Neural Crest-derived Frontal Bone Research Articles

Cranial Neural Crest Cells and Their Role in the Pathogenesis of Craniofacial Anomalies and Coronal Craniosynostosis.

Craniofacial anomalies are among the most common of birth defects. The pathogenesis of craniofacial anomalies frequently involves defects in the migration, proliferation, and fate of neural crest cells destined for the craniofacial skeleton. Genetic mutations causing deficient cranial neural crest migration and proliferation can result in Treacher Collins syndrome, Pierre Robin sequence, and cleft palate. Defects in post-migratory neural crest cells can result in pre- or post-ossification defects in the developing craniofacial skeleton and craniosynostosis (premature fusion of cranial bones/cranial sutures). The coronal suture is the most frequently fused suture in craniosynostosis syndromes. It exists as a biological boundary between the neural crest-derived frontal bone and paraxial mesoderm-derived parietal bone. The objective of this review is to frame our current understanding of neural crest cells in craniofacial development, craniofacial anomalies, and the pathogenesis of coronal craniosynostosis. We will also discuss novel approaches for advancing our knowledge and developing prevention and/or treatment strategies for craniofacial tissue regeneration and craniosynostosis.

Role of embryonic origin on osteogenic potential and bone repair capacity of rat calvarial osteoblasts.

The aim of this study was to evaluate the in vitro osteogenic potential of osteoblasts from neural crest-derived frontal bone (OB-NC) and mesoderm-derived parietal bone (OB-MS) and the bone formation induced by them when injected into calvarial defects. Calvarial bones were collected from newborn Wistar rats (3-day old) and characterized as frontal and parietal prior to OB-NC and OB-MS harvesting. The cells were cultured, and several parameters of osteoblast differentiation were evaluated. These cells, or PBS without cells (control), were locally injected into 5-mm rat calvarial defects (5 × 106 cells/defect) and after 4weeks bone formation was evaluated by morphometric and histological analyses. The characterization of frontal and parietal bones assured the different embryonic origin of both cell populations, OB-NC and OB-MS. The OB-NC presented higher proliferation while the OB-MS presented higher alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of runt-related transcription factor 2, Alp, bone sialoprotein and osteocalcin revealing their high osteogenic potential. µCT analysis indicated that there was higher amount of bone formation in defects injected with both OB-NC and OB-MS compared to the control. Moreover, the bone tissue formed by both cells displayed the same histological characteristics. Despite the distinct in vitro osteogenic potential, OB-NC and OB-MS induced similar bone repair in a rat calvarial defect model. Thus, osteoblasts, irrespective of their in vitro osteogenic potential linked to embryonic origins, seem to be suitable for cell-based therapies aiming to repair bone defects.

Regional difference in microRNA regulation in the skull vault.

The murine calvaria has several membrane bones with different tissue origins (e.g., neural crest-derived frontal bone vs. mesoderm-derived parietal bone). Neural crest-derived frontal bone exhibits superior osteogenic activities and bone regeneration. MicroRNA (miRNA) has been emerged as a crucial regulator during organogenesis and is involved in a range of developmental processes. However, the underlying roles of miRNA regulation in frontal bone and parietal bone is unknown. Total of 83 significantly expressed known miRNAs were identified in frontal bones versus parietal bones. The significantly enriched gene ontology and KEGG pathway that were predicted by the enrichment miRNAs were involved in several biological processes (cell differentiation, cell adhesion, and transcription), and multiple osteogenic pathways (e.g., focal adhesion, MAPK, VEGF, Wnt, and insulin signaling pathway. Focal adhesion and insulin signaling pathway were selected for target verification and functional analysis, and several genes were predicted to be targets genes by the differentially expressed miRNAs, and these targets genes were tested with significant expressions. Our results revealed a novel pattern of miRNAs in murine calvaria with dual tissue origins, and explorations of these miRNAs will be valuable for the translational studies to enhance osteogenic potential and bone regeneration in the clinic.

Autophagy Regulates Craniofacial Bone Acquisition.

Increasing evidence has demonstrated the important role of autophagy in skeletal homeostasis; however, the role of autophagy in craniofacial bone development and acquisition is largely unknown. In this study, we investigated the effect of autophagy suppression on craniofacial bone acquisition by deleting Fip200 or Atg5, two essential autophagy genes, using Osterix-Cre (Osx-Cre). We found that the Osx-Cre transgene mildly decreased the bone mass of parietal bone but not frontal bone, and did not affect cranial base bone mass in adult mice. In the cranial vault, Fip200 or Atg5 deletion similarly decreased 50% bone mass of neural crest-derived frontal bone; Atg5 deletion decreased 50% and Fip200 deletion decreased 30% bone mass of mesoderm-derived parietal bone. In the cranial base, Fip200 or Atg5 deletion similarly decreased 30% bone mass of neural crest-derived presphenoid bone; Atg5 deletion decreased 30% and Fip200 deletion decreased 16% bone mass of mesoderm-derive basioccipital bone. Lastly, we used doxycycline treatment to inhibit the Osx-Cre expression until 2months of age and showed that postnatal Fip200 deletion led to cranial vault bone mass decrease in association with a small increase in both bone volume/tissue volume and tissue mineral density. Altogether, this study demonstrated the important role of autophagy in craniofacial bone acquisition during development and postnatal growth.

Defining a critical period in calvarial development for Hedgehog pathway antagonist-induced frontal bone dysplasia in mice

The Hedgehog (Hh) signalling pathway is essential for cellular proliferation and differentiation during embryonic development. Gain and loss of function of Hh signalling are known to result in an array of craniofacial malformations. To determine the critical period for Hh pathway antagonist-induced frontal bone hypoplasia, we examined patterns of dysmorphology caused by Hh signalling inhibition. Pregnant mice received a single oral administration of Hh signalling inhibitor GDC-0449 at 100 mg•kg−1 or 150 mg•kg−1 body weight at preselected time points between embryonic days (E)8.5 and 12.5. The optimal teratogenic concentration of GDC-0449 was determined to be 150 mg•kg−1. Exposure between E9.5 and E10.5 induced frontal bone dysplasia, micrognathia and limb defects, with administration at E10.5 producing the most pronounced effects. This model showed decreased ossification of the frontal bone with downregulation of Hh signalling. The osteoid thickness of the frontal bone was significantly reduced. The amount of neural crest-derived frontal bone primordium was reduced after GDC-0449 exposure owing to a decreased rate of cell proliferation and increased cell death.

Contribution of cranial neural crest cells to mouse skull development.

The mammalian skull vault is a highly regulated structure that evolutionally protects brain growth during vertebrate development. It consists of several membrane bones with different tissue origins (e.g. neural crest-derived frontal bone and mesoderm-derived parietal bone). Although membrane bones are formed through intramembranous ossification, the neural crest-derived frontal bone has superior capabilities for osteoblast activities and bone regeneration via TGF, BMP, Wnt, and FGF signaling pathways. Neural crest (NC) cells are multipotent, and once induced, will follow specific paths to migrate to different locations of the body where they give rise to a diverse array of cell types and tissues. Recent studies using genetic mouse models have greatly advanced our knowledge of NC cell induction, proliferation, migration and differentiation. Perturbations or disruptions of neural crest patterning lead to severe developmental defects or diseases. This review summarizes recent discoveries including novel functions of genes or signaling molecules that are capable of governing developmental processes of neural crest patterning, which may function as a gene regulatory network in controlling skull development. The proposed regulatory network will be important to understand how the signaling pathways and genes converge to regulate osteoblast activities and bone formation, which will be beneficial for the potential identification of molecular targets to prevent or alleviate human diseases or disorders involving defective neural crest development.

A novel ciliopathic skull defect arising from excess neural crest

The skull is essential for protecting the brain from damage, and birth defects involving disorganization of skull bones are common. However, the developmental trajectories and molecular etiologies by which many craniofacial phenotypes arise remain poorly understood. Here, we report a novel skull defect in ciliopathic Fuz mutant mice in which only a single bone pair encases the forebrain, instead of the usual paired frontal and parietal bones. Through genetic lineage analysis, we show that this defect stems from a massive expansion of the neural crest-derived frontal bone. This expansion occurs at the expense of the mesodermally-derived parietal bones, which are either severely reduced or absent. A similar, though less severe, phenotype was observed in Gli3 mutant mice, consistent with a role for Gli3 in cilia-mediated signaling. Excess crest has also been shown to drive defective palate morphogenesis in ciliopathic mice, and that defect is ameliorated by reduction of Fgf8 gene dosage. Strikingly, skull defects in Fuz mutant mice are also rescued by loss of one allele of fgf8, suggesting a potential route to therapy. In sum, this work is significant for revealing a novel skull defect with a previously un-described developmental etiology and for suggesting a common developmental origin for skull and palate defects in ciliopathies.

Enhanced Activation of Canonical Wnt Signaling Confers Mesoderm-Derived Parietal Bone with Similar Osteogenic and Skeletal Healing Capacity to Neural Crest-Derived Frontal Bone.

Bone formation and skeletal repair are dynamic processes involving a fine-tuned balance between osteoblast proliferation and differentiation orchestrated by multiple signaling pathways. Canonical Wnt (cWnt) signaling is known to playing a key role in these processes. In the current study, using a transgenic mouse model with targeted disruption of axin2, a negative regulator of cWnt signaling, we investigated the impact of enhanced activation of cWnt signaling on the osteogenic capacity and skeletal repair. Specifically, we looked at two calvarial bones of different embryonic tissue origin: the neural crest-derived frontal bone and the mesoderm-derived parietal bone, and we investigated the proliferation and apoptotic activity of frontal and parietal bones and derived osteoblasts. We found dramatic differences in cell proliferation and apoptotic activity between Axin2 -/- and wild type calvarial bones, with Axin2 -/- showing increased proliferative activity and reduced levels of apoptosis. Furthermore, we compared osteoblast differentiation and bone regeneration in Axin2 -/- and wild type neural crest-derived frontal and mesoderm-derived parietal bones, respectively. Our results demonstrate a significant increase either in osteoblast differentiation or bone regeneration in Axin2 -/- mice as compared to wild type, with Axin2 -/- parietal bone and derived osteoblasts displaying a “neural crest-derived frontal bone-like” profile, which is typically characterized by higher osteogenic capacity and skeletal repair than parietal bone. Taken together, our results strongly suggest that enhanced activation of cWnt signaling increases the skeletal potential of a calvarial bone of mesoderm origin, such as the parietial bone to a degree similar to that of a neural crest origin bone, like the frontal bone. Thus, providing further evidence for the central role played by the cWnt signaling in osteogenesis and skeletal-bone regeneration.

Mesodermal expression of Fgfr2S252W is necessary and sufficient to induce craniosynostosis in a mouse model of Apert syndrome

Coordinated growth of the skull and brain are vital to normal human development. Craniosynostosis, the premature fusion of the calvarial bones of the skull, is a relatively common pediatric disease, occurring in 1 in 2500 births, and requires significant surgical management, especially in syndromic cases. Syndromic craniosynostosis is caused by a variety of genetic lesions, most commonly by activating mutations of FGFRs 1–3, and inactivating mutations of TWIST1. In a mouse model of TWIST1 haploinsufficiency, cell mixing between the neural crest-derived frontal bone and mesoderm-derived parietal bone accompanies coronal suture fusion during embryonic development. However, the relevance of lineage mixing in craniosynostosis induced by activating FGFR mutations is unknown. Here, we demonstrate a novel mechanism of suture fusion in the Apert Fgfr2S252W mouse model. Using Cre/lox recombination we simultaneously induce expression of Fgfr2S252W and β-galactosidase in either the neural crest or mesoderm of the skull. We show that mutation of the mesoderm alone is necessary and sufficient to cause craniosynostosis, while mutation of the neural crest is neither. The lineage border is not disrupted by aberrant cell migration during fusion. Instead, the suture mesenchyme itself remains intact and is induced to undergo osteogenesis. We eliminate postulated roles for dura mater or skull base changes in craniosynostosis. The viability of conditionally mutant mice also allows post-natal assessment of other aspects of Apert syndrome.

Inactivation of Msx1 and Msx2 in neural crest reveals an unexpected role in suppressing heterotopic bone formation in the head

In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1 and Msx2 conditionally in neural crest. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx1/2 alleles, the large defect is filled in with mispatterned bone consisting of ectopic islands of bone between the reduced frontal bones, just anterior to the parietal bones. The bone is derived from neural crest, not mesoderm, and, from DiI cell marking experiments, originates in a normally non-osteogenic layer of cells through which the rudiment elongates apically. Associated with the heterotopic osteogenesis is an upregulation of Bmp signaling in this cell layer. Prevention of this upregulation by implantation of noggin-soaked beads in head explants also prevented heterotopic bone formation. These results suggest that Msx genes have a dual role in calvarial development: They are required for the differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of pathological conditions in which calvarial bone development is compromised.

Differential FGF Ligands and FGF Receptors Expression Pattern in Frontal and Parietal Calvarial Bones

The mammalian skull vault consists mainly of 5 flat bones, the paired frontals and parietals, and the unpaired interparietal. All of these bones are formed by intramembranous ossification within a layer of mesenchyme, the skeletogenic membrane, located between the dermal mesenchyme and the meninges surrounding the brain. While the frontal bones are of neural crest in origin, the parietal bones arise from mesoderm. The present study is a characterization of frontal and parietal bones at their molecular level, aiming to highlight distinct differences between the neural crest-derived frontal and mesodermal-derived parietal bone. We performed a detailed comparative gene expression profile of FGF ligands and their receptors known to play crucial role in skeletogenesis. This analysis revealed that a differential expression pattern of the major FGF osteogenic molecules and their receptors exists between the neural crest-derived frontal bone and the paraxial mesoderm-derived parietal bone. Particularly, the expression of ligands such as Fgf-2, Fgf-9 and Fgf-18 was upregulated in frontal bone on embryonic day 17.5, postnatal day 1 and postnatal day 60 mice. Frontal bone also elaborated higher levels of Fgf receptor 1, 2 and 3 transcripts versus parietal bone. Taken together, these data suggest that the frontal bone is a domain with higher FGF-signaling competence than parietal bone.

Tissue Origins and Interactions in the Mammalian Skull Vault

During mammalian evolution, expansion of the cerebral hemispheres was accompanied by expansion of the frontal and parietal bones of the skull vault and deployment of the coronal (fronto-parietal) and sagittal (parietal–parietal) sutures as major growth centres. Using a transgenic mouse with a permanent neural crest cell lineage marker, Wnt1-Cre/R26R, we show that both sutures are formed at a neural crest–mesoderm interface: the frontal bones are neural crest-derived and the parietal bones mesodermal, with a tongue of neural crest between the two parietal bones. By detailed analysis of neural crest migration pathways using X-gal staining, and mesodermal tracing by DiI labelling, we show that the neural crest–mesodermal tissue juxtaposition that later forms the coronal suture is established at E9.5 as the caudal boundary of the frontonasal mesenchyme. As the cerebral hemispheres expand, they extend caudally, passing beneath the neural crest–mesodermal interface within the dermis, carrying with them a layer of neural crest cells that forms their meningeal covering. Exposure of embryos to retinoic acid at E10.0 reduces this meningeal neural crest and inhibits parietal ossification, suggesting that intramembranous ossification of this mesodermal bone requires interaction with neural crest-derived meninges, whereas ossification of the neural crest-derived frontal bone is autonomous. These observations provide new perspectives on skull evolution and on human genetic abnormalities of skull growth and ossification.