A molecular analysis of the isolated rat posterior frontal and sagittal sutures: differences in gene expression

Although it is one of the most commonly occurring craniofacial congenital disabilities, craniosynostosis (the premature fusion of cranial sutures) is nearly impossible to prevent because the molecular mechanisms that regulate the process of cranial suture fusion remain largely unknown. Recent studies have implicated the dura mater in determining the fate of the overlying cranial suture; however, the molecular biology within the suture itself has not been sufficiently investigated. In the murine model of cranial suture fusion, the posterior frontal suture is programmed to begin fusing by postnatal day 12 in rats (day 25 in mice), reliably completing bony union by postnatal day 22 (day 45 in mice). In contrast, the sagittal suture remains patent throughout the life of the animal. Using this model, this study sought to examine for the first time what differences in gene expression--if any--exist between the two sutures with opposite fates. For each series of experiments, 35 to 40 posterior frontal and sagittal suture complexes were isolated from 6-day-old Sprague-Dawley rat pups. Suture-derived cell cultures were established, and ribonuicleic acid was derived from snap-frozen, isolated suture tissue. Results demonstrated that molecular differences between the posterior frontal and sagittal suture complexes were readily identified in vivo, although these distinctions were lost once the cells comprising the suture complex were cultured in vitro. Hypothetically, this change in gene expression resulted from the loss of the influence of the underlying dura mater. Significant differences in the expression of genes encoding extracellular matrix proteins existed in vivo between the posterior frontal and sagittal sutures. However, the production of the critical, regulatory cytokine transforming growth factor beta-1 was equal between the two suture complexes, lending further support to the hypothesis that dura mater regulates the fate of the overlying cranial suture.     

Craniofacial sutures: morphology, growth, and in vivo masticatory strains.

The growth and morphology of craniofacial sutures are thought to reflect their functional environment. However, little is known about in vivo sutural mechanics. The present study investigates the strains experienced by the internasal, nasofrontal, and anterior interfrontal sutures during masticatory activity in 4-6-month-old miniature swine (Sus scrofa). Measurements of the bony/fibrous arrangements and growth rates of these sutures were then examined in the context of their mechanical environment. Large tensile strains were measured in the interfrontal suture (1,036 microepsilon +/- 400 SD), whereas the posterior internasal suture was under moderate compression (-440 microepsilon +/- 238) and the nasofrontal suture experienced large compression (-1,583 microepsilon +/- 506). Sutural interdigitation was associated with compressive strain. The collagen fibers of the internasal and interfrontal sutures were clearly arranged to resist compression and tension, respectively, whereas those of the nasofrontal suture could not be readily characterized as either compression or tension resisting. The average linear rate of growth over a 1-week period at the nasofrontal suture (133.8 micrometer, +/- 50.9 S.D) was significantly greater than that of both the internasal and interfrontal sutures (39.2 micrometer +/- 11.4 and 65. 5 micrometer +/- 14.0, respectively). Histological observations suggest that the nasofrontal suture contains chondroid tissue, which may explain the unexpected combination of high compressive loading and rapid growth in this suture.     

Cytochemical localization of tartrate-resistant acid phosphatase, alkaline phosphatase, and nonspecific esterase in perivascular cells of cartilage canals in the developing mouse epiphysis

Cytochemical localization of tartrate-resistant acid phosphatase (TRAP), tartrate-sensitive acid phosphatases (TSAP), alkaline phosphatase, and nonspecific esterase was used to characterize perivascular cells within cartilage canals. In the distal femoral epiphyses of 5- to 7-day-old mice, three stages of canal development can be distinguished, and at each developmental stage different perivascular cells were present with morphological characteristics of degradative cells. Vacuolated cells resembling macrophages, fibroblastic cells, and chondroclasts were present adjacent to the matrix in superficial, intermediate, and deep canals, respectively. In order to characterize these perivascular cells cytochemically, nonspecific esterase and TSAP staining was used to identify macrophages, alkaline phosphatase staining was used to identify fibroblastic cells, and TRAP staining was used to identify chondroclasts. There were no cells present in the canals at any developmental stage that were positive for TSAP or strongly positive for nonspecific esterase, placing doubt on the identity of the vacuolated cells as macrophages. Alkaline phosphatase-positive perivascular cells were present in the intermediate and deep canals adjacent to matrix containing alkaline phosphatase-positive chondrocytes. These alkaline phosphatase-positive cells were found in the same location within canals as the fibroblastic cells. Tartrate-resistant acid phosphatase was localized in chondroclasts at the tips of deep canals but was not confined exclusively to chondroclasts. Except for the very early stage of canal development prior to chondrocyte hypertrophy, TRAP-positive cells were present at the tips of superficial and intermediate canals as well as at the tips of the deep canals.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro murine posterior frontal suture fate is age-dependent: implications for cranial suture biology

In CD-1 mice, the posterior frontal suture (analogous to the human metopic suture) fuses while all other cranial sutures remain patent. In an in vitro organ culture model, the authors previously demonstrated that posterior frontal sutures explanted immediately before the onset of suture fusion (at 25 days old) mimic in vivo physiologic fusion. In the first portion of this study, the authors defined how early in development the posterior frontal suture fuses in their tension-free, serum-free organ culture system by serially analyzing posterior frontal suture fusion from calvariae explanted at different stages of postnatal development. Their results revealed a divergence of suture fate leading to abnormal patency or physiologic fusion between the first and second weeks of life, respectively, despite viability and continued growth of the calvarial explants in vitro. From these data, the authors postulated that the gene expression patterns present in the suture complex at the time of explant may determine whether the posterior frontal suture fuses or remains patent in organ culture. Therefore, to elucidate potentially important differences in gene expression within this "window of opportunity," they performed a cDNA microarray analysis on 5-day-old and 15-day-old posterior frontal and sagittal whole suture complexes corresponding to the age ranges for unsuccessful (1 to 7 days old) and successful (14 to 21 days old) in vitro posterior frontal suture fusion. Overall, their microarray results reveal interesting differential expression patterns of candidate genes in different categories, including angiogenic cytokines and mechanosensitive genes potentially important in cranial suture biology.     

A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plastiCity among diplomonads and significant lateral gene transfer in eukaryote genome evolution

Background

Craniosynostosis, the premature fusion of calvarial sutures, is a common craniofacial abnormality. Causative mutations in more than 10 genes have been identified, involving fibroblast growth factor, transforming growth factor beta, and Eph/ephrin signalling pathways. Mutations affect each human calvarial suture (coronal, sagittal, metopic, and lambdoid) differently, suggesting different gene expression patterns exist in each human suture. To better understand the molecular control of human suture morphogenesis we used microarray analysis to identify genes differentially expressed during suture fusion in children with craniosynostosis. Expression differences were also analysed between each unfused suture type, between sutures from syndromic and non-syndromic craniosynostosis patients, and between unfused sutures from individuals with and without craniosynostosis.

Results

We identified genes with increased expression in unfused sutures compared to fusing/fused sutures that may be pivotal to the maintenance of suture patency or in controlling early osteoblast differentiation (i.e. RBP4, GPC3, C1QTNF3, IL11RA, PTN, POSTN). In addition, we have identified genes with increased expression in fusing/fused suture tissue that we suggest could have a role in premature suture fusion (i.e. WIF1, ANXA3, CYFIP2). Proteins of two of these genes, glypican 3 and retinol binding protein 4, were investigated by immunohistochemistry and localised to the suture mesenchyme and osteogenic fronts of developing human calvaria, respectively, suggesting novel roles for these proteins in the maintenance of suture patency or in controlling early osteoblast differentiation. We show that there is limited difference in whole genome expression between sutures isolated from patients with syndromic and non-syndromic craniosynostosis and confirmed this by quantitative RT-PCR. Furthermore, distinct expression profiles for each unfused suture type were noted, with the metopic suture being most disparate. Finally, although calvarial bones are generally thought to grow without a cartilage precursor, we show histologically and by identification of cartilage-specific gene expression that cartilage may be involved in the morphogenesis of lambdoid and posterior sagittal sutures.

Conclusion

This study has provided further insight into the complex signalling network which controls human calvarial suture morphogenesis and craniosynostosis. Identified genes are candidates for targeted therapeutic development and to screen for craniosynostosis-causing mutations.     

Regional differentiation of rat cranial suture-derived dural cells is dependent on association with fusing and patent cranial sutures

A significant body of literature supports a role for the dura mater underlying cranial sutures in the regulation of sutural fate. These studies have implicated regional differentiation of the dura mater based on association with fusing and patent rat cranial sutures. The purpose of these experiments was to isolate and characterize dural cells associated with fusing (posterior frontal) and patent (sagittal) rat cranial sutures. Six-day-old rats were killed, and the dura mater underlying the posterior frontal and sagittal sutures was harvested. Dural cells were briefly trypsinized and allowed to reach confluence. Two litters (10 animals per litter) were used for each set of experiments. Cells were harvested after the first and fifth passages for analysis of vimentin and desmoplakin expression (characteristic of human meningeal cells), cellular proliferation, density at confluence (a measure of cellular contact inhibition), and alkaline phosphatase production. In addition, bone nodule formation and collagen I production were analyzed in first passage cells. The results indicate that suture-derived dural cells can be established and that these cells coexpress vimentin and desmoplakin. In addition, it is demonstrated that first-passage sagittal suture-derived dural cells proliferate significantly faster and have decreased cellular contact inhibition than posterior frontal suture-derived cells (p < 0.01). Finally, it is shown that suture-derived dural cells have osteoblast-like properties, including alkaline phosphatase production, collagen I expression, and bone nodule formation in vitro. The possible mechanisms by which regional differentiation of suture-derived dural cells occur are discussed.     

Growth of calvarial width. An experimental investigation in rabbits

This investigation was conducted to analyze growth in width of the rabbit calvarium. 15 male New Zealand white rabbits were subjected to regular stereometric examinations from 31 to 141 days of age after implantation of tantalum bone markers. The sagittal suture complex, i.e. the interfrontal and interparietal sutures, and the bilateral temporal sutures demonstrated similar growth rates in magnitude which moderately decelerated throughout the observation period. Transverse growth exhibited local growth fluctuations and short-term negative growth values in a well-balanced manner.     

Adenovirus-mediated transmission of a dominant negative transforming growth factor-beta receptor inhibits in vitro mouse cranial suture fusion

Recent studies have implicated the transforming growth factor (TGF)-beta family in the regulation of pathological sporadic cranial suture fusion. In addition, these studies have shown that TGF-beta is highly expressed by the dura mater underlying fusing murine cranial sutures. The purpose of the present experiments was to analyze the effects of disrupting TGF-beta signaling during programmed mouse cranial suture fusion. Using recombinant DNA technology, a replication-deficient adenovirus encoding a defective TGF-beta receptor (Ad.DN-TbetaRII) capable of blocking TGF-beta biological activity was constructed. Mouse posterior frontal sutures were harvested before the initiation of suture fusion (postnatal day 25), and the dura mater underlying the suture was infected with vehicle, Ad.DN-TbetaRII, or control virus (Ad.LacZ; n = 10 each). Sutures were cultured for 14 or 30 days in an organ culture system and analyzed macroscopically and histologically.X-gal staining of Ad.LacZ-infected sutures 14 days after culture revealed strong staining of cells localized to the dura mater. Macroscopic analysis revealed complete sutural fusion in vehicle and Ad.LacZ-infected sutures. In contrast, Ad.DN-TBRII-infected sutures demonstrated nearly complete patency. Histological analysis confirmed our macroscopic observations with sutural fusion in 81.3 +/- 10 percent and 74.5 +/- 9 percent of vehicle and Ad.LacZ-infected sutures, respectively, versus 38.1 +/- 12 percent (p < 0.001) in Ad.DN-TbetaRII-infected sutures. In addition, transfection with the Ad.DN-TbetaRII virus resulted in a significant attenuation of anterior-to-posterior suture fusion, with the majority of fused sections localized to anterior sections. These data strongly implicate TGF-beta biological activity in the dura mater underlying the posterior frontal suture in the regulation of programmed sutural fusion. In addition, this study demonstrates the utility of adenovirus-mediated gene transfer in preventing programmed sutural fusion.     

Increased IGF-I and IGF-II mRNA and IGF-I peptide in fusing rat cranial sutures suggest evidence for a paracrine role of insulin-like growth factors in suture fusion

Premature cranial suture fusion, or craniosynostosis, can result in gross aberrations of craniofacial growth. The biology underlying cranial suture fusion remains poorly understood. Previous studies of the Sprague-Dawley rat posterior frontal suture, which fuses at between 12 and 20 days, have suggested that the regional dura mater beneath the cranial suture directs the overlying suture's fusion. To address the dura-suture paracrine signaling that results in osteogenic differentiation and suture fusion, the authors investigated the possible role of insulin-like growth factors (IGF) I and II. The authors studied the temporal and spatial patterns of the expression of IGF-I and IGF-II mRNA and IGF-I peptide and osteocalcin (bone morphogenetic protein-4) protein in fusing posterior frontal rat sutures, and they compared them with patent coronal (control) sutures. Ten Sprague-Dawley rats were studied at the following time points: 16, 18, and 20 days of gestation and 2, 5, 10, 15, 20, 30, 50, and 80 days after birth (n = 110). Posterior frontal and coronal (patent, control) sutures were analyzed for IGF-I and IGF-II mRNA expression by in situ hybridization by using 35S-labeled IGF-I and IGF-II antisense riboprobes. Levels of IGF-I and IGF-II mRNA were quantified by counting the number of autoradiograph signals per cell. IGF-I and osteocalcin immunoreactivity were identified by avidin-biotin peroxidase immunohistochemistry. IGF-I and IGF-II mRNA were expressed in dural cells beneath fusing sutures, and the relative mRNA abundance increased between 2 and 10 days before initiation of fusion. Subsequently, IGF-I and IGF-II mRNA were detected in the suture connective tissue cells at 15 and 20 days during the time of active fusion. In contrast, within large osteoblasts of the osteogenic front, the expression of IGF-I and IGF-II mRNA was minimal. However, IGF-I peptide and osteocalcin protein were intensely immunoreactive within these osteoblasts at 15 days (during the period of suture fusion). These data suggest that the dura-suture interaction may be signaled in a paracrine fashion by dura-derived growth factors, such as IGF-I and IGF-II. These peptides, in turn, stimulate nearby osteoblasts to produce bone-promoting growth factors, such as osteocalcin.     

The correlation between craniofacial and long bone growth: an experimental investigation in normal rabbits

The present study was undertaken to elucidate the relationships between craniofacial and long-bone growth. Nine male New Zealand white rabbits received spherical tantalum bone markers in the tibial epiphyses and in the nasal, frontal, and parietal bones. The animals were followed from 30 to 143 days of age. Growth changes were calculated with a roentgen stereometric system, and the results statistically evaluated. Except for the final interval when all variables varied at random, high correlations between tibial and frontonasal or coronal sutural growth were demonstrated; and the respective linear regression lines were homogeneously assembled. The relationship between the tibia and the sagittal suture displayed great variations between individual animals as well as between the suture's parts, although growth at the interfrontal suture was clearly correlated to tibial growth upon exclusion of the time factor. The first principal component of the three neurocranial sutures was calculated and seemed accurately correlated to long-bone growth. The present study concluded that growth at the frontonasal and coronal sutures normally seems to parallel general somatic development, while growth at the sagittal suture appears individually displaced in time. Nevertheless, when the principal component of the combination of the coronal suture and the neurocranial section of the sagittal suture was computed, this was highly correlated to body growth.     

Mechanisms of murine cranial suture patency mediated by a dominant negative transforming growth factor-beta receptor adenovirus

Using a physiologic model of mouse cranial suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the suture complex of the fusing posterior frontal compared with the patent sagittal suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior frontal suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the suture mesenchyme and subjacent dura mater during the early period of predicted posterior frontal suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.     

Restricted growth at the frontonasal suture: alterations in craniofacial growth in rabbits

Apposition of bone at the sutural margin is generally thought to be a compensatory adjustment to growing soft-tissue organs such as the brain or eyes within the skull. The frontonasal suture which is located at the interface between the cranial and facial skeletons is a site of extremely active growth in the young rabbit. Recently, we showed that premature closure of a cranial suture, the coronal suture, can alter the growth not only at the adjacent frontonasal suture but also of the basicranium and midface. This study examines the effects of restricted growth at the frontonasal suture on both growth at adjacent cranial sutures and linear growth of the basicranium and midface. Thirty newborn New Zealand White rabbits were subdivided into experimental and sham-treated groups of equal size and distribution for sex and birth weight. At 9 days of age, the frontonasal suture of each experimental animal was immobilized by bilateral application of methyl-cyanoacrylate adhesive across the frontonasal suture. Growth and morphometric changes were monitored by radiocephalometric methods through 120 days of age by bilateral implantation of radiopague markers on each side of frontonasal, coronal, and anterior lambdoid sutures. Results indicate that restricted growth at the frontonasal suture results not only in a significant shortening of the midface but also in significant decreases in growth at the coronal and internasal sutures. Growth at the interfrontal and sagittal sutures is increased. Furthermore, growth at the anterior portion of the nasal bones is significantly increased, thereby offsetting a portion of the decreased nasal bone length resulting from frontonasal restriction.     

Strain in the braincase and its sutures during function

The skull is distinguished from other parts of the skeleton by its composite construction. The sutures between bony elements provide for interstitial growth of the cranium, but at the same time they alter the transmission of stress and strain through the skull. Strain gages were bonded to the frontal and parietal bones of miniature pigs and across the interfrontal, interparietal and coronal sutures. Strains were recorded 1) during natural mastication in conjunction with electromyographic activity from the jaw muscles and 2) during stimulation of various cranial muscles in anesthetized animals. Vault sutures exhibited vastly higher strains than did the adjoining bones. Further, bone strain primarily reflected torsion of the braincase set up by asymmetrical muscle contraction; the tensile axis alternated between +45 degrees and -45 degrees depending on which diagonal masseter/temporalis pair was most active. However, suture strains were not related to overall torsion but instead were responses to local muscle actions. Only the coronal suture showed significant strain (tension) during jaw opening; this was caused by the contraction of neck muscles. All sutures showed strain during jaw closing, but polarity depended on the pattern of muscle usage. For example, masseter contraction tensed the coronal suture and the anterior part of the interfrontal suture, whereas the temporalis caused compression in these locations. Peak tensile strains were larger than peak compressive strains. Histology suggested that the skull is bent at the sutures, with the ectocranial surface tensed and the endocranial surface predominantly compressed. Collectively, these results indicate that skulls with patent sutures should be analyzed as complexes of independent parts rather than solid structures.     

Timing of Egf treatment differentially affects Tgf-beta2 induced cranial suture closure

Premature suture obliteration results in an inability of cranial and facial bones to grow, with craniofacial dysmorphology requiring surgical correction as a consequence. Understanding signaling pathways associated with suture morphogenesis might enable non-invasive treatment of patients with fused sutures. Tgf-beta 2 induces premature suture fusion associated with increased cell proliferation both in vitro and in vivo. Tgf-beta 2 and Egf signal transduction pathways use some signaling proteins in common to regulate proliferation and differentiation, leading to speculation that these two pathways converge to regulate normal suture development. It was therefore hypothesized that Egf could induce suture fusion, and that Tgf-beta 2-induced suture closure occurred via an Egf-dependent pathway. A well-established fetal calvarial organ culture system was used to expose developing E19.5 fetal rat coronal sutures to Egf, Tgf-beta 2 and SC-120, a blocker of Egf receptor activity. Co-culture experiments examined the effect of Egf on Tgf-beta 2-induced suture closure when Egf was given either prior to or after Tgf-beta 2 treatment. Histomorphometric measurement of suture width was done on sagittal sections through coronal sutures harvested after 5 days in culture. Western blotting using phospho-antibodies against Egf receptors was used to confirm Egf receptor activity. Suture width increased with increasing concentrations of Egf, demonstrating that Egf-induced cell activity alone was not sufficient to cause premature suture obliteration. Egf administered prior to Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced suture obliteration, demonstrating that pre-exposure of cells to this powerful mitogen prevented their response to signals induced by Tgf-beta 2. However, Egf added after Tgf-beta 2 treatment had no effect on Tgf-beta 2-induced suture closure. Blocking Egf activity after Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced obliteration, indicating that Tgf-beta 2 required Egf activity to induce suture obliteration. Appropriate timing of signal generation by Egf and Tgf-beta 2 is critical for normal suture development and maintenance of suture patency.