From shape to cells: mouse models reveal mechanisms ... · mutation or the other, it has been proposed that patients carrying the S252W mutation present with more severe palatal dysmorphology

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INTRODUCTIONApert syndrome [OMIM 101200] is a rare congenital disorder withdisease prevalence of 15-16 per million live births. Patients withApert syndrome are characterized by premature fusion of thecoronal suture(s) and severe craniofacial dysmorphology (Cohenand MacLean, 2000), but also exhibit many other developmentaldefects, including limb abnormalities, heart and lung defects, aswell as neural malformations that could lead to cognitiveimpairment (Cohen and MacLean, 2000). In humans, there are atleast 70 nucleotide substitutions that alter the amino acid sequenceof fibroblast growth factor receptor (FGFR) genes (Hébert, 2011).Two of these, Ser252Trp (S252W) and Pro253Arg (P253R), occuron neighboring amino acids on the linker region between thesecond and third extracellular immunoglobulin domain onfibroblast growth factor receptor 2 (FGFR2) and together areresponsible for almost 99% of reported cases of Apert syndrome.Approximately 67% of individuals with Apert syndrome have aFGFR2 S252W mutation, while the remaining 33% carry the FGFR2P253R mutation (Park et al., 1995; Wilkie et al., 1995). Thephenotypic outcome of Apert syndrome mutations is generally

similar, such that genetic testing is required to identify the causativemutation. Comparative analyses suggest only highly localizedphenotypic differences between the two FGFR2 mutations (Slaneyet al., 1996). Cleft palate is 3.5 times more frequent in patientscarrying the S252W mutation, whereas digit fusion (i.e. syndactyly)is more severe in patients with the P253R mutation (Slaney et al.,1996). These qualitative analyses are, however, based on relativelysmall samples of patients and details of differential effects of FGFR2mutations on patients with Apert syndrome remain poorlyunderstood.

Given the relatively low prevalence of Apert syndrome inhumans, analysis of appropriate and representative mouse modelsfor Apert syndrome, such as Fgfr2+/S252W and Fgfr2+/P253R mice(Wang et al., 2005; Wang et al., 2010), is crucial for an understandingof the developmental mechanisms underlying the phenotypicdifferences among patients carrying one FGFR2 mutation or theother. The correspondence between Apert syndrome mouse modelsand human patients with Apert syndrome has been demonstratedat the morphological, histological and molecular levels (Chen etal., 2003; Wang et al., 2005; Wang et al., 2010; Holmes et al., 2009;Aldridge et al., 2010; Du et al., 2010; Martínez-Abadías et al., 2010;Martínez-Abadías et al., 2011), validating the use of largeexperimentally controlled samples of mouse models to elucidatethe complex etiology of Apert syndrome.

Using a comparative sample of newborn Fgfr2+/S252W andFgfr2+/P253R Apert syndrome mouse models and their unaffectedlittermates (n=73) we found that murine skull dysmorphologiesparallel those described in Apert syndrome patients, and detectedsignificant morphological differences between the two Apertsyndrome mouse models (Martínez-Abadías et al., 2010). Aparticular highly localized difference at the posterior region of thepalatine bone prompted us to perform the current detailed analysisof the palate of Apert syndrome mouse models. Here, we analyzewith higher precision the palatal dysmorphologies of Fgfr2+/S252W

Disease Models & Mechanisms 6, 768-779 (2013) doi:10.1242/dmm.010397

1Department of Anthropology, Pennsylvania State University, 409 CarpenterBuilding, University Park, PA 16802, USA2Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine.One Gustave L. Levy Place, New York, NY 10029, USA*Present address: European Molecular Biology Laboratory (EMBL)-Center forGenomic Regulation (CRG) Systems Biology Research Unit, CRG, Barcelona 08003,Spain‡Author for correspondence ([email protected])

Received 18 June 2012; Accepted 27 February 2013

© 2013. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms.

SUMMARY

Apert syndrome is a congenital disorder characterized by severe skull malformations and caused by one of two missense mutations, S252W andP253R, on fibroblast growth factor receptor 2 (FGFR2). The molecular bases underlying differential Apert syndrome phenotypes are still poorlyunderstood and it is unclear why cleft palate is more frequent in patients carrying the S252W mutation. Taking advantage of Apert syndrome mousemodels, we performed a novel combination of morphometric, histological and immunohistochemical analyses to precisely quantify distinct palatalphenotypes in Fgfr2+/S252W and Fgfr2+/P253R mice. We localized regions of differentially altered FGF signaling and assessed local cell patterns to establisha baseline for understanding the differential effects of these two Fgfr2 mutations. Palatal suture scoring and comparative 3D shape analysis fromhigh resolution μCT images of 120 newborn mouse skulls showed that Fgfr2+/S252W mice display relatively more severe palate dysmorphologies, withcontracted and more separated palatal shelves, a greater tendency to fuse the maxillary-palatine sutures and aberrant development of the inter-premaxillary suture. These palatal defects are associated with suture-specific patterns of abnormal cellular proliferation, differentiation and apoptosis.The posterior region of the developing palate emerges as a potential target for therapeutic strategies in clinical management of cleft palate in Apertsyndrome patients.

From shape to cells: mouse models reveal mechanismsaltering palate development in Apert syndromeNeus Martínez-Abadías1,*, Greg Holmes2, Talia Pankratz1, Yingli Wang2, Xueyan Zhou2, Ethylin Wang Jabs2 and Joan T. Richtsmeier1,‡

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Disease Models & Mechanisms 769

Palatal dysmorphology in Apert syndrome RESEARCH ARTICLE

and Fgfr2+/P253R newborn mice (n=120), identifying further shapedifferences between the two Apert syndrome mouse models, andperform histological and immunohistochemical analyses to identifythe cellular and molecular mechanisms affected by the Fgfr2mutations that contribute to palatal dysmorphology in Apertsyndrome.

Palatal malformations occur in 75% of Apert syndrome patientsand include cleft soft palate or bifid uvula, as well as highly archedand constricted palates with a median furrow, lateral palatalswellings, relatively shorter hard palate and relatively longer andthicker soft palate (Kreiborg and Cohen, 1992; Cohen andKreiborg, 1996). Understanding the consequences of alteredFGF/FGFR signaling in the palates of Apert syndrome mousemodels can help elucidate phenotypic differences among Apertsyndrome patients and might also suggest potential mechanismsunderlying cleft palate, one of the most common human birthdefects (Mossey et al., 2009; Dixon et al., 2011). Palate

development is a complex and incompletely understood processthat requires a fine spatial and temporal orchestration ofmolecular and tissue interactions during embryogenesis thatenable outgrowth, elevation, reorientation, adhesion and fusionof palatal shelves (Gritli-Linde, 2007; Iwata et al., 2011; Yu andOrnitz, 2011). Failure or change in any of these stages of palatalshelf development, or in any associated tissues of the oropharynx(e.g. tongue), can lead to palatal anomalies including cleft palate.Since FGF/FGFR is one of the interacting signaling pathwaysinvolved in palate development (Gritli-Linde, 2007), a detailedmorphological examination of palatal morphogenesis usingmouse models carrying well-defined mutations in FGF/FGFRsignaling will contribute to our growing knowledge of the role ofthese signaling pathways in palate development.

To further investigate how the two mutations on FGFR2 causingApert syndrome in humans differently affect palatal morphologyin mouse models for Apert syndrome, we combined 3D geometricmorphometric methods with histologic and immunohistochemicaltechniques in a novel characterization of palatal development. Weperformed geometric morphometric analysis of 3D landmark dataregistered from microCT (μCT) images of newborn (P0) skulls ofFgfr2+/S252W and Fgfr2+/P253R mouse models and assessed the patternof fusion of five palatal sutures to precisely define differential palataltraits among Apert syndrome mouse models. Our analyses of largesamples of Fgfr2+/S252W and Fgfr2+/P253 Apert syndrome mousemodels and unaffected littermates guided detailed histologicalanalysis and immunohistochemical assays that defined the relativeroles of cell proliferation, differentiation and apoptosis that lead topalatal dysmorphogenesis.

Our results indicate a differential phenotypic effect of the S252Wand P253R FGFR2 mutations in palatal morphogenesis and confirmthat, as observed in humans, the FGFR2 S252W mutation isassociated with more severe palatal dysmorphology. Histologicaland immunohistochemical analyses performed at the anatomicalsites identified by the morphometric analyses provide suture-specific mechanistic explanations of the cellular and molecularprocesses leading to the differential palatal traits of Fgfr2+/S252W

Apert syndrome mouse models. Translated into the clinicalpractice, our results could help to improve the management ofpatients with these craniofacial disorders.

RESULTSTo comparatively assess the effects of FGFR2 mutations on palatemorphology, we analyzed patterns of palatal shape variation usinggeometric morphometric (GM) methods (Dryden and Mardia,1998; Lele and Richtsmeier, 2001). Three-dimensional (3D)coordinates of a set of ten landmarks located along the medialaspects of the horizontal plate of the right and left palatine boneswere collected from the reconstructed isosurfaces of the μCTimages of each specimen (Fig. 1, Table 1) using heads of newbornFgfr2+/S252W (n=24) and Fgfr2+/P253R (n=41) Apert syndrome mice,as well as their unaffected littermates that do not carry the mutationand that we use as controls (Fgfr2+/+ S252W, n=26; Fgfr2+/+ P253R,n=29). We assessed the pattern of fusion of five palatal sutures(inter-premaxillary, inter-maxillary, inter-palatine, right and leftmaxillary-palatine), qualitatively scoring the sutures as patent,partially fused or completely fused as visualized on μCT (see Fig. 1)(see Materials and Methods for more details).

TRANSLATIONAL IMPACT

Clinical issueApert syndrome is a rare congenital disorder characterized by skullmalformations and facial abnormalities. Almost 100% of Apert syndromecases are caused by one of two amino acid substitutions, S252W or P253R, ina protein involved in FGF/FGFR signaling, fibroblast growth factor receptor 2(FGFR2). Though the causative FGFR2 mutations have been identified, we stillhave little understanding of how they contribute to abnormal craniofacialphenotypes. Despite overall similarity between patients carrying one FGFR2

mutation or the other, it has been proposed that patients carrying the S252Wmutation present with more severe palatal dysmorphology than patientscarrying the P253R mutation. In addition, cleft palate occurs 3.5 times morefrequently in the former group. In humans, it is difficult to differentiate thespecific effects of each mutation because of the relatively low incidence ofthe disease and high level of phenotypic variation. For this reason, mousemodels have become a valuable tool for determining the molecularmechanisms that alter palate development and lead to cleft palate in Apertsyndrome.

ResultsTo determine how the two major Apert syndrome mutations lead to differentpalatal dysmorphologies, this study utilizes Fgfr2+/S252W and Fgfr2+/P253R mousemodels. The authors report anatomical differences between the two mutantmouse models in terms of size and shape of the palatine bones, anddemonstrate that the most striking abnormalities are associated with the Fgfr2

S252W mutation. These include patency of the inter-premaxillary suture, awider separation of the palatine shelves and fusion of the suture between thepalatine bones and maxilla, bones that together define the secondary palate.Furthermore, the authors’ cellular analysis reveals suture-specific aberrantcellular proliferation, differentiation and apoptosis in newborn mice.

Implications and future directionsThis work shows that the two Apert syndrome mutations affect thedevelopment of the mouse palate in different ways. The results provide furtherevidence that the S252W mutation causes more severe palatal phenotypesthan the P253R mutation. However, in line with previous analyses ofcraniofacial phenotypes, these results also suggest that ‘severity’ may be ahighly localized phenomenon and that the local effects of altered FGF/FGFRsignaling should be considered cell- and tissue-specific. Among the localizedanatomical sites demonstrating perturbed FGF/FGFR signaling as a result ofthe S252W mutation is the posterior region of the palate, revealing a potentialtarget for new therapeutic strategies in the clinical management of Apertsyndrome.

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Palatal dysmorphology in Apert syndromeRESEARCH ARTICLE

Differential palate dysmorphologies in Fgfr2+/S252W and Fgfr2+/P253

Apert syndrome micePalate shape information was extracted from the landmarkcoordinates using a General Procrustes Analysis (GPA), a procedurethat superimposes configurations of landmarks and adjusts for theeffects of orientation and scale (Dryden and Mardia, 1998).Allometric shape variation related to size was accounted forstatistically by computing a multivariate regression of shape on size(see Materials and Methods for details). We used the allometry-adjusted Procrustes coordinates as input data for canonical variatesanalysis (CVA), a discriminant analysis used to explore shapevariation and to maximize separation between the four pre-definedgroups of newborn mice, including: Fgfr2+/S252W and Fgfr2+/P253R

Apert syndrome mice and their respective unaffected littermates.The first two canonical variates (CV1 and CV2) explain almost

all the morphological variation of the sample (95.92%) anddistribute the specimens into three different clusters: a cluster ofFgfr2+/P253R Apert syndrome mice, a cluster of unaffected littermates

from both models and a cluster of Fgfr2+/S252W Apert syndromemice (Fig. 2A). The CVA showed that the main differences in palatalshape occur between Fgfr2+/S252W and Fgfr2+/P253R Apert syndromemice, which are distributed at the positive and negative extremesof the first canonical axis, respectively (Fig. 2A). Unaffectedlittermates show an intermediation position along CV1 (Fig. 2A).

Comparison of the associated shape changes corresponding tothe extreme negative and positive CV1 values (Fig. 2B) shows thatthe palatine bones are contracted along the antero-posterior andmedio-lateral axes in Fgfr2+/S252W Apert syndrome mice (Fig. 2Bb).In comparison to the mean shape, the anterior landmarks locatedalong the outline of the palatine bones of Fgfr2+/S252W mice areshifted to a more posterior position (landmarks 1-4), whereas theposterior landmarks are shifted to a more anterior position(landmarks 5-10), and all landmarks of the palatal shelves aredisplaced laterally (Fig. 2Bb). These shape changes lead to palatinebones that are laterally displaced, with an associated increase inthe width of the inter-palatine suture in Fgfr2+/S252W mice (Fig. 2B,top right) relative to Fgfr2+/P253R mutant mice (Fig. 2Ba).

Fgfr2+/S252W and Fgfr2+/P253R Apert syndrome mice are separatedfrom their unaffected littermates along CV2 (Fig. 2A), though thereis some overlap among groups. Shape differences between mutantand unaffected mice associated with this axis are localized to theposterior aspect of the bony palate (Fig. 2Bc,Bd). In unaffectedlittermates (positive end of CV2), the posterior aspect of thepalatine bones show an arched outline (Fig. 2Bd), that graduallycurves laterally and posteriorly away from the midline. This is inmarked contrast to the outline of the palatine bones of Fgfr2+/S252W

and Fgfr2+/P253R Apert syndrome mice that reveal a markedposterior shift of the most posteromedial landmarks (landmarks5, 6) and an anterior shift of the most posterolateral landmarks(landmarks 9, 10) (Fig. 2Bc).

Fig. 1. Sutures and landmarks displayed on the palate of a P0unaffected littermate. The palate is shown from an inferiorview of a μCT reconstruction of the mouse skull in which themandible has been removed. Sutures are shown by colored lines:red, inter-premaxillary suture; purple, inter-maxillary suture;green, right and left maxillary-palatine sutures; yellow, inter-palatine suture. p, premaxilla; m, maxilla; pa, palatine. Landmarksare indicated by red dots; codes and definitions are provided inTable 1.

Table 1. Anatomical definitions of collected palatal landmarks

displayed in Fig. 1

Landmark

(right, left) Anatomical definition

(1) ralp, (2) lalp Most anterolateral point on the posterior palatine plate

(3) ramp, (4) lamp Most anteromedial point on the posterior palatine plate

(5) rpmp, (6) lpmp Most posteromedial point on the posterior palatine plate

(7) rpns, (8) lpns Most anterolateral indentation at the posterior edge of the palatine plate

(9) rplpp, (10) lplpp Most posterolateral point on the posterior palatine plate For more information visit http://www.getahead.psu.edu/LandmarkNewVersion/ P0_Mouse_Palate.html.

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GDMA confidence interval testing (Lele and Richtsmeier, 1995;Lele and Richtsmeier, 2001) reveals significant differences in theway that the horizontal plates of the palatine bones of Fgfr2+/S252W

and Fgfr2+/P253R Apert syndrome mice differ from their respectiveunaffected littermates (α=0.10). These analyses support the resultsof Procrustes-based analyses and provide statistical evidence ofthe differences in the localized effects of these two neighboringFGFR2 mutations on palatal development. GDMA confidenceintervals demonstrate that the distance between the mostanterolateral aspect of the horizontal plate of the palatine bone(where the palatine plates meet and eventually fuse with themaxillary alveolus; between landmarks 1 and 2) is increasedrelative to unaffected littermates in the Fgfr2+/S252W mutant micebut reduced relative to unaffected littermates in Fgfr2+/P253R mice.Posteriorly (between landmarks 7 and 8), the palate of Fgfr2+/S252W

Apert syndrome mice is wider, but there is no difference betweenFgfr2+/P253R mice and unaffected littermates. The horizontal platesof the palatine bones are also more profoundly reduced along theanteroposterior axis in Fgfr2+/S252W Apert syndrome mice.Together these observations suggest that the positioning of themaxillary alveolus and deficiency in development of the palatine

plate contribute to the differences in the effects of the two FGFR2mutations.

More severe palate dysmorphologies in Fgfr2+/S252W Apertsyndrome miceTo statistically test the degree of differentiation in palatal shapebetween groups, we computed the Mahalanobis distances betweenall possible pairs of groups (Klingenberg and Monteiro, 2005).Results indicate that based on palatine morphology, Fgfr2+/S252W

and Fgfr2+/P253R Apert syndrome mice are significantly differentfrom each other as well as from their unaffected littermates, evenafter correcting for multiple testing by Bonferroni (Table 2).Unaffected littermates from S252W and P253R models are notsignificantly different from each other (Table 2). If we consider theMahalanobis distance from each mutant Apert mouse model totheir respective unaffected littermates as a measure of severity ofthe palatine dysmorphology, our results confirm that the palatesof Fgfr2+/S252W Apert syndrome mice are more severely affectedthan those of Fgfr2+/P253R Apert syndrome mice, because the theMahalanobis distance between mutant and unaffected littermatesis 1.4 times greater in Fgfr2+/S252W mice (Table 2).

Fig. 2. Canonical variates analysis. (A)Scatterplot corresponding to the first two canonical variates (CV1 and CV2). Ellipses account for 90% of within-groupvariation. Fgfr2+/S252W Apert syndrome mice (extreme positive CV1 values) show a greater range of variation and include those cases representing the mostextreme variation of the sample. (B)Shape changes (shown as wireframes) associated with extreme positive and negative values of CV1 and CV2 after adjustingfor allometry superimposed on 3D μCT surface reconstructions of the newborn mouse palate. Shape changes associated with CV values are represented by solidred wireframes. Black dotted wireframes represent the palatine morphology of the mean shape. (Ba)Negative values of CV1 (Fgfr2+/P253R); (Bb) positive values ofCV1 (Fgfr2+/S252W); (Bc) negative values of CV2 (Fgfr2+/S252W and Fgfr2+/P253R Apert syndrome mice); (Bd) positive values of CV2 (Fgfr2+/+ unaffected littermates).

Table 2. Pairwise Mahalanobis distances between Fgfr2+/S252W and Fgfr2+/P253R Apert syndrome mice and their unaffected littermate controls Fgfr2+/S252W Fgfr2+/+S252W Fgfr2+/P253R Fgfr2+/+P253R

Fgfr2+/S252W <0.0001 <0.0001 <0.0001

Fgfr2+/+S252W 2.95* <0.0001 0.29

Fgfr2+/P253R 4.07* 2.70* <0.0001

Fgfr2+/+P253R 3.40* 1.31 2.06*

Lower left off-diagonal matrix: Mahalanobis distance values. Upper right off-diagonal matrix: associated P-values estimated after 10,000 permutation rounds. *Statistically significant values after Bonferroni’s correction (P=0.05/6=0.008).

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Differential fusion patterns of palatal sutures in Apert syndromemouse modelsComparison of the qualitative scoring of palatal suture patencyshowed that the inter-maxillary and the inter-palatine sutures arepatent (~75-100% of cases) or just partially fused (~0-25%) in allfour groups (Fig. 3). Although there are no significant differencesbetween mutant and unaffected littermates in terms of the fusionpattern of these two midline sutures, Fgfr2+/S252W Apert syndromemice show a greater degree of patency of the inter-palatine suture(Fig. 3).

Fgfr2+/S252W and Fgfr2+/P253R Apert syndrome mice show a highertendency towards patency (~60%) or partial fusion (~20-40%) ofthe inter-premaxillary suture relative to their unaffected littermates,in which the inter-premaxillary suture is usually fused (~40-95%)or partially fused (~60-5%) at P0 (Fig. 3). Apparent differences inthe pattern of fusion of the inter-premaxillary suture between thetwo groups of unaffected littermates are not relevant. Completeinter-premaxillary suture fusion (FFF) was observed in 22 out of23 unaffected littermates of the S252W model, whereas 26 out of30 unaffected littermates of the P253R model have the inter-premaxillary suture completely (FFF) or almost completely fused(PFF). The palates of unaffected littermates from both Apert

syndrome mouse models are thus morphologically similar andcomparable, as confirmed by the rest of the morphometric analyses(Fig. 2; Table 2).

Finally, we identified a difference in fusion patterns of themaxillary-palatine sutures between Fgfr2+/S252W and Fgfr2+/P253R

Apert syndrome mice. In unaffected littermates of both models andin Fgfr2+/P253R Apert syndrome mice, the right and left maxillary-palatine sutures are patent (~70-95%) or just partially fused (~5-30%) (Fig. 3B-D), whereas these sutures are either partially (~35%)or completely fused (~25%) in Fgfr2+/S252W Apert syndrome miceat P0 (Fig. 3A).

Abnormal palate development in Fgfr2+/S252W Apert syndromemicePatterns of cell proliferation, differentiation and apoptosis wereassessed to establish a baseline for understanding the effects ofthe FGFR2 mutations on localized cellular behaviors that underlieApert syndrome. We focused our comparative histologicalanalysis and immunohistochemical assays on the most significantdifferential effects detected on the palate morphology ofFgfr2+/S252W Apert syndrome mice at P0: the greater degree offusion of the maxillary-palatine sutures, the greater separation ofpalatine bones at the midline and the higher tendency towardsinter-premaxillary suture patency. Similar analyses of Fgfr2+/P253R

Apert syndrome mice were not performed because the fusionpattern of the right/left maxillary-palatine in Fgfr2+/P253R mutantmice mimics the pattern of unaffected littermates (Fig. 3) andpalatal shape changes in Fgfr2+/P253R mice are relatively mild, withmorphologies that overlap with those of unaffected littermates(Fig. 2A).

Histological analyses confirmed that the maxillary-palatinesutures in Fgfr2+/S252W Apert syndrome mice commonly presentedwith localized partial premature fusion, showing variably fusedosteogenic fronts with deposition of osteoid as compared with thedistinct osteogenic fronts separated by undifferentiatedmesenchymal cells in the unaffected littermates (Fig. 4A,B). InFig. 4, a maxillary palatine suture of a Fgfr2+/S252W Apert syndromemouse displays various aspects of early fusion. The opposingosteogenic fronts meet initially at a center of fusion midwaybetween the dorsal and ventral aspects of the suture (Fig. 4B). Inregions of the suture that remain patent, the mutant palatine andmaxillary bones show the overlap typical of the unaffectedlittermates, although the mesenchymal interface between the twobones is often less regular in mutants (Fig. 4A,B and not shown).Alkaline phosphatase (ALP) activity, an early marker ofosteogenesis, is low or absent in normal suture mesenchyme(Fig. 4C). In the mutant suture, ALP is similarly absent from halfthe suture mesenchyme dorsal to the central locus of fusion, butappears in the half ventral to the point of fusion (Fig. 4D). Cellproliferation assessed by Ki67 staining showed similar numbers ofpositive cells in the unfused mutant suture mesenchyme andadjacent osteogenic faces as compared with sutures of unaffectedlittermates. Proliferation was maintained even as ALP activity rosewithin the mesenchyme of fusing sutures (Fig. 4E,F,O). Runx2expression, an additional marker of osteoblast differentiation, wassimilar in mutants and unaffected littermates, being higher inosteoblasts closer to bone and lower in the suture mesenchyme(Fig. 4G,H). As revealed by TUNEL staining, there was almost no

Fig. 3. Comparison of palatal suture patency. (A-D)Stacked columnsrepresent the percentage of specimens with complete patent (green), partiallyfused (yellow) or completely fused (blue) palatal sutures for (A) Fgfr2+/S252W

Apert syndrome mice, (B) Fgfr2+/+ unaffected littermates of the S252W model,(C) Fgfr2+/P253R Apert syndrome mice and (D) Fgfr2+/+ unaffected littermates ofthe P253R model.

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apoptosis in the suture mesenchyme of unaffected littermates, buta low frequency of apoptosis was evident within the palatine andmaxillary bones (Fig. 4I). In mutant sutures, the distribution andfrequency of apoptotic cells was similar to sutures of unaffectedspecimens, even in the area of increased ALP activity. However,focal areas of intense apoptosis occurred in fusing sutures at thepoint where osteoid deposition bridges the overlapping bones inFgfr2+/S252W mice (Fig. 4J). Antibody detection of thephosphorylated forms of the MAPK p38 and ERK1/2, majordownstream effectors of FGFR signaling, showed no appreciabledifferences between mutant and unaffected littermates, althoughsignals were decreased in the area of fusion dominated by apoptoticcells (Fig. 4K-N).

Histological analyses of the inter-palatine suture revealed palatinebones of Fgfr2+/S252W mice to be thicker and more robust relativeto unaffected littermates (Fig. 5A,B). Both the osteogenicmesenchyme of the suture and the non-osteogenic mesenchymeventral to the palatal bones were also thicker. Patency of the midline

inter-palatine suture in both mutant and unaffected littermates atP0 (Fig. 5A,B), as well as the relatively wider separation of thepalatine shelves in mutants detected by the CVA analysis wasevident histologically (Fig. 5C,D). Ki67 staining showed noproliferation differences in either the osteogenic fronts of thepalatine shelves or the intervening suture mesenchyme(Fig. 5E,F,O). Runx2 expression levels and distribution were similarin mutants and unaffected littermates, although the increasedthickness of the mutant osteogenic mesenchyme is evidenced bythe wider domain of Runx2 expression (Fig. 5G,H). Apoptotic cells,indicated by TUNEL staining, were infrequent amongmesenchymal cells or within osteogenic fronts of both Fgfr2+/S252W

Apert syndrome mice and unaffected littermates (Fig. 5I,J).Immunohistochemical staining for phosphorylated p38 and ERK1/2showed no difference between mutants and unaffected littermates(Fig. 5K-N).

Histological analyses of the inter-premaxillary suture revealedstriking differences between unaffected littermates andFgfr2+/S252W mutants. The paired premaxillary bones in unaffectedlittermates are broad, consisting of trabecular bone ventrally, andare separated at the midline by a thick mesenchyme. Eachpremaxilla extends vertically as a thin compact sheet of bonecurving around the overlying vomeronasal cartilage to endadjacent to the cartilage of the nasal septum. The mesenchymeseparating these vertical sheets is sparse and flattened osteoblastsline their surface, separated from the vomeronasal cartilage onthe opposite face by a mesenchyme a few cells in thickness(Fig. 6A). In contrast to unaffected littermates, the mutantpremaxillary bones are thicker ventrally and extend vertically asbroad trabecular wedges tapering to points adjacent to thecartilage of the nasal septum. The thin compact sheets of bone

Fig. 4. Maxillary-palatine suture at P0. (A,B)H&E staining shows a similaroverlap of maxillary (max) and palatal (pal) bones in unaffected littermates(Fgfr2+/+) and Fgfr2+/S252W sutures, but secreted osteoid bridges the two bones(arrowhead) in a mutant suture beginning to fuse. (C,D)Osteoblast-specificALP activity is absent from normal suture mesenchyme (C, arrow). In the earlystages of fusion, ALP is still absent in mutant mesenchyme (D, arrow) dorsal tothe point of fusion (white arrowhead), but has risen throughout mesenchymeventral (black arrowhead) to this point (D). ALP activity is imaged fluorescentlyand shown in gray channel. (E,F)Immunofluorescent staining for Ki67 (green)shows a similar frequency of positive cells in the suture mesenchyme andadjacent osteogenic surfaces (stained for ALP activity; red) of the maxillaryand palatal bones between unaffected littermates and mutant mice.(G,H)Immunofluorescent staining for Runx2 (green) shows similar expressionlevels between unaffected littermates and mutant mice, with expressionbeing generally lower in the suture mesenchyme than in osteoblasts withinosteogenic fronts (stained for ALP activity; red). (I,J)TUNEL staining (green)shows that apoptosis was negligible within the suture mesenchyme andinfrequent in the bones of wild-type sutures. In mutant sutures, apoptosis wasintense specifically in areas undergoing fusion (arrowhead). (K,L)Phospho-p38levels within bone and suture mesenchyme were similar in mutant andunaffected littermate sutures, although expression was decreased in the areaof mutant suture dominated by apoptotic cells (arrowhead). (M,N)Phospho-ERK 1/2 expression was concentrated around bone at similar levels in mutantand unaffected littermate sutures. (O)The percentage of suture mesenchymecells expressing Ki67 was not significantly different between mutant andunaffected littermates. All images are from near-adjacent parasagittal sectionsfrom the same littermate pair. Rostral direction is to the right. Scale bar:100 μm.

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seen in unaffected littermates seem to be retained in thesewedges, forming the lateral side curving along the vomeronasalcartilages, from which trabecular bone extends medially. Themesenchyme of Fgfr2+/S252W mice separating these bones remainsthick as it extends dorsally, but typically thins below the nasalseptum. The septal cartilage is often misaligned with the inter-premaxillary suture mesenchyme in the mutant suture. Only asingle cell layer typically separates the premaxillary bone fromthe vomeronasal cartilage. The vertical thickening of the inter-premaxillary bones and intervening mesenchyme greatly widensthe distance between the paired vomeronasal organs in mutantscompared with unaffected littermates (Fig. 6B).

Ki67 staining showed that the proportion of proliferating cellswas significantly increased within the midline mesenchymeseparating premaxillary bones in Fgfr2+/S252W mice compared with

unaffected littermates. This difference was especially notablecomparing the dorsal portion of the sutures (Fig. 6C,D,M). Theexpression levels of Runx2 were similar in Fgfr2+/S252W mice andunaffected littermates, with high expression in osteoblasts and lowexpression in the intervening mesenchyme, but this mesenchymewas more extensive in the mutant suture. The difference in thecellularity of the medial and lateral faces of the premaxillary bonesis also readily appreciated with Runx2 imaging, with more Runx2-expressing osteoblasts lining the medial surfaces and fewer liningthe lateral surfaces of mutant premaxillary bones compared withunaffected littermates (Fig. 6E,F). ALP activity levels were alsosimilar in Fgfr2+/S252W mice and unaffected littermates, butdecreased strongly in the sparse mesenchyme separating thevertical bone extensions in unaffected littermates (Fig. 6G,H).Apoptotic cells detected by TUNEL staining were infrequent inthe suture mesenchyme but were common in the adjacenttrabecular bone of both mutants and unaffected littermates andoccurred with significantly increased frequency in the Fgfr2+/S252W

mice (Fig. 6G,H,N). The levels of phosphorylated p38 (Fig. 6I,J)and phosphorylated ERK1/2 (Fig. 6K,L) were similar in mutantsutures and unaffected littermates.

DISCUSSIONOur results demonstrate the differential effects of the S252W andP253R FGFR2 mutations on palatal morphogenesis and reveal theaberrant cellular behaviors associated with the phenotypicdifferences between Fgfr2+/S252W and Fgfr2+/P253R Apert syndromemice. The morphometric analyses and the detailed scoring of palatalsuture patency detected significant palatal shape differencesbetween Fgfr2+/S252W and Fgfr2+/P253R Apert syndrome mice andunderscored what has been reported in humans: individualscarrying the S252W mutation show more severe palataldysmorphologies. The histological and immunohistochemicalanalyses highlighted that these palatal defects are highly localizedand suture-specific and can be associated with dynamiccombinations of abnormal patterns of cellular proliferation,differentiation and apoptosis. The extent of these changes at P0suggests their beginnings earlier in embryogenesis.

Fig. 5. Inter-palatine suture at P0. (A,B)H&E staining shows that mutantpalatal (p) bones are more robust than in unaffected littermates. Boxed areasare shown in C and D. (C,D)Within the suture, mutant palatal bones areseparated further apart and both the osteogenic mesenchyme and underlyingconnective mesenchyme are thicker. Boxed areas are shown in E-N.(E,F)Immunofluorescent staining for Ki67 (green) shows a similar frequency ofproliferating cells concentrated in the osteogenic fronts (stained for ALPactivity; red) and lower in the intervening suture mesenchyme betweenunaffected littermates and mutant mice. (G,H)Immunofluorescent staining forRunx2 (green) shows similar expression levels between unaffected littermatesand mutant mice (ALP activity; red). (I,J)TUNEL staining (green) was negligiblewithin the suture mesenchyme of unaffected littermates and mutant mice.(K,L)Phospho-p38 levels and (M,N) phospho-ERK 1/2 levels within osteogenicfronts and suture mesenchyme were similar in mutant and unaffectedlittermate sutures. (O)The percentage of proliferating (Ki67-positive) cells inthe left and right osteogenic fronts (OF) and intervening mesenchyme was notsignificantly different between mutant and unaffected littermates. Images inA-J are from near-adjacent sections from the same littermate pair. Images in K-N are from other littermate pairs. Scale bars: (A,B) 500 μm; (C,D) 100 μm; (E-N)50 μm.

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From bone palatal defects to cellular and molecular alterations inApert syndrome mouse modelsDifferential palatal defects in Fgfr2+/S252W and Fgfr2+/P253R mutantmice involve gross shape changes of the palatine bones (Fig. 2) anddifferences in fusion patterns of palatal sutures (Fig. 3). The moststriking palatal defects of Fgfr2+/S252W mice include palatal shelvesthat were contracted and reduced in length with an enlarged inter-palatine distance (Fig. 2B), a greater tendency to fuse the right andleft maxillary-palatine sutures and a tendency for the inter-premaxillary suture to remain patent (Fig. 3). Analysis of severallitters and specimens per genotype was crucial in revealing highlylocalized differences in dynamic and variable developmentalprocesses such as suture closure.

Our data confirm that premature fusion of the maxillary-palatine suture is a feature of palatal dysplasia in Apert syndrome(Purushothaman et al., 2011; Holmes and Basilico, 2012) andprovide a snapshot of the process of closure of this suture(Fig. 4B,D). Apoptosis in the mutant maxillary-palatine suture isspecifically increased in the areas of bone deposition that bridgethe two bones, but not in areas of the suture with increased ALPactivity that presumably precede fusion, or along bone surfacesin other regions of the maxillary and palatine bones (Fig. 4I,J).Increased apoptosis appears to be a later event in maxillary-palatine suture fusion, similar to its late occurrence in the fusingcoronal suture in this Apert mouse model (Holmes et al., 2009).

Regarding the inter-palatine suture, no significant changes in cellproliferation (Fig. 5E,F), differentiation (Fig. 5G,H) or death (Fig. 5I,J)were found. However, the histological analysis revealed that the suturemesenchyme and the palatal bones were thicker in Fgfr2+/S252W micerelative to unaffected littermates. The greater midline separation ofthe right and left palatine bones observed in Fgfr2+/S252W mice(Fig. 2B; Fig. 5A,B) might be the result of changes occurring earlierin embryogenesis and undetectable at P0 and/or of other cellularprocesses and mechanisms not analyzed here. Considering that Fgfr2is expressed in the outer layer of immature osteoblasts of thepalatine bones during development (Rice et al., 2003), a possibleexplanation that requires further testing is that altered growthkinetics bestowed by a mutant Fgfr2 might affect bone shapethroughout development, causing the localized thickening of palatinebones and the wider separation in the midline suture.

Our results reveal significantly abnormal cell patterns ofproliferation, differentiation and apoptosis local to the inter-premaxillary suture, causing a lack of fusion and maldevelopmentof the anterior palate in Fgfr2+/S252W mice. In contrast to what wasobserved at the maxillary-palatine suture, a high incidence ofapoptosis was common to both the unaffected and mutant inter-premaxillary suture, revealing a distribution largely restricted tothe bone-forming surfaces adjacent to the suture mesenchyme(Fig. 6G,H,N), occurring in the absence of suture fusion. Althoughthe incidence was significantly higher in the mutant suture, thiswould appear to be a normal developmental process at this location,

Fig. 6. Inter-premaxillary suture at P0. (A,B)H&E staining shows that mutant sutures present with aberrant trabecular overgrowth of the vertical extensions ofthe premaxillary bones (pm), thickening of the intervening suture mesenchyme and misalignment of the suture with the cartilage of the nasal septum (s).Widening of the mutant suture is evident in the greater separation of the cartilages (c) encapsulating the vomeronasal organs. (C,D,M) Immunofluorescentstaining for Ki67 (gray channel shown) shows increased proliferation in mutant suture mesenchyme compared with unaffected littermates.(E,F)Immunofluorescent staining for Runx2 (gray channel shown) shows similar expression levels between unaffected littermates and mutant mice, althoughmutant suture mesenchyme is more abundant and highly Runx2-positive cells are more numerous along the bone-mesenchyme interface. (G,H,N) TUNELstaining (green) shows minimal apoptosis within the suture mesenchyme of unaffected littermates and mutant mice but a high frequency in the adjacentosteogenic domains (stained for ALP activity; red). In these regions, apoptosis is significantly higher in mutants compared with unaffected littermates. (I-L)Phospho-p38 and phospho-ERK 1/2 levels are similar in mutant and unaffected littermate sutures. (M)Proliferation is significantly increased in mutant suturemesenchyme relative to unaffected littermates; *P=0.035. (N)Apoptosis is significantly increased in bone bordering the suture mesenchyme in mutants relativeto unaffected littermates; *P=0.026. Images from A-L are from near-adjacent sections from the same littermate pair. Scale bar: 100 μm.

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accentuated by aberrant FGF/FGFR signaling, and perhaps relatedto the differentiation of osteoblasts from mesenchyme with thefunction of limiting osteoblast numbers. Coupled with theincreased proliferation seen in mutant mesenchyme, the inter-premaxillary suture could provide an in vivo example of the findingin vitro that chronic FGF signaling or the expression of FGFR2craniosynostosis mutant proteins induces proliferation in immatureosteoblasts but apoptosis in mature osteoblasts (Mansukhani et al.,2000). The little-known roles of apoptosis in normal andpathological facial suture formation appears to be suture-specificand requires further study.

Finally, immunohistochemical staining suggested that alteredFGF/FGFR signaling in palatal sutures did not trigger significantdifferences in activation of MAPK, p38 and ERK1/2 in any of thesutures analyzed, at least at P0. However, p38 and ERK1/2 are majoreffectors of the FGF signaling pathway and it has been shown thatinhibition of ERK1/2 in mice carrying the S252W mutationsignificantly improves facial development, suggesting theimportance at least of ERK1/2 activity in the Apert facial phenotype(Shukla et al., 2007). Although we did not see significant differencesin FGFR2 signaling within sutures as measured by detection ofphosphorylated p38 and phosphorylated ERK1/2, such changesmight be subtle in vivo, generating aberrant phenotypesincrementally over time.

Palate is a preferential target of the FGFR2 S252W Apert syndromemutationOur results suggest that the S252W mutation leads to more severepalate dysmorphologies than the P253R mutation, confirming thepattern already described in patients with Apert syndrome (Slaneyet al., 1996). Larger Mahalanobis distances (Table 2) and greaterranges of palatal shape variation (Fig. 2A) show that palates ofFgfr2+/S252W mutant mice are more severely affected. The palatalphenotype of Fgfr2+/P253R mice was not unaffected, however. Rather,the palates of the two Apert syndrome mouse models were affecteddifferently (Fig. 2), resulting in varying palatal dysmorphologies thatwere more intense in Fgfr2+/S252W mutant mice. Fgfr2+/P253R micemight show more severe malformations at other anatomicallocations or have other tissues or developmental systems moreseverely affected. In fact, patients carrying the P253R mutationpresent with more severe syndactyly (Slaney et al., 1996).

Perturbed FGF/FGFR signaling leads to palate dysmorphology inApert syndromeCompelling evidence suggests that FGF/FGFR signaling plays anessential role in palatal development mediating cell communicationand interaction (Wilke et al., 1997). Misregulation of FGF/FGFRsignaling can result in severe dysmorphogenesis and disease,including malformations of the palate that occur as a part of asyndrome or contribute to the occurrence of isolated cleft palate(Dixon et al., 2011). For instance, 3-5% of human cases ofnonsyndromic cleft lip and palate are associated with mutationsand single nucleotide polymorphisms affecting genes directlyinvolved in the FGF/FGFR pathway (Riley et al., 2007). Mousemodels carrying mutations on Fgf10, Fgf18, Fgf8 and Fgfr1, Fgfr2display abnormal or cleft palate providing further support of theinvolvement of FGF/FGFR signaling in the pathogenesis of cleftpalate (De Moerlooze et al., 2000; Abu-Issa et al., 2002; Liu et al.,

2002; Ohbayashi et al., 2002; Trokovic et al., 2003; Rice et al., 2004;Wang et al., 2005; Martínez-Abadías et al., 2010; Snyder-Warwicket al., 2010; Wang et al., 2010; Purushothaman et al., 2011).

Several mechanisms involving alternative splicing of the FGFR2molecules and abnormal interaction of mutant receptors withinappropriate ligands have been proposed to explain thepathogenesis of cleft palate in Apert syndrome. Ibrahimi andcolleagues suggested that the differential increase in FGFR2-IIIcaffinity for a specific ligand such as FGF2, which is expressed inboth facial ectoderm and mesenchyme, could explain the higherfrequency of cleft palate in Apert syndrome patients with theFGFR2 S252W mutation (Ibrahimi et al., 2001). Britto andcolleagues proposed that the incomplete expressivity of cleft palatein humans with Apert syndrome is consistent with a dose-dependent effect of FGFR2 mutations on palatal shelf fusion viathe FGFR2-IIIb isoform (Britto et al., 2002). In this case, the Apertmutations might confer a dominant-negative functional effect onFGFR2-IIIb in human palatal epithelia, resulting in failure of fusionof the palatal shelves (Britto et al., 2002). Hajihosseini and colleagueshypothesized that cleft palate in Apert syndrome is the result ofloss-of-FGFR2-IIIb function in epithelial cells, secondary to allelesthat cause a gain-of-FGFR2 function in mesenchymal cells(Hajihosseini et al., 2009).

These varying lines of evidence highlight altered cross-talkbetween epithelial and mesenchymal cells, mediated by FGF/FGFRsignaling and causing palate malformations. Our findings in Apertsyndrome mouse models suggest that cellular miscommunication,evidenced by local variation in patterns of cell proliferation,differentiation and apoptosis at P0, is associated with highlylocalized palate dysmorphologies that might be associated withaberrant signaling specific to early undifferentiated cell populations,formative sutures and/or bony precursors.

Insights into altered palate development in Apert syndromePatterns of palatal suture fusion scored in unaffected littermatessuggest that in normal palate development the fusion progressesfrom anterior to posterior, with anterior palatal sutures fusing firstbefore birth (i.e. inter-premaxillary suture) and posterior palatalsutures fusing postnatally (i.e. inter-maxillary, inter-palatine andmaxillary-palatine sutures) (Fig. 3). This gradient is reminiscent ofthat described for normal development of the palate and tongueduring elevation of the palatal shelves (Yu and Ornitz, 2011),suggesting a continuation of gradients of morphogenetic processesduring early and late palatal development. Here we show that palatedevelopment in Fgfr2+/S252W Apert syndrome mice is altered byaberrant cellular behaviors that cause a premature fusion of anteriortransverse palatal sutures (e.g. right and left maxillary-palatinesutures) (Fig. 4) as well as failure of closure of palatal midlinesutures, such as the inter-palatine (Fig. 5) and the inter-premaxillarysutures (Fig. 6), which are patent or show osteogenic fronts thatare more separated in Fgfr2+/S252W mutant mice. Interestingly, Apertsyndrome FGFR2 mutations also cause premature fusion of othernon-midline sutures of the craniofacial complex, such as thecoronal and the premaxillary-maxillary sutures (Wang et al., 2005;Wang et al., 2010; Martínez-Abadías et al., 2010). This abnormalpattern of suture patency and closure is associated with the typicalwide and short head shape (brachycephaly) and midfacial retrusionof Apert syndrome patients.

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Differences in fusion patterns of palatal sutures could stem fromstructural and/or suture-specific differences in localized FGF/FGFRsignaling environments. Overall, the three sutures examinedhistologically at P0 have strikingly different fates and structuralcharacteristics. The maxillary-palatine suture contains broadlyoverlapping bone edges separated by a reasonably narrowmesenchyme, similar to the coronal suture, where the degree ofoverlap is much shorter but which consistently fuses in Apertsyndrome mice (Holmes et al., 2009; Wang et al., 2005). The inter-palatine suture, on the other hand, has a butted structure similarto the sagittal suture, which fuses infrequently if at all in Apertsyndrome mice. The inter-premaxillary suture is unique, having abutted structure but also an extensive region of proximity betweenthe two premaxillary bones, and it is the suture that in unaffectedlittermates more readily fuses. Although the basis for suture-specificdifferences in FGF signaling are unknown, in the mutant inter-premaxillary suture the result is a more extensive and proliferativesuture mesenchyme relative to unaffected littermates, which mightprevent close contact and fusion between the paired premaxillarybones at P0.

Considering the potential differential sensitivity of mesenchymalcells occupying specific sutures to the presence of mutant receptorsand the selective distribution or concentration of FGF ligands(Hajihosseini et al., 2004), it is not surprising that the same FGFR2mutation appears to have different or even opposite effects on thepatency of varying cranial sutures. FGF/FGFR signaling is knownto be complex, and tissue-specific responses are widespread andvariable (Wilke et al., 1997; Ornitz and Marie, 2002; Dorey andAmaya, 2010; Hébert, 2011; Martínez-Abadías et al., 2011;Martínez-Abadías et al., 2013). The identity of FGFs expressedduring early craniofacial morphogenesis and palatal formation havebeen extensively characterized (Britto et al., 2002; Bachler andNeubüser, 2001; Welsh et al., 2007), but the complement of FGFsexpressed in the facial sutures during late embryologicaldevelopment is undefined. A broad range of FGFs is identifiable inthe coronal suture during late gestation (Hajihosseini and Heath,2002), so the repertoire of FGF signaling within the various facialsutures, modified by their particular affinity for the FGFR2 S252Wand P253R mutant receptors, could be similarly complex and leadto such diverse phenotypic outcomes.

Our results add to the accumulating evidence that althoughFGF/FGFR signaling contributes to the development of manycranial tissues, the effects can be highly localized, varying accordingto the tissue considered and the time of development. Our analysisreveals several localized sites of altered palatal development andconfirms that the posterior palate is one of the palatal regions mostaffected by perturbed FGF/FGFR signaling caused by the FGFR2S252W mutation. These anatomical sites might serve as a potentialtarget for therapeutic strategies in clinical management of cleftpalate in Apert syndrome patients.

MATERIALS AND METHODSMouse modelsFgfr2+/S252W and Fgfr2+/P253R Apert syndrome mouse models werebred on C57BL/6J genetic background for 20 generations tominimize phenotypic variation and were generated, euthanized,fixed and imaged in compliance with animal welfare guidelinesapproved by the Johns Hopkins University, the Mount Sinai School

of Medicine and the Pennsylvania State University Animal Careand Use Committees. Further details on generation of targetingconstruct can be found elsewhere (Wang et al., 2005; Wang et al.,2010).

Skull imagingHigh resolution micro-computed tomography (μCT) images of themouse heads were acquired by the Center for Quantitative Imagingat the Pennsylvania State University (www.cqi.psu.edu) using theHD-600 OMNI-X high-resolution X-ray computed tomographysystem (Bio-Imaging Research Inc., Lincolnshire, IL). Pixel sizesranged from 0.015 to 0.020 mm, and slice thickness from 0.016 to0.025 mm. Image data were reconstructed on a 1024×1024 pixelgrid as a 16-bit TIFF but reduced to 8 bit for image analysis. Basedon hydroxyapatite phantoms imaged with the specimens, theminimum thresholds used to create isosurfaces from these 8-bitimages ranged from 70 to 100 mg/cm3 partial density ofhydroxyapatite. To visualize skull morphology, isosurfaces werereconstructed from the μCT images using the software packageAvizo 6.0 (Visualization Sciences Group). A set of 10 palatallandmarks (Fig. 1, Table 1) were collected twice by the sameobserver to minimize measurement error; deviations between thetwo trials were restricted to 0.05 mm.

Scoring patterns of suture fusionWe assessed the pattern of fusion of five palatal sutures (inter-premaxillary, inter-maxillary, inter-palatine, right and leftmaxillary-palatine), qualitatively scoring the sutures as patent,partially fused or completely fused (Fig. 1). For the inter-premaxillary suture, we divided the whole length of the suture intothree different segments (anterior, middle, posterior) and scoredthe degree of fusion at each segment as open (O), partial (P) orfused (F) independently. Once a code was obtained for each of thethree sections of the inter-premaxillary suture, the three codes wereassembled into a pattern of suture fusion. For example, individualspresenting with a completely open inter-premaxillary suture werecoded as ‘OOO’ and were included in the analysis as ‘patent’,whereas individuals with a totally fused inter-premaxillary suturewere coded as ‘FFF’ and were included in the analysis as ‘completelyfused’. All those cases in which at least one segment was partiallyfused (P) were pooled into the ‘partially fused’ category.

Geometric morphometric analysesGeneral Procrustes Analysis (GPA), a procedure that superimposesconfigurations of landmarks by shifting them to a common position,rotating and scaling them to a standard size until a best fit ofcorresponding landmarks is achieved, was used to extract shapeinformation (Dryden and Mardia, 1998). GPA, however, does noteliminate the allometric shape variation that is related to size. Weadjusted for the effect of allometry (Drake and Klingenberg, 2008)by computing a multivariate regression of shape on centroid size,measured as the square root of the summed distances between eachlandmark coordinate and the centroid of the landmarkconfiguration (Dryden and Mardia, 1998).

Canonical variates analysis (CVA) was performed to exploreshape variation and to maximize discrimination between Apertsyndrome mouse models and their unaffected littermates. CVAderives discriminant functions called canonical variates (CVs) that

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result from an optimal combination of variables (the Procrustescoordinates), so that the first one provides the most overalldiscrimination between groups; the second provides the second-most, and so on (Manly, 2004). The discriminant functionscorrespond to shape features that are independent or orthogonal,that is, their contributions to the discrimination between groupsdoes not overlap.

Mahalanobis distances, a distance measure used in clusteranalysis and classification techniques, were computed from thepooled within-group covariance matrix for all groups after adjustingfor allometry to assess the degree of differentiation between allpossible pairs of groups of Apert syndrome mouse models and theirunaffected littermates (Klingenberg and Monteiro, 2005). Statisticalsignificance for all pairwise comparisons was tested usingpermutation tests based on 10,000 iterations under the nullhypothesis of complete dissimilarity. The P values obtained wereadjusted for the lack of independence using the Bonferroni method:the total comparisons were c=k(k–1)/2 (where k is the number ofpopulations) and the significance level 0.05 was divided by c toguarantee real significance. All geometric morphometric methodsbased on Procrustes analysis were performed using MorphoJ(Klingenberg, 2011).

To statistically determine differences in the way the two mutantphenotypes differed from their respective unaffected littermates,we used a confidence interval procedure developed within EDMA,Euclidean Distance Matrix Analysis (Lele and Richtsmeier, 1995;Lele and Richtsmeier, 2001). EDMA converts 3D landmark datainto a matrix of all possible linear distances between uniquelandmark pairs and tests for statistical significance of differencesbetween shapes using non-parametric confidence intervals (Leleand Richtsmeier, 1995; Lele and Richtsmeier, 2001). An averageform is estimated using the linear distance data and differencesin 3D size and shape are statistically compared as a matrix of ratiosof all like linear distances in the two samples. We tested formorphological differences in each mutant group as compared withits unaffected littermates, and for differences in themutant/unaffected contrasts between the two mouse models forApert syndrome. The null hypothesis for each comparison is thatthere is no difference in shape between groups (or no differencein shape contrasts). Statistical tests for differences in specific lineardistances were evaluated by a non-parametric bootstrappingprocedure (Lele and Richtsmeier, 1995). For each linear distance,a ratio between the average values of that distance for each groupwas computed and confidence intervals for the null hypothesisof similarity in shape were estimated from 100,000 pseudo-samples generated from the data using a non-parametricbootstrapping algorithm. For each linear distance, the nullhypothesis was rejected if the 90% confidence interval producedfrom the bootstrapping method did not include 1.0. Rejection ofthe null hypothesis enables localization of differences to specificlandmarks and linear distances (Lele and Richtsmeier, 1995; Leleand Richtsmeier, 2001). EDMA analyses were performed usingWinEDMA (Cole, 2002).

Histological and immunohistochemical analysesHistological sections (5 μm) were prepared from selected tissuesthat had been fixed in 4% paraformaldehyde for 24 hours,demineralized in 10% EDTA in PBS for 5 days, and embedded in

paraffin. We studied the maxillary-palatine sutures by sectioningthe head in the sagittal plane using the anterior-posterior axis fromthe nose to vertebra; whereas the inter-palatine and inter-premaxillary sutures were analyzed by sectioning the head in thecoronal plane. Sections were stained with haematoxylin and eosin(H&E) for histological analysis. Alkaline phosphatase (ALP) stainingwas performed as described (Miao and Scutt, 2002). Antibodiesused were rabbit anti-Ki67 (1:200; Vectorlabs, VP-RM04), rabbitanti-Runx2 (1:200; Sigma, HPA022040), rabbit anti-phospho-MAPK ERK1/2 (1:200; Cell Signaling, 4376) and rabbit anti-phospho-p38 MAPK (1:100; Cell Signaling, 4631). Antibodies weredetected by the sequential application of biotinylated anti-rabbitIgG (1:200; Vectorlabs, BA-1000) and streptavidin-Alexa Fluor 488conjugate (1:200 of a 1 mg/ml stock; Invitrogen, S-11223) forimmunofluorescence or by the SignalStain Boost IHC DetectionReagent and Signalstain DAB Substrate (Cell Signaling, 8115 and8059) for MAPK immunohistochemistry. Nuclei were stained withHoechst 33258 (1:20,000 of a 100 mg/ml stock; Invitrogen, H1398).The TUNEL assay was carried out using the In Situ Cell DeathDetection Kit, POD (Roche Applied Science, 11684817910) todetect apoptotic cells. For each staining, at least two litters of micewere examined. Brightfield and fluorescent photomicrographswere taken on a Nikon Eclipse 80i microscope with a Q ImagingRetiga 4000R camera using QCapture software. Images wereprocessed in Adobe Photoshop. For statistical analysis, at least threeanimals per genotype were used, and data was analyzed using theunpaired, two-tailed Student’s t-test. Differences with a P valuebelow 0.05 were considered significant.ACKNOWLEDGEMENTSWe gratefully acknowledge Colin Shaw, Tim Ryan and Tim Stecko from CQI(Pennsylvania State University) for their excellent technical skill and support inmicroCT scanning. We thank Yann Heuzé for fruitful discussions of earlier versionsof this manuscript, as well as editorial suggestions. We thank the editor and twoanonymous reviewers whose comments and suggestions greatly improved theoverall quality of the manuscript.

COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

AUTHOR CONTRIBUTIONSN.M.-A., J.T.R., Y.W. and E.W.J. conceived and designed the experiments. N.M.-A.,G.H., T.P., X.Z. and Y.W. collected data and performed the experiments. N.M.-A. andJ.T.R. analyzed the data. G.H., Y.W. and E.W.J. contributed reagents and materials.N.M.-A. and J.T.R. wrote the paper. T.P., G.H., Y.W., E.W.J. and J.T.R. provided criticalrevision and final approval of the manuscript.

FUNDINGThis work was funded by the National Institutes of Craniofacial and DentalResearch, National Institutes of Health and the American Recovery andReinvestment Act [grant numbers R01 DE018500, 3R01 DE018500-02S1, 5R01DE022988]; and Comissionat per a Universitats i Recerca, Generalitat de Catalunya(Spain) [grant number 2008 BP A 00170].

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From shape to cells: mouse models reveal mechanisms ... · mutation or the other, it has been proposed that patients carrying the S252W mutation present with more severe palatal dysmorphology