© 2018. Published by The Company of Biologists Ltd.

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Increased FGF8 Signaling Promotes Chondrogenic Rather Than Osteogenic Development

in the Embryonic Skull

Linnea Schmidt a, b, Aftab Taiyab b, I, Vida Senkus Melvin b, II, Kenneth L. Jones c, Trevor Williams b,d,e*

a Program of Reproductive Sciences and Integrated Physiology, University of Colorado Anschutz Medical Campus,

Aurora, CO 80045, USA b Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA c Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045,

USA d Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO

80045, USA e Department of Pediatrics, University of Colorado Anschutz Medical Campus, Children's Hospital Colorado, Aurora,

CO 80045, USA I Present address: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

L8S 4N5 II Present address: Department of Biology, Metropolitan State University of Denver, Denver, CO 80217, USA

*Corresponding Author: Trevor Williams; Email Address: Trevor.Williams@ucdenver.edu

Summary Statement:

Cranial ossification responds to Fgf8 overexpression in a dose dependent manner with moderate

levels leading to craniosynostosis and higher levels shifting cranial vault ossification to abnormal

cartilage formation.

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.031526Access the most recent version at First posted online on 10 May 2018 as 10.1242/dmm.031526

Highlights

Cranial ossification responds to Fgf8 overexpression in a dose-dependent manner.

Moderate levels of Fgf8 overexpression lead to craniosynostosis.

High levels of Fgf8 overexpression shifts cranial vault ossification to abnormal cartilage

formation.

The formation of intramembranous bone is generally impaired more by Fgf8

overexpression than the generation of endochondral bone.

Fgf8 overexpression inhibits intramembranous ossification through the Wnt signaling

pathway.

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Abstract

The bones of the cranial vault are formed directly from mesenchymal cells through

intramembranous ossification rather than via a cartilage intermediate. Formation and growth of

the skull bones involves the interaction of multiple cell:cell signaling pathways, with Fibroblast

Growth Factors (FGFs) and their receptors exerting prominent influence. Mutations within this

pathway are the most frequent cause of craniosynostosis, which is a common human craniofacial

developmental abnormality characterized by the premature fusion of the cranial sutures. Here,

we have developed new mouse models to investigate how different levels of increased Fgf

signaling can impact the formation of the calvarial bones and associated sutures. While moderate

Fgf8 overexpression resulted in delayed ossification followed by craniosynostosis of the coronal

suture, higher Fgf8 levels promoted a loss of ossification and favored cartilage over bone

formation across the skull. In contrast, endochondral bones were still able to form and ossify in

the presence of increased Fgf8, though the growth and mineralization of these bones were

impacted to varying extents. Expression analysis demonstrated that abnormal skull

chondrogenesis was accompanied by changes in genes required for Wnt signaling. Moreover,

further analysis indicated that the pathology was associated with decreased Wnt signaling since

the reduction in ossification could be partially rescued by halving Axin2 gene dosage. Taken

together, these findings indicate that mesenchymal cells of the skull are not fated to form bone

but can be forced into a chondrogenic fate via manipulation of FGF8 signaling. These results

have implications for evolution of the different methods of ossification as well as for therapeutic

intervention in craniosynostosis.

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Keywords

Intramembranous Ossification; Fgf8; Cranial Vault; Craniosynostosis; Osteogenesis;

Chondrogenesis

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1. Introduction

Bone forms via two processes: intramembranous or endochondral ossification. The

majority of the skeleton, including the long bones, vertebrae, and basicranium, forms via

endochondral ossification during which condensed mesenchyme cells first differentiate into

chondrocytes that form cartilage tissue. This intermediate cartilaginous template is then replaced

by bone, formed through osteogenesis. In contrast, most of the skull, including the jaw and

cranial vault, is generated via intramembranous ossification, in which condensed mesenchyme

cells directly differentiate into osteoblasts that form bone without any cartilaginous precursor

(Ornitz and Marie, 2015). The anterior cranial vault, comprising the frontal bones, is derived

from cranial neural crest cells (Couly et al., 1993; Jiang et al., 2002), whereas the posterior

cranial vault bones are derived from either the paraxial mesoderm (parietal) or a combination of

paraxial mesoderm and cranial neural crest (interparietal/occipital) (Jiang et al., 2002; Noden,

1992; Yoshida et al., 2008). Together, these cranial neural crest and paraxial mesoderm derived

cells form the intramembranous bones and sutures of the skull (Evans and Noden, 2006; Jiang et

al., 2002; Noden and Trainor, 2005; Opperman, 2000). Intramembranous ossification of the

skull vault involves direct bone matrix deposition to form calvarial plates, which expand during

development but do not fuse with other cranial bones during embryogenesis and infancy (Hall

and Miyake, 2000). Instead, sutures connect the individual intramembranous bones and serve as

growth centers that regulate the expansive growth of the skull (Nie et al., 2006). Because sutures

are the major sites of bone growth during cranial vault development, signaling at the sutures is

essential for the regulation of intramembranous ossification (Cohen, 2000; Ornitz and Marie,

2002). Several signaling pathways are implicated in proper skull ossification and growth

including fibroblast growth factor (FGF), hedgehog (HH), and WNT signaling (Katsianou et al.,

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2016). The role of FGF signaling in ossification is of particular interest as mutations in the FGF

signaling pathway, composed of four FGFRs (Fibroblast Growth Factor Receptors) and 22 FGF

ligands (Brewer et al., 2016; Pownall and Isaacs, 2010), cause a number of skeletal disorders

(Nie et al., 2006; Ornitz and Marie, 2015), including those that affect cranial vault ossification;

such as craniosynostosis.

Craniosynostosis is the second most common human craniofacial abnormality, occurring

in 1 in 2,100 to 2,500 births (Boulet et al., 2008; Lajeunie et al., 1995). It is characterized by the

premature fusion of the metopic, sagittal, lambdoid, and/or coronal sutures which causes a

distortion in skull shape that frequently requires surgical correction. Activating mutations in the

FGF signaling pathway, mostly heterozygous mutations of FGFRs, account for the majority of

the known causes of craniosynostosis (Johnson and Wilkie, 2011). The etiology of

craniosynostosis is complicated, however, by the fact that activating mutations in the same gene

can cause a variety of different syndromes and/or types of craniosynostosis. For example,

mutations in FGF receptor 2 (FGFR2 ) can cause seven of the eight FGFR-related

craniosynostosis disorders, including Apert, Crouzon, and Pfeiffer syndromes (Moosa and

Wollnik, 2016; Robin et al., 2011). This diversity in part reflects how each mutation impacts the

multiple signaling pathways that are directly regulated by FGFRs as well as the potential for

genetic interactions to affect the phenotype (Ratisoontorn et al., 2003).

Detailed studies using genetically modified mice with dominant or loss-of-function

FGFR mutations have also confirmed the central role of this receptor family in bone formation,

skeletal development, and craniosynostosis (Su et al., 2014). With respect to the FGF ligands,

FGF 2, 9, and 18 have been shown to act in normal mouse cranial vault ossification (Coffin et

al., 1995; Hung et al., 2016; Liu et al., 2002; Ohbayashi et al., 2002). Moreover, mouse

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mutations that increase the diffusion properties and effective range of FGF9 also lead to

craniosynostosis (Spicer, 2009). This latter result may reflect the mechanism underlying human

multiple synostoses syndrome, which is caused by rare autosomal mutations in FGF9 (Wu et al.,

2009). Taken together, these studies indicate that the molecular properties of FGF ligands and

receptors, along with dosage, location, and timing, likely all contribute to the complexity of

craniosynostosis etiology.

However, the specific mechanisms by which FGF signaling affects skull development

and craniosynostosis remain poorly understood. Notably, mice heterozygous or homozygous for

activating mutations in FGFRs have different craniofacial phenotypes, with homozygous gain-

of-function mutations often phenocopying aspects of the loss-of-function phenotype (Mai et al.,

2010; Snyder-Warwick et al., 2010). Moreover, activating mutations in FGFRs can lead to both

ossification of the sutures as well as thinner calvaria (Tholpady et al., 2004). To further

investigate the mechanisms underlying these somewhat contradictory developmental

abnormalities, we have generated mouse models that can be induced to overexpress different

levels of Fgf8b in the ectoderm of the developing skull. The choice of the FGF8 ligand was

based both on its abilty to influence development and patterning in multiple contexts as well as

its homology to FGF18, which has an important role in chondrogenesis and osteogenesis (Hung

et al., 2016; Liu et al., 2007, 2002; Ohbayashi et al., 2002). Further, we employed the Fgf8b

isoform since this is more potent than Fgf8a in many developmental processes, a finding which

correlates with the higher affinity of FGF8b for FGF receptors (Meyers et al., 1998; Olsen et al.,

2006; Zeller et al., 2009). By comparing skeletal formation in these new mouse models we

demonstrate that the dosage of Fgf8 expression differentially affects development of the calvaria

and the associated sutures. Low Fgf8b expression mimics the craniosynostosis seen with

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particular FGFR2 activating mutations. In contrast, higher Fgf8b expression severely disrupts

the process of intramembranous bone formation. Overall, these results provide insight into the

mechanisms and potential treatment of craniosynostosis as well as the function of FGFs in

skeletal development and ossification.

2. Results

2.1 Generation of new alleles for differential expression of Fgf8

Two new alleles were generated to manipulate the expression of Fgf8 transcripts in vivo.

The first allele incorporates the Fgf8b cDNA into the Gt(ROSA)26Sor locus under the control of

a Lox-Stop-Lox cassette (Fig. 1A and Fig. S1A). Breeding mice with this R26LSL Fgf8b allele to

mice expressing a Cre recombinase transgene allows the deletion of the Stop cassette to generate

a new allele expressing the Fgf8b cDNA (R26Fgf8b), hereafter termed R26F8. To ascertain the

effects of higher levels of Fgf8 overexpression, we generated an additional construct - R26LSL CAG

Fgf8b (Fig. 1A and S1B). Following Cre-mediated recombination this would generate the R26CAG

Fgf8b allele, hereafter termed CAGF8. We initially focused on how elevated FGF ligand

expression in the overlying ectoderm could influence the development of the underlying

mesenchyme at different timepoints and in different locations by testing a number of Cre

recombinase transgenes that target the embryonic ectoderm. In the current study we focused on

formation and patterning of the craniofacial skeleton and determined that Msx2-Cre had a

spatiotemporal pattern of expression that was the most suitable to study calvarial development

((Sun et al., 2000), (Choe et al., 2012) and Fig. S2). Specifically, when used in combination with

R26 LacZ reporter mice, Msx2-Cre activity was detected in the head ectoderm beginning around

E10.5, but was not detected in the underlying mesenchyme derivatives of the skull including the

bones and sutures (Fig. S2). We next used qRT-PCR to examine how Fgf8b mRNA levels were

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altered when Msx2-Cre was employed with either of the new Fgf8 alleles (Fig. 1B). At E18.5, in

the presence of Msx2-Cre, Fgf8b expression was increased ~8 times in R26F8 mutants (Fig. 1B,

p=0.0013) and ~21 times in CAGF8 mutants compared with controls (Fig. 1B, p=0.0002).

Therefore, both the R26F8 and CAGF8 alleles increase Fgf8b mRNA expression when activated

by a Cre recombinase transgene, with the latter generating ~2.5 fold more transcript than the

former (Fig. 1B, p=0.022).

2.2 Moderately increased levels of Fgf8 transcripts cause craniosynostosis

We next studied development in mice with a moderate increase in Fgf8b expression.

These Msx2-Cre;R26Fgf8b mice, hereafter termed MR26F8, were born at normal size and in

normal Mendelian ratios, but were easily identifiable from birth onwards by their gross

morphology. The MR26F8 mice had both striking craniofacial defects (Fig. 2A-F, Fig. S3A, B)

as well as limb defects including sirenomelia and post-axial polydactyly (Fig. S3C-L), the latter

phenotypes consistent with the expression of the Msx2-Cre transgene in the limb bud ectoderm

(Sun et al., 2000). The facial abnormalities included shortened snout, abnormal skin with raised

rounded protrusions that extended to cover the eye (Fig. 2D-F, Fig. S3B), and patchy or absent

hair on top of the head, as well as on the limbs, and underbelly (Fig. 2F; Fig. S3 A-H). Thus,

although the MR26F8 mice were viable into adulthood, they already had significant craniofacial

abnormalities by birth. Therefore, we compared the underlying skeleton of control and MR26F8

mice during early post-natal development using bone and cartilage staining (Figure 2G-L; Fig.

S3M-P). In the P0 wildtype skull, the bony plates of the frontal, parietal, and interparietal bones

had not yet met at the dorsal midline and the coronal and lambdoid sutures were clearly visible

as an overlapping region between adjacent bony plates (Fig. 2G). In contrast, in MR26F8s, the

sagittal and interfrontal sutures were wider except in the region where the coronal and midline

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sutures should intersect (Fig. 2J). Here, there was a narrowing of the sagittal and interfrontal

sutures (Fig. 2J). With respect to the lambdoid suture, the interparietal and parietal bones did not

overlap to the same extent as in the controls. Most strikingly, though, the coronal suture

separating the frontal and parietal bones, was abnormal (Fig. 2J). In some areas, where a coronal

suture would be expected, there were instead wider and irregular regions where the bones did not

overlap. Such irregular fissures were a common feature in the MR26F8s, whereas a typical

coronal suture was never observed in this genotype. In other regions, particularly nearer the

dorsal midline, there was no apparent suture and the frontal and parietal bones were fused

together seamlessly. These suture defects were accompanied by decreased ossification of the

ventral portions of the parietal bones (Fig. S3N). Despite the numerous defects in the cranial

vault, ossification of the cranial base remained largely unaffected (Fig. S3P).

From P12 onwards, some cranial defects in the MR26F8s had resolved, whereas others

had materialized, including ectopic bone development in the base of the eye socket (Fig. 2J-L).

With respect to the calvaria, in the P12 and adult wildtype skulls, the bony plates of the frontal,

parietal, and interparietal bones had met at the dorsal midline to form distinguishable sagittal and

interfrontal sutures, and the coronal and lambdoid sutures were still apparent (Fig. 2H, I).

Similarly, in the MR26F8s, the skull bones had now met at the midline to form the sagittal and

interfrontal sutures. However, craniosynostosis of the coronal suture in the mutants was now

complete (Fig. 2K, L) and the lambdoid suture was now narrower than in the wildtype. MR26F8s

also exhibited misalignment of the frontonasal region, a condition that was associated with variable

defects in the interactions between the frontal, nasal and premaxillary bones, including the presence of

Wormian bones, partial fusion of the frontonasal suture, and a lack of interdigitation of the

frontopremaxillary suture (Fig. 2K, L and data not shown).

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To examine the craniosynostosis of the coronal suture in more detail, we sagittally

sectioned both P0 and P12 craniums and then employed von Kossa and Goldner’s Trichrome

stains to assess mineralization and bone differentiation, respectively. The von Kossa stained

neonate samples showed that, compared to controls, the MR26F8 mice had a wider, more open

region between the mineralized bone fronts, giving the superficial appearance of a

broader coronal suture (Fig. 3C, D). Goldner’s Trichome staining of MR26F8 samples revealed

that this wider area was composed of an unorganized combination of mature and immature bone

matrix as opposed to undifferentiated suture mesenchyme (Fig. 3F), indicating that there is

abnormal or premature osteoblast differentiation in this area. By P12, all MR26F8s exhibited

premature fusion of the coronal suture (Fig. 3H, J) compared to the control sutures, which were

not fused (Fig. 3G, I). Therefore, the coronal suture in the MR26F8s displayed ectopic matrix

deposition with wider separation of both the osteoid and mineralized fronts followed by

mineralization of this intervening matrix and subsequent craniosynostosis. The same phenomena

of relatively wide separation between the mineralized bone fronts at early stages, with

subsequent narrowing compared to controls, was also observed in the MR26F8 lambdoid sutures,

although these structures did not undergo overt fusion during the timepoints examined (Fig.

S4). In summary, moderately increased Fgf8 expression caused cranial vault ossification

defects, most notably, synostosis of the coronal suture.

2.3 Abnormal cartilage replaces intramembranous bone at higher Fgf8 levels

As with the MR26F8 mice, the Msx2-Cre;CAGFgf8b mice, hereafter termed MCAGF8,

were born at normal size and Mendelian ratios. However, unlike the MR26F8s, the MCAGF8s

did not survive beyond the first day, likely due to altered facial shape and clefting (Fig. 4A-F).

Thus, our analysis was limited to P0 and embryological time points. MCAGF8 neonates

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exhibited several striking craniofacial phenotypes: a shortened snout, domed skull, cleft

secondary palate and loss of the eyes. Other morphological defects included limb deformities,

particularly post-axial polydactyly, and more complex fusion phenotypes (Fig. S5).

We next compared E18.5 skeletons of MCAGF8 and control embryos using standard

bone and cartilage staining. These studies revealed that MCAGF8s had a surprising skeletal

phenotype. Specifically, while the calvaria of controls were composed of bone (Fig. 4C), the

bone of the MCAGF8 calvaria was mostly replaced with a matrix that stained with neither alcian

blue nor alizarin red, but was still durable enough to persist through a staining protocol that

degrades soft tissue. In comparison, the endochondral bones and cartilage throughout the rest of

the body stained normally (Fig. 4I). Dorsal views of the skull revealed a ring of bone

surrounding the midline, with non-stained matrix radiating ventrally from there (Fig. 4J). Any

sutures, if present, were difficult to identify due to the prevalence of the non-stained matrix. The

skeletal defect was not uniformly distributed throughout the calvaria, as there was a greater

amount of ossification in the region where the parietal bones would normally form (Fig. 4F, J).

One possibility for the presence of bone in this region is that there was reduced activity of Msx2-

Cre in this location as shown by fate mapping (Fig. S2C). While the primary affected area was

the cranial vault, there were also defects within other components of the craniofacial skeleton

(Fig. 4H, K). Several skeletal elements were smaller, including the palatal process of the maxilla

and palatal process of the palatine (Fig. 4K), consistent with the cleft secondary palate (Fig. 4E).

There was also an overall shortening of the snout and its associated skeletal elements in the

mutants (Fig. 4F, J, K) as well as a reduced supraoccipital bone (Fig. 4J, K). Additionally, like

the MR26F8s, the MCAGF8s have ectopic bone growth within the eye sockets (Fig. 4J, K).

Overall, the MCAGF8s exhibit several abnormalities, including craniofacial patterning defects,

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loss of the eye, and limb defects. Most strikingly, however, was the replacement of the cranial

vault bones with an abnormal non-stained matrix.

To determine the origins and composition of this unusual matrix, we examined critical

stages in embryological development when bone and cartilage are being formed. At E14.5, prior

to extensive bone mineralization, we utilized a cartilage staining protocol with Bouin’s fixative

and alcian blue (Fig 5A, B). This staining regimen was also used at E16.5 alongside a standard

protocol that uses alcian blue and alizarin red to stain cartilage and bone, respectively (Fig 5C-

F). Finally, the standard protocol was used to stain E18.5 samples (Fig. 5G, H). Differences

between the controls and MCAGF8s were first apparent at E14.5 (Fig 5A, B). In the E14.5

controls, cartilage was forming at the site of the jaw, snout, cranial base and otic capsule (Fig.

5A). The MCAGF8s also had cartilage in these locations but further displayed ectopic cartilage

throughout almost the entire cranial vault which was still apparent at E16.5 (Fig. 5D).

Surprisingly, however, this same matrix did not stain at E16.5 or E18.5 when alcian blue and

alizarin red were used for skeletal staining, possibly due to the difference in pH between the two

staining solutions (Fig. 5F and Fig 4F, respectively). Notably, though, this matrix did stain when

we employed toluidine blue, another cartilage stain, alongside alizarin red and alcian blue on

E18.5 preparations (Fig. 5G-H, Fig. S6F-G). Histological analysis of sectioned material

confirmed that there was a loss of both ossification and mineralization in the MCAGF8 skulls

compared with controls (Fig. 5I, J, Fig. S6A-C). Instead, there was development of tissue

comprised of nuclei within lacunae surrounded by a matrix that stains strongly with toluidine

blue, typical of hyaline cartilage (Fig. S6D-E). We next used further histological analysis to

compare this skull cartilage to control cartilage from the cranial base to probe for differences that

might reflect the unusual staining properties of this ectopic tissue in the MCAGF8s. A notable

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difference in matrix composition then became apparent when a dual alcian blue Periodic Acid-

Schiff (PAS) staining protocol was employed (Fig. 5J-L). Specifically, MCAGF8 cartilage

stained weaker with alcian blue and stronger with PAS indicating that this cartilage has less

acidic mucins (i.e. glycosaminoglycans) and more neutral mucins (i.e. polysaccharides and

mucosubstances) than control samples. Thus, overall, the high Fgf8b levels cause cranial

chondrogenesis to be favored over osteogenesis in the MCAGF8s. However, the resulting

cartilage is abnormal with an altered ratio of neutral to acidic mucins.

2.4 Differential effects of increased Fgf8 transcript levels on intramembranous and

endochondral bones

While the expression pattern of the Msx2-Cre transgene was suitable for studying

development of the calvaria, it was not efficient at directing Fgf8b to other elements of the

craniofacial skeleton that form by intramembranous ossification, such as the dentary bone. At

the same time, the expression of Msx2-Cre in the limb ectoderm did not appear to affect

ossification of endochondral bones in the limb, whereas the supraoccipital bone in the skull,

which forms via endochondral ossification, was greatly reduced in the MCAGF8s (Fig. 4). Thus,

to ascertain how increased Fgf8b expression affects both types of ossification more globally

throughout the embryo, we used the two new Fgf8b alleles alongside OC-Cre (osteocalcin-Cre).

OC-Cre is expressed in osteoblasts in both endochondral and intramembranous forming bone

beginning around E14.5 (Zhang et al., 2002) (Fig. S7). Once again, the increase in Fgf8b

expression resulted in developmental defects, with the resulting OR26F8 (OC-Cre;R26Fgf8b) and

OCAGF8 (OC-Cre;CAGFgf8b) mice being readily distinguishable from controls by gross

morphology at E18.5, due to shortened mandibles and limbs (Figs. S8, S9 and data not shown).

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Both OR26F8 and OCAGF8 mice had a number of similar skeletal defects, which were

more severe in the latter (Fig. 6). The calvaria were mostly replaced by an unstained cartilage

matrix, in common with Msx2-Cre-based mice, although there were some notable differences in

the most affected regions. Thus, whereas Msx2-Cre-based mice had some ossification

surrounding the midline sutures, this top most region of the cranial vault consisted solely of non-

stained matrix in the OC-Cre mutants (Fig. 6E, F, and Fig. S10). In contrast, there was more

lateral ossification in both OC-Cre mutants (Fig. 6B, C), than there was in the MCAGF8s (Fig.

4F), likely due to the later initiation of OC-Cre expression. One notable similarity, though, was

that the parietal bones had the most ossification and were least affected when either Msx2-Cre or

OC-Cre were employed. With respect to additional craniofacial bones that form via

intramembranous ossification, both OR26F8 and OCAGF8 mandibles were shorter than the

controls and did not ossify normally, but instead were partially replaced by non-stained matrix

(Fig. 6J-L). Similarly, the bones of the maxilla and palate had severely decreased ossification,

and were mostly replaced by non-stained matrix, particularly in the OCAGF8s (Fig. 6D-I). In

contrast, for bones formed via endochondral ossification we did not observe a replacement of

bone by cartilage or by an abnormal unstained cartilage matrix (Fig. 6G-I, M-O). Nevertheless,

many endochondral bones, such as the basioccipital in the cranial base, were less mineralized

than controls as judged by Alizarin red staining (Fig. 6G-I), and there were also size and shape

differences, most notably for the limb skeleton. Therefore, increased Fgf8b expression, derived

from either adjacent tissues or from within the developing bone itself, significantly impacted

intramembranous ossification, but also caused additional but variable defects wih respect to

endochondral bone formation.

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2.5 Dysregulation of bone and cartilage related genes in the MCAGF8 skull

Given the differential effect of increased Fgf8b levels on the process of intramembranous

ossification, we next explored the basis of this phenomenon by comparing gene expression in the

developing cranial vault between MCAGF8s and controls using RNAseq (Fig. 7A). We dissected

cranial mesenchymal tissue between the skin and brain from the controls and MCAGF8 mutants

at E14.5, when cranial bone development is initiating. We also sampled tissue from E14.5

control cranial base as a baseline for hyaline cartilage gene signatures. The results from these

comparisons are presented in Tables S1 and S2, respectively, and summarized in Fig 7B. Note

that, consistent with the removal of the skin where the MCAGF8 allele was expressed prior to

RNAseq analysis, Fgf8 was not differentially expressed between MCAGF8 and control skull

tissue (Table S1). In contrast, both Dusp6 and Spry4, which are reporters - as well as feedback

regulators - of FGF signaling, were upregulated in the MCAGF8 mesenchyme. Additionally,

HtrA1, which directly cleaves and deactivates FGF8 in the extracellular environment, was

upregulated in the mutants, potentially indicative of attempted feedback regulation of FGF8

signaling. These observations are consistent with FGF8 signaling from the ectoderm affecting

gene expression in the underlying mesenchyme.

Next, to identify important categories of differentially expressed genes between the

control and MCAGF8 cranial vault samples, we used DAVID functional annotation clustering

and functional annotation charting to analyze these datasets (Huang et al., 2009). Comparison of

MCAGF8 and control cranial vaults yielded differentially-expressed genes annotated to

categories including bone development, ossification, osteoblast differentiation, regulators of

bone mineralization, Wnt signaling, and categories likely reflective of the shift to cartilage

differentiation - such as extracellular matrix, glycoproteins, glycosylation, and disulfide bonds

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(Table S1). Based on this DAVID analysis, we next focused on the expression of transcripts

encoding major structural components of bone and cartilage in the calvaria samples of controls

and MCAGF8s. These analyses showed MCAGF8s had major increases in the expression of

Col2a1 and Col9a3, two collagens typically found together in hyaline cartilage (Mayne, 1989)

(Fig. 7C, left). Conversely Col24a1, a gene involved in osteoblast differentiation and bone

formation (Koch et al., 2003) was down-regulated (Fig. 7C, Table S1). Two additional genes

involved in bone formation, Ibsp and Spp1 were down-regulated by >30-fold in the MCAGF8s

while others associated with this process were decreased to a lesser extent including Sparc,

Ifitm5, Fkbp11, Phospho1, Itm2a, Cthrc1, and Clec11a (Fig. 7C, Table S1). Together, these

findings indicate that the mutants favor a cartilage rather than a bone program of gene

expression. However, the effects of ectopic Fgf8b on the transcription of genes involved in bone

development were not uniform (Fig. 7C), implying that specific aspects of osteogenesis were

more severely affected. Alongside these structural components, genes responsible for collagen

processing, and extracellular matrix (ECM) formation and modification, including Sulf1, Ost4,

and Ostc, were also dysregulated in the MCAGF8 skulls (Fig. 7C, right; Table S1). The aberrant

expression of genes involved in collagen modification, as well as the changes in genes encoding

enzymes that alter glycosylation and sulfation of ECM proteins, provides a possible explanation

for the altered staining properties of the cartilage found in the MCAGF8s.

Next, we determined if there were gene expression differences between the cartilage of

the MCAGF8 skull versus control cranial base cartilage (Table S2). This analysis revealed that

more genes were dysregulated in this comparison, and dysregulated at higher levels, than

between the control and mutant cranial vault samples (Fig. 7B). The most striking observation

was that Col10a1, which is critical for endochondral bone formation (Gu et al., 2014), was

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expressed at >100 fold lower levels in the MCAGF8s (Table S2). Several additional genes

involved in endochondral ossification were also differentially expressed between these two

samples, with higher levels in the control cranial base compared with the MCAGF8 cranial vault

cartilage including Mmp13 (>10 fold), 3110079O15Rik /Snorc (>10 fold), Ihh (~10 fold), Spp1

(>5 fold), and Ibsp (>5 fold) (Table S2). Thus, although differentiation in the cranial vault favors

a chondrogenic versus osteogenic trajectory in the presence of upregulated Fgf8b signaling in

MCAGF8 mice, it is not undergoing normal endochondral ossification and displays distinct

histochemical properties from other cartilage in the body (as shown in Figure 5).

2.6 MCAGF8 mutants have impaired differentiation and display dysregulated Wnt signaling

We next examined the balance between regulators of chondrogenesis and osteogenesis in

the control and MCAGF8 skulls to determine how Fgf8b was exerting its mechanistic effects.

These analyses indicated that several regulators of cartilage/bone differentiation were

dysregulated in the MCAGF8s (Fig. 7D). First, there was a shift towards expression of

transcription factors that are linked with a chondrocyte cell fate and inhibit ossification. In

particular, Sox9, which stimulates chondrocyte differentiation (Akiyama et al., 2002; Lefebvre et

al., 1997; Lefebvre and de Crombrugghe, 1998), Twist2, an inhibitor of osteoblast differentiation

(Bialek et al., 2004; Lai et al., 2011; Lee et al., 1999), and Irx1 and Irx5 - markers of immature

chondrocytes - were all upregulated (Fig. 7D, Table S1). Conversely, mRNAs for the

Sp7/Osterix, Mef2c, and Satb2, transcriptional regulators that stimulate bone differentiation, were

down-regulated. Comparing the MCAGF8 skull vault with the control cranial base indicated that

the latter was more similar to the control skull vault with respect to the expression profiles of

these same transcription factors (Table S2). This finding was again consistent with the cranial

base initiating endochondral bone formation, whereas the MCAGF8 skull vault has an expression

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profile that favors cartilage over bone, reinforcing the difference between these two types of

cartilage. We also examined the expression of Sox9 and Sp7 using in situ hybridization in

control and MCAGF8 skulls (Fig. 7F-I). These results demonstrated that Sox9 was upregulated

in the MCAGF8 skulls as early as E12.5, before overt ossification occurs, and this was mirrored

by a later decrease in Sp7 expression at E14.5. Next, to determine if dysregulation of osteoblast

regulatory genes correlated with impaired differentiation, we examined alkaline phosphatase

activity, a marker of bone differentiation, at E16.5 using a liquid Fast-Red staining protocol. The

MCAGF8s had very little alkaline phosphatase activity (Fig. 7K), compared to controls (Fig. 7J).

Together, these data indicate that Fgf8b-induced changes early in the differentiation process are

likely responsible for favoring chondrogenesis over osteogenesis in the skull vault.

It has been previously shown that loss of β-catenin protein in the skull can switch

development of the calvaria from bone to cartilage (Goodnough et al., 2012; Tran et al., 2010).

WNT signaling was also highlighted by the DAVID functional annotation clustering in the

current analysis where it occurred as the 5th annotation cluster with an enrichment score of 4.2

(Table S1). Therefore, we next investigated Wnt signaling pathway aberrations in the MCAGF8

skull. These analyses revealed that multiple inhibitors and negative regulators of Wnt signaling

were upregulated in the MCAGF8s, including Apcdd1, Axin2, Kremen2, Nkd1, Nkd2, and

Prickle1 (Fig. 7E and Table S1). At the same time, several genes that positively regulate WNT

signaling were upregulated in the MCAGF8s, including those encoding the Wnt ligands 4, 5a, 6,

7b. Additionally, members of the canonical Wnt signaling pathway, including frizzled receptors

(Fzd9, Fzd10) and the transcription factor Lef1 (Fig. 7E) were also upregulated. In contrast, only

one Wnt ligand, Wnt2, and one WNT pathway inhibitor, Dkk1, were significantly

downregulated. In addition to the striking impact of Fgf8 overexpression on Wnt signaling, there

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were also clear effects on other signaling pathways, notably the Hedgehog, Insulin-like Growth

Factor, and TGF-β/BMP pathways that might all be predicted to alter bone growth and

development (Table S1).

2.7 Reducing Axin2 gene dosage partially rescues the cranial skeleton phenotype

While the expression of many signaling genes were altered in the MCAGF8 model, the

RNAseq data alone do not provide a clear indication of how these alterations could impact

intramembranous ossification. However, given the many alterations in the Wnt signaling

pathway, as well as the connections between β-catenin and intramembranous ossification, we

further probed the interaction between FGF and WNT signaling in vivo. Specifically, we bred an

Axin2lacZ allele into the MCAGF8 background (Fig. 8). Axin2 expression is stimulated by the

canonical Wnt signaling pathway and also acts as a feedback regulator of the pathway via

degradation of β-catenin (Jho et al., 2002). The Axin2lacZ allele replaces the normal gene with

NLS-lacZ transgene (Lustig et al., 2002), so that normal Axin2 expression decreases in

heterozygotes, theoretically increasing Wnt signaling. Initial gross morphological analysis of the

MCAGF8;Axin2+/- indicated that several MCAGF8 phenotypes still persisted, including the

missing eye, the shortened snout, and the cleft palate (data not shown). However, subsequent

skeletal staining indicated a clear difference in skull ossification between the two models. Thus,

in contrast to the MCAGF8 skulls, which had some ossification near the midline suture with non-

stained matrix throughout most of the skull (Fig 8B, E), the MCAGF8;Axin2+/- mice all showed

an expansion of ossification midway down the skull, with non-stained matrix only on the lateral

part of the skull, nearest the cranial base (Fig. 8C, F). The expansion of bone development in

MCAGF8;Axin2+/- mice also led to the reappearance of the lambdoid suture (Fig. 8F), a feature

not apparent in the MCAGF8s due to the absence of bone (Fig. 8E). However, increased bone

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development did not lead to rescue of all sutures, as the MCAGF8;Axin2+/- mice displayed

craniosynostosis of the coronal suture (Fig. 8F). Indeed, the suture pattern in the

MCAGF8;Axin2+/- mice, with an open lambdoid suture and craniosynostosis of the coronal

suture, was similar to that of the MR26F8 mice (Fig. 2). Interestingly, the reduced Axin2 gene

dosage also rescued the hypoplasia of the supraoccipital bone seen in MCAGF8s, but consistent

with the gross morphology, it did not alter the skeletal defects associated with the eye, snout and

secondary palate. Thus, increasing Wnt signaling by reducing Axin2 gene dosage partially

rescued the shift from osteogenesis to chondrogenesis in the skull vault of MCAGF8s, but did not

rescue the coronal craniosynostosis or craniofacial shape phenotypes.

3 Discussion

Craniosynostosis is a relatively common human craniofacial defect that frequently

involves FGF signaling pathway mutations, particularly in the FGF receptors. Here we have

probed this pathway further by manipulating the dosage of an FGF ligand during mammalian

embryonic development. Our characterization of how increased Fgf8b expression impacts

skeletal formation, particularly within the cranial vault, yields several interesting and previously

undocumented findings. First, the cranial vault has a dose-dependent response to FGF8

signaling, with moderate levels of Fgf8b overexpression leading to coronal craniosynostosis and

high levels of Fgf8b overexpression leading to ectopic cartilage formation. Second, a balance

between FGF and WNT signaling is critical for driving the decision between an osteoblast or

chondrocyte cell fate in vivo for the mesenchymal cells of the calvaria that would normally form

bone via intramembranous ossification. Third, the effect of ectopic Fgf8b on craniofacial

development is partially dependent on the timing and location of expression. Finally, Fgf8b

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overexpression affected both intramembranous and endochondral bone development, although

the effects were overall more severe for the former skeletal elements.

Cranial suture patency is normally maintained via a delicate balance of proliferation and

differentiation. Moderate overexpression of Fgf8 in the cranial ectoderm of MR26F8 mice

affects this balance resulting in coronal craniosynostosis and narrowing of the lambdoid suture.

However, premature suture closure was not simply caused by accelerated ossification at the

sutures. Instead, the coronal and lambdoid sutures were wider at birth than in the controls.

However, by P12, the coronal suture had completely fused and the lambdoid suture was narrower

than in the controls. Therefore, in this model there is a pattern of slower bone maturation

accompanied by a failure to develop appropriate suture architecture that subsequently results in

overgrowth and craniosynostosis. Notably, such delayed ossification followed by catch-up

growth and subsequent obliteration of the sutures has also been observed in two mouse models

with either loss or gain of function alterations in FGFR2 (Eswarakumar et al., 2002; Mai et al.,

2010). In fact, the majority of mouse craniosynostosis models resulting from FGF signaling

aberrations have been generated using activating mutations in FGF receptors, particularly Fgfr2

and Fgfr3 (Flaherty et al., 2016; Su et al., 2014). Such mutations may cause ligand independent

dimerization and activation of the receptors with downstream intracellular signaling

consequences dependent on the position and nature of the mutation within the gene (Burke et al.,

1998; Monsonego-Ornan et al., 2000; Neilson and Friesel, 1996; Sarabipour and Hristova, 2016).

Alternatively, some FGFR activating mutations, including ones that cause Apert Syndrome, do

not lead to ligand independent receptor binding, but instead cause increased affinity for different

FGF ligands (Anderson et al., 1998). This is striking as patients with Apert Syndrome, as well as

those with Crouzon and Pfeiffer syndrome, often exhibit craniofacial phenotypes similar to those

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found in our mouse model of moderate Fgf8b overexpression - a characteristic appearance that

includes a shortened face and premature fusion of the coronal sutures. Additionally, like in our

model, patients with Apert syndrome frequently exhibit other symptoms such as cleft palate

and/or limb defects, including syndactyly as well as occasional polydactyly (Katsianou et al.,

2016; Ko, 2016).

Using a second allele that produces higher levels of Fgf8b expression, an unexpected

phenotype emerged in which extensive cartilage formation replaced ossification across the

cranial vault. Moreover, this ectopic hyaline cartilage did not have a gene expression profile

indicative of control tissue fated to undergo endochondral ossification, but instead had several

unusual properties. In particular, the cartilage extracellular matrix had an abnormal balance

between acidic and basic mucins. We postulate that this switch may be a feedback control

mechanism to alter the ability of the extracellular milieu to interact with the unusually high

levels of Fgf8b in our model since FGFs normally require heparan sulfate proteoglycans for

optimal interaction with the FGFRs (Ornitz, 2000).

The extensive switch from osteogenesis to an abnormal-type of chondrogenesis in the

MCAGF8 calvaria is a novel phenotype associated with altered Fgf signaling. Previous studies

using a transgene to express Fgf9 in the cranial mesenchyme also induced extensive cartilage

formation in the cranial vault, particularly for the region normally occupied by the parietal bones,

but in this instance the cartilage was eventually replaced by bone (Govindarajan and Overbeek,

2006). We suspect that one reason for the difference between the MCAGF8 strain and the Fgf9

transgenic strain is that increased levels of Fgf9 only occur transiently in this latter system

potentially allowing osteogenesis at later stages, although there might also be additional

differences related to the Fgf ligand used in each model. In contrast, once activated by Cre

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recombinase, the MCAGF8 strain will continue to express ectopic Fgf8b constitutively, and we

postulate that this maintains the abnormal cartilage phenotype and prevents bone formation.

Since the MCAGF8 mice die perinatally, we cannot rule out the possibility that Fgf8-induced

cartilage might eventually undergo delayed endochondral ossification. However, we do not

favor this possibility as in a related model system, in which Fgf8b is expressed only within the

frontonasal prominence, the white matrix is never replaced by bone even though these mice

survive into late adulthood (Fig. S13).

In addition to the aforementioned Fgf9 transgenic mice, previous studies have shown that

more limited cartilage formation can also occur in mice expressing certain Fgfr2 mutations. In

particular, mice homozygous for an activating mutation in FGFR2 (W290R), mimicking human

Crouzon Syndrome, exhibit thickened cartilage underlying the cranial bones (Mai et al., 2010).

Similarly, in a mouse model for Apert Syndrome, a heterozygous activating mutation in FGFR2

(S252W) leads to coronal synostosis and ectopic cartilage at the sagittal suture (Wang et al.,

2005). Beyond the FGF signaling pathway, ectopic cartilage formation has also been observed

due to aberrations in WNT signaling (Day et al., 2005; Goodnough et al., 2012; Hill et al., 2005;

Tran et al., 2010). Specifically, removal of β-catenin from cranial bone progenitors results in

near complete transformation of the skull bones to cartilage – a phenotype that is more akin to

the MCAGF8s than that seen with the above mentioned W290R and S252W Fgfr2 alleles.

In support of this latter observation gene expression analysis of MCAGF8 skulls revealed

dysregulation of WNT signaling in the Fgf8b-induced cartilage. The alterations in the Wnt

pathway are accompanied by an upregulation of chondrogenic differentiation markers during a

period that would normally reflect early bone development. Furthermore, an inhibitor of

osteoblast differentiation, Twist2, was also increased in MCAGF8 calvaria concomitant with

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reduced expression of osteogenic markers and genes critical for osteoblast differentiation.

Together, these processes may shift the fate of mesenchymal stem cells from an osteogenic to

chondrogenic potential. Alternatively, the ectopic Fgf8b may block a set of cells that would

normally form the bony calvaria and instead stimulate adjacent underlying cells that can then

adopt a cartilage fate. Although we cannot currently distinguish between these models, it is

apparent that these decisions are at least partly due to altered WNT signaling. However, since

both positive and negative WNT signaling pathway regulators were upregulated it was difficult

to predict if the influence of increased FGF ligand expression was to activate, repress, or else

leave the overall throughput of the canonical WNT pathway unaffected. To distinguish between

these possibilities, and determine if WNT dysregulation was contributing to the cranial

ossification phenotype, we utilized mice containing Axin2lacZ. Since Axin2 is an inhibitor of the

Wnt pathway, mutations in this gene can increase WNT signaling compared with controls

(Minear et al., 2010). Notably, although homozygous loss of Axin2 can cause craniosynostosis

of the interfrontal/metopic suture, heterozygotes were unaffected (Yu et al., 2005). Therefore, a

heterozygous Axin2LacZ mutation was bred into the MCAGF8 background to ascertain if there

was a genetic interaction between the WNT and FGF pathways in our model. Significantly,

MCAGF8;Axin2lacZ mice had a less severe phenotype than MCAGF8s with substantial rescue of

ossification of the cranial vault, suggesting that high levels of Fgf8b overexpression causes a

down-regulation of WNT signaling. Supporting this hypothesis, previous studies in tissue culture

using an osteoblast cell line also demonstrated that Fgf signaling was antagonistic to the role of

the WNT pathway in driving bone differentiation (Ambrosetti et al., 2008). Although

ossification was partially rescued by the presence of the Axin2LacZ allele, additional patterning

defects observed in MCAGF8 mice were not, including a shortened snout, domed skull, and

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missing eye, indicating that these aspects of Fgf8b function are not as responsive to, or do not

rely on, WNT signaling as an intermediate. These latter conclusions were also supported by

studies in which we bred the Axin2LacZ mutation onto the MR26F8 background. At the level of

gross morphology, the MR26F8 and MR26F8; Axin2lacZ mice had equivalent craniofacial defects,

including coronal suture synostosis, ectopic bone within the orbit, and a misaligned snout

accompanied by frontonasal suture defects. Therefore, these pathologies are not rescued in the

MR26F8 model by the presence of the Axin2LacZ allele, despite the lower levels of Fgf8b

expression compared with the MCAGF8 model (L.S. and T.W. unpublished).

The impact of Fgf8b overexpression on ossification of bones other than the calvaria was

examined using OC-Cre transgenic mice to target all developing bones. This approach enabled

any differential impact of Fgf8b overexpression on intramembranous versus endochondral

ossification to be analyzed. In the appendicular and axial skeleton, OR26F8 and OCAGF8 mice

had a moderate impact on the overall process of osteogenesis, although – in common with the

Msx2-Cre Fgf8b strains - there was an impact on limb outgrowth and patterning. These defects,

including polydactyly and forelimb-hindlimb fusions, were similar to those previously observed

with other models that overexpress the FGF ligands FGF2, FGF4 and FGF8 in the limb (Coffin

et al., 1995; Lin et al., 2013; Lu et al., 2006). In the craniofacial skeleton, using either Cre

transgene caused a transformation from intramembranous forming skull bone to abnormal

unstained matrix, indicating that this transformation can occur regardless of whether Fgf8b is

expressed from an earlier timepoint in the ectoderm or from later in the bone progenitors.

Interestingly, both transgenes also had similar differential effects on particular bones of the skull

vault, with the parietal bones less affected than the frontal bones. In this respect, previous

studies suggest that osteoblast location within the skull can impact the degree of FGF signaling

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activation. Specifically, osteoblasts from neural crest-derived bones, such as the frontal bone,

express FGF osteogenic ligands and their receptors at higher levels than osteoblasts from

paraxial mesoderm-derived bones, such as the parietal (Behr et al., 2010; Quarto et al., 2009).

Alternatively, since trasgenic Fgf9 expression has the greatest impact on parietal bone

ossification it is possible that these findings reflect the differential activity of particular FGF

ligands on skull development (Govindarajan and Overbeek, 2006). Thus, overexpression of

Fgf8b appears to be more effective at altering cell fate in neural crest-derived bones than paraxial

mesoderm-derived bones. Future gene expression studies on individual sutures and their

associated progenitors may help to further elucidate why particular bones and sutures respond

differentially to alteration of specific signaling pathways to cause the various types of

craniosynostosis (Brinkley et al., 2016; Teven et al., 2014).

The ability of OC-Cre to target other bones in the craniofacial skeleton, such as those of

the jaw and cranial base, also extended the observation that the increased Fgf8b levels had a

greater overall impact on the intramembranous bones as opposed to those that form via

endochondral ossification. One notable exception to the general model of Fgf8b action was the

supraoccipital bone, which forms via endochondral ossification. Furthermore, this bone showed

a differential ossification response depending on whether Fgf8b was activated by Msx2-Cre

versus OC-Cre. Thus, while this endochondral bone was not affected in the OC-Cre mutants, it

was severely hypoplastic in the MCAGF8 mice. We speculate that the ossification defect in

MCAGF8 mice is caused by an early decrease in Mef2c expression that we detect using RNAseq

analysis (Fig 7D, Table S1), as previous studies have shown that the supraoccipital is particularly

sensitive to loss of this gene (Arnold et al., 2007).

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Currently, the molecular mechanisms responsible for the early transition from progenitor

cell to chondrocyte or pre-osteoblast are not well understood. The availability of these new

alleles will allow for further analysis of how different Fgf8b expression levels can impact cell

fate decisions during skeletogenesis. Furthermore, alongside other mouse models that

manipulate FGF ligands and receptors, they may help tease apart the combinatorial manner by

which specific ligand:receptor interactions can lead to the different forms of craniosynostosis,

with potential therapeutic applications. These alleles can also be used to assess how other

tissues, such as the dura mater, can influence skull ossification and development (Senarath-Yapa

et al., 2012). Finally, our results on the dose-dependent effects of Fgf8b on the cranial vault

have implications for disease treatment. Some craniosynostosis patients require repeat surgeries

due to the re-fusion of the sutures and so a detailed understanding of the molecular pathways

downstream of FGF signaling could lead to the rational design of treatments to prevent re-fusion.

Unraveling the role of FGF signaling in cranial ossification and its downstream molecular

consequences greatly expands our understanding of human craniofacial disorders and provides

the possibility of novel treatments for those pathologies.

4 Materials and Methods

4.1 Mice

4.1.1 Mouse Strains

All mouse experiments were done with the approval of the Institutional Animal Care and

Use Committee of the University of Colorado Denver. Embryonic day 0.5 was considered to be

noon on the day a copulatory plug was found.

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We generated two new ROSA26-based Lox-Stop-Lox alleles that can be used to regulate

the levels of Fgf8 expression under the control of Cre recombinase transgene activity—R26F8

and CAGF8. R26F8 GOF mice have an allele with the endogenous ROSA-26 promoter

separated from Fgf8 by a LoxP STOP LoxP cassette. When the Stop cassette is removed by a

Cre recombinase, mice express moderate levels of Fgf8 expression in a tissue specific manner.

Similarly, CAGF8 GOF mice have an allele with the strong CAG promoter separated from Fgf8

by a LoxP STOP LoxP cassette. Given the stronger promoter activity of CAG (Qin et al., 2010),

Cre recombinase mediated removal of the stop cassette leads to mice expressing high levels of

Fgf8.

For R26LSL Fgf8b, an ~0.8kb mouse Fgf8b cDNA was PCR amplified using Elongase

(Thermo Fisher Scientific Waltham, MA) from a plasmid vector provided by Dr. Mark

Lewandoski (NCI) using the forward primer Fgf8b FWD and the reverse primer Fgf8b REV (all

primer sequences are provided in Table S3). This procedure introduced an XhoI site just prior to

an ATG start codon and a HindIII site downstream of the stop codon. Following subcloning into

TA vector (Thermo Fisher Scientific Waltham, MA) and sequence confirmation, the insert was

digested with HindIII and this site blunted with Klenow fragment of DNA polymerase I in the

presence of all 4 dNTPs (all enzymes for cloning were obtained from New England Biolabs,

Ipswich, MA). Subsequently, the insert was released using XhoI digestion and cloned into the

XhoI and SmaI digested pBTG vector (pBigT-IRES-GFP; Addgene, Cambridge, MA). Next, this

insert was released with PacI and AscI and subcloned into the vector pROSA26PAm1 (Addgene)

using the same enzymes, to generate the R26F8 GOF targeting vector.

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For R26LSL CAG Fgf8b, an ~1.7kb SalI – EcoRI fragment containing the CAG promoter

sequence was isolated from the plasmid CAG-GFP (Addgene) and cloned into a vector

containing a PacI site upstream of the SalI site as well as NotI and NheI sites downstream of the

EcoRI site. Subsequently, the NotI – NheI restriction fragment from PGKneotpAlox2 (Addgene)

which contains the floxed selection cassette was cloned downstream of the CAG promoter.

Finally, the new PacI – NheI insert fragment was inserted into the previous Fgf8 GOF targeting

vector to generate the CAGF8 GOF targeting vector. Both targeting vectors were then linearized

with MluI, gel purified, and then electroporated into 129S1/Sv W9.5 ES cells.

R26LSL Fgf8b ES cell clones were screened using the primer pairs (RF5 + BTR) and (GFP F

+ R3R) to detect appropriate homologous recombination with the endogenous R26R locus at the

5' and 3' end respectively. PCR reactions employed KOD Hot Start DNA Polymerase as

recommended by the manufacturer (EMD Millipore, Billerica, MA). Only correctly targeted

clones produced a 1.35kb band (5' end) and ~4.8kb band (3' end) and these were karyotyped

prior to injection into blastocysts. R26LSL CAG Fgf8b ES clones were screened in analogous fashion

using the primer pairs (RF5 + CMV R1) and (GFP F + R3R) and produced similar sized bands at

both the 5' and 3' ends following homologous recombination.

Following germline transmission, both the R26F8 and CAGF8 mice were maintained on

an outbred Black Swiss background and eventually bred to homozygozity and then maintained as

homozygous colonies. Msx2-Cre mice ((Msx2-Cre)5Rem) were supplied and originally

described for their use in limb studies by Rob Maxson (Sun et al., 2000). ROSA26 Cre reporter

mice (B6.129S4-Gt(ROSA)26Sortm1Sor/J), Axin2lacZ (B6.129P2-Axin2 tm1Wbm/J), and OC-Cre

(B6N.FVB-Tg(BGLAP-cre)1Clem/J) were obtained from Jackson Laboratory (Bar Harbor,

ME). Note that: (1) as mice with Cre alleles are being bred to homozygous GOF mice, all

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offspring will inherit a GOF allele and mutants with an activated GOF allele can be identified by

the presence of a Cre allele; and (2) the combination of the Cre alleles and the GOF alleles leads

to sirenomelia (R26LSL Fgf8b) or perinatal lethality (R26LSL CAG Fgf8b) therefore it is not possible to

generate mice containing these Cre recombinase transgenes with homozygous recombined GOF

alleles.

4.1.2 Genotyping

PCR based genotyping was performed using DNA extracted from yolk sacs or tail clips

using DirectPCR Lysis Reagent (Viagen Biotech. Inc, Los Angeles, CA) plus 10 ug/ml

Proteinase K (Roche, Basel, Switzerland) followed by heat inactivation at 85 °C for 45 min.

Mutants were identified via PCR using the Qiagen DNA polymerase kit, including the optional

Q Buffer solution (Qiagen, Valencia, CA). Mice carrying Msx2-Cre or OC-Cre transgenes were

identified using the primer pair Cre1 and Cre3 at an annealing temperature of 70°C, yielding a

band at ~450bp. R26R mice and the Axin2lacZ allele were identified using the primer pairs

oIMR0039 and oIMR0040. The R26LSL Fgf8b allele was identified using the primer pair ROSA F

and BTR, whereas the primer pair ROSA F and CMV R1 was used to identify the R26LSL CAG Fgf8b

allele. In both cases, ROSA F + ROSA R was used to identify for the presence of the wildtype

allele.

4.2 Skeletal Analysis

4.2.1 Bone & Cartilage Staining

Embryos, pups, and adult mice were collected at appropriate time points and processed as

previously described (McLeod, 1980). In brief, following euthanasia and the removal of the skin

and organs, the mice were dehydrated in ethanol for a minimum of three days before being

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incubated in acetone (at least two days). Subsequently, they were incubated in staining solution

composed of ethanol (70%), acetic acid (5%), alcian blue (0.3%), and alizarin red (0.1%) at 37°C

for a minimum of 5 days before being cleared in 2% KOH. In skeletal preparations where

toluidine blue was utilized, 0.1% toluidine blue was added to the above staining solution for one

hour before clearing.

4.2.2 Cartilage Staining

Embryos (E13.5-E16.5) were collected and processed as previously described (Jegalian

and De Robertis, 1992). In brief, embryos were fixed in Bouin’s solution for two hours followed

by a series of washes in a solution of 70% ethanol plus 0.1% NH4OH until there was no

remaining yellow (Bouin’s) color. Next, the embryos were equilibrated in 5% acetic acid (2x 1

hr washes) and incubated overnight in a solution of alcian blue (0.05%) and acetic acid (5%).

The embryos were then washed twice with 5% acetic acid (~1 hr washes) and twice with

methanol (minimum 1 hr washes). Finally, the embryos were cleared in BABB (1:2 benzyl

alcohol/benzylbenzoate).

4.2.3 Section Histochemistry

For sections from E18.5 embryos, as well as P0 and P12 pups, the embryos/pups were

fixed in 70% ethanol (skin removed from P12 pups first). They were then sent to the Yale

Orthopedic Histology and Histomorphometry Laboratory for plastic embedding and sectioning,

as well as staining. Four stains were utilized: Goldner’s Trichome (Gruber, 1992), von Kossa

(George Clark, 1981), Alcian Blue + PAS (Mowry, 1956), and Toluidine Blue (Sridharan and

Shankar, 2012). Suture widths were calculated as the average of 6 sections—2 sections each

from 3 biological replicates.

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For E16.5 sections, the embryos were fixed overnight in 4% PFA. Following fixation,

tissue was dehydrated in a graded series of ethanol and xylene and subsequently embedded in

paraffin. After embedding, the embryos were sectioned with a Leica RM 2235 at 10µm onto

charged glass slides. After drying, sections were deparaffinized though a graded series of xylene

and ethanol to 70% ethanol. The sections were then stained in 0.04% toluidine blue O in 0.1 M

sodium acetate buffer (pH 4.0), rinsed briefly and counterstained with Methyl Green (Vector

Laboratories, Inc., Burlingame, CA) as per the manufacturer’s instructions. Coverslips were

applied with Fluoromount-G (Southern Biotech, Cat no. 0100-01)

.

4.3 In Situ Hybridization/Immunofluorescence

4.3.1 Section In Situ Hybridization

Probes were generated by cloning a unique fragment (primer sequences given upon

request) into a TOPO vector (Life Technologies, Grand Island, NY), using cDNA synthesized

from mouse embryonic mRNA, as a template. cDNA was generated using the Superscript® III

First-Strand Synthesis System (Life Technologies, Grand Island, NY), as per manufacturer’s

instructions. Sequence verified plasmids were linearized and antisense probes synthesized using

an appropriate DNA-dependent RNA polymerase (T7/T3/SP6) and DIG RNA labeling mix

(Roche, Basel, Switzerland).

Embryos were fixed in 4% paraformaldehyde overnight at 4°C and then incubated in

30% sucrose in PBS at 4° until the embryos sank to the bottom of the well (overnight to several

days). The embryos were then incubated in a 1:1 solution of 30% sucrose: OCT (Sakura Finetek,

Torrence, CA), rocking at 4°, overnight for several days. Finally, the embryos were transferred

to 100% OCT and rocked at 4° for at least half an hour before being embedded in OCT on dry

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ice and stored at -80°C until sectioning. Sections were cut at 10μm on a Leica CM cryostat

(Leica Biosystems Inc., Buffalo Grove, IL) and mounted on APES (aminopropyltriethoxysilane)

coated slides. APES coated slides led to better retention of sections during processing than

traditional charged glass slides and were made as previously described (Matt Lewis,

www.methodbook.net/probes/insitu.html).

Subsequently, sections were pre-hybridized in slide mailers as follows: 1) Fixed 10

minutes in 4% PFA/PBS at room temperature, followed by 3 washes in PBS for 3 minutes each;

2) Digested in Proteinase K (1 µg/ml in 50mM Tris pH 7.5, 5mM EDTA) for 4 (E12.5), 6

(E14.5), or 8 (E16.5) minutes; 3) Refixed in 4%PFA/PBS for 5 minutes at room temperature,

followed by 3 washes in PBS, 3 minutes each; 4) Acetylated (1.36% triethanolamine, 0.178%

HCL, 0.2544% acetic anhydride in water) for 10 minutes at room temperature, followed by 3

washes in PBS for 5 minutes each; 5) Incubated in hybridization buffer (50% formamide, 5x

SSC pH4.5, 50 µg/ml yeast tRNA, 1% SDS, 50 µg/ml heparin) at 55°C for 1-2 hours. Next,

sections were hybridized as follows: 1) Incubated in hybridization buffer plus 1 ng/µl probe

overnight at 70°C; 2) Submerged in prewarmed (70°C) 5x SSC, pH7 and incubated on rocker for

30 minutes at room temperature; 3) Incubated in prewarmed (70°C) 0.2x SSC (pH7) for 3 hours

at 70°C, followed by incubation in fresh 0.2x SSC for 5 minutes at room temperature; 4)

Incubated in 1x MAB (Maleic Acid Buffer, pH 7.5) for five minutes at room temperature; 5)

Incubated in blocking solution (2% Blocking reagent (Roche Ref:11096176001), 10% heat

inactivated sheep serum, 0.1% Tween-20, all in 1x MAB) for 1 hour room temperature, followed

by incubation in blocking solution plus anti-DIG antibody (1:5000, FAB fragments, Roche)

overnight at 4°C. Finally, sections were washed and stained as follows, all at room temperature:

1) Washed 3x in 1X MAB with 0.1% Tween-20 for 15-30 minutes; 2) Washed in DEPC- H2O

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with 0.1% Tween-20 for 20 minutes; 3) Slides were removed from mailers and 200 µl of BM

Purple (Roche) with 0.1% Tween-20 was added to each slide; 4) Slides were kept in dark until

desired signal observed; 5) Slides were counterstained with Nuclear Fast Red (Vector

Laboratories, Inc., Burlingame, CA) for 10 minutes and rinsed with water. Finally, coverslips

were applied with Fluoromount-G (Southern Biotech, Cat no. 0100-01).

4.4 Alkaline Phosphatase Staining

4.4.1 BM Purple

Sections were first washed in PBS at room temperature for 10 minutes and then washed

in DEPC- H2O with 0.1% Tween-20 for 20 minutes. Next, 200 µl of BM Purple (Roche) with

0.1% Tween-20 was added to each slide and the slides were kept in dark until the desired signal

was observed. Finally, slides were counterstained with Nuclear Fast Red (Vector Laboratories,

Inc., Burlingame, CA) for 10 minutes and rinsed with water. Coverslips were applied with

Fluoromount-G (Southern Biotech, Cat no. 0100-01).

4.4.2 Liquid Fast-Red

Sections were first washed in PBS at room temperature for 10 minutes. Next, 200 µl of

liquid Fast-Red solution (abcam, product code ab64254) was added to each slide and the slides

were kept in dark until the desired signal was observed. Finally, slides were counterstained with

Methyl Green (Vector Laboratories, Inc., Burlingame, CA) as per the manufacturer’s

instructions. Coverslips were applied with Fluoromount-G (Southern Biotech, Cat no. 0100-01).

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4.5 β-galactosidase Staining

4.5.1 Whole mount

β-galactosidase staining of whole embryos was carried out as follows. Embryos were

fixed for 1 hr at 4°C in 4% paraformaldehyde in PBS. Next, they were rinsed 3 x 10-30 minutes

at room temperature in lacZ rinse buffer (0.2 M sodium phosphate, pH 7.3; 2 mM magnesium

chloride; 0.02% NP40; 0.01% sodium deoxycholate). They were then rocked overnight in the

dark at room temperature in lacZ staining solution (lacZ rinse buffer containing 5 mM potassium

ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal). Finally, the reaction was

stopped by transferring the embryos to PBS and fixing in 4% paraformaldehyde.

4.5.2 Sections

β-galactosidase staining of frozen sections was carried out as described previously (Chai

et al., 2000). In brief, embryos were fixed in 0.2% glutaraldehyde for 30 minutes at room

temperature. They were then soaked at 4°C in 10% sucrose in PBS for 30 minutes, followed by

PBS plus 2 mM MgCl2, 30% sucrose, and 50% OCT for two hours, at 4°C. Next, the embryos

were embedded in 100% OCT on dry-ice. Sections were cut at 10 μm and mounted on charged

glass slides, after which the slides were fixed in 0.2% glutaraldehyde for 10 min on ice, rinsed

briefly with 2 mM MgCl2 in PBS, and washed in 2 mM MgCl2 in PBS for 10 min, again on ice.

Finally, the sections were incubated in detergent rinse solution (0.005% NP40, 0.01% sodium

deoxycholate in PBS) for 10 min at 4°C and stained in X-gal staining solution (detergent rinse

solution plus 1 mg/ml X-Gal, Invitrogen/Life Technologies, Carlsbad, CA) for two- three days in

the dark at room temperature. Following staining, sections were counterstained with Nuclear Fast

Red (Vector Laboratories, Inc., Burlingame, CA) for 10 minutes and rinsed with water. Finally,

coverslips were applied with Fluoromount-G (Southern Biotech, Cat no. 0100-01).

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4.6 RNA Quantification

4.6.1 Realtime PCR (qRTPCR)

Three embryos from each group -- 1) MR26F8 mutants, 2) MR26F8 littermate controls,

3) MCAGF8 mutants, and 4) MCAGF8 littermate controls—were collected (12 total). Skin

samples were taken from the region between the eyes and the ears of E18.5 embryos. This

region correlates with the region where Msx2-Cre was expressed (Fig. S2C) and, thus, the levels

of Fgf8 should vary between the controls and mutants. RNA was extracted from the skin using

the RNeasy Fibrous Tissue Mini Kit (Qiagen Cat. No. 74704), following the manufacturer’s

instructions, including the DNAse digestion step. Additionally, to ensure DNA removal, the

extracted RNA was treated using the TURBO DNA-free Kit (ThermoFisher Scientific Cat. No.

AM1907). For each sample, 500 ng of RNA was reverse-transcribed to complementary DNA

(cDNA) using SuperScript III First-Strand synthesis kit (Invitrogen Cat. No. 18080051). Real-

time PCR reactions were run using SYBR Select Master Mix (Applied Biosystems Cat. NO

4472908; Austin, TX) on CFX Connect Real-time System (BioRad, Hercules, CA). The

expression of Fgf8 was normalized to that of corresponding Actb (β-actin), a housekeeping gene;

primers utilized are found in Table S3. Controls (Msx2-Cre negative embryos) from MR26F8

and MCAGF8 litters had similar Fgf8 expression levels that were not found to be statistically

significantly different (p=0.678) and thus all six littermate controls were pooled and expression

normalized to one. The expression of Fgf8 in the MR26F8 and MCAGF8 mutants was

normalized to the combined control sample and presented as fold expression change. Error bars

show standard deviation of the fold change.

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4.6.2 RNAseq

For the bone and cartilage RNAseq, E14.5 embryos, generated from an Msx2-Cre x CAGF8

cross, were dissected in ice-cold PBS. From the mutant and control embryos, the skin was

peeled away from the skull and the underlying cranial vault tissue was collected. Subsequently,

for the control embryos, the brain was removed and the cartilaginous sections of the cranial base

were carefully collected as well. All tissues from the mutant (cranial vault) and control embryos

(cranial vault and cartilaginous cranial base) were stored in RNAlater (Ambion/Life

Technologies) until RNA extraction. RNA was extracted from 9 mutant and control cranial

vaults as well as 9 cranial bases using RNeasy Lipid Tissue Mini Kit (Qiagen Cat. NO 74804)

following the manufacturer’s protocol. Within each group, the 9 samples were pooled into three

groups, with 3 samples making up each group, so that the RNA concentrations were similar.

Samples were submitted to the University of Colorado Denver Genomic and Microarray Core

and sequenced using the Illumina Illumina HiSeq4000 Platform and single end reads (1x151).

Reads generated were mapped to the mouse genome by gSNAP, expression derived by

Cufflinks, and differential expressed analyzed by ANOVA in R, as described previously

(Bradford et al., 2015). DAVID (Huang et al., 2009, 2008), with default parameters, was used

for functional annotation clustering of significantly upregulated and downregulated genes. Data

are available at GEO with the accession number GSE112413.

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Acknowledgements

This work was supported by Fellowship TL1 TR001081 from the Colorado Clinical

Translational Science Institute (LS) and NIH grant R01 DE019843 from the National Institute of

Dental and Craniofacial Research (TW). We would like to thank Xin Sun and Mark Lewandoski

for their advice and assistance with developing the Fgf8 alleles, Tim Nottoli for ES cell work to

generate the Fgf8 clones, and Rob Maxson for the Msx2-Cre mice. Additionally, we would like

to thank Nancy Troiano and her colleagues at the Yale Orthopædic Histology and

Histomorphometry Laboratory for sectioning and staining the E18.5- P12 skulls and guidance on

the interpretation of those sections. Finally, we would like to thank Eric Van Otterloo and Hong

Li for troubleshooting and advice throughout this project.

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References

Akiyama, H., Chaboissier, M.-C., Martin, J.F., Schedl, A., de Crombrugghe, B., 2002. The transcription

factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is

required for expression of Sox5 and Sox6. Genes Dev. 16, 2813–28. doi:10.1101/gad.1017802

Ambrosetti, D., Holmes, G., Mansukhani, A., Basilico, C., 2008. Fibroblast Growth Factor Signaling

Uses Multiple Mechanisms To Inhibit Wnt-Induced Transcription in Osteoblasts. Mol. Cell. Biol.

28, 4759–4771. doi:10.1128/MCB.01849-07

Anderson, J., Burns, H.D., Enriquez-Harris, P., Wilkie, A.O., Heath, J.K., 1998. Apert syndrome

mutations in fibroblast growth factor receptor 2 exhibit increased affinity for FGF ligand. Hum.

Mol. Genet. 7, 1475–83. doi:10.1093/HMG/7.9.1475

Arnold, M.A., Kim, Y., Czubryt, M.P., Phan, D., McAnally, J., Qi, X., Shelton, J.M., Richardson, J.A.,

Bassel-Duby, R., Olson, E.N., 2007. MEF2C Transcription Factor Controls Chondrocyte

Hypertrophy and Bone Development. Dev. Cell 12, 377–389. doi:10.1016/j.devcel.2007.02.004

Behr, B., Panetta, N.J., Longaker, M.T., Quarto, N., 2010. Different endogenous threshold levels of

Fibroblast Growth Factor-ligands determine the healing potential of frontal and parietal bones. Bone

47, 281–294. doi:10.1016/J.BONE.2010.05.008

Bialek, P., Kern, B., Yang, X., Schrock, M., Sosic, D., Hong, N., Wu, H., Yu, K., Ornitz, D.M., Olson,

E.N., Justice, M.J., Karsenty, G., 2004. A twist code determines the onset of osteoblast

differentiation. Dev. Cell 6, 423–35. doi:10.1016/S1534-5807(04)00058-9

Boulet, S.L., Rasmussen, S.A., Honein, M.A., 2008. A population-based study of craniosynostosis in

metropolitan Atlanta, 1989-2003. Am. J. Med. Genet. A 146A, 984–91. doi:10.1002/ajmg.a.32208

Bradford, A.P., Jones, K., Kechris, K., Chosich, J., Montague, M., Warren, W.C., May, M.C., Al-Safi, Z.,

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

Kuokkanen, S., Appt, S.E., Polotsky, A.J., 2015. Joint MiRNA/mRNA Expression Profiling Reveals

Changes Consistent with Development of Dysfunctional Corpus Luteum after Weight Gain. PLoS

One 10, e0135163. doi:10.1371/journal.pone.0135163

Brewer, J.R., Mazot, P., Soriano, P., 2016. Genetic insights into the mechanisms of Fgf signaling. Genes

Dev. 30, 751–71. doi:10.1101/gad.277137.115

Brinkley, J.F., Fisher, S., Harris, M.P., Holmes, G., Hooper, J.E., Jabs, E.W., Jones, K.L., Kesselman, C.,

Klein, O.D., Maas, R.L., Marazita, M.L., Selleri, L., Spritz, R.A., Van Bakel, H., Visel, A., Chai, Y.,

2016. The FaceBase Consortium: a comprehensive resource for craniofacial researchers.

Development 143, 2677–2688. doi:10.1242/dev.135434

Burke, D., Wilkes, D., Blundell, T.L., Malcolm, S., 1998. Fibroblast growth factor receptors: lessons

from the genes. Trends Biochem. Sci. 23, 59–62. doi:10.1016/S0968-0004(97)01170-5

Chai, Y., Jiang, X., Ito, Y., Bringas, P., Han, J., Rowitch, D.H., Soriano, P., McMahon, A.P., Sucov,

H.M., 2000. Fate of the mammalian cranial neural crest during tooth and mandibular

morphogenesis. Development 127, 1671–1679.

Choe, Y., Siegenthaler, J.A., Pleasure, S.J., 2012. A Cascade of Morphogenic Signaling Initiated by the

Meninges Controls Corpus Callosum Formation. Neuron 73, 698–712.

doi:10.1016/j.neuron.2011.11.036

Coffin, J.D., Florkiewicz, R.Z., Neumann, J., Mort-Hopkins, T., Dorn, G.W., Lightfoot, P., German, R.,

Howles, P.N., Kier, A., O’Toole, B.A., 1995. Abnormal bone growth and selective translational

regulation in basic fibroblast growth factor (FGF-2) transgenic mice. Mol. Biol. Cell 6, 1861–73.

Cohen, M.M., 2000. Merging the old skeletal biology with the new. II. Molecular aspects of bone

formation and bone growth. J. Craniofac. Genet. Dev. Biol. 20, 94–106.

Couly, G.F., Coltey, P.M., Le Douarin, N.M., 1993. The triple origin of skull in higher vertebrates: a

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

study in quail-chick chimeras. Development 117.

Day, T.F., Guo, X., Garrett-Beal, L., Yang, Y., 2005. Wnt/β-Catenin Signaling in Mesenchymal

Progenitors Controls Osteoblast and Chondrocyte Differentiation during Vertebrate Skeletogenesis.

Dev. Cell 8, 739–750.

Eswarakumar, V.P., Monsonego-Ornan, E., Pines, M., Antonopoulou, I., Morriss-Kay, G.M., Lonai, P.,

2002. The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 129.

Evans, D.J.R., Noden, D.M., 2006. Spatial relations between avian craniofacial neural crest and paraxial

mesoderm cells. Dev. Dyn. 235, 1310–1325. doi:10.1002/dvdy.20663

Flaherty, K., Singh, N., Richtsmeier, J.T., 2016. Understanding craniosynostosis as a growth disorder 5,

429–459. doi:10.1002/wdev.227

George Clark, 1981. Staining procedures, 4th ed. Williams & Wilkins, Baltimore, MD.

Goodnough, L.H., Chang, A.T., Treloar, C., Yang, J., Scacheri, P.C., Atit, R.P., 2012. Twist1 mediates

repression of chondrogenesis by β-catenin to promote cranial bone progenitor specification.

Development 139.

Govindarajan, V., Overbeek, P.A., 2006. FGF9 can induce endochondral ossification in cranial

mesenchyme. BMC Dev. Biol. 6, 7. doi:10.1186/1471-213X-6-7

Gruber, H.E., 1992. Adaptations of Goldner’s Masson trichrome stain for the study of undecalcified

plastic embedded bone. Biotech. Histochem. 67, 30–4.

Gu, J., Lu, Y., Li, F., Qiao, L., Wang, Q., Li, N., Borgia, J.A., Deng, Y., Lei, G., Zheng, Q., 2014.

Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte

hypertrophic differentiation. Cell Death Dis. 5, e1469. doi:10.1038/cddis.2014.444

Hall, B.K., Miyake, T., 2000. All for one and one for all: condensations and the initiation of skeletal

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

development. BioEssays 22, 138–147. doi:10.1002/(SICI)1521-1878(200002)22:2<138::AID-

BIES5>3.0.CO;2-4

Hill, T.P., Später, D., Taketo, M.M., Birchmeier, W., Hartmann, C., 2005. Canonical Wnt/β-Catenin

Signaling Prevents Osteoblasts from Differentiating into Chondrocytes. Dev. Cell 8, 727–738.

doi:10.1016/j.devcel.2005.02.013

Huang, D.W., Sherman, B.T., Lempicki, R.A., 2009. Bioinformatics enrichment tools: paths toward the

comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13.

doi:10.1093/nar/gkn923

Huang, D.W., Sherman, B.T., Lempicki, R.A., 2008. Systematic and integrative analysis of large gene

lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57. doi:10.1038/nprot.2008.211

Hung, I.H., Schoenwolf, G.C., Lewandoski, M., Ornitz, D.M., 2016. A combined series of Fgf9 and

Fgf18 mutant alleles identifies unique and redundant roles in skeletal development. Dev. Biol. 411,

72–84. doi:10.1016/j.ydbio.2016.01.008

Jegalian, B.G., De Robertis, E.M., 1992. Homeotic transformations in the mouse induced by

overexpression of a human Hox3.3 transgene. Cell 71, 901–910. doi:10.1016/0092-8674(92)90387-

R

Jho, E., Zhang, T., Domon, C., Joo, C.-K., Freund, J.-N., Costantini, F., 2002. Wnt/beta-catenin/Tcf

signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol.

Cell. Biol. 22, 1172–83. doi:10.1128/MCB.22.4.1172-1183.2002

Jiang, X., Iseki, S., Maxson, R.E., Sucov, H.M., Morriss-Kay, G.M., 2002. Tissue Origins and

Interactions in the Mammalian Skull Vault. Dev. Biol. 241, 106–116. doi:10.1006/dbio.2001.0487

Johnson, D., Wilkie, A.O.M., 2011. Craniosynostosis. Eur. J. Hum. Genet. 19, 369–76.

doi:10.1038/ejhg.2010.235

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

Katsianou, M.A., Adamopoulos, C., Vastardis, H., Basdra, E.K., 2016. Signaling mechanisms implicated

in cranial sutures pathophysiology: Craniosynostosis. BBA Clin. 6, 165–176.

doi:10.1016/j.bbacli.2016.04.006

Ko, J.M., 2016. Genetic Syndromes Associated with Craniosynostosis. J. Korean Neurosurg. Soc. 59,

187–91. doi:10.3340/jkns.2016.59.3.187

Koch, M., Laub, F., Zhou, P., Hahn, R.A., Tanaka, S., Burgeson, R.E., Gerecke, D.R., Ramirez, F.,

Gordon, M.K., 2003. Collagen XXIV, a Vertebrate Fibrillar Collagen with Structural Features of

Invertebrate Collagens: SELECTIVE EXPRESSION IN DEVELOPING CORNEA AND BONE. J.

Biol. Chem. 278, 43236–43244. doi:10.1074/jbc.M302112200

Lai, W.-T., Krishnappa, V., Phinney, D.G., 2011. Fibroblast growth factor 2 (Fgf2) inhibits differentiation

of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular regulated kinase

activation, and altering Fgf receptor expression levels. Stem Cells 29, 1102–11.

doi:10.1002/stem.661

Lajeunie, E., Le Merrer, M., Bonaïti-Pellie, C., Marchac, D., Renier, D., 1995. Genetic study of

nonsyndromic coronal craniosynostosis. Am. J. Med. Genet. 55, 500–4.

doi:10.1002/ajmg.1320550422

Lee, M.S., Lowe, G.N., Strong, D.D., Wergedal, J.E., Glackin, C.A., 1999. TWIST, a basic helix-loop-

helix transcription factor, can regulate the human osteogenic lineage. J. Cell. Biochem. 75, 566–77.

Lefebvre, V., de Crombrugghe, B., 1998. Toward understanding SOX9 function in chondrocyte

differentiation. Matrix Biol. 16, 529–40.

Lefebvre, V., Huang, W., Harley, V.R., Goodfellow, P.N., de Crombrugghe, B., 1997. SOX9 is a potent

activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene. Mol. Cell. Biol.

17, 2336–46.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

Lin, C., Yin, Y., Bell, S.M., Veith, G.M., Chen, H., Huh, S.-H., Ornitz, D.M., Ma, L., 2013. Delineating a

Conserved Genetic Cassette Promoting Outgrowth of Body Appendages. PLoS Genet. 9, e1003231.

doi:10.1371/journal.pgen.1003231

Liu, Z., Lavine, K.J., Hung, I.H., Ornitz, D.M., 2007. FGF18 is required for early chondrocyte

proliferation, hypertrophy and vascular invasion of the growth plate. Dev. Biol. 302, 80–91.

doi:10.1016/j.ydbio.2006.08.071

Liu, Z., Xu, J., Colvin, J.S., Ornitz, D.M., 2002. Coordination of chondrogenesis and osteogenesis by

fibroblast growth factor 18. Genes Dev. 16, 859–869. doi:10.1101/gad.965602

Lu, P., Minowada, G., Martin, G.R., 2006. Increasing Fgf4 expression in the mouse limb bud causes

polysyndactyly and rescues the skeletal defects that result from loss of Fgf8 function. Development

133, 33–42. doi:10.1242/dev.02172

Lustig, B., Jerchow, B., Sachs, M., Weiler, S., Pietsch, T., Karsten, U., van de Wetering, M., Clevers, H.,

Schlag, P.M., Birchmeier, W., Behrens, J., 2002. Negative feedback loop of Wnt signaling through

upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell. Biol. 22, 1184–93.

Mai, S., Wei, K., Flenniken, A., Adamson, S.L., Rossant, J., Aubin, J.E., Gong, S.-G., 2010. The

missense mutation W290R in Fgfr2 causes developmental defects from aberrant IIIb and IIIc

signaling. Dev. Dyn. 239, 1888–900. doi:10.1002/dvdy.22314

Matt Lewis, n.d. In situ hybridisation to alpha satellite sequences (chromosome specific) [WWW

Document]. URL http://www.methodbook.net/probes/insitu.html (accessed 5.14.17).

Mayne, R., 1989. Cartilage collagens. What Is Their Function, and Are They Involved in Articular

Disease? Arthritis Rheum. 32, 241–246. doi:10.1002/anr.1780320302

McLeod, M.J., 1980. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue

and alizarin red S. Teratology 22, 299–301. doi:10.1002/tera.1420220306

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

Meyers, E.N., Lewandoski1, M., Martin, G.R., Lewandoski, M., Martin, G.R., 1998. An Fgf8 mutant

allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18, 136–141.

doi:10.1038/ng0298-136

Minear, S., Leucht, P., Jiang, J., Liu, B., Zeng, A., Fuerer, C., Nusse, R., Helms, J.A., 2010. Wnt Proteins

Promote Bone Regeneration. Sci. Transl. Med. 2.

Monsonego-Ornan, E., Adar, R., Feferman, T., Segev, O., Yayon, A., 2000. The transmembrane mutation

G380R in fibroblast growth factor receptor 3 uncouples ligand-mediated receptor activation from

down-regulation. Mol. Cell. Biol. 20, 516–22.

Moosa, S., Wollnik, B., 2016. Altered FGF signalling in congenital craniofacial and skeletal disorders.

Semin. Cell Dev. Biol. 53, 115–125. doi:10.1016/J.SEMCDB.2015.12.005

Mowry, R.W., 1956. Alcian blue technics for the histochemical study of acidic carbohydrates. J.

Histochem. Cytochem. 4, 407.

Neilson, K.M., Friesel, R., 1996. Ligand-independent activation of fibroblast growth factor receptors by

point mutations in the extracellular, transmembrane, and kinase domains. J. Biol. Chem. 271,

25049–57. doi:10.1074/JBC.271.40.25049

Nie, X., Luukko, K., Kettunen, P., 2006. FGF signalling in craniofacial development and developmental

disorders. Oral Dis. 12, 102–11. doi:10.1111/j.1601-0825.2005.01176.x

Noden, D.M., 1992. Vertebrate craniofacial development: novel approaches and new dilemmas. Curr.

Opin. Genet. Dev. 2, 576–581. doi:10.1016/S0959-437X(05)80175-3

Noden, D.M., Trainor, P.A., 2005. Relations and interactions between cranial mesoderm and neural crest

populations. J. Anat. 207, 575–601. doi:10.1111/j.1469-7580.2005.00473.x

Ohbayashi, N., Shibayama, M., Kurotaki, Y., Imanishi, M., Fujimori, T., Itoh, N., Takada, S., 2002.

FGF18 is required for normal cell proliferation and differentiation during osteogenesis and

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

chondrogenesis. Genes Dev. 16, 870–9. doi:10.1101/gad.965702

Olsen, S.K., Li, J.Y.H., Bromleigh, C., Eliseenkova, A. V, Ibrahimi, O.A., Lao, Z., Zhang, F., Linhardt,

R.J., Joyner, A.L., Mohammadi, M., 2006. Structural basis by which alternative splicing modulates

the organizer activity of FGF8 in the brain. Genes Dev. 20, 185–98. doi:10.1101/gad.1365406

Opperman, L.A., 2000. Cranial sutures as intramembranous bone growth sites. Dev. Dyn. 219, 472–85.

doi:10.1002/1097-0177(2000)9999:9999<::AID-DVDY1073>3.0.CO;2-F

Ornitz, D.M., 2000. FGFs, heparan sulfate and FGFRs: complex interactions essential for development.

BioEssays 22, 108–112. doi:10.1002/(SICI)1521-1878(200002)22:2<108::AID-BIES2>3.0.CO;2-M

Ornitz, D.M., Marie, P.J., 2015. Fibroblast growth factor signaling in skeletal development and disease.

Genes Dev. 29, 1463–86. doi:10.1101/gad.266551.115

Ornitz, D.M., Marie, P.J., 2002. FGF signaling pathways in endochondral and intramembranous bone

development and human genetic disease. Genes Dev. 16, 1446–65. doi:10.1101/gad.990702

Pownall, M.E., Isaacs, H. V., 2010. FGF Signalling in Vertebrate Development. Morgan & Claypool Life

Sciences, San Rafael, CA.

Qin, J.Y., Zhang, L., Clift, K.L., Hulur, I., Xiang, A.P., Ren, B.-Z., Lahn, B.T., 2010. Systematic

Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter. PLoS One 5,

e10611. doi:10.1371/journal.pone.0010611

Quarto, N., Behr, B., Li, S., Longaker, M.T., 2009. Differential FGF ligands and FGF receptors

expression pattern in frontal and parietal calvarial bones. Cells. Tissues. Organs 190, 158–69.

doi:10.1159/000202789

Ratisoontorn, C., Fan, G.-F., McEntee, K., Nah, H.-D., 2003. Activating (P253R, C278F) and dominant

negative mutations of FGFR2: differential effects on calvarial bone cell proliferation, differentiation,

and mineralization. Connect. Tissue Res. 44 Suppl 1, 292–7.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

Robin, N.H., Falk, M.J., Haldeman-Englert, C.R., 2011. FGFR-Related Craniosynostosis Syndromes,

GeneReviews(®). University of Washington, Seattle.

Sarabipour, S., Hristova, K., 2016. Mechanism of FGF receptor dimerization and activation. Nat.

Commun. 7, 10262. doi:10.1038/ncomms10262

Senarath-Yapa, K., Chung, M.T., McArdle, A., Wong, V.W., Quarto, N., Longaker, M.T., Wan, D.C.,

2012. Craniosynostosis: molecular pathways and future pharmacologic therapy. Organogenesis 8,

103–13. doi:10.4161/org.23307

Snyder-Warwick, A.K., Perlyn, C.A., Pan, J., Yu, K., Zhang, L., Ornitz, D.M., 2010. Analysis of a gain-

of-function FGFR2 Crouzon mutation provides evidence of loss of function activity in the etiology

of cleft palate. Proc. Natl. Acad. Sci. 107, 2515–2520. doi:10.1073/pnas.0913985107

Spicer, D., 2009. FGF9 on the move. Nat. Genet. 41, 272–273. doi:10.1038/ng0309-272

Sridharan, G., Shankar, A.A., 2012. Toluidine blue: A review of its chemistry and clinical utility. J. Oral

Maxillofac. Pathol. 16, 251–5. doi:10.4103/0973-029X.99081

Su, N., Jin, M., Chen, L., 2014. Role of FGF/FGFR signaling in skeletal development and homeostasis:

learning from mouse models. Bone Res. 2, 14003. doi:10.1038/boneres.2014.3

Sun, X., Lewandoski, M., Meyers, E.N., Liu, Y.H., Maxson, R.E., Martin, G.R., 2000. Conditional

inactivation of Fgf4 reveals complexity of signalling during limb bud development. Nat. Genet. 25,

83–6. doi:10.1038/75644

Teven, C.M., Farina, E.M., Rivas, J., Reid, R.R., 2014. Fibroblast growth factor (FGF) signaling in

development and skeletal diseases. Genes Dis. 1, 199–213. doi:10.1016/j.gendis.2014.09.005

Tholpady, S.S., Abdelaal, M.M., Dufresne, C.R., Gampper, T.J., Lin, K.Y., Jane, J.A., Morgan, R.F.,

Ogle, R.C., 2004. Aberrant bony vasculature associated with activating fibroblast growth factor

receptor mutations accompanying Crouzon syndrome. J. Craniofac. Surg. 15, 431-5-8.

Dis

ease

Mo

dels

& M

echa

nism

s •

DM

M •

Acc

epte

d m

anus

crip

t

Tran, T.H., Jarrell, A., Zentner, G.E., Welsh, A., Brownell, I., Scacheri, P.C., Atit, R., 2010. Role of

canonical Wnt signaling/β-catenin via Dermo1 in cranial dermal cell development. Development

137.

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., Yu, K., Ornitz, D.M., Elisseeff, J., Huso, D.L., Jabs, E.W., 2005. Abnormalities in

cartilage and bone development in the Apert syndrome FGFR2+/S252W mouse. Development 132,

3537–3548. doi:10.1242/dev.01914

Wu, X., Gu, M., Huang, L., Liu, X., Zhang, H., Ding, X., Xu, J., Cui, B., Wang, L., Lu, S., Chen, X.,

Zhang, H., Huang, W., Yuan, W., Yang, J., Gu, Q., Fei, J., Chen, Z., Yuan, Z., Wang, Z., 2009.

Multiple Synostoses Syndrome Is Due to a Missense Mutation in Exon 2 of FGF9 Gene. Am. J.

Hum. Genet. 85, 53–63. doi:10.1016/j.ajhg.2009.06.007

Yoshida, T., Vivatbutsiri, P., Morriss-Kay, G., Saga, Y., Iseki, S., 2008. Cell lineage in mammalian

craniofacial mesenchyme. Mech. Dev. 125, 797–808. doi:10.1016/j.mod.2008.06.007

Yu, H.-M.I., Jerchow, B., Sheu, T.-J., Liu, B., Costantini, F., Puzas, J.E., Birchmeier, W., Hsu, W., 2005.

The role of Axin2 in calvarial morphogenesis and craniosynostosis. Development 132, 1995–2005.

doi:10.1242/dev.01786

Zeller, R., López-Ríos, J., Zuniga, A., 2009. Vertebrate limb bud development: moving towards

integrative analysis of organogenesis. Nat. Rev. Genet. 10, 845–858. doi:10.1038/nrg2681

Zhang, M., Xuan, S., Bouxsein, M.L., von Stechow, D., Akeno, N., Faugere, M.C., Malluche, H., Zhao,

G., Rosen, C.J., Efstratiadis, A., Clemens, T.L., 2002. Osteoblast-specific Knockout of the Insulin-

like Growth Factor (IGF) Receptor Gene Reveals an Essential Role of IGF Signaling in Bone Matrix

Mineralization. J. Biol. Chem. 277, 44005–44012. doi:10.1074/jbc.M208265200

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Figures

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Figure 1. Conditional alleles modulate Fgf8b expression levels.

(A) Cartoon diagram of the two Fgf8b conditional alleles placed within the mouse

Gt(ROSA)26Sor locus before and after Cre-mediated recombination with exons (Ex), splice

acceptors (SA) and LoxP sites (black triangles) illustrated. The abbreviations R26F8 (top) and

CAGF8 (bottom) are used to refer to these recombined alleles throughout the text of the

manuscript.

(B) Relative Fgf8b expression levels in E18.5 dorsal head skin measured by qRT-PCR,

normalized to Actb levels, following Msx2-Cre mediated recombination. Control indicates data

from littermates lacking Msx2-Cre transgene. Bars show average fold change for each group

compared to control with error bars indicating standard error. All three groups are statistically

significant from each other (Anova: p<0.001; T-test: controls vs R26F8 p= 0.0013 (**), controls

vs CAGF8 p= 0.0002 (***), R26F8 vs CAGF8 p=0.022 (*)). Controls, n=6; R26F8 and CAGF8,

n=3.

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Figure 2. MR26F8 mice exhibit cranial defects including coronal craniosynostosis.

(A-F): Gross images of the control (A-C) and MR26F8 (D-F) heads. Neonate pups (P0) are

shown in both a lateral (A, D) and dorsal (B, E) view. P12 heads are shown laterally (C, F). (G-

L): Dorsal views of bone and cartilage staining of control (G-I) and mutant (J-L) skulls at P0 (G,

J), P12 (H, K), and P36 (I, L). G’-L’ show magnification of the coronal and lambdoid sutures of

each respective skull. The expected location of the coronal suture is shown by a green arrow.

This suture is present in controls, abnormal in MR26F8s at P0, and absent at P12 and P36. The

narrower region of the MR26F8 lambdoid suture at P12 and P36 (green arrowhead) and ectopic

bone (red arrowhead) are shown. The yellow dashed lines in (G, J) outline the edges of

ossification. The yellow dashed lines in (K, L) align with the sagittal and interfrontal sutures.

Abbreviations: Fr, frontal bone; Lb, lambdoid suture; IP, interparietal bone; Pr, parietal bone;

SO, supraoccipital bone. Scale Bars: D-E, J-L: 1mm; F: 5mm. Aged-matched controls and

mutants are at the same scale. A-F, n=10; G-L, n=5.

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Figure 3. Delayed ossification, followed by craniosynostosis, of the MR26F8 coronal suture.

Low magnification images in (A, B) show the approximate position of the coronal suture (Cr)

from P12 sagittal sections. Sagittal sections from P0 (C-F), and P12 (G-J) skulls for control (A,

C, E, G, I) and MR26F8 (B, D, F, H, J) pups. (A-D, G-H) von Kossa staining with mineralized

bone appearing dark red/black. (E-F, I-J) Goldner’s Trichome staining highlighting mature

(green) and immature (red) bone matrix. In the P0 sections, blue arrowheads (C-D) mark the

limits of mineralization; red arrowheads (E-F) show the extent of the unmineralized mature

osteoid, with a mixture of mature and immature bone matrix occurring between them in the

mutant as shown in greater detail in the inset (F, red arrow). Scale Bars: A-B: 1mm; C-J: 100µM.

Aged-matched controls and mutants are at the same scale. N=3.

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Figure 4. MCAGF8 mice exhibit cranial defects including loss of ossification.

(A-B, D-E): Gross images of control (A-B) and mutant (D-E) E18.5 embryos showing lateral

views of whole head (A, D) or ventral views of secondary palate after removal of lower jaw (B,

E). Note, bluer staining of palate in E versus B results from an examination of skin permeability

using toluidine blue staining. (C, F-K): Bone and cartilage staining of the control (C, G-H) and

mutant (F, J-K) skulls, as well as the entire mutant body (I). (C, F, I): Lateral view; (G, J): dorsal

view of the cranial vault; (H,K): ventral view of cranial base after removal of the lower jaw.

Yellow dashed line (J) notes the boundary between bone and non-stained matrix formation. Red

stars denote MCAGF8 ossification gains or losses. Abbreviations: BS, basisphenoid; Fr, frontal;

IP, interparietal; Md, mandible; Ns, nasal; PPMX, palatal process of the maxilla; PPPL, palatal

process of the palatine; Pr, parietal; SO, supraoccipital. Scale Bars: 1mm. N=15.

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Figure 5. Cartilage replaces bone in the MCAGF8 cranial vault.

Lateral (A-H) views of control (A, C, E, G) and MCAGF8 (B, D, F, H) skulls. (A-D): Bouin’s

fixed skulls stained for cartilage (alcian blue) at E14.5 (A-B) and E16.5 (C-D). (E-F): Bone and

cartilage staining with alizarin red (bone) and alcian blue (cartilage) at E16.5. (G-H): Bone and

cartilage staining with alizarin red, alcian blue, and toluidine blue (cartilage) at E18.5. (I-L):

Alcian blue + PAS stain of coronal sections from control cranial vault (I), MCAGF8 lateral

cranial vault (J, L), and control cranial base cartilage (primordium of the presphenoid bone,

K). Acidic mucins stain blue, neutral mucins stain magenta. Abbreviations: Bn, bone; Br, brain;

Ct, cartilage; Fr, frontal, IP, interparietal; MC, Meckel’s cartilage; Md, mandible; Ns, nasal bone;

Pr, parietal; SE, surface epithelium; SO, supraoccipital bone. Scale Bars: A-H: 1mm, I-L 40µM.

A-F, n=5; G-K, n=3.

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Figure 6. Intramembranous ossification is more severely impacted by increased Fgf8b

expression than endochondral ossification.

(A-O): Bone and cartilage staining of E18.5 control (A, D, G, J, M), OR26F8 (B, E, H, K, N),

and OCAGF8 (C, F, I, L, O) samples. (A-C): lateral view of head, (D-F): dorsal view of cranial

vault (G-I): ventral view of cranial base after mandible removal, (J-L): dorsal view of mandibles,

(M-O): hindlimbs. Blue arrow shows parietal bones. Abbreviations: BO, basioccipital; Fb,

fibula; Fe, femur; Fr, frontal; IP, interparietal; Md, mandible; Ns, nasal; PG, pelvic girdle; Pr,

parietal; SO, supraoccipital; Ti, tibia. Scale Bars = 1mm. Controls, OR26F8, and OCAGF8

samples are shown at the same magnification. N=5,

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Figure 7. MCAGF8 cranial vault differentiation shifts from osteogenic to chondrogenic.

(A) Schematic of RNAseq experimental design. E14.5 tissue was removed from control (left)

and MCAGF8 (center) cranial regions outlined by the red boxes, after skin removal and avoiding

underlying brain tissue. Control cranial base cartilage was removed from areas shown by red

dots (right). (B) Scatterplot depicting average RPKM values. Colored points represent

transcripts that are statistically significant between the groups: MCAGF8 cranial vault (Y-axis),

and either control cranial vault (X axis, left) or control cranial base (X axis, right). Yellow

represents genes upregulated in the MCAGF8 compared to the control (light yellow = p<0.05,

dark yellow= p<0.05 and q<0.1). Blue represents genes downregulated genes in MCAGF8

compared to the control (light blue = p<0.05, dark blue= p<0.05 and q<0.1). (C) Histograms

plotting normalized RPKM values of genes associated with collagen (left), cartilage/bone

differentiation (middle) and ECM (right) significantly dysregulated in MCAGF8 cranial vaults

(orange) as compared to control vaults (black). Error bars represent standard error. (D-E)

Histograms plotting expression fold change in MCAGF8 cranial vault samples as compared to

controls, where control values are normalized to 1 (black bars) whereas mutant samples are

represented by green or red, respectively, for genes involved in differentiation (D) or Wnt

signaling (E). Error bars represent standard error. (F-I) RNA in situ hybridization in the cranial

vault for expression (purple) of Sox9 at E12.5 (F, G) or Osterix/Sp7 at E14.5 (H, I) for control (F,

H) and MCAGF8 (G, I) embryos. (J-K) Alkaline phosphatase activity (red) in the cranial vault

of E16.5 control (J) and MCAGF8 (K) embryos. The cranial vault (red arrows) and surface

epithelium (black arrows) are shown. Abbreviations: Br= brain. Scale Bars= 60µM. N=3.

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Figure 8. Reduced Axin2 gene dosage improves MCAGF8 cranial vault ossification

(A-I): Bone and cartilage staining of control (A, D, G), MCAGF8 (B, E, H), and

MCAGF8;Axin2lacz +/- mutants (C, F, I); n = 5 per genotype. A-C show a lateral view, D-F show

dorsal view of the cranial vault, and G-I show the cranial base. The boundary between bone and

non-stained matrix (yellow dashed line), the presence or absence of the coronal suture (green

arrow), and lambdoid suture (green arrowhead) are shown. Red stars denote regions of the

cranial base that differ from the control; the green star in (I) shows the rescue of the

supraoccipital bone. Abbreviations: BS, basisphenoid; Fr, frontal bone; IP, interparietal bone;

Md, mandible Ns, nasal bone; PPPL, palatal process of the palatine; PPMX, palatal process of

the maxilla; Pr, parietal bone; SO, supraoccipital. Scale Bars= 1mm. N=5.

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Supplementary Figure 1. Detailed Map of R26F8 and CAGF8 constructs.

Disease Models & Mechanisms 11: doi:10.1242/dmm.031526: Supplementary information

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A: Schematic of the R26F8 allele

Top. The targeting vector showing the standard homology arms used for targeting the Gt(ROSA)26Sor locus (ROSA26). The ROSA26 locus provides a promoter and a non-coding first exon to generate mRNA transcripts. The splice acceptor site (SA), LoxP sites (orange triangles), the drug selection cassette (PGK-Neo), and multiple poly A addition sequences to prevent transcription read-through (tpA), along with the IRES-GFP and the bovine growth hormone poly A addition sequences (bpA) are derived from the pBTG vector (see Materials and Methods). DTA indicates the diphtheria toxin A negative selection cassette. The sites of restriction enzymes used for cloning and linearization are shown, but note that these are not necessarily unique sites within vector sequences. Middle. The allele after homologous recombination with the sites of primers used for ES cell screening and genotyping. Note that the primers R5F and R3R used to screen for appropriate homologous recombination lie outside the homology arms. For standard mouse genotyping ROSA F + ROSA R give an ~165bp wildtype band. ROSA F + BTR give an ~250bp band for the targeted allele.

Bottom. The allele after Cre mediated recombination that allows generation of a functional Fgf8b transcript. Note, this allele is abbreviated as R26F8 in the text

Also note that in all instances the various DNA elements are not shown to scale.

B: Schematic of the CAGF8 allele

Top. The targeting vector as in A. except that a CAG enhancer/promoter element has been inserted upstream of the positive selection cassette. The sites of restriction enzymes used for cloning and linearization are shown, but note that these are not necessarily unique sites within vector sequences.

Middle. The allele after homologous recombination with the sites of primers used for ES cell screening and genotyping. Note that the primers R5F and R3R used to screen for appropriate homologous recombination lie outside the homology arms. For standard mouse genotyping ROSA F + ROSA R give an ~165bp wildtype band. ROSA F + CMV R1 give an ~230bp band for the targeted allele.

Bottom. The allele after Cre mediated recombination that allows generation of a functional Fgf8b transcript. Note, this allele is abbreviated as CAGF8 in the text

Also note that in all instances the various DNA elements are not shown to scale.

Disease Models & Mechanisms 11: doi:10.1242/dmm.031526: Supplementary information

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Supplemental Figure 2. Embryonic expression of Msx2-Cre during craniofacial development.

Msx2-Cre mediated recombination was visualized using β-galactosidase staining (blue/green) of ROSA26 LacZ Reporter embryos. (A-C) Lateral views of whole mount β-galactosidase staining on E10.5 (A) and E11.5 (B) embryos. (C) At E16.5, almost the entire dorsal aspect of the head showed β-galactosidase staining (lateral view on the left, dorsal view on the right). Red and blue triangles denote regions over part of the parietal and intraparietal bones, respectively, where Msx2-Cre mediated recombination did not occur as efficiently. Black lines in B and C indicate plane of section in D, F and E, G respectively. (D-G) β-galactosidase staining of frozen sections at E11.5 in a frontal plane (D, F) and E16.5 in a sagittal plane (E, G), counterstained with nuclear fast red. F and G are magnifications of the regions outlined by black boxes in D and E,

Disease Models & Mechanisms 11: doi:10.1242/dmm.031526: Supplementary information

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respectively. Sections are shown at both 10x (D, E) and 40x (F, G) magnification. Note in E, bone (arrow) and cartilage (ct), do not show β-galactosidase staining, whereas staining occurs in both ectoderm (boxed) and nasal epithelium (arrowhead). (H-J): β-galactosidase staining of frozen sagittal sections of the cranial vault of E16.5 ROSA26 LacZ Reporter embryos showing (H): the anterior cranial vault, (I): the coronal suture, and (J): the lambdoid suture. Sections were stained with liquid fast red to highlight bone (magenta). Note that β-galactosidase staining is found only in the ectoderm, not the cranial bone or sutures. Scale bars: A-C: 500µM; D-E: 100µM; F-G: 50µM; H-J: 200 µM.

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Supplementary Figure 3. MR26F8 Cranial and Limb Phenotypes

(A-H): Gross morphological view of P12 heads (A-B) and hindlimbs (C-D) of the control (A, C, E, G) and MR26F8s (B, D, F, H). (I-L): Bone and cartilage staining of P16 limbs of controls (I, K) and MR26F8s (J, L). (E-F, I-J): hindlimb; (G-H, K-L): Forelimb. Red arrowheads denote extra digits on the posterior side of the MR26F8 limb (postaxial polydactyly). (M-P): Skeletal staining of P0 control (M, O) and MR26F8 (N, P) mice showing lateral views of skulls and forelimbs (M, N) and ventral view of the cranial base after removal of the mandible (O, P). Red star in N denotes loss of ossification of the parietal. Scale Bars: 1mm.

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Supplemental Figure 4. MR26F8 mutants have delayed ossification, followed by over ossification, of the lambdoid suture.

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Low magnification images in (A, B) show the approximate position of the lambdoid suture (Lb) from P12 sagittal sections. Sagittal sections from skulls of P0 (C-F) and P12 (G-J) control (C, E, G, I) and MR26F8 (D, F, H, J) pups. Sections (A-D, G-H) were stained with von Kossa, such that mineralized bone appears dark red/black. Sections (E-F, I-J) were stained with Goldner’s Trichome stain such that mature bone matrix appears green whereas immature bone matrix stains red. In the P0 sections, blue arrowheads mark the limits of mineralization (C-D); red arrowheads show the extent of the unmineralized mature osteoid (E-F). In F, only one side of the mature osteoid is shown, with a mixture of mature and immature bone matrix occurring between the mature osteoid in the mutant as shown in greater detail in the inset (red arrow). The bar graph (K) shows the width of the lambdoid sutures at P0 and P12. Comparison of the width between the controls and MR26F8s is significantly different at both P0 (p<0.0001) and P12 (p<0.0003). Bars show average suture width (μM); error bars denote standard error within the group. Scale Bars: A-B: 1mm; C-J: 100µM. Aged-matched controls and mutants are at the same scale.

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Supplementary Figure 5. MCAGF8 Limb Phenotypes

E18.5 gross (A, E, I) and skeletal (B-D, F-H, J-K) limb phenotypes. (A-D): controls, (E-K) MCAGF8s. The latter exhibit a wide range of phenotypes from polydactyly in the forelimb (G, compared to control, C) and hindlimb (H, compared to control, D) to fused forelimbs and hindlimbs (less severe, J; more severe, K). Red arrowheads denote various gross limb phenotypes. Red arrows denote postaxial polydactyly with 1 indicating the anterior most digit and 5 indicating the posterior most digit. Abbreviations: Fb, fibula; Fe, femur; Hu, humerus; Ra, radius; Ti, tibia; Ul, ulna. Scale Bars= 1mm.

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Supplemental Figure 6. Abnormal cartilage replaces intramembranous bone in MCAGF8 mice.

(A-E) Frontal sections through the parietal bones of the E18.5 controls and equivalent location in MCAGF8s; regions shown are midway between the dorsal and ventral most aspects of the parietal. (A, C) von Kossa stain of the control (A) and mutant (B, C) sections. Mineralized bone is stained in dark red/black. (D, E) Toluidine blue staining of control (D) and MCAGF8 (E) sections. The nuclei stain blue and the glycosaminoglycans that make up the cartilage matrix stain reddish purple. (F-G): Dorsal view of E18.5 skulls after bone and cartilage staining with alizarin red, alcian blue, and toluidine blue (cartilage). The red rectangle (G) outlines a region where cartilage and bone overlap. G’ shows that same region magnified. Abbreviations: Bn, bone; Br, brain; Ct, cartilage; SE, surface epithelium. Scale bars: A, D: 20µM, F: 1mm. Aged matched controls and mutants are at the same scale.

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Supplemental Figure 7. Osteocalcin-Cre expression from E14.5-E16.5

Osteocalcin-Cre (OC-Cre) mediated recombination was visualized using β-galactosidase staining (blue) of ROSA26 LacZ Reporter embryos. (A-E) Whole embryo β-galactosidase staining was performed at E14.5 (A), E15.5 (B), and E16.5 (C-E). Skin was removed prior to staining in the E15.5 and E16.5 embryos to allow stain penetration. (C): Lateral view of the ribs and limbs. (D): Lateral view of the head. (E): Dorsal view of the cranial vault. Scale Bars= A, DE: 1mm; B, C: 2mm.

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Supplemental Figure 8. Gross morphology and skeletal analysis of forelimbs in E18.5 OR26F8 and OCAGF8 embryos

(A-F) Gross morphology of E18.5 control (A-C) and OCAGF8 (D-F) embryos. (A, D) Lateral view of head. (B, E) Dorsal view of the head. (C, F) Lateral view of the body. (G-I): Bone and cartilage staining of E18.5 forelimbs from control (G), OR26F8 (H), and OCAGF8 (I) embryos. Mutants are shown at the same magnification as the controls. Abbreviations: FL, forelimb; HL, hindlimb. Hu, humerus; Ra, radius; Sc, scapula; Ul, ulna. Scale Bars = A-B, D-E, G-I: 1mm; C, F: 2mm.

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Supplemental Figure 9. OR26F8 and OCAGF8 mice have craniofacial and skeletal defects.

(A-B): Gross morphology of P0 control (A) and OR26F8 (B) heads, lateral view. (C-D): Bone and cartilage staining of E16.5 control (C) and OR26F8 (D) heads, lateral view. (E-F): Gross morphology of E16.5 control (E) and OCAGF8 (F) heads, lateral view. (G-H) Bone and cartilage staining of E16.5 control (G) and OCAGF8 (H) heads, lateral view. Note that for the bone and cartilage staining shown in panels C, D, G, H a reduced concentration of alcian blue was employed so that bone stained with alizarin red could be more readily visualized in the

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mutants. In addition, the skulls of the mutants were more fragile than the controls and so it was only possible to clear the embryos for a limited time before they were too damaged for photodocumentation. This leads to greater trapping of alcian blue, especially in (H), than would normally occur. Scale bars= 1mm.

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Supplemental Figure 10. Cartilage formation in the OCAGF8 dorsal skull at E16.5

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(A-C): Sagittal sections of E16.5 control (A) or OCAGF8 (B, C) skulls stained with toluidine blue and methyl green. Red lines superimposed on E16.5 embryo heads show plane of dissection. Note the cartilage in the dorsal (C), but not lateral (B) OCAGF8 E16.5 skull. In contrast, both the dorsal (A) and lateral skull sections (not shown) were similar in the controls. In C, the cartilage/lacunae morphology can be visualized in the lighter stained cartilage (top), whereas the characteristic reddish purple toluidine cartilage stain can be seen in the darker stained cartilage (bottom). Abbreviations: Ct, cartilage; SE, surface epithelium. Scale Bars = 80 µM.

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Supplemental Figure 11. Alkaline Phosphatase staining in OCAGF8 intramembranous and endochondral bones

Sections of E16.5 control (A, C, E) and OCAGF8 (B, D, F) stained with BM Purple to detect alkaline phosphatase activity and counterstained with nuclear Fast Red. (A, B) Sagittal sections of cranial vault; (C, D) sagittal sections of trunk showing ribs; (E, F) frontal sections of forelimb digits. Black arrows indicate regions of alkaline phosphatase activity (blue) in the control cranial vault (intramembranous bones) as well as the ribs and digits (endochondral bones) in both the controls and mutants. Red arrow indicates absence of activity in cranial vault of mutant. Scale Bars = A-B, 200 µΜ; C-F, 500 µΜ.

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Supplemental Figure 12. MR26F8 E14.5 and E15.5 phenotypes.

(A-D): Gross morphology (A-B) and cartilage stain (C-D) of E14.5 control (A, C) and MR26F8 (B, D) embryos. (E-F): Gross morphology of E15.5 control (E) and MR26F8 (F) heads. Note embryos in (A-B) have been fixed prior to photography. Scale Bars= A-D, 2mm; E-F, 1mm.

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Supplemental Figure 13. FaceCAGF8 mice skeletal defects.

Bone and cartilage staining of control (A, C, E) and FaceCAGF8 (B, D, F) skulls at P0 (A-B), P12 (C-D), and P20 (E-F), dorsal view. Yellow dotted lines note the boundary of the bone. Black arrowheads denote abnormal tissue in the region that should be occupied by the nasal bones. Abbreviations: Fr, frontal; Pr, parietal. Scale Bars= 1 mm.

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Supplemental Tables

Supplemental Table 1. Gene comparison of Wildtype Cranial Vault vs. MCAGF8 Unless otherwise specified, all data are derived from comparing wildtype (WT, Control) to MCAGF8 (Mutant, Treatment) cranial vault. Cranial vault tissue was collected from 9 E14.5 control and mutant embryos as outlined in Fig. 7.

Tab1: All genes reported from RNA-seq sorted by ENSEMBL ID. Means are expressed as RPKM.

Tab2: Significant genes (p<0.05), sorted by fold change. Blue highlighted cells are genes downregulated <-1.5; yellow highlighted cells are genes upregulated >1.5.

Tab3: Genes listed in text, sorted by order of appearance. Left: wildtype cranial vault vs mutant cranial vault; Right: wildtype cranial base vs mutant. Blue highlighted cells are genes downregulated <-1.5; yellow highlighted cells are genes upregulated >1.5. Significant P-values (p<0.05) highlighted in green.

Tab4: Significant genes (p<0.05), sorted by control mean, with a control mean RPKM >100 and have an expression fold change of either >1.35 (upregulated) or <-1.35 (downregulated). Blue highlighted cells are genes downregulated <-1.5; yellow highlighted cells are genes upregulated >1.5.

Tab5: Significant genes (p<0.05), sorted by treatment (mutant) mean, with an MCAGF8 mean RPKM >100 and an expression fold change of either >1.35 (upregulated) or <-1.35 (downregulated). Blue highlighted cells are genes downregulated <-1.5; yellow highlighted cells are genes upregulated >1.5.

Tab6: Relevant values and histogram plot of normalized RPKM values of genes associated with BMP signaling and significantly dysregulated in MCAGF8 cranial vault samples (orange) as compared to controls (blue). Error bars represent relative standard error calculated from 3 replicates.

Tab7: Relevant values and histogram plot of normalized RPKM values of genes associated with Hedgehog signaling and significantly dysregulated in MCAGF8 cranial vault samples (orange) as compared to controls (blue). Error bars represent relative standard error calculated from 3 replicates.

Tab8: Top functional annotation clusters as calculated by DAVID using all significant genes (p<0.05).

Tab9: Top functional annotation charting as calculated by DAVID using significant genes (p<0.05) that have a fold change of >1.5 or <-1.5.

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Click here to Download Table S1

Supplemental Table 2. Gene comparison of Wildtype Cranial Base vs. MCAGF8 cranial vault

Unless otherwise specified, all data are derived from comparing wildtype cranial base (WT, Control) and MCAGF8 (Mutant, Treatment) cranial vault tissue. Cranial tissue was collected from 9 E14.5 control and MCAGF8 embryos as outlined in Fig. 7.

Tab1: All genes reported from RNA-seq sorted by ENSEMBL ID. Means are expressed as RPKM.

Tab2: Significant genes (p<0.05), sorted by fold change. Blue highlighted cells are genes downregulated <-1.5; yellow highlighted cells are genes upregulated >1.5.

Tab3: Significant genes (p<0.05), sorted by control mean, with a control mean RPKM >100 and have an expression fold change of either >1.35 (upregulated) or <-1.35 (downregulated). Blue highlighted cells are genes downregulated <-1.5; yellow highlighted cells are genes upregulated >1.5.

Tab4: Significant genes (p<0.05), sorted by treatment (mutant) mean with a MCAGF8 mean RPKM >100 and an expression fold change of either >1.35 (upregulated) or <-1.35 (downregulated). Blue highlighted cells are genes downregulated <-1.5; yellow highlighted cells are genes upregulated >1.5.

Tab5: Top functional annotation clusters as calculated by DAVID using all significant genes (p<0.05).

Tab6: Top functional annotation charting as calculated by DAVID using significant genes (p<0.05) that have a fold change of >1.5 or <-1.5.

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Click here to Download Table S2

Primer Name Purpose Sequence

Fgf8b FWD Cloning GGATC CCTCG AGCGC GCCAT GGGCA GCCCC CGCTC C Fgf8b REV Cloning CGAGC TGAAG CTTCG CCTAT CGGGG CTCCG GGGCC CAAG BTR ES Screening/

Genotyping CTAGA GCGGC CTCGA CTCTA CGATA CCGTC GATCC CC

R5F ES screening, outside 5' homology

GGCTG TGCTT TGGGG CTCCG GCTCC TCAG

GFP F ES screening CCAAC GAGAA GCGCG ATCAC ATGGT CCTGC TGGAG TTCGT G

R3R ES screening, outside 3' homology

CCTCA GAGAA ATGGA GTAGT TACTC CACTT TCAAG TTCC

CMV R1 ES screening/ Genotyping

CGTTG GGCGG TCAGC CAGGC GGGCC ATTTA CCG

Rosa F Genotyping GGGAG TTCTC TGCTG CCTCC TGGCT TCTGA GG Rosa R Genotyping CCTGC AGGAC AACGC CCACA CACCA GG Cre1 Cre

Genotyping GCTGG TTAGC ACCGC AGGTG TAGAG

Cre3 Cre Genotyping

CGCCA TCTTC CAGCA GGCGC ACC

oIMR0039 LacZ Genotyping

ATCCT CTGCA TGGTC AGGTC

oIMR0040 LacZ Genotyping

CGTGG CCTGA TTCAT TCC

Fgf8-F qRT-PCR CCGGA CCTAC CAGCT CTACA Fgf8-R qRT-PCR GGCAA TTAGC TTCCC CTTCT Bactin-qF qRT-PCR GCGAG CACAG CTTCT TTG Bactin-qR qRT-PCR CCATG TTCAA TGGGG TACTT C

Supplemental Table 3. Primer Names and Sequences

Primer name, purpose, and sequences for all primers utilized.

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