The Pennsylvania State University Schreyer Honors College

THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE

DEPARTMENT OF ANTHROPOLOGY

FGFR3 P244R MUTATION: SKULL MORPHOLOGY OF NEWBORN MICE

KATHERINE RHODES SPRING 2015

A thesis Submitted in partial fulfillment Of requirements for baccalaureate degrees in Biological Anthropology and Biology with honors in Biological Anthropology

Reviewed and approved* by the following:

Joan Richtsmeier Distinguished Professor of Anthropology Thesis Supervisor

Timothy Ryan Associate Professor of Anthropology Honors Advisor

*Signatures are on file in the Schreyer Honors College i

ABSTRACT

Craniosynostosis is a birth defect involving premature fusion of one of more skull sutures during development and is involved in a variety of syndromes. Of these syndromes, Muenke

Syndrome is the most common in humans and occurs in approximately 1 in every 30,000 human births. Muenke syndrome is caused by a point mutation in the fibroblast growth receptor 3

(FGFR3) gene and results in a gain-of-function amino acid substution in the FGFR3 protein.

FGFR3 is a part of the tyrosine kinase family and abnormal function leads to a variety of diseases involving skeletal dysmorphology such as achondroplasia and thanatophoric dysplasia.

Individuals with Muenke syndrome experience a broad range of phenotypes often involving premature fusion of skull sutures in addition to other morphological changes of the skull. In this study, a mouse model for Muenke Syndrome was studied in order to quantify shape change induced by the Muenke Syndrome mutation. It was found that newborn mice who are either heterozygous or homozygous for this mutation exhibit shortening of the cranial base, elongation of the palate, and shortening of nasal bones, with homozygous mice showing a more severe phenotype. This pattern of dysmorphology in mice aligns with many of the phenotypic traits of

Muenke Syndrome observed in humans.

TABLE OF CONTENTS

LIST OF FIGURES ...... iii

LIST OF TABLES ...... iv

ACKNOWLEDGEMENTS...... v

Chapter 1: Introduction to Craniosynstosis and Muenke Syndrome ...... 1 The History of Muenke Syndrome ...... 2 Human Phenotype ...... 3 Molecular Basis ...... 6 Related diseases ...... 9 Mouse models ...... 11 Chapter 2: P244R murine mutation and the Cranial Morphology of Newborn Mice...... 14 Research Questions and Goals ...... 14 Materials and Methods ...... 14 Results ...... 20 Chapter 3: Conclusions ...... 29 Discussion ...... 29 Future Directions ...... 30 Chapter 4. Supplemental Information ...... 32 Bibliography ...... 34 Academic Vita ...... 38

iii

LIST OF FIGURES

Figure 1. Human Phenotypes of Muenke Syndrome6 ...... 4

Figure 2. Cranial Phenotype of Fgfr3P244R/+ Adult (P60) Mice34 ...... 12

Figure 3. Landmarks Taken and Regions of the Skull ...... 16

Figure 4. Skull Morphology of Fgfr3P244R Mutant Mice and Unaffected Littermates at P0 ...... 20

Figure 5. Variation in Landmark Location Across the Entire Sample ...... 21

Figure 6. Principle Components Analyses of 41 Global Landmarks ...... 23

Figure 7. Multivariate Regression on Size in MorphoJ ...... 24

Figure 8. Fgfr3P244R/P244R vs. Unaffected littermates Shape Change ...... 27

Figure 9. Fgfr3P244R/+ vs . Unaffected littermates Shape Change ...... 28

Figure 10. Fgfr3P244R/P244R vs. Fgfr3P244R/+ Shape Change ...... 28

LIST OF TABLES

Table 1. FGFR-Related Craniosynostosis Syndromes ...... 1

Table 2. Description of the full set of global landmarks taken on mouse crania ...... 32

ACKNOWLEDGEMENTS

Thank you to Dr. Joan Richtsmeier who made this work possible through her guidance and resources. Special thanks to Dr. Susan Perrine for her time and effort in teaching me all of the skills required to complete this project and her continued direction throughout.

Chapter 1: Introduction to Craniosynstosis and Muenke Syndrome

Craniosynostosis is an overarching term referring to the plethora of conditions which include premature fusion of skull sutures during development. Many individuals also show dysmorphology of the cranial base, fusion of facial sutures, and display dysmorphology of soft tissues or aberrant development of multiple organ systems. Over 100 craniosynostosis syndromes have been described and affect approximately 1 in 2,500 live births, making it a common birth defect1. A subset of these syndromes are all caused by single point, autosomal dominant mutations in fibroblast growth receptor genes (FGFRs) and are collectively referred to as FGFR- related craniosynostosis syndromes (see Table 1).

Table 1. FGFR-Related Craniosynostosis Syndromes

Syndrome Name Mutated Gene OMIM Reference # FGFR1, 1. Pfeiffer Syndrome FGFR2 101600 2. Apert Syndrome FGFR2 101200 3. Jackson-Weiss Syndrome FGFR2 123150 4. Beare-Stevenson Syndrome FGFR2 123790 5. Crouzon Syndrome FGFR2 123500 Crouzon Syndrome w/ acanthosis 6. nigricans FGFR3 612247 8. Muenke Syndrome FGFR3 602849

The following thesis will focus specifically on Muenke syndrome—a condition that arises due to a point mutation, or the substitution of a single base pair in the DNA coding region for this protein, in the FGFR3 gene. Muenke syndrome occurs in approximately 33.5 individuals per 1,000,000 births, accounting for about 8% of craniosynostosis cases2,3. Being such a prevalent condition, research into Muenke Syndrome has clinical significance and has the

2 potential to improve care and treatment of affected individuals. In addition, research into this disease can improve understanding of the role of FGFR3 signaling during development.

The History of Muenke Syndrome

The vast majority of craniosynostosis syndromes were first described clinically through observations of specific patterns of dysmorphology. By the 1990s, when genetic techniques had become widely used and new approaches became available, there was a movement to identify the genetic basis for already known diseases. Such findings would provide information about the heritability of these conditions and could play a role in genetic counseling such that potential parents can be aware of the risks that might affect their offspring. Dr. Maximillian Muenke led a group of researchers in the search for the causative mutation for Pfeiffer syndrome, a craniosynostosis syndrome that can be distinguished by broad thumbs and great toes4. The group succeeded in their goal and described two distinct gain-of-function mutations in the genes for

FGFR1 and FGFR24. During this research, Dr. Muenke’s group found certain individuals who had been diagnosed with a craniosynostosis syndrome, but did not carry any of the identified causative mutations. Bellus et. al. (1996) had recently discovered a mutation in the FGFR3 gene in a set of 10 unrelated families5. When Dr. Muenke’s team evaluated 61 individuals diagnosed with a craniosynostosis syndrome from 20 unrelated families, they found that 53 of the individuals were positive for the P250R mutation in the FGFR3 gene.

And thus, a new craniosynostosis syndrome was defined. Today, presence of this mutation is most commonly referred to as Muenke Syndrome. However, it should be noted that some other names exist such as “FGFR3-related coronal synostosis syndrome” or “Muenke nonsyndromic coronal craniosynostosis”5. Usage of alternate names may in part be due to the idea that diseases and illnesses should not be named after researchers and should instead be

3 descriptive. For the remainder of this thesis, this syndrome will be referred to as Muenke

Syndrome.

Today, 20 years after its discovery, Muenke Syndrome is diagnosed solely upon genetic testing to show presence of the FGFR3 Pro250Arg mutation and it is recommended that any incidence of coronal synostosis be checked for this mutation.

Human Phenotype

Muenke Syndrome manifests in a broad range of phenotypes, even within families, due to variation in penetrance and variable expressivity of the affected gene. In about 14% of cases, there is no distinguishable phenotype6. For this reason, any diagnosis of the disease requires genetic testing. There are, however, some common phenotypic changes that have been observed throughout the clinical history of this syndrome. Many of the most notable morphological changes occur in the skull. Premature fusion of the coronal suture either bilaterally (47% of cases) or unilaterally (22% of cases)is a defining feature of the condition, though not present in every individual carrying the mutation6,7. Fusion of other sutures is very uncommon and has been found in less than one percent of cases7. Macrocephaly is observed in about 3 percent of cases and some affected individuals exhibit brachycephaly. Interestingly, craniosynostosis is found more frequently in females and is much more likely to be severe than in males6. And, of the females carrying the mutation that exhibit craniosynostosis, a higher proportion exhibit bilaterally coronal synostosis that unilateral; of the males carrying the mutation that exhibit craniosynostosis, approximately equal numbers exhibit bilateral coronal synostosis as unilateral coronal synostosis7.

A suite of craniofacial features are also associated with Muenke Syndrome, including facial asymmetry, hypertelorism, downslanting palpebral fissures, ptosis of the eyelids, temporal bossing, dental malocclusion and a highly arched palate and midfacial hypoplasia6. These characteristics overlap with some of the facial feature observed in other craniosynostosis syndromes (e.g. Saethre-Chotzen, Pfeiffer, and Crouzon Syndrome).

Muenke syndrome has also been Figure 1. Human Phenotypes of Muenke Syndrome6 . associated with some abnormalities of the

postcranial skeleton. While long bone growth and

stature remain unaffected, subtle differences in

extremities are sometimes observed in Muenke

Syndrome. Calcaneo-navicular and capitate-

hamate coalition have been associated with

Muenke syndrome, as have brachydacty,

clinodactyly, and cone-shaped epiphyses5.

This disease also manifests outside of the

skeletal system. It is estimated that between 62-

95% of cases involve sensorineural hearing loss6.

This series of images, taken from Nah et. al. 2012, Sensorineural hearing loss is caused by shows three pre-operative individuals with Muenke Syndrome. A, B. show brachycephaly abnormalities in hair cells and the cells supporting and a bulging forehead C. shows brachycephaly, a high forehead, bulging temporal squama as well as hair cells which are all located of the organ of mild hypertelorism. D. shows an individual with left unilateral coronal synostosis. Corti, a structure that forms part of the cochlea.

Normally, these hair cells transmit the physical vibrations of sound as nerve impulses. FGFR3 is highly expressed in the cochlea and normal function is necessary for proper cellular

5 differentiation in this location. In Muenke Syndrome, abnormal function of FGFR3 receptors results in the differentiation of cells that would normally have become Dieters’ cells into Pillar cells; both of these cell types play a role in supporting outer hair cells in the ear8. Hearing loss is also common in a host of other craniosynostosis syndromes including Apert, Crouzon, and

Saethre-Chotzen. Muenke Syndrome can also lead to cognitive impairment in some individuals.

However, most cases of the disease retain normal or borderline intelligence9.

Surgery is often required in craniosynostosis cases to release intracranial pressure, allow for rapid brain growth, and aesthetically reshape the head6,10. Surgery was first attempted as a treatment for craniosynostosis in 1890 by the French surgeon Lannelongue, who was renowned for his work with other bone diseases11. Unfortunately, this attempt was a failure and resulted in post-operative death of the patient. Craniosynostosis surgery was not attempted again until 1921, when Mehner performed the first successful removal of a fused suture using a strip craniectomy11. Since then, surgical techniques have greatly advanced and take place at younger ages; currently, many surgeons recommend operation before the age of 6 months10. Endoscopic synostosis repair, first developed in 1998, has made craniosynostosis surgery less invasive, has reduced blood loss, and shortened hospitals stays11. This surgery requires only two small incisions through which the excess bone within a suture is removed. Following the surgery, the infant is fitted with a helmet that helps to reshape the skull. Muenke Syndrome patients often require additional operations to improve aesthetic appearance—a factor which can be crucial in ensuring a normal life the for the patient6.

Molecular Basis

Muenke syndrome is caused by a point mutation in the FGFR3 gene, located on the short arm of chromosome 4 in humans (4p16). The mutation constitutes a cytosine to guanine transversion (C749G) which results in a proline for arginine amino acid change (Pro250Arg) in exon 7 of FGFR3 12,13. The Pro250Arg mutation is inherited in an autosomal dominant pattern and can occur within families or sporadically. It has been estimated that 61% percent of all

Muenke Syndrome cases are sporadic with mutations arising almost exclusively in the paternal germ line14,15. The mutation rate for this site has been estimated to be about 7.6 x 10-6; however, this figure is likely to be an underestimate due to the high probability of clinically undocumented incidences of the mutation15. That this site shows one of the highest transversion rates in the human genome may be due in part to the fact that this mutation occurs at a CpG site, which collectively show 2.8 to 8-fold higher mutation rates than non-CpG sites. However, this particular CpG site shows a 300- to 450-fold higher mutation rate. It has been suggested that this extremely high rate may be due to enrichment of the mutation via clonal expansion over time and confer a selective advantage to spermatogonial cells despite their detrimental effect on development16. Whatever the cause may be, this site has one of the highest known transversion rates within the human genome.

The FGFR3 gene codes for a receptor which binds specifically with FGFs 1,2,4,8 and 9.

Members of the FGF family are collectively responsible for a wide array of developmental processes, including but not limited to limb development, brain patterning and branching morphogenesis17. The FGF and FGFR signaling system is highly conserved through metazoan evolution18. There are 22 known FGFs in humans and mice which are most commonly involved in paracrine signaling18. There are 4 FGFR genes with are alternatively spliced to produced that

7 range of specificity required. In humans, the full range of FGFRs contain 56-71% similarity in their amino acid sequences18. Most FGFRs are tyrosine kinase receptors comprised of about 800 amino acids with three extracellular immunoglobulin domains17. When a specific FGF ligand becomes bound in this extracellular domain, two receptors will dimerize, become phosphorylated, and initiate downstream reactions in a variety of biochemical pathways.

FGFR3 and its ligands are responsible for a variety of developmental processes and as of yet, the exact pathogenesis of Muenke syndrome is unknown. Mice with the FGFR3 gene knocked out had a shortened lifespan and overgrowth of bone, indicating that a function of this gene may be to regulate the extent of bone differentiation19. This finding makes sense in the context of Muenke syndrome in that craniosynostosis may be determined by bone differentiation within sutures. We can further investigate possible effects of FGFR3 mutation in cranial morphogenesis by understanding the known roles of its ligands, FGFs 1,2,4,8,and 9, during development of the head. It should be noted that FGFR3 is alternatively spliced into two isoforms, FGFR3b and FGFR3c. FGFR3b is expressed in epithelial cells and binds to FGF1and

FGF920. FGFR3c is largely expressed in mesenchymal cells and binds preferentially to FGFs

1,2,4,8 and 920. Alternative splicing of mRNA occurs at exon 8 and exon 9; because the mutation causative of Muenke syndrome occurs in exon 7, both FGFR3 isoforms are affected20.

The FGFR3 IIIc isoform is expressed in cartilage and at low quantities in the cranial suture mesenchyme6. The same isoform of this receptor is expressed in the cochlea6. The locations correspond with the characteristic traits of Muenke Syndrome, so it is likely that the FGFR IIIc isoform is largely responsible for the pathogenesis of this disease.

FGFR3 shows high expression in the proliferating zone of the epiphyseal growth plate— the primary center of ossification for longitudinal growth of the long bone via endochondral

8 ossification6. Inactivation of FGFR3 lead to increased chondrocyte proliferation and elongated chondrocyte columns; In mice with the Fgfr3 gene knocked out, this increased proliferation lead to lengthening of long bones and decrease bone density19. Knock-in mice who have mutations in

Fgfr3 that make the receptor constitutively active show decreased proliferation of chondrocytes due to loss of control over the Indian hedgehog hormone related peptide feedback signal and

STAT signaling pathways21,22. This constitutive activation studied in mouse models results in phenotypes similar to those seen in cases of achondroplasia, hypochondroplasia, and thanatophoric dysplasia6. This indicates that the Fgfr3 gene plays an important role in regulating the elongation of long bones by regulating chondrocyte proliferation and endochondral ossification. The cranial base is formed via endochondral ossification and is known to be altered in cases of Muenke Syndrome in both humans and mice. Dysmorphology of the cranial base may be shortened by similar mechanisms as limb development in cases of achondroplasia and could in turn lead to dysmorphology in other regions of the head and fusion of sutures in the cranial vault.

Other hypotheses about the molecular mechanism influencing abnormal morphology in

Muenke Syndrome can be built by understanding the interactions of FGFR3 with various ligands in certain locations during development. For instance, some studies have implicated FGF2 interactions in premature suture closure. FGF2 is ubiquitously expressed in the developing head and has been shown to be much more abundant than any other FGF in day 16 mouse embryos23.

Research has shown that FGF2 plays a role in bone differentiation and is known to influence the localization of FGFR2 expression23. In Pfeiffer and Apert syndromes, mutated FGFRs have been shown to have enhanced binding with FGF21. These mutations are analogous to the

Pro250Arg mutation in FGFR3, which also exhibits enhanced binding with FGF224. FGF2

9 binding is known to repress FGFR3 activity25. It is further understood that FGF2 has varying effects depending on neural crest cells depending on the concentration of exogenous exposure; added FGF2 at low doses induces increased proliferation and higher doses, greater than 10 ng/ml, induces cartilage differentiation26. Neural crest cells grown in culture and exposed to

FGF2 formed both endochondral and membrane bone26. Based on these observations, it is conceivable that increased FGF2 binding by FGFR3 could lead to increased ossification in cranial vault sutures.

FGF8 is known to play a role in regulation of brain development through repression of

FGFR327. FGF8a, one of the differentially spliced forms of the protein, is important in controlling the expansion of the midbrain. FGF8b marks cells for cerebellar fate and reduces the size of the midbrain27. FGF8 plays an additional role in forebrain development; it is secreted by the anterior neural ridge and is required for cell survival and proper formation patterning of the telencephalon28. Development of the brain and the skull are highly integrated. Any morphological change in either of these tissues must be accommodated by the other. It may be that interactions of mutated FGFR3 with ligands like FGF8, are primarily responsible for dysmorphogenesis of the brain and that craniosynostosis simply follows in order to accommodate this change28,29. The dynamics of the co-development of the brain and skull are not fully understood at present, but future research into the huge number of pathways involved in the development of the head may shed further light onto this phenomenon.

Related diseases

Mutations in the FGFR3 gene cause a variety of diseases marked by abnormal skeletal development. The group of FGFR3 related disorders includes achondroplasia,

10 hypochondroplasia, thanatophoropic dysplasia, Crouzon Syndrome with Acanthosis Nigricans, and Muenke syndrome30. Understanding the disorders that result from abnormal function of the

FGFR3 protein helps illuminate the normal role of this receptor in development and the ways in which normal function can be disrupted.

Achondroplasia, hypochondroplasia, and thanatophoric dysplasia are three forms of short-limbed dwarfisms that are referred to collectively as the ‘achondroplasia family’ due to their close relation both clinically and molecularly. Achondroplasia is the most common form of dwarfism and is the most severe of the disorders in the achondroplasia family. It can be caused by several point mutations of FGFR3 which are all inherited in an autosomal dominant pattern with complete penetrance31. Homozygosity for the causative mutation is lethal. The most common mutation causative of achondroplasia, Gly380Arg, is present in 97% of achondroplasia cases and constitutes and amino acid change in the transmembrane domain of the FGFR3 protein; this mutation causes increased dimerization of the receptor which in turn leads to premature chondrocyte differentiation and a shortened period of bone elongation32. In the postcranial skeleton, achondroplasia is often marked by short stature, shortening of the limbs, exaggerated lumbar lordosis, limited extension of the elbow and trident hands31. In the head, achondroplasia usually causes frontal bossing, midfacial hypoplasia, and a shortened cranial base31,32.

Similar to achondroplasia, mutations which cause thanatophoric dysplasia (TD), which occur in either the intra- or extracellular domain of the FGFR3 protein, result in dimerization of the receptor without a ligand present33. Individuals with TD commonly have shortened limbs, normal trunk length, and a narrow thorax with relatively large heads that show frontal bossing and a low nasal bridge31. Two forms of TD exist, type I and type II, which have distinct

11 phenotypic characteristics but are both usually lethal31. Cases of TD type 1 often have curved humeri and femora while type II is more likely to present with cloverleaf skull31.

Hypochondroplasia is the mildest disease in the achondroplasia family and is characterized by short stature, shortened limbs, and lumbar lordosis. In cases of hypochondroplasia, the head often exhibits some degree of macrocephaly but the face is normal30,31.

Crouzon syndrome with acanthosis nigricans (CAN), the other craniosynostosis syndrome caused by a mutation in FGFR3, results in a facial phenotype which is largely distinct from Muenke Syndrome. Individuals with CAN often exhibit hypertelorism, parrot-beaked nose, hypoplastic maxilla, and relatively prognathic maxilla. CAN is also distinct from ‘Crouzon

Syndrome’, which is caused by a mutation in Fgfr230. The mutation causing CAN is thought to be a G-to-A transition resulting in an Ala391Glu substitution in the transmembrane region of the receptor30. The mutated receptor in CAN is thought to be normally expressed and activated, but may have a higher response to lower concentrations of ligand, indicating possible auto-activation of the receptor30.

Studies of FGFR3-related diseases have yielded information on the normal role of

FGFR3 in skeletal development and have provided insights on the mechanisms by which different mutations alter the normal path of development. Considering Muenke Syndrome in the context of these related diseases has provided the basis for hypotheses about the cause of suture closure and the molecular implications of this disease.

Mouse models

Mice are widely used as models for human genetic diseases due to their high degree of phylogenetic similarity to humans. With a fully sequenced genome, knock-in and knock-out mice are easily produced and bred. In the case of Muenke syndrome, the use of mouse models has been used to study pathogenesis of the murine FGFR3 P244R mutation, homologous to the human P250R mutation, and localized phenotypic effects.

In mice, the Fgfr3 gene is located on chromosome 5 and is caused by P244R Figure 2. Cranial Phenotype of Fgfr3P244R/+ Adult (P60) Mice34 . mutation. Muenke Syndrome mouse models Superior Lateral (Right) Posterior Inferior do not show the limb characteristics observed in humans with the disease and, like human, have normal stature and limb length6. All mice with the P244R mutation seem to have sensorineural hearing loss, as is common in humans24. The cranial phenotype of these mutant mice also shows some similarity to the human phenotype of

Muenke Syndrome. In adult P28, or 4 week old mice, homozygous for the P244R

FGFR3 mutation have smaller, more rounded skulls with shortened, twisting snouts34. One study noted that neither Figure 2, taken from Twigg et. al. (2009) shows MicroCT images of C57BL/6 mice. The mouse in A. is unaffected (Fgfr3+/+) and B-D are heterozygous for the murine Muenke Syndrome mutation heterozygous nor homozygous mutants at (Fgfr3P244R/+). A. shows normal phenotype with arrows indicating the coronal suture. B. shows a rounded skull with an abnormal this age appeared to have any coronal suture, the interparietal bone posteriorly displaced and ventrally displaced occipital bone. This mouse also exhibits molar malocclusion. C. shows a severely shortened snout which is twisted to the right and a pronounced underbite. D. also shows a twisted snout. 13 dysmorphology in the cranial base34. A different study indicated that mutant mice did exhibit shortening of the anterior cranial base6. The mutation has been bred into several different genetic backgrounds including BALB/c, C57BL/6, 129/SvEv, and CBA/Ca. Balb/c mice never show an abnormal phenotype, while the other backgrounds show abnormal phenotype in 7% of heterozygotes and 74% of homozygotes34. Interestingly, female mice appear to be more likely than males to have a severe phenotype, the reverse of the trend observed in humans6. The information that has already been gathered from analyses of Muenke Syndrome mouse models provides a strong foundation for further studies on the progression and presentation of this disease.

The use of mouse models provides the added benefit of allowing study of early stages in development that cannot be studied in humans. Mice are commonly used for analysis of embryological development and therefore allow for a more complete understanding of timing of certain events and the trajectory of growth. While existing studies of cranial morphology in murine Muenke Syndrome have focused on adult mice, studies of earlier ages have the potential to illuminate timing and localization of pathological morphogenesis.

Chapter 2: P244R murine mutation and the Cranial Morphology of Newborn

Mice

Research Questions and Goals

While several publications to date describe and analyze the cranial morphology of

Muenke Syndrome mouse models, very little has been published about morphology at younger ages and during development in these mice. This study seeks to quantify morphological differences between homozygous Fgfr3P244R/P244R mice, heterozygous Fgfr3P244R/+, and their unaffected littermates, Fgfr3+/+, at the day of birth (P0). The goals of this research are numerous; results of this research could provide a more complete understanding of the pathological morphogenesis of the skull in Muenke Syndrome, can identify regions of the skull that are specifically affected, and can illuminate differences between the homozygous and heterozygous condition. It was hypothesized that Fgfr3P244R/P244R and Fgfr3P244R/+ Muenke

Syndrome mice would have a significantly different cranial shape compared to their unaffected littermates, and that the homozygotes would show a more severe phenotype that the heterozygotes. It was further hypothesized that shape changes at this age would mirror the observations made in previous studies about of Muenke Syndrome mice, such as shortened nasal bones and shortened anterior cranial base.

Materials and Methods

Breeding

The sample for this study consisted of a total of 60 newborn mice; 20 mice were heterozygotes (Fgfr3P244R/+), 20 mice were homozygous (Fgfr3P244R/P244R), and 20 unaffected littermates. These groups include mice from 12 litters and include mice of both sexes. Mice

15 were bred at Johns Hopkins Medical Institutions and Mount Sinai Medical Center. The knock-in mouse model was backcrossed onto the C57BL/6 background for more than 10 generations to increase genetic homogeneity and to allow for direct comparison between litters. Specimens were harvested at P0 (day of birth after a gestation period of 19.0±0.5 days), euthanized by inhalation of anesthetics, and fixed using 4% paraformaldehyde. All litters were produced, sacrificed, and processed according to animal welfare guidelines approved by the Johns Hopkins

University, the Mount Sinai Medical Center, and the Pennsylvania State University Animal Care and Use Committees (IACUC).

Imaging

High-resolution micro-computed tomography images were acquired by the Center for

Quantitative X-Ray Imaging at the Pennsylvania State University using HD-600 OMNI-X high- resolution X-ray computed tomography system (Bio-Imaging Research Inc, Lincolnshire, IL).

Images of Muenke Syndrome model mice had a pixel size and slice thickness ranging from

0.0136 to 0.0157mm. Image data were reconstructed on a 1024 x 1024 pixel grid as a 16-bit

TIFF, but were reduced to 8-bit for image analysis. Isosurfaces representing all cranial bones were recreated in Avizo 6.3 (FEI, Hillsboro, OR) from 8-bit images and were based on hydroxyapatite phantoms imaged with the specimens. Careful attention was paid to the proper voxel sizes of the images to ensure the accuracy of comparison.

Morphometric Analysis

Using the isosurfaces in Avizo software, the three dimensional coordinates of 64 landmarks representing locations of biological significance on the skull were collected on the

16 isosurface representing each sample skull in an effort to capture global shape. Of these 64 landmarks, 41 were used for the final analysis. This quantity of landmarks was chosen in order to adequately represent global skull shape. A description of the final 41 landmarks used in analysis can be found in Table 2 and their location is illustrated in Figure 3. The 3D locations of the full set of landmarks was recorded twice on each skull by the same researcher in order to eliminate inter observer bias in landmark positioning. Error was further reduced by averaging the coordinates of each landmarking trial. If any landmark had a difference in location that differed more than 0.05 mm between the two trials, the point was retaken thereby decreasing intra observer error. Error checking was performed using an R code to evaluate the distances in all three dimensions of each of the 41 landmarks on each skull. If no large error was found (e.g., confusing left and right sides), the average of the two trials was used as representative of the landmark location in analysis.

Figure 3. Landmarks Taken and Regions of the Skull .

Figure 3 shows the full set of global landmarks taken on P0 mouse skulls. Green points define the face, blue points define the cranial vault, orange points define the cranial base, and purple points define the palate.

Statistical Analysis

Variation in skull shape between the 3 different genotypes was first explored using two types of principle components analysis (PCA) which are used to generate a summary of the variation of a large quantity of variables. For this study, a linear distance PCA was completed using SAS 9.3 (SAS Institute, Cary, NC) and a Procrustes superimposition-based PCA was done using MorphoJ35. In morphometrics, PCA is a data exploration technique and the plots generated can be used to determine the degree of shape variation in the different genotype groups through separation of the clusters of genotype groups. The data are subjected to a spectral composition that reduces the high dimensionality of the original data to a set of linearly uncorrelated variables, the Principle Components (PCs). This transformation is ordered so that the first

Principal Component accounts for largest amount of variance in the data, the second Principal

Components accounts for the second largest amount of variation in the data given the constraint of orthogonality, and so one through the remaining Principal Components.

Generalized Procrustes Analysis using MorphoJ

To extract shape information, a global skull configuration of landmarks were superimposed using MorphoJ35. The generalized Procrustes analysis procedure minimizes the effects of scale, but does not eliminate the allometric shape variation related to size36. A full

Procrustes fit was performed on 3D landmark data imported into MorphoJ, and these data projected to the tangent space by orthogonal projection37. To analyze shape variation within and among groups, we computed a Principal Components Analysis (PCA) of the global skull landmark configuration. PCA performs a coordinate rotation that aligns the transformed axes

(PCs) with the directions of maximum variation. In order to test how allometry affected the

18 sample, we computed a multivariate regression of shape (represented by Procrustes coordinates) on centroid size38. Centroid size is computed as the square root of the summed distances between each landmark coordinate and the centroid of the landmark configuration37. The regression residual estimates for each specimen were used to compute another PCA that represents the distribution of data after the effects of allometry are removed.

Principal Components Analysis of Form and Shape using SAS

Variation in skull shape was also assessed using principal component analysis based of all unique linear distances among the 3D coordinates of the landmarks, followed by a PCA based on shape variation alone39–41. To explore variation in form (size and shape together), 820 unique inter-landmark distances, defined by 41 global landmarks, were ln-transformed and their variance-covariance matrix was used as the basis for the PCA. To explore variation in shape alone by eliminating differences in scale, we performed a PCA based on variation in shape. For shape alone, the linear measures were used to define dimensionless variables, where all information about the absolute size of the measurements was removed and only information about proportions remained. The shape variables for an observation were defined as the ln- transformed ratios of its linear distances to the geometric mean of all of its distances (where the geometric mean serves as a measure of overall size42,43. All PCAs based upon ln-transformed linear distances and ratios were formed using SAS 9.4.

Euclidean Distance Matrix Analysis and confidence intervals for differences in form

Euclidean Distance Matrix Analysis (EDMA), a 3D morphometric technique that is invariant to the group of transformations consisting of translations, rotations, and reflections44.

This method was used to statistically evaluate shape differences between samples of homozygous mutant mice, heterozygous mutant mice, and unaffected littermates. All EDMA analyses were executed using EDMAware45 . The original 3D landmark coordinates are converted into a matrix of the measurements of the unique inter-landmark linear distances; this matrix is referred to as the Form Matrix, or FM. The different genotype groups were then compared by creating a ratio of the corresponding linear distances between groups. This produces a matrix of rations referred to as the Form Difference Matrix, or FDM and represents the relative difference between the inter-landmark distances which were chosen to define the form. If a particular linear distance is similar in two samples, that ratio will equal 1.

Form difference is statistically evaluated using estimates of the mean FMs and variance- covariance structure for each sample. Nonparametric statistical techniques test the null hypothesis of similarity in form between the samples to determine whether a difference in form exists, and if it does, whether it is due solely to size (scaling), or if there is a shape component (P

≤ 0.05 is traditionally used as the level of significance). This nonparametric test for overall similarity in shape uses the original data to generate random samples, each containing the same number of specimens as the true sample. A FDM is calculated for each pair of bootstrapped samples, and a test statistic (maximum ratio of inter-landmark distances divided by minimum ratio, or max/min) is calculated for each pair. This is done an adequate number of times (200–

300). The test compares the true max/min calculated from the original data to the distribution of the max/min values for the bootstrapped samples. If the true max/min lies outside of 95% of the bootstrapped max/min values, we reject the null hypothesis of similarity in shape44. Further examination of the FDM identifies patterns of localized differences in form between the two samples. To statistically test for localized differences in form, an alternate nonparametric

20 bootstrap procedure calculates the 100(1-α)% confidence interval for each linear distance. If this interval contains the value 1.0, the null hypothesis of similarity for that linear distance is accepted. Confidence intervals enable the identification of those linear distances that are most similar or different between the samples. The linear distances that differed significantly between groups were then plotted using Avizo software and can be observed in Figures 6-8.

Results

Skull morphology

Figure 4. Skull Morphology of Fgfr3P244R Mutant Mice and Unaffected Littermates at P0 .

Figure 4 shows isosurfaces of the individual mice of each genotype and can be used as a reference for typical morphology of mice of each genotype at age P0. There are no defining differences that can easily identify any of these individuals as mutant or unaffected based solely on visual inspection.

Cranial morphology of P0 Muenke Syndrome model mice were analyzed using 3D

coordinates of skull landmarks (Table 2, Figure 3). The morphology of three genotypes,

Fgfr3P244R/P224R, Fgfr3P224R/+, and Fgfr3+/+, were compared through analysis of a sample of 20

mice of each genotype.

Principal Components Analysis

First, MorphoJ was used to visualize the variation in location of each landmark across the

entire sample of 60 mice (Figure 5). This allowed for identification of any landmarks that show

a great shift in location than the rest and may therefore be less ideal for use in further analysis.

Figure 5. Variation in Landmark Location Across the Entire Sample .

Figure 5 is a lollipop graph showing a superior/inferior view of the complete set of landmarks used for analysis and represents the total variation in the location of each point across the entire sample. “A lollipop graph shows the shifts of landmark positions with straight lines. Each line starts with a dot at the location of the landmark in the starting shape (often a mean shape, etc.). The length and direction of the line indicates the movement of the respective landmark from the starting shape to the target shape (e.g. the mean shape plus the shape change that corresponds to an increase of 0.1 units of Procrustes distance 48 in the direction of the PC1).” . Points 16 and 34 show the highest degree of variation in location, but this variation is likely due to varying degrees of ossification in the cranial vault. This image was created in MorphoJ. A scaling factor of .025 was chosen as the most accurate representation of the magnitude of the shift in location each landmark. 22

Relatively high amounts of variation in the location of a single landmark may be caused by normal biological variation in the degree of bone ossification and could cause falsely indicate a high degree of shape change at a specific inter-landmark distance analysis. In this sample, some cranial vault landmarks were particularly variable (landmarks 16 and 34 in Figure 5, representing lpfm and rpfm landmarks) due to increased variation in the progression of ossification of cranial vault bones among individual mice. These two landmarks were less variable than other landmarks taken on the cranial vault, and were kept for analysis in an effort to capture the full shape of the skull.

Two Principle Components Analyses (PCAs) were performed to explore variation in morphology. Figure 6 shows the results of these analyses; Figure 6a displays the results of a

Procrustes-based PCA generated using MorphoJ software while Figure 6b shows an ln- transformed linear distance measurement-based PCA that was generated using SAS 9.3.

A plot of principle components 1 and 2 from the Procrustes-based PCA shows that while genotype groups do not completely separate, the areas of overlap are relatively small and the cluster of homozygous mutant mice is almost separated from unaffected littermates along PC1.

The homozygous mutant and the heterozygous mutant groups each show clustering of individuals, with a region of overlap between the two. PC1 accounts for 19.00% of the total variation in form. Both heterozygote and homozygote mutants show some separation from unaffected littermates along PC2, but the mutant groups are not distinct from one another along this principle component. Separation along PC2, which accounts for 8.52% of the total variation, may therefore be caused by changes in the anterior cranial base; distances between many of these landmarks differ significantly between mutants and unaffected littermates, but do not differ greatly between the two mutant groups (Figure 8-10). This plot also indicates that between PC1

23 and PC2 the mutant genotypes have more overlap and more similarity in form that either has with the unaffected mice. PC3 accounts for 8.20% of the total variation. In the plot of PC2 and

PC3, there is very little separation of genotypes. However, homozygous mice are slightly less variable along PC3 than the heterozygous or unaffected mice. Collectively, the first three principle components only convey 35.7% of the variation in morphology, which is a very small

Figure 6. Principle Components Analyses of 41 Global Landmarks .

A. Procrustes-based PCA B. EDMA-based PCA

Figure 6 shows the principle component analyses of shape. A. shows PCA plots generated using Procrusted-based analysis in MorphoJ. Ellipses mark the 75% confidence interval for the variation of each genotype. B. shows the EDMA-based PCA analysis generated using SAS. In both A and B, Red points represent unaffected littermates, green points represent heterozygous individuals, and blue points represent homozygous mutants.

24 amount of the total variation.

Similar patterns of clustering and separation were observed in the EDMA-based PCA generated in SAS (Figure 6b). The groups absolutely DO NOT separate on PC1. In the plot of

PC1 and PC2, there is no separation of the groups along PC1. Homozygous mutants show the smallest degree of variation along this axis while heterozygous mice show most variation along this axis. The three genotypes do separate into fairly distinct groups along PC2. The plot of PC2 and PC3 generated by this method again shows some clustering of the genotypes into groups, with some overlap. In the PC1 vs. PC2 and PC2 vs. PC3 plots, the heterozygous mutants are intermediate between the homozygous mutants and the unaffected littermates.

Both types of PCA analyses indicate an outlier—one of the unaffected individuals groups with the heterozygous mutants in the plot of PCs 1 and 2, and with the homozygous mutants in the plot of PCs 2 and 3. This result has a number of possible causes. It is possible that this individual was genotyped incorrectly. The specimen could also have been damaged during processing, but the CT scans do not show any clear indications of denting or crushing. It is also

Figure 7. Multivariate Regression on Size in MorphoJ .

Figure 7 shows results for the multivariate regression on size of the sample generated in MorphoJ. In the plots, the x axis represents variation in the principle component and the y axis is based on centroid size. The patterns of clustering and separation in these plots closely resemble the clustering and separation in the PCA plots generated without accounting for size, indicating that size variation is not strongly influencing differences in shape.

25 possible that that individual varies slightly in age relative to the other mice in the sample such that differences in the degree of ossification are being interpreted as difference in shape. This possibility highlights the need for better standardization for monitoring age and relative developmental stage.

A multivariate regression on size was performed in MorphoJ in order to interpret shape differences without the influence of size (Figure 7). This analysis resulted in plots displaying similar patterns of clustering and separation of the genotype groups. Along PC1, there is complete overlap of the unaffected and heterozygous mutant groups. The homozygous mutants show the least variation along PC1 and partially separate from the other mice. Along PC2, the homozygous and heterozygous mutants completely overlap, but are largely distinct from the unaffected mice. And finally, along PC3 we see complete overlap of the genotypes. Because these plots so closely resemble the plots generated in normal Procrustes-based PCA, we know that differences in size are not dictating the observed changes in shape.

Euclidean Distance Matrix Analysis (EDMA) and confidence intervals for differences in form

Euclidean Distance Matrix Analysis (EDMA) was used to evaluate patterns of shape difference between heterozygous mutant mice and unaffected mice, between homozygous mutant mice and unaffected mice, and between homozygous and heterozygous mutant mice.

Overall, the homozygous mutant mice were statistically different from both the unaffected littermates and the heterozygous mutants. Testing of overall difference in shape resulted in a statistic with an associated p-value of 0.001 in both analyses; the null hypothesis of similarity in shape can be rejected. Overall, the heterozygous mice and unaffected littermates were not

26 statistically significantly different. In this analysis, the null hypothesis of similarity in shape cannot be rejected (p=0.217). However, all comparisons showed localized differences in inter- landmark differences that differed significantly. Between homozygous Fgfr3P244R/P244R mice and unaffected Fgfr3+/+ mice, a total of 10 inter-landmarks distances differed by more than 10% and

15 inter-landmark distances differed between 5 and 10%. Collectively, these localized differences indicate that the distance between the basioccipital bone and the palate is significantly shorter in the homozygotes and the basioccipital bone itself is elongated in the homozygotes. Additionally, the palate of the homozygotes seems elongated relative to the unaffected mice. Nasal bones of the homozygous mutants are shorter than their unaffected littermates. All distances that were significant different between the Fgfr3P244R/P244R and Fgfr3+/+ mice are shown in Figure 8.

A comparison of heterozygous (Fgfr3P244R/+) and their unaffected littermates (Fgfr3+/+) using EDMA is shown in Figure 9. In this comparison, 16 inter-landmark distance differed between 5 and 10%, and 2 distances differed by more than 10%. The most notable localized difference in morphology indicated by this comparison was a shortening of nasal bone in the heterozygous mice relative to the unaffected mice. This comparison also indicated that fewer inter-landmark distances were significantly different between the heterozygotes and unaffected mice than between the homozygous and unaffected mice. This suggests that morphology may be more severely affected in mice homozygous for the mutation.

Lastly, EDMA was used to compare the heterozygous and homozygous mutant mice

(Figure 10). This comparison revealed 6 inter-landmark distances that differed between 5 and

10%, and only one distance that differed by more than 10% . Most of these differences involve landmarks of the cranial vault; this result is likely to be due to slightly different amount of

27 ossification in the region of the skull between the genotypes and not due to a difference in shape.

Two of these distances indicate shortening of the mid-palate in the homozygous mutants relative to the heterozygotes.

Figure 8. Fgfr3P244R/P244R vs. Unaffected littermates Shape Change .

Figure 8 shows the differences in cranial morphology based on the distances between 3D anatomical landmarks generated using 3D coordinate analyzed using data from EDMA44. Distances marked in blue are statistically significantly larger in the Fgfr3P244R/P244R mutant mice in comparison to the Fgfr3+/+ unaffected littermates. Fuchsia lines indicate distances that are statistically significantly smaller in mutants relative to their unaffected littermates.. Bones have been made partially transparent in order to aid visualization of distances.

Figure 9. Fgfr3P244R/+ vs . Unaffected littermates Shape Change .

Figure 9 shows the differences in cranial morphology based on the distances between 3D anatomical landmarks generated using 3D coordinate data from EDMA44. Distances marked in blue are statistically significantly larger in the heterozygous Fgfr3P244R/+ mutant mice in comparison to the Fgfr3+/+ unaffected mice. Fuchsia lines indicate distances that are significantly smaller in mutants relative to their unaffected littermates. Bones have been made partially transparent in order to aid visualization of distances.

Figure 10. Fgfr3P244R/P244R vs. Fgfr3P244R/+ Shape Change .

Figure 10 shows the differences in cranial morphology based on the distances between 3D anatomical landmarks generated using data from EDMA44. Distances marked in blue are statistically significantly larger in the homozygous Fgfr3P244R/P244R mutant mice in comparison to the Fgfr3P244R/+ heterozygous mice. Fuchsia lines indicate distances that are significantly smaller in homozygotes relative to heterozygotes. Bones have been made partially transparent in order to aid visualization of distances. 29

Chapter 3: Conclusions

Discussion

This study has shown that significant differences in cranial morphology in mice carrying a single copy or two copies of the mutation that causes Muenke syndrome in humans and their unaffected littermates are present at birth. Results indicate that homozygous Fgfr3P244R/P244R mice show a great number of interlandmark distances that are significantly different from the unaffected littermates than do the heterozygous Fgfr3P244R/+ mice; homozygous mice show more severe dysmorphology.

In many ways, the results of this study of newborn mice align with previous studies of adult mice with the Fgfr3 P244R mutation and with the human phenotype for Muenke

Syndrome. This study found that at birth both mutant genotypes, Fgfr3P244R/P244R and

Fgfr3P244R/+ show some degree of shortening of the anterior cranial base with the homozygotes being more severely affected. This matches findings in adult Muenke Syndrome mice described by Nah et. al. (2012) and is a phenomenon that has been reported in 50-70% of human cases6.

Shortening of the anterior cranial base is thought to contribute to midface hypoplasia and a depressed nasal bridge, which are also common elements of the human phenotype of this disease.

It has been proposed that changes in the cranial base are the primary disturbance in craniosynostosis syndromes and actually drive all other cranial manifestations of these diseases, including suture fusion46; however, while changes in the cranial base certainly have broad spanning effects, they may or may not account for the full dysmorphologies observed in craniosynostosis syndromes46. Through the basilar coronal ring, changes in the anterior cranial base have the power to influence the shape of the face and the coronal suture. However, the

30 molecular effects of FGFR3 signaling are so diverse that the cause of dysmorphology is unlikely to emanate from changes in just one area of the head.

Homozygous mutant mice in this sample also showed significant elongation of the palate, a phenomenon which has not been documented in studies of the adult mouse phenotype. In about

71% of humans affected with Muenke Syndrome, highly arched palate is observed47. Cleft palate also occurs, but is rarer in cases of Muenke Syndrome than in other craniosynostosis syndromes.

Mutant mice in this study also showed shorted nasal bones relative to their unaffected littermates as has been observed in the adult mouse models6,34. Adult mice tend to also show twisting of the snout, which was not observed in P0 mice.

While changes in growth patterns are not currently predictable with these data, studies like these which consider both local and global changes in cranial morphology are key to understanding development of the head. Simple studies of changes in molecular mechanisms in highly localized regions cannot hope to explain the full morphogenesis of the head where many localized differences converge to produce phenotype.

Future Directions

The results of this study provide the foundation for a wealth of future research. An important step will be to increase sample sizes. Because the pathological phenotype is so variable in both mice and humans, a greater sample size may be necessary to refine descriptions of patterns in shape.

Further understanding of growth trajectory and the timing of abnormal morphogenesis may be gained by analyzing shape of Fgfr3 P244R mutant mice at more ages to include both embryonic stages and early postnatal stages.

Although the effects of this mutation have been documented in comparison of adult mice carrying these mutations and unaffected adult mice, we do not have a quantitative morphological account of how the mutation affects the growth and development of the head when bred onto different backgrounds. Previous studies have shown highly variable phenotypes of the mutation in different backgrounds of mice, including an apparent lack of phenotypic effect on BALB/c mice34. Applying precise quantitative methods to analysis of a range of ages of several different genetic backgrounds of mice may indicate whether certain backgrounds truly experience no phenotype and when and if developmental differences occur. This analysis will also allow for estimation of growth trajectories for each background and can subsequently be compared to the growth trajectories of other genetic backgrounds to gain an understanding of the way each develops differently. Results of this nature could be useful in understanding the mechanisms of pathogenesis of this disease and could potentially have implications for treatment in humans.

Chapter 4. Supplemental Information

Table 2. Description of the full set of global landmarks taken on mouse crania

Vault rplp Most postero-lateral point on the parietal, left side lplp Most postero-lateral point on the parietal, right side rsqu Most superior point on the squamous temporal, intersection of the coronal suture, right side lsqu Most superior point on the squamous temporal, intersection of the coronal suture, left side rpsq Most posterior point on the posterior extension of the forming squamosal, right side lpsq Most posterior point on the posterior extension of the forming squamosal, left side rpfm Most lateral intersection of the frontal and parietal bones, taken on the parietal, right side lpfm Most lateral intersection of the frontal and parietal bones, taken on the parietal, left side rpto Most postero-medial point on the parietal, right side lpto Most postero-medial point on the parietal, left side rfpi Most medial intersection of the frontal and parietal bones, taken on the frontal, right side lfpi Most medial intersection of the frontal and parietal bones, taken on the frontal, left side intpar Most antero-medial point on the interparietal bone

Face rnsla Most antero-medial point of the nasal bone, right side lnsla Most antero-medial point of the nasal bone, left side rnslp Most postero-medial point of the nasal bone, right side lnslp Most postero-medial point of the nasal bone, left side rnsll Most postero-lateral point of the nasal bone, right side lnsll Most postero-lateral point of the nasal bone, left side riohi most inferior point on the infra-orbital hiatus, right side liohi most inferior point on the infra-orbital hiatus, left side rflac Intersection of the frontal process of the maxilla with the frontal and lacrimal bones, right side lflac Intersection of the frontal process of the maxilla with the frontal and lacrimal bones, left side rzya Intersection of the zygoma with the zygomatic process of the maxillar, taken on the maxilla, right side lzya Intersection of the zygoma with the zygomatic process of the maxillar, taken on the maxilla, left side raalf Most anterior point of the anterior palatine foramen, right side laalf Most anterior point of the anterior palatine foramen, left side

Palate ralp Most antero-lateral point on the posterior palatine plate, right side lalp Most antero-lateral point on the posterior palatine plate, left side rplpp Most postero-lateral point on the posterior palatine plate, right side lplpp Most postero-lateral point on the posterior palatine plate, left side rpns Most antero-lateral indentation at the posterior edge of the palatine pate, right side lpns Most antero-lateral indentation at the posterior edge of the palatine pate, left side

Base rocc Most infero-lateral point on the squamous occipital, right side locc Most infero-lateral point on the squamous occipital, left side cpsh Most anterior point of the indentation in the center of the presphenoid rsyn Most antero-lateral point on the corner of the basioccipital, right side lsyn Most antero-lateral point on the corner of the basioccipital, left side bas Mid-point on the anterior margin of the foramen magnum, taken on the basiooccipital bone rasph Postero-medial point of the inferior portion of the left alisphenoid, right side lasph Postero-medial point of the inferior portion of the left alisphenoid, left side amsph Most antero-medial point on the body of the sphenoid

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Academic Vita

Katherine Rhodes 39 San Souci Drive | South Hadley, MA 01075 | 215-206-0350| [email protected] EDUCATION The Pennsylvania State University at University Park Anticipated Graduation in May of 2015 Schreyer Honors College

Majors Minors Biological Anthropology, B.S. College of Liberal Arts Spanish Biology (Genetics and Development Option) B.S. Eberly College of Science

EXPERIENCE

Research Assistant with Dr. Joan Richtsmeier Fall 2012- Present - Executed Geometric Morphometric analyses of mouse crania in order to understand shape variation in genetic syndromes causing craniosynostosis - Worked in Avizo, R, EDMA, MorphoJ, and Excel

Dmanisi Field School—Dmanisi, Georgia Summer 2012 - Participated in excavation - Learned bone reconstruction and conservation techniques - Attended lectures on Geology, Paleoanthropology, and Taphonomy

Field School For Quaternary Anthropology—Caravaca de la Cruz, Spain Summer 2012, 2014 - Participated in excavation - Classified and sorted finds - Attended lectures about dating techniques, Experimental Archeology, and Geoarcheology

Counselor at El Lago del Bosque)- Bemidji Minnesota Summer 2013 - Supervised campers - Created lesson plans for teaching Spanish vocabulary - Taught Spanish in small language learning groups

Study Abroad- Oxford University, St. Catherine’s College______Spring 2014 - Took tutorials in the following subjects: Traditional Medicine in Ancient and Modern Societies, Forensic Anthropology, History and the Philosophy of Science, Playwriting

Internship with the Malini Foundation______Summer 2014 - Title: EFL Recruitment Intern

- Responsibilities: co-wrote a white paper on teacher recruitment strategies, prepared vocabulary, definitions, and quiz questions for a quiz app, networked with career services at target UK universities.

Reviewing Team: International Journal for Student Research in Archaeology Fall 2014- Present - Current responsibilities: Work with team members to plan the goals and structure of this fledgling academic journal. - Future Responsibilities: Review articles written by students around the world for publication.

VOLUNTEERING

Archive Assistant at the Wisteriahurst Museum—Holyoke, MA Summer 2012 - Compiled inventory sheets - Created folders of information contained in the archives about specific topics (e.g. Mill River Flood 1874) for community use.

Alternative Spring Break, Volunteer Hurricane Relief—Atlantic City,NJ Spring 2013 - Duration: 1 week - Rebuilt homes, cleaned up wreckage

EXTRACURRICULAR ACTIVITIES

 Anthropology Club (Secretary Fall 2012-Spring 2013)  Penn State Fencing Club  No Refund Theater  International Special Living Option: The GLOBE (Fall 2011-Fall 2013)  Stand-up and Sketch Comedy

SCHOLARSHIPS AND HONORS

Paterno Fellow Inducted Fall 2011

Dean’s List All Semesters Summer Discovery Grant Summer 2013 Phi Beta Kappa Award Summer 2013 Science Travel Grant Spring 2014 Schreyer Honors College Travel Grants Spring 2014, Summer 2014 Liberal Arts Enrichment Funding Spring 2014