Unexpected role of SIX1 variants in craniosynostosis: expanding the phenotype of SIX1-related disorders.

BACKGROUND: Pathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported. METHODS: We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1nLacZ/+ reporter mouse. RESULTS: From 1629 unrelated cases with craniosynostosis we identified seven different SIX1 variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of SIX1 loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme. CONCLUSION: Craniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis.

The developing mouse coronal suture at single-cell resolution.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

Aberrantly activated Wnt/ß-catenin pathway co-receptors LRP5 and LRP6 regulate osteoblast differentiation in the developing coronal sutures of an Apert syndrome (Fgfr2S252W/+ ) mouse model.

BACKGROUND: Apert syndrome is an autosomal, dominant inherited disorder characterized by craniosynostosis and syndactyly caused by gain-of-function mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. Wnt/ß-catenin signaling plays critical roles in regulating the skeletal development. Here, we analyzed the role of this pathway in the developing coronal sutures (CS) of a murine Apert syndrome model (Fgfr2S252W/+ ). RESULTS: We observed aberrantly increased mRNA expression of Lrp5 and Lrp6 in CS of Fgfr2S252W/+ mice, whereas both wild type (WT) and Fgfr2S252W/+ mice showed similar expression of other Wnt/ß-catenin-related genes, such as Wnt3, Wnt3a, Fzd4, Fzd6, Axin2, and Dkk1 as evidenced by in situ hybridization. Significantly increased Lrp5 and Lrp6 mRNA expression was observed by quantitative PCR analysis of cultured cells isolated from CS of Fgfr2S252W/+ mice. Phospho-LRP5, phospho-LRP6, and non-phospho-ß-catenin were upregulated in Fgfr2S252W/+ CS compared with that in WT CS. Short-interfering RNA targeting Lrp5 and Lrp6 significantly reduced runt-related transcription factor 2, collagen type 1 alpha 1, and osteocalcin mRNA expression, and alkaline phosphatase activity in cultured cells. CONCLUSIONS: The Wnt/ß-catenin pathway was activated in the CS of Fgfr2S252W/+ mice during craniofacial development, suggesting the involvement of the Wnt/ß-catenin pathway in the pathogenesis of CS synostosis in Fgfr2S252W/+ mice.

Integrated Transcriptome and Network Analysis Reveals Spatiotemporal Dynamics of Calvarial Suturogenesis.

Craniofacial abnormalities often involve sutures, the growth centers of the skull. To characterize the organization and processes governing their development, we profile the murine frontal suture, a model for sutural growth and fusion, at the tissue- and single-cell level on embryonic days (E)16.5 and E18.5. For the wild-type suture, bulk RNA sequencing (RNA-seq) analysis identifies mesenchyme-, osteogenic front-, and stage-enriched genes and biological processes, as well as alternative splicing events modifying the extracellular matrix. Single-cell RNA-seq analysis distinguishes multiple subpopulations, of which five define a mesenchyme-osteoblast differentiation trajectory and show variation along the anteroposterior axis. Similar analyses of in vivo mouse models of impaired frontal suturogenesis in Saethre-Chotzen and Apert syndromes, Twist1+/- and Fgfr2+/S252W, demonstrate distinct transcriptional changes involving angiogenesis and ribogenesis, respectively. Co-expression network analysis reveals gene expression modules from which we validate key driver genes regulating osteoblast differentiation. Our study provides a global approach to gain insights into suturogenesis.

Recent Advances in Craniosynostosis.

Craniosynostosis is a pathologic craniofacial disorder and is defined as the premature fusion of one or more cranial (calvarial) sutures. Cranial sutures are fibrous joints consisting of nonossified mesenchymal cells that play an important role in the development of healthy craniofacial skeletons. Early fusion of these sutures results in incomplete brain development that may lead to complications of several severe medical conditions including seizures, brain damage, mental delay, complex deformities, strabismus, and visual and breathing problems. As a congenital disease, craniosynostosis has a heterogeneous origin that can be affected by genetic and epigenetic alterations, teratogens, and environmental factors and make the syndrome highly complex. To date, approximately 200 syndromes have been linked to craniosynostosis. In addition to being part of a syndrome, craniosynostosis can be nonsyndromic, formed without any additional anomalies. More than 50 nuclear genes that relate to craniosynostosis have been identified. Besides genetic factors, epigenetic factors like microRNAs and mechanical forces also play important roles in suture fusion. As craniosynostosis is a multifactorial disorder, evaluating the craniosynostosis syndrome requires and depends on all the information obtained from clinical findings, genetic analysis, epigenetic or environmental factors, or gene modulators. In this review, we will focus on embryologic and genetic studies, as well as epigenetic and environmental studies. We will discuss published studies and correlate the findings with unknown aspects of craniofacial disorders.

Premaxillary-maxillary suture development in the first trimester : An ultrasound study.

PURPOSE: Our study was designed to investigate premaxillary-maxillary suture growth in fetuses from the first trimester of pregnancy using the B­ultrasound technique in order to determine the suture fusion time. METHODS: We selected 169 healthy Han singleton pregnancies as subjects. All subjects received routine pregnancy tests and were divided into three groups based on the gestational age of the fetus: group 1, the 11th gestational week; group 2, the 12th gestational week; and group 3, the 13th gestational week. Fetal biometric measurements were recorded during consecutive prenatal ultrasonographic examinations. These measurements included nuchal translucency thickness, crown-rump length, and premaxillary-maxillary length. Intergroup comparisons were performed using analysis of variance (ANOVA). RESULTS: The premaxillary-maxillary suture grows gradually and its measured length at the 11th, 12th and 13th week was 0.54 cm, 0.65 cm, and 0.74 cm, respectively. We observed a significant linear correlation between the premaxillary-maxillary length and the week of gestation in the first trimester. The growth rate of the maxilla at the 11th, the 12th and the 13th week are significantly different with a descending order of growth rates being week 12, week 11 and week 13, with the 12th week rate being the most rapid. CONCLUSION: The premaxillary and maxillary growth at 11 and 12 gestational weeks in the first trimester steadily accelerated, peaking at the 12th week. The rate of growth slows down after week 12 which may be associated with the fusion of the premaxillary-maxillary suture.

The Brain Shadowing Sign: A Clue Finding for Early Suspicion of Craniosynostosis?

Antenatal imaging of craniosynostosis mainly relies on the demonstration with 2D ultrasound of the abnormal contour of the calvarium and of the loss of hypoechogenicity of the synostotic sutures and on indirect signs of premature closure of the skull sutures; however, isolated craniosynostosis is detected only sporadically at prenatal ultrasound. In this article, we present the first case to our knowledge in which the "brain shadowing sign," a recently described indirect sign of craniosynostosis, noted at 24 weeks in a structurally normal fetus, was the first clue for the diagnosis of isolated bilateral coronal craniosynostosis, which became evident at late gestation.

Rapamycin rescues BMP mediated midline craniosynostosis phenotype through reduction of mTOR signaling in a mouse model.

Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during embryonic stages, but not at the newborn stage. Our study demonstrated that inhibition of mTOR signaling via rapamycin in a time specific manner partially rescued the loss of the suture stem cell population. This study provides insight into how enhanced BMP signaling regulates suture stem cells via mTOR activation.

Dysregulated PDGFRα signaling alters coronal suture morphogenesis and leads to craniosynostosis through endochondral ossification.

Craniosynostosis is a prevalent human birth defect characterized by premature fusion of calvarial bones. In this study, we show that tight regulation of endogenous PDGFRα activity is required for normal calvarium development in the mouse and that dysregulated PDGFRα activity causes craniosynostosis. Constitutive activation of PDGFRα leads to expansion of cartilage underlying the coronal sutures, which contribute to suture closure through endochondral ossification, in a process regulated in part by PI3K/AKT signaling. Our results thus identify a novel mechanism underlying calvarial development in craniosynostosis.

An HDAC Inhibitor, Entinostat/MS-275, Partially Prevents Delayed Cranial Suture Closure in Heterozygous Runx2 Null Mice.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder caused by mutations in RUNX2, coding a key transcription factor of early osteogenesis. CCD patients suffer from developmental defects in cranial bones. Despite numerous investigations and clinical approaches, no therapeutic strategy has been suggested to prevent CCD. Here, we show that fetal administration of Entinostat/MS-275, a class I histone deacetylase (HDAC)-specific inhibitor, partially prevents delayed closure of cranial sutures in Runx2+/- mice strain of C57BL/6J by two mechanisms: 1) posttranslational acetylation of Runx2 protein, which stabilized the protein and activated its transcriptional activity; and 2) epigenetic regulation of Runx2 and other bone marker genes. Moreover, we show that MS-275 stimulates osteoblast proliferation effectively both in vivo and in vitro, suggesting that delayed skeletal development in CCD is closely related to the decreased number of progenitor cells as well as the delayed osteogenic differentiation. These findings provide the potential benefits of the therapeutic strategy using MS-275 to prevent CCD. © 2017 American Society for Bone and Mineral Research.

An anatomical study of wormian bones from the eastern part of India: is genetic influence a primary determinant of their morphogenesis?

Over the years a number of investigators have analysed the morphology of wormian bones in different population groups across the world. There have been significant variations between findings reported in these studies, and this has prompted researchers to focus on the influence of genetic factors on the morphology of these bones. In the light of the above observation, we considered it justified to conduct anatomical studies on wormian bones in different population groups; hence, we undertook the present study to look into the morphological details of these bones among a population in the eastern part of India. We observed a total of 120 adult dry human skulls of unknown age and sex, and noted the anatomical details of wormian bones when present. It was observed that wormian bones were present in 45 % of skulls, and that 30 % of skulls had more than one wormian bone. We also found that 2.5 % of the skulls had ten or more wormian bones, which is considered as pathognomonic. Maximum incidence (53.33 %) was observed at the lambdoid suture and minimum incidence at the bregma and metopic suture (0.61 % in each case). We noted a high incidence (21.21 %) of Inca bone/lambdoid ossicle, and bilaterally symmetrical wormian bones were present in 12.5 % study skulls. There were statistically significant (P < 0.05) variations between the findings of the present study and values reported in previous studies conducted in other regions of India and different parts of the world. Our observations favour the view that genetic influence primarily determines the morphology of wormian bones.

FGFR2c-mediated ERK-MAPK activity regulates coronal suture development.

Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor's gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis.

Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures.

During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures.

Flat bones and sutures formation in the human cranial vault during prenatal development and infancy: A computational model.

The processes of flat bones growth, sutures formation and interdigitation in the human calvaria are controlled by a complex interaction between genetic, biochemical and environmental factors that regulate bone formation and resorption during prenatal development and infancy. Despite previous experimental evidence accounting for the role of the main biochemical factors acting on these processes, the underlying mechanisms controlling them are still unknown. Therefore, we propose a mathematical model of the processes of flat bone and suture formation, taking into account several biological events. First, we model the growth of the flat bones and the formation of sutures and fontanels as a reaction diffusion system between two proteins: TGF-ß2 and TGF-ß3. The former is expressed by osteoblasts and allows adjacent mesenchymal cells differentiation on the bone fronts of each flat bone. The latter is expressed by mesenchymal cells at the sutures and inhibits their differentiation into osteoblasts at the bone fronts. Suture interdigitation is modelled using a system of reaction diffusion equations that develops spatio-temporal patterns of bone formation and resorption by means of two molecules (Wnt and Sclerostin) which control mesenchymal cells differentiation into osteoblasts at these sites. The results of the computer simulations predict flat bone growth from ossification centers, sutures and fontanels formation as well as bone formation and resorption events along the sutures, giving rise to interdigitated patterns. These stages were modelled and solved by the finite elements method. The simulation results agree with the morphological characteristics of calvarial bones and sutures throughout human prenatal development and infancy.

BCL11B regulates sutural patency in the mouse craniofacial skeleton.

The transcription factor BCL11B plays essential roles during development of the immune, nervous, and cutaneous systems. Here we show that BCL11B is expressed in both osteogenic and sutural mesenchyme of the developing craniofacial complex. Bcl11b(-/-) mice exhibit increased proliferation of osteoprogenitors, premature osteoblast differentiation, and enhanced skull mineralization leading to synostoses of facial and calvarial sutures. Ectopic expression of Fgfr2c, a gene implicated in craniosynostosis in mice and humans, and that of Runx2 was detected within the affected sutures of Bcl11b(-/-) mice. These data suggest that ectopic expression of Fgfr2c in the sutural mesenchyme, without concomitant changes in the expression of FGF ligands, appears to induce the RUNX2-dependent osteogenic program and craniosynostosis in Bcl11b(-/-) mice.

The Development of the Calvarial Bones and Sutures and the Pathophysiology of Craniosynostosis.

The skull vault is a complex, exquisitely patterned structure that plays a variety of key roles in vertebrate life, ranging from the acquisition of food to the support of the sense organs for hearing, smell, sight, and taste. During its development, it must meet the dual challenges of protecting the brain and accommodating its growth. The bones and sutures of the skull vault are derived from cranial neural crest and head mesoderm. The frontal and parietal bones develop from osteogenic rudiments in the supraorbital ridge. The coronal suture develops from a group of Shh-responsive cells in the head mesoderm that are collocated, with the osteogenic precursors, in the supraorbital ridge. The osteogenic rudiments and the prospective coronal suture expand apically by cell migration. A number of congenital disorders affect the skull vault. Prominent among these is craniosynostosis, the fusion of the bones at the sutures. Analysis of the pathophysiology underling craniosynostosis has identified a variety of cellular mechanisms, mediated by a range of signaling pathways and effector transcription factors. These cellular mechanisms include loss of boundary integrity, altered sutural cell specification in embryos, and loss of a suture stem cell population in adults. Future work making use of genome-wide transcriptomic approaches will address the deep structure of regulatory interactions and cellular processes that unify these seemingly diverse mechanisms.

A Genetic-Pathophysiological Framework for Craniosynostosis.

Craniosynostosis, the premature fusion of one or more cranial sutures of the skull, provides a paradigm for investigating the interplay of genetic and environmental factors leading to malformation. Over the past 20 years molecular genetic techniques have provided a new approach to dissect the underlying causes; success has mostly come from investigation of clinical samples, and recent advances in high-throughput DNA sequencing have dramatically enhanced the study of the human as the preferred "model organism." In parallel, however, we need a pathogenetic classification to describe the pathways and processes that lead to cranial suture fusion. Given the prenatal onset of most craniosynostosis, investigation of mechanisms requires more conventional model organisms; principally the mouse, because of similarities in cranial suture development. We present a framework for classifying genetic causes of craniosynostosis based on current understanding of cranial suture biology and molecular and developmental pathogenesis. Of note, few pathologies result from complete loss of gene function. Instead, biochemical mechanisms involving haploinsufficiency, dominant gain-of-function and recessive hypomorphic mutations, and an unusual X-linked cellular interference process have all been implicated. Although few of the genes involved could have been predicted based on expression patterns alone (because the genes play much wider roles in embryonic development or cellular homeostasis), we argue that they fit into a limited number of functional modules active at different stages of cranial suture development. This provides a useful approach both when defining the potential role of new candidate genes in craniosynostosis and, potentially, for devising pharmacological approaches to therapy.

Metopic suture and RUNX2, a key transcription factor in osseous morphogenesis with possible important implications for human brain evolution.

BACKGROUND: Overall, the comparative data available on the timing of metopic suture closure in present-day and fossil members of human lineage, as well as great apes, seem to indicate that human brain evolution occurred within a complex network of fetopelvic constraints, which required modification of frontal neurocranial ossification patterns, involving delayed fusion of the metopic suture. It is very interesting that the recent sequencing of the Neanderthal genome has revealed signs of positive selection in the modern human variant of the RUNX2 gene, which is known to affect metopic suture fusion in addition to being essential for osteoblast development and proper bone formation. It is possible that an evolutionary change in RUNX2, affecting aspects of the morphology of the upper body and cranium, was of importance in the origin of modern humans. Thus, to contribute to a better understanding of the molecular evolution of this gene probably implicated in human evolution, we performed a comparative bioinformatic analysis of the coding sequences of RUNX2 in Homo sapiens and other non-human Primates. RESULTS: We found amino-acid sequence differences between RUNX2 protein isoforms of Homo sapiens and the other Primates examined, that might have important implications for the timing of metopic suture closure. CONCLUSIONS: Further studies are needed to clear the potential distinct developmental roles of different species-specific RUNX2 N-terminal isoforms. Meantime, our bioinformatic analysis, regarding expression of the RUNX2 gene in Homo sapiens and other non-human Primates, has provided a contribution to this important issue of human evolution.

Cranial sutures: a multidisciplinary review.

INTRODUCTION: Progress in cranial suture research is shaping our current understanding of the topic; however, emphasis has been placed on individual contributing components rather than the cranial sutural system as a whole. Improving our holistic view helps further guide clinicians who treat cranial sutural abnormalities as well as researchers who study them. MATERIALS AND METHODS: Information from anatomy, anthropology, surgery, and computed modeling was integrated to provide a perspective to interpret suture formation and variability within the cranial functional and structural system. RESULTS: Evidence from experimental settings, simulations, and evolution suggest a multifactorial morphogenetic process associated with functions and morphology of the sutures. Despite molecular influences, the biomechanical cranial environment has a main role in both the ontogenetic and phylogenetic suture dynamics. CONCLUSIONS: Furthering our holistic understanding of the intricate cranial sutural system promises to expand our knowledge and enhance our ability to treat associated anomalies.