mandibular dysmorphology due to abnormal embryonic ... · department of genetics and genomic...
TRANSCRIPT
© 2019. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
Mandibular dysmorphology due to abnormal embryonic osteogenesis in FGFR2-related
craniosynostosis mice
Susan M. Motch Perrine1,*, Meng Wu2,*,‡, Nicholas B. Stephens1, Divya Kriti2, Harm van Bakel2,
Ethylin Wang Jabs2, Joan T. Richtsmeier1
1Department of Anthropology, Pennsylvania State University, University Park, PA, USA
2Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New
York, NY, USA
*These authors contributed equally to the manuscript.
‡Author for correspondence ([email protected])
ORCID ID: Meng Wu, 0000-0001-5512-9651
KEY WORDS: Apert syndrome, Crouzon syndrome, Pfeiffer syndrome, Osteoclast, Cartilage,
Transcriptome
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
http://dmm.biologists.org/lookup/doi/10.1242/dmm.038513Access the most recent version at First posted online on 7 May 2019 as 10.1242/dmm.038513
SUMMARY STATEMENT
Mandibular dysmorphology was observed in FGFR2-related craniosynostosis mouse models.
FGFR2 gain-of-function mutations differentially affect cartilage formation and
intramembranous ossification of dermal bone, resulting in abnormal embryonic osteogenesis of
the mandible.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
ABSTRACT
One diagnostic feature of craniosynostosis syndromes is mandibular dysgenesis. Using three
mouse models of Apert, Crouzon, and Pfeiffer craniosynostosis syndromes, we investigated
how embryonic development of the mandible is affected by fibroblast growth factor receptor 2
(FGFR2) mutations. Quantitative analysis of skeletal form at birth revealed differences in
mandibular morphology between mice carrying Fgfr2 mutations and their littermates that do
not carry the mutations. Murine embryos with the mutations associated with Apert syndrome
in humans showed an increase in the size of the osteogenic anlagen and Meckel’s cartilage
(MC). Changes in the microarchitecture and mineralization of the developing mandible were
visualized using histological staining. The mechanism for mandibular dysgenesis in the Apert
Fgfr2+/S252W mouse resulting in the most severe phenotypic effects was further analyzed in
detail and found to occur to a lesser degree in the other craniosynostosis mouse models. Laser
capture microdissection and RNA-Seq analysis revealed transcriptome changes in mandibular
bone at E16.5, highlighting increased expression of genes related to osteoclast differentiation
and dysregulated genes active in bone mineralization. Increased osteoclastic activity was
corroborated by TRAP assay and in situ hybridization of Csf1r and Itgb3. Upregulated expression
of Enpp1 and Ank was validated in the mandible of Fgfr2+/S252W embryos resulting in elevated
inorganic pyrophosphate concentration. Increased proliferation of osteoblasts in the mandible
and chondrocytes forming MC was identified in Fgfr2+/S252W embryos at E12.5. These findings
provide evidence that FGFR2 gain-of-function mutations differentially affect cartilage formation
and intramembranous ossification of dermal bone contributing to mandibular
dysmorphogenesis in craniosynostosis syndromes.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
INTRODUCTION
The mandible has been used as a model for investigating how complex morphological
structures arise during development and how they are altered during evolution (Atchley and
Hall, 1991) providing insight into how the spatial and temporal organization underlying the
development of a separate morphological component assimilates into a functioning whole.
Each hemimandible (or dentary) is composed of two functional areas that are mineralized
proximate to Meckel’s cartilage (MC): the anterior body (tooth bearing portion of the
hemimandible) and ramus (containing three prominent processes: coronoid process on the
dorsal aspect, the condylar (or condyloid) process caudally, and the angular process
caudoventrally). Mandible and maxilla are dermal bones derived from neural crest cells that
migrate to the first pharyngeal arch and are the result of complex developmental patterning
(Couly et al., 1996; Depew et al., 2002; Frisdal and Trainor, 2014; Noden, 1983). Together they
form the lower and upper jaws of the facial skeleton whose synchronous development and
proper occlusion is necessary for feeding, respiration, and craniofacial morphogenesis.
Mutations within fibroblast growth factor receptor 2 (FGFR2) are responsible for aberrant
signaling within the FGF-signaling pathway resulting in midface developmental anomalies that
are features of Apert, Crouzon, Pfeiffer, Beare-Stevenson cutis gyrata, Jackson-Weiss, and Bent
Bone Dysplasia syndromes (Azoury et al., 2017; Cohen and MacLean, 2000; Cunningham et al.,
2007). These complex conditions involve the premature fusion of one or more cranial sutures
and midfacial dysgenesis and are often associated with other skeletal and soft tissue
abnormalities (Flaherty et al., 2016; Heuzé et al., 2014). Midfacial dysgenesis can be severe but
is variable within and across FGFR2-related craniosynostosis syndromes. Surgical correction and
reconstruction are adaptable, targeting the midfacial skeleton, dental arcade, choanae, and/or
airway, often requiring significant and multiple reconstructive procedures. The mandible, the
major skeletal element of the lower face, is an important consideration in surgical planning and
orthodontic management in craniosynostosis syndromes to address severe anomalies affecting
mastication and airway anomalies.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Apert, Crouzon, and Pfeiffer syndromes (MIM #101200, MIM #123500, MIM #101600,
respectively) are autosomal dominant conditions sharing many phenotypic similarities,
including premature suture closure, abnormal facies, exophthalmos, midfacial retrusion, dental
malocclusion of varying intensities, cranial base anomalies, and dysmorphic mandibles whose
configuration is discordant with the upper jaw (Cohen and MacLean, 2000). Although the
mandible is not well-studied in these syndromes, adult mandibular morphology in Apert
patients is usually reported as intrinsically normal, and detected differences in mandibular
length are thought to be secondary to midfacial dysgenesis (Lemire, 2000; Wink et al., 2013).
The apparent mandibular prognathism is thought to be relative, a condition resulting in relation
to anomalies of the cranial base and severe retrusion of the midface (Costaras-Volarich and
Pruzansky, 1984). Why the degree and nature of developmental anomalies of the lower face
would be different from the midface when mandible and maxillae are both dermal bones of
neural crest origin derived from the first pharyngeal arch is not clear.
We have previously reported statistical differences in craniofacial bone morphology, brain
morphology, soft tissue and negative space (nasopharynx, inner ear) volumes, and
morphological integration of brain and skull in mouse models for Apert and Crouzon/Pfeiffer
syndromes relative to their respective littermates that do not carry the mutation and show no
phenotypic effects (Aldridge et al., 2010; Holmes et al., 2018; Martínez-Abadías et al., 2013;
Motch Perrine et al., 2014; Motch Perrine et al., 2017). However, investigations of the mandible
have not been included in any of these studies. To test the hypothesis that FGFR2 mutations
causative for craniosynostosis syndromes target processes and mechanisms of mandibular
genesis, we present data on the developmental and morphological consequences of three
unique FGFR2 mutations associated with syndromic craniosynostosis in the mandible of the
mouse. We performed quantitative morphometric analysis of 3D µCT image data of three
mouse models with differing activating Fgfr2 mutations to determine the differential effects of
these mutations on the mandible: two Apert syndrome mouse models, Fgfr2+/S252W (Wang et al.,
2005) and Fgfr2+/P253R(Wang et al., 2010) on a C57BL6/J background, and a mouse model with a
mutation associated with Crouzon and Pfeiffer syndromes, Fgfr2cC342Y/+ (Eswarakumar et al.,
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
2004) on a CD1 background. These analyses revealed significant differences in mandibular
morphology at P0 in all three of the mouse models. Based on these findings, we performed
histological analysis on the mandible at embryonic stages. Of these three mouse models,
mandibles of Fgfr2+/S252W mice showed the greatest magnitude of morphological change at P0
and histologic differences at embryonic day (E) 16.5 and were further analyzed by
transcriptome analysis to reveal cellular and molecular dysregulation contributing to
mandibular dysgenesis. Increased osteoclastogenesis causes abnormal bone resorption and
overexpression of Enpp1 and Ank that are key regulators for inorganic pyrophosphate levels
inhibiting bone mineralization in Fgfr2+/S252W mandible. FGFR2 S252W mutation was associated
with increased proliferation of osteoblasts and chondrocytes in the mandible as early as E12.5.
We provide new information about the molecular processes affecting the mandible in FGFR2-
related craniosynostosis syndromes to improve our understanding of craniofacial dysgenesis
and move us closer to therapeutic approaches for patients.
RESULTS
Mandibular dysmorphology of FGFR2-related craniosynostosis mouse models
The left and right hemimandibles of 182 newborn (P0) mice of each of the three
craniosynostosis models of interest were analyzed morphometrically using the 3D coordinates
of 32 landmarks (lms) (Fig. S1 and Table S1). Landmark datasets characterizing whole mandibles
(consisting of right and left sides (32 lms)), left hemimandibles (16 lms), and right
hemimandibles (16 lms) were analyzed using the same morphometric methods. Within each
model, morphometric analyses compared mice carrying a specific mutation to littermates that
did not carry the mutation. Results revealed a lack of asymmetry in mandibular dysmorphology
in all models, such that the right and left hemimandibles were similarly affected (Table S2). For
clarity of presentation, analysis of left hemimandible is presented graphically (Fig 1A-C).
Two morphometric methods were used. Euclidean Distance Matrix Analysis (EDMA) (Lele
and Richtsmeier, 2001) revealed significant differences in hemimandible shape between all
mice carrying Fgfr2 mutations and their respective littermates that did not carry the mutation
(referred to as “unaffected littermates”) for the full 32 lms set (P≤0.001) and for all three
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
regional landmark subsets representing major functional regions of the left dentary: left
hemimandible (16 lms) (P<0.001); ramus (10 lms) (P<0.001); body (8 lms) (P<0.05).
Bootstrapped confidence intervals for differences in each linear distance obtained from EDMA
reveal statistically significant differences in the localized patterns of mutational effects on the
3D morphology of the hemimandible (Fig. 1D-F). Fgfr2cC342Y/+ mice show effects that are
generally of a lesser magnitude and of a different pattern when compared to the effects of the
other two mutations (Fig. 1F). There are obvious similarities in the way hemimandibles of
Fgfr2+/S252W and Fgfr2+/P253R mutant mice differ from their respective unaffected littermates.
However, the significant phenotypic effects of the FGFR2 S252W mutation on mandibular
morphology are more numerous, of greater magnitude, and located primarily in the posterior
components of the hemimandible (Fig. 1D, E).
Principal components analysis (PCA) of the scale-free shape data shows obvious separation
of hemimandibles of mice carrying an Fgfr2 mutation from their respective unaffected
littermates for each of the three mutation groups (Fig. 1A-C). PCAs of shape were also
conducted using linear distances estimated from the landmark coordinates that define the
anterior body of the hemimandible and the ramus portion of the hemimandible (Table S1, Fig.
S1). While the anterior body of the Fgfr2cC342Y/+ Crouzon/Pfeiffer syndrome mouse mandible
showed a distinct morphology relative to their unaffected littermates, there was less difference
in the anterior body of Fgfr2+/S252W and Fgfr2+/P253R Apert syndrome mice relative to their
respective unaffected littermates (Fig. S2). All three mutation groups showed differences
between mutant mice and their respective unaffected littermates in the ramus portion of the
hemimandible (Fig. S2).
In every analysis, all mice carrying mutations revealed a mandibular morphology that
differed from littermates that did not carry the mutation in unique ways. The finding that
different Fgfr2 gain-of-function mouse models exhibit different mandibular phenotypes is
consistent with our previous work that shows that the cranial phenotype (not including the
mandible) of each Fgfr2 model is different from their respective unaffected littermates that do
not carry the mutation, and that these changes vary across mouse models.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Quantitative characterization of hemimandible bone
Bone volume and bone surface area were determined using the Material Statistics Module of
Avizo 9.4 by first segmenting the left hemimandible as the region of interest from µCT scans.
Bone volume and bone surface area were compared using the Mann-Whitney U Test and did
not differ significantly between mutant and unaffected littermates in any of the three
craniosynostosis mouse models (Table 1), although the Fgfr2+/S252W mice had the least bone
volume of all genotypes. This is consistent with previous findings indicating no difference in
bone volume of the hemimandibles in Fgfr2+/P253R mice and their controls at E15.5, E16.5, E17.5,
P0 and P2 (Percival et al., 2014). Mean bone mineral density maps of the left hemimandibles
reveal little variation between mutant mice and unaffected littermates across all models (Fig.
S3). Cortical bone thickness was mapped in an identical manner and showed little variation (Fig.
S4).
Impaired microarchitecture in Fgfr2+/S252W mandible
The mandibles of Fgfr2+/S252W Apert syndrome mice showed the greatest difference relative to
their unaffected littermates in our analyses of 3D geometry and physical properties, and
therefore we initially focused on this mouse model for additional analyses of histological
properties of embryonic mandibular bone. We present histological analysis on the mandible at
E16.5 when differences were more obvious and consistent than at earlier stages. Osteogenic
tissue was defined operationally as cells that stain with alkaline phosphatase (ALP) that include
osteoprogenitors, preosteoblasts and differentiated osteoblasts (Huang et al., 2007). We
performed serial coronal sections (Fig. 2A) and stained them with ALP and alcian blue for
cartilage (Fig. 2B). Fgfr2+/S252W embryos showed significant morphological changes in the
developing mandible at E16.5. Both the osteogenic tissue and MC were enlarged in Fgfr2+/S252W
E16.5 embryos relative to Fgfr2+/+ littermates (Fig. 2B, C), prefiguring localization of the more
severe differences determined by morphometric analysis of µCT data of P0 mandibles.
Quantification of ALP-positive areas and cell numbers revealed the ALP-positive area was 38.5%
larger in Fgfr2+/S252W embryos relative to Fgfr2+/+ littermates (Fig. 2D) and cell number was
increased by 61.3% (Fig. 2E) at E16.5, while the cell density was not significantly changed (Fig.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
2F; P=0.135). The area of MC was 94.5% larger in Fgfr2+/S252W embryos relative to Fgfr2+/+
littermates (Fig. 2G) and cell number was increased by 56.6% (Fig. 2H), but there was no
significant change in cell density (Fig. 2I, P=0.354).
It has been reported that postnatal and adult Fgfr2S252W/+ mutant mice showed changes in
mineral apposition rate and microarchitecture of the mandible (Zhou et al., 2013). To analyze
the mineralized tissues of embryonic Fgfr2+/S252W mandibles, we performed alizarin red S and
von Kossa staining to detect calcium deposits and the presence of phosphate, respectively. In
the Fgfr2+/+ littermate, trabecular bone of the mandible was a well-organized network, while
the Fgfr2+/S252W trabecular bone had a disorganized and loose structure (Fig. 2J). Quantitative
analysis of alizarin red S staining showed that, although the total areas of mineralized tissues
were not significantly changed (Fig. 2K, P=0.414), the amount of staining versus osteogenic area
was significantly smaller in Fgfr2+/S252W embryos (Fig. 2L, P=0.043). Von Kossa staining revealed
a similar difference in mineralization of the mandible of Fgfr2+/+ and Fgfr2+/S252W embryos (Fig.
2M) and the quantification of the staining showed the similar result (Fig. 2N-O). Thus, the
FGFR2 S252W mutation is associated with impaired microarchitecture affecting mandibular
morphogenesis as early as E16.5.
To determine if these changes were found in the other FGFR2-related mouse models, we
performed histological analysis for mandibles of Fgfr2+/P253R and Fgfr2cC342Y/+ embryos at E16.5.
Fgfr2+/P253R embryos exhibited similar histological changes in the mandible of Fgfr2+/S252W
embryos, including increased osteogenic tissue, MC (Fig. S5B), and disorganized mineralization
pattern (Fig. S5F, J), consistent with the mandibular dysmorphology observed for these mutants
at P0. Fgfr2cC342Y/+ embryos showed more subtle, localized changes in mineralization in the
mandible (Fig. S5H, L).
Abnormal osteogenesis in Fgfr2+/S252W mandible
To understand the molecular mechanism for the changes of bone formation in the mandible by
the FGFR2 S252W mutation, we collected tissues from the mandibular bone and MC of
Fgfr2+/S252W embryos and their Fgfr2+/+ littermates at E16.5 by laser capture microdissection
(Fig. 3A). Total RNA from the two specific tissues was isolated and used for RNA-Seq analysis.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
We identified 122 genes that were significantly differentially expressed in the mandibular
bone of Fgfr2+/S252W embryos compared to their Fgfr2+/+ littermates (Table S3). Sixty-seven
differentially expressed genes (DEGs) were up-regulated and 55 DEGs were down-regulated in
the mutant (Fig. 3B, C). Gene ontology analysis of DEGs in biological process (Fig. S6A) and
cellular component (Fig. S6B) identified several GO terms including osteoclast differentiation
(GO:0030316), bone remodeling (GO:0046849), bone resorption (GO:0045453), ossification
(GO:0001503), and extracellular matrix (GO:0031012), all relevant to the histological phenotype
of Fgfr2+/S252W embryonic mandibles. No significant DEGs were found in MC of Fgfr2+/S252W
embryos compared to their Fgfr2+/+ littermates (data not shown).
Increased osteoclastogenesis in Fgfr2+/S252W hemimandibles
A group of genes active in osteoclastogenesis, including Acp5, Calcr, Csf1r, Ctsk, Il1r1, Itgb3,
Oscar and Tnfrsf11a (Table S3), were identified by transcriptome analysis as significantly
upregulated in Fgfr2+/S252W embryos relative to their Fgfr2+/+ littermates. To validate our
sequencing analyses, we tested the expression of Csfr1 and Itgb3 that are critical regulators of
the osteoclast lineage by section in situ hybridization (ISH) at E16.5. Csf1r encodes a tyrosine
kinase growth factor that is the receptor for the ligand colony stimulating factor-1 (CSF1).
CSF1R-mediated signaling plays an important role in osteoclastogenesis and Csf1r-/- mice exhibit
severe osteoclast deficiency (Dai et al., 2002). Integrin beta 3, encoded by Itgb3, forms a
complex with integrin alpha V, and integrin αvβ3 is essential for normal osteoclast function
(McHugh et al., 2000). ISH showed that Csf1r exhibited a scattered expression pattern in the
mandibular area of Fgfr2+/+ littermates (Fig. 4A, B), labeling preosteoclasts and osteoclasts in
the mandible. In the mandible of Fgfr2+/S252W embryos, increased Csf1r-positive cells were
detected (Fig. 4C, D), consistent with the results of the transcriptome analysis. Similarly, there
was increased expression of Itgb3 in the mandible of Fgfr2+/S252W embryos (Fig. 4G, H) relative
to their Fgfr2+/+ littermates (Fig. 4E, F), indicating increased expression of osteoclast genes in
mice carrying the FGFR2 S252W mutation. To confirm this result, we analyzed the tartrate-
resistant acid phosphatase (TRAP) activity as a functional osteoclastic marker. TRAP staining
revealed increased osteoclastic activity in the mandibular tissues of Fgfr2+/S252W embryos
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
relative to Fgfr2+/+ littermates at E16.5 (Fig. 4I, J). The numbers of osteoclasts per bone area in
mutant embryos were significantly increased compared to Fgfr2+/+ littermates (Fig. 4K,
38.5±8.7/mm2 for Fgfr2+/+, 117.9±12.3/mm2 for Fgfr2+/S252W, P=0.0158), and the percentage of
osteoclasts in the bone area was significantly increased as well (Fig. 4L, 2.61±0.23% for Fgfr2+/+,
7.19±0.89% for Fgfr2+/S252W, P=0.0288), suggesting higher bone resorption activity in mice
carrying the mutation relative to the controls.
To test if increased osteoclastic activity in the mandible observed in the Fgfr2+/S252W mice
was a common mechanism for mandibular dysmorphogenesis in the other FGFR2-related
mouse models, we performed the TRAP assay (Fig. S7). The Fgfr2+/P253R (Fig. S7C) like the
Fgfr2+/S252W (Fig. 4J and Fig. S7B) embryos showed increased osteoclastic activity compared with
unaffected littermates (Fig. S7A). The Fgfr2cC342Y/+ embryos showed increased staining in a
smaller area of the mandible (Fig. S7E) which also exhibited impaired mineralization (Fig. S5H,
L) compared to the unaffected littermates (Fig. S7D). The increase of osteoclastic activity was
consistent to the relative magnitude of changes in mandibular morphology and histology in the
three mouse models (Fgfr2+/S252W > Fgfr2+/P253R > Fgfr2cC342Y/+), indicating abnormal
osteoclastogenesis in the mandible is an important process affecting the relative
morphogenesis in FGFR2-related mouse models.
Increased expression of Enpp1 and Ank and elevated inorganic pyrophosphate (PPi)
concentration in Fgfr2+/S252W mandible
Gene ontology analysis (Fig. S6) showed that the expression of many genes that contribute to
ossification (GO:0001503) were down regulated in the mandible of Fgfr2+/S252W embryos,
including Aspn, Chrdl1, Igf1, Mgp, Ptn, Sfrp2, Thbs3 and Tnn, suggesting that osteogenesis was
inhibited in the Fgfr2+/S252W mandible. Mineralization plays a pivotal role in bone formation and
is initiated within matrix vesicles (MVs) where Ca2+ ions and inorganic phosphate (Pi) crystallize
to form hydroxyapatite (HA). The extracellular PPi (ePPi) adsorbs tightly to HA and potently
antagonizes the ability of Pi to crystallize with calcium to form HA, inhibiting HA crystal
propagation (Terkeltaub, 2001). Enpp1 and Ank are essential in regulating levels of PPi
(Mackenzie et al., 2012). Enpp1 is expressed in differentiated osteoblasts and encodes
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) protein that influences matrix
mineralization by increasing extracellular levels of PPi and regulates osteoblast differentiation
(Nam et al., 2011). Enpp1 is essential for normal bone development and control of physiological
bone mineralization and Enpp1−/− mice are characterized by severe disruption to the
architecture and mineralization of long bones, dysregulation of calcium/phosphate
homeostasis, and changes in Fgf23 expression (Mackenzie et al., 2012). Ank encodes the
progressive ankylosis protein, which is a highly conserved transmembrane pyrophosphate
transporter that channels PPi into extracellular matrix (Chen et al., 2011). Mutations located in
cytoplasmic domains close to the C-terminus of the human ANK gene (ANKH) were identified
for the autosomal dominant form of craniometaphyseal dysplasia (CMD) (Nürnberg et al., 2001;
Reichenberger et al., 2001). Overexpression of Ank in tissue culture cells leads to an increase in
the total level of ePPi (Ho et al., 2000).
Enpp1 and Ank were among the most significantly upregulated genes in the mandible of
Fgfr2+/S252W embryos at E16.5 (Fig. 3C), with log2 fold-change=1.83 and 1.33, respectively (Fig.
3C and Table S3). The differential expression was validated by ISH (Fig. 5A-H). To test if
upregulated expression of Enpp1 and Ank was associated with elevated PPi levels, the E16.5
mandibles were dissected and the weight and amount of PPi were quantified (Fig. 5I, J). The
weight of Fgfr2+/S252W mandibles (3.75±0.48 mg) were significantly lower than controls
(5.41±0.51 mg), while PPi concentrations in the mutant embryonic mandibles were higher
(4.22±0.93 nmol/mg) relative to those of the Fgfr2+/+ littermates (1.52±0.37 nmol/mg). These
findings suggest that genes that were transcriptionally dysregulated by the FGFR2 S252W
mutation changed the Pi/PPi balance toward reduced bone formation and mineralization.
Increased proliferation of osteoblasts and chondrocytes in the mandible of Fgfr2+/S252W
embryos
The osteogenic tissue and MC of Fgfr2+/S252W embryos at E16.5 were enlarged relative to their
littermates that did not carry the mutation (Fig. 2B). IHC for RUNX2 as an osteoblast marker
confirmed that osteoblasts are increased in the osteogenic region of Fgfr2+/S252W embryos (Fig.
6A). However, there is no indication from RNA-Seq analysis in mandibular bone or MC at E16.5,
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
suggesting that the molecular changes resulting from the FGFR2 S252W mutation might have
occurred at an earlier stage to cause the enlargement. As osteoblasts with FGFR2 S252W
mutation have an increased capacity for proliferation and differentiation in vitro (Holmes et al.,
2009; Yang et al., 2008) and FGF signaling plays an important role in chondrocyte proliferation
(Brewer et al., 2016), we hypothesized that the FGFR2 S252W mutation would increase cell
proliferation during the earlier stages of mandible development, resulting in increased cell
numbers and enlargement of the osteogenic tissue and MC. To test this, the EdU assay was
performed at E12.5 when ALP, an early osteoblast marker, can be detected in the jaw (Funato
et al., 2016) and adjacent mesenchymal cells are condensing to form MC (Parada and Chai,
2015). RUNX2 was used as a marker for osteoblasts (Fig. 6B), and chondrocytes in the MC were
visualized by staining SOX9 (Fig. 6D), which was strongly expressed in immature/proliferating
chondrocytes (Leung et al., 2011). The percentage of proliferating cells (EdU-positive) in
osteoblasts (SOX9-positive) of Fgfr2+/S252W embryos was 59.7±2.1%, significantly increased
compared with 45.7±2.0% in Fgfr2+/+ littermates (Fig. 6C). The percentage of proliferating cells
(EdU-positive) in MC (SOX9-positive) of Fgfr2+/S252W embryos was 57.4±6.1%, significantly
increased compared with 32.6±3.7% in Fgfr2+/+ littermates (Fig. 6E).
As apoptosis is another possible mechanism affecting cell numbers, the TUNEL assay was
performed on the mandible of Fgfr2+/S252W and Fgfr2+/+ embryos at E16.5. TUNEL signal was
detected in the mandibular bone of both groups (Fig. S8A). No apoptotic chondrocytes in MC
were observed but signals were detected in perichondrium (Fig. S8A), similar to a previous
study on normal MC development (Amano et al., 2010). Quantitative analysis shows
insignificant increase of TUNEL-positive cells in mandibular bone (Fig. S8B, 33.1±5.7 for Fgfr2+/+
and 60.3±7.8 for Fgfr2+/S252W, P=0.089) and perichondrium (Fig. S8C, 6.8±0.6 for Fgfr2+/+ and
9.0±0.5 for Fgfr2+/S252W, P=0.083) of Fgfr2+/S252W embryos compared with Fgfr2+/+ littermates,
indicating apoptosis does not contribute to the enlargement of the osteogenic tissue or MC. D
isea
se M
ode
ls &
Mec
hani
sms
• D
MM
• A
ccep
ted
man
uscr
ipt
DISCUSSION
The findings of this study demonstrate that the 3D dysmorphology of hemimandibles of three
FGFR2-related mouse models for craniosynostosis syndromes are easily distinguished from the
mandibles of their respective unaffected littermates at birth. Focusing on the hemimandibles of
Fgfr2+/S252W mice, we demonstrated significant dysmorphology, increase in MC size and
dysmorphic bony microarchitecture in embryos at E16.5. Our analyses showed that these
changes are due in part to inhibited osteogenic activity with increased osteoclastogenesis of the
mandible and earlier increased proliferation of osteoblasts and chondrocytes.
There are comparative data relevant to this study from previous analyses of humans and
adult mice. Most studies of the mandible in humans with craniosynostosis syndromes have
concluded that the mandible is intrinsically normal and that the morphological differences that
are noted likely represent a developmental response to the composite of structural variations
in the basicranium and midface in these conditions (Enlow, 2000; Wink et al., 2013). In humans,
the data are necessarily postnatal and analyses of mandibular shape are often conducted after
individuals have undergone reconstructive surgery of the midface. Because the maxilla and
mandible function as a unit in phonation and mastication, surgically-induced changes of
midfacial morphology could affect mandibular growth and morphology through changes in
functional relationships.
Previous mandibular analyses in mouse models for craniosynostosis include little or no
quantitative or gene expression information for embryonic and newborn mice, although it has
been reported that Fgfr2 is expressed in mandibular osteoblasts (Rice et al., 2003). A recent
study using only mandibular morphometry of adult mice carrying the FGFR2c C342Y mutation
on a mixed genetic background (72% C57BL6/J and 28% Swiss) reported significantly reduced
ascending (measured from the apex of coronoid process to gonion) and descending (measured
from the apex of coronoid process to menton) mandibular heights, mandibular lengths
(measured from condylion to pognion and from condylion to pogonion), and intercoronoid and
intercondylar widths, but increased intergonial widths (Khominsky et al., 2018). Though the
method of measurement differs from ours, these observations generally agree with our data
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
for newborn mice carrying the same mutation on a CD1 background. Mice aged 4 and 8 weeks
carrying the FGFR2 P253R mutation causative for Apert syndrome display globally reduced
mandibular dimensions (Du et al., 2010). Results presented here show that the mandibles of
newborn Fgfr2+/P253R mice are generally reduced in size relative to their normal littermates at
birth, and that the reduction is of greater magnitude in the posterior portion (including the
posterior mandibular body, and coronoid, condylar and angular processes), contributing to a
complex change in shape. We also reported that adult Fgfr2+/S252W Apert syndrome mice have a
very small mandible with a dysmorphic angular process (Wang et al., 2005; Wang et al., 2010).
The molecular changes that result from FGFR mutations are complex and include
constitutive (ligand-independent) or ligand-dependent FGFR activation, loss of function, and
altered cellular trafficking of receptors (Ornitz and Itoh, 2015). The FGFR2c C342Y mutation
associated with Crouzon/Pfeiffer syndrome lies within the Ig‐III domain of FGFR2c, and results
in constitutive activation of the receptor (Mangasarian et al., 1997). FGFR2 S252W and P253R
mutations are in the linker region, resulting in increased ligand affinity and altered specificity
(Andersen et al., 1998; Ibrahimi et al., 2001; Yu et al., 2000). Crystal structures of the FGFR2
S252W and P253R mutations indicate that P253R indiscriminately increases the affinity of
FGFR2 toward any FGF, while the S252W mutation selectively enhances the affinity of FGFR2
toward a limited subset of FGFs (Ibrahimi et al., 2001). These mutations in FGFR2 then
differentially affect FGFR2 intracellular signaling pathways (e.g. ERK1/2, PLCγ/PKCα, and
PI3K/Akt), resulting in alterations in cell proliferation, differentiation, and apoptosis, depending
on the stage of cell differentiation (Ornitz and Marie, 2015), forming the basis for different
mandibular phenotypes.
Zhou et al. demonstrated a significant decrease in mandibular cortical bone, decreased
bone mass, a significant decrease in calcein labeling of mineralizing surfaces, and a reduced
mineral apposition rate in the postnatal mandibles of Fgfr2S252W/+ mice at P28 and P56 (Zhou et
al., 2013). An observed increase in the number of osteoclasts, and a decreased number of
osteoblasts per bone surface area suggested lower bone formation capacities in Fgfr2S252W/+
adult mandibles relative to those of Fgfr2+/+ littermates. Bone modeling increases bone mass
and changes the shape of bones and occurs throughout life while bone remodeling functions to
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
renew bone (Allen and Burr, 2015). If the cellular activities reported in the mandibles of adult
Fgfr2S252W/+ mice (Zhou et al., 2013) are functioning primarily to renew bone, while our results
reflect changes in cellular activities that increase bone mass and change its shape, then the
balance of the amount of tissue resorbed and formed at any particular site may be disrupted by
the effect of FGFR2 S252W in modeling and remodeling.
Our results demonstrate an intrinsic difference in mandibular morphology of newborn mice
carrying FGFR2-related craniosynostosis mutations. We used ALP staining to detect mature
osteoprogenitor cells, preosteoblasts and differentiated osteoblasts (Huang et al., 2007), and
found that cell numbers in ALP-positive areas were increased in Fgfr2+/S252W embryos, indicating
that the FGFR2 S252W mutation promotes osteoblastic proliferation and differentiation,
consistent with results of previous analyses of adult cranial bone response to FGFR2 mutations
(Holmes et al., 2009; Yang et al., 2008). However, the overall mandible is reduced in size in
mutant mice both pre- and post-natally. Our finding of increased osteoclastogenesis is a
mechanism that can account for the overall reduction in size of mandibles of mice carrying
FGFR2 mutations.
Investigation of the transcriptome of the mandible in embryonic mice carrying the FGFR2
S252W mutation revealed dysregulation of genes involved in bone formation, bone
mineralization, and osteoclastogenesis, highlighting increased expression of genes undergoing
osteoclast differentiation and dysregulated genes active in bone mineralization. Bone formation
and bone resorption are important determinants of bone size and shape, whether osteoblast
and osteoclastic activity are coupled (as in remodeling) or uncoupled (as in modeling) (Allen and
Burr, 2015). Altered signaling pathways result in the dysregulation of genes that are involved in
osteoclastogenesis, bone formation, and bone mineralization, contributing to impaired
mandibular morphogenesis and microarchitecture. During normal growth, as bone mass
increases, resorption is required to alter bone shape and maintain a functioning skeletal
element. Our data show that improper regulation of osteoblastogenesis and osteoclastogenesis
can offset the balance required for bone modeling contributing to changes in the
developmental trajectory of individual embryonic bones and resulting in altered bone
phenotypes. The FGFR2 S252W mutation may retard mandibular bone formation, contribute to
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
decreased bone volume and compromised skeletal architecture by regulating both
osteoblastogenesis and osteoclastogenesis.
MC plays a crucial role as a supportive tissue for mandible formation and early growth.
Chondrocytes that form endochondral bone are differentiated from mesodermal cells in
general, whereas cells forming MC are differentiated from cells of neural crest origin (Amano et
al., 2010). In addition, the boundary between neural crest and mesoderm cells of the
chondrocranium lies between the hypophyseal and parachordal cartilages (McBratney-Owen et
al., 2008), indicating that the cartilages caudal to the hypophyseal cartilage are of mesodermal
origin. Abnormal MC development is associated with dysmorphogenesis of the mandible. For
example, activating FGFR3 mutations associated with achondroplasia lead to structural
anomalies of MC and condylar cartilages of the mandible, resulting in mandibular hypoplasia
and dysmorphogenesis (Duplan et al., 2016). We observed dramatically increased size and cell
number in MC of Fgfr2+/S252W embryos, with increased proliferation of chondrocytes detected as
early as E12.5. Previous studies have shown that Fgfr2+/S252W mutant mice have increased
cartilage of the basicranium (posterior to the hypophyseal cartilage) and thickened nasal
cartilage due to increased chondrocyte proliferation (Wang et al., 2005; Holmes et al., 2018).
These results suggest a common mechanism of increased proliferation by the FGFR2 S252W
mutation in these cranial cartilages whether derived from cranial neural crest cells or
mesoderm.
In summary, we quantitatively analyzed prenatal mandibular morphology in mouse models
carrying mutations by variants of Fgfr2 that are associated with craniosynostosis syndromes
when present in humans. Finding that the mandibles of Fgfr2+/S252W mice were quantitatively
the most different from their unaffected littermates we further studied the mandibles of these
mice using histology, immunochemistry and transcriptome analyses to understand the source
of altered morphology and abnormal microarchitecture of these mice with altered ligand
affinity and specificity of FGFR2 (Cunningham et al., 2007). We have previously shown that the
craniofacial phenotype (not including the mandible) of mice carrying the mutation in each Fgfr2
model is different compared to that of littermates not carrying the mutation (Martínez-Abadías
et al., 2013; Motch Perrine et al., 2014; Wang et al., 2005; Wang et al., 2010). We suggest a
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
model that mutation-induced changes in activated FGF signaling and downstream pathways are
associated with dysregulation of osteoblastogenesis, osteoclastogenesis, resorption,
mineralization, and the formation of MC, resulting in dysmorphogenesis of the mandible (Fig.
7). Mandibular development is directly affected by the FGFR2 mutations in these mouse
mutants, as was first suggested morphologically and later shown histologically by our results.
Mandibular dysmorphogenesis in these mouse models for craniosynostosis results, at least in
part, from the intrinsic effects of the mutation, and are not solely related to the functional
relationship of the mandible with the midface and cranial base as previously deduced from
human data.
MATERIALS AND METHODS
Mouse models
The generation of Fgfr2+/S252W, Fgfr2+/P253R and Fgfr2cC342Y/+ models were previously described
(Eswarakumar et al., 2004; Wang et al., 2005; Wang et al., 2010). Fgfr2+/S252W and Fgfr2+/P253R
models were maintained on a C57BL6/J background. The Fgfr2cC342Y/+ model was maintained on
a CD1 background for viability and breeding. Our samples for µCT analyses consisted of 182
newborn (P0) (Fgfr2+/S252W model: 22 mutants (9 females (F):13 males (M)); 24 unaffected
littermates (20 F:4 M); Fgfr2+/P253R model: 35 mutants (20 F:15 M); 28 unaffected littermates
(16 F:12 M); Fgfr2cC342Y/+ model: 34 mutants (18 F:16 M); 39 unaffected littermates (24 F:15
M)). Newborn mice (P0) were euthanized by inhalation anesthetics and fixed in 4%
paraformaldehyde. Gestation time was 19.0±0.5 days. Samples for histological and
transcriptome analysis consisted of 91 embryos (Fgfr2+/S252W model: 29 mutants (17 F:12 M); 37
unaffected littermates (21 F:16 M); Fgfr2+/P253R model: 8 mutants (2 F:6 M); 5 unaffected
littermates (3 F:2 M); Fgfr2cC342Y/+ model: 8 mutants (4 F:4 M); 4 unaffected littermates (3 F:1
M)). Genotyping of tail DNA by PCR was performed to distinguish mutants and unaffected
littermates. Mouse litters were produced in compliance with animal welfare guidelines
approved by Icahn School of Medicine at Mount Sinai and Pennsylvania State University Animal
Care and Use Committees.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Imaging protocols
High resolution micro computed tomography (µCT) images with pixel size and slice thickness
ranging from 0.014 to 0.025 mm were acquired by the Center for Quantitative X-Ray Imaging at
the Pennsylvania State University (www.cqi.psu.edu) using the HD-600 OMNI-X high-resolution
X-ray computed tomography system (Bio-Imaging Research Inc, Lincolnshire, IL, USA). A
minimum threshold of 70-100 mg/cm3 partial density hydroxyapatite (HA) (based on HA
phantoms imaged with specimens) was used to reconstruct isosurfaces in Avizo 6.3
(Visualization Sciences Group, VSG, Burlington, MA, USA). 3D coordinates of 32 biologically
relevant landmarks (Fig. S1 and Table S1) were collected from the isosurfaces. Specimens were
digitized twice, and measurement error was minimized by averaging coordinates of the two
trials (maximum accepted error in landmark placement=0.05 mm).
Statistical evaluation of shape differences
Variation in mandible shape was assessed by principal component analysis (PCA) using SAS 9.4
(SAS Institute, Cary, NC, USA). PCA summarizes the variation of large numbers of variables into
a lower-dimensional space defined by principal component (PC) axes that are mutually-
orthogonal, linear combinations of the linear distance data. The scores of an observation
(mandible or mandibular region) on the PC axes map that observation into the space. We
performed PCA (Darroch and Mosimann, 1985; Falsetti et al., 1993) of form (size and shape
together) using inter-landmark linear distances estimated using the full mandible landmark set
and subsets defining the left hemimandible (results of PCA analyses of left and right
hemimandibles were similar), anterior body, and ramus. Inter-landmark distances were ln-
transformed, and their variance-covariance matrix was used as the basis for the PCA.
Euclidean Distance Matrix Analysis (EDMA) was used to statistically evaluate mandibular
shape differences by hypothesis test and confidence interval estimation (Lele and Richtsmeier,
2001). EDMA is a 3D morphometric technique that is invariant to the group of transformations
including translation, rotation, and reflection (Lele and Richtsmeier, 1995; Richtsmeier and Lele,
1993). Briefly, the original 3D coordinates of landmark locations are re-written and analyzed as
a matrix of all unique linear distances among landmarks called the form matrix (FM). An
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
average FM is estimated for each sample (Lele and Richtsmeier, 1995). The difference between
samples is evaluated by estimating ratios of like-linear distances using sample-specific average
FMs. The resulting matrix of ratios, the form difference matrix (FDM), is a collection of relative
differences among landmarks used to define the forms. A non-parametric bootstrap procedure
(100,000 resamples) is used to obtain confidence intervals for elements (each corresponding to
a linear distance) of the FDM (Lele and Richtsmeier, 2001) that reveals the localized effects of
the mutations on the mandible. We also include a non-parametric bootstrap assessment of the
null hypothesis that the mean forms of two samples are the same (Lele and Richtsmeier, 2001).
We tested for form difference of the entire left hemimandible, the anterior body, and the
ramus portion using WinEDMA (Cole III, 2002).
Bone volume, surface area, and bone mineral density analyses
Bone volumes and surface areas were determined using the high resolution µCT scans
described above using Avizo 9.4 (ThermoFischer Scientific, Materials & Structural Analysis
Division, Hillsboro, OR, USA). The minimum thresholds used to create isosurfaces ranged from
70-100 mg/cm3 partial density hydroxyapatite. The isosurfaces were then analyzed using the
Material Statistics module of Avizo 9.4 software to determine bone volumes and bone surface
area. Stradwin v5.3 (http://mi.eng.cam.ac.uk/~rwp/stradwin) was used to create isosurfaces
from 30 of the hemimandibles (5 of each group) (Treece et al., 2010; Treece et al., 2012). The
tooth was excluded manually by placing guiding contours every five tomographic slices along
the hemimandibles. Density values were determined from the partial density hydroxyapatite
phantom normalized grey values at each isosurface vertex (~89,000-112,000 measurements).
Isosurfaces and their associated density values were registered using wxRegSurf v17
(http://mi.eng.cam.ac.uk/~ahg/wxRegSurf) (Gee et al., 2015; Stephens et al., 2018). A statistical
shape model (SSM) was generated for the full dataset and for each mouse model. Mean density
was calculated for each corresponding vertex and mapped onto the pertinent SSM. Pairwise
statistical differences between littermates were determined by performing a linear model
comparison at each vertex of the full SSM in R, with the resulting P values being mapped for
visual comparison.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Histological analysis
Mouse embryos at E16.5 were dissected and the heads were fixed in 4% paraformaldehyde
overnight at 4°C and then washed for 3 times with PBS. Samples were infused in 0.5 M sucrose
in PBS until tissue sank, and then quick-frozen in OCT. Samples were sectioned at a thickness of
10 μm. For ALP and alcian blue staining, cryosection samples were incubated for 5 min at room
temperature (RT) in 100 mM Tris-maleate buffer, pH 9.2, and then incubated for 5 min at RT in
freshly prepared ALP substrate solution (100 mM Tris-maleate buffer, pH 9.2, 0.2 mg/mL
naphthol AS-MX phosphate and 0.4 mg/mL Fast Red TR). The slides were washed briefly and
then stained with alcian blue solution (1% alcian blue, 3% acetic acid, pH 2.5) for 5 min at RT.
The slides were washed with water and then stained with 1 µg/mL Hoechst 33258 (Invitrogen
Life Technologies, Carlsbad, CA, USA) in PBS for 5 minutes. Images were collected in bright field
for ALP and alcian blue staining and then in UV for Hoechst 33258-stained nuclei. Calcium
deposits were detected with alizarin red S staining solution (MilliporeSigma, Burlington, MA,
USA). Presence of phosphate was detected with von Kossa staining kit (American MasterTech
Scientific, Lodi, CA, USA). TRAP staining was performed using cryosections with Acid
Phosphatase, Leukocyte (TRAP) Kit (MilliporeSigma, Burlington, MA, USA) following the
manufacturer’s instructions. Images were analyzed with ImageJ for stained particles and areas.
Laser capture microdissection (LCM) and RNA sequencing (RNA-Seq)
LCM was performed as previously described in detail (Holmes et al., 2018). The heads of female
Fgfr2+/+ littermates (n=3) and female Apert Fgfr2+/S252W (n=3) embryos at E16.5 were embedded
in OCT without fixation and rapidly frozen. Coronal cryosection was performed for the mandible
with 12 µm thickness. The mandibular tissue and MC were captured and collected respectively.
RNA was isolated with Arcturus Picopure RNA Isolation Kit (Thermo Fisher Scientific, Waltham,
MA, USA). Library preparation with NuGEN Ovation RNA-Seq System v2 (NuGEN Technologies,
San Carlos, CA, USA) and Nextera XT Library Prep kit (Illumina, San Diego, CA, USA) was
performed by the Gene Expression Core Facility at the Cincinnati Children's Hospital Medical
Center as described (Holmes et al., 2018). Library sequencing was performed on an Illumina
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
HiSeq 2500 instrument using standard protocols for paired-end 100 bp sequencing by the
Genetic Resources Core Facility at the Johns Hopkins School of Medicine.
Differential gene expression analysis and gene ontology (GO) enrichment analyses
RNA-Seq data processing, differential gene expression analysis and GO enrichment analyses
were done as described previously (Holmes et al., 2018). Briefly, paired-end reads were
mapped to the mouse (mm10) reference genome using STAR (Dobin et al., 2013) and gene
count summaries were generated using featureCounts (Liao et al., 2014). Only genes with
expression levels above 1 FPKM in at least 50% of samples were retained for further analysis.
Normalization factors were computed on the filtered data matrix using the weighted trimmed
mean of M-values (TMM) method (Robinson and Oshlack, 2010), followed by voom mean-
variance transformation in preparation for Limma linear modeling (Law et al., 2014). Data was
fitted to a design matrix containing all sample groups, and pairwise comparisons were
performed between sample groups. Finally, eBayes adjusted P values were corrected for
multiple testing using the Benjamin-Hochberg (BH) method and used to select genes with
significant expression differences (q<0.05). For GO enrichment analyses, the ‘elim’ algorithm
and ‘Fisher exact’ test were used to identify statistically over-represented GO categories at an
FDR corrected P value threshold of 0.05.
RNA in situ hybridization
Differential gene expression identified by RNA-Seq was validated by RNA in situ hybridization.
Riboprobe templates were generated by PCR using primers from published literature or
designed by Primer3 (http://primer3.ut.ee and Table S4), using cDNA derived from mouse
embryonic total RNA at E11.5. Riboprobes were prepared with DIG RNA Labeling Mix (Roche
Applied Science, Mannheim, Germany) as described by the manufacturer. RNA in situ
hybridization (ISH) was performed as described (Xu and Wilkinson, 1999).
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Quantification of PPi levels
Mandibles were isolated from embryos at E16.5 in cold PBS and dried briefly on delicate task
wipers before the weight was measured. Quantification of PPi in the mandible was performed
as described (Murali et al., 2016) with modification. Briefly, each mandible was incubated in
100 µl of 1.2M HCl at 4°C overnight and neutralized with NaOH and diluted with water.
Extracted PPi was quantified using the PPiLight Inorganic Pyrophosphate Assay (Lonza,
Walkersville, MD, USA) according to the manufacturer's protocol.
EdU assay and immunohistochemistry
EdU in vivo labelling was performed by single intraperitoneal injections of EdU to pregnant mice
at E12.5 at a dose of 50 mg/kg body weight in a solution of 10 mg/mL PBS (pH 7.35)
(Chehrehasa et al., 2009). The dams were sacrificed 30 min after the injection and embryos
were dissected for cryosection. The heads were sectioned at a thickness of 10 μm and EdU-
labeled cells were detected with Click-iT™ EdU Alexa Fluor™ 488 Imaging Kit (Thermo Fisher
Scientific, Waltham, MA, USA) followed by immunostaining for SOX9 (1:500, AB5535,
MilliporeSigma, Burlington, MA, USA) or RUNX2 (1: 200, HPA022040, MilliporeSigma,
Burlington, MA, USA) and Hoechst 33258 staining.
TUNEL assay
TdT-mediated dUTP nick end labeling (TUNEL) staining was performed using the In Situ Cell
Death Detection Kit, Fluorescein (MilliporeSigma, Burlington, MA, USA) according to the
manufacturer's protocol.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Acknowledgements
The authors would like to thank Dr. Tim Ryan and Mr. Tim Stecko at Penn State’s Center for
Quantitative Imaging for obtaining high quality µCT images and assisting in protocol review for
bone mineral density analysis. This work was supported in part through the computational
resources and staff expertise provided by Scientific Computing and special thanks to the
Pathology Core for access to the Leica laser capture microdissection platform at the Icahn
School of Medicine at Mount Sinai.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Imaging studies were performed at Pennsylvania State University and mouse and wet lab
studies were performed at Icahn School of Medicine at Mount Sinai. Study design: S.M.M.P.,
M.W., E.W.J., J.T.R.; Methodology: S.M.M.P., M.W., E.W.J., J.T.R.; Software: S.M.M.P., N.S., D.K.,
H.V.B.; Validation: S.M.M.P., M.W. and N.S.; Formal analysis: S.M.M.P., M.W., N.S., D.K., H.V.B.;
Investigation: S.M.M.P., M.W., N.S.; Resources: J.T.R., E.W.J.; Data curation: S.M.M.P., M.W.,
N.S., D.K., H.V.B.; Writing-original draft preparation: S.M.M.P., M.W., and J.T.R.; Visualization:
S.M.M.P, M.W., N.S., D.K., H.V.B.; Supervision: E.W.J., J.T.R., H.V.B.; Project administration:
S.M.M.P., M.W., E.W.J., J.T.R.; Funding acquisition: E.W.J., J.T.R.
Funding
This work was supported by National Institute of Dental and Craniofacial Research
[R01DE022988 to J.T.R. and E.W.J.] and Eunice Kennedy Shriver National Institute of Child
Health and Human Development [P01HD078233 to J.T.R. and E.W.J.].
Data availability
RNA-Seq data has been deposited in Gene Expression Omnibus (GEO) with accession number
GSE121780. Reviewers can access the private GEO records with the token: shoveicupbkdbsv.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
References
Aldridge, K., Hill, C. A., Austin, J. R., Percival, C., Martinez-Abadias, N., Neuberger, T., Wang,
Y., Jabs, E. W. and Richtsmeier, J. T. (2010). Brain phenotypes in two FGFR2 mouse models
for Apert syndrome. Dev. Dyn. 239, 987–997.
Allen, M. R. and Burr, D. B. (2015). Bone modeling and remodeling. In Basic and applied bone
biology, pp. 75–92. Elsevier Science.
Amano, O., Doi, T., Yamada, T., Sasaki, A., Sakiyama, K., Kanegae, H. and Kindaichi, K. (2010).
Meckel’s cartilage: Discovery, embryology and evolution. J. Oral Biosci. 52, 125–135.
Andersen, J., Burns, H. D., Enriquez-Harris, P., Wilkie, A. O. M. and Heath, J. K. (1998). Apert
syndrome mutations in fibroblast growth factor receptor 2 exhibit increased affinity for
FGF ligand. Hum. Mol. Genet. 7, 1475–1483.
Atchley, W. R. and Hall, B. K. (1991). A model for development and evolution of complex
morphological structures. Biol. Rev. Camb. Philos. Soc. 66, 101–57.
Azoury, S. C., Reddy, S., Shukla, V. and Deng, C.-X. (2017). Fibroblast growth factor receptor 2
(FGFR2) mutation related syndromic craniosynostosis. Int. J. Biol. Sci. 13, 1479–1488.
Brewer, J. R., Mazot, P. and Soriano, P. (2016). Genetic insights into the mechanisms of Fgf
signaling. Genes Dev. 30, 751–771.
Chehrehasa, F., Meedeniya, A. C. B., Dwyer, P., Abrahamsen, G. and Mackay-Sim, A. (2009).
EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. J.
Neurosci. Methods 177, 122–130.
Chen, I. P., Wang, L., Jiang, X., Aguila, H. L. and Reichenberger, E. J. (2011). A Phe377del
mutation in ANK leads to impaired osteoblastogenesis and osteoclastogenesis in a mouse
model for craniometaphyseal dysplasia (CMD). Hum. Mol. Genet. 20, 948–961.
Cohen, M. M. and MacLean, R. E. (2000). Craniosynostosis: diagnosis, evaluation, and
management. Oxford University Press.
Cole III, T. M. (2002). WinEDMA: Softw. euclidean distance matrix Anal. Version 1.0.1 beta.
Kansas City Univ. Missouri – Kansas City Sch. Med.
Costaras-Volarich, M. and Pruzansky, S. (1984). Is the mandible intrinsically different in Apert
and Crouzon syndromes? Am. J. Orthod. 85, 475–487.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Couly, G., Grapin-Botton, A., Coltey, P. and Le Douarin, N. M. (1996). The regeneration of the
cephalic neural crest, a problem revisited: the regenerating cells originate from the
contralateral or from the anterior and posterior neural fold. Development 122, 3393–1407.
Cunningham, M. L., Seto, M. L., Ratisoontorn, C., Heike, C. L. and Hing, A. V. (2007). Syndromic
craniosynostosis: From history to hydrogen bonds. Orthod. Craniofacial Res. 10, 67–81.
Dai, X.-M., Ryan, G. R., Hapel, A. J., Dominguez, M. G., Russell, R. G., Kapp, S., Sylvestre, V.
and Stanley, E. R. (2002). Targeted disruption of the mouse colony-stimulating factor 1
receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased
primitive progenitor cell frequencies, and reproductive defects. Blood 99, 111–120.
Darroch, J. N. and Mosimann, J. E. (1985). Canonical and principal components of shape.
Biometrika 72, 241–252.
Depew, M. J., Lufkin, T. and Rubenstein, J. L. R. (2002). Specification of jaw subdivisions by Dlx
genes. Science 298, 381–5.
Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M.
and Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29,
15–21.
Du, X., Weng, T., Sun, Q., Su, N., Chen, Z., Qi, H., Jin, M., Yin, L., He, Q. and Chen, L. (2010).
Dynamic morphological changes in the skulls of mice mimicking human Apert syndrome
resulting from gain-of-function mutation of FGFR2 (P253R). J. Anat. 217, 97–105.
Duplan, M. B., Komla-Ebri, D., Heuzé, Y., Estibals, V., Gaudas, E., Kaci, N., Benoist-Lasselin, C.,
Zerah, M., Kramer, I., Kneissel, M., et al. (2016). Meckel’s and condylar cartilages
anomalies in achondroplasia result in defective development and growth of the mandible.
Hum. Mol. Genet. 25, 2997–3010.
Enlow, D. H. (2000). Normal craniofacial growth. In Craniosynostosis: diagnosis, evaluation, and
management, 2nd ed. New York: Oxford University Press. p (ed. Cohen, M. M. J.) and
MacLean, R. E.), pp. 35–50. New York: Oxford University Press.
Eswarakumar, V. P., Horowitz, M. C., Locklin, R., Morriss-Kay, G. M. and Lonai, P. (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.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Falsetti, A. B., Jungers, W. L. and Colle III, T. M. (1993). Morphometrics of the callitrichid
forelimb: a case study in size and shape. Int J Primatol 14, 551–72.
Flaherty, K., Singh, N. and Richtsmeier, J. T. (2016). Understanding craniosynostosis as a
growth disorder. Wiley Interdiscip. Rev. Dev. Biol. 5, 429–459.
Frisdal, A. and Trainor, P. A. (2014). Development and evolution of the pharyngeal apparatus.
Wiley Interdiscip. Rev. Dev. Biol. 3, 403–418.
Funato, N., Kokubo, H., Nakamura, M., Yanagisawa, H. and Saga, Y. (2016). Specification of
jaw identity by the Hand2 transcription factor. Sci. Rep. 6, 28405.
Gee, A. H., Treece, G. M., Tonkin, C. J., Black, D. M. and Poole, K. E. S. (2015). Association
between femur size and a focal defect of the superior femoral neck. Bone 81, 60–66.
Heuzé, Y., Holmes, G., Peter, I., Richtsmeier, J. T. and Jabs, E. W. (2014). Closing the gap:
Genetic and genomic continuum from syndromic to nonsyndromic craniosynostoses. Curr.
Genet. Med. Rep. 2, 135–145.
Ho, A. M., Johnson, M. D. and Kingsley, D. M. (2000). Role of the mouse ank gene in control of
tissue calcification and arthritis. 289, 265–271.
Holmes, G., Rothschild, G., Basu, U., Deng, C., Mansukhani, A. and Basilico, C. (2009). Early
onset of craniosynostosis in an Apert mouse model reveals critical features of this
pathology. Dev. Biol. 328, 273–284.
Holmes, G., O’Rourke, C., Perrine, S. M. M., Lu, N., van Bakel, H., Richtsmeier, J. T. and Jabs, E.
W. (2018). Midface and upper airway dysgenesis in FGFR2-craniosynostosis involves
multiple tissue-specific and cell cycle effects. Development dev.166488.
Huang, W., Yang, S., Shao, J. and Li, Y. P. (2007). Signaling and transcriptional regulation in
osteoblast commitment and differentiation. Front. Biosci. 12, 3068–3092.
Ibrahimi, O. A., Eliseenkova, A. V., Plotnikov, A. N., Yu, K., Ornitz, D. M. and Mohammadi, M.
(2001). Structural basis for fibroblast growth factor receptor 2 activation in Apert
syndrome. Proc. Natl. Acad. Sci. U. S. A. 98, 7182–7187.
Khominsky, A., Yong, R., Ranjitkar, S., Townsend, G. and Anderson, P. J. (2018). Extensive
phenotyping of the orofacial and dental complex in Crouzon syndrome. Arch. Oral Biol. 86,
123–130.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Law, C. W., Chen, Y., Shi, W. and Smyth, G. K. (2014). voom: Precision weights unlock linear
model analysis tools for RNA-seq read counts. Genome Biol. 15, R29.
Lele, S. and Richtsmeier, J. T. (1995). Euclidean distance matrix analysis: Confidence intervals
for form and growth differences. Am. J. Phys. Anthropol. 98, 73–86.
Lele, S. and Richtsmeier, J. T. (2001). An invariant approach to statistical analysis of shapes.
Chapman & Hall/CRC.
Lemire, R. J. (2000). Embryology of the skull. In Craniosynostosis: diagnosis, evaluation, and
management (ed. Cohen, M. M. (Meyer M.) and MacLean, R. E.), pp. 24–32. New York:
Oxford University Press.
Leung, V. Y. L., Gao, B., Leung, K. K. H., Melhado, I. G., Wynn, S. L., Au, T. Y. K., Dung, N. W. F.,
Lau, J. Y. B., Mak, A. C. Y., Chan, D., et al. (2011). SOX9 governs differentiation stage-
specific gene expression in growth plate chondrocytes via direct concomitant
transactivation and repression. PLoS Genet. 7, e1002356.
Liao, Y., Smyth, G. K. and Shi, W. (2014). featureCounts: an efficient general purpose program
for assigning sequence reads to genomic features. Bioinformatics 30, 923–930.
Mackenzie, N. C. W., Zhu, D., Milne, E. M., van ’t Hof, R., Martin, A., Quarles, D. L., Millán, J.
L., Farquharson, C. and MacRae, V. E. (2012). Altered bone development and an increase
in FGF-23 Expression in Enpp1−/− Mice. PLoS One 7, e32177.
Mangasarian, K., Li, Y., Mansukhani, A. and Basilico, C. (1997). Mutation associated with
crouzon syndrome causes ligand-independent dimerization and activation of FGF receptor-
2. J. Cell. Physiol. 172, 117–125.
Martínez-Abadías, N., Motch, S. M., Pankratz, T. L., Wang, Y., Aldridge, K., Jabs, E. W. and
Richtsmeier, J. T. (2013). Tissue-specific responses to aberrant FGF signaling in complex
head phenotypes. Dev. Dyn. 242, 80–94.
McBratney-Owen, B., Iseki, S., Bamforth, S. D., Olsen, B. R. and Morriss-Kay, G. M. (2008).
Development and tissue origins of the mammalian cranial base. Dev. Biol. 322, 121–132.
McHugh, K. P., Hodivala-Dilke, K., Zheng, M. H., Namba, N., Lam, J., Novack, D., Feng, X., Ross,
F. P., Hynes, R. O. and Teitelbaum, S. L. (2000). Mice lacking beta3 integrins are
osteosclerotic because of dysfunctional osteoclasts. J. Clin. Invest. 105, 433–440.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Motch Perrine, S. M., Cole, T. M., Martínez-Abadías, N., Aldridge, K., Jabs, E. W. and
Richtsmeier, J. T. (2014). Craniofacial divergence by distinct prenatal growth patterns in
Fgfr2 mutant mice. BMC Dev. Biol. 14, 8.
Motch Perrine, S. M., Stecko, T., Neuberger, T., Jabs, E. W., Ryan, T. M. and Richtsmeier, J. T.
(2017). Integration of brain and skull in prenatal mouse models of Apert and Crouzon
syndromes. Front. Hum. Neurosci. 11, 369.
Murali, S. K., Andrukhova, O., Clinkenbeard, E. L., White, K. E. and Erben, R. G. (2016).
Excessive osteocytic Fgf23 secretion contributes to pyrophosphate accumulation and
mineralization defect in Hyp Mice. PLOS Biol. 14, e1002427.
Nam, H. K., Liu, J., Li, Y., Kragor, A. and Hatch, N. E. (2011). Ectonucleotide
pyrophosphatase/phosphodiesterase-1 (ENPP1) protein regulates osteoblast
differentiation. J. Biol. Chem. 286, 39059–39071.
Noden, D. M. (1983). The role of the neural crest in patterning of avian cranial skeletal,
connective, and muscle tissues. Dev. Biol. 96, 144–165.
Nürnberg, P., Thiele, H., Chandler, D., Höhne, W., Cunningham, M. L., Ritter, H., Leschik, G.,
Uhlmann, K., Mischung, C., Harrop, K., et al. (2001). Heterozygous mutations in ANKH, the
human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal
dysplasia. Nat. Genet. 28, 37–41.
Ornitz, D. M. and Itoh, N. (2015). The Fibroblast Growth Factor signaling pathway. Wiley
Interdiscip. Rev. Dev. Biol. 4, 215–266.
Ornitz, D. M. and Marie, P. J. (2015). Fibroblast growth factor signaling in skeletal development
and disease. 29, 1463–1486.
Parada, C. and Chai, Y. (2015). Mandible and tongue development. Craniofacial Dev. 115, 31–
58.
Percival, C. J., Huang, Y., Jabs, E. W., Li, R. and Richtsmeier, J. T. (2014). Embryonic craniofacial
bone volume and bone mineral density in Fgfr2 +/P253R and nonmutant Mice. Dev Dyn.
243, 541–551.
Reichenberger, E., Tiziani, V., Watanabe, S., Park, L., Ueki, Y., Santanna, C., Baur, S. T., Shiang,
R., Grange, D. K., Beighton, P., et al. (2001). Autosomal dominant craniometaphyseal
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
dysplasia is caused by mutations in the transmembrane protein ANK. Am. J. Hum. Genet.
68, 1321–1326.
Rice, D. P. C., Rice, R. and Thesleff, I. (2003). Fgfr mRNA isoforms in craniofacial bone
development. Bone 33, 14–27.
Richtsmeier, J. T. and Lele, S. (1993). A coordinate-free approach to the analysis of growth
patterns: Models and theoretical considerations. Biol. Rev. Camb. Philos. Soc. 68, 381–411.
Robinson, M. D. and Oshlack, A. (2010). A scaling normalization method for differential
expression analysis of RNA-seq data. Genome Biol. 11, R25.
Stephens, N. B., Kivell, T. L., Pahr, D. H., Hublin, J. J. and Skinner, M. M. (2018). Trabecular
bone patterning across the human hand. J. Hum. Evol. 123, 1–23.
Terkeltaub, R. A. (2001). Inorganic pyrophosphate generation and disposition in
pathophysiology. Am. J. Physiol. - Cell Physiol. 281, C1–C11.
Treece, G. M., Gee, A. H., Mayhew, P. M. and Poole, K. E. S. (2010). High resolution cortical
bone thickness measurement from clinical CT data. Med. Image Anal. 14, 276–290.
Treece, G. M., Poole, K. E. S. and Gee, A. H. (2012). Imaging the femoral cortex: Thickness,
density and mass from clinical CT. Med. Image Anal. 16, 952–965.
Wang, Y., Xiao, R., Yang, F., Karim, B. O., Iacovelli, A. J., Cai, J., Lerner, C. P., Richtsmeier, J. T.,
Leszl, J. M., Hill, C. A., et al. (2005). Abnormalities in cartilage and bone development in
the Apert syndrome FGFR2(+/S252W) mouse. Development 132, 3537–3548.
Wang, Y., Sun, M., Uhlhorn, V. L., Zhou, X., Peter, I., Martinez-Abadias, N., Hill, C. A., Percival,
C. J., Richtsmeier, J. T., Huso, D. L., et al. (2010). Activation of p38 MAPK pathway in the
skull abnormalities of Apert syndrome Fgfr2+P253R mice. BMC Dev. Biol. 10, 22.
Wink, J. D., Bastidas, N. and Bartlett, S. P. (2013). Analysis of the long-term growth of the
mandible in apert syndrome. J. Craniofac. Surg. 24, 1408–1410.
Xu, Q. and Wilkinson, D. G. (1999). In situ hybridization of mRNA with hapten labelled probes.
In In Situ Hybridization: a Practical Approach. (ed. Wilkinson, D. G.), pp. 87–106. Oxford:
Oxford University Press.
Yang, F., Wang, Y., Zhang, Z., Hsu, B., Jabs, E. W. and Elisseeff, J. H. (2008). The study of
abnormal bone development in the Apert syndrome Fgfr2+/S252W mouse using a 3D
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
hydrogel culture model. Bone 43, 55–63.
Yu, K., Herr, A. B., Waksman, G. and Ornitz, D. M. (2000). Loss of fibroblast growth factor
receptor 2 ligand-binding specificity in Apert syndrome. Proc. Natl. Acad. Sci. U. S. A. 97,
14536–14541.
Zhou, X., Pu, D., Liu, R., Li, X., Wen, X., Zhang, L., Chen, L., Deng, M. and Liu, L. (2013). The
Fgfr2(S252W/+) mutation in mice retards mandible formation and reduces bone mass as in
human Apert syndrome. Am. J. Med. Genet. Part A 161, 983–992.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Figures
Fig. 1. Morphological differences in newborn (P0) mice carrying mutations associated with
three FGFR2-related craniosynostosis syndromes and their unaffected littermates. Results of
PCA of mandibles based on unique linear distances among 3D landmarks (A-C) and Euclidean
Distance Matrix Analysis (EDMA) of landmark coordinates (D-F). Scatter plots of individual
scores on first and second Principal Components axes (PC1 and PC2) of linear distance based
PCAs of the hemimandibles of mutant and unaffected littermates of Fgfr2+/S252W and Fgfr2+/P253R
Apert syndrome mouse models (A, B, respectively) and Fgfr2cC342Y/+ Crouzon/Pfeiffer syndrome
mouse model (C). Results of EDMA of each craniosynostosis mouse model and unaffected
littermates showing linear distances within each model that are significantly different by at
least 5% between mutant and unaffected littermates (D, E, F). Blue lines are significantly larger
in mutant mice relative to unaffected littermates; fuchsia lines are significantly smaller in
mutant mice. Thin lines indicate linear distances that are increased/decreased by 5-10% in mice
carrying one of the Fgfr2 mutations while thick lines indicate linear distances that differ by
>10% between mutant and unaffected mice. The buccal aspects of the left hemimandibles of
the models were used for illustration. Hemimandibles were segmented into an anterior portion
(anterior body, shown in blue) and posterior portion (ramus, shown in red) to indicate
functional areas. Scale bar=1 mm.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Fig. 2. Histological analysis of mandible of Fgfr2+/S252W embryos at E16.5. (A) Schematic
embryonic mouse head at E16.5 modified from the e-Mouse Atlas Project
(http://www.emouseatlas.org/emap/eHistology). The red line indicates the location of sections
used for B-O. (B) Cryosections of Fgfr2+/+ and Fgfr2+/S252W embryos were stained with the ALP
assay (red) and alcian blue. MB, mandibular bone. MC, Meckel’s cartilage. T, tongue. (C) The
ALP-positive regions (red) were selected to quantify the areas and numbers of nuclei stained
with Hoechst 33258 (blue). (D-I) The areas (D, G), cell numbers (E, H) and cell density (F, I) in the
ALP-positive regions for Fgfr2+/+ (n=6) and Fgfr2+/S252W (n=6) embryos and MC of Fgfr2+/+ (n=6)
and Fgfr2+/S252W (n=6) embryos. (J, M) Alizarin red S staining (J) and von Kossa staining (M)
showed ossification in the mandible of Fgfr2+/+ and Fgfr2+/S252W embryos. The areas and the
percentages of the stained area in osteogenic tissue were measured for (K, L) alizarin red S
staining and (N, O) von Kossa staining. Scale bar=100 µm. The experimental data were analyzed
by two-tailed Welch’s t-test and expressed as the mean±standard error of the mean (s.e.m.).
*P<0.05.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Fig. 3. Laser capture microdissection (LCM) and RNA-Seq analysis of mandibular bone of
Fgfr2+/S252W embryos at E16.5. (A) A representative mandibular region in cryosection was
dissected by laser and collected for RNA-Seq (left, before LCM; right, after LCM). Scale bar=400
µm. (B) Hierarchical clustering of 122 genes significantly differentially expressed in the
mandibular bone between Fgfr2+/S252W and Fgfr2+/+ littermate embryos. Three biological
replicates were used for each genotype. (C) Volcano plot shows P values and fold changes of
DEGs in the mandibular bone between Fgfr2+/S252W and Fgfr2+/+ littermate embryos. Some of
the most significantly differentially expressed genes (-log10(P-value)>4.5) implicated in
mandibular dysmorphology are shown in blue.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Fig. 4. Increased osteoclastogenesis in the mandibular bone of Fgfr2+/S252W embryos at E16.5.
(A-H) The differential expression of Csf1r and Itgb3 in the mandible of Fgfr2+/+ (A, B, E, F) and
Fgfr2+/S252W embryos (C, D, G, H) were validated by in situ hybridization (ISH). The areas in the
boxes in A, C, E and G are shown in B, D, F and H, respectively, with higher magnification. (I-J)
TRAP assay stained osteoclasts (purple) in the mandible of Fgfr2+/+ (I) and Fgfr2+/S252W (J)
embryos. Scale bar=100 µm. (K-L) Quantitative measurements of the density (K) and
percentage (L) of osteoclasts in the bone area of Fgfr2+/+ (n=3) and Fgfr2+/S252W (n=3) embryos.
The experimental data were analyzed by two-tailed Welch’s t-test and expressed as the mean±
s.e.m. *P<0.05.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Fig. 5. Increased expression of Enpp1 and Ank and elevated PPi concentration in the mandible
of Fgfr2+/S252W embryos at E16.5. RNA expression of Enpp1 and Ank in the mandible of Fgfr2+/+
littermate (A, B, E, F) and Fgfr2+/S252W embryos (C, D, G, H) was validated by in situ hybridization
(ISH). The areas in the boxes in A, C, E and G are shown in B, D, F and H, respectively, with
higher magnification. Scale bar=100 µm. The weight of the mandible (I) and PPi concentration
in the mandible (J) were measured for Fgfr2+/+ littermates (n=7) and Fgfr2+/S252W (n=11)
embryos at E16.5. The experimental data were analyzed by two-tailed Welch’s t-test and
expressed as the mean±s.e.m. *P<0.05.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Fig. 6. Increased cell proliferation of osteoblasts and chondrocytes in the mandible of
Fgfr2+/S252W embryos. (A) The osteoblasts in the mandibular bone at E16.5 were visualized by
IHC for RUNX2. The areas in the boxes are shown on the right in higher magnification,
respectively. (B) Double staining with EdU assay (green) and IHC for RUNX2 (red) at E12.5. (C)
The percentage of proliferating osteoblasts (EdU-positive) in the total osteoblasts (RUNX2-
positive) is shown for Fgfr2+/+ (n=4) and Fgfr2+/S252W (n=4) embryos. (D) EdU assay (green) with
IHC for SOX9 (red) in MC at E12.5. (E) The percentage of proliferating cells (EdU-positive) in MC
(SOX9-positive) is shown for Fgfr2+/+ (n=4) and Fgfr2+/S252W (n=4) embryos. Scale bar=100 µm.
The experimental data were analyzed by two-tailed Welch’s t-test and expressed as the
mean±s.e.m. *P<0.05.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Fig. 7. Proposed model of mandibular dysmorphogenesis in prenatal development of
Fgfr2+/S252W mice.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Table
Table 1. Bone volume (mm3) and bone surface area (mm2) of newborn craniosynostosis
mouse models and their unaffected littermates.
Craniosynostosis Mouse Model
Mean Standard
Deviation
P value*
Statistic Std. Error
Fgfr2+/S252W
Fgfr2+/S252W Bone Volume 2.029372 0.1870266 0.5610799 0.606
Bone Area 51.1714 1.47767 4.43300 1.000
Fgfr2+/+ Bone Volume 2.174143 0.1742227 0.4609499
Bone Area 51.7267 2.22106 5.87638
Fgfr2+/P253R
Fgfr2+/P253R Bone Volume 2.2272 0.13911 0.31105 0.310
Bone Area 45.3520 2.12721 4.75659 1.000
Fgfr2+/+ Bone Volume 2.1741 0.17422 0.46095
Bone Area 51.7267 2.22106 5.87638
Fgfr2cC342Y/+
Fgfr2cC342Y/+ Bone Volume 2.378689 0.1105776 0.3317327 0.258
Bone Area 39.5811 1.77999 5.33997 0.258
Fgfr2c+/+ Bone Volume 2.203444 0.1123060 0.3369180
Bone Area 36.4152 1.04272 3.12815
*Exact significance is displayed for the independent samples Mann-Whitney U Test. The chosen
significance level is 0.05.
Dis
ease
Mo
dels
& M
echa
nism
s •
DM
M •
Acc
epte
d m
anus
crip
t
Fig. S1. Thirty-two anatomical landmarks located on left and right hemimandibles of newborn
(P0) mice. Bilateral landmarks are annotated only on the left hemimandible. Landmark
definitions are listed in Table S1. 3D coordinates of landmarks were collected from the buccal
view (A) and lingual (B) view of an isosurface of the left hemimandible. Landmarks shown in the
rostral (C) and caudal (D) views of the left and right hemimandibles are noted. Scale bar=1 mm.
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Fig. S2. Principal component analysis (PCA) of the anterior body and ramus portion of
hemimandibles of three mouse models of craniosynostosis. Scatter plots of individual scores
based on PCA from left hemimandibles of mutant and unaffected littermates of Fgfr2+/S252W (A)
and Fgfr2+/P253R (B) Apert syndrome mouse models; and Fgfr2cC342Y/+ (C) Crouzon/Pfeiffer
syndrome mouse model along first and second Principal Components axes (PC1 and PC2).
Separate PCAs were estimated for the anterior portion (anterior body, shown in blue in inset
image) and posterior portion (ramus, shown in red in inset image) of the hemimandibles. PCA
of the anterior body of the left hemimandible is shown in the top panel of each column while
PCA of the left ramus portion is shown in the bottom panel.
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Fig. S3. Bone mineral density mapping. Bone mineral density (BMD) was mapped for each of
the genotypes of interest: (A, B) buccal view of left hemimandibles of mice carrying Fgfr2
mutations (B) and their respective unaffected littermates (A); (C, D) lingual view of left
hemimandibles of mice carrying Fgfr2 mutations (D) and their respective unaffected littermates
(C). Though there are differences across the models, the contrast between mutant and
respective unaffected littermates is greatest in the Fgfr2+/S252W mice.
Fig. S4. Cortical bone thickness mapping. Cortical bone thickness was mapped for each of the
genotypes of interest: (A, B) buccal view of left hemimandibles of mice carrying Fgfr2 mutations
(B) and their respective unaffected littermates (A); (C, D) lingual view of left hemimandibles of
mice carrying Fgfr2 mutations (D) and their respective unaffected littermates (C).
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Fig. S5. Histological analysis of the mandibles of Fgfr2+/P253R and Fgfr2cC342Y/+ embryos at
E16.5. (A-D) ALP and alcian blue staining showed osteogenic tissues (red) and Meckel’s cartilage
(blue) in the mandible of Fgfr2+/P253R (B), Fgfr2cC342Y/+ (D) and their Fgfr2+/+ littermate embryos
(A, C, respectively). MB, mandibular bone. MC, Meckel’s cartilage. (E-H) Alizarin red S staining
and (I-L) von Kossa staining showed ossification in the mandible of Fgfr2+/P253R (F, J) and Fgfr2+/+
littermates (E, I), and Fgfr2cC342Y/+ (H, L) and Fgfr2+/+ littermates (G, K). Scale bar=100 µm. Boxes
in G, H, K and L indicate the small area that exhibits impaired microarchitecture at a higher
magnification in Fgfr2cC342Y/+ embryos compared with Fgfr2+/+ littermates.
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Fig. S6. Gene ontology analysis of DEGs in mandibular bone of Fgfr2+/S252W embryos at E16.5.
(A) Biological process. (B) Cellular component. (C) Molecular function.
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Fig. S7. Osteoclast activity in the mandibles of three FGFR2-related craniosynostosis mouse
models at E16.5. (A-E) TRAP staining (purple) in mandibular bone of Fgfr2+/+ (A), Fgfr2+/S252W (B)
and Fgfr2+/P253R (C) embryos on a C57BL6/J (B6) background; and Fgfr2c+/+ (D) and Fgfr2cC342Y/+
(E) on a CD1 background. Scale bar=100 µm. The boxes in D and E indicate the area that shows
increased signal in Fgfr2cC342Y/+ embryos compared with unaffected littermates.
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Fig. S8. Apoptosis in the mandible of Fgfr2+/S252W embryos at E16.5. (A) TUNEL assay in the
mandibular area. TUNEL signal was detected in the mandibular bone (MB) and the
perichondrium of Meckel’s cartilage (MC) of both Fgfr2+/+ and Fgfr2+/S252W embryos. Scale
bar=100 µm. (B-C) Quantification shows the number of TUNEL-positive cells in the mandibular
bone (B) and the perichondrium (C) of Fgfr2+/+ (n=3) and Fgfr2+/S252W (n=3) embryos. The
experimental data were analyzed by two-tailed Welch’s t-test and expressed as the
mean±s.e.m.
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Table S1. Anatomical definitions of landmarks displayed in Fig. 1.
Landmark (Left, Right)
Anatomical Definition Region
1, 2 Inferior-most point on incisor alveolar rim at midline of the incisor at bone-tooth junction
AB
3, 4 Junction of the rim of the alveolar process with incisor, most lateral and centered along the cranial-caudal axis (taken on bone, not tooth)
AB
5, 6 Superior-most point on incisor alveolar rim at midline of bone-tooth junction
AB
7, 8 Anterior lip of the mental foramen AB
9, 10 Antero-lateral edge of the molar alveolar process AB
11, 12 Intersection of molar alveolar rim and base of coronoid process (posterior molar alveolus)
R, AB
13, 14 Most dorsal point of the coronoid process R
15, 16 Most cranio-ventral point between the coronoid and condyloid processes
R
17, 18 Most caudal point on the cranial angle of the condyloid process
R
19, 20 Most caudal point on the ventral angel of the condyloid process
R
21, 22 Most anterior point between the angle that separates the condyloid and angular processes
R
23, 24 Midpoint on the cranial caudal axis of the most posterior aspect of the angular process
R
25, 26 Cranial-most point of the angular process along the ventral surface of the mandible
R, AB
27, 28 Dorsal mandibular foramen R
29, 30 Most ventral edge of the molar alveolar process, toward the midline
R
31, 32 Ventral mandibular foramen AB
Landmarks are classified as located on Anterior Body (AB; n=8) or Ramus (R; n=10) to create
subsets for analysis. Additional landmarks defined on the embryonic mouse skull can be found
at: http://www.getahead.la.psu.edu/landmarks.
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
Table S2. Results (P values) of nonparametric null hypothesis tests for form differences
(EDMA) between mice (P0) carrying a mutation for a specific craniosynostosis syndrome and
their littermates without the mutation.
Craniosynostosis
Model
Left and Right
Hemimandibles
(32 landmarks)
Left Hemimandible
(16 landmarks)
Right Hemimandible
(16 landmarks)
Fgfr2+/S252W 0.001 0.001 0.001
Fgfr2+/P253R 0.001 0.001 0.001
Fgfr2cC342Y/+ 0.001 0.001 0.001
Table S3. Differentially expressed genes in the mandibular bone of Fgfr2+/S252W embryos at
E16.5 compared to their Fgfr2+/+ littermates.
Ensembl ID Gene
Symbol
log2FC Average
Expression
t P Value Adjusted P
Value
ENSMUSG00000030257 Srgap3 1.27673267 6.610930486 9.640369417 5.20142E-07 0.003057916
ENSMUSG00000037370 Enpp1 1.831773279 8.59074871 10.20061207 2.82417E-07 0.003057916
ENSMUSG00000045573 Penk -1.928167528 4.158086959 -9.22055957 8.37469E-07 0.003188606
ENSMUSG00000034573 Ptpn13 1.133258416 6.830424831 8.998531675 1.08474E-06 0.003188606
ENSMUSG00000069917 Hba-a2 -1.622438006 4.106158794 -8.558032456 1.83915E-06 0.003902154
ENSMUSG00000022265 Ank 1.326616334 7.721340364 8.493163202 1.99123E-06 0.003902154
ENSMUSG00000073940 Hbb-bt -1.628190337 8.121203499 -8.263608921 2.64729E-06 0.004446698
ENSMUSG00000042436 Mfap4 -1.15168023 6.836719917 -8.135654118 3.11049E-06 0.004571637
ENSMUSG00000024593 Megf10 0.927309625 6.425005115 7.528605255 6.85637E-06 0.008957467
ENSMUSG00000036905 C1qb -2.263114164 2.013981763 -6.999425722 1.41583E-05 0.011193739
ENSMUSG00000052305 Hbb-bs -1.57470114 9.839937637 -7.157779 1.13549E-05 0.011193739
ENSMUSG00000035783 Acta2 -1.163348891 5.560032653 -6.993321688 1.42802E-05 0.011193739
ENSMUSG00000006369 Fbln1 0.877748065 5.903300431 7.109264313 1.21449E-05 0.011193739
ENSMUSG00000054594 Oscar 1.217936999 5.141501095 7.270388572 9.72461E-06 0.011193739
ENSMUSG00000040703 Cyp2s1 1.331780479 3.815520063 6.998767749 1.41714E-05 0.011193739
ENSMUSG00000031351 Zfp185 -2.478906784 3.159340528 -6.742466291 2.03933E-05 0.011887187
ENSMUSG00000033491 Prss35 -1.351275459 5.889560643 -6.721057207 2.1031E-05 0.011887187
ENSMUSG00000040569 Slc26a7 -1.106708548 6.18519432 -6.763093111 1.97983E-05 0.011887187
ENSMUSG00000037664 Cdkn1c -0.880989857 8.705576379 -6.714493501 2.12307E-05 0.011887187
ENSMUSG00000024621 Csf1r 0.932343075 7.271710056 6.790660552 1.90318E-05 0.011887187
ENSMUSG00000020340 Cyfip2 1.222456601 6.830403391 6.838067414 1.77866E-05 0.011887187
ENSMUSG00000028047 Thbs3 -1.146915292 4.350320931 -6.66151792 2.29185E-05 0.012248919
ENSMUSG00000052512 Nav2 0.954836312 6.560031841 6.618351607 2.43993E-05 0.012473328
ENSMUSG00000014846 Tppp3 -2.657022312 2.498697296 -6.399689044 3.36333E-05 0.015732053
ENSMUSG00000055013 Agap1 0.824170057 6.949305293 6.376950077 3.47877E-05 0.015732053
ENSMUSG00000023964 Calcr 1.39086817 4.385746777 6.386112042 3.43176E-05 0.015732053
ENSMUSG00000044667 Plppr4 -1.64880105 3.281452634 -6.337299974 3.69024E-05 0.016070294
ENSMUSG00000021388 Aspn -1.565468808 7.851433763 -6.235000826 4.30138E-05 0.017532802
ENSMUSG00000026321 Tnfrsf11a 0.878575936 6.328271017 6.231469435 4.3243E-05 0.017532802
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
ENSMUSG00000069516 Lyz2 -1.48397449 5.657315666 -5.984140315 6.30167E-05 0.021283798
ENSMUSG00000030218 Mgp -1.221549215 4.443285577 -6.029622637 5.87632E-05 0.021283798
ENSMUSG00000041559 Fmod -0.935657006 6.937668196 -6.047249988 5.71975E-05 0.021283798
ENSMUSG00000026469 Xpr1 0.756418685 8.588488909 5.965430507 6.48599E-05 0.021283798
ENSMUSG00000035311 Gnptab 0.877553017 7.078081271 6.048247317 5.71102E-05 0.021283798
ENSMUSG00000022303 Dcstamp 1.019517844 5.581854423 5.945315992 6.69054E-05 0.021283798
ENSMUSG00000040061 Plcb2 1.240168118 3.840512082 5.944636192 6.69757E-05 0.021283798
ENSMUSG00000021214 Akr1c18 1.299377329 4.04982874 5.9744297 6.39663E-05 0.021283798
ENSMUSG00000020053 Igf1 -1.273626277 6.98869413 -5.913249668 7.03087E-05 0.021754992
ENSMUSG00000028238 Atp6v0d2 0.857306076 8.426331011 5.891337145 7.27394E-05 0.021929993
ENSMUSG00000031283 Chrdl1 -0.834377044 5.949540909 -5.813408207 8.21296E-05 0.021947271
ENSMUSG00000001270 Ckb 0.899333325 8.11410426 5.841953781 7.85492E-05 0.021947271
ENSMUSG00000050390 C77080 1.100606755 4.008047025 5.824856679 8.06733E-05 0.021947271
ENSMUSG00000058897 Col25a1 1.145484143 6.457814805 5.817728948 8.15768E-05 0.021947271
ENSMUSG00000025089 Gfra1 1.467929152 4.267875815 5.853457969 7.71533E-05 0.021947271
ENSMUSG00000012405 Rpl15 -0.906856775 6.677200075 -5.773069762 8.74862E-05 0.022362233
ENSMUSG00000021728 Emb 0.914199758 5.859278388 5.781838677 8.62911E-05 0.022362233
ENSMUSG00000032948 Lipi -0.936729854 5.444723773 -5.663403914 0.000104002 0.025438301
ENSMUSG00000022324 Matn2 -0.694566276 5.740143219 -5.640097671 0.000107918 0.025438301
ENSMUSG00000052459 Atp6v1a 0.718305056 8.851933228 5.638604413 0.000108174 0.025438301
ENSMUSG00000044447 Dock5 1.048477353 6.571337781 5.639942804 0.000107945 0.025438301
ENSMUSG00000017737 Mmp9 1.19764316 9.755281083 5.569229207 0.000120814 0.027853587
ENSMUSG00000035296 Sgcg -4.411710792 -0.922779598 -5.517657881 0.000131215 0.028010594
ENSMUSG00000032291 Crabp1 -2.155264427 3.115239671 -5.497116825 0.000135618 0.028010594
ENSMUSG00000052854 Nrk -1.487662892 6.623819756 -5.547251564 0.000125136 0.028010594
ENSMUSG00000001348 Acp5 0.793398575 8.627342467 5.516153703 0.000131533 0.028010594
ENSMUSG00000021306 Gpr137b 0.861604003 6.046324047 5.519626396 0.000130802 0.028010594
ENSMUSG00000032122 Slc37a2 1.191073369 7.077239901 5.496333193 0.000135789 0.028010594
ENSMUSG00000069919 Hba-a1 -1.337990689 7.815219545 -5.422264917 0.000153024 0.030909916
ENSMUSG00000020689 Itgb3 1.110567359 7.880369008 5.41393388 0.000155102 0.030909916
ENSMUSG00000027562 Car2 0.745533042 6.189698692 5.394819185 0.000159983 0.031351269
ENSMUSG00000022519 Srl -0.874711972 4.426953711 -5.369870117 0.000166598 0.03211249
ENSMUSG00000040037 Negr1 -2.06759258 3.207329165 -5.347098779 0.000172888 0.032266944
ENSMUSG00000041362 Shtn1 0.856606818 5.623009126 5.348913307 0.000172378 0.032266944
ENSMUSG00000029765 Plxna4 1.049654389 5.846000814 5.319457787 0.000180861 0.033227474
ENSMUSG00000035258 Abi3bp -1.564181951 4.695670152 -5.29502116 0.000188231 0.033533688
ENSMUSG00000027962 Vcam1 -1.033135854 4.531254283 -5.295700595 0.000188022 0.033533688
ENSMUSG00000021390 Ogn -1.35118064 7.552490868 -5.285796718 0.000191095 0.033535785
ENSMUSG00000047139 Cd24a -0.988039433 5.207433327 -5.274863718 0.000194549 0.033639831
ENSMUSG00000026072 Il1r1 0.782735636 4.957639421 5.248885822 0.000203022 0.034101898
ENSMUSG00000028581 Laptm5 0.879244011 6.642567459 5.25154349 0.000202138 0.034101898
ENSMUSG00000032060 Cryab -1.110150001 3.610742127 -5.220757626 0.000212635 0.035213634
ENSMUSG00000024236 Svil 0.624813843 7.133248611 5.188180982 0.000224371 0.036640971
ENSMUSG00000026576 Atp1b1 1.17396213 4.596809676 5.172977602 0.000230079 0.037058409
ENSMUSG00000042082 Arsb 0.97021764 7.506223253 5.160310942 0.000234951 0.037331753
ENSMUSG00000004151 Etv1 0.730282583 4.988104973 5.132028881 0.000246224 0.038601426
ENSMUSG00000002014 Ssr4 -0.810564086 5.903376453 -5.097609777 0.000260716 0.039301265
ENSMUSG00000034707 Gns 0.727994379 7.459110088 5.104733751 0.000257645 0.039301265
ENSMUSG00000027670 Ocstamp 1.019536701 5.872442547 5.107174041 0.000256601 0.039301265
ENSMUSG00000061731 Ext1 0.663550112 7.436421895 5.069385813 0.000273267 0.04067181
ENSMUSG00000035357 Pdzrn3 -0.775776383 5.557112496 -5.047366705 0.000283499 0.041667193
ENSMUSG00000000290 Itgb2 0.939153536 5.118505724 5.024342179 0.000294628 0.042768391
ENSMUSG00000033220 Rac2 0.7747666 6.466527167 5.001754487 0.000305993 0.043876437
ENSMUSG00000023886 Smoc2 -1.076440655 5.017467359 -4.98683623 0.000313751 0.043917621
ENSMUSG00000039116 Gpr126 -0.981742058 4.952859094 -4.988178407 0.000313044 0.043917621
ENSMUSG00000015134 Aldh1a3 -1.172030326 5.661165159 -4.96080253 0.000327786 0.044910263
ENSMUSG00000026921 Egfl7 -0.844921515 4.284607323 -4.957154752 0.000329805 0.044910263
ENSMUSG00000018567 Gabarap -0.694331619 7.756610472 -4.952676418 0.000332301 0.044910263
ENSMUSG00000037533 Rapgef6 0.614649699 7.303891696 4.92217191 0.000349838 0.046743173
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n
ENSMUSG00000026596 Abl2 0.608326336 6.50724323 4.910707644 0.000356678 0.047121542
ENSMUSG00000044468 Fam46c -0.684580972 6.669624759 -4.897416701 0.000364783 0.047656834
ENSMUSG00000004730 Emr1 -2.239807676 1.348603199 -4.832587645 0.000407175 0.049783334
ENSMUSG00000027313 Chac1 -2.213689517 4.887600415 -4.764393172 0.000457375 0.049783334
ENSMUSG00000027996 Sfrp2 -1.171324394 4.710267004 -4.788885324 0.000438639 0.049783334
ENSMUSG00000062515 Fabp4 -1.05622216 3.509023446 -4.704234021 0.000507036 0.049783334
ENSMUSG00000028583 Pdpn -1.045235352 3.565172539 -4.802701535 0.000428426 0.049783334
ENSMUSG00000044337 Ackr3 -1.019213136 4.030412869 -4.708330855 0.000503482 0.049783334
ENSMUSG00000029838 Ptn -0.936047162 8.138301746 -4.752584264 0.000466706 0.049783334
ENSMUSG00000027204 Fbn1 -0.801254068 6.408244189 -4.808538301 0.000424186 0.049783334
ENSMUSG00000026421 Csrp1 -0.783880408 6.759861112 -4.787747037 0.000439492 0.049783334
ENSMUSG00000026725 Tnn -0.777064263 7.437115497 -4.847170068 0.000397209 0.049783334
ENSMUSG00000071856 Mcc -0.73828315 5.345518229 -4.714396914 0.000498266 0.049783334
ENSMUSG00000024534 Sncaip -0.723674183 4.868083805 -4.694867838 0.000515261 0.049783334
ENSMUSG00000038871 Bpgm -0.682666203 5.249711873 -4.7094474 0.000502517 0.049783334
ENSMUSG00000022912 Pros1 0.663131966 6.459136494 4.749861565 0.000468885 0.049783334
ENSMUSG00000033705 Stard9 0.706268577 6.374308981 4.698304549 0.000512227 0.049783334
ENSMUSG00000061175 Fnip2 0.710174275 5.074804605 4.84910423 0.000395907 0.049783334
ENSMUSG00000028111 Ctsk 0.713395398 9.9623244 4.816083029 0.000418771 0.049783334
ENSMUSG00000075324 Fign 0.737656804 5.609855492 4.780463665 0.000444989 0.049783334
ENSMUSG00000028962 Slc4a2 0.773555819 6.05593102 4.795937482 0.000433394 0.049783334
ENSMUSG00000041235 Chd7 0.793445297 5.723571848 4.736175534 0.000480003 0.049783334
ENSMUSG00000039062 Anpep 0.813927796 8.056912398 4.76404628 0.000457646 0.049783334
ENSMUSG00000068854 Hist2h2be 0.830691553 4.786180283 4.735427416 0.000480619 0.049783334
ENSMUSG00000038665 Dgki 0.831832322 4.196277449 4.707501683 0.000504199 0.049783334
ENSMUSG00000079057 Cyp4v3 0.83836864 4.764351948 4.838702637 0.000402965 0.049783334
ENSMUSG00000022231 Sema5a 0.839094343 9.365098271 4.837629137 0.0004037 0.049783334
ENSMUSG00000026437 Cdk18 0.88555389 3.867251827 4.693416906 0.000516548 0.049783334
ENSMUSG00000022012 Enox1 0.892698352 3.686678832 4.709026013 0.000502881 0.049783334
ENSMUSG00000018774 Cd68 0.959283668 7.245963357 4.703581561 0.000507605 0.049783334
ENSMUSG00000079625 Tm4sf19 1.030522587 3.924772294 4.762574467 0.000458799 0.049783334
ENSMUSG00000038418 Egr1 1.120357726 6.447009919 4.772930692 0.000450751 0.049783334
ENSMUSG00000049493 Pls1 1.373575186 2.360582503 4.733077184 0.000482558 0.049783334
ENSMUSG00000062151 Unc13c 1.540649612 3.574785275 4.723859106 0.000490244 0.049783334
Table S4. Primers used to generate riboprobe templates for RNA in situ hybridization.
Gene Primers References
Ank 5’-GAGTAATACGACTCACTATAGGGTGGGATGTGCCTCAATCTCA-3’
5’-GAGATTAACCCTCACTAAAGGGACACAGAGTTCTGCAAAGGCAA-3’
Uzuki et al., 2014
Csf1r 5’-GAGTAATACGACTCACTATAGGGAGGAGGTGTCTGTGGGTGAC-3’
5’-GAGATTAACCCTCACTAAAGGGATGGTACTTCGGCTTCTGCTT-3’
Designed using
Primer3
Enpp1 5’-GAGTAATACGACTCACTATAGGGGCTGTCTGAGACTCCCTTGG-3’
5’-GAGATTAACCCTCACTAAAGGGAGTCCCCAGACCACGTACACT-3’
Designed using
Primer3
Itgb3 5’-GAGTAATACGACTCACTATAGGGGAAAATGTCGTCAGCCTTTACC-3’
5’-GAGATTAACCCTCACTAAAGGGAGCAGGAGAAGTCATCGCACTC-3’
Diez-Roux et al.,
2011
Disease Models & Mechanisms: doi:10.1242/dmm.038513: Supplementary information
Dis
ease
Mo
dels
& M
echa
nism
s •
Sup
plem
enta
ry in
form
atio
n