Characterization of Williams-Beuren Syndrome Mouse Models: Linking Genes with Cognition and Behaviour

Emily Lam

A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto

Emily Lam

Master of Science

Institute of Medical Science University of Toronto

2012 Abstract

Deletion (Williams-Beuren syndrome (WBS)) and duplication (Dup7q11.23) of a common interval spanning 26 genes on chromosome 7q11.23 cause disorders with a spectrum of clinical, cognitive and behavioural symptoms. Studies of individuals with atypical deletions have implicated two genes, GTF2IRD1 and GTF2I. Here I describe the behavioural characterization of mice hemizygous for Gtf2i, or Gtf2ird1 and Gtf2i together, as well as mice with additional Gtf2i copies. Dosage changes in Gtf2i were associated with working memory impairment and separation anxiety, and possibly with general anxiety and repetitive behaviours. A potential cause of these phenotypes was found in brain tissue, where subcellular localization of the calcium channel TRPC3, which is regulated by GTF2I, was found to be altered. Collectively, these results provide a better understanding of the contributions of GTF2I to the cognitive and behavioural profile of WBS and Dup7q11.23 and identify a potential biological mechanism that may underlie some of the symptoms.

Acknowledgements

I would like to extend my deepest gratitude to my supervisor, Dr. Lucy Osborne, for her continuous guidance and support throughout the duration of my graduate studies. Lucy’s bright laughter in the lab is always an encouraging sign that, despite the bad days, there are always good times to look forward to in grad school!

I would also like to thank the members of my program advisory committee, Dr. Howard Mount and Dr. Vincent Tropepe, for their assistance and advice that guided my research toward the right direction. In particular, I am grateful to Howard for providing not only scientific guidance but also for imparting valuable insights about the diverse paths of life.

Current and former colleagues in the lab have made my time in the Osborne lab an absolute pleasure. Ingredients to fun and laughter included late-night working, storytelling, commiserating, and celebrating in the lab with Jen O’Leary, Ted Young, Amy Oh, Elaine Tam, Eli Brimble, Emma Strong, and Joana Dida. I owe my gratitude to Joana for her expertise in ultrasonic vocalizations of mouse pups, and to Elaine for maintaining the animal colony and assisting with the genotyping of test animals. I would also like to thank our collaborators at the Toronto Centre for Phenogenomics as well as in Dr. John Roder’s lab (especially Laleh Sinai) for their assistance. My proficiency in behavioural assays would not have been possible without the help and invaluable advice of Keith Ho and Beverly Francis, both of whom have become treasured friends.

Outside of the lab, I am grateful to the many friendships that have endured from high school and my undergraduate studies, as well as to the new friendships that I have formed during my graduate studies. The times spent with each and every friend have provided countless memories of fun, relaxation, and social support. Aymun Qayume, in particular, has always been there to share the good and the bad, to empathize about the miseries of adulthood, and to laugh and dream together.

The hardships of grad school were made much easier with the unwavering love and support from my parents and grandparents. They encouraged me to always reach higher and persevere, and thus, I am indebted to them for where I am in life today. My sisters, Pamela and Carmen, are both able to elicit a smile from me even on the gloomiest of days, and their company and care ensured that I came home every weekend while I lived downtown. Lastly, the final stretch of my graduate studies was made brighter by Wilson, whose love, support, and comforting reassurance, as well as chocolates and desserts, have conveyed a myriad of possibilities ahead.

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Table of Contents

Table of Contents ...... iii

List of Abbreviations ...... viii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1 Introduction ...... 1

1.1 Williams-Beuren syndrome ...... 1

1.1.1 History of Williams-Beuren syndrome ...... 1

1.1.2 WBS clinical profile ...... 2

1.1.3 WBS cognitive profile ...... 6

1.1.4 WBS behavioural profile ...... 8

1.1.5 Mutational mechanisms in WBS, duplication and triplication of 7q11.23 ...... 9

1.2 Genotype-phenotype correlations in WBS ...... 11

1.2.1 Atypical deletions in WBS: implications for GTF2I and GTF2IRD1 ...... 12

1.2.2 GTF2I family of transcription factors ...... 14

1.3 Duplication and Triplication of 7q11.23 ...... 20

1.4 Previously studied mouse models ...... 24

1.4.1 Gtf2ird1-knockout mice ...... 24

1.4.2 Gtf2i-heterozygous mice ...... 26

1.4.3 Mice with a deletion encompassing Limk1 to Gtf2i...... 27

1.4.4 Other single-gene deletion mouse models of WBS ...... 28

Chapter 2 Behavioural analyses of Gtf2i and Gtf2i/Gtf2ird1 mouse models ...... 35

2.1 Introduction ...... 35

2.1.1 Generation of Mouse Models ...... 35

2.1.2 Research Aims ...... 37

2.1.3 Hypothesis...... 38

2.2 Materials and Methods ...... 38

2.2.1 Expression Analysis ...... 39

2.2.2 Animals ...... 40

2.2.3 Statistical Analyses ...... 40

2.2.4 Grip Strength ...... 41

2.2.5 Rotarod Performance ...... 42

2.2.6 Contextual and Cued Fear Conditioning ...... 42

2.2.7 Morris Water Maze ...... 43

2.2.8 Barnes Maze...... 44

2.2.9 Resident Intruder ...... 45

2.2.10 Novel Object Recognition ...... 45

2.2.11 Elevated Zero Maze ...... 46

2.2.12 Open Field ...... 47

2.2.13 Maternal Separation-Induced Ultrasonic Vocalizations ...... 48

2.3 Results ...... 48

2.3.1 Gene and protein expression levels ...... 48

2.3.2 Gtf2i+/- and Gtf+/del mice exhibit impaired rotarod performance but normal grip strength ...... 52

2.3.3 Cued, but not contextual, fear conditioning altered in Gtf2i+/- and Gtf+/del mice ...... 55

2.3.4 Gtf2i and Gtf2ird1 likely not involved in visuospatial processing ...... 57

2.3.5 Measures of social interaction are not affected by reduced or increased Gtf2i expression ...... 58

2.3.6 Cognitive impairments associated with altered expression of Gtf2i and Gtf2ird1 ...... 59

2.3.7 Haploinsufficiency of Gtf2ird1implicated in anxiety-related behaviours ...... 62

2.3.8 Alteration in non-social anxiety levels due to increased Gtf2i copy number ...... 64

2.4 Discussion and Conclusions ...... 68

2.4.1 Gtf2i+/- and Gtf+/del mice exhibit neuromotor learning deficits ...... 68

2.4.2 Additive effect of Gtf2ird1 and Gtf2i on anxiety...... 69

2.4.3 Gtf2i expression level is implicated in altered anxiety- and ASD-related behaviours ...... 70

2.4.4 Gtf2i and Gtf2ird1 are essential for object recognition memory ...... 72

Chapter 3 Elucidating pathways affected by reduced or increased expression of Gtf2i ...... 75

3.1 Introduction ...... 75

3.1.1 Research Aims ...... 75

3.1.2 Hypothesis...... 75

3.2 Materials and Methods ...... 76

3.2.1 Subcellular fractionations ...... 76

3.2.2 Western blot analyses ...... 77

3.3 Results ...... 77

3.3.1 Alteration in TRPC3 surface expression ...... 77

3.3.2 Localization analyses in SrcThl/Thl mice ...... 79

3.4 Discussion and Conclusions ...... 80

Chapter 4 Conclusions and Future Directions ...... 85

4.1 Summary ...... 85

4.1.1 Overview ...... 85

4.1.2 Haploinsufficiency of Gtf2ird1 and Gtf2i leads to motor learning deficits, altered anxiety-related behaviours and cued fear conditioning response, and impaired object recognition memory ...... 86

4.1.3 Increased expression of Gtf2i leads to elevated anxiety-related behaviours and spontaneous self-grooming ...... 87

4.1.4 Gtf2i gene dosage affects subcellular localization of TRPC3 ...... 88

4.2 Future Directions ...... 89

4.2.1 TRP channel function ...... 89

4.2.2 Relationship of GTF2I to Autism Spectrum Disorders ...... 90

4.2.3 Altered gene expression ...... 93

4.2.4 Anxiety ...... 94

4.2.5 Genetic background effects ...... 95

4.3 Conclusion ...... 97

References ...... 98

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List of Abbreviations

5-HT Serotonin

ADF Actin dephosphorylation factor

ADHD Attention deficit hyperactivity disorder

ASD Autism spectrum disorder

BAP-135 Bruton’s tyrosine kinase-associated protein-135

BAZ1B Bromodomain adjacent to zinc finger domain 1B

BCR B-cell antigen receptor

BDNF Brain-derived neurotrophic factor

Btk Bruton’s tyrosine kinase

CaMKII Ca2+/Calmodulin-dependent protein kinase II cAMP Cyclic adenosine monophosphate

CCAT Calcium channel-associated transcriptional regulator cGMP Cyclic guanosine monophosphate

ChREBP Carbohydrate response element binding protein

CLIP2 CAP-GLY domain-containing linker protein 2

CRE cAMP response element

CREB cAMP response element binding protein

CSK c-Src tyrosine kinase

D/P Deletion of WBS syntenic region on mouse chromosome 5

DD Distal deletion

DNA Deoxyribonucleic acid

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DNS Down syndrome

EGF Epidermal growth factor

EIF4H Eukaryotic initiation factor 4H

ELN Elastin

ER Endoplasmic reticulum

ERK Extracellular signal-regulated kinase

ES cell Embryonic stem cell

FKBP6 FK506 binding protein-6

FZD9 Frizzled-9

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GTF2I General transcription factor 2I

GTF2I/BAP-135/SPIN/TFII-I Alternate GTF2I symbols

GTF2IRD1 General transcription factor 2I repeat domain 1

GTF2IRD1/BEN/CREAM/GTF3 /MusTRD1/WBSCR11 Alternate GTF2IRD1 symbols

IIH Idiopathic infantile hypercalcemia

Inr Initiator

KO Knock-out

LAT2 Linker for activation of T cells-2

LCR Low-copy repeat

LIMK1 Lin-11/Isl-1/Mec-3 kinase

MAPK Mitogen-activated protein kinase

MECP2 Methyl CpG-binding protein 2

ix mGluR Metabotropic glutamate receptor

MI Memory index

MLXIPL MLX-interacting protein-like mRNA Messenger RNA

PD Proximal deletion

PDGF Platelet-derived growth factor

PH Pleckstrin homology

PLC-γ Phospholipase C-γ qPCR Quantitative polymerase chain reaction

RNA Ribonucleic acid

SH2 Src-homology 2

SPIN Serum response factor – Phox1 interacting protein

SRF Serum response factor

STX1A Syntaxin 1A

SVAS Supravalvular aortic stenosis

TRPC3 Transient receptor potential canonical 3

USV Ultrasonic vocalization

WBS Williams-Beuren syndrome

WBSCR Williams-Beuren syndrome critical region

WT Wildtype

List of Tables

Table 1.1 Clinical features of Williams-Beuren syndrome

Table 1.2 Behavioural profile of individuals with WBS

Table 1.3 Speech characteristics of individuals with Dup7q11.23 syndrome

Table 1.4 Behavioural profile of children with Dup7q11.23 syndrome

Table 2.1 Summary of results from current studies and behavioural comparison to previously studied Gtf2i and Gtf2ird1 mouse models

List of Figures

Figure 1.1 Distinctive facial features of individuals with WBS

Figure 1.2 Example of visuospatial construction differences in age- and IQ-matched WBS and Down syndrome (DNS) individuals

Figure 1.3 Regions of low-copy repeats in the WBS region

Figure 1.4 Atypical deletions in WBS individuals that implicate GTF2I and GTF2IRD1 in the cognitive and behavioural profile

Figure 1.5 Structures of the GTF2I gene family

Figure 1.6 Mild facial dysmorphism in individuals with a duplication of 7q11.23

Figure 1.7 Summary of previously studied WBS mouse models

Figure 2.1 Individual mouse lines used for generation of mouse models

Figure 2.2 Validation of gene and protein expression in the various mouse models

Figure 2.3 Grip strength and motor learning in Gtf2i+/- and Gtf+/del mice

Figure 2.4 Freezing responses in contextual and cued fear conditioning

Figure 2.5 Visuospatial learning using the Barnes maze

Figure 2.6 The resident intruder test as an index of social and aggressive behaviour

Figure 2.7 Novel object recognition memory impairments due to hemizygosity of Gtf2i and Gtf2ird1

Figure 2.8 Memory index is differentially affected by extra copies of Gtf2i in male and female mice

Figure 2.9 Gtf2i+/- mice do not show significant alterations in the zero maze test, but Gtf+/del animals exhibit increased exploratory behaviour

Figure 2.10 Increased expression of Gtf2i affects anxiety-related behaviours

Figure 2.11 Effects of Gtf2i copy number on anxiety-related behaviours in the open field

Figure 2.12 Maternal separation in Gtf2i heterozygous and duplication mice

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Figure 3.1 Western blot analyses of TRPC3 expression in the membrane fraction of whole brain

Figure 3.2 Western blot analyses of TFII-I and TRPC3 protein expression in SrcThl/Thl mice

xiii 1

Chapter 1 Introduction

1.1 Williams-Beuren syndrome

1.1.1 History of Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is a neurogenetic disorder that is becoming increasingly

well-characterized through detailed clinical assessment and accurate laboratory diagnoses of

patients. The incidence of WBS is estimated to be between 1/20,000 to 1/7,500 (Stromme et

al., 2002). The history of WBS is an interesting one that first began in the early 1950s with diagnoses of idiopathic infantile hypercalcemia (IIH) and children who failed to thrive in

Great Britain and Switzerland (Lightwood, 1952). This epidemic was thought to have occurred due to excessive vitamin D intake, and with the reduction of vitamin D in infantile diets, the reports of IIH appeared to subside (Jones, 1990). However, a severe form of IIH persisted, which could not be treated by merely dietary changes, and those with the severe form had lower birth weights compared to the milder cases (Fraser et al., 1966).

In 1961, four patients were reported to have supravalvular aortic stenosis (SVAS) as well as distinct facial features and mental “subnormality”(Williams et al., 1961). In the following year, Beuren et al. (1962) also published four other patients with SVAS who had facial features resembling those previously observed by Williams et al. (1961): broad foreheads, full cheeks and lips (Beuren et al., 1962). Moreover, Beuren and his colleagues described

what would be a prominent feature of a diagnosis of WBS: these children were

uncharacteristically friendly – “they love everyone, are loved by everyone, and are very

charming”(Beuren et al., 1962). Three of the four patients also had lower IQ than normal.

Dental anomalies were reported in another group of patients, and together with the mental

and physical disabilities, distinct craniofacial features and SVAS, the possibility of a unique

syndrome was considered (Beuren et al., 1964).

Through linkage studies, isolated SVAS was associated with chromosome 7 (Ewart et al.,

1993b), and it was discovered that affected individuals in a family with SVAS had a

breakpoint in the elastin gene as did one patient with SVAS and features of WBS (Curran et al., 1993) . It was then determined that hemizygous deletion of the elastin gene is present in individuals with WBS, who also commonly show symptoms of SVAS (Ewart et al., 1993a).

Further genetic mapping of affected individuals revealed that WBS is a contiguous gene disorder that is due to a microdeletion of approximately 1.5 megabase (Mb) pairs resulting in the loss of 26 genes on chromosome 7q11.23 (Peoples et al., 2000).

1.1.2 WBS clinical profile

WBS is a unique developmental disorder that is associated with cardiovascular abnormalities

and distinctive facial features (Mervis and Klein-Tasman, 2000). The disorder is often

diagnosed through fluorescence in situ hybridization, which probes metaphase chromosome

spreads for the number of elastin alleles present. People with WBS present with variable

3 symptoms, but together, these symptoms demonstrate that multiple organ systems throughout the body are affected (Table 1.1).

Table 1.1. Clinical features of Williams-Beuren syndrome (Adapted from Morris et al., 1988 and Pober, 2010)

Affected system Symptom Prevalence (%)

Neurodevelopment Developmental delay and cognitive 97

impairment

Attention deficit hyperactivity disorder 84

Anxiety and phobias 80

Impaired visuospatial construction 67

Cardiovascular Any cardiovascular disease 84

Supravalvular aortic stenosis 69

Supravalvular pulmonary stenosis 34

Hypertension 17

Ocular Strabismus 50

Hyperopia 24

Auditory Hyperacusis 90

Dental Malocclusions 85

Microdontia 55

Genitourinary Urinary tract infections 29

Musculoskeletal Kyphosis 21

Scoliosis 12

Gait abnormalities 60

Gastrointestinal Obesity 29

Constipation 43

Endocrine Glucose intolerance or diabetes mellitus 75

Hypercalcemia 5 – 50 reported

Hypothyroidism 15 – 30 reported

Integumentary Mild premature aging of skin and hair 60

Children with WBS are often recognizable due to their characteristic gestalt of elfin-like

craniofacial features that include a flat nasal bridge, short upturned nose, full cheeks, wide

mouth, periorbital fullness, stellate irises, long philtrum and dental malocclusions (Figure

1.1) (Morris et al., 1988). The cardiovascular system is typically affected in WBS, and a

signature of this disorder is SVAS, which is a rare medical condition outside of WBS (Morris

et al., 1988, Pober, 2010). Patients also present with pulmonary artery stenoses, coronary

artery abnormalities as well as isolated cardiovascular lesions (Zalzstein et al., 1991, Eronen

et al., 2002). Defects in the aortic or mitral valve occur in around 10% of patients.

Infantile hypercalcemia is observed in a minority of individuals with WBS, and it is

associated with symptoms such as colic, vomiting, decreased appetite, and constipation or it

may be asymptomatic (Jurado et al., 1996, Sforzini et al., 2002). Impaired glucose tolerance

and diabetes are also commonly noted in patients (Pober et al., 2010).

Figure 1.1. Distinctive facial features of individuals with WBS (Pober, 2010)

Developmental and growth delay are typically observed, both in utero and postnatally along with a premature and shortened pubertal growth spurt (Pankau et al., 1992, Partsch et al.,

1999). Musculoskeletal problems change over development – hypotonia and joint laxity occur in younger children while joint contractures, lordosis and scoliosis are observed in

older individuals (Cherniske et al., 2004, Amenta et al., 2005). Fine motor skills are affected in over 60% of WBS individuals, and hypertonia as well as gait abnormalities are observed in a majority of adults (Chapman et al., 1996). People with WBS have neurological problems that include nystagmus, strabismus, and sensorineural hearing loss (Meyer-Lindenberg et al.,

2006). Hyperacusis, an increased sensitivity to certain sounds such as firecrackers and

vacuum cleaners, has also been observed in a majority of individuals with WBS (Klein et al.,

1990, Smoot et al., 2005).

1.1.3 WBS cognitive profile

People with WBS usually show mild to moderate intellectual disability (average IQ 55 to 60)

with characteristic peaks and valleys in cognitive function. Language capabilities are

relatively preserved with strengths in auditory rote memory (Mervis et al., 2000), however,

receptive and expressive language skills are almost invariably delayed in children with WBS

– the onset of vocabulary and grammar acquisition as well as production and comprehension

of gestures develop noticeably later than normal (Jarrold et al., 1998, Mervis and Klein-

Tasman, 2000). Visuospatial construction skills are significantly impaired in individuals with

WBS and they have difficulties constructing a pattern in its entirety because they are only

able to visualize components of a pattern individually (Mervis and Klein-Tasman, 2000). In a

comparison of age- and IQ-matched WBS and Down syndrome (DNS) children, individuals

with DNS were able to draw objects as a whole, whereas individuals with WBS focused only

on the different parts of the object (Bellugi et al., 1990) (Figure 1.2). Mathematical skills are

also poor, likely due to the affected visuospatial construction. Also in comparison to

7 individuals with DNS, spatial memory, especially spatial working memory, is impaired in individuals with WBS (Wang and Bellugi, 1994, Jarrold et al., 1999).

Figure 1.2. Example of visuospatial construction differences in age- and IQ-matched WBS and Down syndrome (DNS) individuals (Bellugi et al., 1990).

1.1.4 WBS behavioural profile

Increased sociability due to a lack of social boundaries is typical of people with WBS, and they are described as being overfriendly, highly approachable and empathic (Bellugi et al.,

1999, Jones et al., 2000). This sociable personality may be due to a strength in facial recognition and a strong interest in faces (Schultz et al., 2001). However, non-social anxiety and simple phobias are prominent features of WBS, along with obsessions and irritability

(Davies et al., 1998) (Table 1.2). Attention deficit hyperactivity disorder has also been reported in children and adolescents (Pagon et al., 1987).

Table 1.2. Behavioural profile of individuals with WBS (C. Mervis, University of Louisville, Personal communication, Jun. 30, 2011)

Behavioural characteristics Prevalence in children with Population Prevalence (%) WBS (%) Social phobia 1.4 4.5

Specific phobia 61 1.3

ADHD 63 2.8

Separation Anxiety 4.2 2.3 Disorder

1.1.5 Mutational mechanisms in WBS, duplication and triplication of

7q11.23

WBS is caused by the deletion of approximately 1.55 million nucleotides on a single copy of human chromosome 7q11.23, resulting in the loss of over 25 genes. Low-copy repeats

(LCRs) are repetitive sequences that flank the commonly deleted WBS region, also known as the WBS critical region (WBSCR) (reviewed in (Merla et al., 2010). These LCRs occur centromeric, medial, and telomeric to the WBS locus, and each consists of three blocks of repeats – A, B, and C (Figure 1.3). The mechanism through which the WBS deletion and reciprocal duplication occur is non-allelic homologous recombination during meiosis. The common deletion of approximately 1.5 Mb has breakpoints in block B, whereas the breakpoints in the rarer deletion of 1.8 Mb are located in block A. This deletion is due to the high degree of sequence homology between the centromeric and medial A and B blocks

(98.2% and 99.6%, respectively) (Bayes et al., 2003). The deletion can be caused by intrachromatidal, inter- and intrachromosomal exchange, whereas the reciprocal duplication occurs via inter- and intrachromosomal exchange mechanisms only.

Figure 1.3.The three regions of low-copy repeats (c: centromeric, m: medial, t: telomeric) each contain three blocks of repetitive sequences (A, B, and C) (Merla et al. 2010)

1.2 Genotype-phenotype correlations in WBS

Genotype-phenotype correlations in WBS have been difficult due to the large number of

genes in the deletion region that may act in a combinatorial or additive manner. Individuals

with smaller, atypical deletions of the WBS critical region may provide clues to the

contributions of specific genes to the WBS cognitive and behavioural profile. However,

individuals with smaller deletions are very rare, and each has a different-sized deletion as

well as different breakpoints. To date, only approximately 30 individuals with atypical

deletions have been identified (Osborne, 2010). The difficulty in comparing different individuals is also due to the differential diagnoses of these individuals by different physicians, and they do not undergo the same clinical, cognitive, and psychological tests. The ascertainment bias toward identifying individuals with a deletion of ELN and the resulting cardiovascular phenotype means that individuals who have smaller deletions that do not encompass ELN are not identified.

However, a number of genes have been implicated in genotype-phenotype correlations, including LIMK1 and CLIP2. LIMK1 has been implicated in the visuospatial construction deficits observed in people with WBS (Frangiskakis et al., 1996, Wang et al., 1998) but other

reports did not find this weakness in visuospatial cognition (Tassabehji et al., 1996, Gray et al., 2006). The phenotypes of individuals with atypical deletions that leave CLIP2 intact suggest that CLIP2 may contribute to the motor skill and cognitive deficits in WBS

(Hoogenraad et al., 2002, van Hagen et al., 2007).

1.2.1 Atypical deletions in WBS: implications for GTF2I and

GTF2IRD1

The General Transcription Factor (GTF) 2I gene family has been implicated in the behavioural and cognitive aspects through studies of individuals with atypical deletions in the

WBS region (Tassabehji et al., 1999, Hirota et al., 2003, Antonell et al., 2010) (Figure 1.4).

These genes are located at the telomeric end of the common deletion, and when left intact, individuals often show milder phenotypes that do not include the distinctive facial features, intellectual disability, motor deficits, or overfriendly personality that are typical of WBS

(Morris et al., 2003, Ferrero et al., 2010). As a result, GTF2I and GTF2I repeat domain protein 1(GTF2IRD1) have been implicated in the cognitive and behavioural profile of WBS.

These genes encode transcription factors that may regulate the expression of other genes and molecular pathways during development.

Figure 1.4. Individuals with atypical deletions of 7q11.23 that implicate GTF2I and GTF2IRD1 in the cognitive and behavioural profile of WBS (SVAS: supravalvular aortic stenosis)

1.2.2 GTF2I family of transcription factors

The General Transcription Factor (GTF) 2I family consists of three transcription factors:

GTF2I, GTF2IRD1, and GTF2IRD2 (Figure 1.5). GTF2I was the first to be studied, and thus, most is known about this transcription factor. Each of the proteins in this family contains

DNA-binding I-repeat domains that are 90 amino acids long, a putative leucine zipper that may allow for homomeric dimerization, and a nuclear localization signal for entry into the nucleus (Hinsley et al., 2004).

Figure 1.5. Protein structures of the GTF2I gene family

1.2.2.1 GTF2I

General transcription factor 2I (GTF2I) encodes the protein, TFII-I, which is almost identical in cDNA sequence to Bruton’s tyrosine kinase-associated protein-135 (BAP-135) and SPIN.

TFII-I contains six I-repeat domains with helix-loop-helix motifs, which facilitate binding to

DNA sequences as well as protein-protein interactions (Roy et al., 1997). There are four known splice variants of TFII-I, all of which contain the putative nuclear localization signal

(Perez Jurado et al., 1998, Cheriyath and Roy, 2000). TFII-I has an observed molecular weight of 135kDa, and four spliced isoforms have been characterized by Cheriyath and Roy

(2000): α (977 amino acids), β (978 amino acids), Δ (957 amino acids), and γ (998 amino acids). In neuronal cells, the γ-isoform is predominantly expressed. These TFII-I variants can interact with one another in both homomeric and heteromeric manner, and these complexes may facilitate nuclear localization of the transcription factor. It is thought that the different combinations of these isoforms might regulate promoters differently, leading to differential gene expression.

The TFII-I Δ and β isoforms localize in different subcellular compartments – TFII-Iβ is basally in the nucleus while TFII-I Δ is situated in the cytoplasm, and upon activation signals, TFII-Iβ is exported to the cytoplasm whereas TFII-I Δ is imported into the nucleus

(Hakre et al., 2006). TFII-I Δ binds to Erk1/2 in the cytoplasm, and this complex is then translocated to the nucleus when activated by growth factor signalling, thereby transducing downstream signalling pathways.

TFII-I was initially identified as a regulator of transcription at multiple promoter elements, including initiator (Inr) sequences and E box sites (Roy et al., 1991). Bruton’s tyrosine kinase

(Btk) is a cytoplasmic protein tyrosine kinase associated with B-cell antigen receptor (BCR) signalling pathways and in B-cell development (Tsukada et al., 2001). A target of Bruton’s tyrosine kinase (Btk) was identified as BAP-135 (Yang and Desiderio, 1997). BAP-135 basally interacts with Btk in B-cells, and in response to BCR activation, BAP-135 is tyrosine phosphorylated by Btk. In B-cells, activation of Btk phosphorylates TFII-I and allows for the

dissociation of the transcription factor from Btk and subsequent translocation of TFII-I into

the nucleus (Novina et al., 1999). Transcriptional activation or repression is determined in

part by histone modifications, and deacetylation of histones renders DNA less accessible to

gene-specific activators. TFII-I has been reported to physically interact with histone

deacetylase-3 (HDAC3), and thus may also be involved in regulating transcription via an

HDAC3-associated mechanism (Wen et al., 2003).

Yang et al. (1997) showed that TFII-I binds to Btk via their pleckstrin homology (PH) and

Tec homology (TH) domains. SPIN (Serum response factor – Phox1 Interacting Protein), identical in sequence to TFII-I, was found to form a complex with serum response factor

(SRF) as well as the homeodomain, Phox1. c-fos is an immediate-early gene that is activated rapidly and transiently following cellular stimuli, and the TFII-I–SRF-Phox1 complex contributes to regulating c-fos expression (Curran et al., 1985, Ceccatelli et al., 1989,

Grueneberg et al., 1997). TFII-I is activated in response to a variety of stimuli, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and serum, extracellular signals that stimulate the c-fos promoter (Kim et al., 1998). c-fos is also

regulated by nitric oxide and cGMP as well as cGMP-dependent protein kinase (G kinase),

and TFII-I has been identified as a serine phosphorylation target of G kinase (Casteel et al.,

2002). cGMP causes the nuclear translocation of G kinase, and in the nucleus, TFII-I binds

to G kinase Iβ through its N-terminal leucine zipper and activates transcription of c-fos.

TFII-I has been shown to interact with extracellular signal-regulated kinase (ERK), a

mitogen-activated protein kinase (MAPK), and mediates its downstream effects through the

Ras/ERK and RhoA signalling pathways (Kim and Cochran, 2000). TFII-I binds to ERK via

the D box, a consensus binding motif, and following serine phosphorylation by ERK, TFII-I

activates the c-fos promoter. TFII-I also plays a regulatory role in the endoplasmic reticulum

(ER) stress response (Parker et al., 2001). In response to ER stress, there is increased binding

of TFII-I to the promoter of Grp78, a stress-induced chaperone, with a concomitant increase

in Grp78 expression level. Interestingly, c-Src is also activated by ER stress and enhances

tyrosine phosphorylation of TFII-I, providing a mechanism for the involvement of TFII-I in

the ER stress response (Hong et al., 2005).

1.2.2.1.1 Roles of TFII-I in intracellular Ca2+ signalling

Although TFII-I is known for its involvement in the nucleus as a regulator of transcription, it

is also highly abundant in the dendrites of cerebellar Purkinje cells (Danoff et al., 2004).

Further evidence suggests that TFII-I not only functions as a transcription factor, but may also have a distinct cytosolic role in inhibiting agonist-induced calcium entry (Caraveo et al.,

2006). In the cytosol, TFII-I is phosphorylated by phospholipase C-γ (PLC-γ) through its

interaction with the SH2 (Src-homology 2) domain of PLC-γ. The pleckstrin homology (PH) domain on PLC-γ has been found to bind to TRPC3, a member of the transient receptor potential canonical (TRPC) channel subfamily (van Rossum et al., 2005). PLC-γ-dependent activation of TRPC3 mediates increases in intracellular Ca2+ and is likely involved in the

proliferation and regulation of neuronal stem cells (Wu et al., 2004). TRPC3 is also required

for dendritic spine formation via mediating brain-derived neurotrophic factor (BDNF)-

induced signalling pathways, and these signalling cascades are coupled to PLC-γ (Amaral

and Pozzo-Miller, 2007). It was proposed that, through binding to TrkB receptors, BDNF

increases hippocampal spine density and mobilizes Ca2+ from intracellular stores to promote

changes in neuronal morphology. Since TFII-I is able to sequester PLC-γ from binding to

TRPC3, TFII-I may play an indirect role in regulating TRPC3 membrane localization

(Caraveo et al., 2006). TFII-I is also a phosphorylation target of c-Src, a tyrosine kinase involved in cellular proliferation, differentiation and signalling (Bromann et al., 2004).

Tyrosine phosphorylation of TFII-I by c-Src is required for the nuclear translocation and transcriptional activation of TFII-I (Cheriyath et al., 2002). Interestingly, Src may also have an obligatory role in activation of TRPC3 and perhaps in indirectly mediating Ca2+ entry

pathways (Vazquez et al., 2004).

1.2.2.2 GTF2IRD1

General transcription factor 2I repeat-domain-containing 1 (GTF2IRD1, also known as

MusTRD1, BEN, WBSCR11, CREAM, GTF3) was first identified as MusTRD1, a nuclear

protein that is expressed in skeletal muscle and binds to the human troponin I slow (TnIs)

upstream enhancer B1 in slow-muscle fibres (O'Mahoney et al., 1998). Due to alternative

splicing, 11 isoforms of Gtf2ird1 have been isolated from mouse skeletal muscle (Tay et al.,

2003). In cell culture, the hMusTRD1α1 isoform repressed activation of TnIs by MEF2C, which is a regulator of slow-muscle fibre gene expression (Polly et al., 2003). Subsequently, the gene was found to be one of the genes deleted in the WBS critical region with sequence homology to GTF2I, and was named WBSCR11 (Osborne et al., 1999). GTF2IRD1 was then identified as BEN (binding factor for early enhancer), and similar to TFII-I, it contains six helix-loop-helix domains as well as a leucine zipper-like motif (Bayarsaihan and Ruddle,

2000).

In vitro evidence suggests that GTF2IRD1 is a regulator of both Xenopus and mouse

Goosecoid (Gsc), a homeobox-containing transcription factor that mediates cell differentiation patterning in embryos (Ku et al., 2005). This may be due to reciprocal regulation of the Gsc promoter by GTF2IRD1 and TFII-I, and it has been postulated that these two proteins may interact through their mutual nuclear exclusion of the other (Tussie-

Luna et al., 2001). GTF2IRD1 has also been found to bind to a highly conserved region of its own promoter, and a negative autoregulation mechanism likely explains the higher-than- normal levels of Gtf2ird1 transcript in Gtf2ird1-knock-out mice where part of the gene has been removed (Palmer et al., 2010).

1.3 Duplication and Triplication of 7q11.23

Individuals with a duplication of the WBS critical region (Dup7q11.23) have recently been identified. These individuals often exhibit speech and expressive language delay as well as anxiety and behavioural problems (Somerville et al., 2005, Berg et al., 2007, Osborne and

Mervis, 2007) (Table 1.3). Some children with Dup7q11.23 syndrome learn to develop nonverbal signs and gestures as a means of communication (Berg et al., 2007). Dup7q11.23 individuals also have a relative strength in visuospatial construction. These characteristics are in direct contrast to those of WBS individuals who have strengths in language capabilities but significant impairments in visuospatial cognition (Mervis and Klein-Tasman, 2000).

Table 1.3. Speech characteristics of individuals with Dup7q11.23 syndrome (C. Mervis, University of Louisville, Personal communication, Jun. 30, 2011)

Characteristic Toddler (1.5-3.8 School-age (4.2-14.7 Adult (28-61 yrs) yrs) yrs) Oral Apraxia 70% 61% 43%

Childhood Apraxia of 80% 83% 86% Speech Dysarthria 80% 61% 86%

Phonological disorder 20% 83% 71%

Facial features in these individuals show mild dysmorphism including thin lips, a high and

broad nose, short philtrum, and high palate (Figure 1.6) (Somerville et al., 2005). Some individuals with Dup7q11.23 have been described as having intellectual disability, attention deficit-hyperactivity disorder (ADHD) and a general developmental delay (Van der Aa et al.,

2009). Hypotonia, autism spectrum disorder (ASD), as well as epilepsy have also been reported in patients (Berg et al., 2007, Torniero et al., 2008). Children with a duplication of

7q11.23 often have poor eye contact and show deficits in social interaction (Van der Aa et al., 2009). In a recent study of 1124 autism-spectrum disorder families from the Simons

Simplex Collection (Fischbach and Lord, 2010), a significant association of ASD with de novo duplications of 7q11.23 was found (Sanders et al., 2011). The clinical profile of

Dup7q11.23 appears to be more variable and not as well defined compared to WBS. A triplication of the 7q11.23 WBS region has also been reported in a single individual with similar, but more severe symptoms than those with Dup7q11.23, including severe developmental, language and speech delay, autistic behaviour, and mild dysmorphic facial features (Beunders et al., 2010).

Figure 1.6. Mild facial dysmorphism in individuals with a duplication of 7q11.23

Table 1.4. Behavioural profile of children with Dup7q11.23 syndrome (Adapted from Van der Aa et al., 2009 and C. Mervis, University of Louisville, Personal communication, Jun. 30, 2011)

Characteristics Prevalence in children with Population Prevalence (%) Dup7q11.23 syndrome (%) Social phobia 37 4.5

Specific phobia 74 1.3

Selective mutism 16 -

ADHD 58 2.8

Separation Anxiety 26.3 2.3 Disorder Generalized Anxiety 10.5 3.1 Disorder Any anxiety disorder 79 9.8

Autism Spectrum Disorder <50 1

1.4 Previously studied mouse models

WBS clinical studies are limited due to diagnoses by different physicians, the relatively small number of patients identified with atypical deletions, as well as ascertainment biases toward individuals with deletions encompassing ELN. Thus, it is difficult to correlate genotype to phenotype in these patients. However, genetically altered mouse models of WBS provide clues toward the function of specific genes in the deletion region, and because they can be bred on specific strain backgrounds and compared to control littermates, genetic background differences can be accounted for. Dissecting the contributions of individual genes implicated in WBS has also been made possible due to a region on mouse chromosome 5G that is syntenic, albeit inverted, to the WBS deletion region (Valero et al., 2000). The following are characterizations of mouse models with either deletions of single genes or larger partial deletions in the WBS region.

1.4.1 Gtf2ird1-knockout mice

Several Gtf2ird1-/- mouse models have previously been generated, and behavioural as well as

molecular studies have been reported. Durkin et al. (2001) generated a viable and fertile

transgenic mouse in which the transcription start site and exon 1 of Gtf2ird1 were deleted.

Homozygous knockout mice exhibited growth delay and craniofacial abnormalities such as misalignment of the jaw, a shorter snout than wildtype controls, and periorbital fullness

(Durkin et al., 2001, Tassabehji et al., 2005). However, heterozygous transgenic mice did not

appear to have altered growth development and craniofacial features (Tassabehji et al.,

2005). This suggests that hemizygosity of Gtf2ird1 in mice may not alter the same molecular

25 pathways or to the same extent in human patients, or that other genes in addition or combination with Gtf2ird1 produce the typical WBS phenotype.

Another mouse model was produced by inserting a LacZ cassette into exon 2 of Gtf2ird1

(Palmer et al., 2007). Expression analyses in these mice during adulthood showed highest levels of Gtf2ird1 in the central and peripheral nervous system including nerves of the retina, olfactory epithelium, and cochlea. These mice did not show altered craniofacial features, but instead showed behavioural and neurological deficits (Palmer et al., 2007). Recent analysis of this mouse revealed a growth deficit, epidermal hyperplasia in the nose and lip region as well as decreased fat tissue weight in female animals (Howard et al., 2011). Motor deficits were observed using an accelerating rotarod and an inverted cage lid test as a measure of grip strength. Differential exploratory activity was observed in these mice – males were significantly more active than wildtype animals, whereas females were significantly less active. These Gtf2ird1-knockout mice generated more ultrasonic vocalizations during a swim stress test than their wildtype littermates. However, Gtf2ird1-knockout female mouse pups made shorter, fewer, and different types of vocalizations during maternal separation.

Following the swim stress, c-fos expression, which is indicative of neuronal activity, was increased in regions of the brain implicated in mammalian vocalization, such as the cingulate cortex and ventral region of the lateral septum. This suggests a possible dysfunction in these brain regions with altered neuronal activation (Howard et al., 2011).

A third mouse model was generated by a targeted knockout of Gtf2ird1 exons 2 to 5, and these mice showed decreased aggression and anxiety as well as increased social interactions.

(Young et al., 2008). Both Gtf2ird1 heterozygous and homozygous mice exhibited decreased

fear in an amygdala-dependent cued fear conditioning test. These phenotypes were correlated

with increased levels of the serotonin (5-HT) metabolite, 5-hydroxyindoleacetic acid, in the

amygdala as well as frontal and parietal cortices. This suggests altered serotonergic

transmission in these mice, which was further confirmed by enhanced inhibitory 5-HT

currents mediated by the 5-HT1A receptor in layer V pyramidal neurons of the prefrontal

cortex (Proulx et al., 2010).

Enkhmandakh et al. (2009) generated Gtf2ird1 gene-trap mice carrying a LacZ-neomycin insertion in Gtf2ird1. However, unlike the other mouse models, this Gtf2ird1 mutant mouse had severe phenotypes, and homozygous mice were embryonically lethal (Enkhmandakh et al., 2009). The phenotypic discrepancies may be explained by the technique used to generate these animals – the gene trap insertion was located in intron 22 of Gtf2ird1, thereby leaving most of the protein intact and potentially available for interactions with its targets. The

Gtf2ird1-LacZ fusion protein would also lack the nuclear localization signal, leading to an altered localization of any Gtf2ird1 protein that was expressed.

1.4.2 Gtf2i-heterozygous mice

In addition to the Gtf2ird1 mutant mice, Enkhmandakh et al. (2009) also generated a Gtf2i

gene-trap mouse model with a gene-trap cassette inserted into intron 3 of Gtf2i

(Enkhmandakh et al., 2009). Similar to their Gtf2ird1-/- mouse, Gtf2i-null mice did not

survive past E10.5, and embryonic hemorrhage and cardiovascular malformations were

discerned at E9.5. Neural tube defects were observed in 60% of homozygote embryos, and

growth delay was apparent in the heterozygote mice, which were viable. A similar mouse

model was generated by Sakurai et al. (2010) in which a gene trap cassette was inserted

downstream of exon 3. Mating of heterozygous mice did not produce any homozygous

animals and, as Enkhmandakh et al. (2009) had observed, Gtf2i-null embryos were

exencephalic (Sakurai et al., 2010). In contrast, the latter study did not find developmental

and cardiovascular abnormalities that the previous group reported. In a battery of behavioural

assays, Gtf2i+/- animals did not show any alterations in spatial and non-spatial learning and

memory, anxiety and neuromotor function. However, a lack of habituation to social stimuli

(novel mouse) was observed in heterozygous mice (Sakurai et al., 2010).

1.4.3 Mice with a deletion encompassing Limk1 to Gtf2i

Although single-gene deletion mouse models may enable elucidation of the single gene

function, the contiguous gene deletion in WBS suggests that the combinatorial or additive

effects of these gene deletions are equally important. Recently, partial deletion mouse

models were generated in which Limk1 to Gtf2i was deleted in proximal deletion (PD) mice,

Limk1 to Fkbp6 was deleted in distal deletion mice (DD), and both partial deletions were

used to generate D/P animals (Li et al., 2009). Effectively, due to the locations of the loxP

sites used to generate the deletion mice, D/P mice are homozygously null for Limk1. PD mice

exhibited growth delay along with hernias and rectal prolapse. Only female PD animals showed reduced brain weight, but overall, both males and females displayed a decrease in lateral ventricle volume with increased neuronal density in the somatosensory cortex. There was a significant increase in social interest and interaction in PD mice, but they appeared to have increased anxiety in the open field test. The PD genes also contribute to the motor

coordination deficit as elucidated by the rotarod test. Increased sensitivity to sound

compared to controls was observed in PD mice using the baseline startle response, and these

animals also exhibited attenuated pre-pulse inhibition.

1.4.4 Other single-gene deletion mouse models of WBS

Eln

Elastin (ELN) is the only gene in the WBS deletion region that has been unequivocally linked

to a phenotype in WBS, namely the supravalvular aortic stenosis (Curran et al., 1993, Ewart

et al., 1993b). In mice with a null deletion of Eln, a progressive decrease in aortic diameter

has been observed alongside thickening of the arterial wall due to proliferation of smooth muscle cells (Li et al., 1998a). Eln+/- mice also showed this increase in smooth muscle during

development of the arterial wall with a 25-35% increase in the quantity of elastic lamellar

rings (Li et al., 1998b). The elastin lamellae in Eln+/- mice were also thinner than in wildtype

controls. However, in mice with partial deletions of the WBS region (DD and D/P)

(described in Section 1.4.3), the number of lamellae in the aorta did not appear to be altered

(Goergen et al., 2011). This suggests that other genes in the deletion region may play a developmental role in the vasculature. Hypertension as well as disorganized and fragmented elastin sheets have also been observed in both DD and Eln+/- mice.

Limk1

LIM (Lin-11/Isl-1/Mec-3) kinases (LIMK) are serine kinases that regulate actin dynamics by phosphorylating and inactivating actin dephosphorylation factor (ADF) and cofilin (Kuhn et al., 2000, Hoogenraad et al., 2004). The turnover of actin filaments is regulated by

ADF/cofilin, which depolymerize actin polymers and sequester actin monomers. LIM kinase-

1 (LIMK1) has been implicated in the visuospatial construction deficits observed in individuals with atypical 7q11.23 deletions (Frangiskakis et al., 1996). A Limk1-knockout mouse model showed no changes in gross brain morphology, however neuronal growth cone size was significantly smaller with anomalous accumulation of cofilin and actin (Meng et al.,

2002). Dendritic spine shape abnormalities were observed in Limk1-KO pyramidal neurons

relative to wildtypes while spine density and length remained unaltered. Hippocampal long- term potentiation was enhanced in these mice and they exhibited an increased fear response in the cued-conditioning test of associative learning as well as impairment in the spatial water maze task. These behavioural abnormalities suggest that Limk1 plays a role in mediating synaptic plasticity. Increased exploratory behaviour, including locomotion and rearing, was observed in null mice compared to wildtype controls.

Clip2

CAP-GLY domain-containing linker protein 2 (CLIP2; also known as CLIP-115 and

CYLN2) regulates microtubule dynamics and facilitates binding of microtubules to other cellular structures (Hoogenraad et al., 2002). Hoogenraad et al. (2002) generated heterozygous and homozygous-null Clip2 mice, and these animals exhibited mild growth deficits that began during postnatal development. The ventricle volume of Clip2-/- was

significantly larger than that of wildtypes, although the size of the corpus callosum in both

Clip2+/- and Clip2-/- was smaller than controls. Both heterozygous and homozygous knockout animals showed impaired performance relative to wildtypes on the accelerating rotarod, a measure of motor coordination dependent upon cerebellar function. As evidenced by decreased fear in contextual fear conditioning and significantly smaller evoked post-synaptic potentials in hippocampal slices, hippocampal synaptic plasticity also appeared to be altered.

Stx1a

Syntaxin 1A (STX1A) is a plasma protein believed to be associated with the soluble N- ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) and is highly expressed in neurons (Fujiwara et al., 2006). STX1A is implicated in regulating neurotransmission via presynaptic calcium-induced exocytosis. Stx1a heterozygous and knockout mice do not appear to have altered hippocampal or cerebral cortical structures and have normal spatial memory (Fujiwara et al., 2006). However, these animals may have deficits in contextual and cued fear conditioning, which are indices of hippocampal and amygdala function, respectively. These findings may be explained by the in vivo long-term potentiation impairments observed in hippocampal slices of the mutant mice. Spontaneous locomotor activity and anxiety in Stx1a+/- and Stx1a-/- mice are similar to that of wildtype

littermates. Interestingly, McRory et al. (2008) found that homozygous animals appeared to

die in utero, suggesting that Stx1a is required for embryonic development and that lack of

Stx1a is embryonically lethal. The heterozygous mice as well as the few viable knockout

animals in this study showed reduced body weight, but did not exhibit any behavioural

abnormalities (McRory et al., 2008). The differences between the two studies can be

reconciled by the techniques used to generate the mice – Fujiwara et al. (2006) generated a

truncated version of Stx1a that might be functional with a structure similar to that of syntaxin

1C.

Baz1b

BAZ1B (Bromodomain adjacent to zinc finger domain 1B; also known as Williams

syndrome transcription factor) is a subunit of two different chromatin remodelling complexes

involved in DNA repair and transcriptional regulation (Yoshimura et al., 2009). Ashe et al.

(2008) showed that Baz1b is most abundant in the facial prominences during embryogenesis.

In mice with a mutation in Baz1b, craniofacial abnormalities were observed, including

shorter skulls, decreased parietal and nasal bone length, mandibular hypoplasia (Ashe et al.,

2008). These features were milder in heterozygote than in homozygote mice. Baz1b-deficient

mice died shortly after birth, and both Baz1b+/- and Baz1b-/- embryos and neonates exhibited

cardiac abnormalities including atrial and ventricular septal defects, ventricular hypertrophy,

as well as coarctation of the aorta (Yoshimura et al., 2009). These abnormalitites were observed at a similar frequency as they are found in individuals with WBS.

Fzd9

The Frizzled (Fzd) family of cell surface receptors is a component of Wnt receptors and are involved in the Wnt signalling pathways, which regulate vertebrate development (Ranheim et

al., 2005, Zhao and Pleasure, 2005). Frizzled9 (Fzd9) is first expressed in the medial cortical wall, followed by expression in the hippocampus and dentate gyrus during later embryonic development (Zhao and Pleasure, 2005). Fzd9-knockout mice showed splenomegaly, atrophy

of the thymus gland, enlarged lymph nodes, as well as a significant reduction in B-cell

precursors (Ranheim et al., 2005). Bone formation was also decreased in Fzd9-deficient mice

and resulted in low bone mass relative to wildtype controls, thereby suggesting a role for Fzd

in regulating osteoblast function (Albers et al., 2011). Increased apoptosis in the dentate

gyrus and alterations to hippocampal structure were observed in mice heterozygous and

homozygously null for Fzd9 (Zhao et al., 2005). Both Fzd9+/- and Fzd9-/- mice exhibited a

decreased latency to seizure in response to chemoconvulsants, suggesting impaired hippocampal circuitry. This is supported by the impaired performance of these mice in the

Morris water maze, which provides a measure of spatial learning and memory.

Mlxipl

MLXIPL (MLX-interacting protein-like; also known as WBSCR14) is a carbohydrate response element binding protein (ChREBP) that is activated by glucose in the liver and regulates de novo lipogenesis and glycolysis (Iizuka and Horikawa, 2008). Mlxipl is expressed ubiquitously, but most highly in the liver, brown and white adipose tissue, as well as in skeletal muscle (Iizuka et al., 2004). The Mlxipl-KO mice generated by Iizuka et al.

(2004) appear to have decreased mRNA expression of glycolytic and lipogenic enzymes in the liver, which resulted in a reduction in fatty acid synthesis. Mlxipl deficiency also led to intolerance to simple sugars such as fructose and glucose, and this dysregulation of glucose metabolism contributed to the accumulation of glycogen in the liver.

Fkbp6

FK506 binding protein 6 (Fkbp6) is involved in meiosis during homologous chromosome

synapsis, and deficiency in this protein specifically affects meiosis in male mice (Crackower

et al., 2003). Female mice lacking Fkbp6 were normal and fertile, but male mice were sterile

with reduced male gonad size. Fkbp6-/- males were unable to produce spermatids and

spermatozoa due to impaired spermatogenesis.

Lat2

LAT2 (Linker for activation of T cells 2) is a transmembrane adaptor protein that is

expressed mostly in spleen and hematopoietic cells (Janssen et al., 2004, Iwaki et al., 2007).

This scaffolding molecule has been implicated in negatively regulating mast cell signalling,

as evidenced by the augmented anaphylactic responses observed in Lat2-knockout mice

(Volna et al., 2004, Zhu et al., 2004).

Eif4h

Eukaryotic initiation factor 4h (EIF4H, also known as WBSCR1) encodes a protein involved in regulating protein synthesis through initiating translation (Richter et al., 1999). Eif4h- homozygous knockout mice exhibit growth retardation as well as a decrease in neuronal cell count as well as brain volume. Associative learning and memory deficits in fear conditioning were also observed in these mice (Capossela et al., 2012).

34 Mouse chromosome 5G

Figure 1.7. Summary of previously studied WBS mouse models

Chapter 2

Behavioural analyses of Gtf2i and Gtf2i/Gtf2ird1 mouse models

2.1 Introduction

2.1.1 Generation of Mouse Models

A newly generated Gtf2i+/- mouse model and a combined-Gtf2ird1/Gtf2i-deletion (Gtf+/del)

mouse model were studied to examine the phenotypic effects of hemizygosity for Gtf2i and

Gtf2ird1, either alone or in combination. Two embryonic stem (ES) cell clones on a 129SvEv

background were identified from the International Gene Trap Consortium. Gtf2i+/- mice were generated using the YTA365 mouse line with a gene trap cassette inserted into intron 3 of the

Gtf2i gene, which disrupts the endogenous Gtf2i transcript (Figure 2.1A). This also generates a Gtf2i-LacZ fusion transcript that can be used to study expression from the trapped locus.

The Gtf+/del mice were generated by in vivo Cre-loxP recombination between targeted sites in two individual mouse lines (XS0608 and G10-targeted line) (Figure 2.1B). The gene trap

XS0608 ES cell clones contain a gene trap cassette inserted into intron 4 of Gtf2ird1, and the

G10-derived mouse line contains a loxP site downstream of the last exon of Gtf2i along with the Cre transgene driven by the Sycp1 promoter. The ES cell clones were injected into

C57BL6 blastocysts, which were implanted into pseudopregnant CD1 albino females. Germ

line transmission in resulting offspring was identified by the presence of dark-coloured fur

and confirmed by clone-specific PCR. Mice carrying these two insertions were crossed, and a

Cre transgene introduced by mating with Sycp1 mice (Herault et al., 1998). The resulting

“trans-loxer” males, carrying both genetrap insertions in trans, along with a Cre transgene

expressed during meiosis, were mated with wildtype females. Recombination between the

respective loxP sites during male meiosis resulted in deletion of the interval between them,

thus generating a deletion of 220 kb spanning Gtf2i and Gtf2ird1 (Figure 2.1C). The in vivo

Cre-loxP recombination technique used to generate mice with a deletion also resulted in a

reciprocal duplication product, where Gtf2i was duplicated intact. Heterozygous mice with

the Gtf2i duplication (Gtf2i+/dup) were crossed together to produce homozygous mice with

four copies of Gtf2i (Gtf2idup/dup). Mice used in the following studies were on a mixed

129SvEv/C57Bl/6/CD1 background.

Figure 2.1. Individual mouse lines used for generation of mouse models. A) The gene trap YTA365 ES cell line contained a gene trap cassette inserted into intron 3 of Gtf2i and was used to generate Gtf2i+/- mice. B) To generate the Gtf+/del mice, the XS0608 and G10-targeted (with a loxP site downstream of Gtf2i) mouse lines were used. C) A schematic of the resultant deletion of Gtf2i andGtf2ird1 as well as the reciprocal duplication.

2.1.2 Research Aims

To uncover genotype-to-phenotype correlations related to WBS, single-gene-deletion

Gtf2ird1 and Gtf2i mouse models were used to determine whether the neurological

symptoms of WBS could be recapitulated. Using behavioural studies and molecular

approaches, a mouse model hemizygous for the general transcription factor gene, Gtf2i

(Gtf2i+/-) was characterized, as well as mice with the combined hemizygous deletion of both

Gtf2ird1 and Gtf2i (Gtf+/del). People with a duplication or triplication of the WBS region have also been reported. Thus, to investigate whether or not there is an increased gene dosage

effect of Gtf2i, mice with one and two extra copies of Gtf2i (Gtf2i+/dup and Gtf2idup/dup, respectively) were studied using behavioural and molecular approaches.

2.1.3 Hypothesis

The General Transcription Factor (GTF) 2I gene family has been implicated in the

behavioural and cognitive aspects of WBS through studies of individuals with atypical

deletions of the WBS region. These genes encode transcription factors that may regulate the

expression of other genes during development. We previously generated Gtf2ird1-/- mice and

showed that they had behavioural features similar to the increased sociability and lack of

inhibition seen in people with WBS. We hypothesize that the neurological features of WBS

may be associated with the GTF2I genes. In particular, that alterations in learning and

memory, social behaviour and fear responses can be linked to deletions of GTF2IRD1 and

GTF2I, either alone or in combination. Mice with extra copies of Gtf2i have also been

generated, and we hypothesize that the affective phenotypes observed in these animals may

recapitulate some of the symptoms reported in people with a duplication of 7q11.23

(Dup7q11.23).

2.2 Materials and Methods

Contributions: Along with Wyanne Law, a summer undergraduate student, I performed

analyses of gene and protein expression levels. The Morris water maze, fear conditioning,

grip strength, and resident intruder tests were carried out by Igor Vukobradovic at the

Toronto Centre for Phenogenomics. I carried out the rotarod task, Barnes maze, novel object

recognition, elevated zero maze, and open field assays. The maternal separation-induced

ultrasonic vocalizations were carried out by Joana Dida. I performed genotyping of test

animals and statistical analyses of behavioural data with the exception of ultrasonic

vocalization data.

2.2.1 Expression Analysis

Adult mice were sacrificed by cervical dislocation, and brains were removed immediately.

One hemisphere of the brain was homogenized for approximately 20 seconds in Tri-Reagent

(Sigma-aldrich, Oakville, ON). Samples were treated with DNase (Turbo DNA-free,

Ambion), and 5μg of RNA was used to synthesize cDNA using SuperScript™ II Reverse

Transcriptase (Invitrogen Canada Inc., Burlington, ON). Samples were diluted 1/100 and were used for real-time quantitative PCR using Power SYBR Green PCR Master Mix and the

Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City,

CA). Gene expression was normalized to internal controls, Sdha and Hmbs. These values were then pooled and compared to normalized gene expression levels in wildtype (WT) littermate controls.

For protein analysis, the other brain hemisphere was homogenized in lysis buffer (10mM

TRIS-HCl 8, 100mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,1mM EDTA) with a protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO) and phenylmethylsulfonyl fluoride. For western blotting, 50 μg of protein was separated by SDS-

PAGE on 10% acrylamide gels. Gels were transferred onto 0.2μm nitrocellulose membranes

(Pall, Port Washington, NY) followed by Ponceau S staining of membranes to ensure equal

loading and efficiency of the transfer. Membranes were then blocked in 5% non-fat skim

milk powder in TBS with 0.5% Tween-20 (TBST) for two hours at room temperature.

Following blocking, membranes were incubated in anti-TFII-I mouse monoclonal antibody

(610943, BD Biosciences, Franklin Lakes, NJ) in blocking buffer overnight at 4°C.

Membranes were washed with TBST and then incubated in horseradish peroxidase-

conjugated rabbit anti-mouse secondary antibody (ab6728, Abcam, Cambridge, UK) for 1

hour at room temperature. Membranes were washed again and incubated in enhanced

chemiluminescence reagents to detect the presence of protein by Bioflex Scientific Imaging

film (CLMS810, Clonex Corporation, Markham, ON). The band densities of TFII-I were

normalized against loading control, GAPDH (ab36840, Abcam, Cambridge, UK). Image J

(NIH) was used to quantify the relative band densities.

2.2.2 Animals

Mice were maintained on a 129SvEv/C57BL6/CD1 background. Standard rodent chow and

water were available to mice ad libitum and animals were socially housed. Unless otherwise

specified, adult mice were tested between 3 and 6 months of age. Behavioural experiments

were conducted during the light cycle between 0900 and 1700h.

2.2.3 Statistical Analyses

Data are expressed as means ± SEM and were analyzed by GraphPad Prism (San Diego, CA,

USA). To assess for differences among means, a one-way analysis of variance (ANOVA)

was carried out for grip strength, contextual and cued fear conditioning, resident intruder,

novel object recognition memory, elevated zero maze, and open field test. Where p<0.05 using one-way ANOVA, Student’s t-test was used as a post-hoc test. Morris water maze,

Barnes maze, and rotarod performance were analyzed using two-way ANOVA, and if p<0.05, the Bonferroni test was used in post-hoc analyses. A trend was defined for comparisons having a p value between 0.05 and 0.10. Where no differences between male and female animals were found, data for both genders were pooled.

2.2.4 Grip Strength

Neuromuscular strength was examined using a force transducer grid with which forelimb and

combined forelimb and hindlimb grip strength were measured. To determine forelimb

strength, mice were lowered over the grid and only its forelimbs were allowed to grasp onto

the top of the grid. The maximal grip strength value was recorded when the animal released

the grid. Combined forelimb and hindlimb grip strength was measured by lowering mice over

the grid and both forelimbs and hindlimbs were allowed to grip the grid. Forelimb and

combined limb recordings were repeated four times for each mouse. This testing was carried

out at the Toronto Centre for Phenogenomics.

2.2.5 Rotarod Performance

Sensorimotor learning using the rotarod requires intact cerebellar function (Nolan et al.,

2003). Postural motor balance and coordination (excluding muscle strength) were examined

with a Columbus Instruments rotarod at a constant speed of 12 rpm for 4 trials per day over 5

consecutive days. Mice were placed onto the rotating drum, and latency to fall off of the

drum was measured during a 3-minute trial session, and each trial was spaced at 20-minute

intervals. Mice that remained on the rotarod for the entire trial were scored with a latency of

180 sec.

2.2.6 Contextual and Cued Fear Conditioning

Fear-based learning has been extensively researched, especially through the use and

application of the Pavlovian fear conditioning paradigm. This paradigm allows for

elucidation of altered hippocampal and amygdala function (LeDoux, 2000, Anagnostaras et

al., 2001). A neutral, conditioned stimulus (CS) is paired with an innately aversive,

unconditioned stimulus (US) in fear conditioning (Johansen et al., 2011). In this auditory test

of fear conditioning, an audible tone (CS) is associated with a foot shock (US), and freezing

duration in response to training context or to the tone itself was recorded as previously described (Clapcote et al., 2005) . Mice were placed into a fear conditioning chamber (MED

Associates Inc., Georgia, VT) and allowed to habituate to their surroundings for 120 seconds.

Following this, an audible tone (3,600 Hz, 80 dB) was played for 30 seconds that co-

terminated with a 2-second, 1mA foot shock. Each animal remained in the chamber for

another 30 seconds before being placed back into their home cages. Contextual fear

conditioning memory was examined 24 hours later in which mice were returned to the same

chamber for 300 seconds without any stimulus. After a 2 hour inter-session interval, the same

chamber was modified (i.e. the context was altered) in order to test for cued fear conditioning

memory. Each mouse was placed into the modified chamber for 180 seconds without any

stimulus followed by 180 seconds in the chamber during which the same auditory cue from

the previous day was played. This testing was carried out at the Toronto Centre for

Phenogenomics.

2.2.7 Morris Water Maze

The Morris water maze is a commonly used test to examine spatial learning and memory

dependent on hippocampal function (Logue et al., 1997). Mice are expected to swim to and

learn the location of a hidden platform using fixed spatial cues in the testing room that allows

them to escape out of the water. The water maze consisted of a water pool (diameter: 122cm)

with 3D and high colour contrast visual cues on the surrounding walls. Video tracking of

animals was conducted using Water 2020 software (HVS Image Ltd, Twickenham,

Middlesex, UK). A platform was positioned in one of the four quadrants (designated N, S, E,

and W) of the water maze to allow mice to escape out of the water. On the first day of testing

(the familiarization phase), the platform was visible and placed in the centre (20cm from the

pool wall) of one quadrant. The starting quadrant for each mouse was then pseudo-randomly

pre-determined. Each mouse was first placed onto the platform for 60 seconds followed by 3

trials during which the animal was given 60 seconds to find the platform. If the mouse was

unable to find the platform within the allotted time, it was gently guided onto the platform.

At the end of each trial, animals were allowed to remain on the platform for another 60

seconds before being returned to their cages. On day 2 (the training phase), the platform

remained in the same position as on day 1, but was submerged 0.5cm under water. 4 sessions of 3 trials each were performed. For each trial, mice were again placed into a pseudo-

randomly pre-determined quadrant and given 60 seconds to find the platform or guided onto

the hidden platform if they could not find it within 60 seconds. Each mouse was allowed to

remain for 60 seconds on the platform until the next trial with a 20-min interval between

sessions. On day 3, the probe trial was conducted in which the platform was removed from

the pool, and mice were placed at the centre of the pool and allowed 60 seconds to search for

the platform. This testing was carried out at the Toronto Centre for Phenogenomics.

2.2.8 Barnes Maze

Similar to the Morris water maze, the Barnes maze is also a task of spatial learning and

memory wherein hippocampal lesions lead to impaired performance (Fox et al., 1998, Pompl et al., 1999). This task is dependent upon the innate preference of rodents to escape into a dark compartment rather than remain in a bright, open area. The Barnes maze does not require swimming and is therefore considered to be less anxiogenic than the Morris water maze (Harrison et al., 2006). Each mouse was placed at the centre of a brightly lit, circular platform, ringed by 20 equally spaced holes, but only one of the holes led to a dark “escape box”. Latency to find the escape box and the path taken were recorded daily during 2 trials of 3-minute sessions, and this was repeated over 5 days. On the 6th day, a probe trial in the

absence of the escape box was conducted for 1 minute.

2.2.9 Resident Intruder

Aggression and social interactions were assessed in male mice (residents) that were initially

isolated for 10 days as previously described (Moy et al., 2004). Intruders (unfamiliar male mice that were age- and weight-matched to residents) were introduced to the resident’s home cage for a 10-minute session. The latency, duration and number of aggressive behaviours

(attacking, biting, wrestling partner) and social investigation behaviours (approaching, following, sniffing, grooming of partner) were recorded. A different intruder animal was used for each resident mouse. This testing was carried out at the Toronto Centre for

Phenogenomics. A simple test of olfaction to exclude olfactory dysfunction as a confounding factor was carried out following the resident intruder test. Latency to find food buried in bedding was recorded. One piece of cereal (Kellogg’s Froot Loops, Battle Creek, MI) was placed into each cage overnight to allow mice to habituate to the food piece. All rodent chow was removed the next morning for 24 hours, and the test was conducted the following day in a standard home cage. Food was placed randomly under bedding that measured 2.5cm in depth, and latency to find the Froot Loop piece and whether or not it was consumed were recorded.

2.2.10 Novel Object Recognition

The novel object recognition task examines non-spatial object memory, and it is a task that

relies on the innate tendency of rodents to explore a novel object more than a familiar object

(Ennaceur and Delacour, 1988). Object recognition memory requires an intact sensory

46 information circuitry that includes the neocortex, entorhinal cortex, and the hippocampus

(Parron et al., 2006). It has been suggested that the working memory retention in the novel object recognition task is an index of mainly entorhinal and perirhinal cortical function (Dere et al., 2007, Sipos et al., 2007). Each mouse was first habituated to an empty cage for 15 minutes over 5 consecutive days. Two inanimate objects (LEGO™ construct, Hot Wheels™ car) were used in this recognition task, and these objects were rigorously tested for matched saliency so that animals were able to distinguish between the two objects but did not have a differential preference toward either one. On the day of testing (i.e. the 6th day), mice were exposed to the two objects in an empty cage for 10 min (acquisition trial). To examine working recognition memory, mice were returned to the same cage in which one of the objects were replaced by a novel object following either a 1-hour or a 3-hour inter-trial interval. Different cohorts were used for each of the retention intervals. Mice were then re- exposed to these test objects for 5 minutes. A memory index (MI) was calculated as follows:

MI=(tn-tf)/(tn+tf), where ‘tn’ represents time spent exploring the novel object and ‘tf’ represents time exploring the familiar object. This MI takes into account any differential exploratory activity between the animals, which is a more robust measurement than merely the difference between time spent exploring the novel and familiar objects (Sik et al., 2003).

2.2.11 Elevated Zero Maze

The elevated zero maze is a circular apparatus with two opposing pairs of open and closed quadrants. Mice that are less anxious spend more time in the open quadrants of the maze in approach-avoidance conflict behaviour (Shepherd et al., 1994). Performance on the zero maze is sensitive to serotonergic transmission and has been validated by the effects of

anxiolytic drugs in mice with serotonergic dysfunction (Heisler et al., 1998) Mice were placed in a closed quadrant of the maze and monitored for 5 minutes. Duration spent in the open and closed quadrants as well as the total number of entries into each quadrant were recorded as indices of anxiety-related behaviours. Grooming and rearing behaviours were also scored in a preliminary fashion to validate findings in the open field test.

2.2.12 Open Field

Locomotor activity was monitored by placing each mouse into an open field measuring 40cm x 40cm x 30cm with opaque walls. Horizontal and vertical movements were scored over a

15-minute period. Measurements included the time spent in the centre zone and time spent in the wall zone including thigmotactic behaviour, which is the tendency to remain close to the walls. Rearing was scored when a mouse moved vertically to stand on its hindpaws

(excluding the time spent sitting and grooming with its forepaws), and grooming was recorded when the mouse licked or scratched any part of its body and when it washed its face with its forepaws. Rearing is an exploratory behaviour that provides a measure of anxiety levels in mice, and previous studies have shown that decreased rearing may be a result of increased anxiety levels (van Gaalen and Steckler, 2000, Rodgers et al., 2002). Self- grooming is a restricted, repetitive behaviour that has been reported to be regulated by the basal ganglia and dopaminergic transmission (Aldridge et al., 2004, Berridge et al., 2005).

Increased spontaneous grooming is also often associated with increased anxiety levels

(Smolinsky et al., 2009).

2.2.13 Maternal Separation-Induced Ultrasonic Vocalizations

Separation from maternal contact induces ultrasonic vocalizations (USVs) in the range of 50

kHz to 120 kHz from mouse pups (Branchi et al., 2001). USVs are affected by serotonergic

and GABAergic drugs as well as opioids, and in particular, these vocalizations are sensitive

to selective serotonin reuptake inhibitors (Hodgson et al., 2008). Post-natal day 8 pups were

individually separated from their parents and placed in plastic container under a D1000X

ultrasonic vocalization detector (Pettersson Elektronik AB, Uppsala, Sweden). Pups were

tested in both a clean condition (unsoiled nesting) and in soiled cotton nesting to eliminate

bedding material as a factor influencing USVs emitted from pups. The total number of USVs

emitted was recorded over 4 minutes for each pup and separated into 1-minute intervals for

analyses. This testing was carried out by Joana Dida, an undergraduate student at the

University of Toronto at the time of testing.

2.3 Results

2.3.1 Gene and protein expression levels

mRNA expression levels of Gtf2i and Gtf2ird1 were validated in the newly generated mouse models using quantitative real-time PCR (qPCR). Gtf2i+/- mice expressed Gtf2i mRNA at

levels that were 50% that of WT mice in the brain (Figure 2.2A), while homozygous mice

(Gtf2i-/-) were early embryonic lethal. Expression levels of Gtf2i and Gtf2ird1 transcripts in

Gtf+/del mice were decreased by 50% compared to WT mice. Expression levels of genes

surrounding Gtf2i and Gtf2ird1were also validated by qPCR, and quantification of the levels

of these transcripts show that these genes are unaltered in the mutant mice. The only

exception is the expression level of Gtf2ird1 exon 1 in Gtf+/del mice, which is higher than the

expected 50% compared to WT. This is probably due to the technique used to generate the

mouse model – the loxP site was inserted into intron 4, and therefore exon 1 is still present in

the genome. To compensate for a decrease in mRNA expression, the promoter of Gtf2ird1 is likely upregulating expression of the remaining three exons.

Protein expression levels were determined by western blot analyses whereby expression of

Gtf2i protein (TFII-I) was normalized to the loading control, GAPDH (Figure 2.2B). In a comparison of the band intensities, the amount of TFII-I protein in Gtf2i+/- animals is

approximately half of that found in wildtype controls. In contrast, TFII-I is increased by 50%

in Gtf2i+/dup mice.

Figure 2.2. Validation of gene and protein expression in the various mouse models. A) Expression of surrounding genes in Gtf2i+/- and Gtf+/del mice normalized to the housekeeping gene, Sdha, and relative to wildtype animals. The shaded area represents variation in normal gene expression relative to controls. (n=6 per genotype) B) Gtf2i protein expression in Gtf2i heterozygous and duplication mice relative to loading control, GAPDH. Data are expressed as mean ± SEM (n=3 per genotype)

Table 2.1. Summary of results from current studies and behavioural comparison to previously studied Gtf2i and Gtf2ird1 mouse models

Gtf2ird1-/- or PD mice (Li Gtf2i+/- Gtf2i+/- Gtf+/del Gtf2i+/dup & +/- (Young et et al. 2009) (Sakurai et Gtf2idup/dup al. 2008) al. 2010)

Morris water -- ND ------ND maze impairments

Barnes maze ND ND ND -- -- ND impairments

Reduced fear * ND ND *(m) * ND in cued fear conditioning

Contextual ------ND fear conditioning

Novel object ND ND ND * ** ns learning & memory impairments

Increased * * ND ------sociability in resident intruder test

Grip strength * ND -- *(f) *Increased ND impairment strength (m)

Rotarod * * -- * * ND impairment

Elevated plus * decreased ND ND ND ND ND maze anxiety level

Zero maze ND ND -- -- *decreased *increased anxiety level

Open field *decreased *increased ------*increased anxiety level

-- Not significant * p<0.05 **p<0.01 (f), female only (m), male only ND, Not Determined

2.3.2 Gtf2i+/- and Gtf+/del mice exhibit impaired rotarod performance

but normal grip strength

Forelimb grip strength is not altered in Gtf2i+/- or Gtf+/del mice. However, combined forelimb

and hindlimb grip strength is affected differentially in females and males (p<0.05, Figure

2.3A). Female Gtf2i+/- mice have a mild impairment in this combined grip strength whereas

53 males do not. Gtf+/del males show an increase in this measure of muscle function.

Interestingly, rotarod performance is significantly impaired in Gtf2i+/- males as shown by the shortened latency to fall off of the constant speed rotarod (p<0.05, Figure 2.3B). Although not significant, both Gtf+/del and female Gtf2i-heterozygous mice show a trend toward minimal acquisition improvements over time.

A *

Rotarod Performance; Males only Rotarod Performance; Females Only B * 600 900 Wt (n=6) Wt (n=5) 800 500 * Gtf2i+/- (n=4) 700 Gtf2i+/- (n=6) 400 Gtf+/del (n=5) 600 Gtf+/del (n=4) 500 300 400 200 300 200 100

Sum m ed duration (s) Sum m duration ed (s) Sum100 m duration ed 0 0 Day 1Day 2Day 3Day 4Day 5 Day 1Day 2Day 3Day 4Day 5

Figure 2.3. Grip strength and motor learning in Gtf2i+/- and Gtf+/del mice. (A) Female Gtf2i+/- mice exhibit a slight deficit in combined forelimb and hindlimb grip strength, but Gtf+/del males show a strength in this measure (n=5 per genotype for each gender). (B) Rotarod performance at constant speed is significantly impaired in Gtf2i+/- males and male Gtf+/del mice also show this trend. Female heterozygous mice, however, do not show significant motor deficits (Grip strength: p<0.05 by one-way ANOVA, and *p<0.05 post-hoc by Student’s t-test. Rotarod: p<0.05 by two-way ANOVA, and *p<0.05 by Bonferroni post-hoc analyses).

2.3.3 Cued, but not contextual, fear conditioning altered in Gtf2i+/- and

Gtf+/del mice

In the auditory fear conditioning test, an unconditioned stimulus that is innately aversive, such as a foot shock, is paired with a conditioned stimulus, such as a tone. Contextual fear- conditioning requires intact hippocampal function to associate the aversive stimulus with the context in which it occurred, but this learning is not altered in mice heterozygous for Gtf2i or in those with a combined deletion of Gtf2i and Gtf2ird1 (Figure 2.4). In the cued fear- conditioning component of the test, male and female animals perform differentially. Male

Gtf2i+/- and Gtf+/del mice do not exhibit altered freezing durations in response to the

conditioned stimulus (tone). However, female Gtf+/del mice show an impairment in cued fear-

conditioning (p<0.05). These animals exhibit a lower percentage of time spent freezing in the

test chamber in response to the conditioned stimulus. Although not significant, female

Gtf2i+/- mice also show a trend towards an impaired freezing response. However, in a

comparison between wildtype males and females, it appears that the impairments observed in

females may only be due to the better-performing wildtype females. Thus, the sample size

per genotype should be further increased in order to avoid any overinterpretation of potential

artifacts within the data.

Figure 2.4. Freezing responses in contextual and cued fear conditioning. The amount of time spent freezing during contextual fear conditioning is not significantly different between wildtypes and mutants, however, female Gtf+/del exhibit an impaired cued conditioning freezing response. n=5 per genotype for each gender (p<0.05 by one-way ANOVA, and *p<0.05 by post-hoc comparison using Student’s t-test).

2.3.4 Gtf2i and Gtf2ird1 likely not involved in visuospatial processing

The latency for Gtf2i+/- and Gtf+/del mice to find the escape box was similar to that of wildtype animals (Figure 2.5). Thus, haploinsufficiency of Gtf2i likely does not lead to

significant alterations in visuospatial processing, which supports evidence that other genes in

the WBS region may be involved in the visuospatial deficits seen in people with WBS.

Figure 2.5. Visuospatial learning using the Barnes maze. Neither Gtf2i+/- nor Gtf+/del mice show an altered latency to learn the location of the escape box using visuospatial cues in the room.

2.3.5 Measures of social interaction are not affected by reduced or

increased Gtf2i expression

Measures of aggressive behaviour include tail rattling, fighting, grooming, and digging,

while social interactions were determined by followings and sniffing of the intruder animal.

Neither Gtf2i heterozygous nor duplication mice show any alteration in the number of social

(Figure 2.6A) or aggressive behaviours (Figure 2.6B). The combined deletion of both Gtf2i

and Gtf2ird1 also does not affect social interaction in the resident intruder test.

Figure 2.6. The resident intruder test as an index of social and aggressive behaviour in male animals. (A) Sniffing is used as an indicator of social behaviour and a total number including all other recorded measures of social interaction is calculated. (B) Fighting is an index of aggressive behaviour, and the total number of aggressive behaviours is also calculated. Each behaviour is shown by the number of times that it occurred during the 10-minute test (n=9 per genotype).

2.3.6 Cognitive impairments associated with altered expression of

Gtf2i and Gtf2ird1

In the novel object recognition task, mice are allowed to explore a pair of objects over 2 trials, and during the second trial, one of the objects is switched with a novel object. Mice that have intact recognition memory will explore the novel object more than the familiar object. A memory index is calculated from the difference in these exploration times.

Working memory following a 1-hour inter-trial interval is unaffected in mutants. However,

Gtf2i+/- mice show a significant impairment in novel object memory with a 3-hour retention

interval (p<0.05, Figure 2.7B), and this poor memory index is further exacerbated by the

combined deletion of Gtf2ird1 and Gtf2i in Gtf+/del animals (p<0.01, Figure 2.7B).

A B

Figure 2.7. Novel object recognition memory impairments due to hemizygosity of Gtf2i and Gtf2ird1. (A) Memory index is unaltered following a 1-hour retention interval. (B) Object memory deficits are observed in both Gtf2i+/- and Gtf+/del animals following a 3- hour retention interval (WT: n=11, Gtf2i+/-: n=11, Gtf+/del: n=10) (Following statistical significance in one-way ANOVA calculations, *p<0.05, **p<0.01 by Student’s t-test in a post-hoc comparison to WT mice.)

In contrast, increased expression of Gtf2i appears to affect object memory differentially in males and females following a 3-hour retention interval (Figure 2.8). Whereas Gtf2i+/dup

males show a deficit in this cognitive test (Figure 2.8A), females exhibit a stronger memory

of the familiar object than wildtype controls (Figure 2.8B). Interestingly, female animals with

increased expression of Gtf2i perform significantly better than their male counterparts

(p<0.01, Figure 2.8C). This finding may be explained by a role for TFII-I in regulating

androgens and/or estrogens, particularly because a previous study showed that TFII-I down-

regulates estrogen-responsive genes (Ogura et al., 2006). However, results from the tests of

anxiety (elevated zero maze and open field) do not indicate any gender differences in

exploratory or anxiety-related behaviours.

A B MI for females only MI for males only 0.35 Wt (n=7) 0.30 0.30 0.25 Wt (n=8) Gtf2i+/Dup (n=10) 0.20 Gtf2i+/Dup (n=9) 0.25 Gtf2iDup/Dup (n=7) 0.15 Gtf2iDup/Dup (n=3) 0.20 0.10 0.15 0.05 -0.00 0.10 M em ory Index -0.05 Wt Gtf2i+/Dup Gtf2iDup/Dup

M em ory Index 0.05 -0.10 -0.15 0.00 -0.20 Wt Gtf2i+/Dup Gtf2iDup/Dup

Male vs. Female Gtf2i+/dup Mice C **

0.35 0.30 0.25 0.20 0.15 0.10 0.05 -0.00 -0.05 Memory index -0.10 -0.15 -0.20 Male Female

Figure 2.8. (A & B ) Memory index is differentially affected by extra copies of Gtf2i in male and female mice. (C) Following a 3-hour retention interval, female Gtf2i+/dup show a significantly higher memory index compared to male animals (**p<0.01 by Student’s t-test).

2.3.7 Haploinsufficiency of Gtf2ird1implicated in anxiety-related

behaviours

Gtf2i+/- and Gtf+/del mice do not spend a significantly different amount of time in the open

quadrants of the zero maze (Figure 2.9A) or in grooming (Figure 2.9B). However, an

increased duration in rearing was observed in Gtf+/del mice (p<0.05, Figure 2.9C) and an

opposing trend as observed in Gtf2i+/- animals. In a comparison of total distance travelled in the open field as an index of motor activity, mutant mice do not show differences compared to wildtype controls (Figure 2.9D). This unaltered total motor activity accompanied by elevated rearing in Gtf+/del mice indicate that the rearing is not merely due to an increase in

spontaneous motor activity. Thus, it appears that this increase in rearing is due to

haploinsufficiency of Gtf2ird1 specifically and not Gtf2i.

A B Grooming 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Duration (s) Duration 1.0 0.5 0.0 Wt Gtf2i+/- Gtf+/del

C D Total Distance Travelled in Open Field 35 30 25 20 15

Distance (m) 10 5 0 Wt Gtf2i+/- Gtf+/del

Figure 2.9. Gtf2i+/- mice do not show significant alterations in the zero maze test, but Gtf+/del animals exhibit increased exploratory behaviour. (A) Time spent in the open quadrants of the maze in mutant mice is comparable to wildtype littermates. (B) Grooming duration is unaltered in both mutant animals. (C) Gtf+/del mice exhibit increased rearing behaviour whereas Gtf2i-deficient mice do not. (WT: n=10, Gtf2i+/-: n=11, Gtf+/del: n=10) (p<0.05 by one-way ANOVA, and using Student’s t-test in post-hoc analyses, *p<0.05 compared to WT animals.)

2.3.8 Alteration in non-social anxiety levels due to increased Gtf2i

copy number

In the elevated zero maze test, mice with one or two additional copies of Gtf2i did not differ

significantly in the amount of time spent in the open quadrants of the maze (Figure 2.10A).

However, a trend towards a decrease in rearing duration may indicate a slightly more anxious

state in the Gtf2i heterozygous and homozygous duplication mice (Figure 2.10B). In support

of this, results from the open field test show that Gtf2i+/dup animals spend more time against the walls of the apparatus rather than in the centre (Figure 2.11A). The duration of rearing, which may be indicative of exploratory behaviour, is also significantly decreased in Gtf2i+/dup mice (Figure 2.11B). Interestingly, mice heterozygous for Gtf2i spend more time exploring the centre of the open field, but do not show a parallel increase in rearing duration. Instead,

Gtf2i+/- animals exhibit a significant decrease in rearing behaviour. An increased spontaneous grooming duration was also observed in mice with an extra copy of Gtf2i, suggesting that

increased Gtf2i expression may lead to a concomitant increase in repetitive behaviours

(Figure 2.10C, Figure 2.11C). Since the decreased centre-to-wall ratio in Gtf2i+/dup animals is accompanied by an increase in grooming, it is possible that these animals are merely hypoactive and display an increased level of “comfort” in the wall zone. However, analysis of total distance travelled during the open field test indicates that locomotor activity is comparable between all genotypes (Figure 2.11D). Gtf2i+/- mice exhibited a trend toward

spending less time on self-grooming (Figure 2.11C), and may be evidence that Gtf2i copy number plays a role in repetitive, stereotyped behaviours reported in 7q11.23 duplication and triplication patients. Anxiety levels as determined by ultrasonic vocalizations (USVs) in pups also correspond with Gtf2i gene expression (Figure 2.12). In response to maternal separation,

pups with extra copies of Gtf2i have an increased number of USVs, whereas the number of vocalizations is less in Gtf2i+/- pups. This correlation of lower anxiety in Gtf2i+/- and

increased anxiety in Gtf2i+/dup pups appears to remain over the course of development, as

adult mice exhibit the same gene dosage effect in the zero maze and open field tests.

B C

Figure 2.10. Increased expression of Gtf2i affects anxiety-related behaviours. (A & B) Although not significant, additional copies of Gtf2i lead to a mild decrease in exploratory behaviour such as time spent in the open quadrants of the elevated zero maze and rearing during the test. Increased grooming duration was observed in Gtf2+/dup animals, and although not significant, homozygous duplication mice also exhibit this trend. (WT: n=13, Gtf2i+/dup: n=13, Gtf2idup/dup: n=6) (p<0.05 by one-way ANOVA, and in post-hoc comparisons,*p<0.05 compared to WT mice.)

Centre:wall ratio Rearing A * B * * 120 1.2 110 1.1 100 1.0 90 0.9 80 0.8 0.7 70 0.6 60 50 Ratio 0.5 0.4 40 0.3 (s) Duration 30 0.2 20 0.1 10 0.0 0 Wt Gtf2i+/- Gtf2i+/Dup Wt Gtf2i+/- Gtf2i+/Dup

C Grooming D Total Distance Travelled * 35 80 30 70 60 25 50 20 40 15 30 Duration (s) Duration

Distance (m) 10 20 10 5 0 0 Wt Gtf2i+/- Gtf2i+/Dup Wt Gtf2i+/- Gtf2i+/Dup

Figure 2.11. Effects of Gtf2i copy number on anxiety-related behaviours in the open field. A) Decreases in centre-to-wall ratio and rearing duration in Gtf2i+/Dup mice suggest an increase in non-social anxiety. Gtf2i+/- animals also show a significant decrease in rearing behaviour. B) Gtf2i+/dup mice exhibit increased self-grooming compared to wildtypes, whereas Gtf2i+/- mice show a trend toward decreased grooming behaviour. (WT: n=28, Gtf2i+/-: n=15, Gtf2i+/dup: n=16) (One-way ANOVA: p<0.05, and using Student’s t-test in post-hoc analyses, *p<0.05 compared to wildtypes.)

Figure 2.12. Maternal separation in Gtf2i heterozygous and duplication mice. In a test of maternal separation anxiety, Gtf2i+/- pups emit fewer USVs than littermate controls while increased Gtf2i copy number leads to a corresponding increase in the number of vocalizations.

2.4 Discussion and Conclusions

2.4.1 Gtf2i+/- and Gtf+/del mice exhibit neuromotor learning deficits

Rotarod sensorimotor learning is dependent upon the normal functioning of the cerebellum

(Nolan et al., 2003). Although female Gtf2i+/- mice exhibit a decrease in combined forelimb

and hindlimb grip strength, they do not perform significantly worse than their WT counterparts in the rotarod test. In contrast, although male Gtf2i+/- animals do not have

impaired grip strength, they do show impaired rotarod performance. Gtf+/del males, which

have better grip strength than controls, also show a trend towards poor performance on the

rotarod. Previously studied Gtf2ird1-/- mice also exhibit motor deficits in the rotarod test, thus

suggesting that the GTF2I gene family members may be involved in the neurological

myopathies found in people with WBS (Morris et al., 1990, Young et al., 2008, Howard et

al., 2011). Gtf2ird1 has been postulated to regulate the promoter of Troponin I slow, which is

involved in slow-twitch muscle fibre expression (Polly et al., 2003). Both Gtf2i+/- and Gtf+/del

males exhibit minimal acquisition improvements over time, suggesting an impairment in

cerebellar function that is required for maintaining postural equilibrium (Lalonde et al.,

1995). Behavioural analyses of a similar Gtf2i+/- gene trap mouse model revealed no

significant differences in motor coordination or grip strength (Sakurai et al., 2010), but this may be due to differences in sample size, animal handling, experimental procedure, and inter-experimenter variability.

2.4.2 Additive effect of Gtf2ird1 and Gtf2i on anxiety

Gtf2i+/- and Gtf+/del mice do not exhibit any differences from wildtype mice in the amount of time spent exploring the open quadrants of the elevated zero maze or spontaneous self- grooming. However, rearing behaviour is significantly elevated in Gtf+/del. The total distance

travelled between the different genotypes in the open field test is not different, suggesting

that the elevated rearing in Gtf+/del mice is not due to increased motor activity. Previous

studies propose that rearing is inversely related to anxiety levels (van Gaalen and Steckler,

2000). Thus, rearing as an exploratory activity indicates lower levels of anxiety in Gtf+/del

animals, which is likely due to haploinsufficiency of Gtf2ird1 only since Gtf2i+/- animals

show a trend towards decreased rearing during the test. The decrease in anxiety level is

similar to a Gtf2ird-/- mouse model generated by our lab that exhibits low innate anxiety

(Young et al., 2008). These mice also show increased levels of serotonin (5-HT) metabolite

in the frontal and parietal cortices as well as the amygdala without a concomitant increase in

5-HT content in these brain regions, which is suggestive of an increase in serotonin turnover.

Inhibitory 5-HT1A-mediated serotonin currents are also enhanced in layer V pyramidal

neurons of the prefrontal cortex of Gtf2ird1-/- mice, which may lead to the observed increase

in 5-HT (Proulx et al., 2010). 5-HT1A-KO mice exhibit elevated anxiety in contrast to

Gtf2ird1-deficient mice that show increased 5-HT turnover and a lower innate anxiety

phenotype (Heisler et al., 1998).

2.4.3 Gtf2i expression level is implicated in altered anxiety- and ASD-

related behaviours

Zero maze and open field test results and maternal separation-induced ultrasonic

vocalizations (USVs) revealed a positive correlation between Gtf2i copy number and anxiety levels in both pups and adults. Pups with only one copy of Gtf2i vocalize less than their wildtype littermates, whereas pups with extra copies of Gtf2i vocalize more. USVs have been

studied as a useful behavioural assay to examine maternal-separation induced anxiety in

neonates (Branchi et al., 2001). The increased number of vocalizations in Gtf2i+/dup and

Gtf2idup/dup mice suggests an increase in maternal-separation anxiety in the mouse pups,

comparable to children with a duplication of the WBS region (Somerville et al., 2005, Berg

et al., 2007). People with Dup7q11.23 syndrome also present with non-social anxiety

(Velleman and Mervis, 2011). To examine non-social anxiety in adult mice, the zero maze

and open field tests were used. Gtf2i+/dup mice spend less time rearing, which indicates

decreased motivation to explore their surroundings. In support of this anxiety-related

measure, Gtf2i+/dup animals also exhibit increased thigmotaxis behaviour during which mice

tend to remain close against the opaque walls of the apparatus (Simon et al., 1994).

Thigmotaxis is an index of anxiety that is modulated by altered neurotransmission, such as differential dopamine transmission. A mouse model of Dup15q also shows decreased

exploratory activity and altered serotonin levels, which suggests that serotonergic

dysregulation may contribute to the neurophysiology Dup7q11.23 (Tamada et al., 2010). The

decrease in the number of maternal separation-induced USVs in Gtf2i-heterozygous mice is

similar to that found in a previously studied Gtf2ird1-/- mouse model (Howard et al., 2011).

Analysis of c-fos expression in the Gtf2ird1-/- mice showed decreased neuronal activation of

71 the anterior cingulate cortex, which is associated with cognitive and emotional processes, and the lateral septum, which is implicated in regulating mood and motivation, such as fear

(Howard et al., 2011).

Patients with a duplication of 7q11.23 are often diagnosed with autism or autism spectrum disorder (ASD), which is a neurodevelopmental disorder characterized by impairments in social interactions, communication, and repetitive behaviours (Van der Aa et al., 2009,

Rodgers et al., 2011). In children with ASD, repetitive behaviours are a common symptom that may reflect a mechanism to reduce anxiety or a consequence of anxiety (Rodgers et al.,

2011). Spontaneous grooming in mice is a repetitive, stereotyped behaviour that has been found to be elevated in the BTBR T+tf/J (BTBR) mouse strain, which is considered to be a mouse model of autism (McFarlane et al., 2008). In Gtf2i+/dup mice, self-grooming behaviour in both the open field and elevated zero maze tests was elevated, and Gtf2idup/dup animals also show this trend. This is perhaps the first indicator that Gtf2i may be involved in ASD-related behaviours. Further investigation into other autism-related behavioural tests is necessary to validate these findings. However, it is important to note the caveat that grooming is also considered an anxiogenic behaviour, and thus, other behavioural tests used to explore the role of Gtf2i in ASDs should ideally be able to separate anxiety-related behaviours from ASD- related ones.

In the test of novel object recognition memory, a trend towards a poor memory index was observed in male Gtf2i+/dup mice, thus suggesting that they have memory impairments and thus cannot distinguish between a familiar and a novel object. However, this lack of

exploration of a novel object may also reflect a preference for familiar objects due to a non-

social anxiety towards novelty. The anxiety phenotype in Gtf2i-duplication mice is consistent

with the specific phobias that are observed in over 50% of children diagnosed with a

duplication of 7q11.23 (Velleman and Mervis, 2011).

2.4.4 Gtf2i and Gtf2ird1 are essential for object recognition memory

Object recognition memory is dependent upon a sensory information circuitry that includes the neocortex, entorhinal cortex, and the hippocampus (Parron et al., 2006). Novel object recognition appears to be delay-dependent, whereby mutant animals showed an impaired memory index following a 3-hour inter-trial interval, but not with a 1-hour retention interval.

This is consistent with the delay-dependent failure to discriminate between a familiar and a

novel object (Sik et al., 2003). Object recognition is not significantly altered in Gtf2i+/dup mice, but male and female animals show a differential preference for the novel object.

Female Gtf2i+/dup mice exhibit a significantly higher object recognition memory index than

males. People with a duplication of 7q11.23 sometimes exhibit mild intellectual disability,

and interestingly, although GTF2I is on the boundary of the duplicated region and is thus

present with only two intact copies, gene expression of GTF2I is significantly higher than

expected (Somerville et al., 2005, Van der Aa et al., 2009).

WBS individuals with atypical deletions that leave GTF2I or both GTF2I and GTF2IRD1

intact have been reported (Tassabehji et al., 2005, Antonell et al., 2010, Ferrero et al., 2010).

These studies suggest that GTF2IRD1 is not sufficient to cause the typical WBS visuospatial deficits but that GTF2I may be involved in this cognitive process. Spatial learning and

memory was examined using the Morris water maze, but performance of Gtf2i heterozygous mice was comparable to wildtype littermates. Latency to find an escape box located below one of the twenty holes of the Barnes maze (a dry-land version of the water maze) was recorded as another measure of visuospatial constructive cognition. Gtf2i+/- mice did not

show a delay in learning the location of the escape box using visuospatial cues in the room,

and spatial learning in Gtf+/del animals do not differ from wildtypes. However, to ensure that

visual memory is spared in the mutant mice, perhaps a learning and memory task that does

not carry a tactile component could be used. For example, touch-sensitive computer screen

tests have been devised to examine the effects of hippocampal lesions on spatial and non-

spatial learning and memory in rodents (Talpos et al., 2008). Spatial memory formation is

dependent upon the hippocampus, and the subtle learning delay in the Barnes maze test may

indicate hippocampal dysfunction (Broadbent et al., 2004). However, Sakurai et al. (2010)

reported no differences in the Morris water maze to examine spatial memory in their Gtf2i+/- mouse model. Thus, with respect to the visuospatial deficits observed in people with WBS, it is therefore likely that other genes in the WBS deletion region are also involved.

Auditory fear conditioning is dependent upon intact hippocampal and amygdala function

(Logue et al., 1997, Schafe et al., 2000). Following association of a cued tone to a foot shock, freezing behaviour is recorded as an index of a defensive response. Gtf2i+/- males do not have

abnormal freezing responses, but female Gtf+/del mice exhibit a decreased duration in freezing

during the cued fear conditioning test and Gtf2i+/- females also show this trend. The cued fear

conditioning impairment in Gtf+/del animals is consistent with the impaired freezing response

in previously studied Gtf2ird1-knockout mice that show decreased fear (Young et al., 2008).

These results suggest a dysfunction of the amygdala that supports the neurofunctional

imaging findings in people with WBS (Meyer-Lindenberg et al., 2005). However, it remains

to be determined whether it is the acquisition or consolidation of fear memory that is

disrupted in the Gtf+/del mice. Noradrenergic and dopaminergic transmission have been

implicated in the formation of fear memory where activation of their respective receptors

may be required for acquisition of fear learning (Guarraci et al., 2000, Bush et al., 2010). The cAMP response element binding protein (CREB) is necessary for fear memory consolidation in the amygdala via gene transcription, and phosphorylation of CREB allows for transcriptional activation of cAMP response element (CRE)-dependent genes to form a

memory trace (Josselyn et al., 2001, Zhou et al., 2009). A downstream target of CREB is the neurotrophin brain-derived neurotrophic factor (BDNF), and lower expression of this gene which has been found to impair memory formation (Ou and Gean, 2007). Ca2+/Calmodulin-

dependent protein kinase II (CaMKII) is phosphorylated following an increase in

intracellular calcium, and this enzyme is also essential for synaptic plasticity leading to memory formation as well as the acquisition of fear (Silva, 2003, Rodrigues et al., 2004).

Chapter 3

Elucidating pathways affected by reduced or increased

expression of Gtf2i

3.1 Introduction

3.1.1 Research Aims

Although the genetic basis for WBS/Dup7q11.23 syndrome is due to an established gene dosage effect, the downstream molecular mechanisms of the neurological symptoms of both syndromes have yet to be elucidated. Through subcellular fractionations of whole brain, the localization of TRPC3, a calcium channel postulated to be indirectly regulated by TFII-I was examined. The role of TFII-I in Src-knockout mice (SrcThl/Thl), which show phenotypic similarities to WBS, was also investigated through protein localization studies to determine whether or not TRPC3 localization was altered.

3.1.2 Hypothesis

We hypothesized that the molecular basis of WBS/Dup7q11.23 involves calcium dysregulation, which may explain some of the phenotypes observed in mouse models of these two syndromes. Other studies suggest a cytoplasmic function for TFII-I as an inhibitor

of agonist-induced calcium entry through TRPC3 localization. Thus, we hypothesized that

differential gene dosages of GTF2I may play a role in modulating the subcellular localization

of TRPC3.

3.2 Materials and Methods

Contributions: I carried out all protein fractionations and western blot analyses detailed in this chapter.

3.2.1 Subcellular fractionations

Adult mice were sacrificed by cervical dislocation, and brains were removed immediately.

One hemisphere of the brain was finely diced and homogenized with 2mL homogenizing buffer (HB) containing 25mM KCl, 1mM MgCl2, 20mM HEPES (pH 7.4), 1mM EGTA,

0.2mM DTT, 0.32M sucrose, 1mM PMSF, 1mM protease inhibitor cocktail (Sigma P8340),

and 1mM sodium orthovanadate. Samples were homogenized by 5 strokes of a Teflon homogenizer followed by an incubation of 2mins on ice in HB and then homogenized with

20 strokes of a Dounce homogenizer. All spins were performed at 4⁰C. The homogenate

was first spun at 600g for 10mins. The pellet (P1) was further treated to obtain the nuclear

fraction, and the supernatant (S1) was stored at -20⁰C for later fractionation. Resuspended in

1mL HB, P1 was passed through a 27G syringe and spun again at 600g for 10mins. The

pellet was then resuspended in 1mL nuclear buffer (2.2M sucrose, 1mM MgCl2, 10mM

HEPES (pH 7.4)) and homogenized by 8 strokes of Teflon. After spinning the homogenate

at 75,000g for 75mins, the resulting nuclear pellet was resuspended in RIPA buffer and

stored at -20⁰C. S1 was spun at 10,000g for 15mins, and the supernatant was further spun at

100,000g for 60mins to obtain the cytosolic fraction (supernatant). The pellet was

resuspended in 1mL HB and spun again at 100,000g for 60mins followed by a last wash in

0.5mL HB at 100,000g for 60mins. The pellet was resuspended in RIPA buffer and

constituted the membrane fraction.

3.2.2 Western blot analyses

Fractions were quantified by DC Protein Assay (Bio-Rad), and 30-40ug was used for western

blot analyses by SDS-PAGE on 8% acrylamide gels (see Section 2.2.1 for detailed western blotting procedure). The nuclear fraction was probed with anti-TFII-I mouse monoclonal antibody (610943, BD Biosciences) and normalized against nuclear loading control, lamin

A/C (sc-7293, Santa Cruz). The membrane fraction was probed with anti-Trpc3 rabbit

polyclonal antibody (ab51560, Abcam) and normalized against membrane loading control,

N-cadherin (610920, BD Biosciences) or NOX2/gp91phox (ab80508, Abcam). Image J

(NIH) was used to quantify the relative band densities.

3.3 Results

3.3.1 Alteration in TRPC3 surface expression

Western blot analyses of TRPC3 membrane expression revealed no significant alterations in

Gtf2i+/- and Gtf2i+/dup animals (Figures 3.1A & 3.1B) although a trend toward increased

TRPC3 in the membrane fraction was seen in Gtf2i+/- mice. In mice with two additional copies of Gtf2i, a significant decrease in TRPC3 expression at the membrane was observed

(p<0.05, Figure 3.1C).

Figure 3.1. Western blot analyses of TRPC3 expression in the membrane fraction of whole brain. (A & B) TRPC3 surface expression in Gtf2i+/- and Gtf2i+/dup mice is comparable to wildtype controls. Band densities of TRPC3 in Gtf2i+/- and Gtf2i+/dup are normalized to loading controls, N-cadherin and NOX2/gp91phox, respectively. C) Decreased membrane expression of TRPC3 in Gtf2idup/dup animals normalized to N- cadherin. (*p<0.05 by Student’s t-test)

3.3.2 Localization analyses in SrcThl/Thl mice

Brain tissue of SrcThl/Thl mice and their wildtype littermates was fractionated to examine subcellular protein expression of TFII-I in the nucleus (Figure 3.2A) and TRPC3 in the plasma membrane (Figure 3.2B) by western blotting. A significant decrease was observed in nuclear TFII-I level along with an increase in membrane localization of TRPC3 (p<0.05,

Figure 3.2C).

Figure 3.2. Western blot analyses of TFII-I and TRPC3 protein expression in SrcThl/Thl mice. (WT: n=3, SrcThl/Thl: n=5) A) Gtf2i expression in the nucleus normalized to loading control, nucleolin. B) TRPC3 membrane levels as a ratio to loading control, N- cadherin. C) Expression of Gtf2i is decreased in the nuclear fraction of SrcThl/Thl with a concomitant increase in TRPC3 in the membrane. (*p<0.05 by Student’s t-test compared to wildtype controls)

3.4 Discussion and Conclusions

In addition to its nuclear function, TFII-I is also present in the cytosol, and following tyrosine

phosphorylation, it has been found to interact with the Src-homology-2 domain of PLC-γ

(Caraveo et al., 2006). TRPC3 (transient receptor potential channel 3) is a member of the

TRPC subfamily that is also regulated by PLC-γ, and surface expression of this receptor mediates increases in intracellular Ca2+ (van Rossum et al., 2005). Thus, TFII-I may play an indirect role in regulating TRPC3 by sequestering PLC-γ from binding to TRPC3, thereby decreasing membrane localization of TRPC3 (Caraveo et al., 2006). Accordingly, Gtf2i- deficient mice would be expected to have increased localization of TRPC3 at the membrane.

In this protein localization study, haploinsufficiency or duplication of Gtf2i did not significantly alter the localization of TRPC3 at the membrane, but two additional copies of

Gtf2i in Gtf2idup/dup led to a significant decrease in TRPC3 surface expression. This supports a

corollary in the mechanism proposed by Caraveo et al. (2006) – increased Gtf2i expression

should lead to increased binding of TFII-I to PLC-γ with lower levels of PLC-γ available for

binding to and activation of TRPC3. The decreased surface density of TRPC3 in whole brain

observed in Gtf2idup/dup animals is consistent with this proposed mechanism. Although

significant changes were not observed in Gtf2i+/- mice, there appears to be a trend toward

increased TRPC3 surface expression. As well, the small sample size of Gtf2i+/dup mice may

not be sufficient to reveal subtle differences in TRPC3 localization. It is also possible that

western blotting is not a sufficiently sensitive assay for detecting small alterations in TRPC3

surface expression. Interestingly, only two other factors besides TFII-I are known to have

both cytoplasmic and nuclear roles: calcium channel-associated transcriptional regulator

(CCAT) and DREAM (also known as K+ channel-interacting protein 3) (Naranjo and

Mellstrom, 2007). Both of these factors are also involved in calcium regulation and provide

further support that TFII-I may also be involved in Ca2+-dependent molecular pathways.

During mouse embryogenesis, both Gtf2i and Gtf2ird1 are highly expressed (Enkhmandakh

et al., 2004, Palmer et al., 2007). However, although Gtf2ird1 is not highly expressed in adult

mouse brain, it is found in greater levels in the Purkinje neurons of the cerebellum (Palmer et

al., 2007). Similarly, the expression level of TFII-I in adult mice is highest in cerebellar

Purkinje cells, and interestingly, TRPC3 channels are also highly expressed in these same

neurons (Hartmann et al., 2008). TRPC3-knockout mice exhibit motor coordination deficits

as well as impaired metabotropic glutamate receptor (mGluR) synaptic signalling (Hartmann

et al., 2008). Interestingly, moonwalker mice with a gain-of-function mutation in TRPC3 also

show deficits in motor coordination accompanied by a loss of Purkinje cells (Becker et al.,

2009). The motor incoordination phenotype of both mouse models is conflicting and has yet

to be resolved. Despite these inconsistencies, it appears that TRPC3 plays an important role

in cerebellar-dependent motor function, especially since TRPC3 activation is involved in

protecting cerebellar granule neurons against cell death in rats (Jia et al., 2007). Altered

localization of TRPC3 in this study may reveal a possible molecular basis to explain the

motor deficits in people with WBS (Morris et al., 1988).

TRPC3 may also be implicated in autism-related behaviours through its involvement in

glutamatergic transmission. Cerebellar abnormalities have been identified in individuals with

autism, and these neuropathologies include a decrease in the number of cerebellar Purkinje

neurons and hypoplasia of the brainstem and posterior cerebellum (Courchesne, 1997).

Heterozygous lurcher (Lc/+) mice are deficient in the δ2 glutamate receptor gene, which

leads to the loss of all Purkinje neurons postnatally, and these mice exhibit ataxia and

impaired rotarod performance (Martin et al., 2003). Since TRPC3 is strongly expressed in the cerebellum, examining cerebellar neuroanatomy as well as neuromotor coordination in Gtf2i- duplication mice may provide a further correlation between TRPC3 dysregulation and the phenotypic outcomes associated with duplication of 7q11.23. Calcium influx through TRPC3 channels can activate CREB (cAMP/Ca2+-response element binding protein), which is

required for both neuronal survival and memory formation in the hippocampus (Finkbeiner et

al., 1997, Ou and Gean, 2007). Thus, TRPC3 and Ca2+ dysregulation may also be some of the factors underlying the learning and memory deficits observed in both individuals with WBS and individuals with Dup7q11.23 syndrome.

TFII-I is also regulated by c-Src, a tyrosine kinase involved in cellular proliferation, differentiation and signaling (Bromann et al., 2004). Tyrosine phosphorylation of TFII-I by c-Src is required for the nuclear translocation and transcriptional activation of TFII-I

(Cheriyath et al., 2002). Similar to the observed phenotypes in mouse models of WBS, Src- knockout (SrcThl/Thl) mice have deficits in learning and memory as well as increased sociability and hyperactivity (Sinai et al., Submitted Jan 2012). Craniofacial abnormalities

consist of a shortened cranial base and a “toothless” phenotype that includes a lack of incisors and a variable number of molar teeth. SrcThl/Thl mice exhibit hypersociability with the

introduction of an unfamiliar mouse in a clean cage as well in the tube test of social

dominance. Visuospatial and amygdala-dependent learning and memory deficits in the

Morris water maze and cued fear conditioning test, respectively, were observed in these

mice. An increase in the number of ultrasonic vocalizations was elicited in a female-female social reunion paradigm. SrcThl/Thl animals also have motor coordination impairments as

elucidated by the balance beam test and performance on the accelerating rotarod. Many of

the behavioural phenotypes are similar to those found in the Gtf2i+/- animals as well as in

previously generated Gtf2ird1-/- animals.

c-Src tyrosine kinase (CSK) gene is encompassed in chromosome 15q24, and de novo copy

number variations of this genomic region has also been associated with autism-spectrum

disorder (McInnes et al., 2010). CSK phosphorylates Src and leads to inhibition of Src,

which, in turn, results in decreased phosphorylation of downstream targets. Since TFII-I is a

phosphorylation target of Src, altered function of Src may affect the same molecular

pathways that underlie WBS, which may explain the similar behavioural phenotypes

observed in SrcThl/Thl mice and in our Gtf2i and Gtf2ird1 mouse models. The Src-knockout

mice show craniofacial features that are reminiscent of those found in people with WBS,

such as a shortened posterior cranial and abnormal tooth development (Pober and Filiano,

1995, Morris and Mervis, 2000). The Gtf2i gene family is also expressed during tooth

development (Ohazama and Sharpe, 2007), further supporting a common molecular pathway

that is downstream of Src or Gtf2i in teeth.

In SrcThl/Thl mice, TFII-I levels were lower in the nuclear fraction of brain tissue as compared

to wildtype controls, which may be a result of decreased phosphorylation of cytosolic TFII-I

84 in the absence of Src. Since TFII-I is found in lower levels in the nucleus, expression of this protein in the cytosol is predicted to be increased with a corresponding decrease in surface

TRPC3 expression. However, protein analysis of the membrane fraction revealed an unexpected increase in membrane expression of TRPC3 in the Src-knockout mice. TFII-I is phosphorylated by Bruton’s tyrosine kinase (BTK) in B cells, and this phosphorylation is required for binding of TFII-I to PLC-γ. However, it is not yet known whether there is a kinase with similar function as BTK in neurons. It is also possible that there are distinct pools of cytoplasmic TFII-I that are differentially regulated depending on whether it is meant for nuclear translocation or for modulating Ca2+ entry. Src is also required for TPRC3 activation

(Vazquez et al., 2004), and thus, the increased TRPC3 surface expression in Src-deficient mice may be due to an alternative pathway that does not include TFII-I. Thus, further investigation of a common molecular pathway linking Src, TRPC3, and TFII-I is required in order for any definitive associations to be made.

Chapter 4

Conclusions and Future Directions

4.1 Summary

4.1.1 Overview

Williams-Beuren syndrome (WBS) is a developmental disorder associated with

cardiovascular abnormalities and distinctive facial features (reviewed in (Pober, 2010).

People with WBS show distinct cognitive strengths and weaknesses – intellectual disability

(average IQ of approximately 60), relatively preserved language capabilities but poor

visuospatial and math skills. Their behavioural profile includes overfriendly personalities due

to social disinhibition, attention deficit hyperactivity disorder, increased general anxiety, and

specific phobias. WBS is caused by a deletion of approximately 1.55 Mb on chromosome 7,

resulting in the loss of 26 genes, but the specific genes underlying the neurological symptoms remain unclear. However, studies of individuals with atypical 7q11.23 deletions implicate genes in the telomeric region, GTF2I and GTF2IRD1, in the neurological features of the syndrome (Botta et al., 1999, Tassabehji et al., 2005, Antonell et al., 2010). The reciprocal

duplication of the WBS region is becoming a more recognized syndrome, although it is

diagnosed with a lower prevalence than WBS in part due to milder symptoms (Somerville et

al., 2005). A recent study also revealed an individual diagnosed with a triplication of 7q11.23

(Beunders et al., 2010). People with Dup7q11.23 syndrome have both similar and contrasting

phenotypes in comparison to WBS. Individuals with the microduplication have facial features that contrast those observed in WBS, social and non-social anxiety, speech delay and expressive language impairments. Some individuals also exhibit mild to moderate intellectual disability, or autistic features (Berg et al., 2007, Van der Aa et al., 2009). Behavioural analyses of WBS mouse models, either lacking a single copy of Gtf2i or having a combined

deletion of Gtf2i and Gtf2ird1, have revealed additive effects of the deletions. Behavioural

and molecular studies of mice with one and two extra copies of Gtf2i (Gtf2i+/dup and

Gtf2idup/dup, respectively) elucidated gene dosage effects on anxiety levels and altered surface expression of TRPC3. Together, these results suggest that GTF2I may play an important role in the cognitive and neuromotor phenotypes in WBS, as well as in the affective symptoms of both individuals with WBS and individuals with Dup7q11.23.

4.1.2 Haploinsufficiency of Gtf2ird1 and Gtf2i leads to motor learning

deficits, altered anxiety-related behaviours and cued fear

conditioning response, and impaired object recognition memory

A shortened latency to fall off the fixed-speed rotarod was observed in Gtf+/del mice, and

Gtf2i+/- animals also showed a similar trend. This suggests a cerebellar dysfunction, possibly

due to altered neuroanatomy of the cerebellum or its circuitry, and this may explain the motor

deficits in people with WBS (Mervis and Klein-Tasman, 2000). Gtf+/del mice exhibited an

increase in rearing duration on the zero maze, which is suggestive of increased exploratory

behaviour due to decreased anxiety levels. In contrast, this exploratory behaviour appeared to

be slightly decreased in Gtf2i+/- animals. Thus, it is likely that Gtf2ird1, and not Gtf2i, is

involved in the decreased anxiety observed in the deletion mice, consistent with the reduced anxiety observed in Gtf2ird1-/- mice (Young et al., 2008). A recently generated mouse model

with a larger deletion (from Gtf2i to Limk1) of the proximal region of mouse chromosome

5G2 was studied, and increased anxiety and neuromotor impairments were observed (Li et

al., 2009). Mice lacking both Gtf2i and Gtf2ird1 show decreased fear and cognitive deficits

that strongly mimic WBS symptoms. Gtf+/del females exhibit diminished innate fear, and both

Gtf2i+/- and Gtf+/del mice have severe deficits in object recognition memory. Along with

analyses of individuals with atypical deletions in chromosome 7q11.23, the results from these

mouse studies support the hypothesis that genes in the telomeric region of human chromosome 7q11.23, Gtf2i and Gtf2ird1 in particular, are involved in the cognitive and behavioural profile of WBS.

4.1.3 Increased expression of Gtf2i leads to elevated anxiety-related

behaviours and spontaneous self-grooming

A trend toward reduced duration in rearing behaviour was noted in Gtf2i+/dup and Gtf2idup/dup

mice during the zero maze test, which provides an index of anxiety levels. In the open field

test, Gtf2i+/dup mice exhibited increased thigmotactic behaviour and decreased rearing. These

results indicate possibly elevated anxiety-related behaviours that are comparable to the non-

social anxiety observed in people with Dup7q11.23 (Berg et al., 2007). In an initial study of

social anxiety, maternal-separation-induced ultrasonic vocalizations (USVs) were used as an

index of anxiety in mouse pups. This test provides a tool to examine the potential

mechanisms underlying the significantly elevated prevalence of Dup7q11.23 individuals

diagnosed with Separation Anxiety Disorder (Velleman and Mervis, 2011). Gtf2i+/dup mouse pups had a significant increase in the number of USVs whereas Gtf2i+/- pups vocalized

significantly less than their wildtype littermates. Gtf2i+/dup mice also showed a significant

increase in spontaneous self-grooming, a repetitive behaviour that mimics the ASD feature in

Dup7q11.23 individuals (Van der Aa et al., 2009) . These results implicate a gene dosage

effect of Gtf2i in regulating both social and non-social anxiety-related behaviours.

4.1.4 Gtf2i gene dosage affects subcellular localization of TRPC3

Animals with two extra copies of Gtf2i (Gtf2idup/dup) showed a significantly lower expression level of TRPC3 at the membrane, and although not statistically significant, a trend of decreased surface expression of TRPC3 was detected in Gtf2i+/- mice. This is consistent with previous studies suggesting that Gtf2i indirectly regulates TRPC3 localization through

binding to PLC-γ, thus sequestering PLC-γ and preventing it from shuttling TRPC3 to its

functional location in the cell membrane (Caraveo et al., 2006). TRPC3 is involved in both

cerebellar-dependent neuromotor function and memory formation, and thus, alteration in

surface expression of TRPC3 may underlie the motor deficits of people with WBS as well as

learning and memory impairments observed in both individuals with WBS and individuals

with Dup7q11.23 syndrome.

4.2 Future Directions

4.2.1 TRP channel function

TRPC3 appears to be a likely candidate in modulating cognition and neuromotor function.

TRPC3 forms heterotetrameric complexes with TRPC6 (Tai et al., 2009), and it would thus be of interest to study subcellular localization of TRPC6 as well. In addition to altered localization, these channels may also have altered activity or sensitivity to agonist-induced calcium entry. Measurement of free Ca2+ using Fura2 calcium imaging may prove to be a useful tool in providing a more accurate index of calcium regulation in the available mouse models. TRPC3 and CREB are upstream of a molecular pathway that includes BDNF, a neurotrophin that is likely involved in learning and memory processes (Ou and Gean, 2007).

Since learning and memory appear to be altered in both Gtf2i-heterozygous and Gtf2i- duplication mice, electrophysiology techniques can be used to examine synaptic plasticity in these animals. To study how altered TRPC3 localization may affect cationic currents evoked by BDNF, cultured primary hippocampal neurons could be used for voltage-clamp whole- cell intracellular recordings. This would allow for examination of synaptic transmission and plasticity, including possible changes in long-term potentiation or long-term depression.

BDNF gene and protein expression levels could also be used to confirm electrophysiological findings.

Subcellular localization of the TRPC channels can be studied using different brain regions and at various developmental time points. The spatial distribution and mRNA expression levels of the TRPC channel family (TRPC1 to TRPC7) can be determined using real-time

qPCR and validated by western blotting or immunohistochemistry techniques. CREB is a

downstream target of TRPC3-regulated Ca2+ entry, and since CREB is involved in fear

memory formation, fear conditioning responses should also be examined in Gtf2i+/dup and

Gtf2idup/dup mice. Preliminary results from GTF2IRD1 localization studies in our laboratory

show that this protein is expressed in the cytoplasm, similar to TFII-I localization. This

suggests the possibility that GTF2IRD1 may also have a cytoplasmic function, perhaps in the

regulation of a different TRPC channel or in modulating other receptors involved in Ca2+- dependent pathways. TRPC5 has been implicated in regulating amygdala function as well as fear-related behaviour whereby TRPC5-/- mice have impaired synaptic transmission in the

amygdala and diminished innate fear (Riccio et al., 2009). This is similar to phenotypes

observed in the Gtf2ird1-/- mouse, and thus, perhaps TRPC5 may be regulated by GTF2IRD1

in a similar fashion to TFII-I-regulated TRPC3 localization.

4.2.2 Relationship of GTF2I to Autism Spectrum Disorders

Further behavioural characterization of Gtf2i+/dup and Gtf2idup/dup mice may validate a link between Gtf2i copy number and autism-spectrum-related phenotypes, especially since these mice appear to exhibit elevated levels of repetitive behaviours. The marble burying test

during which marbles are buried under thick bedding may be useful in exploring highly

perseverative or “compulsive” behaviour in mice (Thomas et al., 2009). This is a robust test

of repetitive behaviour that is not correlated with other anxiety-related assays such as the

open field or light-dark exploration, and it may allow for a behavioural separation of anxiety

from autism spectrum disorder (ASD) in a mouse model of Dup7q11.23 syndrome. A variant

of marble burying behaviour is known as the shock-probe burying test in which a probe is

used to deliver a small shock to the animal, following which animals try to “bury” the probe

with bedding (Degroot and Nomikos, 2004). The duration of freezing during this test is used as an index of defensive behaviour and can be correlated with fear conditioning data.

It may also be worthwhile to examine social interaction in these mice using other tests to

uncover subtle behavioural abnormalities that could not be detected by the resident intruder

test. The three-chambered test of social approach and variations of it can be used to measure

the tendency of a mouse to interact with another mouse through social behaviours (Crawley

et al., 2007). Social learning and memory can be examined using the three-chambered test by

placing a novel mouse into one of the chambers along with the familiar mouse in the other

compartment. Social interactions such as sniffing, following, social grooming or exploratory

activity can be examined in juvenile mice since ASD is often first diagnosed in younger

children (Crawley, 2007). ASD is a neurodevelopmental disorder, and thus, these ASD-

related behaviours can also be tested in mice at different ages to examine how social

interactions change through development.

Another candidate gene for ASD is Shank3, and this gene encodes a postsynaptic density

protein at glutamatergic synapses (Peca et al., 2011). Disruption of Shank3 is implicated in

the genetic basis of 22q13 deletion syndrome, which is an autism-spectrum (Wilson et al.,

2003). Similar to our Gtf2i+/dup animals, rearing behaviour is significantly decreased in

Shank3B knockout mice (which lack the α and β isoforms of SHANK3) and these mice

groom excessively to the point of self-injurious skin lesions. These non-social and repetitive behaviour phenotypes are comparable to the generalized anxiety disorders as well as repetitive and restricted behaviours observed in individuals with autism spectrum disorders.

Mutations in the MECP2 gene, which encodes methyl-CpG binding protein 2, lead to the symptoms observed in the neurological disorder, Rett syndrome. Mutations in MECP2 lead to intellectual disability and movement abnormalities as well as autistic behaviours (Pearson et al., 2011). Duplication of chromosome Xq28, which encompasses MECP2, is also associated with autism that is comorbid with anxiety (Ramocki et al., 2009). Mecp2 mutant mice show a decrease in social interactions in nest building and avoidance of an unfamiliar mouse in the home cage, and pups vocalize less in recordings of maternal separation-induced

USVs (Moretti et al., 2005, De Filippis et al., 2010). Deletion and duplication of chromosome 16p11.2 have also been associated with autism, and mice with the corresponding deletion on mouse chromosome 7 display restricted behaviours that mimic autistic features (Horev et al., 2011). When placed into large cages with a ceiling, these mice do not habituate to their surroundings and show a continuous and stereotypic ceiling- climbing behaviour that is localized to specific parts of the cage. Collectively, the behavioural tests used in the aforementioned mouse models of autism provide useful tools to examine autism-related features, such as social interactions and restricted stereotypies, in mice. Examining nest-building and other forms of social interactions as well as restrictive behaviours using assays such as the ceiling-climbing test may elucidate additional autism- related behaviours in the Gtf2i-duplication mice.

Involvement of the glutamatergic system in ASD-related behaviours is also evident through

93 the blocking of repetitive self-grooming in BTBR mice following administration of a metabotropic glutamate receptor (mGluR) antagonist (Silverman et al., 2010). Thus, drug studies targeting the glutamatergic system in the Gtf2i-duplication mice may elucidate neurotransmission alterations responsible for the autism-related behaviours observed in these mice.

4.2.3 Altered gene expression

Since the GTF2I family of genes comprises putative transcription factors, it would be of interest to examine gene expression in the Gtf2ird1 and Gtf2i mouse models to elucidate possible alterations. Previous work in our lab examined global gene expression in Gtf2ird1-/- mice, but no changes were found (O'Leary and Osborne, 2011). However, this may be because whole brain was used in the microarray studies, and if regional or cell-specific alterations are present, these may be masked by heterogeneous tissues and cell populations.

For instance, microarray experiments were performed in MECP2 transgenic mice using only the amygdala since the amygdala is a region that is associated with social and anxiety-related behaviours (Samaco et al., 2012).Thus, regional gene expression could be examined in Gtf2i- heterozygous and duplication mice in regions such as the amygdala, hippocampus, and frontal cortex, which are implicated in learning and memory as well as affective behaviours.

Gene expression in the cerebellum could provide further support for the motor impairments and altered TRPC3 expression observed in the mice, and since Gtf2i is highly expressed in cerebellar Purkinje neurons, this cell population should also be a candidate for the study of gene expression alterations.

4.2.4 Anxiety

ASD is characterized by impaired social interactions and communication, restricted and

repetitive behaviours as well as behaviours that are resistant to change (Rodgers et al., 2011).

With respect to maternal separation anxiety, children with Dup7q11.23 have an elevated

incidence (33%) compared to children with WBS or those in the normal population (less than

5%) (Velleman and Mervis, 2011). Consistent with this, mice with a duplication of Gtf2i

vocalized more when separated from their dams in comparison to WT mice. mRNA

expression levels of TGFα (transforming growth factor-α) have been found to be

significantly decreased in the prefrontal cortex of neonatal mice following maternal

separation (Romeo et al., 2004) and would thus provide another index of separation anxiety

in Gtf2i+/dup pups. Corticosterone levels in the dams could also be examined, as prenatal

stress has been found to lead to altered hippocampal and cortical function in mice

(Mychasiuk et al., 2012).

Generalized anxiety in the Gtf2i-heterozygous and Gtf2i-duplication animals can be

examined using other tests of anxiety-related behaviours such as the light-dark box and the

Vogel thirsty-lick conflict test. Mice that have higher levels of anxiety tend to explore the

dark compartment more than WT animals in a light-dark box (Tamada et al., 2010). The

Vogel thirsty-lick conflict test, in which mice deprived of water for 48 hours are given a

small electrical shock during the test when they lick a water-containing tube, is also sensitive to anxiolytic drugs (Vogel et al., 1971, Crawley, 2007). Neuroanatomical structures and

volumes can also be examined to determine whether or not there are differences due to gross

or regional brain architecture. Serotonin levels in individuals with ASD have been found to

be altered (Lam et al., 2006) and serotonin has also been implicated in anxiety-related

behaviours (Heisler et al., 1998). In rats, altered dopamine transmission may be related to

repetitive self-grooming behaviours (Taylor et al., 2010). Thus, it would be of interest to

examine serotonergic and dopaminergic transmission in mouse brain, examining potential

alterations both spatially and temporally. Monoamine quantification of serotonin and

dopamine as well as its metabolites would provide a measure of neurotransmitter levels and

turnover.

4.2.5 Genetic background effects

Individuals with atypical deletions of 7q11.23 may provide insights into the genetic basis of the disorder, but there are very few of these cases, and those who have been identified have different deletion breakpoints in chromosome 7q11.23. The difference in genetic backgrounds is an additional complexity that makes comparisons difficult between individuals with atypical deletions, although affected individuals from the same family allow for comparisons as a result of identical deletion breakpoints and similar genetic backgrounds

(Osborne, 2010). Fortunately, animal models can be genetically engineered to study specifically targeted genes of interest, and mouse models of WBS allow for the dissection of genotype-to-phenotype correlations using a large number of mice that are on the same genetic background. Many different congenic strains of mice are used to elucidate the function of specific genes, and in studies of behavioural phenotyping, common mouse strains such as 129, CD1, and C57BL/6J are used (Bouwknecht and Paylor, 2002, Crawley, 2008).

However, each mouse strain has a different level of underlying anxiety as well as other background genes that may be confounding factors in determining behavioural performance.

Serotonin transporter knockout mice display anxiety-like behaviours when backcrossed onto

C57BL/6J, but these phenotypes are absent in mice with a 129/SvEvTac background

(Holmes et al., 2003). The phenotypic discrepancies (e.g. social interactions and anxiety- related behaviours) between the Gtf2i gene trap mice generated by Sakurai et al. (2010) and our Gtf2i gene trap mice may also be explained by genetic background differences – Sakurai et al. maintained their mice on a C57Bl/6Tac background, whereas our mice were backcrossed onto a CD1 background. However, since both mouse models have mixed backgrounds, there may be behavioural variability due to the mixed backgrounds and the influence of flanking genes that remain from the embryonic stem cell lines. Thus, it would be ideal to generate mutant mice on a pure genetic background to eliminate any confounding factors in order to provide unbiased behavioural interpretations and comparisons between mouse models of WBS.

4.3 Conclusion

This study has elucidated genotypic correlations of Gtf2i and Gtf2ird1 haploinsufficiency as well as increased Gtf2i copy number to phenotypic outcomes. However, the molecular bases underlying these phenotypes have not yet been examined in detail. Thus, the wide array of behavioural and mechanistic experiments that can be used to examine Gtf2i copy number and its downstream pathways will certainly prove to be useful in further dissecting the genotype- to-phenotype correlations in WBS and Dup7q11.23 syndrome.

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