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.

Cranial Suture Mesenchymal Stem Cells: Insights and Advances.

The cranial bones constitute the protective structures of the skull, which surround and protect the brain. Due to the limited repair capacity, the reconstruction and regeneration of skull defects are considered as an unmet clinical need and challenge. Previously, it has been proposed that the periosteum and dura mater provide reparative progenitors for cranial bones homeostasis and injury repair. In addition, it has also been speculated that the cranial mesenchymal stem cells reside in the perivascular niche of the diploe, namely, the soft spongy cancellous bone between the interior and exterior layers of cortical bone of the skull, which resembles the skeletal stem cells' distribution pattern of the long bone within the bone marrow. Not until recent years have several studies unraveled and validated that the major mesenchymal stem cell population of the cranial region is primarily located within the suture mesenchyme of the skull, and hence, they are termed suture mesenchymal stem cells (SuSCs). Here, we summarized the characteristics of SuSCs, this newly discovered stem cell population of cranial bones, including the temporospatial distribution pattern, self-renewal, and multipotent properties, contribution to injury repair, as well as the signaling pathways and molecular mechanisms associated with the regulation of SuSCs.

Skeletal stem and progenitor cells maintain cranial suture patency and prevent craniosynostosis.

Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/- mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/- mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis.

Differential Responsiveness to BMP9 between Patent and Fused Suture Progenitor Cells from Craniosynostosis Patients.

BACKGROUND: Several studies have verified that bone morphogenetic proteins (BMPs) may be involved in the development of craniosynostosis; little attention has been focused on the role of BMP9 in cranial suture biology. The authors investigated the role of BMP9 in suture progenitor cells. METHODS: The authors isolated and cultured prematurely fused and internal control patent suture progenitor cells from patients with nonsyndromic craniosynostosis. Overexpression of BMP9 was mediated by adenoviral vectors. Osteoblast and osteoclast differentiation-related markers were evaluated by staining techniques and touchdown quantitative polymerase chain reaction analysis. In vivo analysis of BMP9-induced suture progenitor cell osteogenesis was performed in an ectopic bone formation model. RESULTS: The authors demonstrated that the prematurely fused sutures have a higher endogenous expression of the osteogenic differentiation-related genes than patent sutures, whereas the same pattern of gene expression exists between fused and patent suture progenitor cells. Importantly, both patent and fused suture progenitor cells undergo osteogenic differentiation and express multiple lineage regulators and NELL-1 on BMP9 stimulation, whereas fused suture progenitor cells have a higher basal osteogenic potential than patent suture progenitor cells. BMP9 regulates the expression of osteoclast differentiation-related genes in suture progenitor cells. Forced BMP9 expression enhances the mineralization and maturity of ectopic bone formation of suture progenitor cells implanted in vivo. CONCLUSIONS: The authors' findings suggest that fused suture progenitor cells have elevated osteogenic potential. BMP9 could regulate the expression of multiple osteoblast and osteoclast differentiation-related genes, and NELL-1, in both suture progenitor cells, indicating that BMP9 may play a role in craniosynostosis.

MicroRNA-21 affects mechanical force-induced midpalatal suture remodelling.

OBJECTIVES: miR-21 can promote osteoblast differentiation of periodontal ligament stem cells. However, the effect of miR-21 on bone remodelling in the midpalatal suture is unclear. This study aimed to elucidate the effects of miR-21 on the midpalatal suture bone remodelling by expanding the palatal sutures. MATERIALS AND METHODS: miR-21 deficient (miR-21-/- ) and wild-type (WT) mice were used to establish animal models by expanding the palatal sutures. Micro-CT, haematoxylin-eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, fluorescence labelling and immunohistochemistry were used to investigate the function of miR-21 in midpalatal suture bone remodelling. Besides, bone mesenchymal stem cells (BMSCs) derived from both miR-21-/- and WT mice were cultured. The MTT, CCK8, EdU analysis, transwell and wound healing test were used to assess the effects of miR-21 on the characteristics of cells. RESULTS: The expression of ALP was suppressed in miR-21-/- mice after expansion except 28 days. The expression of Ocn in WT mice was much higher than that of miR-21-/-  mice. Besides, with mechanical force, miR-21 deficiency downregulated the expression of Opg, upregulated the expression of Rankl, and induced more osteoclasts as TRAP staining showed. After injecting agomir-21  to miR-21-/- mice, the expression of Alp, Ocn and Opg/Rankl were rescued. In vitro, the experiments suggested that miR-21 deficiency reduced proliferation and migration ability of BMSCs. CONCLUSIONS: The results showed that miR-21 deficiency reduced the rate of bone formation and prolonged the process of bone formation. miR-21 regulated the bone resorption and osteoclastogenesis by affecting the cell abilities of proliferation and migration.

Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration.

Development of the skeleton is mediated through two distinct ossification mechanisms. Craniofacial bones are formed mainly through intramembranous ossification, a mechanism different from endochondral ossification required for development of the body skeleton. The skeletal structures are quite distinct between the two, thus they are likely to have their unique stem cell populations. The sutures serve as the growth center critical for healthy development of the craniofacial skeleton. Defects in suture morphogenesis cause its premature closure, resulting in development of craniosynostosis, a devastating disease affecting 1 in ~2,500 individuals. The suture mesenchyme has been postulated to act as the niche of skeletal stem cells essential for calvarial morphogenesis. However, very limited knowledge is available for suture biology and suture stem cells (SuSCs) have yet to be isolated. Here we report the first evidence for identification and isolation of a stem cell population residing in the suture midline. Genetic labeling of SuSCs shows their ability to self-renew and continually give rise to mature cell types over a 1-year monitoring period. They maintain their localization in the niches constantly produce skeletogenic descendants during calvarial development and homeostastic maintenance. Upon injury, SuSCs expand drastically surrounding the skeletogenic mesenchyme, migrate to the damaged site and contribute directly to skeletal repair in a cell autonomous fashion. The regeneration, pluripotency and frequency of SuSCs are also determined using limiting dilution transplantation. In vivo clonal expansion analysis demonstrates a single SuSC capable of generating bones. Furthermore, SuSC transplantation into injured calvaria facilitates the healing processes through direct engraftments. Our findings demonstrate SuSCs are bona fide skeletal stem cells ideally suited for cell-based craniofacial bone therapy as they possess abilities to engraft, differentiate.(Presented at the 1980th Meeting, April 16, 2019).

Runx2 regulates cranial suture closure by inducing hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in suture mesenchymal cells.

Cleidocranial dysplasia (CCD, #119600), which is characterized by hypoplastic clavicles, open fontanelles, supernumerary teeth and a short stature, is caused by heterozygous mutations in RUNX2. However, it currently remains unclear why suture closure is severely impaired in CCD patients. The closure of posterior frontal (PF) and sagittal (SAG) sutures was completely interrupted in Runx2+/- mice, and the proliferation of suture mesenchymal cells and their condensation were less than those in wild-type mice. To elucidate the underlying molecular mechanisms, differentially expressed genes between wild-type and Runx2+/- PF and SAG sutures were identified by microarray and real-time reverse transcription polymerase chain reaction analyses. The expression of hedgehog, Fgf, Wnt and Pthlh signaling pathway genes, including Gli1, Ptch1, Ihh, Fgfr2, Fgfr3, Tcf7, Wnt10b and Pth1r, which were directly regulated by Runx2, was reduced in the sutures, but not the calvarial bone tissues of Runx2+/- mice. Bone formation and suture closure were enhanced in an organ culture of Runx2+/- calvariae with ligands or agonists of hedgehog, Fgf, Wnt and Pthlh signaling, while they were suppressed and suture mesenchymal cell proliferation was decreased in an organ culture of wild-type calvariae with their antagonists. These results indicate that more than a half dosage of Runx2 is required for the proliferation of suture mesenchymal cells, their condensation and commitment to osteoblast-lineage cells, and the induction of hedgehog, Fgf, Wnt and Pthlh signaling pathway gene expressions in sutures, but not in calvarial bone tissues, and also that the activation of hedgehog, Fgf, Wnt and Pthlh signaling pathways is necessary for suture closure.

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.

The Morphogenesis of Cranial Sutures in Zebrafish.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

The suture provides a niche for mesenchymal stem cells of craniofacial bones.

Bone tissue undergoes constant turnover supported by stem cells. Recent studies showed that perivascular mesenchymal stem cells (MSCs) contribute to the turnover of long bones. Craniofacial bones are flat bones derived from a different embryonic origin than the long bones. The identity and regulating niche for craniofacial-bone MSCs remain unknown. Here, we identify Gli1+ cells within the suture mesenchyme as the main MSC population for craniofacial bones. They are not associated with vasculature, give rise to all craniofacial bones in the adult and are activated during injury repair. Gli1+ cells are typical MSCs in vitro. Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating that these cells are an indispensable stem cell population. Twist1(+/-) mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells. Our study indicates that craniofacial sutures provide a unique niche for MSCs for craniofacial bone homeostasis and repair.

Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3.

BACKGROUND: Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. RESULTS: A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. CONCLUSIONS: A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.

Tissue interactions between craniosynostotic dura mater and bone.

BACKGROUND: Cells within the dura mater have been implicated in the determination of suture patency and fusion. Craniosynostosis (CS), the premature fusion of 1 or more of the cranial sutures, could result from abnormal control over the differentiation of osteoprogenitor cells from the dura mater. This study tested whether dura mater cells derived from rabbits with congenital CS were different from cells derived from normal rabbits and investigated the effects that CS dura mater had on osteogenic differentiation in vitro and in vivo. METHODS: Cells were derived from the dura mater from wild-type rabbits (WT; n = 23) or CS rabbits (n = 16). Cells were stimulated with bone morphogenetic protein 4, and alkaline phosphatase (ALP) expression and cell proliferation were assessed. Dura mater-derived cells were also cocultured with primary rabbit bone-derived cells, and ALP was assessed. Finally, interactions between the dura mater and overlying tissues were manipulated in vivo. RESULTS: Craniosynostotic dura mater-derived cells proliferated faster than did WT cells but were not more ALP positive. Coculture experiments showed that CS dura mater cells induced increased ALP activity in CS bone-derived cells, but not in WT bone-derived cells. In vivo experiments showed that a physical barrier successfully inhibited dura mater-derived osteogenesis. CONCLUSIONS: Coculture of CS bone- and CS dura mater-derived cells evoked an abnormal phenotype in vitro. Covering the CS dura mater led to decreased bone formation in vivo. Further investigations will focus on the signaling molecules involved in the communication between these 2 CS tissue types in vitro and in vivo.

Retinoic acid enhances osteogenesis in cranial suture-derived mesenchymal cells: potential mechanisms of retinoid-induced craniosynostosis.

BACKGROUND: In utero retinoid exposure results in numerous craniofacial malformations, including craniosynostosis. Although many malformations associated with retinoic acid syndrome are associated with neural crest defects, the specific mechanisms of retinoid-induced craniosynostosis remain unclear. The authors used the culture of mouse cranial suture-derived mesenchymal cells to probe the potential cellular mechanisms of this teratogen to better elucidate mechanisms of retinoid-induced suture fusion. METHODS: Genes associated with retinoid signaling were assayed in fusing (posterofrontal) and patent (sagittal, coronal) sutures by quantitative real-time polymerase chain reaction. Cultures of mouse suture-derived mesenchymal cells from the posterofrontal suture were established from 4-day-old mice. Cells were cultured with all-trans retinoic acid (1 and 5 muM). Proliferation, osteogenic differentiation, and specific gene expression were assessed. RESULTS: Mouse sutures were found to express genes necessary for retinoic acid synthesis, binding, and signal transduction, demonstrated by quantitative real-time polymerase chain reaction (Raldh1, Raldh2, Raldh3, and Rbp4). These genes were not found to be differentially expressed in fusing as compared with patent cranial sutures in vivo. Addition of retinoic acid enhanced the osteogenic differentiation of suture-derived mesenchymal cells in vitro, including up-regulation of alkaline phosphatase activity and Runx2 expression. Contemporaneously, cellular proliferation was repressed, as shown by proliferative cell nuclear antigen expression. The pro-osteogenic effect of retinoic acid was accompanied by increased gene expression of several hedgehog and bone morphogenetic protein ligands. CONCLUSIONS: Retinoic acid represses proliferation and enhances osteogenic differentiation of suture-derived mesenchymal cells. These in vitro data suggest that retinoid exposure may lead to premature cranial suture fusion by means of enhanced osteogenesis and hedgehog and bone morphogenetic protein signaling.

In vitro differentiation of human calvarial suture derived cells with and without dexamethasone does not induce in vivo-like expression.

Osteogenic supplements are a requirement for osteoblastic cell differentiation during in vitro culture of human calvarial suture-derived cell populations. We investigated the ability of ascorbic acid and beta-glycerophosphate with and without the addition of dexamethasone to stimulate in vivo-like osteoblastic differentiation. Cells were isolated from unfused and prematurely fused suture tissue from patients with syndromic and non-syndromic craniosynostosis and cultured in each osteogenic medium for varying lengths of time. The effect of media supplementation was investigated with respect to the ability of cells to form mineralised bone nodules and the expression of five osteodifferentiation marker genes (COL1A1, ALP, BSP, OC and RUNX2), and five genes that are differentially expressed during human premature suture fusion (GPC3, RBP4, C1QTNF3, WIF1 and FGF2). Cells from unfused sutures responded more slowly to osteogenic media but formed comparable bone nodules to fused suture-derived cells after 16 days of culture in either osteogenic media. However, gene expression differed between unfused and fused suture-derived cells, as did expression in each osteogenic medium. When compared to expression in the explant tissue of origin, neither medium induced a level or profile of gene expression similar to that seen in vivo. Overall, our results demonstrate that cells from the same suture that are isolated during different stages of morphogenesis in vivo, despite being de-differentiated to a similar level in vitro, respond uniquely and differently to each osteogenic medium. Further, we suggest that neither cell culture medium recapitulates differentiation via activation of the same genetic cascades as occurs in vivo.

Transforming growth factor-beta1 stimulates chondrogenic differentiation of posterofrontal suture-derived mesenchymal cells in vitro.

BACKGROUND: Evidence from animal studies has associated transforming growth factor (TGF)-beta signaling with both normal and premature cranial suture fusion. However, the mechanisms whereby this pleiotropic cytokine mediates suture fusion remain uncertain. The authors established cultures of suture-derived mesenchymal cells from normally fusing (posterofrontal) and patent (sagittal) sutures and examined the in vitro effects of TGF-beta1 on these distinct cell populations. METHODS: Skulls were harvested from 80 5-day-old mice. Posterofrontal and sagittal sutures were dissected, and cultures of suture-derived mesenchymal cells were established. The mitogenic, osteogenic, and chondrogenic effects of recombinant TGF-beta1 were then assessed on posterofrontal and sagittal suture-derived mesenchymal cells (1 to 10 ng/ml). Quantitative real-time polymerase chain reaction was used to examine the effects of TGF-beta1 on gene expression. RESULTS: TGF-beta1 significantly decreased proliferation of both posterofrontal and sagittal suture-derived mesenchymal cells, by bromodeoxyuridine incorporation assays (n = 6). TGF-beta1 also inhibited osteogenesis in both suture-derived mesenchymal cells determined by alkaline phosphatase activity and mineralization (n = 3 for all assays). During chondrogenic differentiation, TGF-beta1 markedly increased expression of chondrocyte-specific gene markers in posterofrontal suture-derived mesenchymal cells (Sox9, Col II, Aggrecan, and Col X) (p

Proliferation, osteogenic differentiation, and fgf-2 modulation of posterofrontal/sagittal suture-derived mesenchymal cells in vitro.

BACKGROUND: Fibroblast growth factor (FGF) signaling is of central importance in premature cranial suture fusion. In the murine skull, the posterofrontal suture normally fuses in early postnatal life, whereas the adjacent sagittal suture remains patent. The authors used a recently developed isolation technique for in vitro culture of suture-derived mesenchymal cells to examine the effects of FGF-2 on proliferation and differentiation of posterofrontal and sagittal suture-derived mesenchymal cells. METHODS: Skulls were harvested from 40 mice (5-day-old). Posterofrontal and sagittal sutures were dissected, separating sutural mesenchymal tissue from dura mater and pericranium, and cultured. After cell migration from the explant and subculture, differences in proliferation and osteogenic differentiation of these distinct populations were studied. The mitogenic and osteogenic effects of recombinant FGF-2 were then assessed. FGF-2 regulation of gene expression was evaluated. RESULTS: Suture-derived mesenchymal cells isolated from the posterofrontal suture demonstrated significantly higher proliferation rates and a robust mitogenic response to FGF-2 as compared with suture-derived mesenchymal cells isolated from the sagittal suture. Interestingly, posterofrontal suture-derived mesenchymal cells retained a higher in vitro osteogenic potential, as shown by alkaline phosphatase activity and bone nodule formation. FGF-2 significantly diminished osteogenesis in both suture-derived mesenchymal cell populations. Subsequently, Ob-cadherin and Sox9 were found to be differentially expressed in posterofrontal versus sagittal suture-derived mesenchymal cells and dynamically regulated by FGF-2. CONCLUSIONS: In vitro osteogenesis of suture-derived mesenchymal cells recapitulates in vivo posterofrontal and sagittal sutural fates. Posterofrontal rather than sagittal suture-derived mesenchymal cells are more responsive to FGF-2 in vitro, in terms of both mitogenesis and osteogenesis.

Identification of genes differentially expressed by prematurely fused human sutures using a novel in vivo - in vitro approach.

Craniosynostosis is the premature fusion of calvarial sutures. It results from abnormal differentiation or proliferation of cells within the osteogenic fronts of growing calvarial bones. To date, research has focused on animal models and in vitro organ and tissue culture to determine the molecular mechanisms controlling calvarial suture morphogenesis. Here, we test a new, in vivo-in vitro approach based on the hypothesis that calvarial suture cells passaged in minimal medium exhibit a stable gene expression profile similar to undifferentiated osteoblastic cells that can provide a benchmark for comparison with in vivo expression of differentiated tissue. We show that tissue-specific expression is lost after the first passage and, using cDNA microarrays, compare expression between fused suture tissue from craniosynostosis patients and in vitro de-differentiated explant cells. A large number of differentially expressed genes were identified, including novel genes WIF1, LEF1, SATB2, RARRES1, DEFA1, DMP1, PTPRZ1, and PTPRC, as well as those commonly associated with human suture morphogenesis, e.g., FGF2, MSX2, and BMP2. Two differentially expressed genes, WIF1 and FGF2, were further examined in an in vivo-in vivo comparison between unfused and prematurely fused tissue. The same pattern of differential expression was observed in each case, further validating the ability of our in vivo-in vitro approach to identify genes involved in in vivo human calvarial tissue differentiation.

Dlx5 drives Runx2 expression and osteogenic differentiation in developing cranial suture mesenchyme.

Craniofacial bones derive from cephalic neural crest, by endochondral or intramembranous ossification. Here, we address the role of the homeobox transcription factor Dlx5 during the initial steps of calvaria membranous differentiation and we show that Dlx5 elicits Runx2 induction and full osteoblast differentiation in embryonic suture mesenchyme grown "in vitro". First, we compare Dlx5 expression to bone-related gene expression in the developing skull and mandibular bones. We classify genes into three groups related to consecutive steps of ossification. Secondly, we study Dlx5 activity in osteoblast precursors, by transfecting Dlx5 into skull mesenchyme dissected prior to the onset of either Dlx5 and Runx2 expression or osteogenesis. We find that Dlx5 does not modify the proliferation rate or the expression of suture markers in the immature calvaria cells. Rather, Dlx5 initiates a complete osteogenic differentiation in these early primary cells, by triggering Runx2, osteopontin, alkaline phosphatase, and other gene expression according to the sequential temporal sequence observed during skull osteogenesis "in vivo". Thirdly, we show that BMP signaling activates Dlx5, Runx2, and alkaline phosphatase in those primary cultures and that a dominant-negative Dlx factor interferes with the ability of the BMP pathway to activate Runx2 expression. Together, these data suggest a pivotal role of Dlx5 and related Dlx factors in the onset of differentiation of chick calvaria osteoblasts.

Isolation and characterization of posterofrontal/sagittal suture mesenchymal cells in vitro.

BACKGROUND: Craniosynostosis, the premature fusion of cranial sutures, affects one in 2500 children. In the mouse, the posterofrontal suture is programed to fuse postnatally, but the adjacent sagittal suture remains patent throughout life. To study the cellular process of suture fusion, the authors isolated and studied suture-derived mesenchymal cells. METHODS: Skulls were harvested from 80 mice (2 to 5 days old), and posterofrontal and sagittal sutures were dissected meticulously. Suture mesenchymal tissue was separated from the underlying dura mater and overlying pericranium and cultured in growth media. After the cells migrated from the explant tissues, the morphologies of the two cell populations were studied carefully, and quantitative real-time polymerase chain reaction was performed to evaluate gene expression. RESULTS: Both posterofrontal and sagittal cells exhibited highly heterogeneous morphologies, and the posterofrontal cells migrated faster than the sagittal cells. Accordingly, growth factors such as transforming growth factor-beta1 and fibroblast growth factor (FGF)-2 were expressed significantly more highly in posterofrontal compared with sagittal suture mesenchymal cells. In contrast, FGF receptor 2 and FGF-18 were expressed significantly more in sagittal than in posterofrontal suture cells. Importantly, bone morphogenic protein-3, the only osteogenic inhibitor in the bone morphogenic protein family, and noggin, a bone morphogenic protein antagonist, were expressed significantly more in sagittal than in posterofrontal suture cells, suggesting a possible mechanism of suture patency. CONCLUSIONS: To the authors' knowledge, this is the first analysis of mouse suture-derived mesenchymal cells. The authors conclude that isolation of suture-derived mesenchymal cells will provide a useful in vitro system with which to study the mechanisms underlying suture biology.

Histological and histomorphometric study of growth-related changes of cranial sutures in the animal model.

INTRODUCTION: During the early development, numerous histological and morphometric changes occur in the cranial sutures the exact knowledge of which is of fundamental significance for understanding clinically relevant cranial anomalies. In this paper a histological and histomorphometric longitudinal study of the coronal, sagittal and lambdoid sutures in the rat is reported in relation to age. MATERIAL AND METHODS: Forty-eight male Wistar rats (Rattus norvegicus Berkenhout) were raised under standard conditions. Eight animals each were sacrificed at defined time points (10, 14, 28, 42, 70, 98 days post partum) for specimen preparation. Histological preparations of the sagittal, coronal and lambdoid sutures were produced and examined morphologically and histomorphometrically (suture width, height, and area). RESULTS: Histologically, three phases of sutural growth with characteristic structural features were found. Histomorphometry reveals a quasi linear increase in height from the 30th to the 98th day post partum. Suture width remained relatively constant in the area of dura mater and periosteum. CONCLUSION: The sutures of the test animals studied had a similar growth behaviour primarily consisting of an increase in height with almost constant width. The three-phases of development could be demonstrated histologically in all sutures.