Cell fate specification during calvarial bone and suture development
Eva Lana-Elolaa,⁎, Ritva Ricea, Agamemnon E. Grigoriadisa, David P.C. Ricea,b
aDepartments of Craniofacial Development and Orthodontics, Floor 27 Guy’s Tower, King’s College, London, SE1 9RT, UK
bInstitute of Dentistry, PO Box 63, 00014 University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland
Received for publication 10 April 2007; revised 27 July 2007; accepted 13 August 2007
Available online 14 September 2007
In this study we have addressed the fundamental question of what cellular mechanisms control the growth of the calvarial bones and
conversely, what is the fate of the sutural mesenchymal cells when calvarial bones approximate to form a suture. There is evidence that the
size of the osteoprogenitor cell population determines the rate of calvarial bone growth. In calvarial cultures we reduced osteoprogenitor cell
proliferation; however, we did not observe a reduction in the growth of parietal bone to the same degree. This discrepancy prompted us to
study whether suture mesenchymal cells participate in the growth of the parietal bones. We found that mesenchymal cells adjacent to the
osteogenic fronts of the parietal bones could differentiate towards the osteoblastic lineage and could become incorporated into the growing
bone. Conversely, mid-suture mesenchymal cells did not become incorporated into the bone and remained undifferentiated. Thus
mesenchymal cells have different fate depending on their position within the suture. In this study we show that continued proliferation of
osteoprogenitors in the osteogenic fronts is the main mechanism for calvarial bone growth, but importantly, we show that suture mesenchyme
cells can contribute to calvarial bone growth. These findings help us understand the mechanisms of intramembranous ossification in general,
which occurs not only during cranial and facial bone development but also in the surface periosteum of most bones during modeling and
© 2007 Elsevier Inc. All rights reserved.
Keywords: Calvaria; Suture; Intramembranous ossification; Osteoblast; Cell fate
At a molecular level we have a good understanding of what
regulates the commitment of undifferentiated mesenchymal
cells into osteoblasts (reviewed by Cohen, 2006; Karsenty,
2003). We also know a great deal about endochondral
ossification and what controls long bone growth which is a
complex process involving the formation of a cartilaginous
template that is later replaced by bone. Intramembranous bone
ossification accounts for most of the bone growth of the face
and calvaria and in the periosteum of long bones (Kronenberg,
2003; Ornitz, 2005). In contrast to endochondral ossification,
intramembranous bone ossification is a relatively simple pro-
cess with no cartilage anlagen and osteoblasts differentiating
directly from mesenchymal cells. It is therefore surprising that
we know much less about the cellular processes that control
intramembranous bone growth compared to the processes
controlling endochondral bone growth.
In the craniofacial region, intramembranous bone develop-
ment starts with the aggregation of mesenchymal cells into
condensation centers (Hall and Miyake, 2000). These tightly
packed collections of cells expand and once they reach a critical
size the cells at the center of these masses differentiate into
osteoblasts. Growth of immature bones occurs at the leading
edges or osteogenic fronts and when an osteogenic front
confronts its neighbor, the two fronts either merge to create a
single bone or form a suture. Thus a suture is a joint composed
of two osteogenic fronts and the interposed mesenchyme. Once
the basic pattern of bones and sutures has been established
further craniofacial growth primarily occurs in the osteogenic
fronts of the sutures. The processes of osteogenic condensation
formation and function during embryogenesis, to form the first
bony elements, are directly comparable to the processes of cell
aggregation, proliferation, differentiation and function that
Available online at www.sciencedirect.com
Developmental Biology 311 (2007) 335–346
⁎Corresponding author. Fax: +44 2071881674.
E-mail address: Eva.Lana_Elola@kcl.ac.uk (E. Lana-Elola).
0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
occur in the osteogenic fronts of established craniofacial
The rate of calvarial bone growth is determined by the size of
the osteoprogenitor cell population in the perimeter of the
osteogenic condensations and then later by the size of the
osteoprogenitor population in the osteogenic fronts. We have
previously shown that the transcription factor Foxc1 regulates
bone morphogenetic protein (BMP)-mediated osteoprogenitor
proliferation, specifically at the leading edge of the developing
calvarial bones thus regulating bony expansion. Reduced
osteoprogenitor proliferation in Foxc1−/−mice results in small
calvarial bones that do not grow beyond a rudimentary size and
remain at the sites of the initial osteogenic condensations. Foxc1
controls the osteoprogenitor population by regulating the BMP
targets Msx2 and Alx4 (Rice et al., 2005). In both humans and
mice, loss-of-function mutations in Msx2 and Alx4 result in
similar ‘hole in the head’ phenotypes to those exhibited by
Foxc1−/−mutant mice (Antonopoulou et al., 2004; Satokata et
al., 2000; Wilkie et al., 2000). Conversely, mice which
overexpress Msx2 exhibit increased osteoprogenitor prolifera-
tion in osteogenic fronts of the calvarial bones, which results in
enhanced bone growth (Liu et al., 1999b). Consistent with this,
a gain-of-function mutation in MSX2 causes Boston-type
craniosynostosis in humans which is characterized by excessive
calvarial bone growth and an obliteration of the calvarial sutures
(Liu et al., 1995).
In addition to BMP signaling, the size of the calvarial
osteoprogenitor population is regulated by fibroblast growth
factor (FGF) signaling. Several Fgf ligands and receptors are
expressed in the developing calvarial mesenchyme and bones
(Hajihosseini and Heath, 2002; Hajihosseini et al., 2001; Rice
et al., 2003a), and targeted disruption of Fgfr signaling in
mice results in altered calvarial osteoprogenitor proliferation
and abnormal bone growth (Eswarakumar et al., 2004;
Eswarakumar et al., 2002; Wang et al., 2005). It has been
proposed that signaling through Fgfr2 primarily regulates
osteoprogenitor proliferation while signaling through Fgfr1
primarily regulates osteoblast differentiation (Iseki et al.,
1999). In humans, mutations in FGFR1, 2 and 3 cause
several forms of syndromic and non-syndromic craniosynos-
tosis including Apert, Crouzon and Pfeiffer syndromes and
Muenke craniosynostosis. All these conditions are character-
ized by premature craniofacial suture fusion as well as other
skeletal anomalies (Rice, 2005).
We have previously proposed the developing mouse
sagittal suture as an uncomplicated model of osteoblastic
differentiation and intramembranous bone growth (Rice et al.,
2003b). By analyzing the distribution of markers that are
expressed at different stages of osteoblastic differentiation, we
can build up a picture of the suture so that we can see all
stages of differentiation within a single tissue section. In this
study we have expanded and refined this analysis and used it
to study calvarial bone growth. We have previously shown
that embryonic day 15.5 (E15.5) mouse parietal bones grow
towards each other and after 2 days of culture form a normal
patent sagittal suture (Kim et al., 1998). Using this system we
now ask the fundamental questions: what are the cellular
mechanisms controlling calvarial bone growth; and conver-
sely what is the fate of the calvarial mesenchymal cells when
parietal bones approximate and a suture is formed?
In this study we show that proliferation and subsequent
differentiation of osteoprogenitors at the osteogenic fronts,
although important, is not the only cellular mechanism that
contributes to calvarial bone growth. Sutural mesenchymal cells
can differentiate into osteoblasts and become incorporated into
the growing parietal bones, but only if adjacent to the
osteogenic fronts. Thus the fate of calvarial mesenchymal
cells varies depending on their position within the suture. Under
normal conditions we demonstrate that a small percentage of
sutural mesenchymal cells are incorporated into the growing
bones. We can hypothesize that during pathological conditions
such as craniosynostosis the relative contribution of recruitment
from the mesenchyme into the calvarial bones may be altered.
Materials and methods
Calvaria were dissected free from skin and brain from E15.5 mouse
embryos. Explants were placed in Nuclepore polycarbonate filters (Whatman)
andculturedin a Trowell-typeorganculture system for 2 or 5 days dependingon
the experiment. DMEM (Sigma) was supplemented with 20 IU/ml penicillin/
streptomycin (Sigma), 10% fetal bovine serum (Life Technologies), and 100 μg/
ml of ascorbic acid. Media were replaced every 2 days. The antimitotic drug
nocodazole (Sigma-Aldrich) was added to the culture media in different
concentrations (0.1 μg/ml, 0.25 μg/ml, and 1.0 μg/ml) from a stock of 0.5 mg
nocodazole in 2 ml DMSO. Explants treated with 1 μl of DMSO per 1 ml of
media were used as control. At least two explants treated with DMSO as control
and two of each nocodazole dilutions were sectioned and at least 4 sections of
each explant were used to count BrdU positive cells.
Cells were enzymatically isolated from the calvaria of CD1 mice postnatal
day 1 by sequential digestion with trypsin (1 mg/ml), dispase II (2 mg/ml), and
two digestions with collagenase A (2 mg/ml) (Roche). The last two of the four
digestion steps (populations III–IV) were pooled and plated in T75 flasks
containing α-MEM (Sigma), penicillin–streptomycin, glutamine and 15% of
heat activated fetal bovine serum (FBS). After 24 h of incubation attached cells
were then collected by trypsinization. Aliquots were counted and remaining
cells were resuspended in the standard medium described above supplemented
with ascorbic acid (50 mg/ml) and 10 mM of β-glycerol phosphate. The
resuspended cells were plated in tissue culture dishes at approximately 4×103
cells/cm2. The medium was changed every 2–3 days. Cells were incubated at
37 °C in humidified atmosphere at 95% air 5% CO2 incubator. DMSO
(1:50,000) or nocodazole (0.1 μg/ml or 1.0 μg/ml) was added to the media on
day 1 on cell culture (experiment 1), when cells had reached confluence
(experiment 2) or just when nodule formation was staring to occur (experiment
3). Cells were washed in PBS and then stained for alkaline phosphatase using
naphtol-AS-MX phosphate and fast red (Sigma) with 100 mM of Tris pH 8.3.
Vital dye injections
DiI C18 (1-1′-dioctadecyl-3,3,3′,3′-tetramethilindocarbocyanine perchlo-
rate, Molecular Probes, Inc) was dissolved at 5% in absolute ethanol and then
diluted 1:10 in 0.3 M sucrose. CellTracker™CM-DiI (Molecular Probes, Inc)
was used for labeling in the same manner. Using microcapillars filled with DiI,
microinjections were performed under a stereo microscope on the day 0 of
culture. DiI-labeled cells were tracked using an Olympus SZX12 microscope
with a fluorescent light by photographing at day 0, day 1 and day 2 of culture.
336 E. Lana-Elola et al. / Developmental Biology 311 (2007) 335–346
Also, the formation of a sutural blastema has been described
(Markens, 1975). This is a mesenchymal thickening that
develops as intramembranous bones approximate (mouse
E15.5–E17.5). This may, in part, be due to high levels of
cell proliferation in the mesenchyme, concomitant with intense
osteogenesis. However, it may also, in part, be explained by
growth of the adjacent bones firstly compressing the
mesenchyme between the osteogenic fronts and secondly
expelling mesenchymal cells from their path, as we have
observed in our explants (Fig. 3).
In this study we have analyzed calvarial bone development
under normal conditions. The next challenge will be to apply
our findings to abnormal developmental situations such as the
formation of sutural bones, craniosynostosis and delayed
suture closure typified in cleidocranial dysplasia. Within
developing sutures additional ossification centers can arise
which eventually develop into small bones, called sutural or
Wormian bones. Sutural bones most commonly occur in the
lambdoid suture and are seen in a number of conditions
including cleidocranial dysplasia, osteogenesis imperfecta and
hypothyroidism (Gorlin et al., 2001; Hinton et al., 1984).
Sutural bones are also seen in Apert syndrome. Patients with
Apert syndrome are characterized by several abnormalities
including premature fusion of the coronal sutures. In addition,
they present with a wide calvarial defect between the frontal
and parietal bones. Within this region bony islands form in
the calvarial mesenchyme, these later coalesce with each other
and with the neighboring calvarial bones, resulting in the
fusion across the midline and no further calvarial growth in
this plane (Cohen and Kreiborg, 1996). Under normal
conditions we have shown that a small percentage of sutural
mesenchymal cells are incorporated into the growing bones.
However, during craniosynostosis or the formation of sutural
bones the relative contribution of recruitment from the
mesenchyme into the calvarial bones may be abnormally
During normal development the prime mechanism of
calvarial bone expansion is by continued proliferation in the
osteogenic fronts (Fig. 6). As well as studying how intramem-
branous bones grow, we have also examined the fate of the
sutural mesenchyme and found that cells have different fates
depending on their location within the suture. Most mesench-
ymal cells remain undifferentiated in the suture, however a
small proportion are recruited into the developing bones. We
hope that these findings may help us understand the mechan-
isms of intramembranous ossification in general, which occurs
not just in craniofacial bones but also in the periosteum of most
bones during modeling and remodeling.
We thank A Coudert, S Dietrich, T Mitsiadis and A Streit for
their help with the study. We thank S. Pudaruth for her technical
assistance. This work was funded by EU Marie Curie-EST
fellowship, EC contract number MEST-CT-2004-504025 (ELE)
and MRC (DPCR).
Antonopoulou, I., et al., 2004. Alx4 and Msx2 play phenotypically similar and
additive roles in skull vault differentiation. J. Anat. 204, 487–499.
Bialek, P., et al., 2004. A twist code determines the onset of osteoblast
differentiation. Dev. Cell 6, 423–435.
Cohen Jr., M.M., 2006. The new bone biology: pathologic, molecular, and
clinical correlates. Am. J. Med. Genet., A 140, 2646–2706.
CohenJr., M.M.,Kreiborg, S.,1996.Suture formation,prematuresutural fusion,
and suture default zones in Apert syndrome. Am. J. Med. Genet. 62.,
Ducy, P., et al., 1997. Osf2/Cbfa1: a transcriptional activator of osteoblast
differentiation. Cell 89, 747–754.
El Ghouzzi, V., et al., 2001. Mutations in the basic domain and the loop–helix II
junction of TWIST abolish DNA binding in Saethre–Chotzen syndrome.
FEBS Lett. 492, 112–118.
Eswarakumar, V.P., et al., 2002. The IIIc alternative of Fgfr2 is a positive
regulator of bone formation. Development 129, 3783–3793.
Eswarakumar, V.P., et al., 2004. A gain-of-function mutation of Fgfr2c
demonstrates the roles of this receptor variant in osteogenesis. Proc. Natl.
Acad. Sci. U. S. A. 101, 12555–12560.
Gorlin, R., et al., 2001. Syndromes of the Head and Neck. Oxford Univ. Press,
Grigoriadis, A.E., et al., 1988. Differentiation of muscle, fat, cartilage, and bone
from progenitor cells present in a bone-derived clonal cell population: effect
of dexamethasone. J. Cell Biol. 106, 2139–2151.
Fig. 6. Model for parietal bone growth and suture development. The primary
mechanism for parietal bone growth is proliferation and subsequent differentia-
tion of osteoprogenitors at the osteogenic fronts. In addition, sutural
mesenchymal cells can differentiate into osteoblasts and become incorporated
into the growing parietal bones, but only if adjacent to the osteogenic fronts.
Thus the fate of calvarial mesenchymal cells varies depending on their position
within the suture. Yellow figures represent the process of proliferation within the
osteogenic front, in which osteoprogenitors in the osteogenic front undergo
mitosis and remain within the osteogenic front. These cells move with the
osteogenic front into the sutural mesenchyme as the two parietal bones
approximate. Red figures represent the fate of preosteoblasts in the osteogenic
front, which can either advance with the developing bone at its front or can
terminally differentiate into mature osteoblasts and become incorporated in the
parietal bone. Green figures represent the different fate of cells positioned in the
sagittal suture: mesenchymal cells can either remain undifferentiated, getting
expulsed from the suture as parietal bones grow towards each other, or they can
become incorporated by the adjacent parietal bone and consequently express
osteoblast marker genes as Bsp and AP. Neither of these two alternatives
implicates cell migration, the basic movement is the parietal bone growth. of,
345 E. Lana-Elola et al. / Developmental Biology 311 (2007) 335–346
Hajihosseini, M.K., Heath, J.K., 2002. Expression patterns of fibroblast growth
factors-18 and -20 in mouse embryos is suggestive of novel roles in calvarial
and limb development. Mech. Dev. 113, 79–83.
Hajihosseini, M.K., et al., 2001. A splicing switch and gain-of-function
mutation in FgfR2-IIIc hemizygotes causes Apert/Pfeiffer-syndrome-like
phenotypes. Proc. Natl. Acad. Sci. U. S. A. 98, 3855–3860.
Hall, B.K., Miyake, T., 2000. All for one and one for all: condensations and the
initiation of skeletal development. BioEssays 22, 138–147.
Hinton, D.R., et al., 1984. Lambdoid synostosis. Part 1. The lambdoid suture:
normal development and pathology of “synostosis”. J. Neurosurg. 61,
Iseki, S., et al., 1997. Fgfr2 and osteopontin domains in the developing skull
vault are mutually exclusive and can be altered by locally applied FGF2.
Development 124, 3375–3384.
Iseki, S., et al., 1999. Fgfr1 and Fgfr2 have distinct differentiation- and
proliferation-related roles in the developing mouse skull vault. Development
Jordan, M.A., et al., 1992. Effects of vinblastine, podophyllotoxin and
nocodazole on mitotic spindles. Implications for the role of microtubule
dynamics in mitosis. J. Cell Sci. 102 (Pt 3), 401–416.
Karsenty, G., 2003. The complexities of skeletal biology. Nature 423, 316–318.
Kim, H.J., et al., 1998. FGF-, BMP- and Shh-mediated signalling pathways in
the regulation of cranial suture morphogenesis and calvarial bone
development. Development 125, 1241–1251.
Komori, T., 2003. Requisite roles of Runx2 and Cbfb in skeletal development.
J. Bone Miner. Metab. 21, 193–197.
Kronenberg, H.M., 2003. Developmental regulation of the growth plate. Nature
Levine, J.P., et al., 1998. Studies in cranial suture biology: regional dura
mater determines overlying suture biology. Plast. Reconstr. Surg. 101,
Liu, Y.H., et al., 1995. Premature suture closure and ectopic cranial bone in mice
expressing Msx2 transgenes in the developing skull. Proc. Natl. Acad. Sci.
U. S. A. 92, 6137–6141.
Liu, P., et al., 1999a. Regulation of osteogenic differentiation of human bone
marrow stromal cells: interaction between transforming growth factor-beta
and 1,25(OH)(2) vitamin D(3) in vitro. Calcif. Tissue Int. 65, 173–180.
Liu, Y.H., et al., 1999b. Msx2 gene dosage influences the number of
proliferative osteogenic cells in growth centers of the developing murine
skull: a possible mechanism for MSX2-mediated craniosynostosis in
humans. Dev. Biol. 205, 260–274.
Luduena, R.F., Roach, M.C., 1991. Tubulin sulfhydryl groups as probes and
targets for antimitotic and antimicrotubule agents. Pharmacol. Ther. 49,
Malaval, L., et al., 1999. Kinetics of osteoprogenitor proliferation and osteoblast
differentiation in vitro. J. Cell. Biochem. 74, 616–627.
Markens, I.S., 1975. Embryonic development of the coronal suture in man and
rat. Acta Anat. (Basel) 93, 257–273.
Mundlos, S., 1999. Cleidocranial dysplasia: clinical and molecular genetics.
J. Med. Genet. 36, 177–182.
Nakashima, K., et al., 2002. The novel zinc finger-containing transcription
factor osterix is required for osteoblast differentiation and bone formation.
Cell 108, 17–29.
Opperman, L.A., et al., 1995. Cranial sutures require tissue interactions with
dura mater to resist osseous obliteration in vitro. J. Bone Miner. Res. 10,
Ornitz, D.M., 2005. FGF signaling in the developing endochondral skeleton.
Cytokine Growth Factor Rev. 16, 205–213.
Pratap, J., et al., 2003. Cell growth regulatory role of Runx2 during proliferative
expansion of preosteoblasts. Cancer Res. 63, 5357–5362.
Rice, D.P., 2005. Craniofacial anomalies: from development to molecular
pathogenesis. Curr. Mol. Med. 5, 699–722.
Rice, D.P., et al., 1997. Detection of gelatinase B expression reveals osteoclastic
bone resorption as a feature of early calvarial bone development. Bone 21,
Rice, D.P., et al., 1999. Apoptosis in murine calvarial bone and suture
development. Eur. J. Oral Sci. 107, 265–275.
Rice, D.P., et al., 2000. Integration of FGF and TWIST in calvarial bone and
suture development. Development 127, 1845–1855.
Rice,D.P., et al., 2003a.Fgfr mRNAisoforms incraniofacialbone development.
Bone 33, 14–27.
Rice, D.P., et al., 2003b. Molecular mechanisms in calvarial bone and suture
development, and their relation to craniosynostosis. Eur. J. Orthod. 25,
Rice, R., et al., 2003c. Progression of calvarial bone development requires
Foxc1 regulation of Msx2 and Alx4. Dev. Biol. 262, 75–87.
Rice, R., et al., 2005. Foxc1 integrates Fgf and Bmp signalling independently of
twist or noggin during calvarial bone development. Dev. Dyn. 233,
Satokata, I., et al., 2000. Msx2 deficiency in mice causes pleiotropic defects in
bone growth and ectodermal organ formation. Nat. Genet. 24, 391–395.
Vainio, S., et al., 1993. Identification of BMP-4 as a signal mediating secondary
induction between epithelial and mesenchymal tissues during early tooth
development. Cell 75, 45–58.
Wang, T.H., et al., 1998. Microtubule-interfering agents activate c-Jun N-
terminal kinase/stress-activated protein kinase through both Ras and
apoptosis signal-regulating kinase pathways. J. Biol. Chem. 273,
Wang, Y., et al., 2005. Abnormalities in cartilage and bone development in the
Apert syndrome FGFR2(+/S252W) mouse. Development 132, 3537–3548.
Wilkie, A.O., et al., 2000. Functional haploinsufficiency of the human
homeobox gene MSX2 causes defects in skull ossification. Nat. Genet.
Yunker, L.A., et al., 2004. The tyrosine phosphatase, OST-PTP, is expressed
in mesenchymal progenitor cells early during skeletogenesis in the mouse.
J. Cell. Biochem. 93, 761–773.
346 E. Lana-Elola et al. / Developmental Biology 311 (2007) 335–346