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.     

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.     

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.     

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.     

Craniosynostosis and altered patterns of fetal TGF-beta expression induced by intrauterine constraint

Recent work has demonstrated that fusion of the calvarial sutures is mediated by locally elaborated soluble growth factors, including the transforming growth factor-betas (TGF-betas), leading some to speculate that external biomechanical forces play little role in suture development. Clinical evidence has long suggested, however, that fetal head constraint may play a critical role in the pathogenesis of many cases of nonsyndromic craniosynostosis. The purpose of these experiments was to test the hypothesis that intrauterine constraint leads to an alteration in normal patterns of TGF-beta expression and that these alterations are associated with craniosynostosis. Fetal constraint was induced by allowing C57Bl/6 murine fetuses to grow for 2.5 days beyond the normal 20-day gestation by performing uterine cerclage on the eighteenth day. Cranial suture morphology was examined in hematoxylin and eosin-stained sections and in cleared whole-mount specimens, double stained with alizarin red S and Alcian blue. Expression patterns of TGF-beta1 and TGF-beta3 were examined by immunohistochemical techniques. Gross and microscopic examination of the cranial sutures of 17 constrained fetuses revealed changes that ranged from narrowing to complete osseous obliteration of the coronal and squamosal sutures. All sutures of 14 nonconstrained control pups remained patent. Fetal head constraint was associated with increased TGF-beta1 immunoreactivity within the new bone and the underlying dura when compared with nonconstrained age-matched controls. TGF-beta3 immunoreactivity was associated with the dura underlying patent, nonconstrained sutures, whereas constraint-induced synostosis was characterized by down-regulation of dural TGF-beta3 expression. These experiments confirm the ability of intrauterine constraint to induce premature fusion of the cranial sutures and provide evidence that intrauterine head constraint induces the expression of osteogenic growth factors in fetal calvarial bone and the underlying dura.     

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.     

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.     

Enzymatic activation associated with programmed fusion of the posterior interfrontal sutures in rats

Fetal rat coronal sutures in culture undergo fusion in the absence of their dura mater. Coinciding with the period of fusion are marked cellular enzymatic changes. Alkaline phosphatase, a marker of osteoblastic activity, and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclastic activity, both increase significantly within fusing sutures and indicate changes in the control of bone synthesis and breakdown. Other enzymes not specifically related to bone formation or degradation also show activation within these fusing sutures. These enzymes include tartrate-sensitive acid phosphatase (TSAP), a marker of lysosomal activity; hexokinase, a glycolytic enzyme; glucose 6-phosphate dehydrogenase (G6PD), an enzyme of the pentose monophosphate shunt; and glutathione reductase, an enzyme of the antioxidant pathway.In the present study, we compared the enzymatic changes previously seen ex vivo with those occurring in vivo during the programmed closure of the posterior interfrontal suture of the rat. This suture fuses between postnatal days 10 and 30 in the rat. The sagittal suture, which remains patent during this period, was used to establish baseline enzymatic activities in a comparable midline suture. Neonatal rats were killed at postnatal days 2, 4, 5, 8, 10, 12, 15, 20, and 30, and posterior interfrontal and sagittal sutures with bone plates on either side were removed. The suture regions of the samples were isolated, dura mater was removed, and suture regions were assayed by microanalytical techniques. Activities of alkaline phosphatase, TRAP, TSAP, hexokinase, G6PD, and glutathione reductase were measured. DNA content was also assayed, and enzyme activities were expressed per amount of DNA. Three pups were killed at each time point, and three to five assays were performed per suture (posterior interfrontal or sagittal) for each time point assayed.Alkaline phosphatase and TRAP activities showed marked increases in fusing sutures compared with nonfusing controls, similar to the increases demonstrated ex vivo. TSAP and hexokinase also showed elevations in the fusing posterior interfrontal sutures, with the greatest differences predominantly during the period of fusion, comparable to the changes seen ex vivo. However, G6PD and glutathione reductase, enzymes of the antioxidant pathway, did not demonstrate the same degree of activation seen ex vivo in fusing sutures. In fact, the levels were actually higher in the patent sagittal samples for the majority of time points examined.Alkaline phosphatase and TRAP activity elevations indicated both osteoblastic and osteoclastic activation during fusion, as seen in the ex vivo phenomenon. TSAP and hexokinase increases also reflected activation in lysosomes and in cellular metabolism during fusion, paralleling the ex vivo situation. However, a less clear pattern of activation in the antioxidant pathway, in contrast to the pattern seen ex vivo, was present. These differences may reflect the different environments of sutures in vivo and ex vivo. Alternatively, oxidative stress may play a more central role in the pathologic process of induced suture fusion ex vivo than in programmed suture fusion in vivo.     

Osteogenesis in cranial defects: reassessment of the concept of critical size and the expression of TGF-beta isoforms

Transforming growth factor-betas (TGF-beta) have been demontstrated to be upregulated during osteoblast function in vitro and during cranial suture fusion in vivo. The authors hypothesized that spontaneous reossification of calvarial defects was also associated with upregulation of TGF-beta. The present study was designed to (1) evaluate the concept of a critical-size defect within the calvaria in an adult guinea pig model and (2) investigate the association between the ossification of calvarial defects and TGF-beta upregulation. Paired circular parietal defects with diameters of 3 and 5 mm and single parietal defects with diameters of 8 or 12 mm were made in 45 six-month-old skeletally mature guinea pigs. Three animals per defect size were killed after survival periods of 3 days, 1 week, 4 weeks, 8 weeks, or 12 weeks. New bone ingrowth was evaluated by assessing for linear closure by a traditional linear method and by a modified cross-sectional area method using an image analysis system in which the thickness of new bone was taken into account. Immunohistochemistry was performed using rabbit polyclonal antibodies to localize TGF-beta1, -beta2, and -beta3. All specimens were photographed, and the intensity of immunostaining was graded based on subjective photographic assessment by three independent reviewers. No defect demonstrated any measurable bone replacement after a survival period of 3 days. All 3- and 5-mm defects were completely reossified after 12 weeks based on the linear analysis of new bone, indicating these defects to be less than critical size. However, new bone formation in the 5-mm defects never exceeded a mean of 40 percent by cross-sectional area of new bone. Percent of new bone formation by cross-sectional area was significantly higher within 3-mm defects than in all larger defects 4 weeks after the craniotomy, reaching a mean of 89 percent new bone by 12 weeks. Persistent gaps were noted on linear analysis of the 8- and 12-mm wounds by 12 weeks, and mean percent new bone by cross-sectional area remained below 30 percent. Immunolocalization demonstrated osteogenic fronts at the advancing bone edge and the endocranial side, in which the osteoblasts stained strongly for all isoforms of TGF-beta. The intensity of osteoblast expression waned considerably after the majority of the defect had reossified. These data indicate that histometric analysis based on cross-sectional area more accurately reflects the osteogenic potential of a cranial defect than does linear inspection of defect closure. Although the interpretation of immunolocalization studies is highly subjective, independent assessment by three reviewers indicates that isoforms of TGF-beta were upregulated during a limited "window" of time corresponding to the period of active calvarial reossification, and expression of TGF-beta corresponded to osteoblast activity within osteogenic fronts.     

Basic fibroblast growth factor and transforming growth factor beta-1 expression in the developing dura mater correlates with calvarial bone formation

Numerous studies have found dura mater-calvarial mesenchyme interactions during calvarial bone induction; however, the exact molecular mechanisms governing these inductive events remain unknown. Recent studies have implicated basic fibroblast growth factor (FGF-2) and transforming growth factor-beta1 (TGF-beta1) in regulating bone formation. The purpose of this study was, therefore, to investigate the expression of FGF-2 and TGF-beta1 during calvarial bone formation in rats. Eight rats were killed on embryonic days 14, 18, and 20 and neonatal day 1 (n = 32). Four animals at each time point were analyzed by in situ hybridization, and the remainder were analyzed by immunohistochemistry. The results indicated that the dura mater underlying the developing calvarial bone strongly expressed FGF-2 and TGF-beta1 mRNA at all time points examined. In contrast, minimal growth factor expression was noted in the overlying calvarial mesenchyme until embryonic day 18, but it increased significantly with increasing age. Importantly, FGF-2 and TGF-beta1 mRNA expression in the dura mater underlying the developing calvarium preceded and was significantly greater than expression in the calvarium mesenchyme (p < 0.05). Interestingly, minimal expression of FGF-2 and TGF-beta1 mRNA was noted for all time points in the dura mater underlying the posterior frontal suture and within the posterior frontal suture connective tissue (p < 0.01 when compared with the dura mater underlying the developing calvarium). Immunohistochemical findings closely paralleled mRNA expression, with intense staining for FGF-2 and TGF-beta1 in the dura mater underlying the developing calvarial mesenchyme. Increasing FGF-2 and TGF-beta1 staining was noted within calvarial osteoblasts with increasing age, particularly in cells located near the endocranial surface (i.e., in contact with the developing dura mater). These findings, together with the known biologic functions of FGF-2 and TGF-beta1, implicate these growth factors in the regulation of calvarial bone growth by the developing dura mater. The possible mechanisms of this interaction are discussed.     

Cranial suture obliteration is induced by removal of transforming growth factor (TGF)-beta 3 activity and prevented by removal of TGF-beta 2 activity from fetal rat calvaria in vitro

Cranial suture morphogenesis requires soluble, heparin-binding factors secreted by the dura mater to resist premature osseous obliteration. Elevated levels of transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3 have previously been noted in cranial sutures undergoing normal and premature sutural obliteration. To examine the role of TGF-beta s in regulating cranial suture morphogenesis, an established in vitro, serum-free, calvarial culture system was used. In this system, fetal rat coronal sutures undergo apparently normal suture morphogenesis in the presence of dura mater, but undergo osseous obliteration in the absence of dura mater. Neutralizing polyclonal antibodies to TGF-beta 1, TGF-beta 2, or TGF-beta 3 were added to cultures of fetal day 19 rat calvaria, which were harvested at 3, 4, or 5 days, processed for histology, sectioned, and examined. Coronal sutures from calvaria cultured in the presence of dura mater resisted obliteration, either alone or in the presence of TGF-beta 1 or TGF-beta 2 neutralizing antibodies. However, sutures from calvaria cultured in the presence of TGF-beta 3 neutralizing antibodies became obliterated. Conversely, sutures from calvaria cultured in the absence of dura mater became obliterated by bone, either alone or in the presence of neutralizing antibodies to TGF-beta 1 or TGF-beta 3. However, those sutures cultured in the presence of neutralizing antibodies to TGF-beta 2 were rescued from osseous obliteration.     

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

Abstract 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.     

TGF-beta2 stimulates cranial suture closure through activation of the Erk-MAPK pathway

Cranial sutures are important growth sites of the skull. During suture closure, the dura mater is one of the most important sources of various positive and negative regulatory signals. Previous results indicate that TGF-beta2 from dura mater strongly accelerates suture closure, however, its exact regulatory mechanism is still unclear. In this study, we confirmed that removal of dura mater in calvarial organ culture strongly accelerates sagittal suture closure and that this effect is further enhanced by TGF-beta2 treatment. TGF-beta2 stimulated cell proliferation in the MC3T3-E1 cell line. Similarly, it stimulated the proliferation of cells in the sutural space in calvarial organ culture. Furthermore, TGF-beta2-mediated enhanced cell proliferation and suture closure were almost completely inhibited by an Erk-MAPK blocker, PD98059. These results indicate that TGF-beta2-induced activation of Erk-MAPK is an important signaling component that stimulates cell proliferation to enrich osteoprogenitor cells, thereby promoting their differentiation into osteoblasts to achieve a rapid calvarial bone expansion.     

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.     

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.