© 2024. Published by The Company of Biologists Ltd | Development (2024) 151, dev202116. doi:10.1242/dev.
202116
STEM CELLS AND REGENERATION
Cranial suture lineage and contributions to repair of the
mouse skull
Daniel Doro1,2, *, Annie Liu1, Jia Shang Lau1, Arun Kumar Rajendran2, Christopher Healy1, Marko Krstic1,
Agamemnon E. Grigoriadis1, Sachiko Iseki2, * and Karen J. Liu1,*
ABSTRACT
The cranial sutures are proposed to be a stem cell niche, harbouring
skeletal stem cells that are directly involved in development,
homeostasis and healing. Like the craniofacial bones, the sutures
are formed from both mesoderm and neural crest. During cranial bone
repair, neural crest cells have been proposed to be key players;
however, neural crest contributions to adult sutures are not well
defined, and the relative importance of suture proximity is unclear.
Here, we use genetic approaches to re-examine the neural crest–
mesoderm boundaries in the adult mouse skull. These are combined
with calvarial wounding experiments suggesting that suture proximity
improves the efficiency of cranial repair. Furthermore, we
demonstrate that Gli1 + and Axin2 + skeletal stem cells are present
in all calvarial sutures examined. We propose that the position of the
defect determines the availability of neural crest-derived progenitors,
which appear to be a key element in the repair of calvarial defects.
KEY WORDS: Sutures, Stem cells, Cranial repair, Neural crest,
Mesoderm, Bone
INTRODUCTION
Craniofacial bone repair, which can be necessary as a result of
accidents, congenital anomalies, or diseases such as cancer, is a
daunting clinical challenge and a significant biomedical burden.
Current treatment strategies include replacements; however, these do
not truly mimic bone and can lead to difficulties in integration with
other tissues, such as muscle. Poor healing can have severe impact on
function and aesthetics, and multi-fragment breaks can be difficult to
reconstruct (Lanza et al., 2020). Therefore, there is a need to improve
our understanding of the endogenous craniofacial repair process,
including a clearer definition of the osteogenic stem cell niche.
In craniofacial structures, the calvarial and facial bones form via
intramembranous ossification, in which osteoblasts coalesce and
differentiate directly within a membrane, without a cartilaginous
1
Centre for Craniofacial and Regenerative Biology, Faculty of Dentistry, Oral &
Craniofacial Sciences, King’s College London, London SE1 9RT, UK. 2Department
of Molecular Craniofacial Embryology and Oral Histology, Tokyo Medical and
Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
*Authors for correspondence (daniel.doro_pereira@kcl.ac.uk;
sach-dev@tmd.ac.jp; karen.liu@kcl.ac.uk)
D.D., 0000-0001-5058-273X; A.L., 0009-0002-8362-6983; J.S.L., 0009-0003-
1889-5439; A.K.R., 0000-0001-6443-6063; C.H., 0009-0006-9720-5185; M.K.,
0009-0008-7806-0210; A.E.G., 0000-0002-5941-8132; S.I., 0000-0001-8448-9410;
K.J.L., 0000-0002-2483-2165
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Handling Editor: Patrick Tam
Received 27 June 2023; Accepted 8 January 2024
RESEARCH REPORT
intermediate (Nakashima and de Crombrugghe, 2003). Growth of
these skull bones is organised at fibrous joints called sutures and
occurs at the leading edges of the separate bones (Doro et al., 2017).
Throughout embryonic and postnatal development, the sutures
remain as active sites of bone formation. By early adulthood, when
growth of the skull is complete, sutures become quiescent and
gradually fuse. Nevertheless, cells residing in the sutures or adjacent
periosteum retain the potential to heal calvarial bone in adults,
following injury or disease (Doro et al., 2017). The healing capacity
in the skull appears to rely on the sutural mesenchyme, which has
recently been proposed to act as a stem cell niche (Zhao et al., 2015).
In response to wounding, the skeletal mesenchyme rapidly
undergoes proliferation, with sutural cells expanding toward the
wound. Several groups have demonstrated that ablation of these
resident skeletal stem cells blocks the healing capacity of the skull
(Zhao et al., 2015; Maruyama et al., 2016; Wilk et al., 2017).
Here, we make use of a crucial and underappreciated observation:
that adult frontal bones in the mouse heal more efficiently than
parietal bones (Quarto et al., 2010; Doro et al., 2019). Studies from
mouse models have demonstrated that the embryonic frontal bones
are of neural crest origin, whereas the parietal bones are mesodermally
derived (Jiang et al., 2002), raising the possibility that developmental
history influences osteogenic potential. Indeed, we have demonstrated
that adult neural crest-derived osteoblasts have an increased
osteogenic capacity when cultured in vitro (Doro et al., 2019). In
contrast, parietal osteoblasts rarely generate osteogenic nodules (Doro
et al., 2019); however, osteogenesis could be restored by co-culturing
with neural crest-derived cells, either from frontal bones, or from the
dura mater (Doro et al., 2019). Anecdotal evidence suggests a
similarly improved healing capacity in human frontal bones (Skogh
et al., 2013). These observations raise the possibility that there are
increased numbers (or activity) of calvarial stem cells in sutures
adjacent to the frontal bones. Most of the original studies comparing
frontal and parietal healing efficiency do not account for the role of
suture-derived osteoprogenitors, or the relevance of defect position in
relation to the sutures. More recently, it has been demonstrated that
bone healing is not an evenly distributed event across the parietal bone
surface (Park et al., 2016). Although these studies show a correlation
between repair efficiency and suture proximity, they disregard the
dual embryonic origin of the cranial sutures and the possibility that
distinct lineage contributions may influence repair. Furthermore,
DEVELOPMENT
several studies suggest that neural crest-derived cells, and not the
mesoderm, play key roles in the pathogenesis of craniofacial
malformations (Hari et al., 2002; Brault et al., 2001; Wu et al.,
2017), but also that neural crest may account for differences in cranial
healing efficiency upon injuries of every sort (Quarto et al., 2010;
Li et al., 2010; Senarath-Yapa et al., 2013).
Based on historical lineage-tracing assays using Wnt1::cre-driven
ROSA26-lacZ reporters (Jiang et al., 2002; Wu et al., 2017;
Yoshida et al., 2008), both interfrontal and sagittal sutures have
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STEM CELLS AND REGENERATION
been shown to be neural crest-derived, whereas the coronal suture is
mesodermal. These studies focused on late embryonic/early post-
natal stages, when cranial sutures are not yet clearly defined. To
date, no definitive description of the neural crest–mesoderm
boundary in the murine cranial vault has been established. Our
overall hypothesis is that, in addition to suture proximity, the
embryonic origins of the cranial sutures are also relevant when
assessing repair. We hypothesise that calvarial sutures, notably
those with majority neural crest contributions, may harbour more
substantial skeletal stem cell populations. Thus, for non-neural-
crest-derived bone, such as the parietal bones, proximity to specific
sutures is crucial for homeostasis as well as cranial repair.
In this study, we use genetic labelling to compare Wnt1::cre+ and
Mesp1::cre+ lineages, which define the proposed neural crest-
mesoderm domains of the calvarium. We confirm that murine
frontal bones heal more efficiently than parietal bones and note that
the position of the defect in relation to the sutures is paramount for
the outcome. We then demonstrate that Wnt1::cre+ cells are found
in every subcritical defect regardless of the embryonic origin of the
wounded bone, or the proximity to a neural crest suture. We find the
presence of Axin2+ and Gli1+ stem cell populations therein, noting
the contributions of Gli1-CreERT2-positive cells in the healing
wounds. This suggests that localised sources of neural crest cells and
skeletal stem cells play a crucial role in healing of mesodermal skull
bones.
RESULTS AND DISCUSSION
Neural crest-mesoderm boundary separates cranial sutures
into distinct domains
In mouse, Wnt1::cre-dependent lineage labelling has demonstrated
the neural crest-origin of the frontal bones whereas Mesp1::cre-
labelled mesoderm contributes to the parietal bones (Jiang et al.,
2002; Wu et al., 2017; Yoshida et al., 2008). However, the
embryonic origins of the cranial sutures are debatable, as they rely
on early observations that lacked resolution. Nevertheless, we and
others have repeatedly confirmed a higher osteogenic potential of
neural crest osteoblasts compared with mesoderm osteoblasts
(Quarto et al., 2010; Doro et al., 2019; Behr et al., 2010a; Li
et al., 2013). We sought to determine the neural crest-mesoderm
domain and the presence of stem cell lineages in calvarial sutures
using high-resolution lineage tracing.
Transgenic Wnt1::cre mice were crossed with mice carrying a
Cre-responsive Rosa26RmTmG reporter. In these mice, Wnt1::cre
expression begins approximately at embryonic day (E) 8.5 in the
dorsal neural tube, just as cranial neural crest cells are induced
(Danielian et al., 1998). Soon after, these cells migrate into the
cranial vault and can be tracked with membrane green fluorescent
protein (mGFP). Cells lacking Wnt1::cre are labelled with
membrane tomato (mTom) and are presumptive mesoderm. As
expected, at postnatal day (P) 40, frontal bone shows exclusive
neural crest contribution (mGFP+) as opposed to the parietal bones,
which are of mesodermal origin, lacking mGFP (Fig. 1A). When we
examined the midline sutures, we observed a clearly defined neural
crest–mesoderm boundary, which lies right at the middle of the
sagittal suture, delimiting a neural crest domain (interfrontal,
coronal and anterior-sagittal suture) and a mesoderm domain
( posterior-sagittal and lambdoid suture) (Fig. 1A,B). A coronal
section at a more anterior part of the sagittal suture shows complete
absence of mesoderm (Fig. 1B,C-E), contrasting a more posterior
section, which shows very few neural crest cells with abundant
mesoderm (Fig. 1F-K). Likewise, the interfrontal suture exhibited
only neural crest (Fig. 1C-E), whereas the lambdoid suture showed
Development (2024) 151, dev202116. doi:10.1242/dev.202116
only mesoderm (Fig. 1B,L-N). Altogether, this shows that the
sagittal suture has dual embryonic origin with a well-defined neural
crest–mesoderm boundary that extends past the anterior edge of the
parietal bones, recapitulating the pattern observed in early
development. This separates the sutures into two domains: the
neural crest-derived sutures (interfrontal, coronal, squamous and
anterior sagittal) and the mesodermal sutures (lambdoid and
posterior sagittal).
We then performed the parallel experiment with Mesp1::cre
males bred with females carrying a Cre-responsive Rosa26RmTmG
reporter (Saga et al., 1999; Muzumdar et al., 2007). The Mesp1::cre
transgenic animals carry Cre recombinase under the control of the
endogenous Mesp1 promoter, which activates initially a
gastrulation, and later cranial mesenchyme (Yoshida et al., 2008).
Yoshida and colleagues used Mesp1::cre animals to demonstrate
the mesodermal contributions to the calvarial skeleton. We were
able to confirm the putative neural crest–mesoderm boundary in
mouse skulls at P40 (Fig. 1). Mesp1::cre; Rosa26RmTmG mice
clearly showed boundaries at the midpoint of the sagittal suture
(Fig. 1B′,F′-K′). We observed no positive labelling within the most
rostral domains (Fig. 1C′-E′) adjacent to the interfrontal suture,
except the minimal marrow cavity. The posterior sections at the
lamboid suture (Fig. 1L′-N′) were entirely positive for Mesp1::cre.
Of note, using this approach we did see both Mesp1::cre-positive
and -negative cells in the rostral part of the sagittal suture (‘SAG1’;
Fig. 1F′-H′), consistent with this domain having dual contributions
from neural crest and mesoderm.
Suture proximity determines the outcome of a subcritical
cranial defect
Although regional differences in repair efficiency have previously
been reported, the dura mater and periosteum were thought to be the
main sources contributing osteoprogenitors for cranial repair
(Ochareon and Herring, 2011; Greenwald et al., 2000). Here, we
set out to determine the role of proximity to the sutural niche.
Subcritical defects (termed midfrontal and midparietal) of 1 mm
width were made in the frontal bone of adult P40 mice, equidistant
to the interfrontal and coronal sutures, as well as in the parietal bone,
equidistant to the lambdoid, squamous, sagittal and coronal sutures
(Fig. 2A, unfilled, dotted outlines; 2B). After 4 weeks, we observed
a striking difference between midfrontal and midparietal repair
(Fig. 2D), which accords with previous observations (Quarto et al.,
2010; Li et al., 2010; Senarath-Yapa et al., 2013; Behr et al., 2010b).
When the defects were made proximally to the sutures ( peri-coronal
frontal, peri-coronal parietal and peri-lambdoid) (Fig. 2A, filled,
dotted outlines; Fig. 2C), no significant difference in repair was seen
between frontal and parietal bones (Fig. 2D), whereas the peri-
coronal parietal defect healing was comparable to that of the frontal
bones (Fig. 2D). Our findings corroborate recent studies that show
that subcritical parietal defects are more likely to heal the closer they
are to the cranial sutures (Park et al., 2016). However, we did not see
any specific increase in repair in the vicinity of a specific suture in
relation to the other. This shows that, even though the sutures may
DEVELOPMENT
contribute differently to cranial repair, the proximity to any suture is
enough to provide subcritical healing, whereas the more distant
parietal defect (here termed midparietal) seems to exceed the
maximum critical distance from a suture.
Neural crest lineage contributes progenitors to any
cranial defect
We then assessed the extent to which Wnt1::cre-expressing neural
crest cells infiltrate the repair site after injury. Subcritical defects
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STEM CELLS AND REGENERATION Development (2024) 151, dev202116. doi:10.1242/dev.202116

Fig. 1. Embryonic origins of the cranial sutures. (A) Top view of P40 Wnt1Cre; Rosa26R mTmGmouse. Grey, brightfield; green, neural crest; red, non-
neural-crest tissue. Dashed box shows area of the midline explant in B. Scale bar: 2 mm. Schematic depicts the neural crest-mesoderm domain in the cranial
vault based on the linage tracing shown in A. (B) Confocal scan of the midline explant confirming the neural crest–mesoderm boundary at the middle of the
sagittal suture. Scale bar: 1 mm. (C-N) Confocal scans of coronal sections at different regions of the midline showing non-neural-crest tissue (C,F,I,L; red),
neural-crest tissues (D,G,J,M; green) and merged channels (E,H,K,N). Nuclear staining is shown in blue. Dashed lines in B show estimated planes of section
in C-N. IF, interfrontal suture (C-E), SAG1, sagittal suture region 1 (F-H), SAG2, sagittal suture region 2 (I-K), LAMB, lambdoid suture (L-N). Scale bar:
DEVELOPMENT

50 µm. n=3. (A′) Top view of P40 Mesp1::cre; Rosa26R mTmGmouse. Green, mesoderm; red, non-mesodermal tissue. Dashed box shows area of the midline
explant in B′. Scale bar: 2 mm. Schematic depicts the neural crest-mesoderm domain in the cranial vault based on the linage tracing shown in A′.
(B′) Confocal scan of the midline explant confirming the neural crest–mesoderm boundary at the middle of the sagittal suture. Scale bar: 1 mm. (C′-N′)
Confocal scans of coronal sections at different regions of the midline showing non-mesodermal tissue (C′,F′,I′,L′; red), mesodermal tissues (D′,G′,J′,M′;
green) and merged channels (E′,H′,K′,N′). Nuclear staining is shown in blue. Dashed lines in B′ show estimated planes of section in C′-N′ (abbreviations as
in B-N). Scale bar: 50 µm. n=3. Dashed lines in C-N,C′-N′ outline bone.

were made at six distinct locations in Wnt1::cre; Rosa26R +/mTmG interfrontal and coronal sutures, to lambdoid-parietal (O), which
mice (Fig. 2E). Defects varied from midfrontal (J) and coronal- is only surrounded by mesoderm-derived sutures. One week after
frontal (K), which are surrounded by neural crest-derived craniotomy, Wnt1::cre+ cells were detected around and/or within all

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STEM CELLS AND REGENERATION
Fig. 2. See next page for legend.
defects (Fig. 2F-I). This was clearly observed in coronal sections
(Fig. 2J-O′). Even the defects in bones that are mesodermal in origin
were filled with a large proportion of mGFP+ cells, suggesting the
infiltration of neural crest-derived progenitors (Fig. 2M-O′).
Importantly, within the frontal bone outlines, every cell was
Development (2024) 151, dev202116. doi:10.1242/dev.202116
DEVELOPMENT
expected to be GFP+ (white arrows) as these bones are of neural
crest origin (Fig. 2J′,L′). In contrast, within parietal bone outlines
we observed a mix of GFP− (yellow arrows) and GFP+ (white
arrows) cells (Fig. 2L′,M′,O′), suggesting that new bone was
formed from neural crest-derived progenitors, given that parietal
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STEM CELLS AND REGENERATION
Fig. 2. Subcritical repair efficiency correlates with suture proximity
irrespective of the calvarial bone, and neural crest cells are recruited to
every defect. (A) Schematic showing the position of cranial defects in
relation to adjacent sutures. Green outline, midfrontal; solid green, peri-
coronal frontal; solid red, peri-coronal parietal; red outline, midparietal; solid
pink, peri-lambdoid. (B,C) Top view microCT scan of CD1 mouse heads
4 weeks after 1 mm subcritical defect surgery. (B) Defects equidistant from
surrounding sutures in right frontal and left parietal bones. (C) Defects
proximal to coronal and interfrontal suture (right), coronal and sagittal suture
(top left), and sagittal and lambdoid suture (bottom left). (D) Bone volume
fraction (BVF) analysis over a 4-week period post-subcritical defect surgery.
Lower and upper box limits indicate the lower and upper quartile of BVF
values, respectively. Horizontal line indicates the median. Upper and lower
whiskers indicate minimum and maximum BVF values. n=6. *P<0.05,
**P<0.001 (two-tailed, unpaired t-test). (E) Schematic showing the position
of six subcritical defects (1 mm). Sutures are indicated in the picture as
follows: COR, coronal suture; IF, interfrontal suture; SQ, squamous suture;
SAG, sagittal suture; LAMB, lambdoid suture. (F-I) Top view of 40-day-old
Wnt1-Cre; Rosa26RGFP mice 1 week after the surgical procedure. Green
staining indicates neural crest-derived tissue. (J-O′) Coronal sections at the
centre of each defect described in E. Neural crest-derived cells are shown in
green, nuclear staining in blue. Dashed lines outline the bones, and the
boxed area is shown at high magnification in J′-O′. Yellow arrowheads
indicate non-neural crest-derived cells, white arrowheads neural crest-
derived cells. n=4.
bones are expected to be mesodermal. One exception is the
midparietal defect (Fig. 2N′), which maintained its original flat
edges, as obtained by the cylindrical nature of the drill bit, indicating
minimal repair. This wound had an absence of green cells within the
bone outline. This is consistent with previous observations of poor
healing capacity of the mid-parietal bone region.
Although the osteogenic potential of neural crest versus
mesoderm sutures has not yet been defined, it is intriguing that
Wnt1::cre + neural crest cells seem to contribute to the repair of
every defect, including those in a mesoderm-dominant area
(Fig. 2). Although we propose that the suture proximity is a key
factor in repair capacity, it is also worth noting that the underlying
dura mater is also neural crest derived, and a recent study has
shown contributions of dura mater cells to suture regeneration
(Yu et al., 2021). Regardless, neural crest-derived cells appear to
be required for efficient repair, supporting previous observations
that neural crest osteoprogenitors have higher osteogenic
capabilities than other mesodermally derived calvarial cell
populations.
Stem cell populations are present in every calvarial suture
and contribute to calvarial repair
Several putative calvarial stem cell markers have been identified:
the Hedgehog-pathway transcription factor Gli1 (Zhao et al., 2015),
the Wnt-responsive gene Axin2 (Maruyama et al., 2016) and
the transcription factor Prx1 (Prrx1) (Wilk et al., 2017). Here, we
investigate the presence of Gli1+ and Axin2+ stem cell populations
using Gli1::creERT2 or Axin2::creER drivers in combination with
Rosa26 Tomato and Rosa26R mTmG reporters, respectively. To label
cells derived from Gli1 + and Axin2 + populations, 38-day-old mice
were given tamoxifen and the heads were collected 2 days after
injection. Top view and coronal sections of Gli1-CreERT2;
Rosa26 Tomato/+ heads revealed the abundant presence of Gli1+
cells in all calvarial sutures (Fig. 3A-F). The analogous experiment
with Axin2-CreERT2; Rosa26R+/mTmG animals revealed the
presence of Axin2+-derived cells also in every suture; however,
they were sparse in comparison with Gli1+ (compare Fig. 3G-L with
3A-F). Overall, we observed no apparent difference in Gli1+ and
Development (2024) 151, dev202116. doi:10.1242/dev.202116
Axin2+ cell content in the cranial sutures examined. These findings
are consistent with prior proposals that the Axin2+ cells could be a
subset of Gli1+ progenitors (Maruyama et al., 2016; Wilk
et al., 2017). Further assessment of Axin2 mRNA expression on
the Gli1+-derived cells would be necessary to define definitively
these subpopulations of skeletal stem cells.
To investigate whether Gli1+ osteoprogenitors could populate all
healing defects, we labelled Gli1::creERT2; Rosa26 Tomato mice as
described. Subcritical defects were made at five sites of Gli1-
CreERT2; Rosa26 Tomato/+ mice as indicated (Fig. 4A) and Gli1+
cells were examined in healing wounds 1-week post-surgery.
Coronal sections of the wounds shown in Fig. 4B,C reveal that
Gli1+ cells (in red) were present in all wounds (Fig. 4D-H). Cells
were present regardless of whether wounds were made in neural
crest-derived frontal bone (Fig. 4D,E) or parietal (Fig. 4G,H). In the
parietal bones, Gli1+ cells were also seen regardless of proximity to
the sagittal suture (Fig. 4G, compared with Fig. 4F,H). This suggests
that, although midparietal defects (Fig. 4G) were substantially more
distant to the sutures than the other defects were, they were still
supplied with suture-derived osteoprogenitors, revealing the
reparative capability of the suture mesenchyme in relation to
distant defects. In the future, a quantitative assessment of the
signalling events and cell content will be important reveal the
mechanistic links between distance, cell identity and healing.
Conclusion
Our data suggest that the embryonic origins of the cranial bone may
predict the outcome of defect repair based on the neural crest
composition of the intervening sutures and the presence of suture-
residing osteoprogenitors relative to the calvarial wound site.
However, it is important to note that our observations are based on
several crucial assumptions. First, the genetic approaches used in
our studies, although powerful, cannot exclude the possibility that
these transgenic lines are not reactivated later in development, or
subject to unknown transcriptional influences, or are simply not Cre
responsive. Second, it is important to note that meninges, notably
the dura mater, are also neural crest derived (Yoshida et al., 2008;
Gagan et al., 2007; Dasgupta and Jeong, 2019). Indeed, we and
others have demonstrated that the dura mater has osteogenic
capacity (Petrie et al., 2008; Peptan et al., 2007), and when
co-cultured with parietal cells are capable of nucleating
osteogenesis (Doro et al., 2019). Finally, the composition of the
sutural mesenchyme is surely dynamic, as the biological
requirements change from embryogenesis to adult aging. Cell
migration and mixing in adult life has been reported to occur in the
sutures and in the meninges (Gagan et al., 2007; Deckelbaum et al.,
2012). Furthermore, the meninges, which are comprised of neural
crest cells during development, seem later to be invaded by
mesodermal derivatives, leaving the remaining neural crest cells to
act as resident stem cells in later life.
In the future, it will be important to track more localised cell
contributions, such as dura mater-derived cells or the adjacent
periosteal cells. It will also be important to assess the environment
DEVELOPMENT
unique to the frontal versus parietal bones: for example, Marghoub
and colleagues have demonstrated distinct mechanical forces in
the anterior versus the posterior sutures, owing to differences in
bone size, anatomy and underlying brain morphology (Marghoub
et al., 2018). Altogether, although we cannot definitively state
that availability of neural crest cells is the key element, we
can nevertheless conclude that lineage identity and spatial
positioning are both important in relation to the healing of
calvarial defects.
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STEM CELLS AND REGENERATION
MATERIALS AND METHODS
Animal procedures
All procedures were approved by King’s College London ethical review
process and performed in accordance with UK Home Office guidelines
Project Licence P8D5E2773 (K.J.L.) or by the Institutional Animal Care
Development (2024) 151, dev202116. doi:10.1242/dev.202116
Fig. 3. Gli1+-derived and Axin2+-derived
progenitors are found in all cranial sutures.
(A) Top view of 40-day-old Gli1CreERT/+;
Rosa26R TdTomato mouse 48 h after tamoxifen
induction. Grey, brightfield; red, Gli1+ domain. Red
staining on the right side is autofluorescence of
opaque tissue remaining in the sample. COR,
coronal suture; IF, interfrontal suture; SQ,
squamous suture; SAG, sagittal suture; LAMB,
lambdoid suture. Scale bar: 2 mm. Schematics
show the Gli1+ domain in the transgenic mouse
calvarium. (B-F) Coronal sections of the calvarial
sutures at the regions marked in A. Dashed lines
show the bone outline. White arrows show red
Gli1+ cells. Scale bar: 250 µm. n=3. (G) Top view
of 40-day-old Axin2CreERT/+;
Rosa26RmTmGmouse 48 h after tamoxifen
induction (red channel not shown). Grey,
brightfield; green, Axin2+ domain. Abbreviations as
in A-F. Scale bar: 2 mm. Schematics show the
Axin2+ domain in the transgenic mouse calvarium.
(H-L) Coronal sections of the calvarial sutures at
the regions marked in G. Dashed lines show the
bone outline. Axin2+ cells are in green. White
arrows show green Axin2+ cells. bone marrow
(bm), muscle (m), dura mater (dm) and periosteum
( p) are notoriously autofluorescent tissues.
Scale bar: 250 µm.
DEVELOPMENT
and Use Committee of Tokyo Medical and Dental University [A2019-
060C3 (S.I.)].
Mouse lines used were CD-1 mice (obtained from Charles River
Laboratories), and Rosa26RmTmG (MGI ID 3716464), Rosa26RTdTomato
(MGI ID 3809523), Rosa26R-eGFP (MGI ID 2136519) mouse
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STEM CELLS AND REGENERATION Development (2024) 151, dev202116. doi:10.1242/dev.202116

Fig. 4. Gli1+-derived cells are found in every subcritical defect after 1 week. (A) Schematics show sites of 1 mm subcritical defects in P40
Gli1CreERT/+; Rosa26R TdTomatomice. Circular outlines mark the positions of the wounds in relation to the different bones and sutures. (B,C) Top-view CT
scans of heads after 1 week from the surgical procedure. Red dashed lines correspond to the approximate plane of sections in D-H. (D-H) Confocal scans of
coronal sections at different wound sites 1-week post-surgery. Red, Gli1+ cells. Dashed lines outline bone. if, interfrontal suture; c, coronal suture; s, sagittal
suture; l, lambdoid suture. Scale bar: 250 µm. n=3.
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reporter lines (all described previously: Muzumdar et al., 2007;
Madisen et al., 2010; Mao et al., 2001). The following Cre drivers
were used: Wnt1::cre (MGI ID 2386570), Axin2::CreERT2 (MGI ID
5433373), Gli1::CreERT2 (MGI ID 3053957), Mesp1::cre (MGI ID
2176467) (Danielian et al., 1998; Saga et al., 1999; van Amerongen
et al., 2012; Ahn and Joyner, 2004).
Subcritical defects
P40 mice were weighed and anaesthetised with an appropriate dose (10 μl/g of
body weight) of a 10 mg/ml ketamine/2 mg/ml xylazine cocktail (Vetalar®,
Zoetis; Rompun®, Dechra). The state of deep anaesthesia was confirmed
through tail flick test and hind paw withdrawal response. Once the animals
were heavily sedated, the fur on the top of the head was shaved using a hair

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STEM CELLS AND REGENERATION
trimmer. A sagittal incision was performed along the midline with a scalpel.
The periosteal layer was then removed with the help of a cotton bud and 1-
mm-width defects were drilled at top speed (50,000 rpm) into frontal and
parietal bones of the mouse skull using a dental hand drill (Handy-ECO 1000,
Marathon®). The defects were carefully performed to avoid injuring the dura
mater. The bone surface was then rinsed with sterile PBS to remove any debris
and the sagittal incision was sutured with 6-0 ETHILON® nylon absorbable
suture. Finally, the mice were moved to a 28°C incubator until full recovery.
For the CD1 mice shown in Fig. 2, six mice received two wounds distant from
the sutures and six mice received three wounds proximal to the sutures. Wnt1-
Cre; Rosa 26RGFP mice (n=4) all received six wounds each, according to the
schematics. The Gli1CreERT; Rosa26RtdTomato shown in Fig. 4 (n=3) received
separate wounds similarly to CD1 in Fig. 2, i.e. three animals with two
wounds and the other three with three wounds.
Tamoxifen injection
Cre induction in CreERT mice was performed by peritoneal injection of a
10 mg/ml tamoxifen solution (Sigma-Aldrich) in the adult mouse at the
desired stage. The solution was previously prepared by dissolving 10 mg of
tamoxifen into 100 μl of absolute ethanol and 900 μl of corn oil. The dosage
was determined according to the following: 1.5 mM/g of body weight in a
volume of 7.5 μl/g of body weight.
Micro-CT scanning
All head samples were fixed for 48 h at room temperature in 4%
paraformaldehyde and scanned using a Scanco Medical µCT50® with the
following settings: energy 70 kV, intensity 114 µA, resolution 10 µm/voxel.
The images were then reconstructed on Parallax Microview® with isosurface
image threshold set to 6000 and surface quality factor set to 40% with
decimation factor of 0%.
To determine the bone volume fraction of each subcritical defect, a
cylindrical region of interest calibrated to the volume of a 1 mm defect was
used as total volume (mm3) and bone volume (mm3) was then measured
using the Bone Analysis Tool on MicroView®.
Sample fixation and sectioning
Samples were fixed in 4% paraformaldehyde for 48 h at 4°C. After three
PBS washes, the skull cap was dissected and decalcified in 10% formic
acid. After three more PBS washes, the samples were moved to a 30%
sucrose solution in PBS until they sunk. The embedding solution was then
replaced with a 30% sucrose solution mixed (1:1) with OCT compound
(CellPath®). The samples were incubated at 4°C for another 48 h. Finally,
the samples were moved and oriented in a plastic Tissue-Tek® Cryomold®
filled with OCT compound. The mould was quickly moved into a dry-ice bath
with absolute ethanol until the OCT block was fully solidified. Cryosections
of 15 μm thickness were taken using OFT5000® cryostat microtome (Bright
Instruments®) and mounted onto Superfrost Ultra Plus® slides (Thermo Fisher
Scientific®), which were then stored at −80°C for future use.
Immunostaining
GFP and RFP (Tomato) fluorescence in all experiments was assessed
by immunofluorescence staining. Primary antibodies were: anti-GFP
(ab13970, Abcam®), anti-RFP (5F*, ChromoTek®). Secondary antibodies
were: Alexa Fluor™ 488 goat anti-chicken (A-11039, Invitrogen®), Alexa
Fluor™ 568 goat anti-rat (A-11077, Invitrogen®). After dissolution of the
OCT in PBS, slides were blocked with 1% bovine serum albumin (Sigma-
Aldrich®), 10% goat serum (Gibco®), 0.05% Tween 20 (Sigma-Aldrich®)
in PBS. Primary antibodies were diluted to 1:100 in blocking buffer and
incubated overnight at 4°C. Secondary antibodies were diluted to 1:500 in
the same buffer and incubated for 1 h at room temperature. After both
primary and secondary antibody incubation steps, slides were washed three
times for 10 min each wash in PBS with 0.05% Tween 20 (Sigma-Aldrich).
After the immunostaining procedure, sections were mounted with
Fluoroshield™ mounting medium with DAPI (F6057, Sigma-Aldrich®).
Microscopy
A stereoscope (Nikon SMZ1500) with an attached camera (Nikon digital
sight DS-Fi1) was used to take top view fluorescent images of whole heads.
Development (2024) 151, dev202116. doi:10.1242/dev.202116
Confocal microscopy was performed on a Leica Microsystems CMS TCS
SP5 DM16000. Image sequences were reconstructed using Fiji (ImageJ)
analysis software.
Acknowledgements
We thank members of the Liu and Iseki labs and colleagues at the Centre for
Craniofacial and Regenerative Biology for support with experiments and scientific
discussions. We thank William Barrell and Jade Desjardins support with mouse
work. We also thank the Biological Services Unit at Guy’s Hospital for all the support
with the mouse work.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: D.D., A.E.G., S.I., K.J.L.; Methodology: D.D., A.L., J.S.L., C.H.,
M.K.; Validation: D.D.; Formal analysis: D.D., K.J.L.; Investigation: D.D., A.L., J.S.L.,
A.K.R., C.H., M.K.; Resources: S.I., K.J.L.; Data curation: D.D., K.J.L.; Writing -
original draft: D.D., A.E.G., K.J.L.; Writing - review & editing: D.D., A.L., J.S.L.,
A.K.R., C.H., M.K., A.E.G., S.I., K.J.L.; Visualization: D.D.; Supervision: A.E.G., S.I.,
K.J.L.; Project administration: D.D., S.I., K.J.L.; Funding acquisition: S.I., K.J.L.
Funding
This work was supported by King’s College London Dental Institute Seed Funding
(K.J.L.), the Biotechnology and Biological Sciences Research Council (BB/I021922/
1 and BB/R015953/1 to K.J.L.), the Medical Research Council (PC21044 to K.J.L.),
funding from Brazil CAPES (Coordenaçao ̃ de Aperfeiçoamento de Pessoal de Nı́vel
Superior) (D.D.), a Japan Society for the Promotion of Science Short-term
Postdoctoral Fellowship (D.D.) and grants-in-aid from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (MEXT) 21H03098 (S.I.). Open
Access funding provided by Biotechnology and Biological Sciences Research
Council and the Medical Research Council. Deposited in PMC for immediate
release.
Data availability
All relevant data can be found within the article.
The people behind the papers
This article has an associated ‘The people behind the papers’ interview with some of
the authors.
Peer review history
The peer review history is available online at https://journals.biologists.com/dev/
lookup/doi/10.1242/dev.202116.reviewer-comments.pdf
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