it is made available under a cc-by-nc 4.0 international ... · 1 quantification of gene expression...

1

Quantification of gene expression patterns to reveal the origins of abnormal morphogenesis 1

2

N. Martínez‐Abadías1,2,3,*,†, R. Mateu3, J. Sastre4, Motch Perrine S5, Yoon M5, A. Robert‐Moreno1,2,†, J. 3

Swoger1,2,†, L. Russo1,2, Kawasaki K5, J. Richtsmeier5, J. Sharpe1,2,6,† 4

5

1 Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 6

88, 08003 Barcelona, Spain 7

2 Universitat Pompeu Fabra (UPF), Barcelona, Spain 8

3 Universitat de Barcelona, Barcelona, Spain 9

4 Universitat de les Illes Balears (UIB), Palma de Mallorca, Spain 10

5 Pennsylvania State University, University Park (PA), USA 11

6 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 12

* Corresponding author: [email protected]. Dr. Aiguader, 88, ‐1, 08003, Barcelona (Spain). 13

†Current address: European Molecular Biology Lab (EMBL), Barcelona, Spain 14

15

Abstract 16

The earliest developmental origins of dysmorphologies are poorly understood in many congenital 17

diseases. They often remain elusive because the first signs of genetic misregulation may initiate as 18

subtle changes in gene expression, which can be obscured later in development due to secondary 19

phenotypic effects. We here develop a method to trace back the origins of phenotypic abnormalities by 20

accurately quantifying the 3D spatial distribution of gene expression domains in developing organs. By 21

applying geometric morphometrics to 3D gene expression data obtained by Optical Projection 22

Tomography, our approach is sensitive enough to find regulatory abnormalities never previously 23

detected. We identified subtle but significant differences in gene expression of a downstream target of 24

the Fgfr2 mutation associated with Apert syndrome. Challenging previous reports, we demonstrate that 25

Apert syndrome mouse models can further our understanding of limb defects in the human condition. 26

Our method can be applied to other organ systems and models to investigate the etiology of 27

malformations. 28

29

.CC-BY-NC 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted January 11, 2018. . https://doi.org/10.1101/246256doi: bioRxiv preprint

https://doi.org/10.1101/246256

http://creativecommons.org/licenses/by-nc/4.0/

2

Keywords: Developmental defects, Optical Projection Tomography (OPT), Whole‐Mount‐in‐Situ 30

Hybridization (WISH), Geometric Morphometrics (GM), quantitative shape analysis, limb development, 31

Apert syndrome. 32

33

34

Morphogenesis is guided by dynamic spatio‐temporal regulation of gene expression patterns1,2, the 35

regions within tissues where genes are expressed at specific developmental times. Critical changes in 36

the space, time or intensity of gene expression patterns can result in organ malformation, with reduced 37

or even loss of function. These errors of morphogenesis, which occur in approximately 3% of live births3, 38

are induced by environmental and/or genetic insults that alter the normal process of development. The 39

common approach to reveal the origin of these alterations is to look for phenotypic abnormalities in 40

animal models and visually assess the overall patterns of gene expression. However, this qualitative 41

approach has not been able to identify the earliest signs of dysmorphogenesis in many diseases because 42

the first changes in the gene expression patterns may be subtle, limited in time, and later abnormalities 43

may obscure the original genetic cause. To reveal the primary etiology of congenital malformations, 44

more rigorous methods are needed to quantify the 3D phenotypes of gene expression patterns. A useful 45

tool to trace back development should be able to perform a quantitative statistical comparison of 46

normal and disease‐altered embryogenesis and detect the earliest signs of genetic misregulation leading 47

to organ malformation. 48

Developing organs are shaped by regulating gene activity in space and time1. Cascades of gene 49

regulatory networks provide the detailed instructions to organize cell behaviour and orchestrate tissue 50

growth and differentiation. Gene expression patterns can be readily mapped within tissues in a true 51

three‐dimensional framework combining Whole‐Mount‐in‐Situ Hybridization (WISH)4 with Optical 52

Projection Tomography (OPT)5. Whereas WISH is a standard molecular technique for detecting the 53

expression of a specific gene using a labelled complementary RNA probe6,7, OPT is a mesoscopic imaging 54

procedure that can produce high resolution 3D reconstructions of whole developing embryos processed 55

by WISH8,9. These technologies represented breakthroughs in developmental biology and have provided 56

invaluable qualitative insights into gene function and development8. However, methods for quantifying 57

the 3D gene expression distributions in a systematic, objective manner are still lacking. 58


https://doi.org/10.1101/246256


3

Expanding the potential of OPT from qualitative to quantitative analysis of gene expression patterns is 59

challenging. The expression of genes is characterized by highly dynamic patterns, with fast rates of 60

change over time and fuzzy boundaries that usually do not correspond to well‐defined anatomical 61

structures but to tissue regions where cells are dynamically up‐ and down‐regulating genes. The 62

quantification of gene expression patterns has been rarely done10–16. We propose to quantify the shape 63

of developing organs in association with their underlying gene expression patterns applying Geometric 64

Morphometrics (GM), a set of statistical tools for measuring and comparing shapes with increased 65

precision and efficiency17–22. This approach provides the ability to replace qualitative observations with 66

quantification of subtle yet significant biological differences that underlie the processes of 67

morphogenesis altered by disease. 68

Here we illustrate how our method can reveal the genetic origin of developmental defects by 69

investigating limb malformations in Apert syndrome [OMIM 101200]. Apert syndrome is a rare 70

congenital disease, with disease prevalence of 15–16 per million live births, that is characterized by 71

cranial, neural, limb, and visceral malformations23. Over 99% of Apert cases are associated with one of 72

two missense mutations, S252W and P253R, on Fibroblast Growth Factor Receptor 2 (FGFR2)24,25. The 73

mutations occur on neighbouring amino acids on the linker region between the second and third 74

extracellular immunoglobulin domains of FGFR2, and alter the ligand‐binding specificity of the 75

receptors26,27. Thus, the FGF receptors are activated inappropriately, altering the entire FGF/FGFR 76

signalling pathway and causing dysmorphologies of different organs and systems28. Apert syndrome 77

shares craniofacial dysmorphologies with other craniosynostosis syndromes but is differentiated on the 78

basis of limb defects of fore‐ and hindlimb digits. The craniofacial dysmorphology of Apert syndrome 79

(i.e. premature closure of cranial sutures and patent anterior fontanelle associated with atypical head 80

shape, midfacial retrusion and palatal defects)23 has been intensively investigated, especially because 81

mouse models show cranial phenotypes that correspond with the human condition29–33. However, the 82

associated limb defects are less well studied, in part because mouse models for Apert syndrome present 83

only subtle limb anomalies34–36, even in those carrying the FGFR2 P253R mutation34 that is associated 84

with the more severe limb malformations in Apert syndrome37,38. 85

Here we present precise phenotyping of limbs of newborn and embryonic specimens of the Fgfr2+/P253R 86

Apert syndrome mouse model and reveal significant differences that can be traced to as early as one 87

day after the initiation of limb development. To explore the molecular basis of these initial signs of limb 88

dysmorphology we applied our method combining OPT and GM to assess the expression pattern of a 89


https://doi.org/10.1101/246256


4

direct target of Fgf signalling, Dusp6, a relevant gene for limb morphogenesis39,40. Our quantitative 90

analyses demonstrate that the Apert syndrome Fgfr2 P253R mutation induces changes in the expression 91

pattern of Dusp6 and that these genetic changes are associated with significant phenotypic alterations. 92

These results provide insight into the origins of limb malformations in Apert syndrome. 93

94

Results 95

Apert syndrome mice present limb malformations at birth. Previous studies reported that Apert 96

syndrome mice do not show obvious abnormalities of the limb35, and thus focused their molecular 97

analyses on the skull34. Histopathological analyses in Apert syndrome mice only revealed overall limb 98

shortening due to abnormal osteogenic differentiation, but no signs of limb disproportion or 99

syndactyly34,36. As a further test of whether or not the Fgfr2 P253R mutation affects limb development in 100

mice, we first performed an extensive quantitative analysis of the size and shape of individual forelimb 101

bones using data from high resolution microCT images of newborn (P0) mutant and unaffected 102

littermates (Fig. 1a‐f). Our results revealed many more significant differences between P0 unaffected 103

and Fgfr2+/P253R mutant littermates than previously reported. We found that the humerus, radius and 104

ulna were statistically significantly shorter in length but had increased bone volumes in Fgfr2+/P253R 105

mutant mice in comparison to unaffected littermates (Fig. 1g and Table SI_1). More localized size 106

differences were detected in the bones derived from the autopod that give rise to the hands. The distal 107

phalanx of digit I, the proximal phalanx of digit V, and metacarpals II, III and IV were significantly longer 108

in Fgfr2+/P253R mutant mice (Fig. 1g and Table SI_1). In contrast, the proximal phalanx of digit III was 109

shorter and lower in bone volume in Fgfr2+/P253R Apert syndrome mice relative to unaffected littermates 110

(Fig. 1g and Table SI_1). The scapula and the clavicle, the bones that form the shoulder girdle, were also 111

significantly affected: the scapula was longer, the clavicle was shorter and both bones showed increased 112

bone volumes in Fgfr2+/P253R Apert syndrome mice (Fig. 1g and Table SI_1) compared to unaffected 113

littermates. 114

The PCA based on the shape of the humerus did not show marked shape differences between 115

unaffected and Fgfr2+/P253R Apert syndrome mice (Fig. 1h). However, the PCA of the scapula indicated a 116

clear morphological differentiation between groups (Fig. 1i). The scapula of Fgfr2+/P253R Apert syndrome 117

mice presented a more robust phenotype, with wider and longer scapulae in comparison to their 118

unaffected littermates. 119


https://doi.org/10.1101/246256


5

Overall, these size and shape differences demonstrate that Fgfr2+/P253R Apert syndrome mice present 120

widespread and significant limb dysmorphologies at P0 that were not previously reported and would not 121

have been revealed without quantitative statistical testing. Some defects have a direct correspondence 122

with the human phenotype, such as shoulder anomalies and short humeri24. However, in newborn mice 123

we did not detect any clear sign of syndactyly, which is the most prominent limb defect in people with 124

Apert syndrome41,42. Since the forelimb of mice is not yet completely ossified at P0 and Fgfr2+/P253R 125

mutant littermates die shortly after birth, we could not assess whether other limb abnormalities appear 126

later in development. 127

128

A quantitative morphometric method to assess embryonic gene expression patterns. To determine the 129

developmental basis of the limb anomalies quantified in newborn mice, we developed a quantitative 130

method to explore early embryonic limb development. First, to visualize the expression pattern of a 131

downstream target of Fgfr2, we obtained OPT scans of Fgfr2+/P253R Apert syndrome mouse embryos 132

analysed with WISH to reveal Dusp6 expression (Fig. 2). Qualitative assessment of the 3D 133

reconstructions showed that Dusp6 was widely expressed throughout the embryo from embryonic day 134

(E) 10.5 to E11.5, with highest intensity in the limbs, the head and the spinal cord (Fig. 2). Dusp6 was 135

also expressed in the heart with moderate intensity. Visually comparing the distribution of the Dusp6 136

gene expression pattern it was possible to distinguish between embryos at E10.5 and E11.5 stages of 137

development. At E10.5, Dusp6 was prominently expressed in the facial prominences and along the 138

entire spinal cord, whereas at E11.5 the expression of Dusp6 was more widespread in the brain and 139

limited to the tail. Focusing on the limbs, the expression of Dusp6 at the two different stages was also 140

readily distinguishable, with Dusp6 expression domains thinning into a more extended domain along the 141

limb outline as the limb buds grow from E10.5 to E11.5 (Fig. 2). However, due to large amount of 142

developmental variation within litters, Fgfr2+/P253R mutant and unaffected littermates were not 143

distinguishable from each other (Fig. 2). 144

Quantitative testing was thus required to more accurately evaluate limb alterations potentially 145

associated with Apert syndrome but undetectable by eye. We developed a method for 3D shape analysis 146

of the limb and associated gene expression pattern of Dusp6 (Fig. 3 and Video SI_1). This protocol 147

enabled us to determine differences in limb size and shape between genotype groups and whether 148

these phenotypic differences are associated with altered gene expression patterns (Fig. 3). Our 149

approach uses GM methods to directly measure the limb anatomy and gene expression domains 150


https://doi.org/10.1101/246256


6

segmented from the 3D reconstructions of the embryo OPT scans. As expression of Dusp6 showed a 151

fuzzy spatial gradient, multiple thresholding was used to consistently define a high gene expression 152

pattern (Fig. 3, steps from 1 to 5). After manual and semiautomatic recording of 3D coordinates of 153

landmarks on the surfaces of the limb and the gene expression domains blinded to group allocation (Fig. 154

3, step 6), multivariate statistical analyses were performed to explore shape and size variation and 155

covariation patterns between the limb morphology and the Dusp6 domain (Fig. 3, steps 7 and 8). 156

The first signs of limb dysmorphology in Apert syndrome. Since gene expression patterns are highly 157

dynamic and rapidly change in size, shape and position within a few hours of development15, individual 158

limb buds from Fgfr2+/P253R mutant embryos and their unaffected littermates aged between E10.5 and 159

E11.5 were staged using a fine‐resolution staging system (http://limbstaging.crg.es)43. The staging 160

results showed that the analysed limbs represent a temporal continuum over development, with no 161

significant differences between the staging of unaffected and mutant littermates of the same litter (Fig. 162

SI_1). We partitioned the time span from E10 to E11.5 into four periods, each one approximately 163

representing 12 hours of development (see Table SI_2 and Methods for further details on sample 164

composition). The analysis of the complete dataset that considers hindlimbs and forelimbs from each 165

litter separately is available as Supplementary Information. 166

We first focused on analysing limb dysmorphology, aiming to determine the youngest stage which 167

showed morphological differences between mutant and unaffected limbs. To trace limb development 168

back in time we first analysed embryos from the oldest period (as the differences would be easier to 169

find) and from there proceeded towards the earlier (younger) periods. In this way we should confidently 170

identify the initiation of limb dysmorphogenesis associated with the Fgfr2 P253R mutation determining 171

using geometric morphometric methods. 172

During the “Late” period, we detected that Fgfr2+/P253R mice were already clearly separated from their 173

unaffected littermates in the morphospace defined by the Principal Component Analysis (PCA) (Fig. 4a). 174

Relative to their unaffected littermates, limbs of Fgfr2+/P253R mice presented subtle phenotypic limb 175

differences: limbs were shorter, wider and more robust, with limited development of the wrist (Fig. 4a). 176

Quantitative comparison of limb size showed that the limbs of mutant mice were also significantly 177

smaller (Fig. 5a and Table SI_3). Overall, these results confirmed that the Fgfr2 P253R Apert syndrome 178

mutation has an effect on limb development, altering both the size and shape of the limbs. Most likely, 179

these subtle but significant phenotypic differences would have remained undetected by a qualitative 180


https://doi.org/10.1101/246256


7

approach. Our quantitative approach revealed their statistical significance and pointed to the origin of 181

the Apert syndrome limb malformation prior to E11.5, before the “Late” period. 182

At the “Mid late” period, the limbs of Fgfr2+/P253R mutant mice were still distinguishable from the limbs 183

of unaffected mice in the morphospace of the PCA (Fig. 4b). At this period, the limbs of Fgfr2+/P253R mice 184

lacked the antero‐posterior asymmetry and the narrowing of the wrist region more typical of unaffected 185

littermates. Instead, Fgfr2+/P253R mice showed a limb phenotype that was elongated in the proximo‐distal 186

axis and thickened in the dorso‐ventral axis (Fig. 4b), resembling the limb shape of younger unaffected 187

embryos. This shape difference coincided with reduced growth in Fgfr2+/P253R mice, as the limbs of 188

Fgfr2+/P253R mice tended to be smaller than unaffected limbs (Fig. 5a and Table SI_3). Therefore, 189

significant differences between unaffected and mutant limbs still could be detected at the “Mid late” 190

period of development and the origins of limb defects associated with Apert syndrome should be sought 191

earlier in development. 192

The first period where no significant differences could be detected between unaffected and Fgfr2+/P253R 193

Apert syndrome mice was at the “Mid early” period (Fig. 4c). Despite internal variation in limb shape, 194

with Fgfr2+/P253R mice spreading throughout the morphospace and unaffected littermates concentrated 195

on one region, mutant and unaffected littermates completely overlapped. Therefore, limb shape 196

differences could no longer be detected between groups. Limb size differences were not significant 197

either (Fig. 5a and Table SI_3). Therefore, our results suggest that the critical time point of limb 198

dysmorphogenesis associated with Apert syndrome occurred between the “Mid late” and “Mid early” 199

periods, corresponding to the transition period from E10.5 to E11 (Fig. 4b‐c). 200

Finally, no further sign of limb shape dysmorphology was detected at the “Early” period of development 201

(Fig. 4d). At this period there was a great range of developmental variation, with unaffected and 202

Fgfr2+/P253R mutant mice completely overlapping in the morphospace and all limbs displaying similar 203

incipient bud shapes (Fig. 4d). The limbs of Fgfr2+/P253R mice were significantly larger than the limbs of 204

their unaffected littermates (Fig. 5a and Table SI_3), suggesting that at this early time point there is a 205

significant effect of the Fgfr2 P253R mutation on limb size but not on limb shape (Fig. 5a). 206

Fgfr2 Apert syndrome mutation leads to aberrant overexpression of Dusp6 domains. We decided to 207

obtain direct evidence of altered genetic regulation that could explain the observed limb phenotype by 208

analyzing the shape dynamics of Dusp6 expression, a direct target gene of Fgf signaling. As with limb 209

shape, we first examined the gene expression of Dusp6 in the embryos from the latest period. We found 210


https://doi.org/10.1101/246256


8

that at the “Late” period, Dusp6 expression was already different between Fgfr2+/P253R mutant mice and 211

unaffected littermates. The differences were significant both in shape (Fig. 4e) and size (Fig. 5b and 212

Table SI_3). In the limbs of unaffected mice, the Dusp6 expression domain appeared as a thin domain 213

underlying the apical ectodermal ridge, whereas in Fgfr2+/P253R mutant mice the shape of the Dusp6 214

domain was expanded in all directions (Fig. 4e). Accordingly, the volume of the Dusp6 expression 215

domain was significantly larger in Apert syndrome mice (Fig. 5b and Table SI_3), even when these mice 216

presented significantly smaller limbs (Fig. 5a). The Dusp6 expression domain thus grew 217

disproportionately in the limbs of Fgfr2+/P253R mutant mice in the latest period of development (Fig. 4e), 218

which is consistent with a reported whole‐body size reduction in Fgfr2+/P253R Apert mice and the over‐219

activation of Fgfr2 signaling by the Apert syndrome mutation27,44. 220

At the “Mid late” period, an expanded Dusp6 expression domain persisted on the dorsal and ventral 221

sides of mutant limbs, but was reduced on the anterior and posterior sides (Fig. 4f). The overall volume 222

of the Dusp6 expression domains remained larger in Fgfr2+/P253R mutant mice, but the difference was no 223

longer statistically significant (Fig. 5b and Table SI_3). 224

At the “Mid early” period, the separation between unaffected and Fgfr2+/P253R mutant mice was 225

maintained in the PCA analysis (Fig. 4g). Unaffected mice showed a Dusp6 expression domain expanded 226

towards the anterior and posterior edges of the expression domain (Fig. 4g). In contrast, Fgfr2+/P253R 227

mutant mice did not show the extension and the posterior asymmetry of the Dusp6 expression domain 228

typical of normal limb development, suggesting a lack of differentiation in the Dusp6 expression of 229

mutant limbs (Fig. 4g). 230

Finally, the “Early” period was the only time point where we did not detect a significant separation 231

between unaffected and Fgfr2+/P253R mice (Fig. 4h). The PCA showed variation in the expression domains 232

of Dusp6, with similar gene expression patterns in both shape (Fig. 4h) and size (Fig. 5b and Table SI_3) 233

across all mice. Therefore, the first observation of an alteration in the gene expression pattern (Fig. 4g) 234

occurred earlier than the alteration in the limb shape change (Fig. 4b). Our analyses provide evidence 235

that differences in the Dusp6 gene expression pattern occurred first, at the “Mid early period”, 236

preceding the phenotypic limb differentiation, which occurred a few hours later in development, during 237

the “Mid late period”. Overall, the time course showing the dynamics of limb and gene expression shape 238

changes over development (Figs. 4, 5 and SI_3, 4 and 5) confirmed that the Fgfr2 Apert syndrome 239

mutation causes an aberrant overexpression of Dusp6 early in development that could later lead to 240

significant limb malformations. 241


https://doi.org/10.1101/246256


9

Altered Dusp6 expression and limb dysmorphology are highly associated. Finally, we explored the 242

correlation patterns between the limb phenotype and the gene expression pattern to further test 243

whether altered Fgf signaling underlies the limb malformations induced by Apert syndrome Fgfr2 P253R 244

mutation. First, we assessed the relationship between the size of the limbs and the volume of the Dusp6 245

expression domain pooling all the forelimbs and hindlimbs and assessing the correlation between these 246

two traits (Fig. 5c, d). The trend line showed that for the same limb size, Fgfr2+/P253R mutant mice showed 247

larger Dusp6 expression domains, both in forelimbs (R2=0.4) and hindlimbs (R2=0.6). If the extension of 248

the Dusp6 expression only depended on limb growth, a high correlation between the size of the limb 249

and the gene would be expected. However, the moderate correlation found here suggests that the size 250

of the Dusp6 gene expression is not dependent solely on limb size but is also influenced by other factors 251

and could be under further genetic regulatory control. 252

Second, we assessed the morphological integration between the shape of the limbs and the shape of the 253

Dusp6 expression domain. The statistical analysis of the covariance pattern between these shapes can 254

reflect the interaction of the phenotype and the gene expression pattern during limb development. As 255

shown by analysis of additional genes expressed during limb development15, even when a gene is 256

expressed within the limb, the shape of the limb and the shape of the gene expression domain are not 257

correlated by definition, and the integration pattern can change from a strong association to no 258

significant correlation within few hours of development15. The dynamics of the integration pattern can 259

identify the key periods during which the expression of a gene is relevant for determining the shape of 260

the limb. If the morphological integration is low, the expression of the gene will not be as relevant for 261

the shape of the limb as if the integration is high. If the integration is low, the impact of the altered gene 262

expression on the phenotype will be minimal. If the morphological integration is high, the impact of the 263

genetic mutation will be maximized (i.e., changes of the gene expression pattern will produce changes in 264

the limb shape). Our results showed that the shape of the limb and the shape of the Dusp6 domain were 265

indeed highly correlated (RV=0.88 in forelimbs; RV=0.91 in hindlimbs). This is evidence that altered Fgf 266

signaling induced by the Fgfr2 P253R Apert syndrome mutation will have a direct effect on the limb 267

phenotype. 268

By comparing the morphological integration pattern in mutant and unaffected littermates at different 269

periods, we can test whether this interaction is maintained or disrupted by the disease during 270

development. If the pattern or magnitude of morphological integration was different in mutant mice, it 271

would reveal further mechanisms underlying the etiology of the disease. Our analyses showed that the 272


https://doi.org/10.1101/246256


10

pattern of morphological integration of the shape of the limbs and the shape of the Dusp6 expression 273

domains was similar in unaffected and Fgfr2+/P253R mutant mice (Fig. 5e and Fig SI_6). Our results 274

confirmed that the Fgfr2 P253R mutation does not disrupt the strong association between limb shape 275

and the Dusp6 expression domain. Therefore, the alteration of the Dusp6 expression pattern caused by 276

the Fgfr2 mutation between E10 and E11.5 will produce the limb dysmorphologies associated with Apert 277

syndrome. Overall, the high correlation between the shapes of the limb and the Dusp6 expression 278

domain provides further evidence that altered Fgf expression due to the Fgfr2 mutation is strongly 279

associated with limb defects in Apert syndrome. 280

281

Discussion 282

By definition, to reveal the primary etiology of an abnormality requires going back in time to the earliest 283

moment when abnormal development can be found. Typically, the earliest changes will be the most 284

subtle, and so the most statistically sensitive techniques are necessary. The methods currently used to 285

assess gene expression patterns are mainly qualitative and only focus on the shape and size differences 286

that can be simply detected by eye. Therefore, slight changes in gene expression domains, even if they 287

may have large effects on the phenotype45, can remain undetected. To reveal these subtle changes we 288

have developed a precise method combining OPT and GM for quantifying embryo morphology and the 289

underlying 3D gene expression patterns in a systematic, objective manner. This enables visualization and 290

quantification of how the genotype translates into the phenotype during embryonic development, to 291

compare normal and disease‐altered patterns of genetic and phenotypic variation and, eventually, 292

identify the origins of abnormal morphogenesis. This approach can further our understanding of the 293

etiology of genetic diseases in research using animal models46,47, even in those that do not seem to 294

recapitulate the human disease faithfully48. 295

Our study of the Fgfr2 P253R mouse model for Apert syndrome is an exemplary case of how 296

quantitative assessment can overcome the shortcomings of traditional qualitative morphological 297

assessment and lead to new discoveries. So far, the molecular and developmental mechanisms 298

underlying the limb defects associated with Apert syndrome remained obscure, even when these limb 299

abnormalities clinically differentiate Apert syndrome from other craniosynostosis syndromes (e.g. 300

Pfeiffer, Crouzon and Saethre‐Chotzen syndromes)23,24,41. Most Apert syndrome research focused on 301

premature fusion of cranial sutures and craniofacial malformations23,29–33, clinical traits that are 302


https://doi.org/10.1101/246256


11

consistently phenocopied in mouse models. However, little research about the limb defects in Apert 303

syndrome has been done using the same animal models, mainly because previous research reported the 304

absence or subtle malformation of the limbs in the different mouse models for Apert syndrome34–36. 305

Contrary to these previous results, our quantitative morphometric analyses demonstrate that the limbs 306

of Fgfr2+/P253R Apert syndrome mice present with significant defects that are detectable in newborn mice 307

and can be traced back to early embryogenesis (Figs. 1, 4 and 5). 308

Our analyses provide insight into the genetic origins of these limb defects, showing that altered gene 309

expression patterns in the Fgf signaling pathway precede and contribute to limb dysmorphogenesis in 310

Fgfr2+/P253R Apert syndrome mice. In fact, we detected that Dusp6 expression patterns were different in 311

unaffected and mutant littermates a few hours before the first limb dysmorphologies appeared (Fig. 4), 312

and confirmed that limb shape and Dusp6 expression patterns were highly correlated (Fig. 5e). The 313

altered Fgf signaling observed was due to the Fgfr2 P253R Apert syndrome mutation, which causes loss 314

of ligand specificity of the Fgfr2 isoform and increased affinity of various Fgfs to Fgfr2. Our analyses 315

showed that over‐activation of the Fgf signaling pathway results in more expanded (Fig. 4e‐g) and larger 316

(Fig. 5b) expression domains of Dusp6, a gene that acts as a negative‐feedback control of Fgf signaling49. 317

A delay in misregulation of the expression of Dusp6 may explain the lack of antero‐posterior asymmetry 318

and the shape deficiencies observed in Fgfr2+/P253R mutant mice (Fig. 4). We found evidence that limb 319

shape and Dusp6 expression were highly associated (Fig. 5c‐e), but it is likely that other downstream 320

genes of the Fgf signaling pathway also contribute to the limb shape malformations associated with 321

Apert syndrome. 322

Our analyses also demonstrated that the embryonic limb defects persisted until birth (Fig. 1) and thus 323

did not disappear over development. For instance, we found that Fgfr2+/P253R Apert syndrome mice 324

presented postnatal limb malformations involving the shape, length and volume of many bones of the 325

forelimb, including the scapula, humerus, ulna, radius, metacarpals and phalanges (Fig. 1 and Table 326

SI_1). Though subtle, these malformations suggest that further research into the origins and causes of 327

limb dysmorphologies in Apert syndrome using these and other mouse models is warranted. 328

Our quantitative approach could be similarly applied to investigate other developmental defects and 329

dysmorphologies 50. OPT and GM can be potentially used to analyze any organ and animal model that 330

can be visualized during development using light microscopy, and any gene whose gene expression 331

pattern can be detected by WISH and show a continuous expression domain over development15. We 332

exemplified the method analyzing limb defects in mouse models, but it could be applied to 333


https://doi.org/10.1101/246256


12

malformations affecting other organs such as the face, the brain, the heart, etc, in mouse models as well 334

as in any other vertebrate animal model, such as zebrafish, chicken and Xenopus. This is relevant as 335

major developmental defects represent a leading cause of infant mortality and affect a small but 336

relevant percentage of the population, severely compromising their quality of life3. 337

Through its increased quantitative sensitivity, our method has allowed us to reveal that the mouse 338

model for Apert syndrome does indeed show early defects in limb development. We detected that 339

dysregulation of an Fgf target gene precedes measurable changes in limb bud morphology, thus 340

identifying a relevant component of its genetic etiology. Quantitative evaluation of size and shape 341

should thus be performed before discarding any animal models as useful for investigating human 342

congenital malformations51. Our method has the potential to become a high‐throughput tool for 343

biomedical research, providing insight into the processes that cause malformations and lead to 344

malfunction, which is essential for understanding diseases and discovering potential therapies. 345

346

Methods 347

Mouse model. We analysed the Fgfr2+/P253R Apert syndrome mouse model, an inbred model backcrossed 348

on C57BL/6J genetic background for more than 10 generations carrying a mutation that in humans with 349

Apert syndrome is associated with more severe syndactyly. This gain of function mutation, which is 350

embryonically lethal in homozygosis, involves a proline to arginine amino acid change at position 253 of 351

the Fgfr2 protein that alters the ligand binding specificity of the receptor and causes a stronger receptor 352

signaling. Further details of the mouse model on generation of targeting construct can be found 353

elsewhere34. All the experiments were performed in compliance with the animal welfare guidelines 354

approved by the Pennsylvania State University Animal Care and Use Committees (IACUC46558, 355

IBC46590). 356

Micro‐CT imaging. High resolution micro‐computed tomography (μCT) scans were acquired by the 357

Center for Quantitative Imaging at the Pennsylvania State University (www.cqi.psu.edu) using the HD‐358

600 OMNI‐X high‐resolution X‐ray computed tomography system (Varian Medical Systems, Palo Alto, 359

CA). Pixel sizes ranged from 0.01487 to 0.01503 mm, and all slice thicknesses were 0.016 mm. Image 360

data were reconstructed on a 1024×1024 pixel grid as a 16‐bit TIFF. To reconstruct forelimb morphology 361

from the μCT images, isosurfaces were produced with median image filter using the software package 362

Avizo 6.3 (Visualization Sciences Group, VSG) (Fig. 1a‐f). 363


https://doi.org/10.1101/246256


13

Morphometrics in P0 mice. We assessed forelimb morphology at P0 using unaffected (n=10) and 364

mutant (n=12) littermates of Apert syndrome mouse models. A set of 16 landmarks were collected on 365

each left forelimb, including points on the main bones of the forelimb (phalanges, metacarpals, radius, 366

ulna, humerus, scapula and clavicle), as shown in Fig. 1b‐f. To minimize measurement error, each 367

landmark was collected twice by the same observer restricting the deviations between the two trials to 368

0.05 mm. 369

At P0, we estimated the dimensions of the long bones of the forelimbs using the 3D coordinates of the 370

landmarks located at the proximal and distal ends of the bones (Fig. 1b‐f). We also estimated the bone 371

volumes from volume data collected from the μCT scans. To assess size differences in bone length and 372

bone volume between mutant and unaffected P0 mice of the Fgfr2+/P253R Apert syndrome mouse model, 373

we performed a two‐tailed one‐way ANOVA on those variables showing a normal distribution, and the 374

non‐parametric Mann‐Whitney U‐Test on those variables that deviated from a normal distribution. The 375

shape of the humerus and the scapula was comparatively assessed in unaffected and Fgfr2+/P253R Apert 376

syndrome littermates using Geometric Morphometrics. Shape information was extracted using a 377

General Procrustes Analysis (GPA)52, in which configurations of landmarks are superimposed by shifting 378

them to a common position, rotating and scaling them to a standard size until a best fit of corresponding 379

landmarks is achieved53. The resulting Procrustes coordinates from the GPA were the input for further 380

statistical analysis to compare the shape of the bones in unaffected and Fgfr2+/P253R mice. A Principal 381

Component Analysis (PCA) was used to explore the morphological variation within each bone. PCA 382

performs an orthogonal decomposition of the data and transforms variance covariance matrices into a 383

smaller number of uncorrelated variables called Principal Components (PCs), which successively account 384

for the largest amount of variation in the data20. Each specimen is scored for every principal component 385

and the specimens can be plotted using these scores along the morphospace defined by the principal 386

axes. 387

WISH and OPT scanning. To examine early embryonic mouse limb development in Apert syndrome we 388

bred 4 litters of the Fgfr2+/P253R Apert syndrome mouse model and collected them between E10.5 and 389

E11.5. In total, 32 mouse embryos were harvested and classified by PCR genotyping into unaffected 390

(n=16) and mutant (n=16) littermates (see Fig. SI_1 and Table SI_2 for further details on sample size and 391

composition). Dusp6 gene expression was assessed by Whole‐Mount‐In‐Situ Hybridization (WISH). 392

Mouse embryos were dissected in cold phosphate‐buffered saline, 0.1% tween 20 (PBT), fixed overnight 393

in 4% paraformaldehyde (Sigma), dehydrated in a graded PBT/methanol series and stored at ‐20ºC in 394


https://doi.org/10.1101/246256


14

methanol. The mouse embryos recovered their original size after rehydration in decreasing series of 395

methanol/PBT. WISH was carried out using Dusp6 antisense RNA probes labelled with digoxigenin‐UTP 396

(Roche), following standard protocols7. Alkaline phosphatase coupled anti‐digoxigenin (anti‐DIG‐AP, 397

Roche) and NBT/BCIP staining (Roche) were used to reveal the expression pattern for Dusp6. To 398

minimize variations during experimental procedures, all embryos were processed systematically within 399

the same batch, processing unaffected and mutant littermates from different litters in separate tubes, 400

but simultaneously using the same probe, timings and concentration reagents. 401

After embedding in agarose, dehydrating in methanol and chemically clearing the samples with benzyl 402

alcohol and benzyl benzoate (BABB), the stained whole embryos were scanned with both fluorescence 403

and transmission light with a CFP (Cyan Fluorescent Protein) filter using our home‐build OPT imaging 404

system mounted on a Leica MZ 16 FA microscope5. The embryos were 3D‐reconstructed from the 405

resulting 2D images using Matlab and visualized using Amira 6.3 (Visualization Sciences Group, FEI). 406

From the OPT fluorescence scans we produced 3D reconstructions of the embryo surface and we 407

dissected the available right and left fore‐ and hind limbs of each specimen, resulting in a sample of 101 408

embryonic limbs (Table SI_2). From the OPT transmission scans, we recovered the Dusp6 expression 409

domain. As Dusp6 is expressed in a fuzzy spatial gradient, as already shown by other genes15, we used 410

3D multiple thresholding to visualize the gene expression domain at different intensities (Fig. 3 and 411

Video SI_1). To comparatively analyse the gene expression domains across the sample, we inspected for 412

each limb the whole range of threshold values under which the gene expression could be visualized, 413

from the threshold showing its first appearance to the threshold under which it disappeared and was no 414

longer detectable. We analysed the 3D reconstruction based on a threshold computed as 1/3 of the last 415

grey value showing the Dusp6 expression domain, which displayed a Dusp6 domain at high gene 416

expression (Fig. 3, step 3). Finally, we obtained 80 limbs (46 forelimbs and 34 hindlimbs) with associated 417

gene expression patterns for Dusp6. 418

Embryo staging. To account for breeding and developmental variation, individual limb buds were staged 419

using our publicly available web‐based staging system (http://limbstaging.crg.es)43. Considering the 420

spline curve along the outline of the limb, this tool provides a stage estimate with a reproducibility of ± 2 421

hours. According to the staging results, the different mouse litters were ordered following a continuous 422

temporal sequence from E10 to E11.5 (Fig. SI_1). To minimize high developmental variation within and 423

among litters of mice, hindlimbs and forelimbs from each litter were analysed separately, except for 424


https://doi.org/10.1101/246256


15

those from two litters from the earliest stage that were pooled into the same group because their 425

temporal distribution completely overlapped, as shown in Fig. SI_1. 426

Morphometrics from E10 to E11.5. To capture the size and shape of the limbs and the expression 427

domains of Dusp6, we collected a set of anatomical landmarks as well as curve and surface 428

semilandmarks (Fig. SI_2), as recommended in structures devoid of homologous landmarks. 429

Semilandmarks are mathematical points located along a curve54 or a surface55 within the same object 430

that can be slid to corresponding equally spaced locations across the sample. We used Amira 6.3 431

(Visualization Sciences Group, FEI) to record the anatomical landmarks and Viewbox 4 (dHAL software, 432

Kifissia, Greece) to construct a limb template of surface and curve semilandmarks and to interpolate 433

them onto each target shape (Fig. SI_2). 434

The 3D landmark coordinates defining the shape of the limb and the Dusp6 expression domain were 435

analysed using Procrustes‐based landmark analysis54. Semilandmarks were allowed to slide in the GPA 436

by minimizing the bending energy54–56. Quantitative shape analyses based on PCA were performed as 437

explained above. 438

We estimated the size of the limb as the centroid size, calculated as the square root of the summed 439

squared distances between each landmark coordinate and the centroid of the limb configuration of 440

landmarks53. The volumes of the Dusp6 domains were estimated from the 3D reconstructions of the OPT 441

scans. Size differences in limb size and gene volume between mutant and unaffected embryonic mice 442

were tested for statistical significance using a two‐tailed Welch Two Sample t‐test. 443

We quantified the integration between the limb and the Dusp6 expression pattern and produced 444

visualizations of the patterns of associated shape changes between them using Two‐Block Partial Least 445

Squares analysis (PLS)57. This method performs a singular value decomposition of the covariance matrix 446

between the two blocks of shape data (i.e., the limb and the Dusp6 expression domain). Uncorrelated 447

pairs of new axes are derived as linear combinations of the original variables, with the first pair 448

accounting for the largest amount of inter‐block covariation, the second pair for the next largest amount 449

and so on. The amount of covariation is measured by the RV coefficient, which is a multivariate 450

analogue of the squared correlation58. Statistical significance was tested using permutation tests under 451

the null hypothesis of complete independence between the two blocks of variables. Separate analyses 452

for each developmental period, as well as for forelimbs and hindlimbs of all stages were computed. 453


https://doi.org/10.1101/246256


16

All the analyses were performed using R (R Development Core Team, 2013; http://www.R‐project.org); 454

the R package geomorph59 ( http://cran.r‐project.org/web/packages/geomorph), SPSS Statistics 22 (IMB, 455

2013) and MorphoJ60. 456

457

458

References 459

1. Andrey, G. & Mundlos, S. The three‐dimensional genome: regulating gene expression during 460

pluripotency and development. Dev. Camb. Engl. 144, 3646–3658 (2017). 461

2. Chan, C. J., Heisenberg, C.‐P. & Hiiragi, T. Coordination of Morphogenesis and Cell‐Fate 462

Specification in Development. Curr. Biol. CB 27, R1024–R1035 (2017). 463

3. Toxicology, N. R. C. (US) C. on D. Developmental Defects and Their Causes. (National Academies 464

Press (US), 2000). 465

4. Rosen, B. & Beddington, R. S. Whole‐mount in situ hybridization in the mouse embryo: gene 466

expression in three dimensions. Trends Genet. TIG 9, 162–167 (1993). 467

5. Sharpe, J. et al. Optical projection tomography as a tool for 3D microscopy and gene expression 468

studies. Science 296, 541–545 (2002). 469

6. Correia, K. M. & Conlon, R. A. Whole‐mount in situ hybridization to mouse embryos. Methods 470

San Diego Calif 23, 335–338 (2001). 471

7. de la Pompa, J. L. et al. Conservation of the Notch signalling pathway in mammalian 472

neurogenesis. Dev. Camb. Engl. 124, 1139–1148 (1997). 473

8. Sharpe, J. Optical projection tomography as a new tool for studying embryo anatomy. J. Anat. 474

202, 175–181 (2003). 475

9. Boot, M. J. et al. In vitro whole‐organ imaging: 4D quantification of growing mouse limb buds. 476

Nat. Methods 5, 609–612 (2008). 477

10. Xu, Q. et al. Correlations Between the Morphology of Sonic Hedgehog Expression Domains and 478

Embryonic Craniofacial Shape. Evol. Biol. 1–8 (2015). doi:10.1007/s11692‐015‐9321‐z 479

11. Hu, D. et al. Signals from the brain induce variation in avian facial shape. Dev. Dyn. Off. Publ. Am. 480

Assoc. Anat. (2015). doi:10.1002/dvdy.24284 481

12. Jernvall, J., Keränen, S. V. & Thesleff, I. Evolutionary modification of development in mammalian 482

teeth: quantifying gene expression patterns and topography. Proc. Natl. Acad. Sci. U. S. A. 97, 14444–483

14448 (2000). 484

13. Salazar‐Ciudad, I. & Jernvall, J. A computational model of teeth and the developmental origins of 485

morphological variation. Nature 464, 583–586 (2010). 486


https://doi.org/10.1101/246256


17

14. Mayer, C., Metscher, B. D., Müller, G. B. & Mitteroecker, P. Studying developmental variation 487

with Geometric Morphometric Image Analysis (GMIA). PloS One 9, e115076 (2014). 488

15. Martínez‐Abadías, N., Mateu, R., Niksic, M., Russo, L. & Sharpe, J. Geometric Morphometrics on 489

Gene Expression Patterns Within Phenotypes: A Case Example on Limb Development. Syst. Biol. 65, 194–490

211 (2016). 491

16. Airey, D. C., Wu, F., Guan, M. & Collins, C. E. Geometric morphometrics defines shape 492

differences in the cortical area map of C57BL/6J and DBA/2J inbred mice. BMC Neurosci. 7, 63 (2006). 493

17. James Rohlf, F. & Marcus, L. F. A revolution morphometrics. Trends Ecol. Evol. 8, 129–132 494

(1993). 495

18. Wiley: Statistical Shape Analysis ‐ Ian L. Dryden, Kanti V. Mardia. Available at: 496

http://eu.wiley.com/WileyCDA/WileyTitle/productCd‐0471958166.html. (Accessed: 14th September 497

2015) 498

19. Klingenberg, C. P. Evolution and development of shape: integrating quantitative approaches. 499

Nat. Rev. Genet. 11, 623–635 (2010). 500

20. Hallgrimsson, B. et al. Morphometrics, 3D Imaging, and Craniofacial Development. Curr. Top. 501

Dev. Biol. 115, 561–597 (2015). 502

21. Adams, D. C., Rohlf, F. J. & Slice, D. E. A field comes of age: geometric morphometrics in the 21st 503

century. Hystrix Ital. J. Mammal. 24, 7–14 (2013). 504

22. Klingenberg, C. P. Morphometrics and the role of the phenotype in studies of the evolution of 505

developmental mechanisms. Gene 287, 3–10 (2002). 506

23. Craniosynostosis: Diagnosis, Evaluation, and Management. (Oxford University Press, 2000). 507

24. Park, W. J. et al. Analysis of phenotypic features and FGFR2 mutations in Apert syndrome. Am. J. 508

Hum. Genet. 57, 321–328 (1995). 509

25. Wilkie, A. O. M. et al. Apert syndrome results from localized mutations of FGFR2 and is allelic 510

with Crouzon syndrome. Nat. Genet. 9, ng0295‐165–165 (1995). 511

26. Yu, K., Herr, A. B., Waksman, G. & Ornitz, D. M. Loss of fibroblast growth factor receptor 2 512

ligand‐binding specificity in Apert syndrome. Proc. Natl. Acad. Sci. U. S. A. 97, 14536–14541 (2000). 513

27. Yu, K. & Ornitz, D. M. Uncoupling fibroblast growth factor receptor 2 ligand binding specificity 514

leads to Apert syndrome‐like phenotypes. Proc. Natl. Acad. Sci. U. S. A. 98, 3641–3643 (2001). 515

28. McIntosh, I., Bellus, G. A. & Jab, E. W. The pleiotropic effects of fibroblast growth factor 516

receptors in mammalian development. Cell Struct. Funct. 25, 85–96 (2000). 517

29. Martínez‐Abadías, N. et al. Beyond the closed suture in apert syndrome mouse models: 518

evidence of primary effects of FGFR2 signaling on facial shape at birth. Dev. Dyn. Off. Publ. Am. Assoc. 519

Anat. 239, 3058–3071 (2010). 520


https://doi.org/10.1101/246256


18

30. Hill, C. A. et al. Postnatal brain and skull growth in an Apert syndrome mouse model. Am. J. Med. 521

Genet. A. 161A, 745–757 (2013). 522

31. Holmes, G. et al. Early onset of craniosynostosis in an Apert mouse model reveals critical 523

features of this pathology. Dev. Biol. 328, 273–284 (2009). 524

32. Heuzé, Y. et al. Morphological comparison of the craniofacial phenotypes of mouse models 525

expressing the Apert FGFR2 S252W mutation in neural crest‐ or mesoderm‐derived tissues. Bone 63, 526

101–109 (2014). 527

33. Holmes, G. & Basilico, C. Mesodermal expression of Fgfr2S252W is necessary and sufficient to 528

induce craniosynostosis in a mouse model of Apert syndrome. Dev. Biol. 368, 283–293 (2012). 529

34. Wang, Y. et al. Activation of p38 MAPK pathway in the skull abnormalities of Apert syndrome 530

Fgfr2(+P253R) mice. BMC Dev. Biol. 10, 22 (2010). 531

35. Chen, L., Li, D., Li, C., Engel, A. & Deng, C.‐X. A Ser252Trp [corrected] substitution in mouse 532

fibroblast growth factor receptor 2 (Fgfr2) results in craniosynostosis. Bone 33, 169–178 (2003). 533

36. Wang, Y. et al. Abnormalities in cartilage and bone development in the Apert syndrome 534

FGFR2(+/S252W) mouse. Dev. Camb. Engl. 132, 3537–3548 (2005). 535

37. Slaney, S. F. et al. Differential effects of FGFR2 mutations on syndactyly and cleft palate in Apert 536

syndrome. Am. J. Hum. Genet. 58, 923–932 (1996). 537

38. von Gernet, S., Golla, A., Ehrenfels, Y., Schuffenhauer, S. & Fairley, J. D. Genotype‐phenotype 538

analysis in Apert syndrome suggests opposite effects of the two recurrent mutations on syndactyly and 539

outcome of craniofacial surgery. Clin. Genet. 57, 137–139 (2000). 540

39. Kawakami, Y. et al. MKP3 mediates the cellular response to FGF8 signalling in the vertebrate 541

limb. Nat. Cell Biol. 5, 513–519 (2003). 542

40. Li, C., Scott, D. A., Hatch, E., Tian, X. & Mansour, S. L. Dusp6(Mkp3) is a negative feedback 543

regulator of FGF stimulated ERK signaling during mouse development. Dev. Camb. Engl. 134, 167–176 544

(2007). 545

41. Holten, I. W., Smith, A. W., Bourne, A. J. & David, D. J. The Apert syndrome hand: pathologic 546

anatomy and clinical manifestations. Plast. Reconstr. Surg. 99, 1681–1687 (1997). 547

42. Cohen, M. M. & Kreiborg, S. Hands and feet in the Apert syndrome. Am. J. Med. Genet. 57, 82–548

96 (1995). 549

43. Boehm, B. et al. A landmark‐free morphometric staging system for the mouse limb bud. Dev. 550

Camb. Engl. 138, 1227–1234 (2011). 551

44. Ibrahimi, O. A. et al. Structural basis for fibroblast growth factor receptor 2 activation in Apert 552

syndrome. Proc. Natl. Acad. Sci. U. S. A. 98, 7182–7187 (2001). 553

45. Honeycutt, R. L. Small changes, big results: evolution of morphological discontinuity in 554

mammals. J. Biol. 7, 9 (2008). 555


https://doi.org/10.1101/246256


19

46. Rosenthal, N. & Brown, S. The mouse ascending: perspectives for human‐disease models. Nat. 556

Cell Biol. 9, 993–999 (2007). 557

47. Spradling, A. et al. New Roles for Model Genetic Organisms in Understanding and Treating 558

Human Disease: Report From The 2006 Genetics Society of America Meeting. Genetics 172, 2025–2032 559

(2006). 560

48. Guénet, J.‐L. Animal models of human genetic diseases: do they need to be faithful to be useful? 561

Mol. Genet. Genomics 286, 1–20 (2011). 562

49. Ekerot, M. et al. Negative‐feedback regulation of FGF signalling by DUSP6/MKP‐3 is driven by 563

ERK1/2 and mediated by Ets factor binding to a conserved site within the DUSP6/MKP‐3 gene promoter. 564

Biochem. J. 412, 287–298 (2008). 565

50. Winter, R. M. & Baraitser, M. The London Dysmorphology Database. J. Med. Genet. 24, 509–510 566

(1987). 567

51. Zuniga, A., Zeller, R. & Probst, S. The molecular basis of human congenital limb malformations. 568

Wiley Interdiscip. Rev. Dev. Biol. 1, 803–822 (2012). 569

52. Rohlf, F. J. & Slice, D. Extensions of the Procrustes Method for the Optimal Superimposition of 570

Landmarks. Syst. Zool. 39, 40–59 (1990). 571

53. Dryden, I. L. & Mardia, K. V. Statistical Shape Analysis. (Wiley, 1998). 572

54. Bookstein, F. L. Morphometric Tools for Landmark Data: Geometry and Biology. (Cambridge 573

University Press, 1997). 574

55. Gunz, P., Mitteroecker, P. & Bookstein, F. L. Semilandmarks in Three Dimensions. in Modern 575

Morphometrics in Physical Anthropology 73–98 (Springer, Boston, MA, 2005). doi:10.1007/0‐387‐27614‐576

9_3 577

56. Mitteroecker, P. & Gunz, P. Advances in Geometric Morphometrics. Evol. Biol. 36, 235–247 578

(2009). 579

57. Rohlf, F. J. & Corti, M. Use of Two‐Block Partial Least‐Squares to Study Covariation in Shape. 580

Syst. Biol. 49, 740–753 (2000). 581

58. Klingenberg, C. P. Morphometric integration and modularity in configurations of landmarks: 582

tools for evaluating a priori hypotheses. Evol. Dev. 11, 405–421 (2009). 583

59. Adams, D. C. & Otárola‐Castillo, E. geomorph: an r package for the collection and analysis of 584

geometric morphometric shape data. Methods Ecol. Evol. 4, 393–399 (2013). 585

60. Klingenberg, C. P. MorphoJ: an integrated software package for geometric morphometrics. Mol. 586

Ecol. Resour. 11, 353–357 (2011). 587

588

589


https://doi.org/10.1101/246256


20

Acknowledgements 590

We acknowledge support of the Spanish Ministry of Economy and Competitiveness to the EMBL 591

partnership* and ‘Centro de Excelencia Severo Ochoa, and the CERCA Programme / Generalitat de 592

Catalunya. We acknowledge the support of the CERCA Programme / Generalitat de Catalunya. We 593

acknowledge Ethylin Wang Jabs for access to the Fgfr2+/P253R Apert syndrome mouse model. The 594

research leading to these results received funding from the following grants: European Union Seventh 595

Framework Program (FP7/2007‐2013) under grant agreement Marie Curie Fellowship FP7‐PEOPLE‐2012‐596

IIF 327382, National Institutes of Health NICHD P01HD078233 and NIDCR R01DE02298, and a Burroughs‐597

Welcome Fund 2013 Collaborative Research Travel Grant. 598

599

Author contributions 600

NM‐A, JS and JR conceived the project. JR contributed with experimental mouse models; AR‐M, JS, LS 601

and KK conducted experiments and scanning; and NM‐A, RM, JS, SMP and MY collected and analysed 602

the data. NM‐A and JS wrote the paper and all authors critically reviewed the manuscript. 603

604

Figure legends 605

606

Figure 1. Quantitative size and shape comparison of forelimb bones in Fgfr2+/P253R newborn mice (P0) 607

and unaffected littermates (P0). a) Mouse skeleton at P0. 3D isosurface reconstruction of the skeleton 608

of an unaffected littermate obtained from a high resolution µCT scan. b‐f) Anatomical landmarks 609

recorded on µCT scans of Apert syndrome mice at P0. b) Autopod (hand). Landmarks were recorded at 610

the midpoint of the proximal and distal tips of the distal, mid and proximal phalanges (P1‐P28) and the 611

metacarpals (P29‐P38). Proximal phalanx I, middle phalanx V, and metacarpal I are not displayed 612

because these bones have not yet mineralized at P0 and could not be visualized in many specimens. c) 613

Zeugopod. Landmarks were recorded at the midpoint of the proximal and distal tips of the radius (R1‐614

R2) and ulna (U1‐U2). d) Stylopod. Landmarks were recorded at the midpoint of the proximal and distal 615

tips of the humerus (H1‐H2), as well as the tip of the deltoid process (H3). e) Scapula. Landmarks were 616

recorded at the most superior and inferior lateral points of the scapula (S1‐S2), the most posterior point 617

of the spine (S3), most antero‐medial point of the acromion process (S4) and the medial, superior and 618


https://doi.org/10.1101/246256


21

inferior points of the glenoid cavity (S5‐S7). f) Clavicle. Landmarks were recorded at the medial point of 619

the sternal and the acromial ends (C1‐C2). g) Length and volume differences in the forelimbs of Apert 620

syndrome mouse models. Schematic representation of the forelimb of a P0 mouse showing in different 621

colours statistically significant differences in bone length and volume as measured by two‐tailed one‐622

way ANOVA or Mann‐Whitney U‐test in Fgfr2+/P253R mice relative to unaffected littermates as specified in 623

Table SI_1. Longer/shorter refer to length, whereas larger/smaller refer to volume. Blue: Longer and 624

larger, green: longer, purple: shorter and larger, pink: shorter and smaller, orange: no difference, yellow: 625

not assessed because bones were not visible/ossified. h, i) Shape differences in the forelimbs of Apert 626

syndrome mouse models. Scatterplots of PC1 and PC2 scores based on Procrustes analysis of anatomical 627

landmark locations representing the shape of the left humerus (h) and the left scapula (i) of unaffected 628

(n=10) and mutant (n=12) littermates of Apert syndrome mouse models. Convex hulls represent the 629

ranges of variation within each group of mice. 630

631

Figure 2. Qualitative visualisation of gene expression of Dusp6 in unaffected and Fgfr2+/P253R mouse 632

embryos at E10.5 and E11.5. OPT scans of embryos WISH stained for Dusp6 revealed the anatomical 633

location of gene expression (shown in yellow). For each stage, the main expression domains are 634

highlighted on the left for anatomical reference on a lateral view of a 3D reconstruction of a Fgfr2+/+ 635

unaffected embryo (fb: forebrain, is: isthmus, hb: hindbrain, mx: maxillary prominence, md: mandibular 636

prominence, lnp: lateral nasal process, mnp: medial nasal process, ht: heart, sc: spinal cord, fl: forelimb, 637

hl: hindlimb). On the right, 3D reconstructions of three unaffected and three Fgfr2+/P253R mutant 638

embryos from the same litter are displayed to represent the high degree of variation in developmental 639

age within litters. Embryos are not to scale. 640

641

Figure 3. New quantitative analysis method for 3D gene expression data, based on geometric 642

morphometrics. Mouse embryos between E10.5 and E11.5 were analysed with WISH to reveal the 643

expression of Dusp6 (1), and then cleared with BABB and OPT scanned using both fluorescence and 644

transmission light (2). The external surface of the embryo was obtained from the 3D reconstruction of 645

the fluorescence scan (2). Multiple thresholding of the transmission scan by choosing different levels of 646

grey values as shown by the histogram allowed to visualize gene expression patterns at different 647

intensities (3). Moderate gene expression (shown in green) was displayed as the isosurface obtained 648


https://doi.org/10.1101/246256


22

using as a threshold the grey value computed as 2/3 of the last grey value showing the Dusp6 expression 649

domain (3). High gene expression (shown in yellow) was displayed as the isosurface obtained using as a 650

threshold the grey value computed as 1/3 of the last grey value showing the Dusp6 expression domain 651

(3). From the whole mouse embryo isosurfaces, all four limb buds were segmented (5a). From the high 652

gene expression isosurface, the Dusp6 domains from all the available limbs were segmented (5b). 653

Maximum curvature patterns were displayed to optimize landmark recording (5). For each limb we 654

captured the shape and size of the limb bud (6a) and the underlying high Dusp6 gene expression pattern 655

(6b) recording the 3D coordinates of anatomical landmarks (yellow dots), curve semilandmarks (red 656

dots) and surface semilandmarks (pink dots). Anatomical and curve landmarks were recorded manually 657

on each limb. Surface landmarks were recorded on one template limb and interpolated onto target 658

limbs. Landmark coordinates were the input for Geometric Morphometric (GM) quantitative shape 659

analysis (7, 8) to superimpose the landmark data (GPA, General Procrustes Analysis), compute limb size 660

(Centroid size), and explore shape variation within limbs and gene expression domains by litters (PCA, 661

Principal Component Analysis). Finally, the covariation patterns between the shape of the limb and the 662

shape of the gene expression domain were also explored (PLS, Partial Least squares). 663

664

Figure 4. Tracing of limb phenotypes (anatomical and molecular) back through developmental time to 665

the earliest moment of appearance. Principal Component Analyses based on the Procrustes‐based 666

semilandmark was used to analyse the shape of the limbs and the corresponding Dusp6 expression 667

domains at each developmental period. Each period was analysed separately for the shape of the limb 668

(a‐d) and the Dusp6 expression domain (e‐h), as specified in Methods and Table SI_2. Scatterplots of PC1 669

and PC2 axes with the corresponding percentage of total morphological variation explained are 670

displayed for each analysis, along with morphings associated with the negative, mid and positive values 671

of the PC axis that separates mutant and unaffected littermates (PC1 or PC2, as highlighted in bold black 672

letters in the corresponding axis). Morphings are displayed in grey tones when the analysis showed no 673

differentiation between mutant and unaffected littermates. Morphs are displayed in colour when the 674

analysis revealed differentiation between mice (blue: unaffected littermates; pink: mutant littermates). 675

Limb buds and Dusp6 domains are not to scale and oriented with the distal aspect to the left, the 676

proximal aspect to the right, the anterior aspect at the top and the posterior aspect at the bottom of all 677

images. Convex hulls represent the ranges of variation within each group of mice. 678

679


https://doi.org/10.1101/246256


23

Figure 5. Quantitative correspondence between the size and shape of the limbs and the Dusp6 680

expression pattern. a‐d) Comparison of limb bud size and Dusp6 volume in unaffected and Fgfr2+/P253R 681

mutant littermates across development. Limb size was measured as limb centroid size (a), whereas the 682

size of the Dusp6 expression was measured as the volume of the gene domain (b), as specified in 683

Methods and Tables SI_2 and 3. The association between the size of the limbs and the volume of the 684

Dusp6 domain was assessed separately for forelimbs (c) and hindlimbs (d). Statistical significant 685

differences as revealed by two‐tailed t‐tests are marked with asterisks, representing the degree of 686

significance: *P‐value=0.03, ** P‐value=0.01. e) Time course assessing the morphological integration 687

pattern between the limb phenotype and the shape of the gene expression pattern using Partial Least 688

Squares analyses. Associated shape changes from late to early limb development are shown from 689

morphings associated with the negative, mid and positive values of PLS1, which accounted for almost all 690

the covariation (97.6% in forelimbs and 99.5% in hindlimbs) between the limb buds and the Dusp6 gene 691

expression domains. Limb buds and Dusp6 domains are not to scale and oriented distally to the left, 692

proximally to the right, anteriorly to the top and posteriorly to the bottom. On the right, representing 693

the positive extreme of PLS1 axis, typical early limb buds showed a protruding shape (i.e. short in the 694

proximo‐distal axis and symmetrical in the antero‐posterior axis) associated with a flat‐bean shaped 695

Dusp6 expression pattern localized in the distal limb region, underlying the apical ectodermal ridge, and 696

spreading proximally towards the dorsal and ventral sides of the limb. On the left, representing the 697

negative extreme of the PLS1 axis, limb buds were elongated in the proximal axis, asymmetric on the 698

antero‐posterior axis, with an expansion of the distal limb region and a contraction of the proximal 699

region, at the wrist level. This typical limb shape of more developed limbs was associated with a Dusp6 700

expression that was extended underneath the apical ectodermal ridge towards the anterior and the 701

posterior ends of the gene expression, but reduced on the dorsal and ventral sides of the limb. 702


https://doi.org/10.1101/246256


24

FIGURE 1 703

704


https://doi.org/10.1101/246256


25

FIGURE 2 705

706

707


https://doi.org/10.1101/246256


26

FIGURE 3 708

709

710


https://doi.org/10.1101/246256


27

FIGURE 4 711

712

.C

C-B

Y-N

C 4.0 International license

was not certified by peer review

) is the author/funder. It is made available under a

The copyright holder for this preprint (w

hichthis version posted January 11, 2018.

. https://doi.org/10.1101/246256

doi: bioR

xiv preprint

https://doi.org/10.1101/246256


28

FIGURE 5 713

714


https://doi.org/10.1101/246256


it is made available under a cc-by-nc 4.0 international ... · 1 quantification of gene expression...

Documents