsequent development of the underlying skeletal structures. The post-surgical changes were largely offset by the administration of a ß2-agonist, formoterol, which attenuated muscle atrophy. However, the administration of the ß2-agonist only, without surgical denervation, did not lead to changes in skeletal facial form. CONCLUSIONS: Denervation atrophy of the masseter muscle results in statistically significant changes in the development of the underlying skeleton. The changes, however, are localised to areas of muscle attachment. The administration of the ß2-agonist, formoterol, despite its effect on muscle anabolism, does not have a significant effect on underlying skeletal growth.

Development and three-dimensional morphology of the zygomaticotemporal suture in primate skulls.

Cranial sutures are an essential part of the growing skull, allowing bones to increase in size during growth, with their morphology widely believed to be dictated by the forces and displacements that they experience. The zygomaticotemporal suture in primates is located in the relatively weak zygomatic arch, and externally it appears a very simple connection. However, large forces are almost certainly transmitted across this suture, suggesting that it requires some level of stability while also allowing controlled movements under high loading. Here we examine the 2- and 3-dimensional (3D) morphology of the zygomaticotemporal suture in an ontogenetic series of Macaca fascicularis skulls. High resolution microcomputed tomography data sets were examined, and virtual and physical 3D replicas were created to assess both structure and general stability. The zygomaticotemporal suture is much more complex than its external appearance suggests, with interlocking facets between the adjacent zygomatic and temporal bones. It appears as if some movement is permitted across the suture in younger animals, but as they approach adulthood the complexity of the suture's interlocking bone facets reaches a level where these movements become minimal.

The effects of denervation and formoterol administration on facial growth.

OBJECTIVE: To identify and demonstrate possible alterations of skeletal structures which might follow either unilateral surgical denervation of the masseter muscle, unilateral intramuscular injection of formoterol directly into the masseter muscle, or intramuscular formoterol injection after surgical denervation. MATERIALS AND METHODS: Male Sprague Dawley rats (N = 16; four weeks of age) were prepared as four groups: 1. surgical sham + saline injection into the masseter muscle (sham); 2. surgical denervation of the masseter muscle only (den.); 3. surgical denervation of the masseter muscle plus intramuscular formoterol injection into the affected muscle (den.+form.); 4. intramuscular formoterol injection into the masseter muscle only (form.). The specimens were submitted for CT examination, the skulls and hemimandibles were photographed and measurements of craniofacial bones were made. RESULTS: In this relatively small sample, comparisons between non-experimental and experimental sides revealed differences, both within the groups and for the same measurements between groups, with the den. and den.+form. groups showing the most change. Relative increases in the gonial angle shown in these groups occurred bilaterally, with the change on the experimental side always greater in magnitude than the change on the contralateral side. CONCLUSIONS: Surgical denervation of the masseter muscle leads to an alteration in the size and shape of the skeletal structures close to the zygoma and the mandible. The intramuscular injection of formoterol into denervated masseter muscle seems to limit this skeletal alteration after surgical denervation.

Malformación venosa intraósea primaria en hueso cigomático: actualización en criterios diagnósticos / Primary intraosseous venous malformation of the zygoma: update in diagnostic criteria

Las anomalías vasculares incluyen dos entidades bien diferenciadas aunque frecuentemente confundidas en la literatura científica: los hemangiomas y las malformaciones vasculares. La localización primaria intraósea cigomática es muy infrecuente. Además, la mayoría de los casos publicados han sido categorizados de «hemangioma» de forma poco rigurosa. Los autores describen un caso de malformación venosa intraósea cigomática y discuten las características clínicas, histológicas e inmunohistoquímicas distintivas de las anomalías vasculares que conducen a un adecuado diagnóstico y un tratamiento eficaz (AU)

Vascular anomalies include two well-differentiated conditions that are nevertheless often confused in the scientific literature: haemangiomas and vascular malformations. Primary intraosseous involvement of the zygoma is particularly rare. Moreover, most reported cases of zygomatic involvement have been categorised as “haemangioma” without appropriate diagnostic criteria. The authors describe a case of intraosseous venous malformation of the zygoma and discuss the specific clinical, histological and immunohistochemical criteria of vascular anomalies that lead to a correct diagnosis and subsequent effective treatment (AU)

Three-dimensional analysis of maxillary changes associated with facemask and rapid maxillary expansion compared with bone anchored maxillary protraction.

INTRODUCTION: Our objectives in this study were to evaluate in 3 dimensions the growth and treatment effects on the midface and the maxillary dentition produced by facemask therapy in association with rapid maxillary expansion (RME/FM) compared with bone-anchored maxillary protraction (BAMP). METHODS: Forty-six patients with Class III malocclusion were treated with either RME/FM (n = 21) or BAMP (n = 25). Three-dimensional models generated from cone-beam computed tomographic scans, taken before and after approximately 1 year of treatment, were registered on the anterior cranial base and measured using color-coded maps and semitransparent overlays. RESULTS: The skeletal changes in the maxilla and the right and left zygomas were on average 2.6 mm in the RME/FM group and 3.7 mm in the BAMP group; these were different statistically. Seven RME/FM patients and 4 BAMP patients had a predominantly vertical displacement of the maxilla. The dental changes at the maxillary incisors were on average 3.2 mm in the RME/FM group and 4.3 mm in the BAMP group. Ten RME/FM patients had greater dental compensations than skeletal changes. CONCLUSIONS: This 3-dimensional study shows that orthopedic changes can be obtained with both RME/FM and BAMP treatments, with protraction of the maxilla and the zygomas. Approximately half of the RME/FM patients had greater dental than skeletal changes, and a third of the RME/FM compared with 17% of the BAMP patients had a predominantly vertical maxillary displacement.

Evaluation of some facial anthropometric parameters in an Iranian population: infancy through adolescence.

OBJECTIVES: By finding the mean value of anthropometric parameters in normal samples of a population, it is possible to create a template for facial analysis. The aim of our study was to measure the anthropometric parameters in 0- to 12-year-old girls of Fars ethnic origin in the Northeast of Iran. STUDY DESIGN: Six hundred sixty-two newborn to 12-year-old girls of Fars ethnic origin participated in the study. A digital camera was used to take frontal full-face photographs of each child. Thirteen measurements were taken with the Smile Analyzer software: al-al, ch-ch, en-en, ex-ex, ft'-ft', go'-go', t-t, zy'-zy', n'-gn', n'-sn, t-g', t-gn', t-sn. Data were analyzed using the SPSS software at the significance level of 0.05. RESULTS: In almost all parameters, we found significant growth acceleration between 2 and 4 years as well as 5 and 6 years of age. Another growth spurt was seen between 9 and 11 years, although it was less noticeable. Comparing the linear regression equations suggests that different craniofacial dimensions do not grow similarly. CONCLUSIONS: By age, craniofacial dimensions change at different rates. Different craniofacial dimensions do not grow at consistent rates. Some parts grow slower compared with others. The intercanthal width has the slowest growth. Facial height shows the fastest growth.

Zygomatic dysmorphology in unicoronal synostosis.

INTRODUCTION: Unicoronal synostosis (UCS) imparts a fronto-facial deformity, the hallmark feature being orbital and forehead dysmorphology. The facial and malar regions also consistently display asymmetry, however, zygomatic structural characteristics have not been investigated. The purpose of this study is to objectively analyze the zygomata of UCS patients compared to normal controls. METHODS: Three dimensional-computed tomographic images and demographic information were obtained from normal control and UCS patients. Volumetric and morphometric analyses were performed and results statistically analyzed. P values<0.05 were considered statistically significant. RESULTS: A total of 68 zygomatic sides were analyzed: twelve control (6 females; mean age: 6.6 months) and 22 UCS patients (10 females; mean age: 5.1 months). The affected side was right in 55% (n=12) and left in 45% (n=10) of UCS patients. The affected zygomata were volumetrically deficient compared to unaffected and normal control zygomata. Unaffected zygomata demonstrated diminished volume compared to norms. Morphometrically, affected zygomata differed, while both the unaffected and control zygomata were similar. Age stratification revealed marked differences in zygomatic volume and morphometry between the affected and unaffected zygomata was greatest at an early age interval. CONCLUSION: The affected UCS zygomata are on average smaller compared to unaffected and normal control zygomata. Moreover, distinct morphometric differences exist on the affected zygomata versus both unaffected and control zygomata. These differences are not addressed by commonly employed treatment approaches. Further studies evaluating the growth effect of UCS zygomatic morphology should be entertained.

Radiation-induced craniofacial bone growth inhibition: acute and long-term effects on bone histopathology with and without cytoprotection.

BACKGROUND: The authors previously established an animal model of radiation-induced craniofacial bone growth inhibition and demonstrated the effectiveness of cytoprotection in preserving growth using amifostine, but the mechanism is unclear. The objective of this study was to investigate the acute and long-term histopathologic effects of single-dose orthovoltage irradiation on craniofacial bone with and without cytoprotection. METHODS: Sixty infant New Zealand White rabbits (7-week-old) were randomized into three groups (n = 20 per group): group 1, 0-Gy, sham irradiation; group 2, 35-Gy single-dose orthovoltage irradiation; and group 3, cytoprotection with amifostine before irradiation. Orbitozygomatic complex bone was harvested from animals 12 hours after irradiation and at skeletal maturity (21 weeks of age). Histologic parameters measured included native bone cell (osteoblast, osteoclast, and osteocyte) populations, periosteal proliferation indices (MIB-1 stains), bone turnover rates [triple fluorochromes: tetracycline administered at 7 weeks of age (before irradiation), alizarin complexone at 12 weeks, and calcein at 16 weeks of age], and endosteal space fibrosis levels. RESULTS: Orthovoltage irradiation significantly (p < 0.05) reduced osteoblast and osteoclast counts 12 hours after irradiation (age, 7 weeks) with or without pretreatment with amifostine but had no effect on osteocyte populations. Long-term analysis at age 21 weeks demonstrated significantly (p < 0.05) increased osteoblast counts, reduced endosteal space fibrosis, reduced periosteal proliferation indices, and improved bone turnover (fluorochrome stains) in amifostine-treated animals. CONCLUSION: This study suggests that amifostine cytoprotection is mediated through a combination of reduced cellular injury with enhanced promotion of cellular bone rebuilding potential.

Early partial monolateral zygomatic arch defect leads to unilateral craniofacial malformation.

OBJECTIVE: This study used reconstructed three-dimensional imaging to examine the influence of early partial monolateral zygomatic arch defect on the craniofacial development in a minipig model. METHODS: Five 7-week-old Chinese minipigs were used in this study. Each of them underwent skull radiography, three-dimensional computed tomography (CT), and surgery at 8 weeks of age. Bilateral zygomatic arches were randomized and divided into the experimental side and the control side. A standard 2-cm-long zygomatic arch defect was made by an electric drill on the experimental side. The contralateral side was left intact. One of them underwent skull radiography and three-dimensional CT 2, 4, 8, and 12 weeks after surgery. The other 3 minipigs underwent scanning 4, 8, and 12 weeks after surgery. The bone defect was observed by radiography and three-dimensional CT. All three-dimensional CT data were examined by Mimics 10.01 software, and three-dimensional images were reconstructed. The length of both zygomatic arches, the length and width of the skull, and the hemicranial angles of both sides were measured and compared. RESULTS: The zygomatic arch developed to a summit at approximately 20 weeks of age. The zygomatic arch length of the experimental side is longer than that of the control side at each time point after surgery. The hemicranial angle of the experimental side is less than that of the control side at each time point after surgery. CONCLUSIONS: Early partial monolateral zygomatic arch defect accelerates its growth in the sagittal plane and impedes the hemicranial growth in the coronal plane. Early reconstruction of zygomatic arch defect may be essential to minimize the developmental craniofacial malformation in children.

A mixed longitudinal anthropometric study of craniofacial growth of Colombian mestizos 6-17 years of age.

The purpose of this study was to evaluate the craniofacial growth of Colombian mestizos. Four age cohorts, including a total of 458 children and adolescents (262 males and 216 females), were included in this mixed-longitudinal study. The cohorts were first measured at ages 6, 9, 12, and 15 and every year thereafter for 3 years. Eight anthropometric measurements were taken, including three cranial (head perimeter, head width, and head length), two craniofacial (maxillary and mandibular length), and three facial (face height, bizygomatic width, and bigonial width). Multilevel analyses showed that all dimensions increased between 6 and 17 years of age. The cranium grew less than the craniofacial, which in turn grew less than the facial dimensions. In addition, vertical dimensions showed more growth than antero-posterior dimensions, which in turn grew more than transverse dimensions. None of the measurement showed statistically significant growth differences between subjects with normal occlusion and Class I or Class II malocclusions. Males were generally larger than females and showed greater growth rates. Except for facial width, whose yearly velocities decreased regularly with age, an adolescent growth spurt was evident for most of the male measurements. Yearly velocities for females followed a simpler decelerating pattern. The results provide reference data for Colombian mestizos, for whom normative data of other ethnic groups are not applicable. While occlusion had little or no effect, there were gender differences, as well as important growth differences between cranial and facial measurements.

Craniofacial growth, maturation, and change: teens to midadulthood.

Despite the attainment of several adult cranial dimensions relatively early in childhood, skeletal maturity and, by consequence, adult form are typically defined by the eruption of the third molars around 17 years of age. This in turn serves as the division between subadults and adults, which is then applied to population studies of biological variation. Specifically, comparative data sets of adult measurements are not directly applied to individuals who do not have complete skeletal growth, as it is believed that the confounding effects of allometry may skew the results. The present study uses geometric morphometrics techniques to investigate the appropriateness of this division with respect to three-dimensional anatomical landmarks. Twenty-six landmarks were collected from a single population of 24 crania partitioned into 4 age groups spanning late adolescence to midadulthood. Generalized Procrustes and multivariate statistical analyses were performed on the landmark data. Results showed no significant morphological differences between the teen and young adult age groups, whereas significant shape and size differences were found in older adults relative to their younger cohorts. Moreover, no growth-related shape variation (ie, allometry) was detected within the sample. These findings suggest that adult form is attained several years earlier than commonly thought and corroborate other research that suggest that subtle changes in cranial morphology continue throughout adulthood.

Superimposition of 3-dimensional cone-beam computed tomography models of growing patients.

INTRODUCTION: The objective of this study was to evaluate a new method for superimposition of 3-dimensional (3D) models of growing subjects. METHODS: Cone-beam computed tomography scans were taken before and after Class III malocclusion orthopedic treatment with miniplates. Three observers independently constructed 18 3D virtual surface models from cone-beam computed tomography scans of 3 patients. Separate 3D models were constructed for soft-tissue, cranial base, maxillary, and mandibular surfaces. The anterior cranial fossa was used to register the 3D models of before and after treatment (about 1 year of follow-up). RESULTS: Three-dimensional overlays of superimposed models and 3D color-coded displacement maps allowed visual and quantitative assessment of growth and treatment changes. The range of interobserver errors for each anatomic region was 0.4 mm for the zygomatic process of maxilla, chin, condyles, posterior border of the rami, and lower border of the mandible, and 0.5 mm for the anterior maxilla soft-tissue upper lip. CONCLUSIONS: Our results suggest that this method is a valid and reproducible assessment of treatment outcomes for growing subjects. This technique can be used to identify maxillary and mandibular positional changes and bone remodeling relative to the anterior cranial fossa.

Fluctuating asymmetry and developmental instability in sagittal craniosynostosis.

OBJECTIVE: To determine whether premature sagittal craniosynostosis is associated with developmental instability in the skull by analyzing fluctuating asymmetry in skull shape. DESIGN: Cranial shape was quantified by collecting coordinate data from landmarks located on three-dimensional reconstructions of preoperative computed tomography (CT) images of 22 children with sagittal craniosynostosis and 22 age-matched controls. A fluctuating asymmetry application of Euclidean distance matrix analysis (EDMA) was used to quantify and compare asymmetry in cranial shape using these landmark data. RESULTS: In contrast to expectations, the sagittal craniosynostosis group did not show a statistically significant increase in the overall level of fluctuating asymmetry relative to the control group. However, we discerned statistically significant localized increases in fluctuating asymmetry in the sagittal craniosynostosis group at pterion and the anterior clinoid processes (alpha = .05). We also determined a significant correlation of fluctuating asymmetry values between the two groups (r = .71). CONCLUSIONS: We conclude that there is no evidence of a role for system-wide developmental instability in the etiology of nonsyndromic sagittal craniosynostosis. However, the localized evidence of asymmetry at the anterior clinoid processes in the sagittal synostosis group suggests an association with the tracts of dura mater that attach there.

Effect of eda loss of function on upper jugal tooth morphology.

The Tabby/eda mice, which bear a loss of function mutation for the eda (ectodysplasinA) gene, are known to display developmental anomalies in organs with an ectodermal origin. Although the lower jugal (cheek) teeth of Tabby/eda mice have been extensively studied, upper teeth have never been investigated in detail. However, this may help us to further understand the function of the eda gene in tooth development. In this work, the shape and size of both the crown and the radicular system were studied in the Tabby/eda mice upper jugal teeth. To deal with the high morphological variability, we defined several morphotypes based on cusp numbers and position. Statistical tests were then performed within and between the different morphotypes to test the correlation between tooth size and morphology. Our analysis reveals that, as in lower teeth, eda is necessary to segment the dental lamina into three teeth with the characteristic size and proportions of the mouse. Nevertheless, since strong effects are observed in heterozygous upper teeth while lower are only mildly affected, it seems that the upper jaw is more sensitive than the lower jaw to the loss of eda function. Modifications in cusp number and the abnormal crown size of the teeth are clearly linked, and our results indicate a role of eda in cusp patterning. Moreover, we found that the Tabby mutation induces variations in the dental root pattern, sometimes associated with hypercementosis, suggesting a newly uncovered role played by eda in root patterning and formation.