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Molecular mechanisms underlying malformations of cortical development

Sabrina Anjum Oishi

Bachelor of Science (Biomedical Sciences), Honours Class I A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2020

Faculty of Medicine

School of Biomedical Sciences

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Abstract

The human cerebral cortex is comprised of billions of neurons and glial cells, all originating from a

pool of neural stem cells (NSCs) within the developing brain. NSCs retain their undifferentiated stem

cell identity through self-renewal while also having the capacity to produce the three main cell types

within the nervous system, namely neurons, astrocytes and oligodendrocytes. The coordinated

proliferation and timely progression of NSC differentiation in both the developing and adult brain determines the growth and functions of the cerebral cortex. A network of molecular mechanisms,

including epigenetic regulators, transcription factors, and post-translational modifiers, temporally and

spatially dictate the fate of NSCs. Disruption to these processes can result in malformations of cortical

development (MCD), characterized by abnormal brain structure and size which in turn often manifest

as intellectual disability, autism, epilepsy or other cognitive deficits. Mutations in numerous genes

have been implicated in MCDs and have since classified as clinical syndromes. This thesis addressed the underlying developmental mechanisms contributing to the abnormal cortical

phenotype, namely macrocephaly and intellectual disability, in patients with mutations in three

different genes using transgenic mice as a model system. Firstly, heterozygous mutations in the transcription factor called nuclear factor one X (NFIX) and histone modifier, nuclear receptor SET domain containing protein 1 (NSD1), cause Malan syndrome (formerly known as Sotos syndrome 2) and Sotos syndrome (also Sotos syndrome 1) respectively. Both neurodevelopmental disorders are

characterized by highly similar cortical abnormalities, including macrocephaly, intellectual disability

and autistic-like traits. Although clinical assessments have determined the underlying symptomology of both syndromes, the fundamental mechanisms contributing to the enlarged head circumference and

intellectual disability in these patients remains undefined. In Chapter 2, I performed volumetric and

diffusion tensor magnetic resonance imaging (DTMRI) analyses on Nfix heterozygous mice to reveal

that these animals have abnormally enlarged brain size and significantly abnormal cortical

connectivity between crucial areas of the brain, which may underlie the learning impairment and

abnormal social behaviour in these animals. Collectively, these data provide a significant conceptual

advance in our understanding of Malan syndrome by suggesting that megalencephaly underlies the enlarged head size of these patients, and that disrupted cortical connectivity may contribute to the

intellectual disability these patients exhibit. In Chapter 3, I examined the role of NSD1 during cortical

development as very little is known about its function in the cerebral cortex. Firstly, I showed that

Nsd1 was predominantly expressed neuronally in both the developing and adult brain. I further analysed the morphological and behavioural characteristics of Nsd1 heterozygous mice, revealing

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behavioural characteristics reminiscent of some of the deficits seen in Sotos syndrome patients. In

Chapter 4, I further analysed the causes of intellectual disability in patients with mutations in the

deubiquitylating enzyme called ubiquitin specific protease 9 (USP9X). Using the rodent system, I showed that deficiency of USP9X activity during neurodevelopment results in the abnormal development of the hippocampus, aberrant neurogenesis in the adult hippocampus, and significant deficits in cognitive function, which subsequently models and provides significant insight into the underlying causes of intellectual disability in patients with USP9X mutations. Overall, this thesis provides significant insight into how abnormal activity in various molecular mechanisms during development of the cerebral cortex lead to highly similar cortical malformations, including abnormal brain size and cognitive deficits.

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Declaration by author

This thesis is composed of my original work, and contains no material previously published or written

by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance,

survey design, data analysis, significant technical procedures, professional editorial advice, financial

support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research

candidature and does not include a substantial part of work that has been submitted to qualify for the

award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright

holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright

holder to reproduce material in this thesis and have sought permission from co-authors for any jointly

authored works included in the thesis.

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Publications included in thesis

Incorporated as Chapter 2

1. Oishi S, Harkins D, Kurniawan N.D, Kasherman M, Harris L, Zalucki O, Gronostajski R.M,

Burne T.J, and Piper M. (2019) Heterozygosity for Nuclear Factor One-X in mice models features of Malan syndrome. EBioMedicine 39, 388-400. doi: 10.1016/j.ebiom.2018.11.044.

Incorporated as Chapter 3

2. Oishi S, Zalucki O, Vega M.S, Harkins D, Harvey T.J, Kasherman M, Davila R.A, Hale L, White

M, Piltz S, Thomas P, Burne T.J, Harris L, and Piper M. (2020) Investigating cortical features of Sotos syndrome using mice heterozygous for Nsd1. Genes, Brain and Behaviour, e12637. doi:

10.1111/gbb.12637.

Incorporated as Chapter 4

3. Oishi S, Zalucki O, Premarathne S, Wood S, and Piper M. (2016). USP9X deletion elevates the

density of oligodendrocytes within the postnatal dentate gyrus. Neurogenesis 3, 1-9.

4. Johnson B.V, Kumar R, Oishi S, et al. (2020). Partial Loss of USP9X Function Leads to a Male

Signaling. Biological Psychiatry, 87, 100-112. doi: 10.1016/j.biopsych.2019.05.028.

Other publications during candidature

5. Zalucki O, Harris L, Harvey T.J, Harkins D, Widagdo J, Oishi S, Matuzelski E, Yong X.L.H,

Schmidt H, Anggono V, Burne T.H.J, Gronostajski R.M, and Piper M. (2018) NFIX-Mediated Inhibition of Neuroblast Branching Regulates Migration Within the Adult Mouse Ventricular- Subventricular Zone. Cerebral Cortex. doi: 10.1093/cercor/bhy233.

6. Harris L, Zalucki O, Clement O, Fraser J, Matuzelski E, Oishi S, Harvey T, Burne T.H, Heng J,

Gronostajski R, and Piper M. (2017). Neurogenic differentiation by hippocampal neural stem and progenitor cells is biased by NFIX expression. Development 145. doi: 10.1242/dev.155689.

7. Harris L, Zalucki O, Oishi S, Burne T.H, Jhaveri D.J, and Piper M. (2017). A morphology

independent approach for identifying dividing adult neural stem cells in the mouse hippocampus.

Developmental Dynamics. doi: 10.1002/dvdy.24545.

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Contributions by others to the thesis

Chapter 2

Magnetic resonance imaging of brains and programming scripts were processed by Nyoman Kurniawan at the Centre for Advanced Imaging, University of Queensland. Danyon Harkins and Maria Kasherman performed the histological analyses presented in Figure 2.1. Danyon Harkins performed the behavioural assays presented in Figures 2.5 and 2.6.

Chapter 3

Oressia Zalucki assisted with cell counts presented in Figure 3.4 and 3.5. Michelle Vega performed the behavioural assays presented in Figures 3.6 and 3.7.

Chapter 4

Susitha Premarathne performed the microdissection of the hippocampus for the microarray presented in Figure 4.1. Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis.

Research Involving Human or Animal Subjects

animal use in research (AEC approval number: QBI383/16 and QBI/351/16). Additionally, all work was carried in accordance to the Australian Code of Practice for the Care and Use of Animals for

A copy of

the ethics approval letter is included in the thesis appendix.

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Conference Abstracts

1. Heterozygosity for NFIX in mice models features of Malan syndrome.

Oishi S, Harkins D, Kurniawan N.D, and Piper M.

Presented at:

Poster - 10th International Brain Research Organisation (IBRO) World Congress of

Neuroscience, South Korea (2019).

Oral - Australian Neuroscience Society Annual Conference, Australia (2018). Poster - International Society of Developmental Neuroscience Biennial Meeting, Japan (2018). Poster - EMBL Australia Postgraduate Symposium, Brisbane (2018). Poster - 9th International Postgraduate Symposium in Biomedical Sciences, Brisbane (2018). Poster - 9th Brisbane Cell and Developmental Biology Meeting, Brisbane (2018).

2. Usp9x-deficiency disrupts the morphology of the postnatal hippocampal dentate gyrus.

Oishi S, Premarathne S, Wood S, and Piper M.

Presented at:

Poster - Australian and New Zealand Society of Cell and Developmental Biology (ANZSCDB)

Annual Combio Conference (2016).

Poster - 7th International Postgraduate Symposium in Biomedical Sciences (2016). Poster - 7th Brisbane Cell and Developmental Biology Meeting (2016).

Awards

PhD Research Excellence Award, School of Biomedical Sciences, UQ (2019) Faculty of Medicine Microscopy Image Award (3rd), UQ (2019) Faculty of Medicine RHD Travel Scholarship, UQ (2019) Cover of the EBioMedicine Journal, January issue, Elsevier (2019) Photography Competition Award (3rd) at 38th ANS Meeting, Brisbane (2018) Winner of SciArt Competition, EMBL Australia Postgraduate Symposium, Brisbane (2018) Best Poster at 9th International Postgraduate Symposium, UQ (2018) Best Poster at 9th Brisbane Cell and Developmental Biology Meeting, Brisbane (2018) Competitive Bursary Award (travel), ISDN conference, Elsevier (2018) Candidate Development Award (travel), UQ Graduate School (2017) Best Poster at 7th International Postgraduate Symposium, UQ (2016) Keith Dixon Poster Prize in Developmental Biology, ANZSCDB Combio2016, Brisbane (2016)

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Acknowledgements

I would like to thank and acknowledge many people who contributed to this work.

Firstly, my deepest gratitude goes first and foremost to my principal supervisor, Michael Piper. Thank

you for your scientific input, and most of all thank you for your steadfast support, encouragement, guidance, and mentorship over the last six years. Thank you for giving me the opportunity, since my first undergraduate research project in 2014 to now as a PhD graduate, to truly grow as a scientist.

Many thanks to the rest of the Piper group, past and present. I will always remember our lunch chats,

coffee runs, and overseas conference travels. Special thanks to Tracey Harvey for your constant support and help with lab work, but also your care outside of the laboratory. The years would have been far less productive and far less enjoyable without our awesome lab members. Thank you to the QBI Animal House team, SBMS and QBI microscopy and histology teams, in particular Shaun Walters and Rob Sullivan, for your generous help and excellent technical support. Thank you to my co-supervisors Oressia Zalucki and Jens Bunt, and advisory committee of Elizabeth Coulson, Daniel Blackmore and Taylor Dick for your advice and support. Most importantly, to my parents, siblings and extended family, thank you for your unwavering support, prayers and encouragement. This process would not have been possible or worthwhile without you.

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Financial support

This research was supported by an Australian Government Research Training Program Scholarship.

Keywords

NFIX, NSD1, USP9X, neurogenesis, macrocephaly, intellectual disability, DTMRI. Australian and New Zealand Standard Research Classifications (ANZSRC)

110902: Cellular Nervous System (80%)

110904: Neurology and Neuromuscular Diseases (20%)

Fields of Research (FoR) Classification

1109: Neurosciences (100%)

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Table of Contents

List of Figures 13

List of Tables 14

List Abbreviations 15

Chapter 1. General Introduction 17

1.1 Overview 17

1.2 Development of the cerebral cortex 17

1.3 Adult neurogenic niches 21

1.4 Malformations of cortical development 23

1.5 Thesis aims and structure 27

Chapter 2. Heterozygosity for NFIX in mice models features of Malan syndrome 30

2.1 Overview of Chapter 30

2.2 Abstract 31

2.3 Introduction 31

2.3.1 Heterozygosity of NFX causes Malan syndrome 31

2.3.2 NFIX as a transcription factor 32

2.3.3 NFIX is crucial for neurogenesis in the embryonic and adult brain

34

2.4 Aims and Hypotheses 35

2.5 Method 35

2.5.1 Animals 35

2.5.2 Magnetic resonance imaging 35

2.5.3 Volumetric and tractography 36

2.5.4 Tissue preparation 37

2.5.5 Haematoxylin 37

2.5.6 Immunofluorescence 37

2.5.7 Di-I labelling 38

2.5.8 Imaging analysis and cell quantification 38

2.5.9 Behavioural assays 38

2.5.10 Statistical analyses 39

2.6 Results 40

2.6.1 Nfix+/- adult mice exhibit increased brain volume

40

2.6.2 Expansion of cortical layers within the neocortex of Nfix+/- adult mice

42

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2.6.3 Nfix+/- adult mice have aberrant microstructure of major forebrain commissure

tracts 46

2.6.4 Abnormal structural connectome of the Nfix+/- adult mouse brain

49

2.6.5 Adult Nfix+/- animals exhibit learning and memory impairments

52

2.6.6 Adult Nfix+/- animals display abnormal social behaviour

55

2.7 Discussion 58

Chapter 3. Investigating cortical features of Sotos syndrome using mice heterozygous for Nsd1. 67

3.1 Overview of Chapter 67

3.2 Abstract 69

3.3 Introduction 69

3.3.1 NSD1 mutations causes Sotos syndrome

69

3.3.2 NSD1 as a histone methyltransferase 70

3.3.3 The role of NSD1 during cortical development

72

3.4 Aims and Hypotheses 74

3.5 Method 75

3.5.1 Animals 75

3.5.2 Tissue preparation and sectioning

75

3.5.3 RNAscope in situ hybridisation and immunofluorescence

76

3.5.4 Generation of the Nsd1 mutant allele 76

3.5.5 Genotyping PCR and qPCR

77

3.5.6 Haematoxylin staining and immunofluorescence labelling

78

3.5.7 Imaging and cell counts

78

3.5.8 Behavioural assays 78

3.5.9 shRNA plasmids

79

3.5.10 Neurosphere assay

80

3.5.11 Neuroblast migration assay 81

3.5.12 Statistical analyses

81

3.6 Results 81

3.6.1 Nsd1 is highly expressed by neurons within the mouse cerebral cortex

81

3.6.2 Generation of Nsd1 mutant allele in mice 85

3.6.3 Characterisation of the Nsd1 heterozygous mouse cerebral cortex

87

3.6.4 Nsd1 heterozygous mice display subtle changes in social behaviour

91

3.6.5 Morphology of the embryonic homozygous Nsd1 cerebral cortex

96

3.6.6 Knockdown of Nsd1 in vitro reveals neuroblast migration deficits 100

3.6 Discussion 102

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Chapter 4. USP9X deletion leads to deficits in hippocampal neurogenesis and function. 113

4.1 Overview of Chapter 113

4.2 Abstract 115

4.3 Introduction 115

4.3.1 USP9X mutations causes intellectual disability in humans

115

4.3.2 Function of USP9X as a deubiquitylating enzyme

116

4.3.2 USP9X is required during hippocampal development and adult neurogenesis 117

4.4 Aims and Hypotheses 118

4.5 Method 119

4.5.1 Animals 119

4.5.2 Microarray

119

4.5.3 Quantitative polymerase chain reaction

119

4.5.4 Tissue preparation 120

4.5.5 Immunofluorescence

120

4.5.6 Imaging analysis

120

3.5.7 Active place avoidance task

121

4.6 Results 121

4.6.1 Usp9x deletion leads to overexpression of oligodendrocyte and myelin genes

121

4.6.2 Usp9x deletion leads to increased density of oligodendrocytes in the dentate hilus

124

4.6.3 Usp9x /Y; Emx1-Cre mice display impaired learning and memory function

128

4.7 Discussion 130

Chapter 5. General Discussion 134

5.1 Modelling human cortical malformations using mouse models 134

5.1.1 Patient phenotype recapitulated by mouse models 135

5.1.2 Limitations and differences between human and mouse brains 135

5.2 Alternative models of cortical development and cortical malformations 142

5.3 Interconnected network in neurodevelopmental disorders 145

5.4 Conclusions 148

References 150

Appendix 183

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