Effects of Altered Gtf2i and Gtf2ird1 Expression on the Growth of Neural Progenitors and Organization of the Mouse Cortex

Effects of altered Gtf2i and Gtf2ird1 expression on the growth of neural progenitors and organization of the mouse cortex

Hyemin Amy Oh

A thesis submitted in conformity with the requirements For the degree of Master of Science

Institute of Medical Science University of Toronto

Effects of altered Gtf2i and Gtf2ird1 expression on the growth of neural progenitors and organizations of the mouse cortex

Hyemin Amy Oh

Master of Science Institute of Medical Science University of Toronto

2013 Abstract

Williams-Beuren syndrome (WBS) and 7q11.23 Duplication Syndrome (Dup7) are rare neurodevelopmental disorders associated with a range of cognitive and behavioural symptoms, caused by the deletion and duplication, respectively, of 26 genes on human chromosome 7q11.23. I have studied the effects of deletion or duplication of two candidate genes, GTF2I and GTF2IRD1, on neural stem cell growth and neurogenesis using cultured primary neuronal precursors from mouse models with gene copy number changes. I found that the number of neuronal precursors and committed neurons was directly related to the copy number of these genes in the mid-gestation embryonic cortex. I further found that in late-gestation embryos, cortical thickness was altered in a similar gene dose-dependent manner, in combination with layer-specific changes in neuronal density. I hypothesize that some of the neurological features of WS and Dup7 stem from these impairments in early cortical development

Acknowledgement

I would like to take this opportunity to thank Dr. Lucy Osborne for her continuous support and guidance. She is not just a supervisor but also a mentor as well as a motherly figure for me. The bright mood of the lab was the reason why I chose this lab for my masters and is the reason to continue to do my studies in the same lab for doctorial degree. I also like to extend my appreciation to supervisory committee members Dr. Freda Miller and Dr. Vincent Tropepe for their insightful suggestions, technical assistance and guidance. I give my deepest gratitude to Dr. Freda Miller for providing a research location, and to David for continuous guidance and feedbacks, without the support I would have not been able to finish my project.

All the former and current members of the Osborne lab have made every corner of my master life pleasurable and exciting, allowing me to persevere despite all the curved bullets thrown by both negative and positive results I have encountered throughout the course of my masters. In particular, I owe a big gratitude to Emma for unconditional support, companionship (let us sail together in this boat and we will reach the new land), and irresistible art on the lab wall, to Elaine for maintaining animal colony and delightful lab- licious lunch, to Jennifer for delicious baked goods and exquisite suggestions for the thesis writing, to Emily for the kindly advices and directions especially when I faced with animosity, to Eli for brining colourful smiles through a stacks of intelligence and refinement, and lastly to Joana for allowing me to grow patience and extend goals to the level that I have never imagined before. I would also like to thank Victor, my previous supervisor, and mentor for inspiring me to become an elegant and insightful scientist. I also like to thank my friends especially BK, for listening to my abysmal concerns and doubts, and giving assurance and mental therapy sessions to solve problems, and for relieving the distress through countless numbers of sweets and food visits.

None of this would have been even possible without support and unconditional love from my family, especially my parents. They encouraged me to be the best of myself and to

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persevere until I achieve my goals. Their love, sacrifice and wisdom are the reason for who I am and how I stand here.

Table of Contents

Abstract------ii

Acknowledgements------v

Table of Contents------vi

List of Tables ------ix

List of Figures ------x

List of Abbreviations ------xii

Chapter 1. Introduction ------1

1.1 Williams-Beuren Syndrome------1 1.1.1 History of Williams-Beuren Syndrome: 1 1.1.2 Williams-Beuren syndrome clinical phenotype 2 1.1.3 Williams-Beuren syndrome cognitive and behavioral phenotype. 6 1.1.4 Genetic Basis of WBS 8

1.2 7q11.23 Duplication Syndrome ------11

1.3 Genotype –Phenotype correlations------14 1.3.1 Implications of GTF2I and GTF2IRD1 in behavioural and

cognitive profiles of WBS 15

1.4 GTF2I gene family ------18 1.4.1 GTF2I 19 1.4.2 GTF2IRD1 22

1.5 GTF2I gene family animal models ------23 1.5.1 Gtf2ird1 mouse model 24 1.5.2 Gtf2i mouse model 26

1.6 Development of the cerebral cortex ------30 1.6.1 Genesis of the cerebral cortex 30 1.6.2 Signaling pathways in cortical development 34

1.7 . Neural mechanisms in WBS ------36 1.7.1 Neural mechanisms of impaired social processing in WBS 36 1.7.2 Role of Serotonin in emotional behaviors 38 1.7.3 Altered brain connectivity in Gtf2ird1 targeted mice 39

1.8 Research Aims and hypothesis------41

Chapter II. Disturbance in early neuronal development 42 in mouse models of WBS

2.1 Introduction ------42 2.1.1 Mouse models 42 2.1.2. Neural Precursor Cells 44

2.2 Material and Methods ------45 2.4.1. Animals 45 2.4.2 Genotyping 46 2.4.3. Cortical Precursor cell culture 47 2.4.4. Immunohistochemistry 48 2.4.5. Microscopy and Quantification 50

2.4.6. Statistical Analysis 51

2.3 Results ------53 2.3.1. Gtf2i and Gtf2ird1 copy number variation regulates cortical precursor physiology 53 2.3.2. Hemizygous deletion of Gtf2i and Gtf2ird1 disrupts cortical precursor physiology and neurogenesis 57 2.3.3 Gtf2i duplication promotes proliferation and differentiation of cortical precursors but does not affect survival 63 2.3.4. Effects of a single gene Gtf2i+/del on precursor maintenance and neurogenesis. 69 2.3.5. Altered cortical layer organization in mouse models of WBS 71

Chapter III. Discussion and conclusions ------74

3.1 Effects of Gtf2i and Gtf2ird1 on precursor cell biology ------74 3.2 Possible interactions between Gt2i and Trk signaling in cortical

development ------77 3.3 Gtf2i and Gtfi2rd1 plays a role in laminar organization and cell density of cortices ------83

Chapter IV. Conclusions and Future Directions 88

4.1 Summary ------88 4.1.1. Overview 88 4.1.2. Gtf2i and Gtf2ird1 play a crucial role in radial glial cells maintenance and differentiation without affecting proliferation and survival 90 4.1.3. Gtf2i and Gtf2ird1 copy number variation regulates cortical architecture and cell density in developing cortex 92

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4.2 Future Directions ------95 4.2.1. Balance between different cellular components: 95 4.2.1.1 defining Precursor differentiation 95 4.2.1.2. Defining precursor population 96 4.2.2. Molecular mechanism: relationship between TFII-I and ERK1/2 in neural precursor physiology 97 4.2.3 Cortical cytoarchitecture 98 4.2.4. Circuits and networks - synaptic function 100 4.2.5. Contribution of individual genes to neural development during prenatal and postnatal periods 103 4.2.6. Human models of WBS - induced pluripotent stem cells 105

4.3 Conclusion ------106

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List of Tables

Table 1.1 Clinical presentations of Williams-Beuren syndrome

Table 1.2 The percentage of diagnosed WBS with different deletion size due to nonallelic homologous recombination between respective LCRs.

Table 1.3 Comparison between WBS Duplication and WBS deletion clinical phenotypes

Table 1.4 The summary of mouse models with varying copy number of Gtf2i and Gtf2ird1.

Table 2.1 List of primers used and its sequence.

Table 2.2 List of observed genotypes in each cross.

Table 3.1 The summary of research findings.

Table 3.2 Altered cortical architecture in different developmental disorders with cognitive impairments.

List of Figures

Chapter I

Figure 1.1 The distinctive facial features of WBS individuals.

Figure 1.2 The drawings of Down syndrome (DS) and WBS individuals displaying dissociation in visuospatial construction cognition.

Figure 1.3 Schematic diagrams of WBS region on chromosomal region 7q11.23.

Figure 1.4 The comparison between individuals with WBS deletion and WBS duplication.

Figure 1.5 The genetic mapping of the reported cases of atypical deletion and typical WBS deletion.

Figure 1.6 The protein structure diagram of the GTF2I gene family

Figure 1.7 Timed genesis of neurons, astrocytes and oligodendrocytes in the developing neocortex.

Figure 1.8 Schematic diagram of neocortex during neurogenesis.

Chapter II

Figure 2.1 Schematic diagrams of individual mice lines used in the study.

Figure 2.2 The representative picture of nucleus staining.

Figure 2.3 The effects of Gtf2i and Gtf2ird1 copy number variants on cortical precursor cells.

Figure 2.4 The effects of Gtf2i and Gtfi2rd1 deletion on single embryo NPCs at 1

d.i.v

Figure 2.5 The effects of Gtf2i and Gtfi2rd1 deletion on single embryo NPCs at 3 d.i.v

Figure 2.6 The effects of Gtf2i duplication on neural stem cell physiology.

Figure 2.7 Gtf2i duplication promotes precursor cell maintenance through increased proliferation

Figure 2.8 The effects of sing gene knockout of Gtf2i on regulation of precursor cell maintenance.

Figure 2.9 Early changes in neural precursor cells persist into later stages of cortical layer formation and organization.

Chapter III

Figure 3.1 Trk Signaling disruptions in NCFC family of syndrome.

Figure 3.2 The involvement of Trk signaling in regulation of cell-fate determination, proliferation, survival, and differentiation of cortical precursors.

List of Abbreviations

ACE Agonist-induced calcium entry ADHD Attention hyperactivity disorder ANOVA Analysis of variance BAP-135 Bruton’s tyrosine kinase associated protein 135 BCR B-cell antigen receptor BDNF Brain-derived neurotrophic factor BP Basal progenitor Btk Bruton’s tyrosine kinase CBP CREB binding protein CC Corpus callosum C/EBP CCAAT/enhancer binding protein cen Centromeric CFC Cardio-facio-cutaneous syndrome CNT Ciliary neurotrophic factor CP Cortical plate CS Costello syndrome cGMP Cyclic guanine monophosphate CLIP2 CAP-Gly domain-containing linker protein 2 d.i.v. Days in vitro DE Distal control element DS Down syndrome EGF Epidermal growth factor ELN Elastin ES Embryonic stem G-kinase Cyclic guanine monophosphate - dependent protein kinase gsc goosecoid GTF General transcription factor GTF2I General transcription factor 1 GTF2IRD1 General transcription factor 2I repeat domain containing I GTF2IRD2 General transcription factor 2I repeat domain containing 2 hES human embryonic stem HLH Helix-loop-helix LIF Leukemia inhibitory factor LIMK LIM kinase 1 LS LEOPARD syndrome LZ Leucine zipper med Medial MEK Mitogen activated Erk kinase MRI Magnetic resonance imaging NAHR Nonallelic homologous recombination NCFC Neuro-cardio-facial cutaneous family of syndromes NE Neural epithelial NGF Nerve growth factor NF1 Neurofibromatosis type 1 xii

NPC Primary neural cortical cells NO Nitrogen oxide NS Noonan syndrome NT-3 Neurotrophin-3 NT-4 Neurotrophin-4 OFC Orbitoforontal cortex PDGF Platelet-derived growth factor PE Proximal control element PH Pleckstrin homology PLC-γ Phospholipase C-γ Rb Retinoblastoma protein RG Radial glial cell RS Rett’s syndrome RT Rubinstein-Tayi syndrome SH2 Src-homology 2 SHP-2 Src homology domain protein tyrosine phosphatase-2 SPIN Serum response –factor – Phox1- interacting protein SRF Serum response factor SVAS Supravalvular aortic stenosis SVZ Subventricular zone tel Telomeric TrkA Tyrosin-kinase receptor-A TrkB Tyrosin-kinase receptor-B TrkC Tyrosin-kinase receptor-C TRPC Transient receptor potential channels VZ Ventricular zone WBS Williams-Beuren syndrome 5-HT Serotonin

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Chapter I. Introduction to WBS

1.1 Williams-Beuren Syndrome

1.1.1 History of Williams-Beuren Syndrome:

The first case of Williams-Beuren syndrome (WBS) was reported 60 years ago in Great

Britain and Switzerland as form of infantile hypercalcemia due to excess intake of vitamin

D (Lightwood, 1952). Most infants with a mild form of infantile hypercalcemia were treated with adjustment of Vitamin D supplementation in food, which was different from the severe form of infantile hypercalcemia, where additional abnormal features were present, including an elfin facial appearance, congenital heart disease, and mental retardation

(Jones, 1990; Lowe et al., 1954). The question of whether these two forms of hypercalcemia were two distinct disorders or variations of the same disorder displaying different degrees of severity was unsolved until the 1960s. In 1961, Williams et al reported four children from New Zealand with supravalvular aortic stenosis (SVAS), a distinct facial appearance, and mental retardation, in the absence of infantile hypercalcemia, and made a note of it as a distinct syndrome (Williams et al., 1961). In 1962, Beuren et al., also documented three patients with similar symptoms and were the first to describe a “friendly nature” as a behavioral feature of the syndrome.

Although clinical phenotypes of WBS have been continuously documented, the etiology and phenotype - genotype relationship only began to be explored in the early 1990s. The

2 genetic basis of WBS was first hinted at by the identification of a disruption of the elastin gene (ELN) by a translocation in a family with SVAS (Currant et al., 1993). A subsequent study revealed haploinsufficiency for ELN in individuals with WBS and it was determined that WBS was caused by a microdeletion mapped to the ELN gene on chromosome 7q11.23

(Ewart et al., 1993). The deletion has now been shown to encompass approximately 1.5 million base pairs of chromosome 7q11.23, resulting in a loss of 26 genes. (Pober 2010).

Although it is clear that hemizygosity for ELN is the cause of SVAS in WBS, the contribution of other genes within the deletion to the multifaceted phenotype of WBS remains less well defined.

1.1.2 Williams-Beuren syndrome clinical phenotype

The characteristic cardiovascular abnormality includes elastin arteriopathy, predominantly

SVAS, which is present in about 70% of WBS individuals, with 30% of those individuals requiring surgical intervention (Collins et al., 2010). Other connective tissue phenotypes proposed to be due primarily to ELN haploinsufficiency include bowel and bladder diverticulitis (leading to increased urinary frequency and structural abnormalities), soft skin, hernias, and a hoarse voice (Sammour et al., 2006).

The distinctive facial features of WBS include long nasal columnella, bitemporal narrowing, full nasal tip, long philtrum, broad forehead, periorbital fullness, wide mouth, full cheeks, and small jaw with widely spaced small teeth (Fig. 1.1).

Figure 1.1. The distinctive facial features of WBS individuals.

Young children with WBS often have short stature with developmental delay, hypercalcemia, chronic constipation, gastroreflux, and failure to thrive (Morris CA., 2010).

Abnormal glucose metabolism is observed in adults with WBS (Pober et al., 2010)

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

Affected system Symptom Prevalence (%)

Cardiovascular Any cardiovascular disease 84

Supravalvular aortic stenosis 69

Supravalvular pulmonary stenosis 34

Hypertension 17

Nervous system Developmental delay and cognitive deficits 97

Attention deficit hyperactivity disorder 84

Specific phobias 96

Visuospatial construction deficit 67

Increased generalized anxiety 57

Hyperreflexia 40-70%

Ocular Esotropia 50

Hyperopia 24

Auditory Hyperacusis 90

Dental Malocclusions 85

Microdontia 55

Genitourinary Urinary tract infections 29

Gastrointestinal Vomiting in infancy 45

Constipation 43

Obesity 29

Endocrine Diabetes mellitus 75

Hypercalcemia 5 - 50

Hypothyrodism 15 -30

Musculoskeletal Curved spine 21

Gait abnormalities 60

Integumentary Dysmorphic facial features 96

Hoarse, low voice 95

Mild premature aging of skin 60

1.1.3 Williams-Beuren syndrome cognitive and behavioral phenotype:

Individuals with WBS have distinct peaks and valleys of cognitive function. They present with mild to moderate intellectual disability, with an average overall IQ of between 55 and

60. (Pober and Dykens, 1996). The neuropsychological profile is characterized by relative preservation of expressive language skills, verbal short-term memory and ability to recognize and process human faces, co-existing with severe deficits in mathematical skills and visuospatial cognition (writing, drawing, pattern construction) (Mervis et al., 2000).

Individuals with WBS fail to integrate individual components into a global whole, which is in direct contrast to the global cognitive deficit seen in individuals with Down syndrome

(DS), who fail to see the individual components and see only the whole (Bihrle et al., 1989)

(Fig 1.2).

Matched in age and IQ

Bellugi et al. 1990, Am J Med genet

Fig 1.2. The drawings of Down syndrome (DS) and WBS individuals displaying dissociation in visuospatial construction cognition.

WBS individuals display very unique social behavior that includes social disinhibition, overfriendliness and empathy (Bellugi et al., 1999; Kelein-Tasman and Mervis, 2003). The characteristic social profile also includes a decreased fear of strangers but increased nonsocial anxieties and a high incidence of specific phobias (Bellugi et al., 1990). Attention deficit hyperactivity disorder (ADHD)is also very common.

Changes in anatomical structures of the brain have been associated with the distinct cognitive and behavioral phenotype. These neuroanatomical abnormalities include volumetric and morphological changes. Grey matter was reduced in the intraparietal sulcus, the orbitofrontal cortex, and the region around the ventricle in a study of adults with WBS and IQs within the normal range (Meyer-Lindenberg et al., 2004). Magnetic resonance imaging (MRI) of WBS individuals with average IQ of 47.87 showed reduction in the corpus callosum (CC) cross sectional area and volume, and changes in CC morphology with larger bending angles and increased thickness (Sampaio et al., 2012). Post mortem analysis of three individuals with WBS showed a decrease in brain size with altered cell size and increased cell density in layer IV of the primary visual cortex of the left hemisphere (Galaburda et al., 2002). The mechanisms underlying how such neuroanatomical changes are expressed as cognitive or behavioural profiles in WBS has yet to be examined.

1.1.4 Genetic Basis of WBS:

WBS is a rare neurodevelopmental disorder that occurs in frequency of 1 in 7,500 to

20,000 in live births (Greenberg 1990; Stromme et al, 2000). WBS is caused by the hemizygous deletion of approximately 1.55 million nucleotides at chromosomal region

7q11.23. The deletion occurs due to unequal homologous recombination between highly

9 similar LCRs flanking WBS region, during meiosis. The interchromosomal rearrangements between homologous chromosomes constitute of two thirds of the recombination and one third is due to intrachromosomal rearrangements between sister chromatids (Cusco et al.,

2008). This results in the loss of between 26 and 28 genes depending on the deletion breakpoint. The WBS deletion region consists of 1.2 Mb of single copy gene and 3 LCRs

(A,B,C) located within the centromeric (cen), medial (med), and telomeric (tel) regions of the WBS interval: A-B-C cen, A-B-C mid, A-B-C tel (Fig 1.3).

Figure 1.3. Schematic diagrams of WBS region on chromosomal region 7q11.23. The centromeric (c), medial (m) and telomeric (t) LCRs are shown as coloured arrow. The direction of arrow indicates the direction of each LCR blocks. Adapted from Merla et al. 2010, Hum Genet.

The common WBS deletion of 1.55Mb occurs between centromeric B and medial B blocks.

A deletion of 1.8Mb can also occur in 5% of typical WBS individuals due to recombination between centromeric A and medial A blocks (Table 1.2). The higher sequence homology of

B-cen and B-mid LCRs and smaller interstitial size between the blocks are thought to be the primary reason for the higher occurrence of the 1.5Mb deletion (Schubert, 2009).

Table 1.2. The percentage of diagnosed WBS with different deletion size due to nonallelic homologous recombination between respective LCRs.

Percentage of Unequal meiotic recombination Deletion Size diagnosed WBS between respective LCRs

> 95% B-cen and B-mid 1.5Mb

3-5% A-cen and A-mid 1.8 Mb

2-3% Atypical deletion Various sizes

1.2 7q11.23 Duplication Syndrome:

The WBS microdeletion arises by nonallelic homologous recombination (NAHR) between

LCRs flanking the WBS region and this recombination can also give rise to the reciprocal duplication of the region, leading to a separate disorder known as 7q11.23 Duplication

Syndrome (Dup7)(Somerville et al., 2005). The clinical phenotype of individuals with Dup7 is distinct from many of the typical WBS deletion clinical features (Table 1.3). The Dup7 individuals display less distinct and contrasting facial features including broad forehead, high and broad nose, short philtrum, and thin lips (Fig 1.4) (Van der Aa et al., 2010). Dup7 individuals often have severe delay in speech and language with preserved visuospatial skills (Berg et al., 2007; Depienne et al., 2007) whereas WBS deletion individuals have relatively preserved expressive language skills with deficits in visuospatial skills (Klein-

Tasman and Mervis 2003). The behavioural features of Dup7 individuals include extreme shyness around strangers, social anxiety, repetitive behaviours and aggressive behavior, which are in direct contrast with social disinhibition and hypersociability observed in individuals with WBS deletion (Berg et al., 2007; Depienne et al., 2007). The inversion of the 7q11.23 region can also occur due to intrachromatid misalignment between LCR blocks with inverted orientation (Osborne et al., 2001). The resulting paracentric inversion leads to no recognizable phenotype, but the inversion carrier is at a higher risk of generating the

WBS deletion in their gametes during meiosis (Scherer et al. 2005; Hobart 2010).

Table 1.3. Comparison between WBS Duplication and WBS deletion clinical phenotypes

WBS Duplication WBS Deletion

Facial features Short philtrum Long philtrum

Thin lips Full lips

Larger teeth Small teeth

Arched palate Normal palate

Narrow forehead Broad forehead

Normal periorbital area Periorbital fullness

Cognitive and Developmental delay Developmental delay Behavioral abnormalities Severe delay in speech and Relative strength in expressive

language language

Relative strength in visuospatial Deficits in visuospatial skills

skills Excessively social

Deficits in social interaction

Children with WBS

Children with 7q11.23 duplication syndrome

Figure 1.4. The comparison between individuals with WBS deletion (top) and WBS duplication (bottom).

1.3 Genotype –Phenotype correlations

Individuals with atypical deletions that do not span the entire WBS region provide an opportunity to study the contribution of specific genes to the WBS phenotype. To date, apart from the direct implication of ELN in SVAS, the specific genes that contribute the many symptoms of WBS have yet to be determined. This is due to the large number of genes deleted in WBS, the relatively small number of individuals with atypical deletions, and the potential for gene interaction to beget combinatorial effects on the phenotype.

However, a number of studies have shown genotype-phenotype correlations of a few genes, including LIM kinase 1 (LIMK1) and CAP-Gly domain-containing linker protein 2 (CLIP2).

Some studies have suggested implication of LIMK1 in visuospatial construction deficits in

WBS (Frangiskakis et al., 1996; Wang et al., 1998). Yet this finding is inconsistent with another study where three individuals with similar deletions including LIMK1 showed no

WBS cognitive abnormalities (Tassabehji et al. 1999). Previous studies of individuals with atypical deletion that did not include CLIP2 gene and who exhibited milder phenotype with absence of visuospatial and motor skills deficits, suggested CLIP2 may contribute to the motor and cognitive phenotype (Hoogenraad et al., 2002; van Hagen et al., 2007). However, the recent identification of a family with an interstitial CLIP2 deletion and no observable phenotype argues against a major role for CLIP2 in WBS (Vandeweyer 2012).

1.3.1 Implications of GTF2I and GTF2IRD1 in behavioural and cognitive profiles of WBS

There are more clinical studies as well as mouse studies that provide evidence that deletion of genes toward the distal end of the WBS region are associated with the neurocognitive features of the disorder (Fig 1.5).

The primary candidates implicated in the neurocognitive profiles of WBS are the General

Transcription Factor (GTF) 2I gene family. Studies of individuals with different-sized deletions of the WBS region implicate these genes as being important in the neurological features of this disorder (Hirota et al. 2003;Tassabehji et al. 2005;Antonell et al., 2010).

Individuals with atypical deletion that leave these genes intact display a milder phenotype than that of individuals with typical WBS.

Three atypical patients carrying smaller deletions that left GTF2I and GFT2IRD1 intact have no distinct WBS facial features and their visuospatial construction skills and abilities to visualize at global and local levels are spared, suggesting contribution of GTF2I and

GTF21RD1 to visuospatial processing deficits in typical WBS (Hirota et al., 2003). This finding is further supported by another study by Antonell et al (2010), where atypical patients with intact GTF2I and GTF2IRD1 have normal visuospatial skills with low-average general intelligence (IQ 70-80) (Antonell et al., 2010).

Deletion of GTF2I also has been implicated in intellectual disability in WBS. In the study by

Morris et al. (2003), five families with various deletion sizes but with no deletion of centromeric FKBP6 or GTF2I were assessed for their phenotypes. Although some WBS cognitive and behavioural features are present in all family members, none had intellectual disability. A patient with an atypical deletion that included FKBP6 but left GTF2I intact, did not exhibit intellectual deficits (Karmiloff-Smith et al., 2003), further reinforcing implication of GTF2I deletion in general intellectual disability.

Deletion either GTF2I or/and GTF2IRD1 may contribute to social behaviour profiles of WBS.

An atypical patient, haploinsufficient for GTF2IRD1 but with normal GTF2I expression levels, exhibited a delay in language acquisition but distinct craniofacial features and hypersociability were not observed (Tassabehji et a., 2005). Unusual social profiles were also observed in two case studies of children with different deletion sizes of the WBS region (Karmiloff-Smith et al., 2010). A child with intact GTF2I and deletion of GTF2IRD1 displayed autistic behaviours including decreased social motivation and deficits in social cognition but social disinhibition was not present. On the contrary, another child with intact GTF2IRD1 and deletion of GTF2I showed hypersociability with strong social motivation and social disinhibition but less than the typical WBS individuals.

1.4 GTF2I gene family

The GTF2I gene family is composed of a three-member group of transcription factors,

General Transcription Factor 2I (GTF2I), GTF2I Repeat Domain containing I (GTF2IRD1) and GTF2I Repeat Domain containing 2 (GTF2IRD2). GTF2I and GTF2IRD1 are consistently deleted in typical WBS, whilst GTF2IRD2 is variably deleted. Proteins of the GTF2I gene family are characterized by multiple helix-loop-helix (HLH) I-repeat domains for DNA- binding and protein interaction, an N-terminal leucine zipper (LZ) for homomeric dimerization, and a nuclear localization signal for entry into the nucleus (Hinsley et al.,

2004)

Fig. 1.6. The protein structure diagram of the GTF2I gene family

1.4.1 GTF2I

GTF2I was the first to be characterized among the GTF2I gene family. It codes for the TFII-I protein with six HLH I-repeats for DNA binding and protein interactions (Roy et al., 1997) and a LZ and basic region, which are important for DNA binding (Cheriyath et al., 2002).

There are four isoforms of TFII-I – α, β, γ, and Δ (Roy et al., 2001). Each isoform has a different expression profile, with the γ-isoform predominantly expressed in neuronal cells.

TFII-I β and Δ also localize at different subcellular compartments – the β-isoform localizes in the nucleus while Δ-isoform localizes in the cytoplasm. Upon activation by growth factor signaling, the Δ-isoform binds to extracellular signal-regulated kinase 1, and 2 (ERK1/2) and translocates into the nucleus, whereas the β-isoform is transported into the cytoplasm

(Hakre et al., 2006). The differential gene expression and localization of proteins infer that all isoforms despite structural similarity may serve non-redundant functions. TFII-I isoforms can form homomeric and heteromeric interactions, which may facilitate nuclear localization and serve as the mechanism for differential gene expression.

TFII-I is an ubiquitously expressed multifunctional transcription factor, which can act both as a basal factor through the initiator element (Inr) and an activator through the upstream promoter element, E-box (Roy et al., 1991). TFII-I is the target of many extracellular signaling pathways and is activated by phosphorylation. TFII-I was originally identified as

BAP-135 (Bruton's tyrosine kinase (Btk) Associated Protein 135 kDa). BAP-135 interacts with Btk in the b-cell antigen receptor (BCR) signalling pathway. The cross-linking of

20 immunoglobulin activates the nonreceptor molecule Btk, which in turn activates BAP-135 through tyrosine phosphorylation (Yang and Desiderio., 1997). Upon activation by Btk, the transcription factor translocates into nucleus enhancing the transcriptional activity

(Novina et al., 1999).

TFII-I is also involved in growth factor signaling through regulation of c-fos expression.

TFII-I was found to be identical in sequence to SPIN (Serum response factor – Phox1

Interacting Protein), which forms protein-protein complexes with serum response factor

(SRF) and the homeodomain Phox 1 to regulate c-fos (Curran et al., 1985; Ceccatelli et al.,

1989). The c-fos promoter is induced by epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and serum, which also activate TFII-I (Kim et al., 1998). c-fos expression is also regulated by nitrogen oxide (NO)/ cyclic guanine monophosphate

(cGMP)/cGMP-dpendent protein kinase (G-kinase) signal transduction pathway. cGMP causes nuclear import of G-kinase, and in the nucleus, G-kinase Iβ directly interacts with and phosphorylates TFII-I, acting synergistically to enhance transactivation of c-fos promoter (Casteel et al., 2002).

TFII-I is both present in the nucleus for its transcriptional function and in the cytosol where it inhibits agonist-induced calcium entry (ACE) (Caraveo et al., 2006). Activation of phospholipase C-γ (PLC-γ) results in an increase in intracellular calcium through the

21 release of calcium from intracellular stores and calcium entry through transient receptor potential channels (TRPC) located at the plasma membrane. PLC-γ binds to TRPC3 subunits through its pleckstrin homology (PH) domain (van Rossum et al., 2005) and increases intracellular calcium by increasing surface accessible TRPC3 channels. It was found that TFII-I also interacts with the Src-homology 2 (SH2) domain of PLC-γ and that

TFII-I can be phosphorylated by PLC-γ. Activated TFII-I act as a competitive inhibitor; it competes with TRPC3 in sequestering PLC-γ, and inhibits ACE (Caraveo et al., 2006).

TRPC3-dependent calcium increase may be involved in the regulation of neural stem cell proliferation and neuronal differentiation (Wu et al., 2004). It is also proposed that TRPC3 channels may regulate synaptic plasticity through a BDNF (Brain Derived Neurotrophic

Factor) induced signaling pathway (Amaral and Pozzo-Miller, 2007). BDNF is known to be a potent mediator involved in brain development and plasticity (Barnabe-Heider and Miller

2003; Jones et al., 1994; Rossi et al., 2006). BDNF triggers activation of TrkB receptors which activates the PLC-γ –IP3 pathway, leading to mobilization of calcium through increasing surface TRPC3. This increase in intracellular calcium results in dendritic remodeling with increased hippocampal dendritic spine density. As a competitive inhibitor of TRPC3 channels, TFII-I may have an indirect role in regulating intracellular calcium and may affect synaptic plasticity. TFII-I is abundant in the dendrites of cerebellar Purkinje cells, hippocampal interneurons and CA1 regions of hippocampus (Danoff et al., 2004 ). In both of brain regions, BDNF are expressed and actively regulate neuronal differentiation and synaptogenesis, and maintenance, and these regions are also affected in WBS. TFII-I may therefore have an influence on neuronal development and its hemizygosity may account for some of the neurological features of WBS.

1.4.2 GTF2IRD1

GTF2IRD1 (identical in its sequence to MusTRD1, BEN, WBSCR11, CREAM, and GTF3) was first identified as MusTRD1, an activator of troponin I in slow muscle fibers (O’Mahoney et al., 1998). Its structure is similar to TFII-I with five HLH I-repeats in human (six HLH I- repeats in mice), and N-terminal LZ and basic region for DNA binding. There were 11 isoforms of Gtf2ird1 found in mouse skeletal muscle (Tay et al., 2003). GTF2IRD1 was subsequently identified as WBSCR11, a gene deleted in WBS (Osborne et al., 1999), as a retinoblastoma protein (Rb) associated protein (CREAM1), where Rb is involved in cell cycle and development (Yan et al., 2000), and as an upstream transcription factor (BEN) that interacted with the Hoxc8 early enhancer in mouse (Bayarsaihan et al., 2000).

The repeat domain of GTF2RID1 is highly conserved across frogs and mammals. Xenopus

GTF2IRD1(XWBCR11) binds to the promoter of goosecoid (gsc) (Ring et al., 2002), the downstream target of activin/nodal-smad signaling pathway which is involved in embryo patterning and induction of mesoderm during early embryo development (Schier et al

2003). XWBCR11 forms a large transcription complex with another transcription factor,

FoxH1 to induce gsc expression. This complex is formed by XWBCR11 binding to a gsc distal control element (DE) and activated Smad, while FoxH1 binds to a gsc proximal control element (PE) and XWBCR11.

GTF2IRD1 may act as an activator in the muscle but it also act as a repressor of TFII-I by preventing nuclear transport through direct interaction with TFII-I (Tussie-Luna et al.,

2001). It has also been shown to negatively regulate itself by binding to its own promoter

(Palmer et al., 2010).

1.5 GTF2I gene family and their animal models

Linking specific genes with the cognitive and behavioural aspects of WBS is difficult because there are few patients reported with atypical deletions and the identification of

WBS deletions is very biased toward those individuals with deletions encompassing ELN because of the recognizable phenotype. To help overcome these problems, different mouse models that carry deletions of different genes from the WBS region have been generated and studied to investigate genotype-phenotype correlations. Mouse chromosome 5 is syntenic to the WBS region, containing all the same genes in the same order (Valero et al.,

2000).

These mouse provides a very important tool in elucidating the contribution of individual genes to the WBS phenotype, as they can be used to generate different deletion lengths – single–gene, combined–gene, and a large multi-gene deletions - that have not yet been found in humans, and can be bred onto a homogeneous genetic background. To help

24 determine the contribution of the GTF genes to cognitive and behavioural aspects of WBS, several mouse models for GTF2I and GTF2IRD1 have been generated.

1.5.1 Gtf2ird1 mouse models

The first Gtf2ird1 mouse model was proudced by a random insertion of the c-myc gene under the control of the albumin promoter and enhancer (Durkin et al., 2001). Transgene insertion resulted in the deletion of about 40 kb encompassing the transcription start site and first exon of Gtf2ird1. Homozygous knockout mice (Gtfi2ird1-/-) were viable and fertile, yet displayed decreased growth and craniofacial abnormalities including periorbital fullness, short snout and a misaligned jaw (Durkin et al., 2001; Tassabehji et al., 2005).

Gtf2ird1-/- mice exhibited hind leg clasping and kicking reflex, and increased anxiety, indicating possible neurological impairments. Gtf2ird1 null animals also showed abnormal motor coordination, decreased locomotor activity and reduced strength (Tassabehji et al.,

2005; Schneider et al., 2012). Heterozygous Gtf2ird1+/- mice had normal growth and craniofacial features, and minor impairments in motor coordination and anxiety

(Tassabehji et al., 2005; Schneider et al., 2012).

Another mouse model of Gtf2ird1 was generated by targeted insertion of a nls-LacZ cassette into exon 2 of the gene (Palmer et al., 2007). This Gtf2ird1 null mutant displayed no distinct craniofacial dysmorphology but showed neurological and behavioural defects

(Palmer et al., 2007). The Gtf2ird1 null mice were further characterized in another study by

Howard et al (2010). These mice exhibited many phenotypes that paralleled WBS including decreased body weight, motor coordination deficit with decreased grip strength, and differential explorative activity where male mice were hyperactive while females were hypoactive. When the mice were exposed to a stressful condition - during the swim test – adult knockout mice emitted more frequent and novel vocalizations that were different from adult wild type mice. Gtf2ird1 knockout mice also had higher serum corticosterone levels after the swim test, indicating elevated stress levels. The novel vocalization was correlated with increased neuronal activation in the ventral side of the lateral septum and cingulate cortex of female knockout mice - brain areas important for mammalian vocalization. Upon maternal separation, the Gtf2ird1 knockout pups had shorter, quieter and fewer vocalizations. These behavioural phenotypes are hypothesized to be comparable to increased anxiety and stress towards specific non-social stimuli observed in WBS

(Howard et al., 2010).

A third model was generated by replacement of Gtf2ird1 exons 2,3,4, and part of 5 with a neomycin-resistance gene cassette (Young et al., 2008). Gtf2ird1 heterozygous and homozygous knockout mice had altered behaviour and impairments in amygdala-based learning and memory. These mice exhibited mild growth retardation, reduced anxiety in the elevated plus maze and open field tests, decreased aggression, and increased sociability towards an intruder mouse, which parallels the hypersociability and disinhibition seen in individuals with WBS. Gtf2ird1 homozygous mutants had increased serotonin metabolite levels in the amygdala, frontal cortex, and parietal cortex (Young et al., 2008). Furthermore,

26 serotonin elicited larger inhibitory, outward current through 5-HT1a receptors in the layer

V of prefrontal cortex, a brain area important for the regulation of anxiety related behaviors (Proulx et al., 2010). These changes in serotonergic transmission may stem from altered anxiety or aggression in these mice.

The last Gtf2ird1 mouse model was generated by insertion of LacZ gene trap cassette into intron 22 (Enkhmandakh et al., 2009). Unlike the other three mouse lines, Gtf2ird1 homozygous mutant mice were embryonic lethal and displayed severe phenotypes including brain hemorrhage, craniofacial abnormalities, abnormal vasculature and neural tube defects. At E10.5, Gtf2ird1-/- embryos had arrested hearts and pale yolk sacs caused by improper formation of vascular networks due to defects in vasculogenesis and angiogenesis. The discrepancies in phenotypes may be due to different mouse genetic backgrounds used in the first three models, and the gene trap technique resulting in a dominant negative protein or indirect disruption of the expression of neighboring genes in the fourth (Schneider et al., 2012).

1.5.2 Gtf2i mouse models

Enkhmandakh et al (2009) also generated three Gtf2i knockout mouse lines by insertion of

Lac Z gene trap cassette into intron 2, 3 or 9 of the Gtf2i locus respectively. All three lines exhibited same severe phenotype: Gtf2i-/- were embryonically lethal with growth

27 retardation, embryonic hemorrhage and 60% of embryos had neural tube defects

(Enkhmandakh et al., 2009). 15% of Gtf2i+/- embryos also had neural defects, with abnormal brain development indicated by smaller brain size, pigmentation defects and growth retardation.

Gtf2i heterozygous deletion mice were generated in our lab by insertion of a gene trap cassette into intron 3 of the Gtf2i gene. Gtf2i homozygous deletion mice were embryonic lethal at a similar stage of development. Gtf2i+/- mice show impaired short-term memory, neuromotor learning deficits and decreased anxiety (Emily Lam, personal communication;

Sakurai 2010).

Gtf2i duplication mice were generated by using Cre-loxP technology through the recombination between lox P sites inserted into intron 4 of Gtf2ird1 and the 3’UTR of Gtf2i.

Recombination between these sites in trans, during meiosis, resulted in offspring with either duplication of an intact copy of Gtf2i (Gtf2i+/dup), or deletion of both Gtf2ird1 and

Gtf2i on the same chromosome copy (Gtf2i+/del). Pups with duplication of Gtf2i showed increased separation-induced anxiety when removed from the nest, as measured by ultrasonic vocalization (Mervis 2012). Gtf2i+/dup mice also showed increased duration of grooming, a repetitive behaviour reminiscent of those often seen in individuals with Dup7

(Emily Lam, personal communication).

Table. 1.4. The summary of mouse models with varying copy number of Gtf2i and Gtf2ird1.

Gtf2ird1-/- Gtf2i+/- or +/- Gtf2i +/dup (Sakurai et Gtf2i+/- Gtf+/del (Young et & dup/dup al. 2010) al. 2008)

Morris water maze ------nd impairments

Barnes maze nd nd -- -- nd impairments

Reduced fear in cued * nd -- *(f) nd fear conditioning

Contextual fear ------nd conditioning

Novel object learning & nd nd * ** -- memory impairments

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

Grip strength * -- *↓(f) *↑(m) nd impairment

Rotarod impairment * -- *(m) *(m) nd

Elevated plus/zero *↓ -- -- *↓ *↑ maze anxiety level

Open field anxiety level *↓ ------*↑

Sections highlighted in blue is recently studied and analyzed in our laboratory.

n -- not significant n * p<0.05 n **p<0.01 n (f) female only n (m) male only n ND, not determined

1.6 Development of the cerebral cortex

1.6.1 Genesis of cerebral cortex

Cerebral cortex is the outer layer of the cerebral hemisphere, which plays an important role in many cognitive functions including planning, execution, language, awareness, memory and behavior. It is anatomically and functionally divided into distinct four lobes – frontal, parietal, occipital and temporal. Many genes regulating cortical development in humans are also conserved in mice, allowing easily accessible analysis of different stages of cortical development. During embryonic development, various intrinsic and extrinsic factors regulate the genesis of the cerebral cortex in a temporarily ordered manner (Miller and

Gauthier, 2007). In mice, neurogenesis occurs first, between embryonic day 12 (E12) to

E18 with its peak at E15, followed by astrogenesis from E18 with its peak in the neonatal period, and lastly oligodendrogenesis occurs postnatally (Fig 1.7) (Bayer and Altman,

1991).

Figure 1.7. Timed genesis of neurons, astrocytes and oligodendrocytes in the developing neocortex. Neurogenesis starts E12 and peaks at E15, followed by astrogenesis which starts at E18 and peaks at neonatal period. Oligodendrogenesis starts during postnatal period until early adulthood.

During development, different populations of neural stem cells give rise to all cellular population of central nervous system, through symmetric proliferative division, asymmetric neurogenic division, and symmetric neurogenic division (reviewed in Gotz and

Huttner, 2005). Upon the onset of neurogenesis, a single layer of neural epithelial cells asymmetrically divides to give rise to radial glial cells (RG). RG cells are neural precursor cells which divide symmetrically and asymmetrically multiple times to generate neurons.

RG cells have processes extending from the pial ventricular surface to the cortical plate, which provide guidance for new born neurons to migrate along the radial fiber to the cortical plate (Fig 1.8). These RG cells in the ventricular zone (VZ) initially divide symmetrically into two daughter RG cells to expand the pool of precursors. Later, RG cells divide asymmetrically to give rise to one RG cell and one neuron, or one RG cell and one basal progenitor (BP) cell. BP cells are more neuronally committed, and occupy the subventricular zone (SVZ) between the VZ and the cortical plate (CP). BP cells divide symmetrically to give rise to two neurons and are thought be responsible for expanding same laminar cortical layer.

Figure 1.8. Schematic diagram of neocortex during neurogenesis. Radial glial (RG) cells in ventricular zone (VZ) initially exhibit symmetric proliferative division to expand the progenitor pool. Then RG divides asymmetrically into one RG cell and one neuron (yellow), or one RG cell and one basal progenitor cell (BP). BP in subventricular zone (SVZ) divides symmetrically to generate two neurons which migrate towards cortical plate (CP).

Timed genesis is also apparent in the generation of cortical layers. Cortical progenitors in the VZ are responsible for generating neurons that are specific to each cortical layer in the appropriate order. The CP, which contains RG- and BP-derived neurons, is generated in an inside-out fashion where early-born neurons occupy the inner cortical layer and later-born neurons migrate past the early born neurons and occupy the most outer cortical layer. The cerebral cortex is organized in 6 layers of neurons with the CP forming layers II-VI, and the marginal zone between the CP and the basal membrane forming layer I (Gotz and Huttner,

2005). The fate of each laminar neuron is determined by the date the cell is born from its cortical progenitor. When the developmental state of cortical progenitors is perturbed, cortical progenitors lose their ability to generate proper laminar specific neurons for that particular developmental stage (McConnelle et al., 1991; Frantz and McConnell,

1996;Mizutani and Saito, 2005). When the differentiation of cortical progenitors into neurons was prohibited at earlier stages and cells were forced to remain at the progenitor state, these progenitors failed to produce early stage of neurons and produced only later stage neurons occupying the upper cortical layers (Mizutani and Saito, 2005). The earlier the progenitor developmental stage, the greater the fate potential of cortical progenitors.

Early progenitors, when transplanted into older brain, are competent to produce upper- layer neurons (McConnelle et al., 1991), whereas late progenitors show restricted fate and are only able to generate upper layer neurons when transplanted into younger brain

(Frantz and McConnell, 1996). Therefore changes in the early stages of development may lead to abnormal brain formation and function.

1.6.2. Signaling pathways in cortical development

During embryonic development, proliferating neural stem cells in the SVZ generate differentiated neurons, astrocytes and oligodendrocytes in that specific sequence. In murine cortical development, neurogenesis commences at E12, followed by astrogenesis at

E17 and oligogenesis occurs postnatally. Remarkably, NPCs collected at E12 recapitulate this timed genesis; neural precursors generate neurons first then glial cells (Miller and

Gauthier, 2007). This cortical genesis is a complex process requiring proper temporal and spatial regulations by various intrinsic signals and extrinsic cues. Although the intrinsic mechanisms are crucial for cell fate determination as seen in timed differentiation pattern of precursors in NPC cultures, the extracellular cues such as growth factors and ligands exert profound regulatory effects on precursor development, survival, proliferation and differentiation (Miller and Gauthier, 2007;Corbin et al., 2008; Rakic, 2009). Growth factors and ligands bind to their own specific receptors, and initiate signal transduction involving secondary messengers. The secondary messengers regulate intracellular proteins, which in turn regulate expression of genes involved in various cellular processes. Depending on which types of ligand, receptors, secondary messengers and intracellular proteins are involved, different signal transduction pathways can be elicited allowing precise spatial and temporal regulation of cellular responses. For instance, neurotrophin-3 (NT-3) increases precursor proliferation and neurogenesis through tyrosin-kinase receptor-C

(TrkC), when injected into lateral ventricle of E13 embryo (Ohtsuka M et al., 2008),NT-4 promotes neuronal maintenance and survival through TrkB (Conover et al., 1995) and NGF regulates neuronal survival and growth through TrkA (Hamburger & Levi-Montakcini).

Moreover, perturbation of extracellular signaling pathways leads to altered neural stem cell biology. Inhibition of brain-derived neurotrophic factor (BDNF) by an antibody decreased survival and proliferation of precursor cells and inhibited neurogenesis

(BarnabeHeider and Miller, 2003). Cytokines such as Leukemia Inhibitory Factor (LIF) increased survival and differentiation of human embryonic stem (hES) cell–derived neural precursors (Majumder et al., 2012). Ciliary neurotrophic factor (CNT) exerts opposite effects on precursor cells; it decreases glial differentiation of undifferentiated glial precursor cells, whereas it promotes glial differentiation of already differentiated glial precursor cells (Shimazaki et al., 2001). Thus, regulation of neuronal development requires a cocktail of intrinsic and extrinsic signaling pathways at specific time points. These disturbances in early development stages may contribute to changes in cortical organization and brain connectivity, abnormal brain structure formation, and impaired brain function, which may contribute to the neurological features of many cognitive disorders.

1.7. Neural mechanisms in WBS

1.7.1 Neural mechanisms of impaired social processing in WBS

Although dramatic phenotypic effects are seen in both humans and mice with deletions of genes from the WBS region, brain structure appears relatively normal. The heterozygous and homozygous Gtf2ird1 mutant mice had no morphological abnormalities (volumes of cerebellum, cerebrum, hippocampus and amygdala were not differ from wild-type mice) and no impairments in hippocampal-based fear conditioning (Young 2008; Hagen et al.,

2007). The neurological features we see could result not only from gross morphology changes in brain structure, but also from changes in brain connectivity.

The amygdala is known for its function in reward and it also codes emotions, especially fear, through continuous monitoring of environmental threats and danger (Adolphs, 2003).

The amygdala is also important in social perception for processing complex social stimuli such as recognition of facial expressions or mental states (Tager-Flusberg and Sullivan,

2000). The amygdala’s interaction with orbitoforontal cortex (OFC) plays an important role in social cognition by establishing social judgments based on the sensory representations of both negatively and positively valenced social salience (Baron-Cohen et al., 1994;

Eslinger and Damasio, 195). Individuals with lesions in amygdala and linked cortical areas,

37 including the OFC, have profound abnormalities in social function and social disinhibition

(Prather et al., 2001; Amaral, 2002).

Individuals with WBS fail to detect social danger signals and acquire social adaptation and social success (Tager-Flusberg and Sullivan, 2000). These impairments in both social perception and cognition in WBS may be due to changes in activation of regions of the frontal cortex and amygdala. Functional MRI studies in people with WBS have shown increased amygdala activation to socially positive stimuli, whilst amygdala activation to negative (threatening) social stimuli is significantly diminished, compared to typically developing controls participants (Haas et al., 2009). Differential amygdala activation is crucial for appropriate avoidance behaviour and abnormal amygdala activity may contribute to the gregariousness, and decreased fear towards strangers and social disinhibition seen in individuals with WBS. The control participants showed increased amygdala response to social stimuli (faces) than to non-social stimuli such as scenes.

However, WBS individuals show heightened amygdala reactivity to scenes compared to faces, which may help explain increased nonsocial anxiety and specific phobias found in

WBS (Meyer-Lindenberg et al., 2005). The control participants recruited different regulatory regions for specific tasks and these regulatory links were impaired in WBS. WBS individuals did not show differential activation of the OFC whereas controls showed increased activation of OFC in response to faces compared to scenes. Conversely, the medial prefrontal cortex, the area associated with empathy, is hyperactive in WBS individuals. This persistent activation of mPFC may contribute to the increased empathy seen in WBS (Meyer-Lindenberg et al., 2005).

1.7.2 Role of Serotonin in emotional behaviors

Serotonin (5-HT) acts as an important modulator for mood, and affect. Abnormal 5-HT levels and neurotransmission have been implicated in emotional disorders and the 5-HT signaling pathway is a target for many psychotherapeutics (Ballenger, 1999). Around one third of children with autism have elevated platelet 5-HT levels (Penn, 2006). In contrast, lower levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), have been documented in the blood and cerebral spinal cord fluid of depressed individuals (Qintana,

1992), whilst individuals with high risk of suicide also have reduced levels of 5-HT and 5-

HIAA in the brain stem (Arango et al., 1997). Decreased 5-HT signaling is also associated with increased aggression in mice as well as in humans (Vergnes et al., 1986; Roy, 2001).

Excitatory 5-HT2A and inhibitory 5-HT1A receptors are located in presynaptic serotonergic neurons in the dorsal raphe nuclei, and postsynatpically in different brain regions including in pyramidal neurons of the prefrontal cortex. The pyramidal neurons in layer V of prefrontal cortex send inhibitory projections to various structures including the raphe nuclei, amygdala, hypothalamus, and striatum (Vertes, 2004; Gabbott et al., 2005;

Goncalves et al., 2009) . The dysregulation of 5HT1A and 5HT2A receptors may lead to abnormal brain function as some of the structures such as the raphe nuclei receive their only cortical inputs from these layer V neurons (Goncalves et al. 2009). Moreover, the

39 prefrontal cortex and amygdala share reciprocal interactions. Inhibition of the prefrontal cortex leads to disinhibition of the raphe nuclei, which in turn increases serotonin release in the prefrontal cortex, and affects its modulatory effects on the amygdala.

Variability in 5-HT receptor signaling within the cortical limbic circuitry has been implicated in the functional coupling and the modulation of the amygdala response to threat related stimuli. Neuroimaging studies in humans showed that reduced mPFC-5-

HT1A receptor binding is correlated with increased amygdala reactivity (Tauscher et al.,

2001), whereas an inverse correlation was observed between mPFC-5HT2A binding and amygdala reactivity (Fisher et al., 2009). Decreased dorsal raphe nucleus-5HT1A binding is correlated with increased amygdala reactivity in response to threat-associated stimuli

(Fisher et al., 2006). 5HT1A receptor knockout mice display increased anxiety (Parks et al.

1998; Ramboz et al. 1998) whereas overexpression of the 5HT1A receptor leads to decreased anxiety (Kusserow et al., 2004).

1.7.3 Altered brain connectivity in Gtf2ird1 targeted mice.

Gtf2ird1-/- mice exhibit hypersociability, low anxiety and less aggression paralleling cognitive behaviours in WBS individuals. Gtf2ird1-/- mice also show hypoactivation of the frontal cortex as indicated by reduced c-fos expression in response to stress (Young, 2010).

The mice display elevated levels of 5-HIAA in the amygdala and prefrontal cortex without

40 significant changes in 5-HT, which suggests increased 5-HT release rather than an increase in overall 5-HT production (Young et al., 2008). The electrophysiological studies in

Gtf2ird1-/- mice have also shown altered neuronal connectivity where 5-HT elicited increased inhibitory, outward currents in layer V of the prefrontal cortex compared to wild-type mice (Proulx et al., 2010). The interaction between the amygdala and prefrontal cortex plays a crucial role in social cognition and altered brain connectivity may contribute the cognitive profile of WBS.

1.8 Research Aims and hypothesis

Functional MRI studies in people with WBS and electrophysiological studies of transgenic mouse models of WBS suggest that the deletion of the GTF2I and GTF2IRD1 genes likely contribute to the neurological features of this disorder through perturbing brain connectivity, rather than causing major structural abnormalities. Although detailed studies of structural deficits at the neuronal level in the mouse or humans with atypical deletion of

GTF2I and GTF2IRD1 have not been explored, these results hint that changes in altered connectivity may be the downstream result of abnormal development. The disturbances incurred during early stages of brain development may result in aberrant postnatal brain formation and maturation leading to the complex neurological features of WBS. To try to understand how the brain is affected by hemizygosity for GTF2I and GTF2IRD1 during embryonic development, I am using primary cortical cell cultures from mouse models with deletion and duplication of these two genes to investigate the effects on neuronal development. I hypothesize that altered GTF2I and GTF2IRD1 expression may affect neurogenesis by altering neural stem cell physiology, more specifically, changes in neural precursor cell proliferation, survival, and neural differentiation, and may subsequently contribute to the neurological features of WBS.

Chapter II. Disturbance in early neuronal development in mouse models of WBS

2.1 Introduction

2.1.1 Mouse models

To try to link specific genes with the cognitive and behavioural aspects of WBS, our lab has generated single-gene- and combined-gene-deletion and duplication mouse models for two strong candidate genes from the commonly deleted region, Gtf2i and Gtf2ird1. Gtf2i+/- mice were generated from YTA365 embryonic stem cell line, that has an insertion of a gene trap cassette into intron 3 of the Gtf2i gene . The Gtf2i duplication and Gtf2i-Gtf2ird1 deletion mice were generated by recombination between targeted loxP sites in the XS0608 and G10- targeted mouse lines. The XS0608 mice line, carrying a gene trap cassette with a loxP insertion into intron 4 of Gtf2ird1, was crossed with the G-10 mouse line that contains a loxP site in the Gtf2i 3’UTR, downstream of the last exon, and a Cre transgene under the control of Sycp1 promoter. Recombination between the respective loxP sites occurred in trans during meiosis, resulting in offspring with either deletion of Gtf2i and Gtf2ird1

(Gtf+/del) or complete duplication of Gtf2i and a partial, non-functional duplication of

Gtf2ird1 (Gtf2i+/dup). Homozygous Gtf2i duplication mice (Gtf2idup/dup) were generated by crossing Gtf2i+/dup mice.

Gene Trap YTA 365 - reporter and loxP inserted into intron 3 of Gtf2i

Gene Trap XS0608 – reporter inserted into intron 4 of Gtf2ird1

G10 targeted line - loxP inserted downstream of Gtf2i

Figure 2.1. Schematic diagrams of individual mice lines used in the study. (A) Gtf2i+/- mice is generated by using the gene trap YTA365 mice line with a gene trap cassette inserted into intron 3 of Gtf2i. (B) Gene trap XS0608 mice line with a gene trap cassette inserted into intron 4 of Gtf2ird1 and G10 targeted mice line with insertion of loxP in 3’UTR downstream of Gtf2i are used for the generation of Gtf+/del mice. (C) The recombination between respective loxP sites results in either deletion of Gtf2i and Gtf2ird1 and reciprocal duplication of Gtf2i.

2.1.2. Neural Precursor Cells

During embryonic development, the cerebral cortex arises in a sequential manner from proliferating neural stem cells. The neural stem cells from the VZ first divide symmetrically, duplicating themselves. Then they divide asymmetrically to produce one neuron and one progenitor cell, which continues to divide until it differentiates into astrocytes and oligodendrocytes at later stages of development. In rodents, neurogenesis occurs from E12 to E18 followed by astrocytes formation from E18 to neonatal period, and lastly oligodendrocytes appear in postnatal period (Bayer and Altman, 1991). The NPCs, which consist of these precursors, can be used as a very useful tools for studying mechanisms underlying nervous system development, because NPCs follow the sequential differentiation time window - generating neurons first followed by astrocytes and then oligodendrocytes (Miller and Gauthier, 2007) - and can be directed to differentiate into particular lineages under different culture conditions. I collected NPCs from Gtf+/del,

Gtf2i+/dup, and Gtf2i+/- mice for the detailed study of effects of copy number variants of these genes on cortical neuronal development.

2.2 Material and Methods

2.2.1. Animals

Mice were maintained on a 129SvEv/C57BL6/CD1 mixed genetic background. All experimental animals were housed at the Medical Sciences Building of the University of

Toronto in polycarbonate cages (30 x 22 x 15 cm) under standard animal housing conditions. Animals were maintained in a light-controlled room on a 12:12 light-dark cycle

(with lights on at 6 am) at a controlled temperature (23 ± 2 °C) and humidity

(approximately 50-60%.) All experimental protocols using animals were performed in accordance with the Guide to the Care and Use of Experimental Animals (Canada), and approved by the Animal Care Committee of the University of Toronto.

For NPC cultures of individual embryos, mixed litters of mutant and WT, Gtf+/del, Gtf2i+/dup, and Gtf2i+/-mice were used. For NPC cultures of pooled embryos, crosses between heterozygous mice - WT x WT, Gtf2idup/dup x Gtf2i+/dup, Gtf+/del x Gtf+/del and Gtf2i+/- x Gtf2i+/- - were used to obtain litters of same genotype. In the case of the Gtf2i +/- crosses, litters were

46 a mixture of WT and Gtf2i +/- embryos since homozygous Gtf2i -/- embryos died before E9.0.

In the case of the Gtf +/del crosses, litters were a mixture of WT and Gtf +/del embryos since homozygous Gtfdel/del embryos died before E9.0. The timed pregnancy was detected by knowing when a mouse is conceived through appearances of a copulatory plug. Mice usually mate four to six hours into the dark cycle, therefore the plug was detected in early morning time in light cycle. The plug may dislodge or dissolve if it is detected later into light cycle. Plug can be located by lifting the female by the base of the tail, opening the vagina gently using a metal probe. The day the plug was found was day 0.5, and 12 days after was noted as E12.5 (when the embryos were collected for NPCs).

2.2.2. Genotyping

Genomic DNA was isolated from the tail tips of E12 and E18 embryos. The tissues were incubated in 400 μL of lysis buffer containing 50 mM Tris, 0.5% SDS, 0.1M NaCl, 0.5 μM

EDTA, 0.25 μg/μl proteinase K) at 62°C water bath until tissues of tail is completely dissolved. The DAN was extracted by adding 75μl of 1.2M potassium acetate and 500 μL of chloroform – the volume that is close to the total volume of the solution. The solution was incubated at -20°C for 20mins and centrifuged for 5mins at 13,000 g at the room temperature. The aqueous phase was then moved to fresh tubes. DNA precipitation was carried out by adding 2 volumes of 100% ethanol, and centrifuging for 5mins at 13,000g at the room temperature. The supernatant was removed and DNA pallet was washed with

70% ethanol, centrifuged for 5mins at 13,000g at the room temperature, and suspended in

200μl of nuclease free water. Genotype was determined by using conventional PCR. 1μl genomic DNA samples are amplified in 2.9μl of PCR Master mix containing 19.95μl water,

25mM MgCl2, 3μl 10X buffer, 2mM dNTP, 0.5μl forward and reverse primers, and 0.25μl of

Tag polymerase. The each DNA samples are loaded into the agarose gel along with a dye for gel electrophoresis and appearances of two bands denotes each specific genotype.

Table 2.1. List of primers used and its sequence.

Primer Name Forward Primer sequence (5’ to 3’) Reverse Primer sequence (5’ to 3’) m2iGTi3 –G GGT GCT ATG GCA ACA TTG TG GGT GGT GGC CTT TGA AGT AA mGT-del-G AAG GGG AGA TGC CA AGA CT GCT GAT CCG GAA CCC TTA AT mGT-dup-G CAA GCA CTG GCT ATG CAT GT GTT TTC CCA GTC ACG ACG TT

2.2.3. Cortical Precursor cell culture

NPCs were collected from E12.5 mouse cerebral cortex by harvesting E12.5 mouse embryos from the uterus. Skin and meninges were removed in cold HBSS (Invitrogen,

Gaithersburg, MD and the dissected cerebral cortices transferred into Neurobasal medium

(Invitrogen) containing 500μl glutamine (Cambrex Biosciences, Hopkinton, MA), 2% B27 supplement (Invitrogen), and 40ng/ml FGF2 (Promega Madison, WI) . For NPC culture for

48 individual embryos, each cortex was transferred separately into a different tube containing culture media whereas for NPC culture of pooled litters, cortices were collected in a single tube containing culture media. The collected tissue was then mechanically dissociated with a plastic pipette and plated on four-well chamber slides (Nunc, Naperville, IL) in

Neurobasal medium at a density of 150,000 cells/plate. The four-well chamber slides were coated with 2% laminin and 1% poly-D-lysine (BD Biosciences, Bedford, MA). Cortical cultures were grown for either 1 or 3 days in vitro (d.i.v.) before immunohistochemistry was performed. These experiments were carried out in Dr. Freda Miller’s laboratory at the

SickKids Research Institute under the guidance of MD/PhD student David Tsui (single embryo culture) and in Dr. Lucy Osborne’s laboratory at the Medical Sciences Building

(pooled embryo culture).

2.2.4. Immunohistochemistry

For immunohistochemistry, cultured cells were washed with HEPES-buffered saline (HBS) and fixed with 4% PFA for 10-15mins. Then cells were permeabalized with 0.2% NP-40

(USB Corporation, Cleveland, OH) in HBS. Using 6% normal goat serum (NGS) (Jackson

ImmunoResearch ,West Grove, PA) and 0.5% Bovine serum albumin (BSA) (Jackson

ImmunoResearch) in phosphate buffer solution (PBS) (Hyclone, Logand, UT), cells were then blocked for 1-2 hours at room temperature. Primary antibodies were prepared at different concentrations in HBS with 3% NGS and 0.25% BSA and cells were incubated with

49 primary antibodies at 4°C overnight. Cells were washed with HBS, and incubated with secondary antibodies in HBS with 3% NGS and 0.25% BSA at room temperature for 1 hour.

Cells were then washed with HBS, Hoeschst 33258 (1:1000; Sigma, St-Louis, MO) was applied for 1-2 minutes and washed again with HBS, followed by immediate application of mounting agent, GelTol (Fisher Scientific, Houston, TX). For immunohistochemistry of tissue sections, sections were dried at 37°C for 10-15 minutes, washed in PBS, and fixed with 4% PFA. Sections were then blocked and permeabalized with PBS containing 10%

BSA at room temperature for 1 hour. Sections were incubated with diluted primary antibodies at 4°C overnight , washed with PBS and incubated with diluted secondary antibodies at room temperature for 1 hour. Hoechst 33258 (1:2000) was applied for nuclear staining for 1-2 minutes at room temperature. Sections were then washed with PBS and mounting agent applied, followed by coverslips.

Primary antibodies used include mouse anti-nestin (1:200, Chemicon), mouse anti-Ki67

(1:200; BD Biosciences), rabbit anti-cleaved caspase 3 (1:1000; Cell Signaling Technology,

Beverly, MA), mouse anti-βIII-tubulin (1:1000; Convance, Princeton, NJ), rabbit anti-βIII- tubulin (1:1000; Covance), and rabbit anti-Pax6 (1:2000; Covance). The secondary antibodies used include indocarbocyanine (Cy3)-conjugated goat anti-mouse and anti- rabbit IgG (1:400; Jackson ImmunoResearch), FITC conjugated anti-mouse and anti-rabbit

IgG (1:200; Jackson Immunoresearch) Alexa Fluor 350 conjugated goat anti-mouse and anti-rabbit (1:500; Moleclar Probes, Eugene, OR), and Alexa Fluor 647 conjugated goat anti- mouse and anti-rabbit (1:1000; Molecular Probes).

2.2.5. Microscopy and Quantification.

For NPC cultures from individual embryos and pooled litters of embryos, approximately

300-400 cells in 5 fields were randomly chosen per experiment. Digital acquisition was performed with a 40x objective using Northern Eclipse software (Empix) with a Sony XC-

75CE CCD video camera. The total live cell numbers were determined by counting cells with cloudy and big round nucleus indicated by DAPI staining. The dead cells, which often constitute of condensed or small round broken nucleus were not counted into the total live cell number (Fig 2.2).

Figure 2.2. The representative picture of nucleus staining.

DAPI staining allows to distinguish live cells (arrow heads) from the dead cells with broken nucleus (arrow).

The total number of stain-positive cells was measured by counting cells stained-positive for specific marker and that had round and cloudy nucleus (DAPI staining). The percentage of specific marker-positive cells was calculated by total number of stain-positive cells divided by total number of live cells in the view.

For quantification of immunocytochemistry on tissue sections, 2-3 fields per brain section were randomly chosen. There were 8 anatomically similar coronal sections per brain encompassing the rostral hippocampus (Bregma, -1.2 to -2.5). Two rectangle counting areas of 0.0482mm2 and 0.2558mm2 per view were used to count DAPI stained cell density of cortical layer V and all layers respectively. ImageJ software (NIH) was used to measure the cortical thickness on images taken with a 40x objective. The quantification for all experiments was performed blindly.

2.2.6. Statistical Analysis

Error bars in all graphs indicate Standard Error of the Mean (SEM). The pair wise comparisons of different genotype mice to wild type control were performed using two- tailed Student' statistics were performed using the Student’s t-test compared to wild type litter mates for single NPCs and compared to wild type litters for pooled NPCs. The statistical analysis for the differences between means of more than two groups were determined by using Tukey analysis of variance (ANOVA) test where indicated using PAST

(PAlaeontological STastics). The null hypothesis was rejected at the 0.05 level for all the data analysis.

2.3 Results

2.3.1. Gtf2i and Gtf2ird1 copy number regulate cortical precursor growth in pooled embryo cultures

To investigate how Gtf2i and Gtf2ird1 regulate neural development, I initially used NPCs from pooled population of embryos. Wild type mice were intercrossed to produce homogeneous population of WT embryos. Gtf2i+/dup and Gtf2idup/dup mice were crossed to generate litters with expected genotypes of Gtf2i+/dup (1/2 of embryos) and Gtf2idup/dup (1/2 of embryos). Gtf+/del mice were intercrossed to produce litters with expected genotypes of

WT (1/3 embryos) and Gtf2i+/del (2/3 of embryos) since Gtf2idel/del embryos are embryonically lethal (Table 2.2).

Table. 2.2 List of observed genotypes in each cross.

Genotypes Expected ratio Observed genotypes

Litter#1 Litter#2 Litter#3

Gtf+/del X Gtf+/del

Gtf+/del 2/3 7 8 9

WT 1/3 5 3 4

Gtf2i+/dup X Gtf2idup/dup

Gtf2i+/dup 1/2 9 6 6

Gtf2idup/dup 1/2 4 5 4

Gtf2i+/del X Gtf2i+/del

Gtf2i+/del 2/3 7 7

WT 1/3 4 3

Immunostaining of NPCs for Nestin, a neural stem cell marker, after 3 d.i.v showed a lower percentage of neuronal precursors in Gtf+/del litters (Fig 2.3 C and G) and higher percentage of neuronal precursors in Gtf2i+/dup litters (Fig 2.3 A and G) compared to NPCs from WT litters (Fig 2.3 B and G). To determine whether Gtf2i and Gtf2ird1 regulated neuronal differentiation, NPCs were stained with βIII tubulin, a post-mitotic neuronal marker, at 3 d.i.v. βIII tubulin labels NPCs that are more committed toward a differentiated neuron. Gtf

+/del precursor cultures showed fewer βIII tubulin-labeled NPCs (Fig 2.3 C and F) than WT cultures. In contrast, Gtf2i+/dup precursors showed more βIII tubulin-labeled NPCs (Fig 2.3

A and F) than WT cultures (Fig 2.3 E and F). These data suggest that two copies of Gtf2i and/or Gtf2ird1 are necessary for maintenance of the neural precursor population and precursor differentiation into neurons.

Fig 2.3. The effects of Gtf2i and Gtf2ird1 copy number variants on cortical precursor cells. Pooled embryo NPCs showed a decreased number of neuronal precursors (C,G) and neurons (F,H) in mice with deletion of Gtf2i and Gtf2ird1 compared to WT mice (B), and an increased number of precursors (A,G) and neurogenesis (A,H)in mice with Gtf2i

56 duplication, compared to NPCs from WT mice (B). nestin = neural stem cell marker, βlll tubulin = neuronal marker. *p <0.05. Error bars denote SEM. Scale bars represent 50μm.

2.3.2. Hemizygous deletion of Gtf2i and Gtf2ird1 disrupts cortical precursor physiology and neurogenesis in single embryo cultures.

Pooled cultures of Gtf+/del embryos did not contain homogeneous embryo populations with same genotype and therefore some effects of Gtf2i and/or Gtf2ird1 hemizygosity may have been masked by a compensatory effect of WT embryos within the same litter pool. To confirm findings from pooled embryo NPC cultures, single-embryo cultures were performed using E12.5 embryos from crosses between Gtf+/del mice. Immunostaining of single embryo cultures with nestin, a neural stem cell marker, demonstrated that Gtf2i and

Gtf2ird1 hemizygosity decreased the total number of Nestin and Pax6 positive precursor cells and neuronal cells (Fig 2.4 K,L,N). These findings suggest the role of Gtf2i and Gtfi2rd1 in the regulation of precursor maintenance and neurogenesis. There was also a decrease in the percentage of neural stem cells present in Gtf+/del cultures after 1 d.i.v (Fig 2.4 B and C), compared to cultures from WT littermates (Fig 2.4 A). Gtf+/del mice also had decreased percentage of cells positive for Pax 6, a marker of RG precursors (Fig 2.4 E and F) and fewer cells that stained with βIII tubulin (Fig 2.4 G-I). There were overlapping populations of cells that labeled as early neural progenitors (Pax 6- and nestin- positive) and as more committed progenitors (co-expressed βIII tubulin) (Fig 2.4 J). This indicates that decreased copy number of Gtf2i and Gtf2ird1 affected both the number of progenitors and more committed neuronal cells.

Figure 2.4. The effects of Gtf2i and Gtfi2rd1 deletion on single embryo NPCs at 1.d.v. Single embryo NPCs after 1 day revealed decreased number of Nestin and Pax6 positive neuronal precursors (B,C,D,F) and neurons in mice with deletion of Gtf2i and Gtf2ird1 (H,I ;n=5) compared to wild type litter mates (A,D,G; n=5). (J) Pax6- and βlll tubulin costained cells indicate overlapping population of more committed neural progenitor cells. (K-M) The total number of nestin-, Pax6- and βlll tubulin-positive cells were reduced in in mice with deletion of Gtf2i and Gtf2ird1 (n=5) compared to wild type (n=5). Nestin = neural stem cell marker, Pax6=radial precursor cell marker, βlll tubulin = neuronal marker. *p <0.05. Error bars denote SEM. Scale bars represent 25μm

Similarly, immunostaining for nestin and Pax6 at 3 d.i.v. demonstrated fewer precursor cells in Gtf+/del embryo cultures (Fig 2.5 B,C,E, and F) compared to WT embryo cultures.

To investigate whether Gtf2i and Gtfi2rd1 were regulating NPC numbers through changes in survival or apoptosis, single embryo cultures were stained with Ki67, a marker for cell proliferation, and cleaved-caspase 3 (CC3), an apoptotic marker, at 3 d.i.v. The deletion of

Gtf2i and Gtf2ird1 had no effect on proliferation or survival of precursor cells (Fig 2.5 I-O).

The regulation of precursor differentiation was also perturbed in after 3 d.i.v; mice with deletion of two genes had decreased percentage of βIII-tubulin positive neuronal cells (Fig

2.5 G-I). Tbr2 staining revealed no changes in the percentage of the basal precursor (BP) population (Fig 2.5 P-R), suggesting that the decrease in neurogenesis may be due to changes in RG precursor physiology rather than than BP physiology.

Figure 2.5. The effects of Gtf2i and Gtfi2rd1 deletion on single embryo NPCs at 3 d.i.v. Single embryo NPCs from Gtf+/- showed disrupted precursor cell physiology. (A-I) The result showed decreased percentage of neuronal precursors and neurons in mice with deletion of Gtf2i and Gtf2ird1 (n=5) compared to wild type littermates (n=4) at 3d.i.v. (J-R) The percentage of Tbr2+ cells, proliferation and survival of precursor cells were not affected. (S)The total number of neural precursor cells, and neurons were decreased (WT n= 3, Gtf+/- n=3). Nestin = neural stem cell marker, Pax6=radial precursor cell marker, CC3 = apoptotic marker, Ki67= proliferative marker, Tbr2 = basal progenitor marker, βlll tubulin = neuronal marker. *p <0.05. Error bars denote SEM. Scale bars in A-H and M-Q represent 25μm and Scale bars in J and K represent 100μm.

2.3.3 Gtf2i duplication promotes proliferation and differentiation of cortical precursors but does not affect survival.

To further confirm and investigate whether Gtf2i copy number affects cortical precursor development in a dose-dependent manner, single-embryo cultures were collected from the progeny of Gtf2i+/dup crosses. Immunostaining for Nestin at 1 d.i.v. showed that the total number of precursor cells and neurons were also increased, indicating overall increase in the proportion of RG precursors that were present in Gtf2i duplication NPC cultures (Fig

2.6 A-C). Moreover, the percentage of neural stem cells was increased in Gtf2i+/dup precursor cultures (Fig 2.6 D,E). Pax6-positive cells were also increased (Fig 2.6 F,G), further confirming a positive role for Gtf2i in neural precursor physiology. Gtf2i increased neurogenesis at 1 d.i.v (Fig 2.6 H,I) as indicated by increased percentage of βIII tubulin- positive cells. The WT from Gtf2i+/dup had lower percentages of nestin-, Pax6- and βIII tubulin-positive cells (Fig2.6) compared to WT from Gtf+/del (Fig 2.5). This discrepancy could be due to slightly different genetic backgrounds of these mouse lines.

Figure 2.6. The effects of Gtf2i duplication on neural stem cell physiology. An increased number of neuronal precursors and neurons 1 day in vitro, compared to wild type littermates (n=4) in single-embryo cultures in Gtf2i+/Dup mice (n=4). Nestin = neural stem cell marker, Pax6=radial precursor cell marker, βlll tubulin = neuronal marker. *p <0.05. **p<0.01. Error bars denote SEM. Scale bars in A-C represent 45 μm and in D,F,H represent 50μm.

To determine whether this enhancement in precursor maintenance persisted, we also used immunohistochemistry with Nestin and Pax6 at 3 d.i.v. The quantification of Nestin and

Pax6-positive cells showed even a greater increase in the percentage of precursor cells in

Gtf2i+/dup compared to WT littermates; there was 2 fold increase in the precursor cell percentage in 3 d.i.v compared to 1.5 fold increase in 1 d.i.v. (Fig 2.7 A,B). Gtf2i duplication had no effect on cell survival at 3 d.i.v, and the percentage of CC3 positive cells did not differ between Gtf2i+/dup and WT (Fig 2.7 G). Then we investigated whether increased copy number of Gtf2i had effects on cortical cell proliferation. Interestingly, Gtf2i+/dup embryos showed a significant increase in Ki67-positive cells (Fig 2.7 E,F), indicating an increase in proliferation. There are different populations of proliferating precursor cells in NPC culture, including primary RG precursors and intermediate basal progenitors, that could be responsible for increased proliferation observed in Gtf2i+/dup culture. To further analyze the population of precursors, NPC cultures were immunostained after 3 d.i.v. with Tbr2, a marker for BP cells. Gtf2i duplication did not affect the percentage of Tbr2-positive cells

(Fig 2.7 C,D). The quantification of βIII tubulin-positive cells revealed a significant increase in the percentage of neurons generated in 3 d.i.v. These finding suggest Gtf2i is important for RG precursor development and maintenance.

Figure 2.7. Gtf2i duplication promotes precursor cell maintenance through increased proliferation. (A,B) Increased percentage of precursors and neurons were observed in NPC culture from Gtf2i+/dup crosses (n=4) compared to wild type littermates (n=4) at 3d.i.v. (C,D) Gtf2i duplication did not have any effect on the percentage of Tbr2 positive- intermediate progenitors. Gtf2i duplication enhanced proliferation of radial precursors (E,F) without affecting survival of precursors (G). Nestin = neural stem cell marker, Pax6=radial precursor cell marker, CC3 = apoptotic marker, Ki67= proliferative marker, Tbr2 = basal progenitor marker, βlll tubulin = neuronal marker. (Student’s t-test) *p <0.05. Error bars denote SEM. Scale bars in A and C represent 15μm, in E represent 25μm.

2.3.4. Effects of hemizygosity for Gtf2i on precursor maintenance and neurogenesis.

To try to separate the contributions of Gtf2i and Gtf2ird1 hemizygosity to the regulation of precursor cell physiology, we performed pooled embryo NPC cultures with embryos from intercross of single gene knockout,mice, Gtf2i+/del. Immunostaining for Nestin at 3 d.i.v revealed that Gtf2i deletion cultures had decreased percentage of neuronal precursors compared to wild type embryos, however to a lesser degree than mice with combined deletion of Gtf2i and Gtf2ird1 (Fig 2.8A). To investigate whether Gtf2i alone had a regulatory effect on neural development, cultures were stained with βIII-tubulin at 3 d.i.v.

There was a trend for a decreased percentage of neurons in Gtf2i+/- precursor cultures (Fig

2.8B). Since Gtf2i null mice are embryonic lethal at a stage before our cortical dissection, progeny of Gtf2i+/del intercross should constitute of 1/3 WT and 2/3 Gtf2i+/- embryos.

However, the ratio of newborns were not strictly 1:2 ratio in our experiment, and the number of wild type mice present in any given litter might have ameliorated the effects of deletion of Gtf2i on the assessment of neuronal precursor cell physiology.

Figure 2.8. The effects of sing gene knockout of Gtf2i on regulation of precursor cell maintenance. (A) Gtf2i deletion results in decreased precursor cells compared to WT but at a lesser extent than Gtf2i and Gtf2ird1 combined deletion. (B) Effects of Gtf2i deletion on neurogenesis were not consistent between different litters but have a trend of decreased neurogenesis compared to WT. Genotypes for each crosses includes: Gtf2i+/dup x Gtf2i+/dup (Gtf2i+/dup n=9; Gtf2idup/dupn=4), Gtf2i+/- x Gtf2i+/- Litter#1 (Gtf2i+/del n=7, WT n=4), Litter#2 (Gtf2i+/del n=7, WT n=3), Gtf+/del x Gtf+/del (Gtf+/del n=9, WT n=4). Tukey ANOVA test * means compared to WT (* p<0.05 ), # means compared to Gtf2i+/dup (# p< 0.05, ### p<0.01, #### p<0.0001) and $ means compared to Gtf+/del ($ p<0.05, $$$ p<0.01)

2.3.5. Effects of Gtf2i and Gtf2ird1 gene dosage on later cortical development

To determine whether the perturbations in the early stages of neural development that we identified in our mouse models persisted into later stages of cortical formation and development, cortical sections of E18.5 embryos for each genotype were studied.

Immunochemistry was performed with Ctip2, a cortical layer V marker, and sections were counterstained with DAPI, a nuclear marker, followed by quantitative analysis of cell density and cortical thickness. The quantification of cell density revealed increased cell packing density in layer V of Gtf+/del embryo cortex (1975.97±78.8 cells) compared to WT embryos (1408.45±76.7 cells) (Fig2.9 A,C), whereas the cell density for the cortex as a whole did not change (Fig2.9 B,D). Gtf2i duplication did not have any effect on layer V or whole cortical cell density (Fig2.9 C,D). To assess the effects of Gtf2i and Gtf2ird1 copy number on cortical thickness, sections were immunostained with layer specific markers,

Ctip2 (layer V) and Cux1 (layer II-III) and compared to whole cortex counterstained with

DAPI. Measuring DAPI staining revealed no changes in the total cortical thickness in Gtf+/del compared WT (Fig2.9 F). In contrast, Gtf2i+/dup had increased total cortical thickness

(Fig2.9 F). Gtf+/del did not affect the thickness of layer II-III (Cux1) and layer V (Ctip2) compared to WT (Fig2.10 G,H). Duplication of Gtf2i not only had a profound effect on total cortex thickness, but also on specific layer thickness; layer V thickness was reduced

(22.47±0.9% compared to 25.6±1.3% in WT) (Fig2.9 G) and layer II-III thickness was increased (25.89± 0.8% compared to 22.96± 0.9% in WT) (Fig2.9 H). Thus, the Gtf2i genes are involved in the regulation of cortical density and thickness.

Fig 2.9. Early changes in neural precursor cells persist into later stages of cortical layer formation and organization. Immunostaining for Ctip2 , cortical layer 2 marker (A) and DAPI, nuclear marker(B) on the cortical sections of E18.5 WT, Gtf+/- and Gtf2i+/dup mice. Scale bar in A represent 100μm and B represent 200μm. Boxed regions were shown at higher magnification on the side. (A-1) The schematic diagram of coronal brain section. Blue rectangle indicates the location of quantification. (C) Quantification of Ctip2-positive cells to measure cortical layer 5 cell density. (D) Quantification of DAPI-positive cells to measure whole cortical cell density. (C/D) Each genotype had n=5. (E) Immunochemistry for Ctip2 and Cux2 on cortical sections of WT and Gtf2i+/dup. Arrow indicates differences in cortical thickness between WT and Gtf2i+/dup. Scale bars denote 100 μm. (F) Measure of the thickness of a whole cortex. (G) Measure of thickness of Ctip2-positive layer 5. (H) thickness of Cux1-positive cortical layer 2-3. (F,G,H) Each genotype had n=6. (Student’s t- test) *P<0.05 compared to WT.

3. Discussion and conclusions

3.1 Roles of Gtf2i and Gtf2ird1 in precursor cell biology.

In this project, I proposed to investigate how changes in early neural development might contribute to aberrant prenatal cortex formation and maturation, which in turn could lead to the neurocognitive profiles of WBS and/or Dup7 in postnatal development. Data from

NPC cultures collected from mouse models with different copy numbers of Gtf2i and Gtf2ird revealed three main conclusions (Table 3.1).

First, experiments on NPC cultures showed that Gtf2i and Gtf2ird1 play a crucial role in precursor cell maintenance. Mouse embryos with hemizygous deletion of Gtf2i and Gtf2ird1 had an overall reduction in the number of cortical precursors compared to WT embryos, but precursor survival and proliferation were not altered. Additional copies of Gtf2i resulted in an increase in the number of cortical precursors caused at least in part by increased proliferation, but did not affect precursor survival. Moreover, the maintenance of the BP population was not altered in NPC cultures from either of the mouse models suggesting that Gtf2i and Gtf2ird1 are mainly involved in RG cell maintenance. NPCs from mouse embryos hemizygous for Gtf2i alone showed intermediate phenotypes between

Gtf+/del and WT, where the number of precursor cells was decreased compared to WT but to a lesser degree than Gtf+/del. This suggests that both genes play a role in maintaining NPC physiology.

Second, Gtf2i and Gtf2ird1 are essential for normal differentiation of cortical precursors into neurons in NPC cultures. Gtf+/del NPC cultures had a decreased number of neurons. In contrast, Gtf2i+/dup NPC cultures had an increased number of neurons, likely due to increased proliferation of RG cells. This suggests that Gtf2i and Gtf2ird1 are not required for

NPC survival but are required for RG precursor maintenance and neurogenesis. The effects of Gtf2i deletion alone on neurogenesis were inconsistent, but one of the pooled litters had a small but significant decrease in neurogenesis in NPC cultures. These findings suggest possible synergism of Gtf2i and Gfi2rd1 in the regulation of precursor maintenance, and

Gtf2ird1 may compensate for some of the effects of loss of Gtf2i on neurogenesis.

Third, Gtf2i and Gtf2ird1 are required for proper formation of the cortex. Changes in early neuronal development (E12.5) affected the cortical organization at later developmental stages (E18.5). Gtf+/del mice showed increased cell packing density in cortical layer V, without changes in cortical thickness. Interestingly, Gtf2i+/dup had a profound effect on cortical organization; the cortical thickness was increased with decreased layer V thickness and increased layer II-III thickness. These findings suggest that changes in Gtf2i and

Gtf2ird1 copy number result in altered cellular composition of precursor cells and cortical structure during early cortical development, and may contribute to abnormal cognitive and behavioral phenotypes of WBS and Dup7.

3.2 Possible interactions between Gtf2i and Trk signaling in cortical development.

Gtf2i and Gtf2ird1 are expressed starting at very early stages of development (E3.5) in the inner cell mass and trophectoderm, and then throughout development (Makeyev et al.,

2012). Both transcription factors play a crucial role in embryonic development. Gtf2i homozygous knockout mice are embryonically lethal due to brain hemorrhage, severe neural tube defects and poor vasculature (Enkhmandakh et al., 2009). TFII-I is also involved in growth factor signaling through regulation of c-fos expression and gets activated by various growth factors including epidermal growth factor (EGF), platelet- derived growth factor (PDGF) and serum (Kim et al., 1998). Based on mouse models of

WBS, I propose a model where TFII-I and GTF2IRD1, in their roles as multi-functional transcription factors, may interact with intracellular proteins in the extracellular signal- regulated pathways to regulate precursor cell maintenance, proliferation and differentiation. There are other neurodevelopmental syndromes that share affected systems with WBS, including the neuro-cardio-facial cutaneous family of syndromes

(NCFC). NCFC includes Noonan syndrome (NS), Costello syndrome (CS), cardio-facio- cutaneous syndrome (CFC), neurofibromatosis type 1 (NF1) and LEOPARD syndrome (LS) all of which are characterized by heart defects, distinct facial features, short stature, and intellectual disability (Bentires-Alj et al., 2006). NCFC involves mutations in different signaling proteins involved in the Trk signaling pathway (SHP2 -Ras-Raf- MEK-Erk)

(Fig3.1). The Trk signaling pathway is initiated by various growth factors including neural

78 growth factor (NGF), BDNF, NT-3 and NT-4. Previous studies by many laboratories have demonstrated the importance of Trk signaling in cell-fate determination, proliferation, survival, and differentiation of cortical precursors (Medina et al., 2004; Lotto et al., 2001;

Bartkowska et al., 2007). Mutations of different intracellular proteins beget different outcomes on neuronal precursor cell biology (Fig.3.2). Inhibition of SHP-2 leads to decreased precursor proliferation and neurogenesis along with enhanced astrogenesis

(Gauthier et al., 2007), whereas inhibition of MEK and CCAAT/enhancer binding proteins

(C/EBPs), downstream targets of ERK, lead to decreased neurogenesis with increased precursor proliferation (Kim and Son, 2006). The conditional knockout of Erk2 results in decreased neurogenesis and increased astrogenesis (Samuels et al., 2008).

Interestingly, TFII-I is involved in Trk signaling as a downstream molecule of Ras (Kim and

Cochran, 2000). Any factors that can activate ERK, Raf, and MEK1 also increased TFII-I activity through phosphorylation, and inhibition of MEK1 led to abolishment of TFII-I function on the c-fos promoter (Kim and Cochran, 2000). Furthermore, TFII-I activates the c-fos promoter through direct interaction with ERK1.2. During basal conditions, the TFII-Iβ isoform is localized in nucleus (where it may serve as a basal repressor for the c-fos gene) and TFII-IΔ is localized in cytoplasm. Upon mitogenic stimulation by growth factors, isoforms reciprocally translocate; TFII-Iβ is exported out into cytoplasm and TFII-IΔ is imported into the nucleus. Phosphorylated TFII-IΔ by Ras/Raf/Mek signaling forms a complex with ERK1/2, binds to c-fos promoters and increases its activity (Hakre et al.,

2006).

Figure 3.1. Trk Signaling disruptions in neuro-cardio-facial cutaneous family of syndrome. (LS) LEOPARD syndrome, (NF1) neurofibromatosis type, (CFC) Cardio- facio-cutaneous syndrome, (CS) Costello syndrome, (NS) Noonan syndrome

Figure 3.2. The involvement of Trk signaling in regulation of cell-fate determination, proliferation, survival, and differentiation of cortical precursors. Down-regulation of different signaling molecules led to changes in proliferation, neurogenesis and astrogenesis. (Down arrow) down-regulation, (+ ) direct interaction of TFII-I with ERK.

Gtf2i deletion may have similar effect as Erk deletion. The decreased neurogenesis without changes in precursor proliferation and survival in Gtf+/del parallels the Erk-/- cortical phenotypes. Altered dynamics of neurogenesis in Gtf+/del might be due to decreased interaction with ERK1/2 which could down regulate genes involved in neurogenesis, such as the C/EBPs induced tα1 α-tubulin. One might hypothesize that given the effects of TFII-

Iβ as a basal repressor on gene regulation, decreased expression of TFII-Iβ should result in increased downstream gene expression. In Gtf+/del mice , since both TFII-Iβ and TFII-IΔ are deleted, there should be no net changes in gene expression as deletion of TFII-I -β should increase while deletion of TFII-I -Δ should decrease gene expression. However, inhibition of

TFII-I β did not cause enhanced expression of the c-fos gene (Hakre et al., 2006). Thus, it is likely deletion of Gtf2i and Gtf2ird1 will lead to decreased gene expression of neuronal genes leading to decreased neurogenesis. This may be due to the fact that the expression of developmental genes requires a complex of transcription machineries, and TFII-I, as one of intracellular proteins, may act in synergy with other molecules to promote gene transcription.

Although the TFII-I and ERK1/2 interaction may explain decreased neurogenesis, the decreased number of Pax6- and Nestin-positive precursor cells but unaffected precursor survival or proliferation in Gtf+/-del neurons cannot be explained by disrupted Trk signaling.

This is because disruption of Trk signaling, either through inhibition of MEK or C/EBP, results in increased proliferation (Kim and Son, 2006). MEK knockdown inhibits differentiation of neurons and forces precursor cells to remain in an undifferentiated state, resulting in an increased population of proliferating precursor cells (Samuels et al., 2008).

Since deletion of Gtf2i and Gtfi2rd1 did not have any effects on Tbr2-positive intermediate progenitor numbers, the affected precursor cells in NPC cultures from Gtf+/del mice should consist of primarily RG cells. This decrease in the number of RG cells is not due to RG cells stuck at a quiescent state because even Ki67-negative quiescent stem cells should also stain positive for Pax6 and Nestin.

The reduction in the number of RG cells may be due to changes at an even earlier developmental stage than E12.5, involving changes in the physiology of neural epithelial

(NE) cells. In the embryonic development, NE cells give rise to RG cells. In Gtf+/del mice, NE cells might remain in an undifferentiated state, giving rise to a smaller number of RG cells at E12.5. A recently published paper by Bayarsaihan (2012) supports the idea that TFII-I may have epigenetic regulatory effects on lineage-specific genes by colocalizing with bivalent chromatin on their promoters to promote embryonic stem (ES) cell differentiation.

This may be the case in the Gtf+/del mice, where deletion of TFII-I inhibits its regulatory role on precursor cell differentiation.

In contrast, the increased neurogenesis observed in Gtf2i+/dup could be due to upregulation of neuronal genes through increased interaction with ERK1/2, or due to an increased number of proliferating precursors resulting in a larger pool of precursors with the potential to differentiate in the NPC culture. No changes in the number of Tbr2-positive BP cells demonstrated increased proliferation of RG cells in Gtf2i+/dup mice. TFII-I may promote

RG proliferation through its interaction with TRPC3. TRPC3 is a crucial receptor in

83 stimulating ACE through PLC-γ signaling (Caraveo et al., 2005). Both TRP3 and TFII-I are activated by PLC-γ. The cytosol TFII-I acts as competitive inhibitor of TRPC3 by sequestering PLC-γ away from TRPC3, which leads to decreased calcium entry. TRPC family receptors have been implicated in neural stem cell proliferation and neuronal differentiation (Wu et al., 2004). In proliferating hippocampal cells, expression of TRPC1 and TRPC3 mRNA and protein was decreased whilst expression of TRPC4 and TRPC7 mRNA and protein was increased. In differentiated neurons, TRPC1 and TRPC3 mRNA and protein expression were enhanced with decreased expression of TRPC4 and TRPC7 mRNA and protein. Since TFII-I is an inhibitor of TRPC3, increased TFII-I in Gtf2i+/dup NPCs may result in decreased ACE, leading to an environment that favors neural stem cell proliferation.

3.3 Gtf2i and Gtfi2rd1 play roles in laminar organization and cell density of the cortex.

Copy number variation in Gtf2i and Gtf2ird1 resulted in disturbances in neural precursor cell biology and neurogenesis. These early perturbations persisted into later stages of cortical development, that manifest as altered cortical laminar architecture and neuronal cell packing. Gtf +/del mice had increased cell-packing density in cortical layer V. This data concurs with other studies carried out in human brain. Some subtle morphological changes

84 in brain structure have been observed in WBS patients. The post-mortem analysis of three

WBS brains showed a reduction in overall brain size and increased cell-packing density in layer IV in left hemisphere as well as an excess of small neurons in layers IV, V, and VI of the visual cortex. (Meyer-Lindenberg et al. 2006; Galaburda, A.M et al. 2002). Another post-mortem analysis of a single WBS subject showed a general increase in the cell packing density without changes in cortical thickness in premotor area (Galaburda et al., 1994).

Increased cell density could result from defects in the migration of neurons from the SVZ to the cortical plate, or from temporal dysregulation of neural differentiation. The fate of each laminar neuron is determined by the cell birth date, and precursor cells may have failed to generate layer-specific neurons during the right time window in Gtf +/del mice. Moreover, increased cell packing density is correlated with reduced dendritic aborization in other mental disorders (Raymond et al., 1996; Kaufmann et al., 1998), suggesting that reduced neurite length in Gtf2i+/del mice could be correlated with increased cell-density.

Gtf2i+/dup mice had an increase in overall cortical thickness, which could be due to increased proliferation of Pax6- and Nestin-positive precursor cells. Previous studies have shown that changes in neuronal proliferation and migration lead to altered cortical organization and cell density (Barth 1987; Kaufmann and Galaburda, 1989). An increase in precursor proliferation and survival in Fgfr3-/- mice resulted in a larger brain with increased cortical thickness (Inglis-Broadgate et al., 2005). Gtf2i+/dup mice also had increased cortical layer II-III thickness with decreased layer V thickness. Interestingly, intense expression of Gtf2i was observed in layer II-III, whereas layer 5 had scattered expression in Gtf2iGT(YTA365)Byg adult mice (Young , 2010 ; Osborne, 2011)The level of Gtf2i

85 expression may be correlated with altered thickness of specific layers, with increased Gtf2i expression resulting in increased cortical thickness. There are many other cognitive disorders that share altered laminar organization and altered cortical thickness, which may contribute to cognitive impairments in these disorders (Table 3.2). In Rett’s syndrome

(RS), cell body size is increased with increased cell packing density (Bauman et al., 1995).

Rubinstein-Tayi syndrome (RT) is caused by mutations in the transcriptional coactivator,

CREB binding protein (CBP) (Petrij et al., 1995). Knockdown of CBP using shRNA results in decreased precursor differentiation into all three neuronal lineages with increased Pax6- positive precursor cells but no changes in precursor proliferation and survival (Wang et al.,

2010). In RT, neuronal size is decreased with increased cell packing density (Pogacar et al.,

1973). Thus, increased neuronal density in Gtf +/del mice and increased cortical thickness in

Gtf2i+/dup mice may contribute to neurological features of WBS despite the lack of major changes in gross morphology of brain structures in these mice.

Table 3.2. Altered cortical architecture in different developmental disorders with cognitive impairments.

Disorder Laminar disturbance Cell packing Neuronal

density Morphology

Down Syndrome Irregular Not present Decreased in

size and length (Takashima et al., 1989)

Rett’s Syndrome Not present Increased cell Increased cell

density body size (Bauman et al., 1995)

Rubinstein-Tayi syndrome Not present Increased cell Decreased

density neuronal size (Pogacar et al., 1973)

Williams Syndrome Decreased cortical Increased cell Decreased

thickness density neuronal size (Galaburda, A.M et al. 2008)

Although additional Gtf2i copies increased neurogenesis, the neuronal physiology in these

NPC cultures could also be altered. As a ubiquitous second messenger, calcium not only plays an important role in neuronal differentiation, but also in neuronal maturation and synaptic plasticity. Copy number variation of Gtf2i likely result in altered cellular calcium levels and potentially neuronal maturation, by altering surface-available TRPC3. Our collaborators in the Department of Physiology have shown that Gtf2i+/dup primary neurons show decreased calcium entry with decreased neurite branching, and Gtf2i+/- neurons show increased intracellular calcium levels with increased neurite branching (Marielle Deurloo, personal communication).. This ties well into this study; changes in Gtf2i and/or Gtf2ird1 copy number result in aberrant neural precursor cell physiology, which can lead to abnormal neuronal maturation. These findings emphasize that neurological impairments in

WBS may result not only through changes in cellular composition (number of cells) but also

87 through imbalance between different populations of cells (neurons VS glial cells), through temporal dysregulation (born too early or too late), through migratory defects (cell packing density), through improper maturation (altered neurite branching), or through abnormal circuit formation (abnormal dendritic spine morphology and density, and changes in synaptic activity).

Very little is known about the mechanisms underlying the neurological features of WBS.

Here, I have shown that Gtf2i and Gtf2ird1 copy number changes result in altered neural precursor cell physiology and differentiation, leading to cortical dysgenesis. Together, these findings demonstrate that one way in which neurological features may be elicited in

WBS is through early developmental perturbation. Thus, further investigation of molecular mechanisms linking Gtf2i and Gtf2ird1 with intracellular proteins such as Trk signaling molecules involved in the regulation of cortical development may provide useful insight into specific avenues for therapeutic intervention in individuals with WBS.

Chapter 4. Conclusions and Future Directions

4.1 Summary

4.1.1. Overview

WBS is a neurodevelopmental disorder that affects cognitive ability and behaviour, as well as causing characteristic cardiovascular abnormalities (predominantly supravalvular aortic stenosis) and facial features. Children with WBS have distinct peaks and valleys of cognitive function with mild to moderate intellectual disability (average IQ of 55 to 60). They have relatively spared language capabilities and face recognition, but extremely poor visuospatial and math skills. They are described as over-friendly with a lack of many normal social boundaries, but they also have increased anxiety and specific phobias.

WBS is caused by the hemizygous deletion of approximately 1.55 million nucleotides at chromosomal region 7q11.23, resulting in the loss of between 26 and 28 genes depending on the deletion breakpoint. Patients with atypical deletions that do not span the entire WBS region provide opportunity to study the contribution of individual genes to WBS phenotype. For instance, based on studies of atypical patients, hemizygosity for the elastin gene was proposed as the contributing factor responsible for the congenital cardiovascular

89 defects of WBS (Tassabehji et al. 1997). However, the specific genes that cause the neurological symptoms have yet to be determined.

To link specific genes with the cognitive and behavioural aspects of WBS, our lab has generated single-gene and combined-gene deletion and duplication mouse models for two candidate genes from the commonly deleted region, GTF2I and GTF2IRD1. These genes encode transcription factors that likely regulate the expression of many other genes during development (Schubert 2009). Studies of patients with different-sized deletions of the

WBS region implicate these genes as being important in the neurological features of this disorder (Tassabehji et al. 2005; Hirota et al. 2003). In our lab, we have already shown that

Gtf2ird1 knockout mice have altered behaviour and impairments in amygdala-based learning and memory. These mice showed reduced fear and aggression and increased social behaviour, that parallels the hypersociability in WBS (Young et al. 2008). Gtf2i heterozygous deletion mice show impaired short-term memory and pups with duplication of Gtf2i show increased maternal separation induced anxiety.

Although dramatic phenotypic effects are seen in both humans and mice with deletions of genes from the WBS region, brain structure appears relatively normal. The heterozygous

Gtf2ird1 mutant mice had no morphological abnormalities (volumes of cerebellum, cerebrum, hippocampus and amygdala was not differ from wild-type mice) and no impairments in hippocampal-based fear conditioning (Hagen et al. 2007). Functional MRI studies in people with WBS and electrophysiological studies in our mouse models have

90 shown hypoactivation of regions of the frontal cortex and amygdala which are the brain areas involved in the modulation of anxiety related social behaviors. Thus, we hypothesized that the neuropsychological phenotypes may be the result of early disturbance in the neuronal development. The current data shows that changes in gene copy number of both

Gtf2i and Gtf2ird1 affect neural precursor development and neurogenesis in a dose- dependent manner and these disturbances translate into aberrant brain formation with altered laminar organization and cell density. These results indicate the proper growth and differentiation of the neural precursor pools into neurons are crucial for the proper development of the cortex and that when this process is disrupted, it may contribute to neurological features of WBS.

4.1.2. Gtf2i and Gtf2ird1 play a crucial role in radial glial cell maintenance and differentiation without affecting proliferation and survival.

Both single embryo and pooled embryo NPC cultures of Gtf+/del and Gtf2i+/dup embryoinic cortex showed the importance of Gtf2i and Gtf2ird1 in the regulation of precursor cell maintenance. Gtf+/del mice had an overall decrease in the percentage of Nestin- and Pax6- positive neural precursor cells compared to WT mice, although the survival and proliferation of precursors were not affected. In contrast, Gtf2i+/dup mice had an overall increase in the Nestin- and Pax6- positive precursor cells with enhanced proliferation but

91 no changes in apoptosis. Thus it is likely that Gtf2i, not Gtf2ird1, is involved in the regulation of precursor proliferation, and neither Gtf2i nor Gtf2ird1 are required for precursor cell survival. Gtf+/del and Gtf2i+/dup cortex showed no effect on maintenance of the

BP population, suggesting that both Gtf2i and Gtfi2ird1 play a crucial role in RG cell maintenance. Deletion of Gtf2i and Gtf2ird1 led to a decreased number of neurons whereas duplication resulted in enhanced neurogenesis. Increased neurogenesis in Gtf2i+/dup was due to increased proliferating neural stem cells. This pool of undifferentiated precursors that can be differentiated upon appropriate neurogenic signals are normally expanded through increased proliferation. Reduction in the number of stem cells in Gtf+/del mice maybe caused by disturbances at an earlier developmental stage, where NE cells remain at an undifferentiated state. Future experiments are necessary to dissect these possible changes, but if this is the case, the number of Sox2-positive NE cells should be increased in

Gtf+/del cultures. Gtf2i+/- mice showed a reduced number of cortical precursor cells compared to WT, though to a lesser extent than Gtf+/del mice, indicating that Gtf2i alone can mediate cortical precursor maintenance. The neurogenesis was not affected in Gtf2i+/- mice suggesting Gtf2ird1 may compensate for the loss of Gtf2i in neurogenesis. These findings for NPCs hint that despite similar structure TFII-I (GTF2I) and GTF2IRD1 have independent and also overlapping function. Analysis of single embryo cultures from Gtf2i+/- and

Gtf2ird1+/- mice will help determine the exact contribution of these two genes to precursor cell production, maintenance and differentiation.

Monogenic deletions and copy number variants have been shown to contribute to structural and functional brain changes in prenatal development, that predispose people to

92 develop specific neurological diseases, and result intellectual disabilities in many neurodevelopmental disorders. The data obtained in this study coincides well with previous studies of other neurodevelopmental disorders where changes in neural stem cell physiology of differentiation, proliferation or/and survival are observed upon knockout or overexpression of genes disrupted (Gauthier et al., 2007; Wang et al., 2010; Dugani et al.,

2010; Pucilowska et al, 2012). When Cbp (haploinsufficient in RT syndrome) is inhibited using siRNA, differentiation of cortical precursors into neurons, astrocytes and oligodendrocytes was reduced, with increased Pax6-positive neural stem cells; precursor proliferation was increased in expense of neurogenesis (Wang et al., 2010). These changes in precursor physiology are somewhat relevant to the decreased precursor pool and neurogenesis seen in Gtf+/del mice. On the other hand, Gtf2i+/dup NPC cultures with increased proliferating precursor cells parallel Fgfr3-/- mice that exhibit increased proliferation and neurogenesis (Inglish-Broadgate et al., 2005). These findings suggest both Gtf2i and

Gtf2ird1 are essential for RG cell maintenance and neurogenesis, and that altered precursor physiology may contribute to some of the neurological features of WBS.

4.1.4. Gtf2i and Gtf2ird1 copy number variation regulates cortical architecture and cell density in developing cortex.

Altered dynamics of neural precursor cell maintenance and neurogenesis in Gtf+/del and

Gtf2i+/dup mice at E12.5 persisted into E18 in the form of altered laminar organization and cell density. Gtf+/del mice that had decreased numbers of neuronal precursors and decreased neurogenesis showed an increase in cell packing density in cortical layer V but no changes in cortical thickness. This is consistent with postmortem analysis of brain tissue from individuals with WBS, where increased cell packing density was observed in layer IV in left hemisphere (Galaburda, A.M et al. 2008) and cortical thickness was not affected

(Galburda et al., 1994).

Gtf2i+/dup mice had a significant increase in the number of neuronal precursors and increased neurogenesis, which resulted in an increase in total cortical thickness. Some children with autism also have a larger brain size, which is relevant to Dup7 individuals as they display autistic behaviour (Stanfield et al., 2008). Early cortical overgrowth is common in children with autism and one recent study of post-mortem brain tissue found increased neuron numbers in the prefrontal cortex of children with autism, compared to controls (Courchesne et al., 2011). Increased proliferation of neural stem cells has been correlated with an increase in cortical thickness. An increase in proliferation and survival of neural precursors in Fgfr3-/- mice resulted in increased cortical thickness and a larger brain (Inglis-Broadgate et al., 2005). Ciliary neurotrophic factor (CNTF) increased proliferation of neural stem cells in lateral ventricle, and resulted in increased cortical thickness postnatally (Shimazaki et a., 2001). Many other mental disorders display altered cortical architecture. In DS individuals, whom display a similar degree of intellectual disability to those with WBS, have an overall increase in cell packing density and irregular

94 laminar organization in the cortex (Takashima et al., 1989). RS and RT also show increased cortical cell density (Pogacar et al., 1973; Bauman et al., 1995). The findings obtained in this study along with other previous studies provide important insight into cortical development. The nervous system is built from the bottom up, from genes to precursors, through differentiation to cortical formation, and disturbances incurred during early stages of brain development can result in aberrant postnatal cortical formation and maturation, as may be the case in WBS.

4.2 Future Directions

4.2.1. Balance between different cellular components:

4.2.1.1 Defining precursor differentiation

During murine neural development, neural stem cells generate neurons first, followed by glial lineages (Miller and Gauthier, 2007). NPC cultures collected from E12.5 embryo also recapitulate this temporal differentiation pattern, generating neurons followed by astrocytes then oligodendrocytes (Qian et al., 2000). This pattern of differentiation can be easily investigated in primary NPC cultures as they can be directed to differentiate into specific lineages by adding different chemical agents. For instance, addition of LIF allows stem cells to be in a proliferative state, whereas serum-free medium allows stem cells to differentiate into less pluripotent lineage-specific cells (Faherty et al, 2005). In the current study, the capacity of NPCs to differentiate into glial lineages was not investigated.

However, many other studies have shown imbalances between different neuronal lineages in NPC cultures upon signaling disruption or enhancement (Bonni et al., 1997;Raballo et al.,

2000; Bonni et al., 1999). The conditional knockout of Erk2 resulted in a decrease in the number of neurons while the number of astrocytes was increased (Samuels et al., 2008).

Constitutively active SHP-2 increased neurogenesis with decreased astrogenesis (Gauthier et al., 2007). CNTF or neuregulin β can be added to NPC cultures to promote gliogenesis.

Then NPC cultures can then be stained with astrocyte marker, glial fibrillary acidic protein

(GFAP) and oligodendrocyte marker, O4, to see whether changes in copy number of Gtf2i and/or Gtf2ird1 have any effects on gliogenesis. The astrocytes provide a supportive role to neurons by releasing growth factors, and oligodendrocytes myelinate neurons for faster signal transduction. Investigating the balance between neurons and glial cells are important because glial cells can affect the function of neurons.

4.2.1.2. Defining precursor population

In the developing nervous system, various populations of neural stem cells are present. The first neural stem cells that arise from neural plate are NE cells. NE cells then give rise to B cells (RG cells). B cells can be at the quiescent state (slowly proliferating B cells) or at the active state, proliferating to expand the RG cell population. Then RG cells asymmetrically divide to give rise to C cells (BP or IP cells) that are also known as transit amplifying progenitors (TA). C cells divide asymmetrically to produce two neurons and are thought be responsible for laminar expansion of cortices (Morshead and van der Kooy, 2004; Gotz and

Huttner, 2005). Intrinsic programs and extrinsic signaling pathways are involved in regulating the expression of different sets of genes, which act as a hallmark for each neural precursor cell type. NPC cultures collected from E12.5 embryo also contain various neural stem cell populations, and we can map different types of precursor cells based on specific gene markers. In the current study, Pax6- and Nestin-positive RG cells were stained along with Tbr2-positivie BP cells. However, effects of Gtf2i and Gtf2ird1 gene dosage on NE cells were not investigated. In Gtf+/del NPC culture, an overall reduction in RG cells and neurons was observed without changes in precursor proliferation and survival, or in BP cell maintenance. This suggests that decreased RG cell number may be accounted for by for

97 changes in NE physiology. Performing immunohistochemistry for Sox2, a NE marker, on

Gtf+/del NPC culture and costaining with Nestin, Ki67 and CC3 will allow further investigation of how deletion of Gtf2i and/or Gtf2ird1 affects different neural stem cells during cortical development.

4.2.2. Molecular mechanism: relationship between TFII-I and ERK1/2 in neural precursor physiology.

TFII-I and GTF2IRD1 are multifunctional, and inducible transcription factors thought to be involved in a variety of cellular responses. Activity of TFII-I is modulated by extracellular cues including growth factors that also activate Trk signaling pathway (SHP-2-Ras-Raf-

MEK-Erk). Trk signaling plays a crucial role in regulating precursor cell-fate determination, proliferation, survival, and neurogenesis (Medina et al., 2004; Lotto et al., 2001;

Bartkowska et al., 2007). Disruption of this signaling pathway can lead to cortical dysgenesis, and is implicated in the NCFC family of syndromes with some clinical characteristics that overlap with WBS including intellectual disability, heart abnormalities and craniofacial features. In each NCFC syndrome, disruption of intracellular proteins involved in Trk signaling pathway was seen, leading to inhibition of neurogenesis with promotion of astrogenesis in NPC cultures (Medina et al., 2004; Lotto et al., 2001;

Bartkowska et al., 2007). In contrast, enhancing Trk signaling resulted in increased neurogenesis (Kim and Son, 2006; Bartkowska et al., 2007; Samuels et al., 2008; Majumder et al., 2012). TFII-I is known to directly interact with ERK1/2 in modulating c-fos promoter

98 activity as well as being regulated by Ras and activated by extracellular cues that also activate Trk signaling (Kim and Cochran, 2000; Hakre et al., 2006). Interaction between

ERK1/2 and TFII-I can be investigated to define whether TFII-I exerts regulatory effects on neural precursor maintenance and neurogenesis through SHP-2-Ras-Raf-MEK-Erk pathway. Inhibition of TFII-I could be carried out in vitro using a functional antibody or siRNA, and expression constructs could be used to increase levels of TFII-I. Both these changes in TFII-I protein levels may change ERK1/2 activity on the promoters of neurological genes.

4.2.3 Cortical cytoarchitecture

Cortical dysplasia can result from disruptions at early steps during cortical development.

Disruptions at early phases of RG proliferative and differentiating cell divisions can lead to global cortical dysgenesis with altered brain size, cortical thickness and lissencephaly

(Crino and Eberwine, 1997). Disruptions at migratory phases of differentiated neurons from the apical side into the CP can lead to focal cortical dysgenesis as seen in the disorders

DS, RT, RS and WBS (Takashima et al., 1989;Bauman et al., 1995,Pogacar et al.,

1973;Galaburda, A.M et al. 2008). The current data showed that changes in gene copy number of both Gtf2i and Gtf2ird1 affect neural precursor physiology and differentiation in a dose dependent manner in the midgestation developing mouse cortex. These effects were translated into altered neuronal density and cortical thickness in the prenatal cortex.

Interestingly, Gtf+/del and , Gtf2i+/dup mice exhibited distinct modifications to different

99 layers. Gtf+/del mice had increased cell packing density at layer V whereas Gtf2i+/dup mice had an increase in the overall cortical thickness with increased thickness for layer II-III and decreased thickness for layer V. Increased cell density at layer V in Gtf+/del mice may accompany decreased cell density in other layers. For detailed studies of laminar specific effects of Gtf2i and Gtfi2rd1, we can obtain cortical sections of E18 Gtf+/del, and Gtf2i+/dup embryos, and stain them with other layer specific markers, Reelin (Layer 1), and Foxp2

(Layer VI) for aberrant cytodifferentiation. Moreover, examining cortical sections at different developmental time points, E14, E18, postnatal day 1 (P1) and P3, will allow investigation of any migratory defects or temporal disturbances in laminar organization that persist into the post-natal period. The analysis at different development time points is necessary, because disturbances may be caused by developmental delay and cytoarchitecture differences may disappear at later stages. A previous study by Wang et al

(2010) showed haploinsufficiency of Cbp resulted in increased Pax6-positive precursors and decreased neurogenesis and astrogenesis at E13 along with a decrease in corpus callosum (CC) thickness at P10. However, CC thickness caught up to WT at P50, indicating developmental delay in Cbp-/- mice. Individuals with WBS and 7q11.23 duplication display developmental delay, hence changes in the density or cortical thickness may “catch up” during later stages of development. In parallel, WBS individuals have altered CC morphology including reduced CC area, larger bending angle and increased thickness

(Sampaio et al., 2012). The CC connects the two hemispheres together, executing various cognitive functions such as selective visual attention, binocular coordination and intelligence (Hines et al., 2002, Johansen-Berg et al.,2007; Luders et al. 2007). Staining P10 and P30 cortical sections with Nissl will allow us to examine effects of Gtf2i and Gtf2ird1 copy number changes on the regulation of CC development, that may contribute to

100 cognitive dysfunction, specifically, visuospatial deficits and intellectual disability observed in WBS.

4.2.4. Circuits and networks - synaptic function

After observing changes in neural precursor physiology, neurogenesis and cortical organization, the functional state of neuronal network can be examined. Once cells migrate to appropriate cortical layer, they start to mature and extend their dendrites and axons to form synapses with other neighboring neurons. Proper growth and branching of dendrites are crucial for nervous system function as neurons receive and integrate synaptic information through those processes. Previous studies have shown that defects in dendrite development and maintenance result in aberrant connectivity, which may contribute to neurocognitive phenotypes in neurodevelopmental disorders (Kaufmann and Moser,

2000). For example, in DS and RS, which both exhibit cognitive dysfunction, dendritic length is reduced with decreased spine density (Kaufmann and Moser, 2000). It has already shown by our collaborators (Zhong-Ping Feng and Marielle Duerloo) that Gtf2i+/- primary cortical neurons show increased axon branching whereas Gtf2i+/dup neurons show decreased axon branching. This was related to increased TRPC3 localization at the membrane in Gtf2i+/- and widespread cellular localization of TRPC in Gtf2i+/dup, which in both of the cases led to abnormal synaptic activity. Abnormal dendritic aborization, dendritic spine morphology or density can lead to impaired synaptic plasticity. Aberrant synapse development may contribute, in part, to the pathophysiology of WBS. The potential

101 role of Gtf2i and Gtf2ird1 in controlling axon branching and synapse numbers can be studied with P1 neuronal cultures to look for changes in height, density and morphology of dendritic spines in Gtf+/del, Gtf2i+/- and Gtf2i+/dup neurons. If there are changes, these may infer changes in synaptic function, and current-clamp whole-cell recording can be performed to measure changes in electrophysiological properties of the neuronal cultures.

4.2.5. Contribution of individual genes to neural development during prenatal and postnatal periods.

Cognitive and behavioral profiles are differentially affected in both atypical individuals and mouse models with single- and combined-gene deletion of Gtf2i and Gtf2ird1. The case studies of individuals with atypical deletion of either GTF2I and/or GTF2IRD1 showed distinct social behaviours; an individual with deletion that included GTF2IRD1 but spared GTF2I displayed strong social motivation, hypersociability, and mild social disinhibition along with craniofacial features, whereas an individual with a deletion including GTF2I but sparing GTF2IRD1 exhibited autistic social behavior with decreased social motivation and a shy personality with no apparent social disinhibition or facial features (Karmiloff-smith et al., 2012). Another individual carrying hemizygous deletion of GTF2IRD1 but not GTF2I displayed impaired language acquisition with no overfriendliness and facial features (Tassabehji et al., 2005).

Distinct phenotypes were also observed in mouse models with deletion of either Gtf2i or

Gtf2ird1. Gtf2ird1+/- mice had reduced fear, decreased levels of anxiety and hypersociability

(Young et al., 2008), while Gtf2i+/- mice from our lab showed impairments in novel object

102 recognition, reduced fear only in male mice, increased grip strength in female mice and no differences in anxiety level (Emily, through personal communication). These behaviors differences indicate that Gtf2i and Gtf2ird1, despite structural similarity, may function independent of each other.. The data from this study showed Gtf +/del mice with a decreased percentage of neural precursors and reduced neurogenesis. Gtf2i+/- mice had reduced number of cortical precursor cells compared to WT but the effect was milder than in Gtf+/del mice, with inconsistent effects on neurogenesis. This demonstrates a possible compensatory role of

Gtf2ird1 on neurogenesis upon deletion of Gtf2i, and possible synergism between Gtf2ird1 and

Gtf2i in NPC maintenance. Only NPC cultures from pooled embryos were carried out on Gtf2i+/- mice in the current study. Pooled NPC cultures consist of embryos with different genotypes and depending on how many WT embryos present, compensation or masking for the loss of Gtf2i could occur, resulting in inconsistent data. Thus, it will be important to segregate the effects of the two genes on neural precursor cell physiology and development using single-embryo NPC cultures from single-gene knockout mouse models, Gtf2i+/- and Gtf2ird1+/-.

Immunohistochemistry for Ki67, CC3, Sox2, Pax6, Tbr2 and βlll tubulin can be performed on these cultures collected from E12.5 embryo to investigate changes in proliferation, apoptosis, number of NE, RG, IP, and neuronal cells respectively. Then same neuroanatomical analysis of developing cortex can be performed on E18.5 cortical sections to unravel each individual gene’s contribution to later brain development.

Time course experiments can be performed to determine how altered expression of Gtf2i or/and Gtf2ird1 may affect neural precursor cell physiology and differentiation at early stages of development, cortical thickness and cell packing density at later stages of development and finally spine morphology , electrophysiological properties and synaptic function during

103 postnatal periods.

4.2.6. Human models of WBS - induced pluripotent stem cells

Understanding the process of neural development through the analysis of transgenic and knockout mouse technology is limited, as it represents only a fraction of diseases with such complex cognitive phenotypes, and may display species- and genetic background- differences in phenotypes. Yamanaka’s groundbreaking reprogramming technique to convert an already specialized cell into pluripotent stem cell – called an induced pluripotent stem cell (iPSC) - by introducing 4 transcription factors (SOX2, OCT4, KLF4, and MYC) opened a new window for patient-specific personalized therapy, disease modeling, and drug discovery (Takashashi et al.,

2007;Marchetto et al., 2010). Recently, modeling of vascular phenotypes in WBS was carried out using patient-derived iPSCs (Kinnear et al., 2013). Deletion of ELN in the WBS region leads to the cardiovascular phenotypes of hypertension, aortic and vascular stenosis and abnormal proliferation of smooth muscle cells (SMC) in the vessel walls (Ewart et al., 1993). In James

Ellis’ lab, iPSCs were generated from fibroblast of an individual with WBS (WBS-iPSCs) and differentiated into SMCs. WBS-iPSC derived SMCs (WBS-iPSC-SMCs) were highly proliferative and remained at the immature state with reduced expression of differentiated SMC markers.

WBS-iPSC-SMCs failed to produce proper tube-like contractile structures that resemble vasculature, paralleling ectopic proliferation of SMCs in individuals with WBS. WBS-iPSC-SMCs recapitulated the disease-state of WBS. Moreover, addition of rapamycin fully rescued the diseased phenotype by reducing proliferation of WBS-iPSC-SMCs, thereby enabling them to

104 differentiate into mature and functional SMCs. Thus, iPSCs derived from individuals with WBS can be used as an alternative tool for studying the mechanisms underlying pathobiology of neural development in WBS, as they can be differentiated into disease-bearing neuronal cell types. Two WBS iPS cell lines have already been generated by the Ellis lab, and we are currently generating iPSC lines from individuals with Dup7. To investigate whether neural precursor cell physiology and subsequent differentiation and maturation are disturbed in the human cellular models of WBS, similar experimental procedures as those carried out in NPC cultures from a mouse model could be undertaken in iPSCs. Immunohistochemistry for Sox2, Pax6, Nestin,

Ki67, CC3, Satb2, and βlll tubulin can be performed on WBS-iPSC derived neural stem cells. This would allow us to assess the number, proliferation, vitality and differentiation capabilities of neuronal precursor cells derived from patient iPSCs. Immunohistochemistry for CC3, NeuN

(early neuronal marker) and βlll tubulin can be performed on WBS-iPSC derived neurons to assess changes in neuronal physiology. Using WBS-iPSC derived neurons, neuronal morphology could be studied to look for for changes in neuronal morphology and synapse formation. The advantage of using iPSCs is that the results obtained from WBS-iPSC-NPCs and WBS-iPSC- neurons can be compared with analyses of NPC cultures from Gtf2i or/and Gtf2ird1 deletion/duplication mouse models to confirm the role of these two genes in regulation of precursor maintenance, proliferation, neurogenesis, and neuronal maturation or reveal novel roles for those genes. Together, these data may provide deeper understanding of how the developing nervous system is perturbed in WBS individuals, and may give insight into an answer of pathological causes in WBS and possibly therapeutic interventions to treat the disorder.

105

4.3 Conclusion

This study has investigated effects of altered copy number of Gtf2i and Gtf2ird1 on neuronal precursor physiology using NPC cultures from mouse models of WBS. The changes in gene copy number of both Gtf2i and Gtf2ird1 affected neural precursor physiology, maintenance, proliferation and differentiation in a dose dependent manner in the midgestation developing mouse cortex. These effects were translated into altered neuronal density and cortical thickness in the post-natal cortex. However, molecular mechanisms underlying these phenotypes are yet to be examined. Further investigation of effects of altered Gtf2i and Gtf2ird1 gene expression on cortical dysgenesis using different mouse models and human cells will provide insight into the molecular pathways underlying abnormal behaviour and cognition in WBS.

106

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