Cranium growth, patterning and homeostasis.

Craniofacial development requires precise spatiotemporal regulation of multiple signaling pathways that crosstalk to coordinate the growth and patterning of the skull with surrounding tissues. Recent insights into these signaling pathways and previously uncharacterized progenitor cell populations have refined our understanding of skull patterning, bone mineralization and tissue homeostasis. Here, we touch upon classical studies and recent advances with an emphasis on developmental and signaling mechanisms that regulate the osteoblast lineage for the calvaria, which forms the roof of the skull. We highlight studies that illustrate the roles of osteoprogenitor cells and cranial suture-derived stem cells for proper calvarial growth and homeostasis. We also discuss genes and signaling pathways that control suture patency and highlight how perturbing the molecular regulation of these pathways leads to craniosynostosis. Finally, we discuss the recently discovered tissue and signaling interactions that integrate skull and cerebrovascular development, and the potential implications for both cerebrospinal fluid hydrodynamics and brain waste clearance in craniosynostosis.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-ß signaling.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-ß signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-ß signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-ß signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

Suture Cells in a Mechanical Stretching Niche: Critical Contributors to Trans-sutural Distraction Osteogenesis.

Trans-sutural distraction osteogenesis has been proposed as an alternative technique of craniofacial remodelling surgery for craniosynostosis correction. Many studies have defined the contribution of a series of biological events to distraction osteogenesis, such as changes in gene expression, changes in suture cell behaviour and changes in suture collagen fibre characteristics. However, few studies have elucidated the systematic molecular and cellular mechanisms of trans-sutural distraction osteogenesis, and no study has highlighted the contribution of cell-cell or cell-matrix interactions with respect to the whole expansion process to date. Therefore, it is difficult to translate largely primary mechanistic insights into clinical applications and optimize the clinical outcome of trans-sutural distraction osteogenesis. In this review, we carefully summarize in detail the literature related to the effects of mechanical stretching on osteoblasts, endothelial cells, fibroblasts, immune cells (macrophages and T cells), mesenchymal stem cells and collagen fibres in sutures during the distraction osteogenesis process. We also briefly review the contribution of cell-cell or cell-matrix interactions to bone regeneration at the osteogenic suture front from a comprehensive viewpoint.

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Sutural fibroblasts exhibit the function of vascular endothelial cells upon mechanical strain.

Midfacial hypoplasia is a type of facial dysplasia. The technique of trans-sutural distraction osteogenesis promotes midface growth so as to ameliorate this symptom. In the process of distraction osteogenesis, the fiber matrix in the suture acts as a mechanical sensor. Compared with osteogenesis, the formation of collagen fibers by fibroblasts is significant in the early stage of sutural distraction. However the transformation of fibroblasts during sutural bone formation induced by tensile force is poorly characterized. Here, we used single-cell RNA sequencing to define the cell classification of the zygomatic maxillary suture and the changes of cell clusters in the suture before and after seven-day distraction. We identified twenty-nine cell subsets spanning monocyte/macrophages, neutrophils, red blood cells, B cells and fibroblasts. Compared with the control group, Monocle analysis revealed the emergence of a unique fibroblast subset (Cdh5+, Col4a1+, Fat1-, and Acta2-) (cluster 27) that expressed vascular endothelial cell genes within the distracted zygomatic maxillary suture. We constructed the differentiation trajectories of the fibroblast population (cluster 23, 27) in the suture before and after distraction. In addition, we clarified that a subset of fibroblasts (cluster 27) lost expression of Fat1, an upregulator of the Hippo pathway, and upregulated Cyr61, a downstream gene of the Hippo pathway, during the distraction process. Further enrichment analysis suggests that cells of the new subset (cluster 27) are undergoing conversion of their identity into a vascular endothelial cell-like state in response to mechanical stimulation, associated with upregulation of angiogenesis genes along the single-cell trajectory. Further immunofluorescence staining confirmed this phenomenon. A combined general transcriptome RNA sequencing data analysis demonstrated that the fibroblasts expressed a number of extracellular matrix-related genes under mechanical strain. These data together provide a new view of the role of fibroblasts in tension-induced sutural angiogenesis via interaction with the Hippo pathway.

Genetic background dependent modifiers of craniosynostosis severity.

Craniosynostosis severity varies in patients with identical genetic mutations. To understand causes of this phenotypic variation, we backcrossed the FGFR2+/C342Y mouse model of Crouzon syndrome onto congenic C57BL/6 and BALB/c backgrounds. Coronal suture fusion was observed in C57BL/6 (88% incidence, p < .001 between genotypes) but not in BALB/c FGFR2+/C342Y mutant mice at 3 weeks after birth, establishing that that the two models differ in phenotype severity. To begin identifying pre-existing modifiers of craniosynostosis severity, we compared transcriptome signatures of cranial tissues from C57BL/6 vs. BALB/c FGFR2+/+ mice. We separately analyzed frontal bone with coronal suture tissue from parietal bone with sagittal suture tissues because the coronal suture but not the sagittal suture fuses in FGFR2+/C342Y mice. The craniosynostosis associated Twist and En1 transcription factors were down-regulated, while Runx2 was up-regulated, in C57BL/6 compared to BALB/c tissues, which could predispose to craniosynostosis. Transcriptome analyses under the GO term MAPK cascade revealed that genes associated with calcium ion channels, angiogenesis, protein quality control and cell stress response were central to transcriptome differences associated with genetic background. FGFR2 and HSPA2 protein levels plus ERK1/2 activity were higher in cells isolated from C57BL/6 than BALB/c cranial tissues. Notably, the HSPA2 protein chaperone is central to craniofacial genetic epistasis, and we find that FGFR2 protein is abnormally processed in primary cells from FGFR2+/C342Y but not FGFR2+/+ mice. Therefore, we propose that differences in protein quality control responses may contribute to genetic background influences on craniosynostosis phenotype severity.

ROCK-TAZ signaling axis regulates mechanical tension-induced osteogenic differentiation of rat cranial sagittal suture mesenchymal stem cells.

Mechanical force across sutures is able to promote suture osteogenesis. Orthodontic clinics often use this biological characteristic of sutures to treat congenital cranio-maxillofacial malformations. However, the underlying mechanisms still remain poorly understood. Craniofacial sutures provide a special growth source and support primary sites of osteogenesis. Here, we isolated rat sagittal suture cells (rSAGs), which had mesenchymal stem cell characteristics and differentiating abilities. Cells were then subjected to mechanical tension (5% elongation, 0.5 Hz; sinusoidal waveforms) showing that mechanical tension could enhance osteogenic differentiation but hardly affect proliferation of rSAGs. Besides, mechanical tension could increase Rho-associated kinase (ROCK) expression and enhance transcriptional coactivator with PDZ-binding motif (TAZ) nuclear translocation. Inhibiting ROCK expression could suppress tension-induced osteogenesis and block tension-induced upregulation of nuclear TAZ. In addition, our results indicated that TAZ had direct combination sites with runt-related transcription factor 2 (Runx2) in rSAGs, and knock-downed TAZ simultaneously decreased the expression of Runx2 no matter with or without mechanical tension. In summary, our findings demonstrated that the multipotency of rSAGs in vitro could give rise to early osteogenic differentiation under mechanical tension, which was mediated by ROCK-TAZ signal axis.

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.

Glycosylation of DMP1 maintains cranial sutures in mice.

Craniosynostosis, a severe craniofacial developmental disease, can only be treated with surgery currently. Recent studies have shown that proteoglycans are involved in the suture development. For the bone matrix protein, dentin matrix protein 1 (DMP1), glycosylation on the N-terminal of it could generate a functional proteoglycan form of DMP1 during osteogenesis. We identified that the proteoglycan form of DMP1 (DMP1-PG) is highly expressed in mineralisation front of suture. But, the potential role of DMP1-PG in suture fusion remain unclear. To investigate the role of DMP1-PG in cranial suture fusion and craniofacial bone development. By using a DMP1 glycosylation site mutation mouse model, DMP1-S89G mice, we compared the suture development in it with control mice. We compared the suture phenotypes, bone formation rate, expression levels of bone formation markers in vivo between DMP1-S89G mice and wild-type mice. Meanwhile, cell culture and organ culture were performed to detect the differences in cell differentiation and suture fusion in vitro. Finally, chondroitin sulphate (CHS), as functional component of DMP1-PG, was employed to test whether it could delay the premature suture fusion and the abnormal differentiation of bone mesenchymal stem cells (BMSCs) of DMP1-PG mice. DMP1-S89G mice had premature closure of suture and shorter skull size. Lack of DMP1-PG accelerated bone formation in cranial suture. DMP1-PG maintained the essential stemness of BMSCs in suture through blocking the premature differentiation of BMSCs to osteoblasts. Finally, chondroitin sulphate, a major component of DMP1-PG, successfully delayed the premature suture fusion by organ culture of skull in vitro. DMP1-PG could inhibit premature fusion of cranial suture and maintain the suture through regulating the osteogenic differentiation of BMSCs.

Cdc42 regulates cranial suture morphogenesis and ossification.

Cdc42 (cell division cycle 42) is ubiquitously expressed small GTPases belonging to the Rho family of proteins. Previously, we generated limb bud mesenchyme-specific Cdc42 inactivated mice (Cdc42 conditional knockout mice; Cdc42 fl/fl; Prx1-Cre), which showed short limbs and cranial bone deformities, though the mechanism related to the cranium phenotype was unclear. In the present study, we investigated the role of Cdc42 in cranial bone development. Our results showed that loss of Cdc42 caused a defect of intramembranous ossification in cranial bone tissues which is related to decreased expressions of cranial suture morphogenesis genes, including Indian hedgehog (Ihh) and bone morphogenetic proteins (BMPs). These findings demonstrate that Cdc42 plays a crucial role in cranial osteogenesis, and is controlled by Ihh- and BMP-mediated signaling during cranium development.

BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients.

The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long-term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long-term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.

In vivo expression and regulation of genes associated with vascularization during early response of sutures to tensile force.

Sutures are fibrous tissues that connect bones in craniofacial skeletal complexes. Cranio- and dentofacial skeletal deformities in infant and adolescent patients can be treated by applying tensile force to sutures to induce sutural bone formation. The early gene expression induced by mechanical stress is essential for bone formation in long bones; however, early gene expression during sutural bone formation induced by tensile force is poorly characterized. In vivo studies are essential to evaluate molecular responses to mechanical stresses in heterogeneous cell populations, such as sutures. In this paper we examined in vivo early gene expression and the underlying regulatory mechanism for this expression in tensile-force-applied cranial sutures, focusing on genes involved in vascularization. Tensile force upregulated expression of vascular factors, such as vascular endothelial growth factor (Vegf) and endothelial cell markers, in sutures within 3 h. The expression of connective tissue growth factor (Ctgf) and Rho-associated coiled-coil containing protein kinase 2 (Rock2) was also upregulated by tensile force. A CTGF-neutralizing antibody and the ROCK inhibitor, Y-27632, abolished tensile-force-induced Vegf expression. Moreover, tensile force activated extracellular signal-related kinase 1/2 (ERK1/2) signaling in sagittal sutures, and the ERK1/2 inhibitor, U0126, partially inhibited tensile-force-induced Ctgf expression. These results indicate that tensile force induces in vivo gene expression associated with vascularization early in tensile-force-induced sutural bone formation. Moreover, the early induction of Vegf gene expression is regulated by CTGF and ROCK2.

Role of Notch Signaling in the Physiological Patterning of Posterofrontal and Sagittal Cranial Sutures.

BACKGROUND: The mutations in a Notch signaling ligand, jagged 1, are associated with unilateral coronal craniosynostosis in humans. However, the underlying mechanisms of Notch signaling in cranial suture biology still remain unclear. METHODS: The temporal and spatial patterns of Notch signaling expression were examined in the posterofrontal and sagittal sutures of Sprague-Dawley rats by real-time quantitative reverse-transcription polymerase chain reaction at postnatal ages of 2, 15, and 25 days. The role of Notch signaling in the proliferation and differentiation of osteoblasts isolated from calvarial was examined in vitro by EdU incorporation assays and real-time quantitative reverse-transcription polymerase chain reaction after activating and inhibiting Notch signaling. RESULTS: The mRNA levels of Notch family members (including Jagged 1, Delta 1, 3, 4, Notch 1-4, Hes 1, and Hes 5) decreased during the posterofrontal cranial suture fusion in rat. However, in the patent sagittal sutures, the mRNA levels of Notch family members (Jagged 2, Delta 1, Notch 1, Notch 3, Hes 5, and Hey 1) increased during suture development. The EdU incorporation assays revealed that the induction of Notch signaling in calvaria osteobalsts using Jagged 1 promoted the proliferation rates in those cells in vitro. Further studies showed that activation of Notch signaling calvaria osteobalsts using Jagged 1 led to the suppression of late osteogenetic markers such as type I collagen and osteocalcin. CONCLUSIONS: The regulation of Notch signaling is of crucial importance during the physiological patterning of posterofrontal and sagittal cranial sutures. Thus, targeting this pathway may prove significant for the development of future therapeutic applications in craniosynostosis.

Application of Laser Capture Microdissection to Craniofacial Biology: Characterization of Anatomically Relevant Gene Expression in Normal and Craniosynostotic Rabbit Sutures.

OBJECTIVE: Fusion of the cranial sutures is thought to depend on signaling among perisutural tissues. Mapping regional variations in gene expression would improve current models of craniosynostosis. Laser capture microdissection (LCM) isolates discrete cell populations for gene expression analysis. LCM has rarely been used in the study of mineralized tissue. This study sought to evaluate the potential use of LCM for mapping of regional gene expression within the cranial suture. DESIGN: Coronal sutures were isolated from 10-day-old wild-type and craniosynostotic (CS) New Zealand White rabbits, and LCM was used to isolate RNA from the sutural ligament (SL), osteogenic fronts (OF), dura mater, and periosteum. Relative expression levels for Fibroblast Growth Factor 2 (FGF2), Fibroblast Growth Factor Receptor 2 (FGFR2), Transforming Growth Factor Beta 2 (TGFß-2), Transforming Growth Factor Beta 3 (TGFß-3), Bone Morphogenetic Protein 2 (BMP-2), Bone Morphogenetic Protein 4 (BMP-4), and Noggin were determined using quantitative real-time PCR. RESULTS: A fivefold increase in TGFß2 expression was detected in the CS SL relative to wild type, whereas 152-fold less TGFß-3 was detected within the OF of CS animals. Noggin expression was increased by 10-fold within the CS SL, but reduced by 13-fold within the CS dura. Reduced expression of FGF2 was observed within the CS SL and dura, whereas increased expression of FGFR2 was observed within the CS SL. Reduced expression of BMP-2 was observed in the CS periosteum, and elevated expression of BMP-4 was observed in the CS SL and dura. CONCLUSIONS: LCM provides an effective tool for measuring regional variations in cranial suture gene expression. More precise measurements of regional gene expression with LCM may facilitate efforts to correlate gene expression with suture morphogenesis and pathophysiology.

A preliminary investigation of the effect of relaxin on bone remodelling in suture expansion.

Background and objectives: Relaxin (RLN) is an insulin-like hormone associated with extracellular matrix degradation, osteoclastogenesis, and osteoblast differentiation. This study aimed to assess the effect of RLN during and after lateral expansion of murine calvarial sagittal sutures. Materials and methods: RLN was injected topically using a nano-sized liposome carrier into the sagittal sutures of 8- to 10-week-old wild type mice just before lateral expansion. Suture morphology, bone mineral density (BMD), and bone volume were analysed by micro-computed tomography. Collagen deposition and osteoclast differentiation were observed by Verhoeff-Van Gieson (VVG) and tartrate-resistant acid phosphatase (TRAP) staining, respectively. Results: Less collagen staining and higher tissue-specific relaxin/insulin-like family peptide receptor (Rxfp)-1 and -2 expression were observed in the RLN-treated samples after 48 hours. Increased BMD and volume, and thick well-organised osteoid tissue, with multinucleated TRAP-positive cells, were observed in RLN-treated samples after 1 week. Increased Rxfp-1 expression was observed in the sagittal sutures in the mid-suture fibrous tissue following RLN treatment. Rxfp-2 was only expressed in the calvarial bone under tensile stimulation and RLN treatment further increased its expression. Limitations: RLN-liposomes were not detected at any instance under the current experimental conditions. This is a preliminary study and the sample number limits the power of its results. VVG staining cannot quantify collagen contents but can provide preliminary information on the presence of collagen fibres. Conclusions: RLN treatment may modify bone remodelling and collagen metabolism during and after suture expansion.

Mice lacking the conserved transcription factor Grainyhead-like 3 (Grhl3) display increased apposition of the frontal and parietal bones during embryonic development.

BACKGROUND: Increased apposition of the frontal and parietal bones of the skull during embryogenesis may be a risk factor for the subsequent development of premature skull fusion, or craniosynostosis. Human craniosynostosis is a prevalent, and often serious embryological and neonatal pathology. Other than known mutations in a small number of contributing genes, the aetiology of craniosynostosis is largely unknown. Therefore, the identification of novel genes which contribute to normal skull patterning, morphology and premature suture apposition is imperative, in order to fully understand the genetic regulation of cranial development. RESULTS: Using advanced imaging techniques and quantitative measurement, we show that genetic deletion of the highly-conserved transcription factor Grainyhead-like 3 (Grhl3) in mice (Grhl3 -/- ) leads to decreased skull size, aberrant skull morphology and premature apposition of the coronal sutures during embryogenesis. Furthermore, Grhl3 -/- mice also present with premature collagen deposition and osteoblast alignment at the sutures, and the physical interaction between the developing skull, and outermost covering of the brain (the dura mater), as well as the overlying dermis and subcutaneous tissue, appears compromised in embryos lacking Grhl3. Although Grhl3 -/- mice die at birth, we investigated skull morphology and size in adult animals lacking one Grhl3 allele (heterozygous; Grhl3 +/- ), which are viable and fertile. We found that these adult mice also present with a smaller cranial cavity, suggestive of post-natal haploinsufficiency in the context of cranial development. CONCLUSIONS: Our findings show that our Grhl3 mice present with increased apposition of the frontal and parietal bones, suggesting that Grhl3 may be involved in the developmental pathogenesis of craniosynostosis.

Fibulin-5 deficiency causes developmental defect of premaxillary bone in mice.

Craniofacial sutures govern the shape of the craniofacial skeleton during postnatal development. The differentiation of suture mesenchymal cells to osteoblasts is precisely regulated in part by signaling through cell surface receptors that interact with extracellular proteins. Here we report that fibulin-5, a key extracellular matrix protein, is important for craniofacial skeletal development in mice. Fibulin-5 is deposited as a fibrous matrix in cranial neural crest-derived mesenchymal tissues, including craniofacial sutures. Fibulin-5-null mice show decreased premaxillary bone outgrowth during postnatal stages. While premaxillo-maxillary suture mesenchymal cells in fibulin-5-null mice were capable of differentiating into osteoblasts, suture cells in mutant mice were less proliferative. Our study provides the first evidence that fibulin-5 is indispensable for the regulation of facial suture mesenchymal cell proliferation required for craniofacial skeletal morphogenesis.

Craniofacial divergence by distinct prenatal growth patterns in Fgfr2 mutant mice.

BACKGROUND: Differences in cranial morphology arise due to changes in fundamental cell processes like migration, proliferation, differentiation and cell death driven by genetic programs. Signaling between fibroblast growth factors (FGFs) and their receptors (FGFRs) affect these processes during head development and mutations in FGFRs result in congenital diseases including FGFR-related craniosynostosis syndromes. Current research in model organisms focuses primarily on how these mutations change cell function local to sutures under the hypothesis that prematurely closing cranial sutures contribute to skull dysmorphogenesis. Though these studies have provided fundamentally important information contributing to the understanding of craniosynostosis conditions, knowledge of changes in cell function local to the sutures leave change in overall three-dimensional cranial morphology largely unexplained. Here we investigate growth of the skull in two inbred mouse models each carrying one of two gain-of-function mutations in FGFR2 on neighboring amino acids (S252W and P253R) that in humans cause Apert syndrome, one of the most severe FGFR-related craniosynostosis syndromes. We examine late embryonic skull development and suture patency in Fgfr2 Apert syndrome mice between embryonic day 17.5 and birth and quantify the effects of these mutations on 3D skull morphology, suture patency and growth. RESULTS: We show in mice what studies in humans can only infer: specific cranial growth deviations occur prenatally and worsen with time in organisms carrying these FGFR2 mutations. We demonstrate that: 1) distinct skull morphologies of each mutation group are established by E17.5; 2) cranial suture patency patterns differ between mice carrying these mutations and their unaffected littermates; 3) the prenatal skull grows differently in each mutation group; and 4) unique Fgfr2-related cranial morphologies are exacerbated by late embryonic growth patterns. CONCLUSIONS: Our analysis of mutation-driven changes in cranial growth provides a previously missing piece of knowledge necessary for explaining variation in emergent cranial morphologies and may ultimately be helpful in managing human cases carrying these same mutations. This information is critical to the understanding of craniofacial development, disease and evolution and may contribute to the evaluation of incipient therapeutic strategies.

Concurrent impact of de novo mutations on cranial and cortical development in nonsyndromic craniosynostosis.

OBJECTIVE: Nonsyndromic craniosynostosis (nsCS), characterized by premature cranial suture fusion, is considered a primary skull disorder in which impact on neurodevelopment, if present, results from the mechanical hindrance of brain growth. Despite surgical repair of the cranial defect, neurocognitive deficits persist in nearly half of affected children. Therefore, the authors performed a functional genomics analysis of nsCS to determine when, where, and in what cell types nsCS-associated genes converge during development. METHODS: The authors integrated whole-exome sequencing data from 291 nsCS proband-parent trios with 29,803 single-cell transcriptomes of the prenatal and postnatal neurocranial complex to inform when, where, and in what cell types nsCS-mutated genes might exert their pathophysiological effects. RESULTS: The authors found that nsCS-mutated genes converged in cranial osteoprogenitors and pial fibroblasts and their transcriptional networks that regulate both skull ossification and cerebral neurogenesis. Nonsyndromic CS-mutated genes also converged in inhibitory neurons and gene coexpression modules that overlapped with autism and other developmental disorders. Ligand-receptor cell-cell communication analysis uncovered crosstalk between suture osteoblasts and neurons via the nsCS-associated BMP, FGF, and noncanonical WNT signaling pathways. CONCLUSIONS: These data implicate a concurrent impact of nsCS-associated de novo mutations on cranial morphogenesis and cortical development via cell- and non-cell-autonomous mechanisms in a developmental nexus of fetal osteoblasts, pial fibroblasts, and neurons. These results suggest that neurodevelopmental outcomes in nsCS patients may be driven more by mutational status than surgical technique.

Distraction force promotes the osteogenic differentiation of Gli1+ cells in facial sutures via primary cilia-mediated Hedgehog signaling pathway.

BACKGROUND: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown. METHODS: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling. RESULTS: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force. CONCLUSIONS: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.