Expression of Cre recombinase in chondrocytes causes abnormal craniofacial and skeletal development

The cranial base synchondroses are growth centers that drive cranial and upper facial growth. The intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS) are two major synchondroses located in the middle of the cranial base and are maintained at early developmental stages to sustain cranial base elongation. In this study, we report unexpected premature ossification of ISS and SOS when Cre recombinase is activated in a chondrocyte-specific manner. We used a Cre transgenic line expressing Aggrecan enhancer-driven, Tetracycline-inducible Cre (ATC), of which expression is controlled by a Col2a1 promoter. Neonatal doxycycline injection or doxycycline diet fed to breeders was used to activate Cre recombinase. The premature ossification of ISS and/or SOS led to a reduction in cranial base length and subsequently a dome-shaped skull. Furthermore, the mice carrying either heterozygous or homozygous conditional deletion of Tsc1 or Fip200 using ATC mice developed similar craniofacial abnormalities, indicating that Cre activity itself but not conditional deletion of Tsc1 or Fip200 gene, is the major contributor of this phenotype. In contrast, the Col2a1-Cre mice carrying Cre expression in both perichondrium and chondrocytes and the mice carrying the conditional deletion of Tsc1 or Fip200 using Col2a1-Cre did not manifest the same skull abnormalities. In addition to the defective craniofacial bone development, our data also showed that the Cre activation in chondrocytes significantly compromised bone acquisition in femur. Our data calls for the consideration of the potential in vivo adverse effects caused by Cre expression in chondrocytes and reinforcement of the importance of including Cre-containing controls to facilitate accurate phenotype interpretation in transgenic research.

     

Patterns of postnatal development in skulls of lynxes,genus Lynx (Mammalia: Carnivora)

Studies on ossification patterns and other ontogenetic events associated with postnatal cranial growth of wild felids are scarce. An analysis of developmental processes undergone by several cranial structures (presphenoidal and sphenooccipital synchondroses, temporal and sagittal crests, and deciduous and permanent teeth) during postnatal growth has been conducted on a sample of 336 specimens belonging to the four Recent species of lynxes (Lynx pardinus, Lynx lynx, Lynx rufus, and Lynx canadensis). Age has been estimated based on tooth replacement, skull size, and by counting the annual lines of cementum growth. Comparison of the results obtained for each of the four species reveal (1) a single pattern for both tooth replacement and ossification of the sphenooccipital synchondrosis, (2) two ossification patterns for the presphenoidal synchondrosis, (3) a common pattern for development of temporal ridges and sagittal crest showing different degrees of morphological expression, and (4) evidence suggesting the involvement of a heterochronic process, neoteny, in the morphological differentiation of several populations and species of the genus Lynx. These data also support the hypothesis that processes involved in the replacement of carnassials are based on functional requirements. © 1996 Wiley-Liss, Inc.     

Postnatal growth of the cranial base in Macaca nemestrina.

Postnatal growth of the cranial base was longitudinally studied in 21 male and 11 female Macaca nemestrina. The basicranium of each animal was marked with tantulum implants in order that the tracings of each serial roentgenogram could be superimposed. Between the ages of 3.0 and 5.0 years the degree of sexual demorphism in both angular and linear dimensions increased. The cranial base flattened as a result of the upward and forward migration of nasion and the upward and backward relocation of basion. The movement of basion was primarily due to differential growth recorded at the spheno-occipital synchondrosis. Sexual difference in the relative growth of this synchondrosis resulted in a longer and somewhat flatter male cranial base. Male and female velocity curves showed accelerations that coincide with their estimated age for the onset of puberty.     

A factor analysis of cranial base and vault dimensions in children

Lengths within the cranial base and vault were measured in cephalometric radiographs of 220 boys and 177 girls ranging in age from 0 to 15 years; all these children are participants in The Fels Longitudinal Growth Study. The present study is based on mixed longitudinal data derived from 1640 radiographs for boys and 1260 radiographs for girls. Factor analysis was applied separately for boys and girls for each age group; i.e., 0–3, 4–6, 7–9, 10–12, and 13–15 years. For the 0–3 year age group, two factors were extracted in each sex, whereas four factors were extracted in the rest of the age groups. The factor structures are similar in the three older age groups of boys (7–9, 10–12, and 13–15 years). The first four factors for these groups are labelled, respectively: cranial vault size, posterior cranial base length, presphenoid length, and basisphenoid length. The order of the third and fourth factors is reversed in the 7–9 year olds. For girls, the factors extracted were also the same in both the 7–9 and 10–12 year age groups, even though the order of factors was different between age groups; i.e., anterior cranial base length, cranial vault size, basisphenoid length, and basioccipital length. Differential growth rates among cranial base dimensions probably cause changes in factor patterns. Obliteration of the spheno-occipital synchondrosis is suggested as the mechanism responsible for the change of factor pattern in the girls. Closure of this synchondrosis would have occurred too late to affect the patterns in boys.     

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.     

Anterior cervical microsurgical approach to the cranial base in the rabbit: technical note.

Current trends in research on craniofacial syndromes have led to enhanced interest in the cranial base as a contributory factor in the development of normal and abnormal midfacial structure. Indeed, attention has focused upon one particular growth plate in the posterior cranial base, the spheno-occipital synchondrosis, since it has been shown that alterations in this structure are associated with profound changes in craniofacial growth. In this report we describe a surgical approach to the cranial base of the rabbit that is safe, simple and reliable. It is applicable to neonatal as well as adult rabbits.     

Cranial suture closure in two species of South American monkeys

Cranial suture closure is examined in two species of South American monkeys, Saimiri sciureus and Saguinus nigricollis. Sequences in closure were sought as indicators of skeletal age. Some sutures seem to be more reliable determinants of skeletal age than others, and these sutures and their sequence of closure are different in the two species examined. The sphenooccipital synchondrosis and the palatal portion of the Interpremaxillary suture show regular fusion associated with age in both species. In Saimiri the maxillopremaxillary sutures are also reliable indicators of age, whereas they are not in Saguinus; however, in the latter the presphenoid-postsphenoid synchondrosis closes regularly whereas it does not in Saimiri. In Saimiri the predictable sequence is (1) maxillo-premaxillary, (2) transverse maxillo-premaxillary, (3) spheno-occipital, (4) interpremaxillary. In Saguinus it is (1) presphenoid-postsphenoid, (2) spheno-occipital, (3) interpremaxillary. It is possible that the sequences of suture closure and the variability in this process may indicate genetic and taxonomic relationships.     

Brief communication: infracranial maturation in the skeletal collection from Coimbra, Portugal: new aging standards for epiphyseal union

Age at death of a single skeletal individual or a group is essential information in archaeological, paleoanthropological, and forensic contexts. Dental remains are the most commonly used age indicators, but when the dentition is not available, or too few teeth are present for an accurate age assessment, other age indicators such as skeletal maturation must be used. Of particular utility in this regard is the fusion of the epiphyses of the infracranial skeleton. Here we present new aging standards based on the infracranial maturation of individuals from the known age and sex collection from Coimbra, Portugal. We scored infracranial epiphyseal fusion and spheno-occipital synchondrosis closure (64 loci of ossification in total) on 137 skeletons from individuals between 7 and 29 years old. We further discuss developmental differences between the sexes and similarities and differences between the Coimbra documented collection and other published aging standards.     

A cranial base of Australopithecus robustus from the hanging remnant of Swartkrans, South Africa

SKW 18, a partial hominin cranium recovered from the site of Swartkrans, South Africa, in 1968 is described. It is derived from ex situ breccia of the Hanging Remnant of Member 1, dated to approximately 1.5-1.8 Mya. Although partially encased in breccia, it was refit to the facial fragment SK 52 (Clarke 1977 The Cranium of the Swartkrans Hominid SK 847 and Its Relevance to Human Origins, Ph.D. dissertation, University of the Witwatersrand, Johannesburg), producing the composite cranium SKW 18/SK 52. Subsequent preparation revealed the most complete cranial base attributable to the species Australopithecus robustus. SKW 18 suffered weathering and slight postdepositional distortion, but retains considerable anatomical detail. The composite cranium most likely represents a large, subadult male, based on the incomplete fusion of the spheno-occipital synchondrosis; unerupted third molar; pronounced development of muscular insertions; and large teeth. Cranial base measures of SKW 18 expand the range of values previously recorded for A. robustus. SKW 18 provides information on anatomical features not previously visible in this taxon, and expands our knowledge of morphological variability recognizable in the cranial base. Morphological heterogeneity in the development of the prevertebral and nuchal muscular insertions is likely the result of sexual dimorphism in A. robustus, while differences in cranial base angles and the development of the occipital/marginal sinus drainage system cannot be attributed to size dimorphism.     

Ossification and midline shape changes of the human fetal cranial base

An appreciation of ontogenetic changes to the cranial base is important for understanding the evolution of modern human skull form. Using geometric morphometric techniques, this study explores midline shape variations of the basicranium and midface during human prenatal ontogeny. In particular, the analysis sets out to explore shape variations associated with endochondral ossification and to reassess shape variations previously observed on the basis of angular measures.Fifty-four formalin-preserved human fetuses were imaged using high-resolution MRI. Coordinates for 10 landmarks defining the midline basicranium and midface were acquired and areas of ossification in the midline basioccipital, basisphenoid, and presphenoid cartilages were measured as percentages of overall cranial base area. The results show shape variations with increasing fetal size that are consistent with cranial base retroflexion, anterior facial projection and dorsal facial rotation. These growth variations are centered on the midsphenoid area and are associated with disproportionate variations of sphenoid height and length. Small but significant correlations were observed between ossification of the presphenoid cartilage and components of shape that described, among other variations, sphenoid shortening. While ossification cannot be directly linked with the shape variations observed, it seems likely that bone formation plays a role in modulating the influence of other factors on the fetal cranial base.     

Ontogeny of the spheno‐occipital synchondrosis in a modern Queensland,Australian population using computed tomography

Due to disparity regarding the age at which skeletal maturation of the spheno‐occipital synchondrosis occurs in forensic and biological literature, this study provides recalibrated multislice computed tomography (MSCT) age standards for the Australian (Queensland) population, using a Bayesian statistical approach. The sample comprises retrospective cranial/cervical MSCT scans obtained from 448 males and 416 females aged birth to 20 years from the Skeletal Biology and Forensic Anthropology Research Osteological Database. Fusion status of the synchondrosis was scored using a modified six‐stage scoring tier on an MSCT platform, with negligible observer error (κ = 0.911 ± 0.04, intraclass correlation coefficient = 0.994). Bayesian transition analysis indicates that females are most likely to transition to complete fusion at 13.1 years and males at 15.6 years. Posterior densities were derived for each morphological stage, with complete fusion of the synchondrosis attained in all Queensland males over 16.3 years of age and females aged 13.8 years and older. The results demonstrate significant sexual dimorphism in synchondrosis fusion and are suggestive of intrapopulation variation between major geographic regions in Australia. This study contributes to the growing repository of contemporary anthropological standards calibrated for the Queensland milieu to improve the efficacy of the coronial process for medicolegal death investigation. As a stand‐alone age indicator, the basicranial synchondrosis may be consulted as an exclusion criterion when determining the age of majority that constitutes 17 years in Queensland forensic practice. Am J Phys Anthropol 157:42–57, 2015. © 2014 Wiley Periodicals, Inc.     

Synchondroseal growth in the first cervical vertebra of the rat

Growth and remodelling of the first cervical vertebra were studied in the rat by means of biometry, vital staining (alizarin red S and oxytetracycline), and histology. The measurements showed a change in the ratio of the dorso-ventral to the transversal diameter in the lumen after obliteration of the dorsal synchondrosis. The pattern of labelling between the three segments of the vertebrae joined by synchondroses in the young animals indicated that expansion of the vertebral lumen took the form of a displacement of all three parts making up the bony ring. After closure of the dorsal synchondrosis the two ventral ones were still active and the lumen size increased more in the dorso-ventral direction than transversally. Alizarin red S and oxytetracycline tended to persist in the ventral part of the vertebra, while the staining almost disappeared from the dorso-lateral segment. Resting lines were found laterally to the ventral synchondroses in haematoxylin and eosin stained sections, but not in the ventral segment between the cartilages. The growth of the rat atlas is the result of an early rapid cartilage-mediated expansion of the vertebral lumen in conjunction with the growth of the spinal cord and, at a later age, mainly of a displacement of the ventral vertebral segment, leading to the final form of the vertebra and its lumen.     

Growth of cranial synchondroses and sutures requires polycystin-1

In vertebrates, coordinated embryonic and postnatal growth of the craniofacial bones and the skull base is essential during the expansion of the rostrum and the brain. Identification of molecules that regulate skull growth is important for understanding the nature of craniofacial defects and for development of non-invasive biologically based diagnostics and therapies.Here we report on spatially restricted growth defects at the skull base and in craniofacial sutures of mice deficient for polycystin-1 (Pkd1). Mutant animals reveal a premature closure of both presphenoid and sphenooccipital synchondroses at the cranial base. Furthermore, knockout mice lacking Pkd1 in neural crest cells are characterized by impaired postnatal growth at the osteogenic fronts in craniofacial sutures that are subjected to tensile forces. Our data suggest that polycystin-1 is required for proliferation of subpopulations of cranial osteochondroprogenitor cells of both mesodermal and neural crest origin during skull growth. However, the Erk1/2 signalling pathway is up-regulated in the Pkd1-deficient skeletal tissue, similarly to that previously reported for polycystic kidney.     

Brief Communication: Timing of spheno‐occipital closure in modern Western Australians

The spheno‐occipital synchondrosis is a craniofacial growth centre between the occipital and sphenoid bones—its ossification persists into adolescence, which for the skeletal biologist, means it has potential application for estimating subadult age. Based on previous research the timing of spheno‐occipital fusion is widely variable between and within populations, with reports of complete fusion in individuals as young as 11 years of age and nonfusion in adults. The aim of this study is, therefore, to examine this structure in a mixed sex sample of Western Australian individuals that developmentally span late childhood to adulthood. The objective is to develop statistically quantified age estimation standards based on scoring the degree of spheno‐occipital fusion. The sample comprises multidetector computed tomography (MDCT) scans of 312 individuals (169 male; 143 female) between 5 and 25 years of age. Each MDCT scan is visualized in a standardized sagittal plane using three‐dimensional oblique multiplanar reformatting. Fusion status is scored according to a four‐stage system. Transition analysis is used to calculate age ranges for each defined stage and determine the mean age for transition between an unfused, fusing and fused status. The maximum likelihood estimates for the transition from open to fusing in the endocranial half is 14.44 years (male) and 11.42 years (female); transition from fusion in the ectocranial half to complete fusion is 16.16 years (male) and 13.62 years (female). This study affirms the potential value of assessing the degree of fusion in the spheno‐occipital synchondrosis as an indicator of skeletal age. Am J Phys Anthropol 153:132–138, 2014. © 2013 Wiley Periodicals, Inc.     

Hyaluronan is essential for the expansion of the cranial base growth plates

Exquisite control of chondrocyte function in the zone of hypertrophy results in expansive growth of cartilaginous growth plates, and is a prerequisite for normal skeletal lengthening. We hypothesize that hyaluronan-mediated hydrostatic pressure causes lacunae expansion in the zone of hypertrophy; an important mechanism in cartilaginous growth plate and associated skeletal expansion. The role of hyaluronan and CD44 in this mechanism was studied using organ culture of the bipolar cranial base synchondroses. Hyaluronan was present in the hypertrophic zones, pericellular to the hypertrophic chondrocytes, while no hyaluronan was detected in the resting, proliferating and maturing zones. This localization of hyaluronan was associated with increased lacunae size, suggesting that chondrocytes deposit and retain pericellular hyaluronan as they mature. In comparison, Toluidine Blue staining was associated with the territorial matrix. Hyaluronidase, the hyaluronan-degrading enzyme, and CD44, the receptor for hyaluronan which also participates in the uptake and degradation of hyaluronan, were co-localized within the zone of ossification. This pattern of expression suggests that cells in the early zone of ossification internalize and degrade hyaluronan through a CD44-mediated mechanism. Treatment of the cultured segments with either Streptomyces hyaluronidase or hyaluronan hexasaccharides inhibited lacunae expansion. These observations demonstrate that hyaluronan-mediated mechanisms play an important role in controlling normal skeletal lengthening.     

Radiographic determination of prenatal basicranial ossification

In a previous investigation on prenatal development of the human cranial base, the sequence in which the bones develop in the midsagittal region was elucidated. The purpose of the present study was to identify fetal ossification on horizontal plane roentgenograms of the occipital and sphenoid bones in the central part of the cranial base, and establish stages in bone appearance related to general fetal developmental parameters. This study is based upon roentgenograms of the cranial base of 145 human fetuses from the first half of the prenatal period. Two different maturation patterns of the sphenoid bone were observed. The first, most common pattern is characterized by a midsagittal centre of ossification and the second by bilateral centres of ossification in the corpus of the sphenoid bone. These bilateral centres might in some cases be connected by a slight bony bridge. It appears that these different maturation patterns are maintained throughout the period investigated. The material was divided into five well-defined developmental stages for both maturation patterns and general parameters of fetal development. Mapping different aspects of ossification in normal cranial development is necessary for understanding deviations of cranial maturation and growth.     

Augmentation of bone morphogenetic protein signaling in cranial neural crest cells in mice deforms skull base due to premature fusion of intersphenoidal synchondrosis

Craniofacial anomalies (CFAs) are a diverse group of disorders affecting the shapes of the face and the head. Malformation of the cranial base in humans leads CFAs, such as midfacial hypoplasia and craniosynostosis. These patients have significant burdens associated with breathing, speaking, and chewing. Invasive surgical intervention is the current primary option to correct these structural deficiencies. Understanding molecular cellular mechanism for craniofacial development would provide novel therapeutic options for CFAs. In this study, we found that enhanced bone morphogenetic protein (BMP) signaling in cranial neural crest cells (NCCs) (P0-Cre;caBmpr1a mice) causes premature fusion of intersphenoid synchondrosis (ISS) resulting in leading to short snouts and hypertelorism. Histological analyses revealed reduction of proliferation and higher cell death in ISS at postnatal day 3. We demonstrated to prevent the premature fusion of ISS in P0-Cre;caBmpr1a mice by injecting a p53 inhibitor Pifithrin-α to the pregnant mother from E15.5 to E18.5, resulting in rescue from short snouts and hypertelorism. We further demonstrated to prevent premature fusion of cranial sutures in P0-Cre;caBmpr1a mice by injecting Pifithrin-α through E8.5 to E18.5. These results suggested that enhanced BMP-p53-induced cell death in cranial NCCs causes premature fusion of ISS and sutures in time-dependent manner.     

Bone abnormalities in latent TGF-[beta] binding protein (Ltbp)-3-null mice indicate a role for Ltbp-3 in modulating TGF-[beta] bioavailability.

The TGF-betas are multifunctional proteins whose activities are believed to be controlled by interaction with the latent TGF-beta binding proteins (LTBPs). In spite of substantial effort, the precise in vivo significance of this interaction remains unknown. To examine the role of the Ltbp-3, we made an Ltbp-3-null mutation in the mouse by gene targeting. Homozygous mutant animals develop cranio-facial malformations by day 10. At 2 mo, there is a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. Histological examination of the skulls from null animals revealed ossification of the synchondroses within 2 wk of birth, in contrast to the wild-type synchondroses, which never ossify. Between 6 and 9 mo of age, mutant animals also develop osteosclerosis and osteoarthritis. The pathological changes of the Ltbp-3-null mice are consistent with perturbed TGF-beta signaling in the skull and long bones. These observations give support to the notion that LTBP-3 is important for the control of TGF-beta action. Moreover, the results provide the first in vivo indication for a role of LTBP in modulating TGF-beta bioavailability.