Cheryl A. Hill

Abnormalities in cartilage and bone development in the Apert syndrome FGFR2+/S252W mouse

Apert syndrome is an autosomal dominant disorder characterized by malformations of the skull, limbs and viscera. Two-thirds of affected individuals have a S252W mutation in fibroblast growth factor receptor 2 (FGFR2). To study the pathogenesis of this condition, we generated a knock-in mouse model with this mutation. The Fgfr2+/S252W mutant mice have abnormalities of the skeleton, as well as of other organs including the brain, thymus, lungs, heart and intestines. In the mutant neurocranium, we found a midline sutural defect and craniosynostosis with abnormal osteoblastic proliferation and differentiation. We noted ectopic cartilage at the midline sagittal suture, and cartilage abnormalities in the basicranium, nasal turbinates and trachea. In addition, from the mutant long bones, in vitro cell cultures grown in osteogenic medium revealed chondrocytes, which were absent in the controls. Our results suggest that altered cartilage and bone development play a significant role in the pathogenesis of the Apert syndrome phenotype.

Activation of p38 MAPK pathway in the skull abnormalities of Apert syndrome Fgfr2+P253R mice

BackgroundApert syndrome is characterized by craniosynostosis and limb abnormalities and is primarily caused by FGFR2 +/P253R and +/S252W mutations. The former mutation is present in approximately one third whereas the latter mutation is present in two-thirds of the patients with this condition. We previously reported an inbred transgenic mouse model with the Fgfr2 +/S252W mutation on the C57BL/6J background for Apert syndrome. Here we present a mouse model for the Fgfr2+/P253R mutation.ResultsWe generated inbred Fgfr2+/P253Rmice on the same C56BL/6J genetic background and analyzed their skeletal abnormalities. 3D micro-CT scans of the skulls of the Fgfr2+/P253Rmice revealed that the skull length was shortened with the length of the anterior cranial base significantly shorter than that of the Fgfr2+/S252Wmice at P0. The Fgfr2+/P253Rmice presented with synostosis of the coronal suture and proximate fronts with disorganized cellularity in sagittal and lambdoid sutures. Abnormal osteogenesis and proliferation were observed at the developing coronal suture and long bones of the Fgfr2+/P253Rmice as in the Fgfr2+/S252Wmice. Activation of mitogen-activated protein kinases (MAPK) was observed in the Fgfr2+/P253Rneurocranium with an increase in phosphorylated p38 as well as ERK1/2, whereas phosphorylated AKT and PKCα were not obviously changed as compared to those of wild-type controls. There were localized phenotypic and molecular variations among individual embryos with different mutations and among those with the same mutation.ConclusionsOur in vivo studies demonstrated that the Fgfr2 +/P253R mutation resulted in mice with cranial features that resemble those of the Fgfr2+/S252Wmice and human Apert syndrome. Activated p38 in addition to the ERK1/2 signaling pathways may mediate the mutant neurocranial phenotype. Though Apert syndrome is traditionally thought to be a consistent phenotype, our results suggest localized and regional variations in the phenotypes that characterize Apert syndrome.

Beyond the closed suture in Apert syndrome mouse models: evidence of primary effects of FGFR2 signaling on facial shape at birth

Apert syndrome is a congenital disorder caused mainly by two neighboring mutations on fibroblast growth factor receptor 2 (FGFR2). Premature closure of the coronal suture is commonly considered the identifying and primary defect triggering or preceding the additional cranial malformations of Apert phenotype. Here we use two transgenic mouse models of Apert syndrome, Fgfr2+/S252W and Fgfr2+/P253R, to explore variation in cranial phenotypes in newborn (P0) mice. Results show that the facial skeleton is the most affected region of the cranium. Coronal suture patency shows marked variation that is not strongly correlated with skull dysmorphology. The craniofacial effects of the FGFR2 mutations are similar, but Fgfr2+/S252W mutant mice display significantly more severe dysmorphology localized to the posterior palate. Our results demonstrate that coronal suture closure is neither the primary nor the sole locus of skull dysmorphology in these mouse models for Apert syndrome, but that the face is also primarily affected. Developmental Dynamics 239:3058–3071, 2010.

Effects of aneuploidy on skull growth in a mouse model of Down syndrome

Adult craniofacial morphology results from complex interactions among genetic, epigenetic and environmental factors. Trisomy causes perturbations in the genetic programmes that control development and these are reflected in morphology that can either ameliorate or worsen with time and growth. Many of the specific changes that occur in Down syndrome can be studied in the Ts65Dn trisomic mouse, which shows direct parallels with specific aspects of adult craniofacial dysmorphology associated with trisomy 21. This study investigates patterns of craniofacial growth in Ts65Dn mice and their euploid littermates to assess how the adult dysmorphology develops. Three‐dimensional coordinate data were collected from microcomputed tomography scans of the face, cranial base, palate and mandible of newborn (P0) and adult trisomic and euploid mice. Growth patterns were analysed using Euclidean distance matrix analysis. P0 trisomic mice show significant differences in craniofacial shape. Growth is reduced along the rostro‐caudal axis of the Ts65Dn face and palate relative to euploid littermates and Ts65Dn mandibles demonstrate reduced growth local to the mandibular processes. Thus, the features of Down syndrome that are reflected in the mature Ts65Dn skull are established early in development and growth does not appear to ameliorate them. Differences in growth may in fact contribute to many of the morphological differences that are evident at birth in trisomic mice and humans.

Brain phenotypes in two FGFR2 mouse models for Apert syndrome

Apert syndrome (AS) is one of at least nine disorders considered members of the fibroblast growth factor receptor (FGFR) ‐1, ‐2, and ‐3–related craniosynostosis syndromes. Nearly 100% of individuals diagnosed with AS carry one of two neighboring mutations on Fgfr2. The cranial phenotype associated with these two mutations includes coronal suture synostosis, either unilateral (unicoronal synostosis) or bilateral (bicoronal synostosis). Brain dysmorphology associated with AS is thought to be secondary to cranial vault or base alterations, but the variation in brain phenotypes within Apert syndrome is unexplained. Here, we present novel three‐dimensional data on brain phenotypes of inbred mice at postnatal day 0 each carrying one of the two Fgfr2 mutations associated with AS. Our data suggest that the brain is primarily affected, rather than secondarily responding to skull dysmorphogenesis. Our hypothesis is that the skull and brain are both primarily affected in craniosynostosis and that shared phenogenetic developmental processes affect both tissues in craniosynostosis of Apert syndrome. Developmental Dynamics 239:987–997, 2010.

Complex Contributions of Ets2 to Craniofacial and Thymus Phenotypes of Trisomic “Down Syndrome” Mice

Ts65Dn mice have segmental trisomy for orthologs of about half of the genes on human chromosome 21, including Ets2. These mice develop anomalies of the cranial skeleton and thymus that parallel those in Down syndrome. Overexpression of the Ets2 transcription factor gene was posited to be sufficient to produce these craniofacial and thymus deficits in transgenic mice that constitutively overexpress a processed Ets2 transcript under a promiscuous promoter [Sumarsono et al. (1996); Nature 379:534–537; Wolvetang et al. (2003); Hum Mol Genet 12:247–255]. Evaluation of trisomic mice with varying copy numbers of a properly regulated Ets2 gene indicated increased dosage of Ets2 was not sufficient to produce effects on thymus and most of the cranial anomalies seen in Ts65Dn mice. However, mesoderm‐derived cranial skeletal elements are significantly more affected in Ts65Dn, Ets2+/− mice compared to Ts65Dn littermates suggesting a differential interaction of Ets2‐related processes with mesoderm‐derived and neural crest‐derived formative tissues. Our results support the growing evidence for interactions among multiple genes contributing to developmental perturbations resulting in variation in complex Down syndrome phenotypes.

A quantitative method for the evaluation of three-dimensional structure of temporal bone pneumatization

Temporal bone pneumatization has been included in lists of characters used in phylogenetic analyses of human evolution. While studies suggest that the extent of pneumatization has decreased over the course of human evolution, little is known about the processes underlying these changes or their significance. In short, reasons for the observed reduction and the potential reorganization within pneumatized spaces are unknown. Technological limitations have limited previous analyses of pneumatization in extant and fossil species to qualitative observations of the extent of temporal bone pneumatization. In this paper, we introduce a novel application of quantitative methods developed for the study of trabecular bone to the analysis of pneumatized spaces of the temporal bone. This method utilizes high-resolution X-ray computed tomography (HRXCT) images and quantitative software to estimate three-dimensional parameters (bone volume fractions, anisotropy, and trabecular thickness) of bone structure within defined units of pneumatized spaces. We apply this approach in an analysis of temporal bones of diverse but related primate species, Gorilla gorilla, Pan troglodytes, Homo sapiens, and Papio hamadryas anubis, to illustrate the potential of these methods. In demonstrating the utility of these methods, we show that there are interspecific differences in the bone structure of pneumatized spaces, perhaps reflecting changes in the localized growth dynamics, location of muscle attachments, encephalization, or basicranial flexion.

Postnatal brain and skull growth in an Apert syndrome mouse model

Craniofacial and neural tissues develop in concert throughout prenatal and postnatal growth. FGFR‐related craniosynostosis syndromes, such as Apert syndrome (AS), are associated with specific phenotypes involving both the skull and the brain. We analyzed the effects of the FGFR P253R mutation for AS using the Fgfr2+/P253R Apert syndrome mouse to evaluate the effects of this mutation on these two tissues over the course of development from day of birth (P0) to postnatal day 2 (P2). Three‐dimensional magnetic resonance microscopy and computed tomography images were acquired from Fgfr2+/P253R mice and unaffected littermates at P0 (N = 28) and P2 (N = 20).Three‐dimensional coordinate data for 23 skull and 15 brain landmarks were statistically compared between groups. Results demonstrate that the Fgfr2+/P253R mice show reduced growth in the facial skeleton and the cerebrum, while the height and width of the neurocranium and caudal regions of the brain show increased growth relative to unaffected littermates. This localized correspondence of differential growth patterns in skull and brain point to their continued interaction through development and suggest that both tissues display divergent postnatal growth patterns relative to unaffected littermates. However, the change in the skull–brain relationship from P0 to P2 implies that each tissue affected by the mutation retains a degree of independence, rather than one tissue directing the development of the other.

Ontogenetic Change in Temporal Bone Pneumatization in Humans

Pneumatization of the temporal bone is often included in descriptions of fossils and as a phylogenetic marker, but a number of questions about the evolution, growth, and development of the trait remain. Many studies have analyzed temporal bone pneumatization from a clinical perspective, but a systematic quantification of normal development of pneumatized spaces has not been conducted. In this study, ontogenetic change in the size and organization of temporal bone pneumatization is analyzed in a cross‐sectional sample of humans. High resolution computed tomography scans of the temporal bone were acquired from a cross‐sectional sample of humans (N = 28). Bone volume fractions, anisotropy, trabecular number, trabecular thickness, surface area, and volume were analyzed to provide information about the organization and size of pneumatized spaces across ontogeny. The results indicate that there are general and region‐specific patterns of ontogenetic changes in the organization of pneumatized spaces. These changes reflect the transition from nonpneumatized bone to pneumatized bone. It also demonstrates that those regions that are pneumatized early in ontogeny (such as the mastoid antrum) continue to remodel after the initial period of pneumatization. The dynamic nature of temporal bone pneumatization over ontogeny suggests that this character requires careful consideration when used as a character for phylogenetic analyses. These results demonstrate the importance of comparing individuals from similar developmental stages, especially when completing quantitative analyses of the extent of pneumatization or organization of the spaces. Anat Rec,, 2011.

Intracranial Volume and Whole Brain Volume in Infants With Unicoronal Craniosynostosis

Objective Craniosynostosis has been hypothesized to result in alterations of the brain and cerebral blood flow due to reduced intracranial volume, potentially leading to cognitive deficits. In this study we test the hypothesis that intracranial volume and whole brain volume in infants with unilateral coronal synostosis differs from those in unaffected infants. Design Our study sample consists of magnetic resonance images acquired from 7- to 72-week-old infants with right unilateral coronal synostosis prior to surgery (n = 10) and age-matched unaffected infants (n = 10). We used Analyze 9.0 software to collect three cranial volume measurements. We used nonparametric tests to determine whether the three measures differ between the two groups. Correlations were calculated between age and the three volume measures in each group to determine whether the growth trajectory of the measurements differ between children with right unicoronal synostosis and unaffected infants. Results Our results show that the three volume measurements are not reduced in infants with right unicoronal synostosis relative to unaffected children. Correlation analyses between age and various volume measures show similar correlations in infants with right unicoronal synostosis compared with unaffected children. Conclusions Our results show that the relationship between brain size and intracranial size in infants with right unicoronal synostosis is similar to that in unaffected children, suggesting that reduced intracranial volume is not responsible for alterations of the brain in craniosynostosis.