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J Anat. May 2005; 206(5): 437–444.
PMCID: PMC1571510

Twist is required for establishment of the mouse coronal suture

Abstract

Cranial sutures are the growth centres of the skull, enabling expansion of the skull to accommodate rapid growth of the brain. Haploinsufficiency of the human TWIST gene function causes the craniosynostosis syndrome, Saethre–Chotzen syndrome (SCS), in which premature fusion of the coronal suture is a characteristic feature. Previous studies have indicated that Twist is expressed in the coronal suture during development, and therefore that it may play an important role in development and maintenance of the suture. The Twist-null mouse is lethal before the onset of osteogenesis, and the heterozygote exhibits coronal suture synostosis postnatally. In this study we investigated the function of Twist in the development of the mouse coronal suture, by inhibiting Twist synthesis using morpholino antisense oligonucleotides in calvarial organ culture. Decreased Twist production resulted in a narrow sutural space and fusion of bone domains within 48 h after the addition of the morpholino oligonucleotides. Proliferation activity in the sutural cells was decreased, and the expression of osteogenic marker genes such as Runx2 and Fgfr2 was up-regulated in the developing bone domain within 4 h. These results suggest that during establishment of the suture area, Twist is required for the regulation of sutural cell proliferation and osteoblast differentiation.

Keywords: calvarial organ culture, craniosynostosis, development, morpholino antisense oligonucleotides, mouse

Introduction

The skull vault consists of several membrane bones: paired frontal and parietal bones, and the upper part of the occipital bone. These are connected by fibrous tissue, forming the sutures, which are the main centres of skull growth. Craniosynostosis – premature loss of the suture – results in impaired growth of the head and face, and leads to physiological problems because of constrained growth of the brain (Cohen, 1993; Wilkie, 1997; Wilkie & Morriss-Kay, 2001).

The coronal (fronto-parietal) suture is predominantly affected in craniosynostosis caused by mutations in the TWIST and fibroblast growth factor receptor (FGFR) genes (Wilkie, 1997; Wilkie & Morriss-Kay, 2001). Haploinsufficiency of the TWIST gene causes Saethre–Chotzen syndrome (SCS), which is characterized by coronal synostosis and soft-tissue syndactyly (El Ghouzzi et al. 1997; Howard et al. 1997). An interaction between Twist and Fgf signalling has been suggested by studies in Drosophila and mouse (Shishido et al. 1993; Zuniga et al. 2002; O'Rourke et al. 2002). In the developing coronal suture, the sutural mesenchyme is flanked by overlapping edges of the frontal and parietal bones, and sequential osteoblast differentiation is observed from the periphery to the inside of the bone domain. Previous studies have revealed that in the developing coronal suture, Twist is expressed in the midsutural mesenchyme and preosteoblasts (Johnson et al. 2000; Rice et al. 2000), Fgfr2 is expressed in proliferating preosteoblasts around the periphery of the bones, and Fgfr1 expression is related to differentiating osteoblasts adjacent to the bone matrix (Iseki et al. 1999).

Twist is a highly conserved transcription factor that belongs to the family of basic helix–loop–helix proteins. Previous studies have suggested that Twist is involved in maintenance of the undifferentiated condition (Lee et al. 1999; reviewed in Castanon & Baylies, 2002; Ishii et al. 2003), including osteoblast differentiation (Yousfi et al. 2001; Bialek et al. 2004). Functions have also been described in apoptosis (Yousfi et al. 2002; Hjiantoniou et al. 2003) and tumorigenesis (Maestro et al. 1999). The Twist-null mouse is lethal around mid-gestation, before the onset of osteogenesis, exhibiting failure of cranial neural tube closure, poorly partitioned somites and an increase in apoptotic cells in somites (Chen & Behringer, 1995). Heterozygotes show mild abnormalities, for instance in the development of the skull (Bourgeois et al. 1998); fusion of the coronal suture, which never closes during the life of the wild-type mouse, is observed postnatally (Carver et al. 2002).

To clarify the role of Twist in development, we inhibited Twist protein synthesis in the mouse fetal calvarium in culture by means of morpholino antisense oligonucleotide (MO). Osteoprogenitor cell proliferation was decreased and differentiation markers up-regulated in the coronal suture, suggesting that Twist is an important regulator of these processes during establishment of the suture.

Materials and methods

Sampling

C57BL/6 mice were used for all experiments (plug day = E0). All experiments were performed in accordance with protocols certified by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University. For fresh frozen sections, samples were directly embedded into Tissue-Tek OCT compound (Sakura, Japan). For fixed frozen sections, samples were fixed in 4% PFA/PBS at 4 °C, overnight, then equilibrated in 25% sucrose/PBS followed by embedding into OCT compound. For paraffin sections, samples were fixed in 4% PFA/PBS at 4 °C overnight, then embedded into paraffin after dehydration.

In situ hybridization

RNA probes used in this study were prepared as previously described (Iseki et al. 1997). In situ hybridization (ISH) on sections was carried out on fresh or fixed frozen sections and probe preparation was carried out as described by Iseki et al. (1997) with some modification. For ISH on fixed frozen sections, specimens were treated with proteinase K at 2 µg mL−1 in PBS for 5 min, then refixed in 4% PFA/PBS for 20 min, followed by acetylation.

Western blotting

A full-length mouse Twist cDNA fragment was inserted into the pIRES-hrGFP-2a (Stratagene, USA). This Twist-expressing vector was transfected to HeLa cells using FuGene 6 transfection reagent (Roche, Germany). Twist morpholino antisense oligonucleotide (MOT) 5′-GACACGTCCTGCATCATCTCGCGG-3′ or standard control morpholino antisense oligonucleotide (MOC) 5′-CCTCTTACCTCAGTTACAATTTATA-3′ (Gene Tools, USA) was supplemented to the medium at 1.4 µm concentration 16 h after the transfection. The morpholino oligo solution was prepared by following the manufacturer's instructions with EPEI (Gene Tools, USA) delivery solution. The cells were harvested 32 h after the delivery of the MOs. MO-untreated cells were harvested 48 h after transfection of the Twist vector. For SDS-PAGE, 100 µg of total protein was loaded in each lane. For the detection of Twist protein, anti-Twist polyclonal antibody (h81, Santa-Cruz, USA) was used.

Histological staining

Histological staining was carried out on paraffin sections. For Mallory's staining, specimens were stained with haematoxylin, 1% acid fuchsin and aniline blue-orange G.

Immunohistochemistry

For the detection of proliferating cell nuclear antigen (PCNA), mouse monoclonal anti-PCNA (Santa Cruz, USA) antibody was applied at 1 : 200 dilution on fixed or fresh frozen sections, followed by application of biotinylated antimouse IgG (Vector Laboratories, USA). For the detection of active caspase3, antiactive-Caspase3 antibody (Promega, USA) was applied at 1 : 100 dilution on fixed frozen sections, followed by application of biotinylated anti-rabbit IgG (Chemicon, USA) at 1 : 200 dilution. They were followed by Vectastain ABC elite (Vector laboratories, USA) at 1 : 100 dilution, then Sigma Fast DAB (Sigma, USA) was used to visualize the signal.

Calvarial culture and MO application to the culture

Calvaria were dissected from E15 fetuses removing the skin and brain, and cultured for 48 h as described by Kim et al. (1998) with the modification that the calvaria were cultured in organ culture dishes (Falcon, USA). The medium was supplemented with 20 units mL−1 of penicillin and 20 g mL−1 of streptomycin (Invitrogen, USA), 10 mmβ-glycerophosphate and 10−7 m dexamethazon. Ascorbic acid was supplemented at 10 g mL−1 every 24 h. After 1 h of preculture in the medium above, the medium was replaced by medium containing MOT or MOC at 14 µm concentration.

Results

Expression of osteogenic markers in the developing mouse coronal suture

First we investigated the expression pattern of Runx2, the earliest marker of osteoblast differentiation, and bone sialoprotein (Bsp), and compared them with those of Twist, Fgfr1 and Fgfr2 on the E16 mouse fetal coronal suture area. As previously reported (Iseki et al. 1997), in the E16 mouse coronal suture area, sequential osteogenic differentiation is observed within a developing bone from the very edge of the domain towards the osteoid, inside the domain. Twist is expressed in the suture as well as in the osteoblastic cell lineage (fig. 1A), which is in agreement with previous data (Johnson et al. 2000; Rice et al. 2000). Twist expression overlaps with that of Runx2 to a certain extent (fig. 1A,B), consistent with the report that Twist inhibits Runx2 function by protein–protein interaction (Bialek et al. 2004). Runx2 is not expressed in the suture; its expression overlaps with Fgfr2 expression associated with preosteoblasts (fig. 1B and C), and it is also transcribed in more differentiated osteoblasts located close to the osteoid, which also express Fgfr1 and Bsp. It is therefore suggested that Fgfr2 expression is as early osteogenic marker as Runx2, and that Runx2 expression is maintained during the maturation of preosteoblasts to osteoblasts, associated with Fgfr1 and Bsp expression. Staining for PCNA shows that cells at the tip of developing bones and in the suture are actively proliferating (fig. 1F, black and white arrowhead, respectively).

Fig. 1
Gene expression and cell proliferation in the developing coronal suture area of E16 mouse. Gene expression was studied by ISH (A–E), and PCNA was detected using immunohistochemistry on fresh frozen sections. (A) Twist is expressed in the sutural ...

Inhibition of Twist protein synthesis results in fusion of the frontal and parietal bone domains in calvarial culture

We studied the effect of Twist morpholino antisense oligonucleotides (Twist MOT) on the developing coronal suture area. We applied MOT or standard control morpholino oligonucleotides (MOC) to HeLa cells transfected with Twist-expressing vector to evaluate the effect of MOT (fig. 2A). With the presence of MOT, the Twist expression level in the Hela cells was strongly decreased. In contrast, MOC had no effect on Twist expression. We then cultured E15 fetal calvaria with the presence of MOC or MOT. At E15, the skull vault is not yet mineralized, but the developing frontal and parietal bones are clearly visible when the calvarium is dissected (data not shown). At the end of culture after 48 h in the medium, the bone domains expanded and became clearer, indicating the progress of osteogenesis (Fig. 2B). In histological sections, the coronal suture area of calvaria cultured with MOC showed overlapping of the frontal and parietal bone domains (Fig. 2C, a and b). The morphology and staining pattern was identical to that of calvaria cultured without additives (data not shown). However, the calvaria cultured with MOT showed contact between osteoid of the frontal and parietal domains (fig. 2C, d and e, arrow), although the extent of overlapping was similar to that of the controls. In the MOC sections there was fibrous tissue at the tip of the bone (fig. 2C, a, arrowhead); this was missing in the MOT specimens. The amount of sutural mesenchyme between the frontal and parietal bones was reduced in the explant cultured with MOT compared with that with MOC (compare fig. 2C, a and b, with fig. 2C, d and e), suggesting that the sutural cells fail to proliferate or undergo cell death. We therefore carried out immunohistochemical detection of PCNA on 18-h cultured calvaria. In the MOC specimen, cell proliferation activity was observed mainly in the preosteoblasts at the tip of the bone domain and in the suture, as in the E16 in vivo section (fig. 2C, c, arrowhead and double arrows; fig. 1F, arrowhead). However, the staining of the MOT specimen showed that there was a decrease in the number of PCNA-positive cells, and this was more significant in the suture (fig. 2C, f, asterisk) than in the bone domain. We also investigated cell death by detecting caspase-3, which is expressed in both apoptotic and differentiating cells (Testa, 2004), using immunohistochemistry. Caspase-3 was detected in the cells around the bone domain after 18 h of culture with MOC, and the MOT sample showed the same result (data not shown). We did not detect pyknotic nuclei at the end of culture with MOT (fig. 2C, a and b). These results suggest that inhibition of Twist synthesis in the suture area results in cell proliferation defects, not cell death. We investigated the effects on osteogenic marker expression, Runx2, Fgfr2 and Bsp, in the suture area of the calvarium cultured with MOT. At the end of culture (48 h), the result of ISH of these genes represented developing bone domains of the explants and we did not observe any difference in the expression pattern between the specimen with MOC and without MO (data not shown). In the explant with MOT, the expression domain clearly indicated that the frontal and parietal domains are thicker than those of controls (fig. 3D–fig. 3F) and have started to fuse (fig. 3D, E, arrow), indicating increased osteogenesis. In addition, expression of these genes in the MOT explants is stronger than that with MOC when the culture and ISH were carried out at the same time and under the same conditions (compare fig. 3A–fig. 3C with fig. 3D–E). This up-regulation is observed in the 4-h cultured specimen (compare fig. 4A–C with fig. 4D–fig. 4D), and Runx2 and Fgfr2 up-regulation is more obvious than Bsp up-regulation.

Fig. 2
MOT induces fusion of frontal and parietal bones and reduction of cell proliferation in the suture area by reducing Twist synthesis. (A) Western blotting of HeLa cell extracts: untreated, transfected Twist-expressing vector only (Twist), or together with ...
Fig. 3
MOT enhances the expression of osteogenic markers and induces fusion of their frontal and parietal expression domains after 48 h. Samples were cultured with MOC (A–C), and MOT (D–F). All of the Runx2, Fgfr2 and Bsp gene expression domains ...
Fig. 4
Acceleration of osteogenesis already starts after 4 h. Samples cultured with MOC (A–C) and with MOT (D–F) for 4 h. The expression of Runx2, Fgfr2 and Bsp in the MOT sample is up-regulated in the bone domain compared with the MOC sample ...

Discussion

In this study we were able to observe that expression of Fgfr2 begins at a relatively early stage of osteogenesis, by comparing it with the Runx2 expression pattern in the developing skull bone. We also showed that inhibition of Twist expression in the calvarium results in fusion of the frontal and parietal bones due to impaired cell proliferation in the suture and accelerated osteogenic differentiation in the bone domain, with no effect on apoptosis.

Antisense morpholino oligonuclotides can be used as a gene knockdown tool in mouse organ culture

Previous studies, mainly on Zebrafish, Xenopus and human cells, have shown that MO can be used as a gene knockdown tool (Summerton & Weller, 1997; Yang et al. 2001; Darken et al. 2002; Ishiwata et al. 2004). In this study we demonstrated that MO could also be used effectively in organ culture of mouse embryonic tissue. In our study, a supplement of MOT into the medium of mouse fetal calvarial culture induced fusion of the bone domains. We observed that the number of PCNA-positive cells was decreased in the suture, indicating that MOT can penetrate the tissue and affect not only the surface but also the interior of the tissue.

Twist is required for regulation of osteoblast differentiation

Inhibition of Twist protein synthesis, with MOT-induced up-regulation of the osteogenic markers Runx2, Fgfr2 and Bsp in the area of the developing bone domain, is indicative of osteogenesis promotion, although it is not clear whether the up-regulation of these genes is due to an increase in the number of expressing cells, or whether transcription is more activated in each cell, or both. However, this observation supports the idea that the human craniosynostosis phenotype resulting from loss-of-function TWIST mutations (SCS) is the result of excess osteogenesis. The results are also consistent with experimental reports that inhibition of TWIST expression with antisense RNA from an expression vector in an osteoblastic cell line induced differentiated osteoblast phenotype (Lee et al. 1999). A recent report showed that Twist inhibits osteoblast differentiation without affecting Runx2 expression (Bialek et al. 2004). A new domain, Twist box, is found in the carboxyterminal of Twist protein; the region interacts with the Runx2 DNA-binding domain, resulting in inhibition of Runx2 function as a transcription factor. We detected up-regulation of Runx2 transcription within 4 h after MOT addition. Because the Runx2 promoter has its own binding site and is self-regulated (reviewed in Otto et al. 2003; Levanon & Groner, 2004), a decreased level of the Twist protein might have promoted Runx2 function both by the lack of inhibitor protein and by the enhancement of its own promoter activity. In the case of Bsp, although there is no direct evidence that its expression is controlled by Twist, its weak up-regulation after 4 h of culture with MOT is consistent with the finding that Bsp expression is regulated by Runx2, and there are Runx2 binding sites in the Bsp promoter region (Ducy et al. 1997; Javed et al. 2001). This is supported by the fact that there is a lower level of up-regulation of Bsp than Runx2 and Fgfr2 after 4 h. There has been no report that Fgfr2 expression is directly controlled by Twist or Runx2. However, considering the previous studies which suggest that Twist controls Fgfr expression (Shishido et al. 1993; Zuniga et al. 2002; O'Rourke et al. 2004), Fgfr2 expression may be regulated by Twist.

Twist is required for suture development

The life cycle of the suture is principally composed of two stages, establishment and maintenance. Johnson et al. (2000) showed that Twist is expressed in the sutural mesenchyme at the highest level around E16, and is then partially down-regulated. Rat coronal suture development during the prenatal stage has been reported (Markens, 1975), and after the frontal and parietal bone domains approach and overlap each other, the suture area is thicker and the suture becomes a blastema-like structure. Concomitantly, a decreased number of undifferentiated mesenchymal cells and an increased number of fibroblastic cells are observed in the suture. When we compare the histological observations and Twist expression, down-regulation of Twist around E17 except at the bone edges (Johnson et al. 2000) coincides with the appearance of the thickened suture area, suggesting suture establishment. In our calvarial culture we observed the dilation of the coronal suture area 48 h after culture as seen in vivo at the equivalent age (E17). MOT treatment clearly inhibits this suture development morphologically, and we observed specific decreased proliferation activity in the sutural cells. Therefore, we suggest that loss of Twist function results in defects in cell proliferation leading to impaired suture establishment. Although it has been suggested that Twist is involved in cell death, we did not detect any increase in caspase-3-positive cells, or any pyknotic nuclei, in the MOT-treated specimen. Apoptotic cell death is first detected in the coronal suture at E16 (Rice et al. 1999) around the time of suture establishment, but our results suggest that Twist is not involved in cell death during the stage of suture establishment. It is not clear whether sutural cells (midsutural mesenchyme cells) have an osteogenic lineage or not. Our data suggest that the fusion of the adjacent bones appears to be the result of accelerated osteogenesis of the bone domain, but is not due to recruitment of osteogenic cells from the suture after inhibition of Twist synthesis, because we observed up-regulation of osteogenic markers in the bone domain but not in the suture. However, we do not have any direct evidence for this hypothesis, and our calvarial culture was carried out during the period leading to suture establishment. It is therefore possible that after suture establishment the sutural cells acquire an osteogenic character. Further investigation is required to determine whether the sutural mesenchyme cells have an osteogenic lineage.

In conclusion, inhibition of Twist synthesis in fetal calvarial organ culture causes fusion of the coronal suture by inhibiting suture establishment and promoting osteoblast differentiation. These results suggest that Twist is required for suture area establishment and maintenance because of the part it plays in inhibiting the premature differentiation of osteoblast and regulating proliferation of the sutural cells.

Acknowledgments

We are grateful to M. Young (Bsp), S. Nomura (Runx2) and J. Heath (Fgfr1, Fgfr2) for providing the plasmids, and K. Morinaka for technical assistance. This work was supported by a program grant from Human Frontier Science Program (L.A.P., J.B.U. and S.I.), Grants in Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 13357015 to K.E., No. 15390554 to S.I.), and A.G. Leventis Foundation grant (L.A.P.).

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