Cranial Vault Development Research Articles

FGF signaling in cranial suture development and related diseases.

Suture mesenchymal stem cells (SMSCs) are a heterogeneous stem cell population with the ability to self-renew and differentiate into multiple cell lineages. The cranial suture provides a niche for SMSCs to maintain suture patency, allowing for cranial bone repair and regeneration. In addition, the cranial suture functions as an intramembranous bone growth site during craniofacial bone development. Defects in suture development have been implicated in various congenital diseases, such as sutural agenesis and craniosynostosis. However, it remains largely unknown how intricate signaling pathways orchestrate suture and SMSC function in craniofacial bone development, homeostasis, repair and diseases. Studies in patients with syndromic craniosynostosis identified fibroblast growth factor (FGF) signaling as an important signaling pathway that regulates cranial vault development. A series of in vitro and in vivo studies have since revealed the critical roles of FGF signaling in SMSCs, cranial suture and cranial skeleton development, and the pathogenesis of related diseases. Here, we summarize the characteristics of cranial sutures and SMSCs, and the important functions of the FGF signaling pathway in SMSC and cranial suture development as well as diseases caused by suture dysfunction. We also discuss emerging current and future studies of signaling regulation in SMSCs.

Fgfr3 Is a Positive Regulator of Osteoblast Expansion and Differentiation During Zebrafish Skull Vault Development.

Gain or loss-of-function mutations in fibroblast growth factor receptor 3 (FGFR3) result in cranial vault defects highlighting the protein's role in membranous ossification. Zebrafish express high levels of fgfr3 during skull development; in order to study FGFR3's role in cranial vault development, we generated the first fgfr3 loss-of-function zebrafish (fgfr3lof/lof ). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. Integrated analyses (in vivo imaging and single-cell RNA sequencing of the osteoblast lineage) of zebrafish fgfr3lof/lof revealed a delay in osteoblast expansion and differentiation, together with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis. We conclude that changes in the extracellular matrix within growing bone might impair cell-cell communication, mineralization, and new osteoblast recruitment. © 2020 American Society for Bone and Mineral Research.

On the traces of tcf12: Investigation of the gene expression pattern during development and cranial suture patterning in zebrafish (Danio rerio).

The transcription factor 12 (tcf12) is a basic Helix-Loop-Helix protein (bHLH) of the E-protein family, proven to play an important role in developmental processes like neurogenesis, mesoderm formation, and cranial vault development. In humans, mutations in TCF12 lead to craniosynostosis, a congenital birth disorder characterized by the premature fusion of one or several of the cranial sutures. Current research has been primarily focused on functional studies of TCF12, hence the cellular expression profile of this gene during embryonic development and early stages of ossification remains poorly understood. Here we present the establishment and detailed analysis of two transgenic tcf12:EGFP fluorescent zebrafish (Danio rerio) reporter lines. Using these transgenic lines, we analyzed the general spatiotemporal expression pattern of tcf12 during different developmental stages and put emphasis on skeletal development and cranial suture patterning. We identified robust tcf12 promoter-driven EGFP expression in the central nervous system (CNS), the heart, the pronephros, and the somites of zebrafish embryos. Additionally, expression was observed inside the muscles and bones of the viscerocranium in juvenile and adult fish. During cranial vault development, the transgenic fish show a high amount of tcf12 expressing cells at the growth fronts of the ossifying frontal and parietal bones and inside the emerging cranial sutures. Subsequently, we tested the transcriptional activity of three evolutionary conserved non-coding elements (CNEs) located in the tcf12 locus by transient transgenic assays and compared their in vivo activity to the expression pattern determined in the transgenic tcf12:EGFP lines. We could validate two of them as tcf12 enhancer elements driving specific gene expression in the CNS during embryogenesis. Our newly established transgenic lines enhance the understanding of tcf12 gene regulation and open up the possibilities for further functional investigation of these novel tcf12 enhancer elements in zebrafish.

Trps1 Regulates Development of Craniofacial Skeleton and Is Required for the Initiation of Palatal Shelves Fusion.

Trichorhinophalangeal syndrome (TRPS) is an autosomal dominant disorder resulting from heterozygous mutations of the TRPS1 gene. Common craniofacial abnormalities in TRPS patients include micrognathia, hypoplastic zygomatic arch, high-arched palate, and, occasionally, cleft palate. Studies have demonstrated that mice with a heterozygous Trps1 mutation (Trps1+/− mice) have similar features to patients with TRPS, including high-arched palates. However, mice with a homozygous Trps1 mutation (Trps1−/− mice) exhibit similar but more severe abnormalities, including cleft palate. Our study aimed to characterize the craniofacial phenotype to understand the role of Trps1 in craniofacial development and gain insight on the cleft palate pathogenesis in Trps1 deficiency. Whole-mount skeletal staining revealed hypoplastic skeletal and cartilaginous elements, steep nasal slope, and missing presphenoid in Trps1−/− mice. Although several craniofacial skeleton elements were abnormal in Trps1−/− mice, the Trps1 deficiency did not appear to disrupt cranial vault development. All Trps1−/− mice presented with cleft palate. Analyses of Trps1 expression during palatogenesis detected Trps1 mRNA and protein in palatal mesenchyme and in specific regions of palatal epithelium, which suggested that Trps1 is involved in palatal fusion. Ex vivo culture experiments demonstrated that Trps1−/− palatal shelves were unable to initiate the fusion process. On the molecular level, Trps1 deficiency resulted in decreased epithelial expression of proteins involved in palatal fusion, including chondroitin sulfate proteoglycan, transforming growth factor-beta 3, Twist1, and beta-catenin. Mesenchymal expression of chondroitin sulfate proteoglycan expression was unaffected, indicating a cell type-specific mechanism of Trps1 regulation on chondroitin sulfate proteoglycan. In conclusion, we demonstrated that Trps1 is involved in the development of craniofacial skeletal elements and in the initiation of the palatal shelves fusion. Furthermore, our studies uncovered that Trps1 is required for epithelial expression of several proteins involved in the palatal shelves fusion.

Exploring Mechanisms of Cranial Vault Development using a Coupled Turing‐Biomechanical Model

Bones of the mouse cranial vault are formed by differentiation of mesenchymal cells into osteoblasts, a process called intramembranous ossification that is primarily thought to be controlled by a cascade of reactions between extracellular molecules and cells that may be significantly guided by mechanical forces. In this study, we use a computational approach and an extensive experimental database to elucidate the role of biomechanics in morphogenesis of the cranial vault. We assume that the biochemical process can be modeled using Turing's reaction-diffusion model, a mathematical model describing the pattern formation controlled by interacting molecules (activators and inhibitors). In addition, we hypothesize that mechanical stimuli of the cells due to growth of the underlying brain contributes significantly to the process of cell differentiation. We have developed a coupled framework that incorporates these modeling constructs. The mathematical formulation was solved using the finite volume method. The computational domain and model parameters are determined using a large collection of experimental data that provides precise three-dimensional measures of mouse cranial geometry and cranial vault bone formation for specific embryonic time points. The results of this study suggest that mechanical strain contributes information to specific aspects of bone formation. Our mechanobiological model predicts some key features of cranial vault bone formation that were verified by experimental observations in normal case, including the relative location of ossification centers of the individual vault bones, the pattern of cranial vault bone growth over time, and the position of cranial vault sutures. Support or Funding Information Computations for this research were performed on the Pennsylvania State University's Institute for Cyber Science Advanced Cyber Infrastructure (ICS-ACI). This work was supported in part through instrumentation funded by a National Science Foundation grant OCI0821527, a Burroughs-Wellcome Fund 2013 Collaborative Research Travel Grant, Pennsylvania Department of Health using Tobacco Cure Funds, and by the National Institutes of Health grants R01DE022988 and P01HD078233. The model predicts the pattern of cranial vault bone growth. a) Computational prediction of the distribution of differentiating osteoblasts and cranial vault bone formation by embryonic day. b) Observed cranial vault bone formation and growth in embryonic mice. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Signaling Mechanisms Underlying Genetic Pathophysiology of Craniosynostosis.

Craniosynostosis, is the premature fusion of one or more cranial sutures which is the second most common cranial facial anomalies. The premature cranial sutures leads to deformity of skull shape and restricts the growth of brain, which might elicit severe neurologic damage. Craniosynostosis exhibit close correlations with a varieties of syndromes. During the past two decades, as the appliance of high throughput DNA sequencing techniques, steady progresses has been made in identifying gene mutations in both syndromic and nonsyndromic cases, which allow researchers to better understanding the genetic roles in the development of cranial vault. As the enrichment of known mutations involved in the pathogenic of premature sutures fusion, multiple signaling pathways have been investigated to dissect the underlying mechanisms beneath the disease. In addition to genetic etiology, environment factors, especially mechanics, have also been proposed to have vital roles during the pathophysiological of craniosynostosis. However, the influence of mechanics factors in the cranial development remains largely unknown. In this review, we present a brief overview of the updated genetic mutations and environmental factors identified in both syndromic and nonsyndromic craniosynostosis. Furthermore, potential molecular signaling pathways and its relations have been described.

Minimally invasive approaches to craniosynostosis.

Craniosynostosis (CS) is defined as the premature fusion of one or more calvarial sutures. This carries several consequences, including abnormal/asymmetric cranial vault development, increased intracranial pressure, compromised neurocognitive development, and craniofacial deformity. Definitive management is surgical with the goal of protecting cerebral development by re-establishing normal cranial vault expansion and correcting cosmetic deformity. In today's practice, CS surgery has advanced radically from simple craniectomies to major cranial vault reconstructive (CVR) procedures. More recently there has been considerable interest in endoscopic assisted surgery (EAS). Theoretical benefits include decreased operative time, morbidity, blood loss, postoperative pain, cost and faster recovery times. In this focused review, we summarize the current body of literature reporting clinical outcomes in EAS and review the data comparing EAS and CVR.

A COMPUTATIONAL ANALYSIS OF BONE FORMATION IN THE CRANIAL VAULT USING A COUPLED REACTION-DIFFUSION-STRAIN MODEL.

Bones of the murine cranial vault are formed by differentiation of mesenchymal cells into osteoblasts, a process that is primarily understood to be controlled by a cascade of reactions between extracellular molecules and cells. We assume that the process can be modeled using Turing's reaction-diffusion equations, a mathematical model describing the pattern formation controlled by two interacting molecules (activator and inhibitor). In addition to the processes modeled by reaction-diffusion equations, we hypothesize that mechanical stimuli of the cells due to growth of the underlying brain contribute significantly to the process of cell differentiation in cranial vault development. Structural analysis of the surface of the brain was conducted to explore the effects of the mechanical strain on bone formation. We propose a mechanobiological model for the formation of cranial vault bones by coupling the reaction-diffusion model with structural mechanics. The mathematical formulation was solved using the finite volume method. The computational domain and model parameters are determined using a large collection of experimental data that provide precise three dimensional (3D) measures of murine cranial geometry and cranial vault bone formation for specific embryonic time points. The results of this study suggest that mechanical strain contributes information to specific aspects of bone formation. Our mechanobiological model predicts some key features of cranial vault bone formation that were verified by experimental observations including the relative location of ossification centers of individual vault bones, the pattern of cranial vault bone growth over time, and the position of cranial vault sutures.

Syndromic craniosynostosis associated with microdeletion of chromosome 19p13.12–19p13.2

Craniosynostosis, a condition in which the cranial sutures prematurely fuse, can lead to elevated intracranial pressure and craniofacial abnormalities in young children. Currently surgical intervention is the only therapeutic option for patients with this condition. Craniosynostosis has been associated with a variety of different gene mutations and chromosome anomalies. Here we describe three cases of partial deletion of chromosome 19p. Two of the cases present with syndromic craniosynostosis while one has metopic ridging. A review of the genes involved in the rearrangements between the three cases suggests several gene candidates for craniosynostosis. CALR and DAND5, BMP regulators involved in osteoblast differentiation, and MORG1, a mediator of osteoclast dysregulation may play a role in abnormal cranial vault development. Additionally, CACNA1A, a gene that when mutated is associated with epilepsy and CC2D1A, a gene associated with non-syndromic mental retardation may contribute to additional phenotypic features seen in the patients we describe. In addition, these findings further support the need for genetic testing in cases of syndromic craniosynostosis.

Presphenoidal synchondrosis fusion in DBA/2J mice

Cranial base growth plates are important centers of longitudinal growth in the skull and are responsible for the proper anterior placement of the face and the stimulation of normal cranial vault development. We report that the presphenoidal synchondrosis (PSS), a midline growth plate of the cranial base, closes in the DBA/2J mouse strain but not in other common inbred strains. We investigated the genetics of PSS closure in DBA/2J mice by evaluating F1, F1 backcross, and/or F1 intercross offspring from matings with C57BL/6J and DBA/1J mice, whose PSS remain open. We observed that PSS closure is genetically determined, but not inherited as a simple Mendelian trait. Employing a genome-wide SNP array, we identified a region on chromosome 11 in the C57BL/6J strain that affected the frequency of PSS closure in F1 backcross and F1 intercross offspring. The equivalent region in the DBA/1J strain did not affect PSS closure in F1 intercross offspring. We conclude that PSS closure in the DBA/2J strain is complex and modified by different loci when outcrossed with C57BL/6J and DBA/1J mice.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-012-9437-8) contains supplementary material, which is available to authorized users.

Quantitative Morphometric Outcomes Following the Melbourne Method of Total Vault Remodeling for Scaphocephaly

The Melbourne method of total vault remodeling was developed at The Royal Children's Hospital (Melbourne) to address all phenotypic aspects of scaphocephaly. To quantitatively evaluate this technique, a retrospective analysis was performed on 33 consecutive patients who underwent the Melbourne procedure between October 2004 and June 2007. To monitor outcomes, three-dimensional digital surface photography was used, obtaining 4 anthropometric measurements (cranial length, cranial width, head circumference, auricular head height) preoperatively and postoperatively. To assess the effect on continuing cranial vault development, cranial volume was calculated with computed tomography. Anthropometric measurements were obtained in 27 patients (81.8%), with a mean postoperative follow-up of 8.9 months. Cranial index (width/length) demonstrated an 11.1% improvement. The mean head circumference in the scaphocephaly group preoperatively remained larger than the normative population postoperatively, although the magnitude of difference was decreased, whereas the mean auricular head height demonstrated a 10.5% increase postoperatively, remaining higher than the normative population. Cranial volume was calculated in 30 patients (91%), with a mean postoperative follow-up of 7.9 months. This demonstrated that the mean intracranial volume was significantly higher in the scaphocephaly group preoperatively, and this difference was maintained postoperatively. These results support our belief that the Melbourne procedure is a technique that may be used to correct all phenotypic aspects of scaphocephaly, with no apparent evidence for a detrimental effect on cranial growth.

BMP signals regulate Dlx5 during early avian skull development

The vertebrate skull vault forms almost entirely by the direct mineralisation of mesenchyme, without the formation of a cartilaginous template, a mechanism called membranous ossification. Dlx5 gene mutation leads to cranial dismorphogenesis which differs from the previously studied craniosynostosis syndromes [Development 126 (1999), 3795; Development 126 (1999), 3831]. In avians, little is known about the genetic regulation of cranial vault development. In this study, we analyze Dlx5 expression and regulation during skull formation in the chick embryo. We compare Dlx5 expression pattern with that of several genes involved in mouse cranial suture regulation. This provides an initial description of the expression in the developing skull of the genes encoding the secreted molecules BMP 2, BMP 4, BMP 7, the transmembrane FGF receptors FGFR 1, FGFR 2, FGFR 4, the transcription factors Msx1, Msx2, and Twist, as well as Goosecoid and the early membranous bone differentiation marker osteopontin. We show that Dlx5 is activated in proliferating osteoblast precursors, before osteoblast differentiation. High levels of Dlx5 transcripts are observed at the osteogenic fronts (OFs) and at the edges of the suture mesenchyme, but not in the suture itself. Dlx5 expression is initiated in areas where Bmp4 and Bmp7 genes become coexpressed. In a calvarial explant culture system, Dlx5 transcription is upregulated by BMPs and inhibited by the BMP-antagonist Noggin. In addition, FGF4 activates Bmp4 but not Bmp7 gene transcription and is not sufficient to induce ectopic Dlx5 expression in the immature calvarial mesenchyme. From these data, we propose a model for the regulatory network implicated in early steps of chick calvarial development.