Maternal and Indirect Genetic Effects on Behavioural And

Maternal and indirect genetic effects on

behavioural and developmental traits in

mammalian disease models

Naorin Sharmin

A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Life Sciences.

2015

1 Contents

Abstract………………………………………………………………...... 10

Declaration…………………………………………………………………… 11

Acknowledgements………………………………………………………… 13

The author……………………………………………………………………. 15

1. Introduction………………………………………………………………….. 17 1.1 Williams-Beuren Syndrome……………………………………………………….. 18 1.1.1 History…………………………………………………………………………...... 18 1.1.2 Clinical Features……………………………………………………………...... 19 1.1.3 WBS and social behaviour……………………………………………………… 24 1.2 Genetic Basis of WBS……………………………………………………………... 27 1.2.1 Genes deleted in WBS………………………………………………………….. 29 1.2.2 GTF gene family and WBS…………………………………………………...... 32 1.3 Mouse models…………………………………………………………………….... 34 1.3.1 Mouse models of Gtf2ird1 and Gtf2ird2 genes……………………………….. 37 1.4 Indirect genetic effects and maternal effects…………………………………… 42 1.4.1 QTL Mapping…………………………………………………………………….. 45 1.5 Aims and objectives………………………………………………………………... 48 1.6 Thesis Chapters……………………………………………………………………. 50

2. General Methods…………………………………………………………………… 52 2.1 Establishment of Gtf2ird1 and Gtf2ird2 knockout and heterozygous models……………………………………………………………………………………. 53 2.1.1 Gtf2ird1……………………………………………………………………………. 53 2.1.2 Gtf2ird2……………………………………………………………………………. 53 2.2 Animal Maintenance……………………………………………………………….. 54 2.3 Cross-fostering protocol…………………………………………………………… 55 2.4 Phenotyping maternal behaviours of Gtf2ird1 mouse model in Chapter-1…... 55 2.5 Phenotyping protocol used in Gtf2ird1 transgenerational study in Chapter-3 and Gtf2ird2 study in Chapter-4……………………………………………………… 58 2.6 Scoring behaviours ……………………………………………………………….... 60 2.7.1 Statistical Analysis………………………………………………………………… 60 2.7.2 Behavioural and Weight Data……………………………………………………. 60 2.7.2 Nesting Experiment……………………………………………………………….. 61 2.7.3 Real-Time PCR Data……………………………………………………………... 2.7.4 Data analysis for QTL Mapping……………………………………………...... 3. Deletion of Gtf2ird1 gene results in abnormal maternal behaviours in a

2 Williams-Beuren Syndrome mouse model- A Pilot study……………………… 63 3.1 Abstract…………………………………………………………………………….... 64 3.2 Introduction………………………………………………………………………….. 65 3.3 Methods……………………………………………………………………………… 66 3.3.1 Experimental Design…………………………………………………………….. 66 3.4 Results………………………………………………………………………………. 67 3.4.1 Behavioural Data…………………………………………………………………. 67 3.4.2 Pup Retrieval Test……………………………………………………………….. 68 3.4.3 Weight data……………………………………………………………………….. 70 3.5 Discussion…………………………………………………………………………… 71 3.5.1 Maternal Behaviour………………………………………………………………. 72 3.5.2 Growth…………………………………………………………………………….. 73 3.6 Conclusion…………………………………………………………………………... 74

4. Social behaviours in a single-gene knockout mouse model of Gtf2ird1 gene………………………………………………………………………………………. 75 4.1 Abstract…………………………………………………………………………….... 76 4.2 Introduction………………………………………………………………………….. 77 4.3 Methods……………………………………………………………………………… 79 4.3.1 Experimental Design……………………………………………………………... 79 Maternal behaviours of Gtf2ird1 mice…………………………………………. 79 Maternal effects on Gtf2ird1 mice……………………………………………… 81 Effect of offspring genotype on maternal behaviour…………………………. 82 4.3.2 Phenotyping………………………………………………………………………. 83 4.4 Results………………………………………………………………………………. 85 4.4.1 Maternal behaviours of Gtf2ird1 mice reared by B6 mothers...... 85 4.4.2 Maternal effects on Gtf2ird1 mouse model……………………………………. 88 4.4.3 Growth of litters reared by Gtf2ird1 mutant mothers…………………………. 92 4.4.4 Maternal effects on behaviours of Gtf2ird1 mouse model…………………… 94 4.4.5 Effect of offspring genotype on maternal behaviours………………………… 97 4.5 Discussion…………………………………………………………………………… 99 4.5.1 Physiological deficits arising from Gtf2ird1 deletion affect crucial maternal behaviours………………………………………………………………………………... 99 4.5.2 Maternal effects are an important regulator of maternal behaviours………... 102 4.6 Conclusions………………………………………………………………………..... 104

5. Gtf2ird2 deleted in Williams-Beuren Syndrome affects social and maternal behaviours in a mouse model system by impairing oxytocin 106 expression in the brain……………………………………………………………….. 5.1 Abstract………………………………………………………………………………. 107 5.2 Introduction…………………………………………………………………………... 108 5.3 Methods……………………………………………………………………………… 110 5.3.1 Genotyping……………………………………………………………………...... 110 5.3.2 Agarose gel electrophoresis…………………………………………………….. 110 5.3.3 Testing nesting ability…………………………………………………………. 111 5.3.4 Experimental design and phenotyping…………………………………………. 113 5.3.5 Tissue collection………………………………………………………………….. 114

3 5.3.6 Sectioning brain regions…………………………………………………………. 114 5.3.7 Histology for identification of brain regions…………………………………….. 116 5.3.8 Transcription of brain regions of Gtf2ird2 mouse model……………………… 117 5.3.9 Reverse transcription…………………………………………………………….. 117 5.3.10 Polymerase chain reaction (PCR)…………………………………...... 118 5.3.11 Real-time quantitative reverse transcriptase polymerase chain reaction (qPCR)……………………………………………………………………………………. 118 5.4 Results……………………………………………………………………………….. 122 5.4.1 Nest Building Study………………………………………………………………. 122 5.4.2 Behavioural study………………………………………………………………… 123 5.4.3 Litter Growth………………………………………………………………………. 126 5.4.4 Pup Retrieval Test………………………………………………………………... 126 5.4.5 Filial cannibalistic behaviour…………………………………………………….. 127 5.4.6 Histology for cryosectioning the right brain region……………………………. 128 5.4.7 PCR………………………………………………………………………………… 129 5.4.8 Gene Expression via qPCR……………………………………………………… 130 5.5 Discussion…………………………………………………………………………… 133 5.5.1 Fine-motor skill……………………………………………………………………. 133 5.5.2 Behaviour………………………………………………………………………….. 133 5.5.3 Filial Cannibalism…………………………………………………………………. 135 5.5.4 Growth……………………………………………………………………………… 136 5.5.5 Neuroendocrinology……………………………………………………………… 137 5.6 Conclusions………………………………………………………………………….. 139

6. Indirect genetic effects contribute to maternal and pup weight traits during early development ……………………………………………………………. 141 6.1 Abstract……………………………………………………………………………..... 142 6.2 Introduction…………………………………………………………………………... 143 6.3 Methods……………………………………………………………………………… 144 6.3.1 QTL Mapping……………………………………………………………………… 146 6.3.2 Identification of Candidate Genes…………………………………………...... 147 6.4 Results………………………………………………………………………………... 149 6.5 Discussion……………………………………………………………………………. 154 6.6 Conclusion…………………………………………………………………………… 157

7. General Discussion…………………………………………………………………. 158 7.1 Gtf2ird1………………………………………………………………………………. 160 7.1.1 Are homozygous female mice more social and empathic than heterozygous and wildtype Gtf2ird1 females? ...... 160 7.1.2 Does offspring genotype affect maternal behaviours? ………………………. 161 7.1.3 Do maternal effects of wildtype and homozygous mothers play an important role in mediating maternal behaviours of mutant females in the next generation? 162 …………………………………………………………………………….... 7.1.4 Future Directions………………………………………………………………….. 163 7.2 Gtf2ird2………………………………………………………………………………. 166 7.2.1 Does Gtf2ird2 affect nest building skills? ……………………………………… 166 7.2.2 Is Gtf2ird2 involved in hyper sociability in female mice? …………………….. 167

4 7.2.3 Does Gtf2ird2 regulate expression levels of neurohormones in the hypothalamic brain regions of the Gtf2ird2 mouse model? ………………………… 168 7.2.4 Future directions………………………………………………………………….. 170 7.3 Gtf2ird1 and Gtf2ird2 as mouse models to study WBS………………………… 171 7.4 Indirect genetic effects……………………………………………………………… 173 7.4.1 Are there maternal effects of BXD maternal genotype on growth of B6 pups and IGEs of BXD litter genotype on weight changes of B6 mothers? ……… 173 7.4.2 IGE and maternal effects QTL and functions………………………………….. 174 7.4.3 Future directions………………………………………………………………….. 175

8. Final Conclusion…………………………………………………………………….. 177

References………………………………………………………………………………. 180

Appendices………………………………………………………………………………. 207 Appendix-1: Protocols ……………………………………………………………….. 208 Behavioural study………………………………………………………………… 208 Setting up breeding cages………………………………………………………. 208 Separating pregnant females……………………………...... 209 Phenotyping maternal behaviours……………………………………………… 209 Nesting experiment………………………………………………………………. 210 Quantifying neuropeptide gene expression……………………………………. 214 Cryosectioning…………………………………………………………………….. 214 RNA extraction……………………………………………………………………. 220 cDNA synthesis…………………………………………………………………… 221 qPCR………………………………………………………………………………. 222 Appendix-2: Primer Spans………………………………………………………...... 224 Appendix-3: qPCR amplification efficiency graphs………………………………... 235 Appendix-4: Graphical representation of behavioural data from Chapter-2 ...... 238 Appendix-5: Graphical representation of behavioural data from Chapter-3 ...... 241 Appendix-6: QTL maps for Chapter-4 …………………………………………...... 248

Word count: 39,109 without references and appendices.

5 List of Figures

1.1 Characteristic features seen in patients…………………………………. 21 1.2 Reduced brain volumes in WBS patients versus controls …………….. 23 1.3 The Williams-Beuren Syndrome Chromosome Region (WBSCR) on chromosome 7 ……………………………………………………………... 28 1.4 Genes deleted in WBS ……………………………………………………. 31 1.5 Mouse orthologue of WBSCR…………………………………………….. 35 1.6 Examples of abnormalities in Gtf2ird1 knockout mice………………..... 40 1.7 Contribution of Direct genetic effects and IGEs to the phenotype of individuals…………………………………………………………………… 43 2.1 Mouse appearance by age ……………………………………………….. 58

3.1 Breeding plan……………………………………………………………….. 67 3.2 Gtf2ird1 deletion affects maternal behavioural phenotypes…………… 69 3.3 Growth of Gtf2ird1 litters reared by homozygous and wildtype mothers ……………………………………………………………………... 71

4.1 Breeding plan used to study Gtf2ird1 maternal care behaviour by controlling litter genotype and maternal effects ………………………… 80 4.2 Breeding plan used to study maternal effect on Gtf2ird1 maternal care behaviour in the next generation……………………………………. 82 4.3 Breeding plan used to study the effect of litter genotype on maternal behaviours of B6 females ………………………………………………… 83 4.4 Effect of Gtf2ird1 gene deletion on maternal behaviours ……………… 87 4.5 Gtf2ird1 knockout mothers show disrupted pup retrieval behaviour …. 88 4.6 Behaviours of females reared by knockout Gtf2ird1 mothers ………… 89 4.7 Pup retrieval is affected by Gtf2ird1 gene deletion …………………….. 91 4.8 Proportional growth of pups reared by mutant mothers ……………….. 92 4.9 Maternal effects on maternal behaviours of Gtf2ird1 mouse model …. 96 4.10 Effect of offspring genotype on behaviours of B6 mothers ……………. 98

5.1 Gtf2ird2 gene PCR products of ear samples of the three genotypic mice ran on 2% agarose gel ……………………………………………… 111 5.2 Scoring the quality of nests ………………………………………………. 113 5.3 Brain regions dissected …………………………………………………… 116 5.4 Example of qPCR optimisation steps ……………………………………. 121 5.5 Gtf2ird2 gene is not a determinant of nest building ability and fine- motor skills …………………………………………………………………. 123 5.6 Gtf2ird2 deletion enhances maternal behaviours which are partly controlled by litter size …………………………………………………….. 125 5.7 Monitoring growth of Gtf2ird2 homozygous and wildtype litters ……… 126 5.8 Pup retrieval in Gtf2ird2 mouse model ………………………………….. 127 5.9 Hemizygous Gtf2ird2 deletion makes heterozygous mothers more prone to filial cannibalism …………………………………………………. 128 5.10 Brain sections of Gtf2ird2 mouse model, stained in Cresyl Violet dye.. 129 5.11 PCR products of PVN and SON samples of Gtf2ird2 gene ran with Oxt and Atp5j ………………………………………………………………. 130 5.12 Expression of social neuropeptides in Gtf2ird2 mouse brain regions... 132

6.1 Experimental design ………………………………………………………. 145 6.2 Example of an interval mapping output …………………………………. 147 6.3 Interval mapping output of weekly growth (P1-6) of B6 pups reared by BXD mother ………………………………………………………………… 150 6.4 Interval mapping of short-term weight changes in B6 pups on day 14.. 150 6.5 Interval mapping of B6 maternal weekly weight change (P1-6) ……… 151 6.6 Interval mapping of B6 maternal short-term weight change on day 6... 152

Figure-39 Ethogram used for Chapter-1 ……………………………………………. 212 Figure-40 Ethogram used for Chapter-2 ……………………………………………. 213 Figure-41 Flow-diagram of cryosectioning step ……………………………………. 218 Figure-42 Flow-diagram of histology step …………………………………………... 219

Appendix-3 Figure-43 Calculating amplification efficiency of primers…………………………... 235

Appendix-6 Figure-44 QTL maps for change in weight in B6 pups …………………………….. 244

List of Tables

Table-1.1 List of phenotypes seen in WBS…………………………………………… 22 Table-1.2 Role of genes in WBS phenotypes………………………………………... 31 Table-1.3 Mouse models of WBS……………………………………………………… 37

Table-2.1 List and description of the maternal behaviours studied………………... 56 Table-2.2 Traits studied during behavioural observations………………………….. 57

Table-4.1 A brief comparison of behavioural phenotypes of Gtf2ird1 knockout mice …………………………………………………………………………... 93

Table-5.1 PCR reaction cycles ………………………………………………………... 118 Table-5.2 List of primer pairs used for the quantitative PCR of PVN and SON samples from Gtf2ird2 mice ……………………………………………….. 119 Table-5.4 Quantitative-PCR reaction cycles …………………………………………. 122

Table-6.1 List of suggestive and significant QTL ……………………………………. 153

7 Abbreviations

Avp Arginine Vasopressin gene

B6 C57Bl/6J

CA Central amygdala

CBA CBA/CaJ

Crh Corticotrophin releasing hormone

CT Cycle threshold

ELN/Eln Elastin gene

FISH Fluorescence in situ hybridization

GLM General Linear Model

Gtf2i General Transcription Factor II-i

Gtf2ird1 General Transcription Factor II-I Repeat Domain-Containing Protein 1

Gtf2ird2 General Transcription Factor II-I Repeat Domain-Containing Protein 2

Het Heterozygous

Homo Homozygous

IGE Indirect genetic effects

IIH Idiopathic infantile hypercalcaemia

KO Knockout

LRS Likelihood ratio statistic

MA Medial amygdala mbp Mega base pairs

MGI Mouse genome informatics mRNA Messenger ribonucleic acid

Oxt Oxytocin gene

Oxtr Oxytocin receptor gene

P/D Postnatal day

8 PCR Polymerase chain reaction

PVN Paraventricular nucleus qPCR Quantitative polymerase chain reaction

QTL Quantitative trait loci

SON Supraoptic nucleus

SPSS Statistical Package for the Social Sciences

SVAS Supravalvular aortic stenosis

WBS Williams-Beuren Syndrome

WBSCR Williams-Beuren Syndrome Chromosome Region

Wt Wildtype

9 Abstract

Maternal and indirect genetic effects on behavioural and developmental traits in mammalian disease models Naorin Sharmin The University of Manchester Doctor of Philosophy Date submitted: 05/08/2015

In the study of social behaviour, a fundamental question is to what degree specific genes and environmental influences predispose individuals to develop certain behaviours. The discovery of maternal effects on phenotypic traits has added a significant factor that needs to be accounted for, along with genetic and other environmental factors, when trying to understand the causes of variation in normal and disease phenotypes. This has been addressed in my PhD by studying the human genetic disorder Williams-Beuren syndrome (WBS) that affects both physiological and behavioural traits. WBS results from a chromosomal micro-deletion, on chromosome seven, and is characterised by an interesting cognitive profile; although compromised in visuospatial abilities and fine-motor coordination, WBS individuals have significantly elevated social behaviours. The aim of my PhD was to assess the functions of two genes, Gtf2ird1 and Gtf2ird2, deleted in WBS. Using single-gene knockout mouse models, I explored the unique hyper-social demeanour in WBS along with fine-motor skill efficiency and underlying neurohormone levels. In addition, I analysed indirect genetic effects (IGEs) where genes expressed in a focal individual have phenotypic effects in an interacting individual. In Chapters One and Two of this thesis, a detailed behavioural protocol with a cross-fostered, transgenerational experimental design was implemented to record any variation in maternal behaviours in Gtf2ird1 knockout mice compared to heterozygous and wildtypes. The study found a number of physiological impairments in Gtf2ird1 knockout mice that are analogous of WBS such as metabolic or anxiety-related traits with previously found phenotypes such as severely misaligned jaws and growth retardation. This study also found significant maternal effects on disease phenotypes thus highlighting the importance of studying maternal effects. Chapter Three of the thesis introduces the very first knockout model of Gtf2ird2. This study found upregulated social behaviour in the knockout mice compared to wildtypes. Further analysis of brain tissues of this model found elevated oxytocin mRNA levels in the knockouts and a similarly low levels of oxytocin receptor mRNA. This finding not only provides an initial link between genes deleted in WBS and social neuropeptides, but the lower level of oxytocin receptor mRNA is also indicative of an oxytocin induced desensitisation; a mechanism which has previously been reported in human myometrium cells. Although further studies are required to confirm these findings, this study certainly provides an insight in the role played by Gtf2ird2 in WBS. Finally, Chapter Four, concentrates on taking genotype-phenotype correlation studies further by mapping quantitative trait loci (QTL) using the largest recombinant inbred panel, BXD mice, to find indirect genetic effects on weight traits in inbred, C57BI/6J (B6) mice. The study found three significant QTL and one suggestive QTL affecting weight changes of B6 pups and mothers.

10 Declaration

The University of Manchester

PhD Candidate Declaration

Candidate Name: Naorin Sharmin

Faculty: Life Sciences

Thesis Title: Maternal and indirect genetic effects on behavioural and developmental traits in mammalian disease models

Declaration to be completed by the candidate:

“I declare that no portion of this work referred to in this thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.”

Signed:

Date:

11 Copyright

I. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the ’Copyright’) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes.

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12 Acknowledgments

The three years of my PhD certainly flew by fast but the breadth of experiences and the amount of fun I have had cannot be enumerated. There are so many people who have assisted in this process with their constant support, guidance and motivation. I would like to take this opportunity to thank all of you from the bottom of my heart for helping me complete this wonderful journey.

My utmost gratitude goes to my parents for encouraging me to pursue this PhD and financially and mentally supporting me throughout with everything you have had. Thank you for loving me selflessly! This thesis is dedicated to you.

Thank you very, very much to my main supervisor, Reinmar Hager, for believing in me, for your constant support, countless meetings, advice about my future ventures and for helping me get a scholarship from the univeristy. It has been a pleasure to know a wonderful person like you.

Thank you so much May Tassabehji, my co-supervisor, for all the long hours of meetings and your constant guidance all throughout the project. I have learnt so much from you; being methodical, thorough and pre-planning before starting any new project. Thank you for letting me in to the world of rare-genetic disorders. An aspect of research I certainly wish to pursue in the future.

Thanks a lot Chris Murgatroyd, from Manchester Metropolitan University (MMU), for mentoring the gene expression experiments (Chapter-3) of my PhD and teaching me how to cryosection mouse brains. Thank you Glenn Ferris for arranging my visits to MMU and making me feel welcomed.

Special thanks to the friends I have made in the Hager lab: Christina Stanley, thank you for being my friend, a sister and a proofreader. I am so lucky to have met you! David Ashbrook thanks for listening to my constant queries and supporting me whenever I needed any advice. Beatrice Gini, thank you for advising me about my project and career choices.

Thank you to my other friends from the Faculty of Life Sciences: Emma Randle thanks for your constant smile and friendship; one of the best times I have had during my PhD was with you. Thank you Marco Smolla, for making me laugh and motivating me to work hard. Xaali O’Reilly thanks for the constant smiles and affection. David Pettifer, Veronica Cowl, Roobini Ragu and Heather Robinson thank you for spending time with me, cheering me up and commending my work when I needed that extra push.

A massive thank you to the Tassabehji lab: Glenda Beaman thanks a lot for being the perfect mentor and friend. Usman Khalid thank you for helping me out with the qPCR optimisations and supporting me throughout. Daniel White thank you for all your suggestions about my data analysis.

My heartfelt thanks to my friends Eleni Spyropoulou, Intahab Hossain, Merve Engin and Mirza Nathoo for being my emotional support and strength and never letting me feel lonely

13 in Manchester. Special thanks to Vedia Shahin for constantly checking up on me and always being there to hear about my PhD experiences.

Thank you to my sister Nahid, brother-in-law Romel and my brother Fahmid for their endless support, love and inspiration. Without your support I would not be able to complete my PhD and I am truly grateful for that.

And finally, thank you to my partner Zeeshan Saeed for living every moment of my PhD with me and for being extremely supportive these three years.

The author

My interest in Genetics was first sparked in school when I learnt how something so small, the human genome can control one’s entire bodily functions! To expand my knowledge and to be at the forefront of Life Sciences, in 2009, I moved to the U.K. and commenced my undergraduate degree, in Biomedical Sciences, in King’s College London (KCL).

My time in KCL was invaluable; I experienced very high standards of education, science and research which not only affirmed my aspiration to become a researcher, but also promoted me to think scientifically and vastly improved my scientific writing skills. I volunteered as a lab assistant at KCL during my second year which helped me gain lab and teaching experiences and also drove me to apply and secure a six-month long research project, to be held in my final year. My final year research project was in an Endocrinology lab, where my project focussed on quantifying levels of transforming growth factor (TGF) in diabetic patient tissues. During this project is when I initially learned to conduct PCRs and qPCRs along with cell culture, RNA and cDNA extraction procedures which were later applicable in my PhD.

My PhD project is the perfect combination of Genetics and Human Disorder; two aspects of Life Sciences that intrigues me the most. During my PhD I have developed a wider understanding of the field of genetics, alongside the practical and analytical skills necessary to investigate genetic disorders by using mouse models. Alongside my research, I had the opportunity to get involved with teaching, outreach programmes and widening participation events at The University of Manchester. I really enjoyed these experiences thus upon submission of my PhD thesis, I am commencing a salaried training programme to become a researcher in school.

1. Introduction

17 Studies on genotype-phenotype correlations in disease models provide a good platform to investigate genes and gene functions (Tassabehji 2003). The focus of my research was to understand the functions of Gtf2ird1 and Gtf2ird2 genes in social phenotypes of the genetic disorder Williams-Beuren Syndrome (WBS). I also aimed to take the understanding on genotype-phenotype correlations further by investigating indirect genetic effects of interacting individual’s genotype on traits of a focal individual.

1.1 Williams-Beuren Syndrome

1.1.1 History

WBS is a rare neurodevelopmental disorder, affecting 1 in 20,000 live births (Domenico et al. 2013). The disorder is caused by a chromosomal microdeletion on chromosome seven

(7q11.23). Typical symptoms of WBS include dysmorphic facial features, short stature, hypercalcaemia and supravalvular aortic stenosis (SVAS) (Tassabehji 2003; Pober 2010).

Initially vitamin D teratogenicity was considered to be the cause of WBS (Friedman and

Roberts 1966). This idea stemmed from empirical work on rabbits exposed to high doses of vitamin D in fetal stage which later developed to conform SVAS and craniofacial abnormalities (Friedman and Roberts 1966; Friedman and Mills 1969). Prior to this, people with WBS used to be diagnosed with idiopathic infantile hypercalcaemia (IIH)

(Lightwood and Stapleton 1953). When dietary change in vitamin levels had no effect on the symptoms observed the differentiation of WBS from IIH was recognised (Stapleton et al. 1957). A few years later, in the early 1960s, cardiologist J. C. P. Williams and his team reported four individuals with SVAS out of which three of them had the characteristic facial features and mental retardation (Williams et al. 1961). This striking correlation of craniofacial features to mental disabilities led the authors to suggest a formerly unrecognized syndrome (Williams et al. 1961; Beuren et al. 1962). Soon after, German physician A. J. Beuren reported three more individuals with the characteristic features as

18 described by Williams et al. (1961) (Beuren et al. 1962). These reports are what led to the nomenclature of the disorder Williams-Beuren Syndrome.

Later, WBS was established to be a genetic disorder through two compelling studies

(Morris et al. 1993; Sadler et al. 1993) which recognised the transmission pattern to be from parents to children in an autosomal dominant manner (Morris et al. 1993).

Cardiovascular abnormalities are common symptoms of WBS (Pober 2010) and familial

SVAS results from mutation in the elastin gene, ELN (Curran et al. 1993). Thus, WBS individuals were screened for mutations in the ELN gene and surprisingly a total loss of one of the ELN alleles was found instead of mutations within the gene (Ewart et al. 1993).

This is what led to the understanding that WBS is in fact a microdeletion disorder as opposed to a point-mutation disorder (Ewart et al. 1993). The loss of ELN and associated symptoms suggested that symptoms of WBS should correlate to genes in the microdeletion. In the next decade studies identified 26 to 28 deleted genes neighbouring the ELN gene in WBS individuals (Tassabehji et al. 1996; Osborne et al. 1999; Tassabehji et al. 1999; Merla et al. 2002; Tipney et al. 2004) and started to identify the roles played by some of these genes in this multifaceted disorder (Hoogenraad et al. 1998; Nakayama et al.1998; Tassabehji et al. 2005; Dai et al. 2009). Similarly, the focus of my research is to investigate two of the genes (Gtf2ird1 and Gtf2ird2) deleted in WBS and their possible roles in the distinct behavioural symptoms of the disorder.

1.1.2 Clinical Features

The genetic disorder WBS is present at birth occurring in all ethnic groups (Domenico et al. 2013). It affects both males and females and is usually diagnosed by preliminary clinical criteria (Committee on Genetics 2001), listed in Table-1.1, followed by confirmation from laboratory tests (Domenico et al. 2013; Morris 2013). Fluorescence in situ hybridization (FISH) is used to diagnose WBS; ELN-specific probes show the presence of a single allele instead of double thus confirming the microdeletion (Pober 2010; Morris

19 2013). Although FISH is the most popular diagnostic test, quantitative polymerase chain reaction assays are frequently used too (Pober 2010).

The phenotypes observed in individuals with WBS are variable. The common features affecting different systems in the body are listed in Table-1.1. The earliest and most critical clinical feature of WBS is the onset of congenital heart disorder from infancy

(Committee on Genetic 2001). SVAS occurs in 75% of affected individuals (Committee on

Genetic 2002) (Figure-1.1). Some individuals also have peripheral pulmonic stenosis which may require heart surgery in infancy (Martin et al. 1984). Another phenotype that is observed in infancy but resolves with age is IIH (Committee on Genetics 2001). Children with WBS suffer from frequent vomiting, constipation and muscle cramps linked with IIH

(Martin et al. 1984; Morris et al. 1988). Although IIH resolves in childhood, individuals may endure vitamin D and calcium metabolism related problems throughout life (Committee on

Genetics 2001).

Dysmorphic facial features are considered to be a major diagnostic phenotype in individuals with WBS (Mass and Belostoky 1993). They have a wide mouth and forehead, spread out dental morphology, puffiness around the eyes, short but broad nose, and a delicate chin (Figure-1.1) (Preus 1984; Morris 2013). With age some of the features become more prominent. For example, round full face in childhood develop to a long face and neck with slanting shoulders in adulthood (Figure-1.1) (Morris 2013).

Growth pattern in people with WBS is another significant noticeable phenotype of the disorder (Figure-1.1). Deficiency in growth starts to appear before birth and the individuals continue to show reduced weight gain and growth during the first four years of life (Preus 1984). Children with WBS tend to be petite and slim and show 75% growth to that of normal in childhood (Morris 2013). There is a brief growth spurt in puberty but the mean adult height is lower than the third percentile (Morris et al. 1988; Morris 2013).

A B C

Figure-1.1: Characteristic features seen in WBS individuals. The Figure shows examples of a few characteristic features found in WBS individuals. A. shows the three dimensional image of an angiography showing prevalence of SVAS (white arrow) in a 44 year old WBS patient (image has been adapted from Pober 2010). B. and C. show examples of the typical WBS face: broad forehead, periorbital fullness, spaced teeth and prominent nose (images from www.williams- syndrome.org). D. shows an example of a WBS adult with typical long face and neck and slanting shoulders (image has been adapted from Morris 2013).

21 Table-1.1: List of phenotypes seen in WBS. The Table lists the symptoms seen in WBS individuals. Morris et al. 19881, Mervis and Klein-Tasman 20002, Committee on Genetics 20013, Pober 20104 and OMIM number 1940505.

Feature Affect Vascular stenosis mainly SVAS (in 75% Cardiovascular WBS individuals) and pulmonic1

Hypertension3

Cognitive Low IQ (average 55)5

Poor visuospatial functioning1,2,5

Gastrointestinal Difficulty feeding in childhood3

Constipation3

Vomiting3

Celiac disease1

Rectal prolapse1

Endocrine Diabetes Mellitus (in 75% WBS individuals)2

Hypercalcaemia1 Osteoporosis from poor vitamin D and calcium metabolism4

Early onset of puberty1

Growth Prenatal growth deficiency 1 Linear growth is 25% lower than normal in childhood3

Mean adult height is below third percentile3

Facial Periorbital fullness4

Wide mouth, full lips and cheeks4

Flat nasal bridge4

Pointy chin4

Dental Small, unusually shaped primary teeth5

Malocclusions5

Enamel hypoplasia5 Musculoskeletal Lax joints4

Scoliosis4

Curved spine4 Motor Poor balance and coordination2 Poor fine motor skills2 Problems with gait and posture4

Behavioural Over friendly3

Attention deficit disorders1

Phobias1

Enhanced empathetic nature3

Generalised anxiety1,3

Ocular Esotropia1 Auditory Oversensitivity to certain frequencies and volumes of sound3 Low to medium loss of sensorineural hearing3,4

Middle ear infection3

22 WBS individuals have a unique social behavioural profile despite of an impaired cognitive and motor profile. Brain imaging studies have found disproportionate distribution of sizes and gray matter volumes in the brains of these people affected by WBS compared to controls (Chiang et al. 2007). From these studies, features observed in WBS individuals such as visuospatial dysfunctioning and lower IQ have been linked to impairments in the parieto-occipital regions (Bellugi et al. 1999; Bellugi et al. 2000; Brown et al. 2003) and certain limbic structures such as the hippocampus (Bush et al. 2000; Wilke et al. 2003).

The total brain volume is reduced in WBS individuals, than in controls, yet the volumes of the frontal lobes and cerebellum are relatively preserved (Chiang et al. 2007) (Figure-1.2).

This explains the enhanced face perception (Mobbs et al. 2004; Meyer-Lindenberg et al.

2005) and verbal abilities (Jarrold et al. 1998) in WBS individuals despite having an uneven cognitive profile.

Parieto-occipital lobes

Cerebellum Frontal lobe

Figure-1.2: Reduced brain volumes in WBS individuals versus controls. The Figure shows a three dimensional image of WBS patient visualized using tensor-based morphometry. The significance of unevenly distributed volumes of brain regions in WBS individuals compared to control subjects have been represented via a heat map (red=smaller; blue=larger than normal). The parieto-occipital lobes have significantly reduced volume whereas frontal lobes and cerebellum are relatively preserved. Image has been adapted from Chiang et al. 2007.

Strengths in verbal and non-verbal reasoning have been highlighted through a series of tests which calculated the IQ in accordance with a differential abilities scale (Elliot 1990;

23 Mervis and Klein-Tasman 2000) and the Kaufman brief intelligence test (Kaufman and

Kaufman 1990; Mervis and Klein-Tasman 2000). These tests assessed the vocabulary, memory span and construction of patterns and drawings thus targeted a number of cognitive abilities (Elliot 1990; Kaufman and Kaufman 1990). It was concluded from the results of these studies that WBS individuals show relatively good specificity to certain cognitive abilities only, such as language and auditory rote memory (Mervis and Klein-

Tasman 2000). People with WBS are described as over friendly (Gosch and Pankau

1997), highly sociable (Dilts et al. 1990) and approachable (Tomc et al. 1990). This is the phenotype that I am interested in studying and before moving to more details about my study I will discuss the existing literature on this phenotype.

1.1.3 WBS and social behaviour

WBS individuals have relatively good language and face processing abilities in comparison to their visuo-spatial domain. Individuals affected with other genetic disorders such a Down syndrome and Prader-Willi syndrome are reported to have similar personality traits too, hence, some studies have tested personality profiles of these individuals against people with WBS (Gosch et al. 1994; Dykens and Rosner 1999; Mervis et al. 2000; Dimitriou et al. 2015). Children with WBS were far less reserved with strangers than children with Down syndrome (Gosch et al. 1994) moreover, adolescents and adults with WBS were highly approachable and empathic compared to individuals with Prader-Willi syndrome (Dykens and Rosner 1999). These characteristics commence early in life when toddlers with WBS interact with their mothers (Mervis et al. 2000;

Weisman et al. 2015). Compared to children with normal development, WBS toddlers spent substantially more time looking and interacting with their mothers during play sessions (Mervis and Klein-Tasman 2000; Mervis et al. 2000). In the same scenario when a stranger was asked to enter the room, toddlers with WBS were found to look intently at the face of the stranger throughout most of the duration of the play session and the

24 toddler did not even notice when their parent left the room (Mervis et al. 2000). Prolonged attention to faces is a means of processing facial information (Riby et al. 2012; Dimitriou et al. 2015), which has been found to correlate with anomalous brain activation in WBS individuals (Mills et al. 2000; Grice et al. 2001). Typically from early infancy, differences in attention levels have an impact on visual cognition (Brown et al. 2003). Cross-syndrome comparison studies on WBS and Down syndrome found attention deficits in both disorders (Karmiloff-Smith et al. 2012). WBS individuals are unable to shift their attention from a certain stimuli (Lincoln et al. 2002; Brown et al. 2003); this is due to deficits in saccade planning resulting in difficulty to visually disengage from individual objects in display (Karmiloff-Smith et al. 2012). Compared to invidividuals with Down Syndrome and controls, WBS individuals also showed significantly longer attention on a certain stimulus

(Brown et al. 2003). Therefore in a social scenario, attention towards strangers can prove to be dangerous as WBS individuals lack the ability to sense social threat (Plesa et al.

2009). Despite their caring and sociable nature, this reduced ability to understand other’s mental states is a major reason why people with WBS find it difficult to make friends and end up isolated in social scenarios (Mervis et al. 2003).

Although WBS indviduals appear to be social, non-social anxiety is another important aspect of the disorder, which has been reported in children, adolescents and adults with

WBS (Dykens 2003; Meyer-Lindenberg et al. 2005; Leyfer et al. 2006; Leyfer et al. 2009;

Porter et al. 2009). The anxiety-related traits mainly observed are phobias or fear of stimuli ranging from injections to alarm clocks and coughing (Dykens and Rosner 1999;

Dykens 2003; Leyfer et al. 2009) and extreme worrying about one’s health and chances of survival (Udwin and Yule 1991; Davies et al. 1998). These studies used a number of different approaches to measure anxiety traits in WBS individuals: reports from parents; scores on Fear Survey Schedule for Children-Revised; Diagnostic and Statistical Manual based interviews (Dykens 2003; Cherniske et al. 2004; Stinton et al. 2010; Woodruff-

25 Borden 2010) and found that approximately 3% of WBS individuals are clinically diagnosed with generalised anxiety disorder (Dykens 2003).

The amygdala has been the key brain region under focus when neural correlates underlying social phenotypes of WBS are studied (Levitin et al. 2003; Meyer-Lindenberg et al. 2005; Meyer-Lindenberg et al. 2006). This is because social functioning can be disinhibited due to lesions in the amygdala and linked cortical regions (Amaral 2002). In tasks requiring perceptual processing of threatening visual stimuli, activity of amygdala in subjects with WBS was significantly low in socially relevant cues such as angry or scared faces but significantly high in socially irrelevant cues such as frightful or menacing scenes

(Meyer-Lindenberg et al. 2005; Meyer-Lindenberg et al. 2006). Hormonal imbalances in

WBS causing diabetes, hypercalcaemia and thyroid problems have been linked to hypothalamic abnormalities (Cammareri et al. 1999; Meyer-Lindenberg et al. 2006) however, clear understanding of the mechanisms is still lacking. Hypothalamic oxytocin is a key regulator of social and emotional phenotypes in mammals (Carter et al. 2008;

Heinrichs et al. 2009) and has been found to be in high levels in blood samples of WBS individuals compared to nonaffected invidiuals (Dai et al. 2012). Oxytocin is a neurohypophysial hormone primarily synthesised within the hypolothalamus of the brain. It has been found to be involved in phenotypes observed in psychiatric disorders such as autism (Guastella et al. 2010; Meyer-Lindenberg et al. 2011), depression (Ebstein et al.

2012) and anxiety disorder (Neumann and Slattery 2015).

My research focussed on the social phenotypes of WBS. I aimed to study the contribution of genes, which may be influencing this interesting phenotype. In the last ten years, genotype-phenotype correlations have provided a good insight in to the microdeletion in

WBS and have identified 26 to 28 genes within the deleted region (Tassabehji 2003; Dai et al. 2009).

26 1.2 Genetic Basis of WBS

The microdeletion on chromosome seven occurs because of the unique genetic architecture in this region (Tassabehji 2003). The deleted region, also known as the

Williams-Beuren syndrome chromosome region (WBSCR) is flanked by highly homologous genes and pseudogene clusters which are organised in to low-copy-repeats called duplicons (Figure-1.3). These flanking duplicons are located very close to each other and have a high degree of sequence homology which promotes the WBSRC to misalign during meiosis due to successive unequal crossing overs of the region (Figure-

1.3) (Francke 1999; Tassabehji 2003; Pober 2010).

27 A

Figure-1.3: The Williams–Beuren Syndrome Chromosome Region (WBSCR) on Chromosome 7. Panel A shows the WBSCR positioned between the flanking duplicons with the most common deletions in the WBSCR, approximately 1.5 mbp in size. The breakpoint regions responsible for the deletions occur in the centromeric B block duplicons and medial A block duplicons. The bottom of Panel A is an enlargement of the WBSCR showing the genes unique in this region with the width of the rectangle roughly corresponding to the size of the gene. Panel B shows the mechanism of WBSRC deletion during meiosis. Adapted from Pober 2010.

The unequal crossing over of WBSCR can also result in duplication (Somerville et al.

2005) or inversion (Tam et al. 2008) of the region. Individuals with the duplication of

WBSCR show different physiological and cognitive symptoms to that of WBS individuals,

28 for example, unlike the increased verbal skills in WBS, individuals with an additional copy of the genes demonstrate phonological disorder (Somerville et al. 2005; Van der Aa et al.

2009). By contrast, individuals with the inversion of the region show no phenotypic abnormality (Tam et al. 2008) but are more likely to have children with WBS due to the higher risk of producing gametes with the deleted WBSCR (Scherer et al. 2005).

Normally, WBS is generated from healthy parents who do not carry the deletion. The deletion ensues sporadically during meiosis and can thus arise in either the inherited maternal or paternal chromosome seven (Pober 2010). Thus, the probability of the second child of healthy parents to have WBS is much less than 1% (Pober 2010). The majority of people with WBS choose not to reproduce; however, if they do there is a 50% chance of the offspring to inherit WBS (Morris et al. 1993; Sadler et al. 1993).

In more than 98% of WBS cases, the deletion is between the medial and centromeric duplicons, mainly the B blocks (Figure-1.3), and spans 1.5 mega base pairs (mbp) in size encoding 26 to 28 genes. In less than 10% cases, there is a slightly larger deletion of 1.8 mbp encoding all 28 genes (Morris 2006; Porter et al. 2012). Although initially no phenotypic differences were found between individuals with the 1.5 mbp and 1.8 mbp deletions (Pober 2010), studies with detailed neuropsychological and genomic array screenings have found significant differences in social and cognitive functioning in individuals with the bigger deletion (Marshall et al. 2008; Porter et al. 2012).

1.2.1 Genes deleted in WBS

The understanding of the loss of an ELN allele on the pathology of cardiovascular symptoms of WBS has inspired investigations on the clinical consequences of loss of other genes in the WBSRC (Frangiskakis et al. 1996; Hoogenraad et al. 1998; Morris et al. 2003; Tassabehji et al. 2005). Table-1.2 lists the genes currently implicated in the phenotypes of WBS and Figure-1.4 shows all the genes deleted. LIMK1 is one of the first genes which was found to be deleted in WBS along with ELN (Tassabehji et al. 1996).

29 LIMK1 encodes LIM domain kinase 1, a cytoplasmic protein, involved in actin regulation in neurones. LIMK1 gene contributes to abnormal visuospatial cognition in WBS

(Frangiskakis et al. 1996) and this has been confirmed through studies on individuals with an atypical deletion of ELN and LIMK1 genes (Frangiskakis et al. 1996; Tassabehji et al.

1999). Similarly, the RFC2 gene within the WBSCR has been linked to growth and developmental deficits in WBS (Francke 1999) because of its involvement in the elongation step of DNA replication (Okumura et al. 1995; Osborne et al. 1996). CLIP2 gene has been highlighted as one of the genes involved in the cognitive and behavioural deficits of WBS (De Zeeuw et al. 1995). It encodes a cytoplasmic linker protein CLIP115, which is responsible for the regulation of microtubule dynamics. CLIP2 is highly expressed in the brain and hemizygous deletion of the gene in WBS has been suggested to lead to an abnormal hippocampal and cerebellar hemispheres functioning (Hoogenraad et al.

2002).

Table-1.2: Role of genes in WBS phenotypes. The Table lists some of the genes deleted in WBS and symptoms related to them.

Gene Proposed role in WBS phenotype Reference ELN (Elastin) Cardiovascular disease and Ewart et al. 1993 connective tissue abnormalities LIMK1 (lim kinase 1) Impairment in visuospatial cognition Frangiskakis et al. 1996 Morris et al. 2003 STX1A (syntaxin 1A) Diabetes mellitus Osborne et al. 1997 Lam et al. 2005 BAZ1B (bromodomain Idiopathic infantile hypercalcaemia Meng et al. 1998 adjacent to a leucine zipper 1B) CLIP2 (cytoplasmic linker 2) Cerebellar abnormalities Hoogenraad et al. 2002 NCF1 (neutrophil cytosolic Hypertension Del Campo et al. 2006 factor 1) GTF2I (general transcription Negative effect on IQ Morris et al. 2003 factor II, I) Separation anxiety Mervis et al. 2012 GTF2IRD1 (General Craniofacial features Osborne et al. 1999, transcription factor II-I Tassabehji et al. 2005 repeat domain-containing Social drive Young et al. 2008 protein 1) GTF2IRD2 (General Executive function Porter et al. 2012 transcription factor II-I repeat domain-containing protein 2) RFC2 (replication factor C, Developmental deficits Francke 1999 subunit 2)

cen tel

Figure-1.4: Genes deleted in WBS. Image has been adapted from Delgado et al. 2013. The image shows all the genes deleted in WBS and gives a representation of the classical deletion (1.55 mbp) and atypical (1.84 mbp). The genes of interest in my study have been circled. Cen and tel represents the centromeric and telomeric ends of the region.

31 1.2.2 GTF gene family and WBS

In recent years, studies have been focussing on genes located near the telomeric end of the deletion breakpoints (Figure-1.4) which are constantly found to be deleted in individuals with the ‘classical’ WBS (Roy 2001; Vullhorst and Buonanno 2003). The GTF family of genes (GTF2I, GTF2IRD1 and GTF2IRD2), also known as the TFII-I gene family, have been found to encode ubiquitously expressed transcription factors with roles in many developmental pathways (Tassabehji et al. 2005; Roy 2006). Because they can bind to both upstream and basal regulatory sites of a variety of promoters, this gene family is a strong candidate for the main neurological phenotypes in WBS (Morris 2013). GTF family members are characterised structurally by the presence of multiple I-repeats, each containing a helix-loop-helix-like domain (Tipney et al. 2004). The I-repeat is thought to be involved in DNA and/or protein binding and signal transduction (Vullhorst and Buonanno

2003). Each of the GTF proteins are expressed in different tissues and thus raise the possibility that hemizygous deletion of these genes might be contributing to a number of different aspects of WBS (Hinsley et al. 2004; Jackson et al. 2005).

Genotype-phenotype correlations on individuals with different types of atypical deletions have found some preliminary understanding of the roles of the GTF genes. Individuals with atypical deletions starting from STX1A gene and finishing at GTF2IRD1 (Tassabehji et al. 2005) or starting upstream from NSUN5 and finishing at GTF2IRD1 gene (Dai et al.

2009) showed the low IQ, facial and visuospatial features of WBS but no apparent behavioural phenotypes (Tassabehji et al. 2005; Dai et al. 2009). Comparatively, individuals with atypical deletions spanning from the ELN gene to the GTF2I gene showed all the behavioural phenotypes of WBS including over friendliness as well as visuospatial and motor deficits (Botta et al. 1999; Heller et al. 2003). From these studies it can be predicted that deletion of both GTF2IRD1 and GTF2I genes is required to develop the characteristic motor and behavioural features of WBS. However, detail on individuals with deletion spanning up to GTF2IRD1 gene lacked in-depth studies on social behaviours

32 (Tassabehji et al. 2005; Dai et al. 2009) thus the role of the gene in regulating this phenotype cannot be completely ruled out without further studies.

GTF2IRD1 and GTF2I show a dynamic pattern of expression in the body. Both genes are expressed in embryonic states and the level of expression changes with different phases of development (Danoff et al. 2004; Palmer et al. 2007). Gtf2ird1 is highly expressed in the smooth muscle tissues (Tassabehji et al. 1999), pituitary regions in the brain and craniofacial tissues (Schneider et al. 2012) thus haploinsufficiency of the gene is compatible with muscle fatigue, facial deformation and neurocognitive features of WBS.

The third member of the GTF gene family, GTF2IRD2, is deleted in only 10% of the reported WBS population (Bayes et al. 2003; Porter et al. 2012) thus the implication of this gene in the disease phenotypes has been ignored partly due to the lack of prevalence of individuals with the bigger deletion (1.8 mbp). However, considering the overall biological role of GTF2IRD2, its shared homology to the GTF family suggests that it may also be involved in contributing to the neurological and social symptoms in individuals (Tipney et al. 2004). Comparisons of sequence homology and exon-intron structures have found striking similarities between GTF2IRD2 and GTF2I genes (Makeyev et al. 2004). The

GTF2I gene plays a significant role in elevating social drive in WBS indivduals (Dai et al.

2009). A multi-level analysis of cognitive, behavioural and psychological functioning in 55

Australian WBS indivduals has provided evidence of a potential role for GTF2IRD2 in cognitive and psychological development (Porter et al. 2012). Thus, it is important to study this gene and understand its role in WBS phenotypes.

The main interest of my research lies in investigating the function of Gtf2ird1 and Gtf2ird2 genes in social phenotypes of the disorder. We created single-gene knockout mouse models of Gtf2ird1 and Gtf2ird2 to study the gene functions in isolation.

33 1.3 Mouse models

To date, the main caveat of studying the contribution of genes deleted in WBS pathogenesis has been the lack of individuals with an atypical deletion. Genotype- phenotype correlation relies on the identification of these individuals and thus the attempt has been moderately successful (Morris 2006). Even though atypical deletion of the

WBSCR provides some understanding of the potential roles of specific genes in the multifaceted phenotype, clear correlations between specific genes and phenotypes are still lacking. This is because there are only a few individuals, each with different small deletions, and in many cases the exact breakpoints of the deletions are not known

(Osborne 2010). In cases where the breakpoints are defined, the effects of deletion of regulatory elements on the expression of the neighbouring genes have not yet been examined (Osborne 2010). Also, it is sometimes difficult to pool and rely on data from atypical studies across the world, mainly due to the variation in methods, environments and parental genes that the WBS individuals are subjected to. These problems can be avoided by studying mouse models of the syndrome. Mice can be genetically modified, for example by knockout of a specific gene, to investigate the function of genes in isolation; both in heterozygous and homozygous states. The sequence homology of human and mouse genome are relatively high, thus gene products and functions are reasonably conserved amongst the two species (Rossant and McKerlie 2001). We can therefore translate the phenotypic changes from genomic alterations in mice in context to human studies and study clinical features of diseases and aspects of evolution on mouse models.

Mice can be easily manipulated genetically, physiologically and environmentally, also, their short gestation period makes them an excellent animal model for genetic studies.

Human chromosome seven, band 7q11.23, is very similar to mouse chromosome 5G1-G2

(Valero et al. 2000; Tassabehji 2003). Although the orthologous region in mouse chromosome five lack the duplicated blocks, it has the all the genes in the region which are arranged in the same order except the region is inverted with respect to the human

34 map (Figure-1.5) (Tassabehji 2003). The Eln mouse model confirmed the pathophysiological mechanism for SVAS from haploinsufficiency of the gene (Li et al.

1998a). Eln knockout mice die after birth but heterozygous mice, although did not develop

SVAS, were hypertensive, indicating a similar mechanism in hypertensive indivduals with

WBS (Li et al. 1998a).

Figure-1.5: Mouse orthologue of WBSCR. Image has been adapted from DeSilva et al. 2002. The dotted line shows syntenic WBSCR region in mouse chromosome 5. Gtf2ird2 gene is not shown in the image but is located proximal to Ncf1 gene by the centromere end.

To target the effect of genes deleted from the proximal and distal end of the WBSCR, Li et al. (2009) created two mouse models one with a deletion spanning from the Trim50 to the

Limk1 gene (distal deletion DD) (Figure-1.4) and the other spanning Limk1 to the Gtf2i gene (proximal deletion PD) (Figure-1.4). Both mutant models weighed less than wildtype litter mates, however, the DD group showed significantly impaired aortic function, reduced skull size, craniofacial abnormalities whilst the PD group showed increased social drive, reduced dominating behaviour, impaired motor coordination and compromised craniofacial features (Li et al. 2009; Osborne 2010). Thus it can be concluded from this study that, genes within the DD group including Eln, Limk1 and Stx1a are involved in regulating the cardiovascular and developmental phenotypes whereas genes within the

PD group including Clip2, Gtf2i, and Gtf2ird1 are more involved in the social phenotypes.

35 Single gene knockout studies can provide further insight in to how specific genes within the deletion can impact on genetic, biochemical and psychological functions.

Eleven single gene knockout models of WBS genes exist and Table-1.3 summarises their phenotypes. Most of the models were generated by conventional gene targeting techniques where homologous recombination in embryonic stem cells is used to replace the target gene thus producing a non-functional allele (Hardouin and Nagy 2000).

36 Table-1.3: Mouse models of WBS. The Table lists the established single gene knockout mouse models of WBS and phenotypes.

Gene Phenotype References Baz1b Heterozygotes: minor impairments in craniofacial features Ashe et al. 2008, and few cases of cardiovascular problems Yoshimura et al. 2009 Homozygotes: neonatal lethality, growth retardation, craniofacial abnormalities, cardiac abnormalities Clip2 Heterozygotes: minor deficits in growth, motor coordination Hoogenraad et al. 2002 and hippocampal functioning Homozygotes: similar to heterozygotes Eln Heterozygotes: high blood pressure, thickening of heart Li et al. 1998a,b muscles, impairments in aorta Homozygotes: prenatal lethality Fkbp6 Heterozygotes: no phenotypes Crackower et al. 2003 Homozygotes: no phenotypes in females. Males are infertile due to reduced semen production Fzd9 Heterozygotes: deformed hippocampus, increased Ranheim et al. 2005, suscepitibility to seizures Zhao et al. 2005 Homozygotes: impaired memory and spatial learning, enlarged spleen, deformed hippocampus, increased susceptibility to seizures Gtf2i Heterozygotes: growth retardation, craniofacial and skeletal Enkhmandakh et al. defects. 2009 Homozygotes: prenatal lethality Sakurai et al. 2011 Gtf2ird1 Heterozygotes: growth retardation, hyper sociability, Tassabehji et al. 2005, impaired memory and learning Young et al. 2008, Homozygotes: craniofacial abnormalities, growth Palmer et al. 2007 retardation, hyper sociability, impaired memory and learning Lat2 Heterozygotes: no reported data Volna et al. 2004, Homozygotes: autoimmune disease Zhu et al. 2004, 2006 Limk1 Heterozygotes: no reported data Meng et al. 2002 Homozygotes: deformed dendritic spinal structure, minor impairment in learning and memory, impaired hippocampus functioning. Mlxipl Heterozygotes: no reported data Iizuka et al. 2004 Homozygotes: decreased body fat formation, reduced glucose metabolism

Stx1a Heterozygotes: normal loco motor activity and anxiety Fujiwara et al. 2006 Homozygotes: prenatal lethality

1.3.1 Mouse models of Gtf2ird1 and Gtf2ird2 genes

There are four main mouse models of Gtf2ird1 gene each have been created on different genetic backgrounds and underwent different types and levels of testing (Tassabehji et al.

2005; Palmer et al. 2007; Van Hagen et al. 2007; Young et al. 2008; Enkhmandakh et al.

37 2009). Studies on the two knockout models Gtf2ird1tm1Lro and Gtf2ird1tm1Hrd have identified increased social behaviours, reduced level of aggression towards strangers, defects in amygdala dependent learning and memory and an increased serotonin metabolite levels in the frontal and parietal cortices (Young et al. 2008; Palmer et al. 2007). The model created by Enkhmandakh et al. (2009), Gtf2ird1Gt(XE465)Byg, resulted in prenatal lethality in the homozygotes and some very severe phenotypes in the heterozygotes such as impaired craniofacial features, curvature of spine, bone loss in the skull and unusual accumulation of cerebrospinal fluid in ventricles. Although some of the phenotypes are reminiscent of WBS, the other severe symptoms have been described to be due to the disruption of adjacent genes as a result of the knockout technique used (Osborne 2010;

Schneider et al. 2012) ; a LacZ gene-trap cassette was inserted in intron 22 of the gene

(Enkhmandakh et al. 2009). Studies on the insertion model used in our lab,

Gtf2ird1Tg(Alb1-Myc)166.8Sst initially found growth retardation and craniofacial abnormalities

(Figure-1.6) in homozygous mice (Tassabehji et al. 2005). All homozygous mice were significantly smaller than wildtypes and showed severely misaligned jaws and twisted snout (Figure-1.6) (Tassabehji et al. 2005). Further analysis on this mouse model has found a few more phenotypes resembling that of WBS. The Gtf2ird1 homozygous mice showed abnormal motor coordination in a number of tests including accelerating rotarod test and showed significantly impaired fine motor skills analysed from nest building efficiency (Figure-1.6) (Schneider et al. 2012; Skitt 2013). Defects in motor coordination and delay in the acquisition of motor milestones are commonly noted in WBS indivduals

(Trauner et al. 1989; Tassabehji 2003) along with abnormal gait (Chapman and Pober

1996) and anxious behaviours (Leyfer et al. 2009). Gtf2ird1 homozygous mice show abnormal gait in a range of analysis (Schneider et al. 2012) and showed increased anxiety in commonly used tests such as the elevated plus maze (Schneider et al. 2012; Skitt

2013). Social behaviours in these studies were mainly monitored using tests such as the resident intruder test and social recognition test where the aggressive behavioural profile

38 of a focal individual was noted in the presence of an intruder and the reaction and ability of the subject to identify the intruder after a brief separation was recorded (Young et al.

2008; Schneider et al. 2012). These tests are accurate in noting the immediate response of the subject animals and have found reduced aggressive behaviour in the homozygous

Gtf2ird1 mice and increased social drive and interest towards novel intruders (Young et al.

2008; Schneider et al. 2012; Skitt 2013). There is yet a lack in data for prolonged social interaction profiles of these Gtf2ird1 mutant mice which could broaden our understanding of this gene deletion on long terms effects on social behaviours.

Figure-1.6: Examples of abnormalities in Gtf2ird1 knockout mice. Image has been adapted from Tassabehji et al. 2005 (A and B) and Skitt 2013 (C). Panel A shows the severe craniofacial deformities in the homozygous mice compared to wildtypes. Panel B shows that Gtf2ird1 mice are smaller than wildtype mice. Panel C shows the severe fine motor skill deficiency in homozygous mice leading to inability to form a nest.

Recent studies on Gtf2ird1 knockouts have identified potential roles of the gene in aiding with hearing and vision (Canales et al. 2014; Masuda et al. 2014). Gtf2ird1 is highly

40 expressed in the cochlea and a knockout of the gene has found mild hearing loss in adult mice (Canales et al. 2014). The high expression of Gtf2ird1 in the spiral ganglion of the cochlea suggests important roles in mediating hearing; neurones in spiral ganglion are the first activator of an action potential which then passes on to other neurones and finally conveys the auditory stimulus to the brain (Canales et al. 2014). Similarly, GTF2IRD1 transcription factor is a key regulator of M cone cell and rod cells functioning in the eye

(Masuda et al. 2014). The GTF2IRD1 gene product interacts with the promoter and enhancer regions of the rod and cone opsin genes in humans and in mice the knockout of

Gtf2ird1 gene showed impaired M cone and rod electrophysiological responses highlighting that Gtf2ird1 plays an important role in retinal functioning (Masuda et al.

2014).

Gtf2ird1 has also been linked to enhancement of inhibitory serotonin receptor currents in the prefrontal cortex (Proulx et al. 2010) and increased serotonin metabolites in the amygdala, frontal and parietal cortices (Young et al. 2008). A study by Dai et al. (2012) has identified significantly high levels of the hormone oxytocin in blood samples of WBS indivduals compared to controls. Oxytocin is a key regulator of social behaviours including bond formation (Andari et al. 2010; Insel 2010; Marlin et al. 2015). Yet studies discussing levels of oxytocin or other social hormones and neurotransmitters in WBS cases are scarce.

Compared to Gtf2ird1, Gtf2ird2 has rarely been studied. There are no reported knockout models of Gtf2ird2 to date. A study by Tipney et al. (2004) has mapped the gene in somatic hybrid cells of WBS indivduals and found a hemizygous deletion of the gene.

Gtf2ird2 is thought to be involved in similar roles to that of Gtf2i and Gtf2ird1 genes along with an executive functioning in cognitive and psychological profiles of WBS indivduals

(Porter et al. 2012).

41 1.4 Indirect genetic effects and maternal effects

So far I have discussed at genotype-phenotype correlations where the phenotype of an individual directly mapped to its own genotype. This is termed a ‘direct genetic effect’

(Figure-1.7). However, in a social environment interactions amongst individuals can affect traits as diverse as behaviour and development (Cheverud and Moore 1994). Studies on species with parental care and/or delayed dispersal have shown that almost every life- history trait in offspring can be influenced by the environment provided by its relatives

(Mousseau and Fox 1999). Studies in both plants and animals have repeatedly demonstrated that the somatic effect of the mother can significantly affect the characteristics of developing embryos (Falconer and Mackay 1996). These effects are referred to as ‘indirect genetic effects’ (IGEs) because the genes influencing the trait act indirectly; that is, they are expressed in an interacting individual and not in the individual whose phenotype is measured (Wolf et al. 1998) (Figure-1.7). In mammals, the environment provided by the mother is the biggest part of social environment and is a significant determining factor of offspring’s traits (Hager et al. 2009; Maestripieri and

Mateo 2009). Maternal effects are the most widely studied type of IGEs where the environment provided by the mother is heritable (Falconer 1965; Mousseau and Fox

1999).

IGEs affect evolution in a non-Mendelian fashion. According to a standard quantitative genetic measure, the phenotype of an individual is affected by additive genetic and environmental effects. When IGEs exist, it acts as an additional genetic component alongside the additive genetic effect and environmental effect and results in changing the overall phenotypic value of the interacting individual (Wolf et al. 1998) (Figure-1.7). In case of maternal effects, the phenotype of the mother in the previous generation affects the expression of trait in the offspring in current generation (Wolf et al. 1998). This in turn acts like an evolving environment (Moore et al. 1997). For example, if higher milk production produces bigger offspring, a change in the mean milk production will result in

42 an evolutionary change in offspring size (Kirkpatrick and Lande 1989; Rossiter 1996).

Offspring size therefore shows a response to selection on milk production (Falconer

1965).

Figure-1.7: Contribution of Direct genetic effects and IGEs to the phenotype of individuals. The image has been adapted from Wolf et al. 1998. (a) According to a standard quantitative genetic measure, phenotype (zj) of an individual is contributed by genetic effect (a´j) and environmental effect (ej). (b) The IGE of phenotype z´j of individual 1 on the phenotype zi of individual 2 e´zj denotes the environmental effect of individual 1 on the expression of trait i in the individual 2 (focal individual). Ψij denotes the coefficient that measures the effect of z´j on expression of zj.

Maternal effects can in fact reverse a response to selection. For example Falconer (1965) found that a negative correlation between litter size in mice in successive generations affected the number of pups produced. Falconer explained that when a female produces a large number of pups, the pups are usually small in size. When these smaller female pups give birth later on, it was observed that the number of pups that they can give birth to was smaller (Falconer 1965). Similar findings have also been established by Holbrook and

Schal (2004) in the Diploptera punctata species of cockroaches who suggested that this negative correlation between the numbers of offspring is for providing a great degree of variation and for the maintenance of the overall litter size. In springtails, Janssen and Jong

(1988) found a negative correlation between the maturation times of mothers and daughters. This correlation has been estimated to be three times higher than the

43 correlation obtained for litter size in mice (Kirkpatrick and Lande 1989), thus having higher chances of significantly affecting the response to selection.

IGEs play an important role in mediating sexual selection and also in making the effects of sexual selection stronger. Griffith and Owens (1999) carried out a study investigating maternal effects on a wild population of sparrows in which after hatching, eighty seven whole litters were cross-fostered and the correlation between the size of badges (black spots on the throat) of offspring and biological parent and offspring and foster parents were measured. The results showed that the correlation was significant only for offspring with foster parents thus demonstrating that sexual traits such as badge size is affected not by the genetic makeup of an individual but by their parents.

The complex interactions during mate choice between males and females in many species indicate that IGEs influence sexually selected traits (Chenoweth and Blows 2006) such as odour in mammals (Meikle et al. 1995), singing in bird (Fortsmeier et al. 2004), however the implications of IGEs in the evolution of sexual display trait is yet to be tested empirically.

When IGEs exist, both direct genetic effects and IGEs affect a phenotype. This might result in amplifying the effects of the phenotype-genotype relationship (Trubenová and

Hager 2012). For example, when one individual becomes aggressive it tends to provoke aggression in the other individual as well (Wolf et al. 1997). If the strength of this influence is very large, this trait (aggression) will evolve at a faster rate than in Mendelian inheritance. IGEs thus help to give an insight in to how social behaviour can evolve at a faster rate than physiological traits.

The sign and correlation between the phenotypes of interacting individuals affects the rate of evolution of traits (Kirkpatrick and Lande 1989; Wolf et al. 1998). If the covariance of traits between two or more individuals is positive it will result in a positive feedback loop which will in turn increase the rate at which evolution occurs (Wolf et al. 1998).

44 Alternatively, a negative covariance will slow down evolutionary change. This was demonstrated by Wolf (2003) in his study on the body size of Drosophila melanogaster.

Pairs of unrelated flies with limited food supply were grown and it was demonstrated that flies which were predisposed to a bigger body size reduced the growth of the other fly; negative value for IGE variance. Also, the number of offspring produced by large flies was greater which means that eventually the mean size of the population would increase as the gene predisposed for large size was increasing in frequency. Thus the majority of flies will be predisposed to a large body size in the subsequent generations leading to diminished change in the mean phenotype and slower rate of evolutionary change. Similar effects were observed in cockroaches on dominance status (Moore et al. 2002).

Despite this wealth of studies showing that social, parental or other biotic environments are ecologically important, there has been little attention paid to the causes of variation in these environments (Cheverud and Moore 1994) and thus to their genetic influences.

There is still an absence of empirical studies that have quantified the occurrence of IGEs and have mapped the genes involved. Theoretical work in quantitative genetics suggests that this neglect has resulted in overlooking potentially wide-ranging effects in evolution

(Wolf et al. 1998). The second part of my PhD focussed on computationally establishing

IGEs and maternal effects on growth and weight data collected from a cross-fostered experimental design in mice. We have used the inbred strain BXD, with variable genetic makeup (Gini and Hager 2012), and C57Bl/6J (B6) mice, with a constant genetic makeup, as models for the study. My aim was to understand IGEs through mapping quantitative trait loci (QTL) of B6 weight phenotypes as a function of the BXD genotype.

1.4.1 QTL Mapping

Quantitative trait loci are stretches of the genome controlling quantitative traits in an individual. Analysis of QTL is a statistical method that links phenotypic data to genotypic data in an attempt to understand the variation in complex traits on a genetic basis

45 (Falconer and Mackay 1996; Kearsey 1998; Lynch and Walsh 1998). For the mapping of

QTL, a marker-linkage approach is generally utilised. The marker linkage analysis aims to identify the location of the QTL by co-segregating markers with genes in the QTL. The basic principle of determining whether a QTL is linked to a marker is by partitioning the mapping population in to different genotypic classes based on the genotypes at the marker locus. QTL analysis is thus undertaken in segregated mapping populations.

Linkage between the phenotype and markers which have already been mapped is tested in these populations in order to determine the position of the QTL.

Mapping data from experimental organisms are searched using statistical methods, to find major QTL (Soller et al. 1976; Lander and Botstein 1989). Interval mapping, by Eric

Lander and David Botstein (1989), is a standard method used for QTL mapping and has also been implemented in my study. Interval mapping uses an estimated genetic map as the framework for the location of QTL. The significance of a hypothetical QTL is assessed at fixed intervals across a chromosome. This is done by using the regression method,

Haley-Knott equations (Haley and Knott 1992) in which phenotypic values are correlated to the genotype between flanking markers (Chesler and Williams 2004).

The main goal of QTL mapping is to identify genes underlying polygenic traits and to understand their biological functions (Korstanje and Paigen 2002). So far QTL mapping has identified numerous chromosomal regions and genes involved in atherosclerosis

(Wang et al. 2005), obesity (Cheverud et al. 2004), hypertension (Zagato et al. 2000), asthma (Symula et al. 1999) and immune responses to T. trichiura, the human whipworm, infection (Hayes et al. 2014). QTL mapping approaches have also been implemented in studying genetic and epigenetic effects of traits (Hager et al. 2005), parent-of-origin dependent effects (Hager et al. 2008), and effects of change in social environment on developmental traits (Hager et al. 2009). My interest lies in identifying the QTL affecting developmental traits in a mother-offspring social environment in mice.

46 Recombinant inbred lines of mice are widely used for QTL mapping studies. Their fixed unique recombination pattern provides a valuable tool to identify QTL associated with quantitative traits (Klein 1978; Gini and Hager 2012). Recombinant inbred BXD lines are produced from inbred B6 mice strains and inbred DBA/2J (DBA) mice. Female B6 were crossed with male DBA. The resulting progeny consisted of individuals each with one allele from the B6 mother and one from the DBA father. The individuals of the first generation were then intercrossed producing heterogeneous, recombinant second generation individuals. The recombination was ‘frozen’ with 20 generations of sib-mating in the second generation progeny (Gini and Hager 2012). This yields a set of isogenic and highly recombinant strains, each with a complex but fixed mixture of recombined chromosome segments inherited from the parental strains. The BXD set is most widely used in mapping studies, and the analytical tools are shared in the community of over 100 labs across the world. The phenotypic data can be analysed and integrated with the largest genome, transcriptome and phenome datasets in existence. This way, pathways from phenotypic variation to physiology down to genetic variants can be identified. The

BXD set is characterized by approximately five million segregating single nucleotide polymorphisms (SNPs), 500,000 insertions and deletions, and 55,000 copy number variants (Gini and Hager 2012) thus capturing a vast amount of genetic variation and reaching the complexity of human populations.

47 1.5 Aims and objectives

Studies on genetic disorders provide a good platform to unravel the functions of genes

involved in different pathways of the body. The rare genetic disorder WBS results from a

microdeletion on chromosome seven which maps to the syntenic chromosome five in

mice. The genes located near the telomeric end of the deleted region have been found to

be involved in the distinct behavioural phenotypes of the disorder (Li et al. 2008). My PhD

has focussed on understanding the effect of the Gtf2ird1 and Gtf2ird2 in knockout mouse

models. Through investigating the effects of single gene deletion on behavioural and

physiological phenotypes, the aim was to understand the aietiology behind WBS

symptoms and also to find the overall functions of the Gtf2i genes. Another focus of my

PhD was to infer IGEs and maternal effects on weight traits of B6 mother and offspring by

finding the chromosomal location of QTL affecting these traits in the genome of BXD mice.

The main hypotheses investigated through my research are:

Gtf2ird1

1. Gtf2ird1 knockout female mice are more social and empathic than heterozygous and

wildtype females.

2. Maternal effects from varied upbrinding (by wildtype and homozygous mothers) play an

important role in mediating maternal behaviours of mutant females (homozygous,

heterozygous and wildtype) in the next generation.

Gtf2ird2

1. Gtf2ird2 deletion affects nest-building skills in pregnant mice.

2. Gtf2ird2 determines social behaviours in the single gene knockout mouse model.

3. Deletion of Gtf2ird2 affects expression levels of neurohormones in the hypothalamic brain

regions of the Gtf2ird2 mouse model

48 IGEs

1. Variable genotype of BXD foster mother has an effect on the growth of B6 pups.

2. IGEs of BXD litter genotype influences weight changes of B6 mother during early

developmental days of litters.

49 1.6 Thesis Chapters

The overall aim of my thesis was to improve the understanding of genetic and indirect genetic effects on behavioural phenotypes of Williams-Beuren syndrome mouse models and BXD/B6 inbred strains. A number of different mouse strains, breeding plans and overall experimental designs had been implemented in this body of work and has been presented in this thesis as four main chapters. Here is a brief summary of what these chapters entail.

The General Methods section follows the Introduction section. This part of the thesis describes the methods of those experiments which are applicable throughout all the chapters. For example how the animals were maintained, behaviours were phenotyped and statistical significance of the results were calculated are all described in this part of the thesis. The methodologies which were unique to a certain chapter have been incorporated within the methods section of that chapter.

Chapter-3 follows the General Methods section. This was the first preliminary maternal behavioural study that was conducted on the Gtf2ird1 mouse model. The results of this study were used to design a transgenerational experiment on the Gtf2ird1 mouse model.

This formed the fourth chapter of my thesis in which the protocols used were modified in accordance to the results from chapter-3.

The results from Chapter-4 found a contribution of maternal effects to offspring traits. To elucidate maternal effects and indirect genetic effects, a quantitative genetics approach study in chapter four was implemented. Although a very different but widely used inbred strain, BXD, was used as the model for this study, the same cross-fostering and phenotyping protocols were used for the data collection of this study.

Chapter-5 is the first reported knockout study on the Gtf2ird2 gene. This chapter provides a comprehensive understanding of the effects of the gene deletion on maternal behaviours and underlying neuroendocrine homeostasis in this mouse model.

50 The general discussion and conclusion brings all the studies together and explains the findings of my PhD with respect to existing literature. The future directions of my work are also described in this part of the thesis.

2. General Methods

52 2.1 Establishment of Gtf2ird1 and Gtf2ird2 knockout and heterozygous models

2.1.1 Gtf2ird1

Gtf2ird1 homozygous and heterozygous mice were created in a transgenic Tg(Alb1-

Myc)166.8 mouse strain on a C57Bl/6J x CBA/J background (Durkin et al. 2001;

Tassabehji et al. 2005). The deletion was induced by integration of c-Myc transgene on distal chromosome 5, syntenic WBS region in mice. The deletion spans 40kb and starts downstream of Cyln2 gene and includes the exon 1 and upstream promoter region of

Gtf2ird1 while preserving translation start site in exon 2 (Tassabehji et al. 2005). This eliminated expression of Gtf2ird1 in various tissues but retained expression of Cyln2 and

Gtf2i genes (Tassabehji et al. 2005).

For my studies wildtype Gtf2ird1 mice were obtained by crossing between a male

C57Bl/6J (in-house breeding stocks and Charles Rivers) and a female CBA/J (house breeding stocks) mouse. Homozygous mice were obtained by crossing between male and female mice of Tg(Alb1-Myc)166.8 strain; original C57Bl/6J x CBA/J background.

Heterozygous group was obtained by crossing between a male wildtype C57Bl/6J x

CBA/J with homozygous Tg(Alb1-Myc)166.8, C57Bl/6J x CBA/J background.

2.1.2 Gtf2ird2

The cohort used was a congenic C57Bl/6j (B6) mouse cohort which had been backcrossed for two years for >30 generations to get pure B6. Backcrossing is a crossing of a hybrid with the individuals being genetically similar to the parent, in order to achieve offspring with a genetic identity which is closer to that of the parent (Doerge 1993). The

Gtf2ird2 knockout was established by inserting a PGK-neo transgene in Gtf2ird2 exon 2 that resulted in a truncated protein (Tassabehji personal communication, unpublished model).

2.2 Animal Maintenance

For all my empirical experiments, all mice have been handled, accommodated and fed in exactly same way. Animals were kept and tested in the Biological Services Facility,

University of Manchester. All animals were treated in accordance with the UK Animals

(Scientific Procedures) Act 1986.

Adult mice were six to eight weeks old at the time of testing. 10 female mice per genotypic group were selected for all behavioural studies. This was to ensure that when comparing between the three genotypic groups an average could be taken for within group results.

Also, during the experiment 12 females were bred instead of 10 this was to control for any unsuccessful breeding. All mice were housed in ventilated cages (Tecniplast

30x10x10cm) with a relative humidity of 55% (±10) and room temperature of 20°C (± 2°).

They were given sterile grain pellets for food and sterile water bags for liquid; Gtf2ird1 homozygous and heterozygous mice were additionally given powdered food to accommodate their craniofacial abnormalities. The cage room was set to a 12-12 light to dark period with all the behavioural experiments being conducted in the dark period (red light) as mice are nocturnal animals. Females were mated with respective males by housing in the same cage. At around day 18 of the gestation period, when the pregnancy signs became obvious (bulge in the belly; significantly increased weight of around 8-10g), they were separated and housed in individual cages containing 3g nesting material, 200g food and a water bag. Cages were cleaned once a week; except during the first six days after the pups were born in order to reduce disturbance thus preventing the mothers from getting stressed. Behavioural study was commenced when litters were born. When the pups were 14 days old, mash (powdered food soaked in water) was provided alongside regular solid food as the teeth of the pups are still underdeveloped at this time. Pups were separated from their mother once they were weaned; around 21 to 24 days after birth.

Refer to Appendix-1 (Page 204) for detailed, step-wise protocol.

2.3 Cross-fostering protocol

Cross-fostering in Chapter-2 was done within the first two days of both litters being born to the different females. All cages were checked on a regular basis to note the date when the pregnant females littered. Once littered, mother and pup weights were recorded from both cages. Before the litters were exchanged, the bedding and nesting material from the respective recipient cage was rubbed on the pups for a few times. This smears the smell of the recipient mother on the litter being cross-fostered and thus reduces the chances of the mother rejecting the pups.

2.4 Phenotyping maternal behaviours of Gtf2ird1 mouse model in Chapter-1

Litters, mothers and food were weighed using the scale Sartorius Entris 2202-1S

Toploader Balance (Cole-Parmer), on postnatal day P2 and thereafter on P4, P7, P9,

P12, P14 and P21. Short-term changes in weight were also recorded on days 4, 9 and 14 as this is the lactation period of the mother mice during which differences in maternal behaviour between individuals are observed. To simulate maternal departure during foraging and standardize maternal and offspring motivation to exhibit maternal and offspring solicitation on postnatal days 4, 9 and 14, the weight of mother and pups were recorded and then the mother was separated from the litter for 4 hours. The mother was put in a new cage containing food and water from the home cage (Hager and Johnstone

2003) thus, during this time the pups had no access to any food sources. The pups were weighed and kept in the home cage, which was placed on a heat mat to retain temperature. The mother was reunited with the pups after 4 hours; weight of the mother, food and pups were recorded. Behavioural observations for 15 minute intervals were then carried out; with the behaviour being recorded every 20 seconds. Table-2.1 lists all the behaviours that had been phenotyped. Behaviours were phenotyped using a defined ethogram (Appendix Figures 39 and 40), which was generated such that it consisted of all

55 the arrays of behaviours being tested (Hager and Johnstone 2005; Lyst et al. 2012).

Table-2.1 lists all the behaviour that has been phenotyped. The pups and mother were then reunited and left undisturbed for 2 hours after which mother and pups were reweighed and the pup retrieval efficiency of mothers was tested. This was done by placing the pups at the furthest end of the cage and away from the nest and placing the mother near the nest and then recording how efficiently mother returned the pups to the nest.

Table-2.1: List and description of the maternal behaviours studied. The right column of the table describes each of the behaviours studied. Behaviours written in bold were recorded every 20 seconds because they were more common and lasted longer than other features during the 15 minutes observation. Behaviours written in italics were recorded every time they were observed as they were less common than other features.

Maternal Behaviour Description of Behaviour

Nursing Feeding half or less of the litter Suckling Feeding more than half of the litter Sniffing Smelling the pups to identify them Licking Licking the pups to groom them Nestbuilding Gathering nesting material to maintain the round shape of the nest Pup Retrieval Successfully being able to carry pups to the nest Time taken for Pup Time spent in retrieving pups; good measure for testing any Retrieval physical deficiency Autogroom Licking and grooming own self Resting Sitting or standing still away from the nest; usually during autogrooming Feed/Drink Feeding on solid food from the food hopper and/or licking the water nozzle to drink water

Other Active Any behaviour which is not specified elsewhere, including exploring the cage, digging, climbing the food rack and hanging upside-down.

2.5 Phenotyping protocol used in Gtf2ird1 transgenerational study in Chapter-3 and Gtf2ird2 study in Chapter-4

Maternal behaviours were recorded using the same protocol as described above except that the behavioural studies were conducted on postnatal days P6, 10 and 14. Moreover

56 the battery of behavioural traits studied, during fifteen minute period of observations, were expanded by adding new features to the ethogram. This included time spent by mothers in pups' quadrant (near the nest), number of times mother dropped pups during pup retrieval, and the way mothers carried pups during retrieval for example: by the head, tail, scruff or ears (Table-2.2).

Table-2.2: Traits studied during behavioural observations. On days 6, 10 and 14, behavioural observations were carried out for 15 minutes following 4 hours of mother-offspring separation. Table lists all the maternal behaviours that have been recorded during each of the behavioural observations. The traits in red highlight the new features that have been added to the studies. The trait in blue highlights an unexpected trait that was observed and added to the phenotype to be recorded. The table also shows the intervals in which the weight data were collected.

Behavioural Phenotypes Weight Data Maternal Nursing Initial Weight Suckling Weight on days 6, 10 and 14 Sniffing Weight before 4 hours separation Autogroom Weight after 4 hours separation Licking Weight after 2 hours of reuniting Time spent in pup’s quadrant (mins) Food consumption No. of times mother visited pups Weight when pups were weaned (day 21) Pup retrieval Mode of pup retrieval No. of times mother dropped pups during retrieval Feeding Drinking Infanticidal and cannibalistic behaviour Offspring Suckling Initial Weight Playfight Weight on days 6, 10, 14 and 21 Feeding Weight before 4 hours separation Weight after 4 hours separation Weight after 2 hours of reuniting

57 2.6 Scoring Behaviours

The behavioural protocol was devised to record maternal behaviours during early pup development. Therefore, postnatal days 4, 9 and 14 in Chapter-1 and 6, 10 ans 14 in

Chapter 2 and 3 were chosen as the observation days. At postnatal day 4 and 6 the pups are immobile, eyes are closed, have no fur and fully depend on the mother to provide food and warmth; at day 10 the fur growth is complete and pups are active although eyes remain closed; at day 14 pups almost double in size and weight, eyes are open, they are very active and start eating solid food (Figure 2.1). These days were also chosen to allow an interval between the observation days to ensure minimal disturbance to the maternal environment.

Figure-2.1: Mouse appearance by age. The figure shows the gradual change in appearance of mouse (B6) pups from postnatal day 1 to 14.

Behaviours were scored using the ethogram in Appendix Figures 39 and 40. Common behaviours such as nursing, suckling, nestbuilding, other activity and feeding/drinking were recorded on the beep. These behaviours generally were found to continue for a longer time than other less common behaviours and thus were recorded every 20 seconds. Each small box in the ethogram represents a minute thus, if one tick was added to the box at 20 second intervals a total of 3 ticks can be added per minute. In the end, the total number of ticks was added to see how frequently the mouse exhibited those behaviours during the 15 minutes observation.

58 Less common behaviours such as sniffing, autogrooming and licking (in italics in table 4 and 5) were recorded whenever they were observed. In the ethogram, a tick was given under the minute at which the observation was seen. Appendix-1 describes the detailed, step-wise protocol.

59 2.7 Statistical Analysis

2.7.1 Behavioural and Weight Data

The data sets of results obtained from my studies consisted of different response variables, such as weight, and many predictor variables, including genotype and food consumption, that might affect the response variable. To analyse such data, the statistical package SPSS was used to perform correlation analyses and general linear model (GLM).

The predictor variable can be either categorical, for example gender, or continuous, for example height. The general linear model was formulated by Nelder and Wedderburn

(1972) as a way of unifying various other statistical models, including analysis of variance

(ANOVA) and regression. GLM defines whether a predictor variable significantly affects a response variable or not. It also allows multiple predictor values to be analysed at a time further giving the opportunity to analyse the effect of interaction between predictor variables on a response variable. For example, when genotype and food consumption are two predictor variables in a model where the response variable is the weight of the mice, using GLM we can find whether genotype of the mice affects food consumption and the impact this has on the weight.

For all behaviours studied, except pup retrieval, the predictor variables that were controlled for in the GLM are litter size and weight of the mother mouse. This is because a bigger litter tends to seek more attention from mothers (Mendl 1988; Chiang et al. 2002), hence, mothers of the same genotype but varied litter size might show varied behaviours and therefore should be controlled for in our model. Similarly, bigger mice produce more milk (Falconer 1965) and this will affect the growth of pups, however, as we aimed to understand how the mothers’ behaviours affected the growth of pups, weight of mothers should be controlled in the statistical analysis. For pup retrieval analysis, along with litter size and maternal weight the initial weight of litters were also added as a predictor

60 variable. This was to configure whether any variation in pup retrieval was due to craniofacial abnormalities in the mother as opposed to variation in pup weight.

2.7.2 Nesting Experiment

Chapter-3 describes the detailed protocol used for testing nesting ability in Gtf2ird2 mice.

When the same study was conducted previously in our lab on the Gtf2ird1 knockout model (Schneider et al. 2012) significantly different results were found for the knockout mice compared to wildtypes. Gtf2ird1 knockout mice were unable to shred the nesting material and make a proper nest. Thus comparing the nests built by Gtf2ird2 mice I investigated whether the underlying genetic mutation compromises physical abilities in this model. Nesting ability of the mice were tested by scoring the nest on a 1-5 scale. The nest score data is hence a catergorical variable. Generalised linear models were thereby used to analyse the results of the nesting experiment. Generalised linear model expands

GLM by allowing the response variable to be categorical. Genotype of the females were the dependent variable in the model and nest score was the response variable.

2.7.3 Real-Time PCR Data

GLM was used to identify any significant differences in expression of oxytocin, oxytocin receptor, vasopressin and corticotrophin-releasing hormone levels between the three genotypic groups: wildtype, heterozygous and homozygous.

2.7.4 Data analysis for QTL Mapping

Before the mapping is carried out, the raw data needs to be analysed first so that any variation due to environment can be controlled statistically. My aim is to find out whether any variation in the B6 phenotype is due to the underlying variation in the BXD genotype, thus it is important to control for any other factors that might cause variation; this is done by calculating the residuals through GLMs. For example, assuming for my weight data I

61 want to know if weight change of B6 pups is affected by predictors such as initial weight of

B6 pups, weight of BXD siblings, and food consumption. The model maybe represented in the following way:

Y = β₀ + β₁x₁ + β₂x₂ + β₃x₃ + ………+ error

Where, Y= variable being tested (weight change of B6 pups)

β= All β-values are coefficients of the variables and determines how the predictor

variable affects the response variable.

x₁= Initial weight of B6 Pups

x₂= weight of BXD siblings

x₃= food consumption

A result of the above GLM might be that B6 pups weight change can be predicted by effects of genes controlling BXD sibling weight and food consumption. These would be the significant predictors with p values < 0.05, but for example if p> 0.05 for a certain variable it would mean that this variable is not significantly affecting the change in B6 pups weight.

As the initial weight of all the pups was not the same, GLM will allow us to control for it and calculate the residuals, which can then be mapped for QTL. Any resulting variation in

B6 phenotype can then be predicted to be due to an underlying variation in the BXD genotype. GLM analyses were carried out in statistical package SPSS.

3. Deletion of Gtf2ird1 gene results in abnormal maternal behaviours in a Williams-Beuren Syndrome mouse model- A pilot study

Data collection for this study was carried out by Z. Hawke and statistical analysis was conducted by N. Sharmin.

63 3.1 Abstract

The transcriptional regulator gene GTF2IRD1 has become a strong candidate for many phenotypes observed in the neurodevelopmental disorder Williams-Beuren Syndrome

(WBS). Studies in mouse models and genotype-phenotype correlations in humans show that deletion of the GTF2IRD1 gene influences important motor, neurocognitive and craniofacial features of the disorder. Here I show the role played by Gtf2ird1 in the behavioural phenotypes of the disorder. To elucidate the effect of the gene deletion on the social features of WBS, I have carried out a pilot study invetigating maternal behaviours of a knockout mouse model of Gtf2ird1. The hypothesis is that deletion of Gtf2ird1 affects maternal behaviours during early development of litters. By phenotyping behaviours during early development, I show that Gtf2ird1 impairs maternal care patterns.

Furthermore, I provide evidence that the underlying physiological problems of this gene deletion play a key factor in influencing the social behaviours studied. The results also suggest an enhanced empathy in Gtf2ird1 knockout mothers and provide a good platform for further studies on social profiles this mouse model.

64 3.2 Introduction

The rare genetic disorder Williams-Beuren Syndrome is characterised by distinctive physical and behavioural features (Morris 1988; Tipney et al. 2004). To understand the pathology of WBS, it is crucial to identify the role played by each of the genes in the deleted region. The elastin gene (ELN) was the first one to be identified in the microdeletion region and haploinsufficiency of this gene causes the characteristic supra vulvar aortic stenosis phenotype in the disorder (Ewart et al. 1993). This led to the understanding that individual genes located in the microdeletion are responsible for certain aspects of this multisystem disorder.

In recent years, researches have increasingly shown interest towards the GTF gene family located near the telomeric end of the deletion breakpoints (Roy 2001; Vullhorst and

Buonanno 2003). The GTF family of genes, GTF2I and GTF2IRD1, encode transcription factors involved in developmental pathways (Tassabehji et al. 2005; Roy 2006). Studies on mouse models and humans with atypical hemizygous deletions in the WBS region, have shown that GTF2I and GTF2IRD1 account for craniofacial abnormalities (Tassabehji

2003), diminished visuospatial construction (Hirota et al. 2003) and social phenotypes of the disorder (Karmiloff-Smith et al. 2003).

In this study I explored the role played by a deletion of Gtf2ird1, in a knockout mouse model of WBS. A number of single gene knock-out models of WBS exist; Gtf2ird1 models have exhibited craniofacial abnormalities, motor skill deficiencies and behavioural phenotypes with varying severity (Tassabehji et al. 2005; Palmer et al. 2007; Young et al.

2008). Using a Gtf2ird1 knockout model, I investigated the effect of gene deletion on the social phenotype of the disorder. In particular I tested the ‘hyper sociability’ symptom of the disorder by phenotyping maternal behaviour towards her pups. I mostly investigated maternal behaviours such as, suckling, nursing and pup retrieval, in order to understand whether the phenotypes are impaired due to the underlying genetic mutation or not.

Nursing at critical points during early development is crucial because mice as altricial

65 species rely fully on maternal care for survival and growth (Latham and Mason 2004). The

Gtf2ird1 null transgenic mice have been described with both physical (craniofacial and growth) and neurological (anxiety, motor, social) abnormalities. To assess the role of

Gtf2ird1 in social behaviour I carried out maternal studies as an extended social test to investigate its impact on mother pup interaction and rearing.

3.3 Methods

3.3.1 Experimental Design

Ten wildtype (Wt) and homozygous (Homo) virgin female mice were selected for the purpose of the study. All mice were adults, eight to nine weeks old, at the time of testing.

Half of the wildtype females and half of the homozygous females were mated to homozygous males whilst the others were mated to wildtype males (Figure-3.1).

Behavioural studies were commenced on birth of litters (General Methods, Page 54).

Using an adapted protocol (Hager and Johnstone 2003) a number of maternal behaviours were recorded, including suckling and licking (Table-3.1, Page 55). Through analysing the differences in maternal behaviours of wildtype and homozygous mothers, the effect of

Gtf2ird1 deletion on social behaviours were identified.

Figure-3.1: Breeding plan. Wildtype (green) and homozygous (Red) females were mated to wildtype (green) males (left) and the other females from both genotypic groups were mated to homozygous (red) males (right). The breeding plan resulted in half of each genotypic group rearing heterozygous (orange) litters and the remaining half rearing their own genotypic litters.

3.4 Results

3.4.1 Behavioural Data

Maternal behaviour towards offspring was analysed during the early developmental stages (Days 4, 9 and 14) of the pups. All mice were maintained in the same environmental conditions thus any differences in behaviour are predicted to be the result of the underlying deletion of Gtf2ird1. The analysis of behavioural data revealed that although there was a clear trend in ‘nursing’ and ‘suckling’ behaviours with homozygous mothers nursing/suckling less than wildtype mothers, the differences were not statistically significant (Nursing: GLM; F1,19=0.56; p=0.47; Suckling: GLM; F1,19=0.43; p=0.65) (Figure-

3.2). However, a significant effect was observed for overall maternal behaviours. Maternal behaviour was calculated as mean sums of nursing and suckling scores on days 4, 9 and

14. Wildtype mothers were seen to exhibit greater maternal behaviours compared to homozygous mothers (GLM; F1,19=4.61; p=0.046) (Figure-3.2, Page 68). But when short term weight changes in litters, also termed as maternal provisioning, were recorded, I

67 found that on day 14, litters of homozygous mothers gained significantly more weight during the two hours post reunion compared to the litters of the wildtype mothers (GLM;

F1,19=7.90; p=0.013). Genotype also affected the licking behaviour in mothers. Wildtype mothers licked their pups significantly more than homozygous mothers (GLM; F1,17=25.62, p<0.0001) (Figure-3.2). Motor dependent behaviours such as nestbuilding and pup retrieval were also affected by the underlying gene deletion in mothers (Figure-3.2).

Gtf2ird1 homozygous mice have significantly impaired fine motor skill and craniofacial features (Tassabehji et al. 2005; Schneider et al. 2012). These deformities affected the mean time taken by homozygous mothers to retrieve their litters (GLM; F1,16=15.96; p=0.001) and also nest building frequency in homozygous mothers compared to the wildtypes on days 9 and 14 (GLM; F1,16=4.55; p=0.05). Behaviours such as drinking/feeding were also affected by the Gtf2ird1 deletion. Homozygous mothers fed/drank significantly more than the wildtype mothers (GLM; F1,19=29.89; p=0.001), during the 15minutes observations on all three days (day 4, 9 and 14). All other behavioural measures including ‘sniffing’ (GLM; F1,17=0.82; p=0.379), ‘autogrooming’

(GLM; F1,17=0.73; p=0.821) and ‘other activities’ (GLM; F1,17=0.45; p=0.543) were not affected by maternal genotype. Also, litter genotype alone had no effect on the maternal behaviours studied (GLM; F1,17=1.04; p=0.341).

3.4.2 Pup Retrieval Test

When assessing the efficiency of homozygous and wildtype mothers, to collect all their pups, one by one, from one corner of the cage to the centre of the nest, no significant differences were found. Both genotypic mothers were capable of gathering all their pups back to the nest, however, homozygous mothers took significantly longer to handle the pups and carry them to the nest, compared to wildtype mothers (GLM; F1,16=15.96; p<0.001). During the pup retrieval test, it was also observed that homozygous mothers were dropping the pups several times during the process of carrying the pups to the nest;

68 this behaviour was however based on observations and was not officially recorded during this set of experiments.

Figure-3.2: Gtf2ird1 deletion affects maternal behavioural phenotypes. Behavioural differences between the two genotypic groups, wildtype (Wt) and homozygous (Homo) are exemplarily presented for 7 different behavioural measures: (A) Mean Maternal Behaviour (B) Licking (C) Nest Building (D) Pup Retrieval Duration (E) Maternal Feeding/Drinking behaviour (F) Nursing behaviour (G) Suckling behaviour. There was a significant difference between the wildtype and homozygous groups for all the behavioural measures from A to E with Homos showing reduced maternal behaviours, licking and nest building behaviours but increased pup retrieval time

69 and feeding/drinking. Panels F and G show reduced nursing and suckling behaviours in Homos but this was not significant. Data are presented as untransformed means ± standard error of the mean, * p<0.05.

3.4.3 Weight data

The proportional growth per pup was calculated from postnatal day P2 and thereafter on

P4, 7, 9, 12, and 14. Taking in to account the differences in birth weight, proportional growth rates of litters nursed by homozygous mothers were not significantly different to that of wildtypes’ (GLM; F1,19=1.60; p=0.29) (Figure-3.3). Furthermore, after being kept with mothers undisturbed for two hours, maternal provisioning of homozygous mothers was significantly high on postnatal day 14. This was reflected by weight gain in litters of homozygous mothers compared to litters of wildtype mothers (GLM; F1,19=5.38; p=0.034)

(Figure-3.3).

A. Growth

70 B. Maternal provisioning

Figure-3.3: Growth of Gtf2ird1 litters reared by homozygous and wildtype mothers Panel A shows the proportional growth of litters nursed by homozygous mothers were comparable to that of litters of wildtype mothers. The graph on the right shows that litters born of and nursed by wildtype (Wt) mothers gained significantly more weight over time compared to litters of homozygous (Homo) mothers. Panel B shows that maternal provisioning, or short term gain in weight of pups after 2 hours of undisturbed rearing by mother is also affected by maternal genotype. A significant difference is observed between the two genotypes only on day 14.

3.5 Discussion

The objective of this pilot study was to investigate the role played by the deletion of

Gtf2ird1 on social phenotypes of the neurogenetic developmental disorder, Williams-

Beuren Syndrome. By phenotyping maternal behaviours during pup rearing I aimed to understand how Gtf2ird1 affects the social phenotypes of a single-gene WBS mouse model. Although a number of studies have been conducted on the Gtf2ird1 deleted mouse model of WBS (Tassabehji et al. 2005; Young et al. 2008), no study to date has particularly discussed the social phenotypes in the context of parent-offspring interaction.

Maternal behaviour in early life is crucial for development and acts as a good means of studying the genetic contributions in the aetiology of social disorders (Franks et al. 2011).

Using an extensive phenotyping protocol we studied a broad spectrum of behavioural phenotypes; this ranged from maternal care to feeding and grooming behaviours. The

71 results show how underlying physical deformity affects nurturing behaviours in a Gtf2ird1 null mouse model. This study provides a good base to explore the effect of varied maternal behaviours of WBS mouse models towards the development of their litters.

3.5.1 Maternal Behaviour

Maternal behaviour was calculated as an average of nursing and suckling behaviours performed by the two genotypic groups (wildtype and homozygous) on postnatal days 4, 9 and 14. As nursing and suckling are the most basic maternal care behaviours, the sum of these scores can give us a measure of the total maternal behaviour. The Gtf2ird1 homozygous mothers exhibit significantly less maternal behaviours during the 15 minutes behavioural observations, than wildtype mothers. However, on day 14, after the mother and pups were left undisturbed for 2 hours, it was observed that the litters of the homozygous mothers gained significantly more weight than the litters of wildtype mothers.

Normally, from day 14 mothers reduce provisioning to promote the pups to start weaning

(Kikusui et al. 2009). This illustrates that although the homozygous mothers show an overall lack of maternal care or attention towards the pups, on day 14, when the pups are physically bigger and seek more attention, due to prior starvation for 4 hours, the homozygous mothers provision them considerably more than wildtypes. This is an indication of increased empathy in the homozygous mothers.

The apparent ‘lack of maternal care’ is also highlighted by the significantly reduced number of times homozygous mothers licked their pups during the 15minutes observations. I propose that this reduction in licking behaviour in Gtf2ird1 homozygous mice is due to the underlying physical problems caused by deletion of this gene. One of the most obvious is the misalignment of the jaw in Gtf2ird1 homozygous mice. Gtf2ird1 null mice have significantly impaired craniofacial features (Tassabehji et al. 2005) which explain the reduced nest building characteristics seen in the homozygotes. This has previously been deciphered in a Gtf2ird1 mouse model (Schneider et al. 2012); the

72 homozygous mice were incapable of tearing nesting material to make a nest, and even when torn nesting material was provided they still built poorly constructed nests compared to the wildtypes (Schneider et al. 2012).

Due to the impairment in jaw alignment, the homozygous mothers also took over twice as long to retrieve the pups back to the nest, whilst the wildtype mothers swiftly retrieved the whole litter. But the fact that the homozygous mothers did retrieve every pup back to the nest demonstrates that Gtf2ird1 homozygous mothers are not ‘bad’ mothers. The underlying physical deformity seems to be affecting the maternal behaviours studied.

3.5.2 Growth

During the early development of pups, weight recordings have established that homozygous litters had a significantly reduced growth rate compared to wildtypes.

Homozygous pups, as well as mothers, were smaller than wildtypes; this has been previously observed (Tassabehji et al. 2005). However, behavioural observations from this study have noted that homozygous females demonstrated significantly elevated feeding/drinking behaviour compared to wildtypes (spent more time at the feeding/drinking stations even during pup retrieval tests). This might be indicative of a high metabolic rate in Gtf2ird1 knockout model (Tassabehji personal communication), however further studies are required to draw firm conclusions. Factors such as litter size or initial weight of mother or litters were controlled for via statistical analysis. This is because I found that litter size has a significant effect on maternal provisioning irrespective of genotypic effects.

Considering the weight differences at birth, I found that litters reared by homozygous mothers had comparable proportional growth to the litters of wildtype mothers. Additionally we observed that maternal provisioning by homozygous mothers is at its peak on postnatal day 14 when pups just commence weaning. Studies on weaning and maternal behaviour have repeatedly shown that mothers start maintaining a physical distance with pups as they reach weaning age (Cramer et al. 1990; Curely et al. 2009) however in my

73 study I observed that Gtf2ird1 homozygous mothers show increased maternal care behaviour at this time leading to significantly increased short term weight gain in pups.

This reflects higher sociality in the Gtf2ird1 homozygous mice and is in line with existing literature (Hirota et al. 2003; Tassabehji 2003; Young et al. 2008; Dai et al. 2012).

3.6 Conclusion

To date, behavioural social studies conducted on Gtf2ird1 models have included only short term social interactions; such as resident intruder test (Young et al. 2008), social recognition tests (Chadman et al. 2009), and social preference tests (Li et al. 2009). The protocol we used recorded, for the first time, a long term study on social interactions in a

Gtf2ird1 knockout model. This study shows dysregulated social behaviours in the Gtf2ird1 mouse model. Maternal behaviours are diminished in this knockout model during early pup rearing but elevated at the start of weaning. This can be explained by the physical deformities observed in homozygous mice which could result in the lack of care behaviour/sociality during the earlier stages. Our study highlights how maternal behaviours such as licking and pup retrieval are compromised by physiological constraints in these homozygous mice. However, the fact that homozygous mothers provisioned more at start of weaning and on average, litters of homozygous mothers did not have significantly reduced growth compared to the litters of wildtype mothers indicate that

Gtf2ird1 homozygous mothers are in fact more empathetic despite their physical disabilities (misaligned jaws, impaired fine motor skills). This is in line with increased social and empathetic phenotypes seen in WBS indivduals (Klein-Tasman and

Mervis 2003; Ouertani et al. 2014) and other mouse models (Tassabehji et al. 2005;

Young et al. 2008; Li et al. 2009). As this was a pilot study, further research with more detailed behavioural protocol controlling for the physical deformities could help to decipher detailed sociality in this model.

4. Social behaviours in a single-gene knockout mouse model of Gtf2ird1 gene

Data collection and statistical analysis for this study was carried out by N. Sharmin.

75 4.1 Abstract

The transcriptional regulator gene GTF2IRD1 plays important roles in social cognition in the neurodevelopmental disorder Williams-Beuren Syndrome (WBS). Research on mouse models and WBS indivduals have found an association of GTF2IRD1 and GTF2I genes with the neurocognitive, motor and craniofacial features of WBS. An earlier pilot study

(Chapter-1) has found that knockout of Gtf2ird1 in mice results in impaired maternal care behaviours due to underlying physiological deformities, but an increased empathy at start of weaning. To understand this effect further, I have carried out further extensive maternal studies with an adapted protocol on the Gtf2ird1 mouse model. By phenotyping maternal behaviours in three distinct experimental setups, I show that Gtf2ird1 gene deletion impairs maternal care irrespective of maternal effects or offspring genotype. Maternal effects, however, do alter behavioural phenotypes when compared within the same genotypic groups which partially counteracts the effect of the gene deletion but cannot fully compensate for it. Furthermore, my study confirms that the underlying physiological problems of this gene deletion play a key factor in influencing the social behaviours studied. I also monitored growth of the litters from birth to weaning and observed a comparable proportional growth in all litters. This suggests an enhanced empathising behaviour in knockout Gtf2ird1 female mice which are able to nurse litters efficiently despite underlying physiological difficulties.

76 4.2 Introduction

Individuals with Williams-Beuren Syndrome (WBS) are known to be hyper sociable and interact highly empathetically with reduced social inhibition (Jarvinen-Pasley et al. 2008;

Dai et al. 2009). This trait is as a result of abnormal neural profiling in the hippocampal brain regions controlling socio-cognitive properties in WBS indivduals (Dykens et al. 2005;

Meyer-Lindenberg et al. 2005). The deletion of GTF genes, located on the telomeric end of chromosome seven, has been linked to social symptoms of WBS (Tassabehji et al.

2005; Roy 2006; Young et al. 2008). Gtf2ird1 is of particular interest because of its contribution to behavioural symptoms in WBS indivduals (Dai et al. 2009). The gene has been found to be associated with craniofacial abnormalities (Tassabehji et al. 2005); growth retardation (Howard et al. 2011); motor deficits (Schneider et al. 2012) and increased social drive in mouse models (Young et al. 2008).

My research aimed to study the contribution of the Gtf2ird1 gene deletion on social symptoms of WBS by phenotyping behaviours in a Gtf2ird1 null mouse model. I previously studied this single-gene knockout mouse model of Gtf2ird1 in a pilot study investigating maternal behaviours (Chapter-1). The purpose of the pilot study was to understand some basic phenotypes of the mouse model before commencing my extensive behavioural studies which are described in this chapter. The results of the pilot study (Chapter-1) indicated impairments in crucial maternal behaviours such as suckling, licking and pup retrieval in the knockout Gtf2ird1 mice. As previously described (Tassabehji et al. 2005;

Schneider et al. 2012), the Gtf2ird1 knockout females had severely misaligned jaws and poor fine motor skills (detected by observing poor nesting behaviour). Although they were smaller than wildtype mice, knockout Gtf2ird1 females spent significantly more time eating/drinking during the behavioural experiments, which was not reflected by a gain in weight or size indicating a possible metabolic defect (Tassabehji personal communication). Despite these physiological difficulties, Gtf2ird1 mothers showed enhanced maternal provisioning and litters reared by knockout mothers had comparable

77 proportional growth to that of the wildtype litters. I thus hypothesised that Gtf2ird1 females are in fact more empathetic but are compromised due to their underlying physical problems.

To take replicate and study this further, I aimed to investigate the effect of the Gtf2ird1 gene deletion on maternal behaviours of mothers reared themselves by either knockout or wildtype mothers. I implemented a cross-fostering protocol to ensure uniformity in genotypes of litters reared by mutant mothers (General methods, page 53). The main purpose of this experimental design was to understand the effect of the gene deletion alone on maternal behaviours in the Gtf2ird1 mouse model; controlling for effects of litter genotype and maternal effects. I also sought to understand whether maternal effects of being reared by knockout mothers alter maternal behaviours of females in the next generation.

The phenotype of an offspring is, in part, a result of the genes they inherit from their parents but can also be highly influenced by parental phenotypic effects. These parental phenotypic effects are referred to as 'maternal effects' when the environment provided by the mother has a phenotypic effect on the offspring (Maestripieri and Mateo 2009).

Maternal effects can have a significant impact on the survival of the litter thus ultimately having important evolutionary consequences. Studies on rodents and nonhuman primates have shown that maternal effects can transmit a trait from one generation to the next without direct effect of genotypes and can even result in mothers and cross-fostered daughters having similar parental care patterns (Francis et al. 1999; Meaney 2005;

Maestripieri et al. 2007). A study on rhesus monkeys, Macaca fuscata, found an inattentive and abusive maternal care patterns in females who were reared by abusive and careless mothers themselves (Maestripieri 2005). Conversely, mothers producing more milk had offspring with bigger size which produced more milk themselves

(Kirkpatrick and Lande 1989; Rossiter 1996), thus, greater provisioning by mothers can lead to an evolutionary change in size of offspring.

78 Through phenotyping maternal care patterns in two consecutive generations I aimed to understand whether maternal effects contribute to phenotypes in the Gtf2ird1 mouse model. Essentially, I wanted to understand whether being reared by wildtype mothers make Gtf2ird1 knockout females more maternal than the females reared by knockout mothers. Furthermore, to elucidate any effect of offspring genotype on maternal behaviours, I monitored the behaviour of wildtype C57BL/6J (B6) mothers towards cross- fostered mutant Gtf2ird1 homozygous, heterozygous and wildtype litters until the litters were of weaning age. In mammals, mothers often tend to reject pups with physical abnormalities (Turgeon and Meloche 2009) and focus on ‘good quality’ pups (Thünken et al. 2010). It will hence be interesting to find whether the B6 mothers display variable maternal care towards the Gtf2ird1 mice.

4.3 Methods

The breeding plans and behaviour monitoring protocol have been updated following the pilot study on maternal behaviours on the Gtf2ird1 mouse model (Chapter-1).

4.3.1 Experimental Design

Maternal behaviours of Gtf2ird1 mice

Following on from my previous study (Chapter-1), I designed an experiment to control for two important factors, which could otherwise interfere with the analysis. Firstly, I reared all three genotypic Gtf2ird1 mothers (wildtype, heterozygous and homozygous) by wildtype

C57Bl/6J (B6) mice to prevent any differences in maternal effects on upbringing. I then cross-fostered B6 litters to the Gtf2ird1 mothers to ensure uniformity in offspring genotype

(Figure-4.1). This study aimed to understand any differences in maternal care behaviour, which solely related to underlying Gtf2ird1 gene deletion.

79 Twelve, eight-week old Gtf2ird1 homozygous, heterozygous and wildtype female mice, reared by B6 mothers, were used in this study. All three genotypic females were mated with males of the same genotype to obtain litters of pure genotype. Simultaneously, 36 B6 females were mated with B6 males to obtain pure B6 litters. Once born, within the first two days of littering, each mutant litter, homozygous, heterozygous and wildtype was cross- fostered with a B6 litter. This experimental set up resulted in mutant mothers fostering B6 pups and B6 mothers fostering mutant pups (Figure-4.1).

Figure-4.1: Breeding plan used to study Gtf2ird1 maternal care behaviour by controlling litter genotype and maternal effects. (I) Wildtype (green), homozygous (red) and heterozygous (orange) females were reared by B6 wildtype mothers until weaning age. (II) When adult, these females were mated with males of same genotype to produce mutant litters of the same genotype. (III) The mutant litters born, were cross-fostered with newly born B6 litters. The breeding plan resulted in all the mutant mothers rearing B6 litters and the B6 mothers rearing mutant litters.

80 Maternal effects on Gtf2ird1 mice

Maternal effects are an important environmental factor which can affect an offspring's phenotype and significantly bias estimates of additive genetic variance and correlations of traits (Mastriprieri and Mateo 2009). Thus an understanding of maternal effects will give a clearer understanding of the genetic basis of traits and potential evolutionary changes.

My main interest from this research was to understand whether maternal effects from

Gtf2ird1 homozygous mothers had an effect on the maternal behaviours of their female offspring towards litters in the next generation, independent of offspring genotype.

Homozygous, heterozygous and wildtype Gtf2ird1 litters were reared by homozygous

Gtf2ird1 mothers. Once the litters were weaned, 12 females from each of the genotypic groups were separated. When these females were eight weeks old they were set up for breeding with males of same genotypes. The litters born were crossfostered with B6 wildtype litters of same age (Figure-4.2); this ensured all mutant mothers nursed pups of same B6 genotype. The behaviour of these second generation Gtf2ird1 females towards wildtype B6 litters was recorded and analysed. The maternal behaviour of homozygous and wildtype mothers from this study were compared against maternal behaviours of homozygous and wildtype mothers from the pilot study. This enabled us to analyse the effect of being reared by homozygous mothers (deficient in some maternal skills) compared to B6 wildtype mothers (normal) on maternal care in the next generation. Table-

6 gives a comparison of the results from the studies (Page 91).

Figure-4.2: Breeding plan used to study maternal effect on Gtf2ird1 maternal care in the next generation. (I) Wildtype (green), homozygous (red) and heterozygous (orange) females were reared by homozygous mothers until weaning age. (II) When adult, these females were mated with males of the same genotype to produce mutant litters of same genotype. (III) The mutant litters were cross-fostered with B6 (black) litters. The breeding plan resulted in all the mutant mothers rearing uniform B6 litters.

Effect of offspring genotype on maternal behaviours

To study whether litter genotype affects maternal behaviours, I cross-fostered homozygous, heterozygous and wildtype Gtf2ird1 litters to B6 wildtype mothers (Figure-

4.3) and recorded maternal behaviours during early pup development.

Figure-4.3: Breeding plan used to study effect of litter genotype on maternal behaviours of B6 females. Litters of wildtype, homozygous and heterozygous Gtf2ird1 breeding cages were cross-fostered with B6 litters of same age. The breeding plan resulted in B6 mothers rearing mutant litters.

4.3.2 Phenotyping

The results from Chapter-1 identified a number of new features which have not been recorded previously. Gtf2ird1 homozygous mice in the previous study were seen to drop their pups very frequently during the pup retrieval process. Homozygous mothers were also seen to be foraging and eating or drinking for a prolonged period of time during the behavioural observations. These eating and drinking behaviours were not differentiated and were documented as a single behaviour. Taking these results in to account the ethogram used to record behavioural phenotypes was updated (Appendix Figure-39 and

40, Pages 208 and 209). The number of times the mothers dropped the pups during the retrieval process was documented in this study along with the mode of retrieval that is

83 whether the pups were pulled by the scruff, legs, tail or head. Mode of retrieval was documented to understand whether homozygous mice, with misaligned jaws, use abnormal means of carrying the pups. The number of times mother visited the nest or the time spent near the nest were also recorded in this study as an additional measure of attentive maternal care pattern. The General Methods section of this thesis lists all the traits recorded in this study and intervals of the weight screenings (Page 55).

84 4.4 Results

4.4.1 Maternal behaviours of Gtf2ird1 mice reared by B6 mothers

The results of my behavioural study showed that although knockout mothers suckled their whole litter the least number of times compared to wildtype and heterozygous mothers

(GLM; F2,29=14.67; p<0.001), they nursed a few pups at a significantly higher rate (GLM;

F2,29=11.51; p<0.001) (Figure-4.4). Similarly, heterozygous mothers suckled the whole litter less frequently than wildtype mothers (GLM; F2,29=16.09; p=0.05) but nursed a few pups at a time significantly more than wildtype mothers (GLM; F2,29=11.51; p=0.02). In fact when maternal provisioning was calculated, I found that on all three days, litters of knockout mothers gained significantly more short term weight during the two hours post reunion with mothers compared to the litters of the wildtype mothers (Day 6: GLM;

F2,26=4.16; p=0.017; Day 10: GLM; F2,26=3.00; p=0.026; Day 14: GLM; F2,26=0.13; p=0.05)

(Figure-14). Moreover, knockout mothers licked their pups a similar number of times as the other two genotypic mothers, except for on day 14 when they were seen to lick them the least number of times (Day 6: GLM; F2,29=5.33; p=0.09; Day 10: GLM; F2,29=0.43; p=0.65; Day 14: GLM; F2,29=5.33; p=0.012) during the 15 minute observations (Figure-

4.4). Although these Gtf2ird1 knockout mice show improved maternal behaviours compared to the pilot study (Chaper-1); knockout mothers were seen to be visiting the pups the least number of times compared to wildtype mothers on days 10 and 14 (GLM;

F2,29=21.66; p<0.001) (Figure-4.4). On these two days, during the behavioural observations, knockout mothers were eating food for a significantly prolonged time (GLM;

F2,29=15.82; p<0.001) (Figure-4.4) while no differences were observed in drinking behaviours (GLM; F2,29=2.35; p=0.115). All other behaviours such as ‘sniffing’ (GLM;

F2,25=2.39; p=0.112), ‘nest building’ (GLM; F2,25=0.73; p=0.49) and ‘other activities’ (GLM;

F2,25=3.94; p=0.32) (Figure-4.4) were not found to be affected by maternal genotype.

85 A. Nursing and Suckling

B. Time spent with pups and feeding

C. Maternal Provisioning

86 D. Licking

E. Nest building and Other activities

Figure-4.4: Effect of Gtf2ird1 gene deletion on maternal behaviours The figure shows the behaviours monitored and how they differed between the three genotypic groups. Panel A shows that Gtf2ird1 knockout mothers (KO) nursed their pups a significantly higher number of times during the behavioural observations but suckled them less compared to wildtype (Wt) and heterozygous (Het) mothers. Panel B shows that on day 10 and 14 Gtf2ird1 knockout mothers spent less time near the pups’ nest during the 15 minutes behavioural observations and instead spent significantly more time feeding themselves. Panel C shows that knockout mothers provisioned their litters significantly more than wildtype mothers on all three days. Panel D shows that there were no significant differences between the mothers for frequency of licking pups on all days except on day 14 when knockout mothers were seen to lick the pups less frequently than wildtypes. Panel E shows that frequency of nest building and other activities did not vary between the three genotypic mothers on all three days.

Results of the pup retrieval test showed that knockout mothers took a significantly longer time to retrieve their pups (GLM; F2,27=3.22; p=0.05) (Figure-4.5) than wildtype mothers.

87 Moreover, knockout mothers took significantly longer to initiate the retrieval process compared to wildtype and heterozygous mothers (GLM; F2,27=2.44; p=0.015) (Figure-4.5).

During pup retrieval all mothers used a uniform mode of retrieval; pulling pups via the scruff, leg, tail, or ear.

Figure-4.5: Gtf2ird1 knockout mothers show disrupted pup retrieval behaviour. Knockout mothers took a significantly long time to retrieve the whole litter back to the nest. Knockout mothers also took longer time to initiate the retrieval process compared to heterozygous and wildtype mothers.

4.4.2 Maternal effects on Gtf2ird1 mice

In this study I phenotyped maternal behaviours of female Gtf2ird1, knockout, heterozygous and wildtype mice that were reared by knockout Gtf2ird1 mothers in the previous generation (Figure-4.6). The results from this study are very similar to the pilot study on Gtf2ird1 knockout model (Chapter-1). Knockout mothers in this study suckled

(GLM; F2,56=8.01; p<0.001) and nursed (GLM; F2,56=3.80; p=0.015) their litters significantly less than both wildtype and heterozygous mothers (Figure-4.6). Knockout mothers also licked their pups significantly less frequently (GLM; F2,56=4.91; p=0.004) and spent the least time near the nest compared to wildtype and heterozygous mothers (GLM;

F2,56=4.82; p=0.005) on all three days (Figure-4.6). In fact in comparison to the other two genotypic groups, knockout mothers spent a considerably longer time eating solid and powdered food followed by drinking water from the pouch (GLM; F2,56=14.93; p<0.001)

88 (Figure-4.6) during the 15 minutes observations; a behavioural pattern that was noticed in my previous study as well. Another result found in this experimental design which mimicked the behaviour of knockout mice in Chapter-1 is maternal provisioning. Knockout mothers in this study were found to provision their litters significantly more than wildtype mothers but only on day 14 (GLM; F2,57=2.65; p=0.05) (Figure-4.6). All other behaviours such as ‘nestbuilding’ (GLM; F2,29=0.73; p=0.490), ‘sniffing’ (GLM; F2,29=0.65; p=0.590) and ‘other activities’ (GLM; F2,29=4.21; p=0.09) were not affected by underlying gene deletion (Figure-4.6). Table-4.1 summarises the behavioural phenotypes recorded from the pilot study (Chapter-3) and the two experimental designs implemented here (reared by

B6 and KO) (Page 91).

A. Nursing and Suckling

B. Time spent with pups and feeding

C. Maternal Provisioning

D. Licking and Sniffing

90 E. Nest building and other activities

Figure-4.6: Behaviours of females reared by knockout Gtf2ird1 mothers Panel A shows Gtf2ird1 knockout mothers nurse and suckle their litters significantly fewer times than wildtype mothers. Panel B shows Gtf2ird1 knockout mothers spent least time with the pups or near the nest than wildtype mothers and instead visited the food hob and powdered food tray very frequently. Panel C shows that on day 14 litters of knockout mothers gained more weight than litters of wildtype mothers, after being reunited with pups and left undisturbed for two hours. Panel D shows that although no differences in frequency of sniffing pups were found between the three genotypic mothers, knockout mothers licked the pups significantly less than both wildtype and heterozygous mothers. Panel E shows that mean frequency of nest building or other activities were not affected by underlying maternal genotype.

The pup retrieval test showed, as expected, a long time taken by the knockout mothers to retrieve the whole litter (GLM; F2,57=1.64; p=0.05) (Figure-4.7). Additionally, knockout mothers took the longest time to initiate the retrieval process (GLM; F2,57=1.64; p=0.05)

(Figure-4.7); this was measured by noting the latency of the first retrieval. No significant differences were observed in the mode of retrieval. All genotypic mothers retrieved their pups by either grabbing them by the scruff or pulling by the tail, leg or ear although scruff was the most common choice.

Figure-4.7: Pup retrieval is affected by Gtf2ird1 gene deletion Knockout mothers took significantly long time to retrieve their whole litter back to the nest. Knockout mothers also initiated the retrieval process much later than the wildtype mothers.

4.4.3 Growth of litters reared by Gtf2ird1 mutant mothers

The proportional growth per pup was calculated from postnatal day 1 and thereafter on days 6, 10, 14, and 21. Birth weight and litter size was taken in to account when calculating the differences in proportional growth per litter. Litters of knockout mothers showed comparable growth to litters of wildtype and heterozygous mothers in both experimental setups: one where mothers were fostered by knockout mothers and the other where mothers were fostered by B6 mothers (Figure-4.8). There were no significant differences on average proportional growth of litters reared by mothers from B6 upbringing

(GLM; F2,29=5.31; p=0.79) and mothers from knockout upbringing (GLM; F2,29=4.22; p=0.81). However, when the weight of litters on postnatal day 21 was compared, litters of knockout mothers were found to weigh significantly lower than litters of wildtype mothers from knockout upbringing (GLM; F2,52=4.69; p=0.03). No weight differences were found between litters of heterozygous mothers and litters of wildtypes or knockouts from knockout bringing (GLM; F2,52=8.75; p=0.206) or between any of the three genotypic groups from the B6 upbringing (GLM; F2,52=8.28; p=0.427). This is evident from growth

92 curves in Figure-4.8 where proportional weight per pup is similar in all three genotypic groups except on day 21 in panel A.

Figure-4.8: Proportional growth of pups reared by mutant mothers. Litters of knockout Gtf2ird1 mothers gained comparable weight to litters of wildtype and heterozygous mothers. Similar results were seen for the two experimental set ups: one where mothers were fostered by KO mothers (A) and the other where mothers were fostered by B6 mothers (B) except on day 21 in panel A when litters of knockout mothers weighed significantly less than litters of wildtype mothers.

Table-4.1: A brief comparison of behavioural phenotypes of Gtf2ird1 knockout mice. The table lists the behaviours of knockout Gtf2ird1 mice recorded from the pilot study and the two experimental designs discussed in this chapter (reared by B6 and reared by KO mice). The table shows any differences in KO mothers’ behaviours compared to wildtype mothers. Key: no sig difference= no significant differences with wildtype; sig decreased/increased= significantly lower/higher than wildtypes; n/a= measure was not recorded in this study.

93 4.4.4 Maternal effects on behaviours of Gtf2ird1 mouse model

The behaviours of Gtf2ird1 mothers were studied in two distinct experimental set-ups where the effect of maternal environment during early upbringing was controlled. In one study all female Gtf2ird1 mothers (homozygous, heterozygous and wildtype) were reared by knockout Gtf2ird1 females and in the other study they were reared by wildtype B6 females (Figure-4.1 and 4.2). The comparison of the results from these studies were used to infer whether maternal effects played a role in mediating nurturing behaviours of the next generation and this has been reported in this section.

All three genotypic mothers, knockout (GLM; F2,36=3.83; p=0.032), heterozygous (GLM;

F2,36=5.21; p=0.02) and wildtype (GLM; F2,36=9.02; p=0.001), from the B6 upbringing have enhanced overall maternal behaviour compared to respective knockout, heterozygous and wildtype mothers from Gtf2ird1 KO upbringing. Maternal behaviour was calculated as the mean sums of nursing and suckling scores (Figure-4.9). Mothers from the B6 upbringing also spent more time with their litters or around the nest compared to mothers from the

KO upbringing (Knockout: GLM; F2,35=9.36; p=0.001; Heterozygous: GLM; F2,35=4.14; p=0.03; Wildtype: GLM; F2,35=9.36; p=0.005) (Figure-4.9). When the interaction effect between genotype (KO and B6) of foster mothers from the previous generation and their own genotype (KO, Het and Wt) was analysed, no significant effect was found for time spent with pups (GLM; F2,36=0.470; p=0.50) or mean maternal behaviours (GLM;

F2,36=3.104; p=0.08). In other words, foster mother’s genotype (GLM; F2,36=2.13; p=0.01) and own genotypes (GLM; F2,36=2.130; p<0.001) independently affected the traits; however, there were no interaction effects between the maternal genotypes. There were also no significant differences in ‘licking’ (GLM; F2,36=2.82; p=0.231) ‘feeding’ (GLM;

F2,36=3.44; p=0.360) or ‘pup-retrieval’ behaviours (GLM; F2,36=4.42; p=0.513) (Figure-4.9).

94 A. Maternal Behaviour

B. Time Spent with pups

C. Licking

D. Feeding

E. Retrieval time

Figure-4.9: Maternal effects on maternal behaviours of Gtf2ird1 mouse model The graphs show the comparison in behavioural traits between the three mutant mothers knockout, heterozygous and wildtypes raised by knockout (KO) mothers and B6 mothers. For example, behaviours of knockout females raised by knockout mothers (KOKO) were compared with behaviours of knockout females raised by B6 mothers (KOB6) and similarly KOWT and KOB6 and KOHet and B6Het were compared. All three genotypic groups raised by B6 mothers demonstrated higher overall maternal behaviours (Panel A) and spent more time with pups (Panel B) compared to females of own genotype reared by KO mothers. Other behavioural phenotypes such as licking (C), feeding (D) and pup-retrieval (E) were not affected by maternal effects of different upbringing.

96 4.4.5 Effect of offspring genotype on maternal behaviours

By cross-fostering mutant litters to B6 mothers and comparing the maternal behaviours of

B6 mothers rearing the three genotypic litters I aimed to investigate any effect due to litter genotype. The results of the behavioural study found no significant differences between any groups for any maternal behaviours. All mothers demonstrated similar patterns of nursing (GLM; F2,22=0.88; p=0.427), suckling (GLM; F2,22=0.34; p=0.714), licking (GLM;

F2,22=0.27; p=0.760), sniffing (GLM; F2,22=0.57; p=0.57), and grooming behaviours (GLM;

F2,22=0.35; p=0.704) (Figure-4.10). Moreover, litters of all genotypes showed a similar growth rate irrespective of litter size (Figure-4.10). Knockout Gtf2ird1 mice are generally smaller in size than heterozygous and wildtype mice (Tassabehji et al. 2005). However, knockout litters gained comparable proportional weight to that of heterozygous and wildtype litters during early upbringing.

A. Nursing and Suckling

97 B. Licking and Sniffing

C. Growth curve

Figure-4.10: Effect of offspring genotype on behaviours of B6 mothers. B6 mothers did not differentiate between the three genotypic litters and demonstrated similar nurturing patterns. Panel A and B shows that B6 mothers rearing KO, Wt and Het litters showed similar frequency of nursing, suckling, sniffing and licking behaviours. Panel C shows that all three genotypic litters gained comparable weight during early pup rearing by B6 wildtype mothers, despite the knockout pups (Homo) being smaller due to the underlying gene deletion.

98 4.5 Discussion

The main objective of my research was to investigate mother-offspring interactions in a mouse model of Gtf2ird1. The study was designed to take the preliminary findings

(Chapter-1) on this mouse model further. Using an extensive behavioural protocol, I phenotyped behaviours of mutant Gtf2ird1 mothers toward cross-fostered litters. The key result of my study is that the physiological abnormalities that arise due to the gene deletion hinder crucial maternal behaviours in this model. Knockout mothers nurtured their pups significantly less and visited them much less frequently than wildtype or heterozygous mothers. Instead, these knockout mothers were regularly seen to be foraging food. Maternal behaviour of both Gtf2ird1 knockout and wildtype mothers were seen to be regulated by maternal effects of the foster mother’s genotype from previous generation. This is an important finding of my study highlighting the importance of maternal effects on traits of the next generation. Both empirical and quantitative work has shown the evolutionary and non-evolutionary implications of maternal effects on traits.

(Kirkpatrick and Lande 1989; Moore et al. 1997; Griffith and Owens 1999). There are reports in rhesus monkeys showing that abusive and inattentive mothers produce daughters who are abusive in their turn (Maestripieri 2005). Maternal effects influence traits as diverse as sexual selection (Griffith and Owens 1999), population size

(Moore et al. 2002) and mate choice (Chenoweth and Blows 2006). It is hence important to study maternal effects and investigate how significantly these may influence the genotype-phenotype correlation.

4.5.1 Physiological deficits arising from Gtf2ird1 deletion affect crucial maternal behaviours

A number of Gtf2ird1 knockout models display physical deficits in mice (Tassabehji et al.

2005; Palmer et al. 2007; Young et al. 2008); these physical deficits interfere with good

99 maternal provisioning. Gtf2ird1 knockout mice have been repeatedly found to be smaller and weigh less compared to both heterozygous and wildtypes (Tassabehji et al. 2005;

Schneider et al. 2012). Weight screenings in my study have found similar effects of the gene deletion on knockout litter weights. However, the study points out an unusually high feeding and drinking habit in the knockout Gtf2ird1 mothers during ongoing behavioural studies. Knockout mothers seemed to be abandoning their litters in both of my experimental setups and instead were visiting the food hopper very frequently and eating approximately double the number of times than nursing or suckling their litters.

Regardless of an increased appetite, body weight of knockout mothers stayed lower than heterozygous and wildtype mothers at all times. This is indicative of a possible disrupted metabolic rate in this Gtf2ird1 knockout model. A recent study has found several metabolic changes in WBS indivduals and mouse models, although no direct association with Gtf2ird1 gene was found, they highlighted that there is a lack in studies focussing on how genes deleted in WBS may result in biochemical changes in individuals (Palacios-

Verdú et al. 2015). Interestingly, previous studies on Gtf2ird1 knockout models have found an abnormal thermoregulatory profile in knockout mice; knockout mice had a lower core body temperature compared to wildtypes (Howard et al. 2012; Skitt 2012). In mice, heat loss per unit size produces significant changes in food consumption (Nielson et al.

1997). Thus I propose that the smaller size of Gtf2ird1 knockout mice in my study led to increased heat loss which was compensated by knockout females eating significantly more. This phenotype might also be as a result of lack of expression of Gtf2ird1 gene in the hypothalamic and pituitary brain regions of the mouse model. These brain regions control homeostatic maintenance, including thermoregulation, and highly express Gtf2ird1 throughout murine embryonic development to adulthood (Palmer et al. 2007).

Crucial maternal behaviours such as nest building and pup retrieval were also significantly impaired in the knockout Gtf2ird1 mothers in my study due to their physical abnormalities.

Mothers were briefly separated from the litters during the study and the pups were

100 scattered at one end of the cage. Under this scenario the mothers were returned to their cages and attempts of looking for pups and retrieving them successfully to the nest were monitored. This was very difficult for Gtf2ird1 knockout mothers who had craniofacial abnormalities characterised by periorbital fullness and a short snout and severely misaligned jaws with overgrowing teeth (Tassabehji et al. 2005; Van Hagen et al. 2007).

Knockout Gtf2ird1 mothers also had very poor fine-motor skills (Schneider et al. 2012) and were incapable of making ‘good’ quality nests (Deacon 2006) for their pups even when aided with shredded nesting materials (Schneider et al. 2012). My study has extended these findings by noting that knockout mothers took significantly longer to start the retrieval process. Studies on maternal care often regard latency of retrieval as indices of maternal responsivity (Champagne et al. 2007). Although variation in strains (Hennessy et al. 1980; Carlier et al. 1982) and lactation (Rosenblatt 1994) can affect the time taken to retrieve, differences in genetic makeup between wildtype, heterozygous and knockout

Gtf2ird1 mice were the most likely explanation for the delayed retrieval seen in my model.

Hearing impairments might also explain the impaired maternal responsivity shown by this mouse model. Maternal responsivity refers to how mothers respond to and provide for offspring (Warren and Brady 2007). Ultrasound vocalisation of pups is a major communicative behaviour directing mothers to elicit nursing, licking and retrieval approaches (Cohen-Salmon et al. 1985; Scattoni et al. 2009). Thus mothers with hearing impairments will fail to show these maternal behaviours. WBS indivduals have noted mild to moderate hearing loss in all ages (Cherniske et al. 2004; Marler et al. 2005; Barozzi et al. 2012). A recent study has found high expression of Gtf2ird1 protein in the inner ear and has found principal hearing deficits in a Gtf2ird1 knockout model of WBS (Canales et al. 2014). Thus if Gtf2ird1 gene is a regulator of hearing loss in WBS, it is indicative of another physical complication that may interfere with maternal provisioning behaviours.

Future studies in this knockout model recording ultrasound vocalisation of both mothers and pups are necessary to draw any further conclusions.

101 Despite such physiological disabilities, Gtf2ird1 knockout mothers demonstrated an enhanced empathetic behaviour as indicated by the growth rates of their litters.

Regardless of a reduced suckling behaviour during the 15 minutes observations, on postnatal days 6, 10, 14 and 21, pups reared by Gtf2ird1 knockout mothers gained similar weights to pups of wildtype and heterozygous mothers. Overall litters of Gtf2ird1 mothers further showed a significantly increased short-term weight gain during the two hours post- reunion with their mother compared to the litters of the wildtype mothers. During these two hours the mother was left undisturbed with the litter and any weight changes in litters was due to suckling by mothers. This shows that Gtf2ird1 mothers are more nurturing when there are no external disturbances such as handling and can in fact provision their litters more than wildtype mothers despite their physical difficulties. This empathetic behaviour is in line with enhanced empathizing/social nature of WBS indivduals (Meyer-Lindenberg

2006) and hyper sociability in other Gtf2ird1 mouse models (Young et al. 2007; Li et al.

2009).

The lack of maternal care can also be due to attention problems in the Gtf2ird1 homozygous mice. WBS individuals generally find it difficult to shift attention to multiple visual stimuli in the background and gives extended attention to individual objects (Brown et al. 2003; Karmiloff-Smith et al. 2012; Dimintriou et al. 2015). The prolonged eating behaviours and lack of attention toward pups might be indicative of such attention deficits in the Gtf2ird1 knockout mouse model.

4.5.2 Maternal effects are an important regulator of maternal behaviours

Maternal effects can be defined as the effect of maternal phenotypes on offspring phenotype (Wolf and Wade 2009). Many aspects of offspring phenotypes including body size and growth, reproduction, or behaviour, can be directly influenced by the mother’s phenotypes such as behaviour or nutritional status (Maestripieri et al. 2007). Thus maternal effect is a mode through which traits can be passed to the next generation

102 without direct effect of genes. In this study I cross-fostered mutant Gtf2ird1 litters, which were reared by either B6 or knockout Gtf2ird1 mothers and then monitored the maternal provisioning behaviours of the females from the mutant litters in the next generation.

Although own genotype significantly affected the behavioural traits, maternal effects seemed to have a positive influence on the behaviours when compared within the same genotypic group. Both wildtype and knockout females reared by B6 mothers later spent more time with their litters and also exhibited more maternal behaviours when compared to respective wildtype and knockout females reared by knockout mothers. Francis et al.

(1999) had reported similar findings in rats; daughters of more nurturing mothers displayed more caring and nurturing parental behaviours themselves. In fact a number of studies to date have shown dramatic changes arising from natural variability in maternal care behaviours ranging from changes in behaviour of offspring, stress mechanism, reproduction profile and neuroendocrine pathways in brain regions (Meaney 2001;

Champagne et al. 2003; Cameron et al. 2005).

Behavioural phenotypes were analysed using general linear models (GLMs) in SPSS.

Using GLMs, interaction effects between own genotype and foster mother’s genotype on parental behaviour were investigated. Although no significant effect was observed on either of the traits a suggestive effect was seen for mean maternal behaviours. This suggests that maternal effects of foster mothers did have an effect on maternal care patterns in mutant mothers. Gtf2ird1 gene deletion poses significant physical challenges in the mouse model making it difficult for them to provision litters efficiently. Thus, although maternal effects of B6 foster mothers partially compensated the reduced nursing skills in knockout females, they were unable to fully balance the physiological effects of the Gtf2ird1 gene deletion. This explains why overall maternal behaviours and time spent with pups were influenced by maternal effects but licking, feeding and retrieval behaviours were not (Figure-4.9).

103 My study has also found that reciprocal cross-fostering of mutant litters (CBAXB6 background) to B6 mothers did not cause any variation in maternal care patterns.

However, knockout Gtf2ird1 litters reared by B6 mothers gained proportional weight at the same rate as that of wildtype and heterozygous Gtf2ird1 pups, despite the overall growth retardation due to underlying genetic effects. A study by Van Der Veen et al.

(2008) has found that the two mouse strains DBA and B6, display similar maternal behaviours to their biological litters, cross-fostered litters of the same strain and cross- fostered litters of the opposite strain (either DBA or B6) (Van Der Veen et al. 2008).

Strain and genotype of cross-fostered litters can have effects on behaviour of foster mothers (Cierpial et al. 1990) but here I show how the behaviour of cross-fostered litters in adulthood is affected due to the nurturing patterns of B6 foster mothers. Cross- fostering is a widely used laboratory practice and has given an insight in to carry-over effects in to adulthood (Winslow et al. 2000; Bartolomucci et al. 2004) as seen in my study as well.

4.6 Conclusions

The aim of this study was to investigate social interactions of Gtf2ird1 knockout mouse model during pup development. By employing a modified protocol I analysed maternal behaviours of Gtf2ird1 mouse model (knockout, heterozygous and wildtype) towards cross-fostered B6 wildtype litters. I can conclude from the results that the Gtf2ird1 gene deletion causes significant physical deformities in the knockout females thus interfering with their maternal care patterns. Litters of knockout mothers still had similar growth patterns to that of wildtype and heterozygous litters. This suggested strong empathy in knockout mothers who were able to overcome their physical impairments and rear their litters successfully (no infant mortality was observed throughout the course of the study).

Maternal effects were found to play an important role in maternal care behaviours in this model. Maternal effects arose from mutant Gtf2ird1 mothers being reared by either B6 or

104 knockout Gtf2ird1 mothers in the previous generation. Although maternal effects of B6 mothers improved maternal care behaviours in the mutant mothers, it was not strong enough to compensate fully for the genetic effects of Gtf2ird1 deletion.

105

5. Gtf2ird2 deleted in Williams-Beuren Syndrome affects social and maternal behaviours in a mouse model system by impairing oxytocin expression in the brain

Data collection and statistical analysis for this study was carried out by N. Sharmin.

106 5.1 Abstract

Human social behaviour is affected by both environmental and genetic factors. The human genetic disorder Williams-Beuren Syndrome (WBS) is characterised by neurological deficits that affect social and cognitive behaviour. The hypothesis is that deletion of the Gtf2ird2 gene, from the Gtf2i-family of transcription factors, influences social and neuropsychological development and consequently affects maternal behaviour during early development. Furthermore I propose that social behaviours are modified by irregular expression levels of the neuroendocrine hormone oxytocin. By phenotyping maternal behaviours in a single-gene knockout mouse model from the WBS region, I show, for the first time, that deletion of Gtf2ird2 gene influences key maternal and social behaviours in homozygous and heterozygous animals, affecting offspring development. In addition, Gtf2ird2 heterozygous mothers exhibit high rates of filial cannibalism compared to either knockout or wildtype mothers. These results show that this single gene mutation has an important role in the behavioural symptoms seen in WBS indivduals. The results of the behavioural studies are further supported by upregulated mRNA expression levels of

Oxt gene in the paraventricular nucleus and supraoptic nucleus of homozygous mothers, and equally down regulated Oxt levels in the heterozygous mothers. The results are also indicative of an oxytocin-induced desensitisation of oxytocin receptor in the brain. The results thus show an association of the Gtf2ird2 gene deletion with social symptoms of

WBS and provide evidence of neuroendocrine implications behind the hyper social behaviours. My study provides a good insight in to the potential role of Gtf2ird2 and highlights the importance of researching the Gtf gene family.

107 5.2 Introduction

The neurogenetic disorder Williams-Beuren syndrome arises due to a hemizygous deletion of around 26 to 28 genes (~1.5Mb) on chromosome 7q11.23 (Wang et al. 1999).

The disorder presents a unique spectrum of physical and behavioural features (Greenberg

1990; Pober 1996); although WBS indivduals have cognitive and visuospatial deficits, they show enhanced verbal and social skills (Udwin and Yule 1991; Bellugi et al. 1994; Jarrold et al. 1998). Phenotypes arising from such chromosomal microdeletions provide a good means to identify genes crucial for cognition and behaviour (Tassabehji et al. 1999). The function of a number of genes deleted in WBS has been deciphered. This includes the elastin gene ELN involved in cardiovascular and connective tissue abnormalities in the disorder (Ewart et al. 1993; Metcalfe et al. 2000), cytoplasmic linker protein encoding gene CLIP115 is involved in neurological aspects of the disorder (Hoogenraad et al.

2002), GTF2I and GTF2IRD1 involved in craniofacial, motor and social symptoms of the disorder (Tassabehji 2005; Young et al. 2008; Enkhmandakh et al. 2009; Palmer et al.

2010).

Some WBS indivduals have a larger atypical microdeletion of the Williams Syndrome region, of size 1.8 mbp, which results in the loss of an additional gene, the transcription factor GTF2IRD2 (Porter et al. 2012). GTF2IRD2 belongs to the GTF gene family clustered near the telomeric end of the deleted region. This gene family, GTF2IRD1,

GTF2I and GTF2IRD2 are all highly expressed in the brain, and GTF2I and GTF2IRD1 are thought to be involved in the pathogenesis of the cognitive and behavioural phenotypes associated with WBS (Tipney et al. 2004; Palmer et al. 2007; Porter et al.

2012; Shirai et al. 2015). When considering the overall biological role of GTF2IRD2, its shared homology to the GTF family suggests that it may contribute to the neurological and social symptoms in indivduals (Tipney et al. 2004). A multi-level analysis of cognitive, behavioural and psychological functioning in 55 Australian WBS indivduals provided the first evidence for the role of GTF2IRD2 in cognitive and psychological development

108 (Porter et al. 2012). This study carried out an array of genomic and neuropsychological studies on the indivduals and compared the differences in results between indivduals with a deletion size of 1.5/1.6 mbp and 1.7/1.8 mbp. The results highlighted significantly impaired social functioning and executive reasoning in indivduals with the bigger deletion of 1.8 mbp, which includes GTF2IRD2. This study has provided support for the potential role of GTF2IRD2 in the pathogenesis of the syndrome and has pointed out the importance of studying this gene further.

To date, the main caveat of studying the contribution of GTF2IRD2 deletion in WBS pathogenesis has been the lack of individuals with a single gene deletion of GTF2IRD2.

Studying a mouse model of the disorder is therefore a good option. The mouse orthologue of GTF2IRD2, Gtf2ird2, has also been isolated and maps to the syntenic WBS region on mouse chromosome 5G. The genes in this critical region in mice are organised in the exact same order to that of humans except that the whole region is inverted (Tassabehji et al. 2005).

My research aimed to demonstrate, the effect of Gtf2ird2 gene deletion on social and fine motor skills in a knockout mouse model. Particularly I investigated maternal behaviours, such as suckling, nursing and pup retrieval, of transgenic adult female mice towards their pups in order to understand whether the social phenotypes are impaired due to the underlying genetic mutation or not. Also, I wanted to decipher if the mutation directly affected behaviour or indirectly by imposing physical constraints such as poor fine-motor skills as observed in Gtf2ird1 mouse models (Schneider et al. 2012). At the end point of the behavioural studies I collected brain tissue from all three genotypic mothers, wildtype, heterozygous and homozygous, and investigated the gene expression of the neuropeptides oxytocin (Oxt), oxytocin receptor (Oxtr), vasopressin (Avp) and corticotrophin-releasing hormone (Crh) in selective brain regions of this Gtf2ird2 knockout model using molecular biology techniques. Growing evidence confirms that oxytocin and vasopressin play key role in parental care and social attachment (Leckman 2011).

109 Moreover, WBS indivduals show impaired endogenous oxytocin levels in social scenarios

(Dai et al. 2012). Thus my study yields a comprehensive understanding to if and how social behaviours are controlled by the transcription factor gene Gtf2ird2.

5.3 Methods

5.3.1 Genotyping

All female mice were genotyped before the behavioural experiments. DNA was extracted from ear punches of the mice using the KAPA mouse genotyping extraction kit (KAPA

Biosystems) and amplified using a transgene specific polymerase chain reaction (PCR).

The PCR products were run on a 2% agarose electrophoresis gel to identify the genotypes of each mouse according to the DNA band size; DNA band size for wildtype mice was 325 base pairs (bp), heterozygous mice was 195bp and 325bp, homozygous mice was 195bp.

5.3.2 Agarose gel electrophoresis

The two percent gel was prepared using two grams of agarose powder (Geneflow, UK) dissolved in 100 ml TAE buffer (400mM Tris, 0.01 M EDTA; pH 8.3) and heated in a domestic microwave until a clear transparent solution had formed. The solution was cooled for three to four minutes in room temperature and 10µl Safeview dye (Applied

Biological Materials) was then added. Safeview is a safer nucleic acid stain and does not possess mutagenic properties like standard ethidium bromide (www.abmgood.com). The solution was then poured into a prepared electrophoresis gel tank, combs inserted and left to solidify. TAE buffer was used to completely submerge the gel before the samples were loaded and a 100 bp ladder, as a molecular weight marker, were loaded into appropriate wells. 5 μl of 100 bp ladder was then loaded to the first well, and in some cases both first and last well, and 10 μl of PCR product was loaded in each well. The gel was

110 electrophoresed at 160 Volts for 15 to 20 minutes. The DNA bands were then visualised using a GeneGenius bioimaging system and Genesnap software (Syngene, UK). Figure-

5.1 shows an image of an electrophoresis gel.

Figure-5.1: Gtf2ird2 gene PCR products of ear samples of the three genotypic mice ran on 2% agarose gel. The picture shows the PCR products of digested ear samples from Gtf2ird2 batch. Three types of primer pairs were used for the PCR: GTF4 exon 3 primer pair for wildtype samples, KOPCR primer pair and loxPR forward primer and BGH reverse primer for detection of the targeted deleted allele in homozygous and heterozygous samples. Lane1: 100bp ladder. Lanes 2 and 4: Samples from homozygous mice. Lane 3, 5, 7 and 8: Samples from wildtype mice. Lane 6: Sample from a heterozygous mouse. Lane 9: PCR mix with no DNA. Lane10 and 11: Heterozygous controls from a different Gtf2ird2 batch.

5.3.3 Testing nesting ability

The protocol used in this experiment has been designed by Deacon (2006). A ‘good’ nest is one which is made of almost fully shredded nesting material, with the walls being higher than the height of the mouse’s body and the nest covering its circumference. Building a good nest requires good craniofacial and fine-motor skills. Thus by scoring and comparing the nests built by Gtf2ird2 homozygous, heterozygous and wildtype mice, I investigated whether the underlying genetic mutation compromises physical abilities in this model, as observed in Gtf2ird1 knockout model (Schneider et al. 2012). If nesting was impaired, shredded nesting material would be provided to aid the mice in building a nest for its pups.

Nests provide shelter and warmth (Van de Weerd et al. 1998), especially for the pups,

111 hence nesting before parturition is an important part of maternal behaviours and thus my study.

On the day pregnant females were separated, three grams (3g) of pressed cotton squares

(Nestlets), were provided to each of their cages. The nests were assessed the next morning (12 hours later) on a one to five rating scale and any unshredded nestlet pieces were weighed and the percentage unused was calculated. Using the measure of the untorn pieces and observing the shape and quality of the nests, the nests were scored on a rating scale of one to five with five being the highest mark (Figure-5.2). If more than 90% nestlet was unused the nest was scored one; if nestlet was partially torn with 50-90% remaining unshredded it was scored two; if the nest was flat and had no defined round shape with some pieces spread all over the cage but >50% nestlet being shredded, it was scored three; if a nest had a round shape with >90% of the nestlet being shredded but was flat, it was scored four; if a nest fell under all the categories of a ‘good’ nest and had >

90% torn nestlet, it was scored five.

112 Figure-5.2: Scoring the quality of nests. The picture shows the differences in nests scored from 1-5. The nest in picture 5 has a well round structure with high walls thus scoring a perfect 5. The nest in picture 1 shows a barely shredded nest thus scoring a poor 1. Adapted from: Deacon (2006).

5.3.4 Experimental design and phenotyping

For the purpose of the study, 12 wildtype, heterozygous and homozygous Gtf2ird2 females were mated with heterozygous males. This breeding strategy allowed all three genotypic mothers to rear fairly uniform genotypic litters. The experimenter was blind to the genotypes. An adapted behavioural protocol (Phentoyping, Page 55) was employed to phenotype maternal behaviour following mother-offspring separation (Page 56).

At the end of the behavioural study, ten of the wildtype, heterozygous and homozygous females were mated again and after littering they were culled via schedule one Home

Office procedure and brain and serum tissue was collected. From the brains collected, specific brain regions such as the paraventricular nucleus (PVN) and supraoptic nucleus

113 (SON) were dissected out. Molecular biology techniques were then applied to measure the expression levels of oxytocin, oxytocin receptor, vasopressin and corticotrophin- releasing hormone genes in these brain regions in order to decipher whether deletion of

Gtf2ird2 impairs the expression of these crucial neuropeptides in this mouse model.

5.3.5 Tissue collection

After the completion of the behavioural study, mothers from each of the three genotypes were mated once again with heterozygous males. The breeding was planned such that all mice were mated on the same day to synchronise births. On postnatal day 3 of the second generation, cages with mother and litters were taken out of racks and put on the table to rest for two hours prior to end point. Mothers were culled, one at a time, as they were suckling the pups. This is because oxytocin level is at peak during maternal bonding

(Fuch et al. 1984). Whole brain was dissected and immediately frozen on dry ice before storing in the -80°C freezer. The three different genotypes were culled randomly throughout the day preventing any effect of the circadian rhythm on the neuropeptide levels.

5.3.6 Sectioning brain regions

This part of the research was conducted under collaboration with Dr. Chris Murgatroyd at

Manchester Metropolitan University.

Using a cryostat microtome (Leica 1800) 30 whole mouse brains were individually cryosectioned (10 µm) at rostral PVN and hippocampus levels (Murgatroyd et al. 2009).

The frozen brain (-80°C) was glued to the cryostat using Cryomatrix (Thermo Scientific) and acclimatised to the internal -21°C temperature of the machine for 20 minutes prior to starting the procedure. A sharp razor blade was positioned in the cryostat microtome which cut through the tissue as the platform with the tightly secured brain was manually moved up and down. With each pass of the blade the tissue was advanced a specified

114 distance such that uniformly thick sections were obtained. The cut slices were picked up on adhesive microscope slides and stained using a basic histology technique (Page 114).

This helped in identifying the required brain regions and mainly gave a direction to how much further the brain needed to be cryosectioned. The brain regions that had been collected were: paraventricular nucleus (PVN) and supraoptic nucleus (SON). The histology sections of mouse brains from Paxinos and Franklin (2001), as shown in Figure-

5.3, was used as a reference. Once the brain regions were located and confirmed using the histology test a tissue micropunching technique was then applied to extract the regions. A micropuncher, also called sample corer (Fine Science Tools), with 0.5mm diameter plunger was injected into that specific part of the brain and the tissue was excavated. This was repeated for all the different regions and the tissues were collected in labelled eppendorf tubes which were cooled to -21°C by placing inside the machine at the beginning of the experiment. The brain punches were stored in a -80°C freezer until required.

PVN

115 SON

Figure-5.3: Brain regions dissected. The figure has been adapted from Paxinos and Franklin (2001). The black arrows point at the regions that I had dissected.

5.3.7 Histology for identification of brain regions

Cresyl Violet stain (FD Neurotechnologies) was used for identifying the different brain regions. Cresyl Violet is a Nissl (endoplasmic reticulum) stain that dyes both neurons and glia in to a bright purple/violet colour (Pilati et al. 2008).

Brain slices were picked up on dry adhesive microscope slides and placed on a heating block for 30 seconds. Warming the frozen brain section and microscope slide prior to staining helped to improve penetration and allowed even staining (Bankcroft and Gamble

2008). The dry slide was then placed in a coplin jar filled with 0.5% Cresyl Violet stain for three to four minutes. The slide was then removed and rinsed in distilled water to wash off any excess stain. The tissue was then dehydrated through soaking in increasing concentrations of alcohol (Fisher Scientific) 70% and 100% respectively for two minutes in each.

116 The stained brain sections were viewed under a light microscope and were compared to illustrations in the mouse brain atlas (Figure-5.3). Appendix-1 (Page 210) gives a stepwise protocol with images.

5.3.8 Transcription of brain regions of Gtf2ird2 mouse model

Micropunches of PVN, SON, CA and MA brain regions from wildtype, homozygous and heterozygous Gtf2ird2 mice were homogenised prior to RNA extraction. Each tissue was vigorously vortexed in 400µl TRIzol reagent (Life Technologies) to break the tissue up and homogenise it. RNA was then extracted from the brain punches of each of the three genotypic groups using the ‘Direct-zol mini prep’ kit (Zymo Research) following the manufacturer’s instructions (Appendix-1, Page 216). This protocol aided total RNA extraction and also constituted a DNase treatment step allowing for removal of any DNA contamination. Upon extraction, the concentration and purity of RNA was measured using a Thermo Scientific Nanodrop™ 1000 Spectrophotometer. A purity value of approximately

2.0 at an absorbance ratio of 260/280nm is considered ‘pure’ for RNA samples (Thermo

Scientific Technical Bulletin). RNA was stored in -80°C until required.

5.3.9 Reverse transcription

The concentrations of RNA samples were obtained from nanodrop readings (ng/µl) and then used to calculate the required volume of RNA for reverse transcription to cDNA. 40ng of RNA was required in a 10μl volume (volume was calculated by dividing 40ng by the sample concentration). Using the calculated RNA volumes and cDNA synthesis kit

(Thermo Scientific), reaction mixtures for cDNA synthesis were made. Once prepared, the synthesis was carried out in a Veriti 96-well thermal cycler using the manufacturer’s instructions (Life Technologies). The cDNAs were stored at -20°C until required (detailed protocol in Appendix-1, Page 217).

117 5.3.10 Polymerase chain reaction (PCR)

PCR was conducted prior to quantitative-PCR (qPCR) step to test whether the PVN and

SON regions were in fact correctly punched during the cryosectioning step. The PCR was carried out in a Veriti 96-well thermal cycler using Oxt gene primers and cDNA of PVN and SON regions of Gtf2ird2 mouse model. Table-5.1 shows the PCR cycles and temperatures used. The hypothalamus of the brain was used as a positive control

(expresses Oxt) and cerebellum as a negative control (does not express Oxt). PCR master mix was pre-ordered from Promega. All cDNA samples were diluted 1:10 (1µl cDNA+ 9µl water) for the purpose of this step.

Table-5.1: PCR Reaction cycles. Shows the different steps involved in PCR reactions. 35 cycles of final denaturation to elongation allows maximum output of amplicons.

Temperature Step Time Cycles 95°C Denaturation 2mins 1 95°C 30secs 60°C Annealing 30secs 35 72°C Elongation 30secs 72°C 10mins 1 4°C Hold ∞

5.3.11 Real-time quantitative reverse transcriptase polymerase chain reaction (qPCR)

Using existing literature on oxytocin, oxytocin receptor, vasopressin and corticotrophin releasing hormones, primer pairs were designed and optimised prior to the experiments

(Kublaoui et al. 2008; Zhang et al. 2011). Table-5.2 shows the list and sequences of the primers used. Atp5j and Rn18s were used as housekeeping genes because their ubiquitous and similar levels of expression provided a reliable normalisation of test genes.

118 Table-5.2: List of primers used for quantitative PCR of PVN and SON samples from Gtf2ird2 mice. At5pj and Rn18s genes were used as housekeeping to normalise the qPCR results. The primer sequences are given from 5’ to 3’ DNA strand. The accession number can be used to obtain full gene information about these primers from NCBI database.

Gene Forward Primer (5'-3') Reverse Primer (5'-3') Amplico Accession n Size Number (bp)

Atp5j TATTGGCCCAGAGTATCAGCA GGGGTTTGTCGATGACTTCAAA 134 NM_001302213 T .1 (ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F)- House Keeping

Rn18s CAGGATTGACAGATTGATAGC ATCACAGACCTGTTATTGCTC 219 NR_003278

(18S Ribosomal RNA) House Keeping

Oxt TGGCTTACTGGCTCTGACCT AGGCAGGTAGTTCTCCTCCTG 214 NM_011025.4

(Oxytocin)

Oxtr ACGTCAATGCGCCCAAAGAA CGACGACTCAGGACGAAGG 226 NM_001081147 .1 (Oxytocin Receptor)

Crh GAGGCATCCTGAGAGAAGTC GTTAGGGGCGCTCTCTTCTC 81 NM_205769.2 C (Corticotrophi n releasing hormone)

Avp TCGCCAGGATGCTCAACAC TTGGTCCGAAGCAGCGTC 173 NM_009732

(Vasopressin)

Prior to the start of the experiments, qPCR reactions were optimised using 1:2 serial dilutions of known cDNA concentrations (control PVN and SON samples) for every primer pair that had been tested. Seven concentrations of cDNA were run in duplicates (0.78,

1.56, 3.125, 6.25, 12.5, 25 and 50 ng/µl) to get a standard curve so that amplification efficiency values for each primer could be determined.

The results obtained from a qPCR reaction are in the form of cycle threshold (CT) values.

A CT value is the number of cycles at which the fluorescent signal surpasses the threshold at which there is a significant rise in detected signals with respect to the

119 baseline (Life Technologies Real-time PCR handbook). CT is used to measure the initial

DNA copy number as the value is inversely proportional to the initial amount of the target; the higher the CT value the lower the gene expression.

The CT value obtained from the qPCR of serial dilutions of known genomic concentrations was used to calculate the efficiency of the primers. Knowing the assay efficiency is critical to accurate interpretation of data. Efficiency was calculated from the slope of a log-linear phase of an amplification plot. Figure-5.4 shows an example. Efficiency was determined using the following equation: Efficiency = 10(-1/slope) -1. Primer pairs with an efficiency of 90-

110% were chosen for the main reactions.

Figure-5.4: Example of qPCR optimisation steps

120 B.

Figure-5.4: Example of qPCR optimisation steps. Panel A lists the serial dilution concentrations of cDNA used for optimising the qPCR. The primer pairs used in this qPCR targeted Oxt and cDNA used was a PVN from a control sample. The concentration was converted to a log-value for obtaining a log-linear plot. qPCR was run in duplicates and the resulting cycle threshold values CT- 1 and CT-2 represents the results for duplicate 1 and 2 respectively. Panel B shows a linear plot drawn using the mean CT value obtained for the serial dilution. The slope of this plot was used to calculate the amplification efficiency of the Oxt primers.

Real-time PCR was performed using GoTaq qPCR master mix (Promega) according to manufacturer’s instructions. Amplification reactions were performed using an Mx3000P thermal cycler (Stratagene). The cycle profiles were programmed as listed in Table-5.4.

The qPCR reactions were run in triplicates and CT values of test genes obtained (Oxt,

Oxtr, Avp and Crh) were normalised against housekeeping genes (Atp5j and Rn18s). The resulting delta-CT value for each test genes were used to calculate the fold change in expression relative to the expression in wildtypes. The percentage of fold change was used to compare between the three genotypic groups to find any statistically significant differences in expression levels.

121 Table-5.4: Quantitative-PCR reaction cycles. Shows the different steps involved in qPCR reaction once in the thermal cycler. qPCR uses fluorescent probes to detect the intensity of fluorescence once a threshold level is surpassed (cycle threshold, CT value). The level of fluorescence exponentially increases with increasing amplicon numbers until finally saturating the detector of the machine. The results of a qPCR are denoted by the CT value.

Temperature Step Time Cycles 95°C Denaturation 10mins 1 95°C 15secs 60°C Annealing 30secs 40 55°C Elongation 30secs 95°C Final Extension 30secs 1 Data Acquisition ∞

5.4 Results

Gtf2ird2, a family member of the Gtf gene family is thought to be involved in the social symptoms of Williams-Beuren Syndrome (Porter et al. 2012). My research aimed to gain a better understanding of how the gene deletion affected crucial maternal behaviours during pup rearing. The results show that Gtf2ird2 gene deleted mothers (homozygous) are in fact more sociable than mothers with the hemizygous deletion or wildtypes. The hemizygous deletion results in the heterozygous mothers demonstrating an unusual cannibalistic behaviour. Furthermore I show that the hyper sociability in the homozygous mothers is in fact due to upregulation of important neuropeptides involved in socio- emotional behaviours in the mouse model.

5.4.1 Nest Building Study All females from the three genotypic groups made good quality nests (Figure-5.5); any differences in scores were not significant according to generalised linear model (Wald Chi- square=0.14, p=0.712). The model was such that the dependent variable was the nest score and the covariate was genotype. The differences in nest scores between each genotype were not significant.

122

Wt Het Homo

Figure-5.5: Gtf2ird2 gene is not a determinant of nest building ability and fine-motor skills. Panel A shows an example of the nests built by the three Gtf2ird2 genotypic models; wildtype (Wt), heterozygous (Het) and homozygous (Homo). All three nests shown were scored 5. Panel B shows a bar-chart of genotype vs nest score. Almost all the nests scored a good 4. The small differences are not significant (Wald Chi-square=0.136, p=0.712).

5.4.2 Behavioural study

Analysis of behavioural data showed that deletion of Gtf2ird2 gene affects the most crucial maternal behaviour, suckling (Figure-5.6). On average homozygous mothers suckled their pups a significantly higher number of times compared to both heterozygous and wildtype mothers (GLM; F2,30=5.15; p=0.01). Overall maternal behaviour was also

123 significantly elevated in homozygous mothers (GLM; F2,36=7.62; p=0.002). Maternal behaviour was calculated as mean sums of nursing and suckling scores on days 6, 10 and 14. Along with genotype, number of pups in a litter also affected suckling (GLM;

F2,30=6.90; p=0.01). This was seen on postnatal days 10 and 14 (GLM; F2,30=7.11; p=0.01)

(Figure-5.6). Litter size also affected maternal behaviour licking (GLM; F2,32=18.73; p<0.001) and pups play fighting behaviour (GLM; F2,32=5.59; p=0.02), however these phenotypes were not directly regulated by underlying gene deletion. All other behavioural measures including ‘sniffing’ (GLM; F2,33=0.36; p=0.70), ‘licking’ (GLM; F2,33=0.166; p=0.85), ‘feeding/drinking’ (GLM; F2,32=1.93; p=0.16) and ‘nest building’ (GLM; F2,33=1.36; p=0.270) (Figure-5.6) were not affected by underlying gene deletion.

Maternal behaviours were studied on postnatal Days 6, 10 and 14. Figure-5.6 shows the mean behaviours where significant differences were seen on all three days between the genotypic groups. Day specific significant data are also shown in this section. All other data are shown in Appendix-5 (page 240).

A. Suckling and maternal behaviour

124 B. Sniffing and licking

C. Feeding/drinking and nest building

Figure-5.6: Gtf2ird2 deletion enhances maternal behaviours which are partly controlled by the litter size. Panel A shows the effect of Gtf2ird2 gene deletion on suckling and maternal behaviours respectively. Homozygous mothers suckled their pups and demonstrated significantly higher maternal behaviours compared to heterozygous and wildtype mothers. Panel B and C shows that underlying gene deletion did not affect maternal sniffing, licking, nest building or feeding behaviours.

125 5.4.3 Litter Growth

Litters were weighed on postnatal days 1, 6, 10, 14 and 21 to monitor growth. Day 10 onwards litters of homozygous and heterozygous mothers weighed significantly more than litters of wildtype mothers (Homozygous: GLM; F2,32=3.24; p=0.05; heterozygous: GLM;

F2,32=3.24; p=0.03) (Figure-5.7). This correlates with increased suckling behaviours seen in the homozygous mothers. Differences in birth weight and litter sizes were statistically controlled.

Figure-5.7: Monitoring growth of Gtf2ird2 homozygous and wildtype litters. Litters of Gtf2ird2 homozygous and heterozygous mothers weigh significantly more than litters of wildtype mothers from postnatal day 10.

5.4.4 Pup Retrieval Test

In order to identify whether the mutation affected pup retrieval the following measures were recorded: the number of pups retrieved, the number of times the mother dropped the pups during the retrieval and also the mode of retrieval that is whether they carried the pups via the scruff, leg, head or tail. By finding the latency of retrieval, maternal responsivity was additionally deciphered by calculating the time taken to start retrieving the first pup.

126 All mothers were capable of retrieving every single pup and swiftly gathering them back to the middle of the nest. No ‘unusual’ method of retrieval was observed and no significant differences were found in latency of retrieval (GLM; F2,33=1.10; p=0.345) (Figure-5.8).

Mode of pup retrieval seemed uniform amongst the three genotypic groups. The mothers either grabbed the scruff of the pups with their mouth, or dragged the pups via the legs or even by the tip of their head/ears. However, during retrieval the homozygous mothers were observed to drop their pups significantly more often than either wildtype or heterozygous mothers. GLM showed that this result is significant (GLM; F2,32=4.27; p=0.017) only for day 10 (Figure-5.8).

Figure-5.8: Pup retrieval in Gtf2ird2 mouse model On postnatal day 10 homozygous mothers dropped the pups, during pup retrieval, significantly high number of times compared to heterozygous and wildtype mothers. However, on all three days homozygous, heterozygous and wildtype mothers initiated the retrieval process at similar times.

5.4.5 Filial cannibalistic behaviour

When the breeding cages were set up and pregnant females were separated, around two to three days after the birth of the pups a significant number of mothers were found to be cannibalistic towards their entire litter, irrespective of litter size. When the number of mothers was counted and matched with their respective genotype five out of 12 (41%)

127 heterozygous mothers were found to be cannibalistic compared to only one out of 12 (7%) wildtype and homozygous mothers (GLM; F2,32=6.37; p=0.005) (Figure-5.9).

Figure-5.9: Hemizygous Gtf2ird2 deletion makes heterozygous mothers more prone to filial cannibalism. 41% of the heterozygous mothers were cannibalistic towards their whole litter (irrespective of litter size) whilst only 7% wildtype mothers and 6% homozygous mothers showed significant infanticidal behaviour.

5.4.6 Histology for cryosectioning the right brain region

The histology test was done to ensure that PVN and SON regions were correctly identified and extracted. Cresyl Violet stains the PVN and SON dark violet which are then identified following the characteristic shape. ‘The Mouse Brain in Stereotaxic Coordinates’ by

Paxinos and Franklin (2001) was used as reference (Plate 37). Figure-5.10 shows examples of stained brain sections from wildtype, heterozygous and homozygous brains of Gtf2ird2 mouse model.

128

Wildtype Gtf2ird2 mouse brain section Heterozygous Gtf2ird2 mouse brain section

Homozygous Gtf2ird2 mouse brain section Example of brain section with no PVN/SON staining

Figure-5.10: Brain sections of Gtf2ird2 mouse model, stained in Cresyl Violet dye. The Figure shows examples of brain sections from each of the three genotypic groups. Brain sections were picked up on microscope slices and stained using basic histology technique and viewed under a light microscope. Cresyl violet stains the PVN and visualises the characteristic ‘palm tree’ like shape and the two bulges of developing SON at the bottom. The fourth slide on the bottom shows an example of cases where PVN/SON did not appear as obvious and resulted in mis-extraction of tissue; these samples were excluded from the study.

5.4.7 PCR PCR was carried out prior to the qPCR step and the products were run on an electrophoresis gel. Oxt gene primers were used on PVN cDNA samples from all genotypic groups. Figure-5.11 shows electrophoresis gel images of the PCR products.

Where no bands were found, the sample identification numbers were noted and used to

129 cross-check with the histology slide images to confirm whether the brain regions were rightly extracted. Also the PCRs gave an idea of whether the cDNA extractions worked.

Figure-5.11: PCR products of PVN and SON samples of Gtf2ird2 gene ran with Oxt and Atp5j primers. Electrophoresis gel with PCR products of PVN samples show that Oxt was present in the cDNA of samples where a band was seen at 214bp. The lanes with no bands were indicative of a cryosectioning error. The PCR with housekeeping gene Atp5j was used a control.

5.4.8 Gene Expression via qPCR qPCR results were used to deduce the expression of Oxt, Oxtr, Crh and Avp genes in

Gtf2ird2 mouse brain samples. The percentage expression was measured relative to wildtype samples. Samples with an abnormally high CT value/low gene expression were cross-checked with expression in histology test and PCR and were not considered in the statistical analysis. Gtf2ird2 deletion significantly elevates Oxt expression in PVN whilst hemizygous deletion of the gene significantly reduces the expression (GLM; F2,21=3.48; p=0.03). Conversely Oxtr expression was seen to be significantly lower in Gtf2ird2 homozygous PVN samples compared to wildtypes (GLM; F2,21=4.51; p=0.023) (Figure-

5.12). Although a similar trend was found in the expression levels in SON, this was not significantly different (Oxt: GLM; F2,21=1.25; p=0.448; Oxtr: GLM; F2,21=2.18; p=0.43). Crh

(GLM; F2,21=2.2; p=0.70; SON: GLM; F2,21=2.20; p=0.57) and Avp (PVN: GLM; F2,21=2.18;

130 p=0.83; SON: GLM; F2,21= 2.26; p=0.33) expression did not vary significantly between the three groups.

A. Oxt

B. Oxtr

C. Crh and Avp

131

Figure-5.12: Expression of social neuropeptides in Gtf2ird2 mouse brain regions. Mean ± SEM relative percentage expression of Oxt (A), Oxtr (B), Crh and Avp (C) mRNA in the PVN and SON regions of wildtype (Wt- Green), heterozygous (Het- Orange) and homozygous (Homo- Red) mice. * indicates a significant difference (p≤0.05) between the three genotypes.

132 5.5 Discussion GTF2IRD2 the least studied gene among the Gtf gene family, influences executive function and social cognition in people with Williams-Beuren Syndrome (Porter et al.

2012). This is the first research that studied the Gtf2ird2 gene in isolation and its role in impairing maternal social behaviour in an animal model. A single gene knockout mouse model of the disorder was created in our lab (Tassabehji unpublished) to which I carried out an array of studies in order to investigate the role played by the gene deletion on maternal behaviours (Adapted from Hager and Johnstone 2003), basic fine motor skill efficiency (Schenieder et al. 2012) and expression of neuroendocrine hormones (adapted from Murgatryod and Nephew 2013).

5.5.1 Fine-motor skill Fine-motor skill was tested by scoring the quality of nests built; a protocol adapted from

Deacon (2006). The same protocol was used by our group previously to monitor fine motor skills in a Gtf2ird1 mouse model (Schneider et al. 2012). Gtf2ird1 mouse models showed significant deficits in motor coordination when unshredded nesting material was provided along with follow up study with shredded nesting material (Schneider et al.

2012). When the test was used in my study for the Gtf2ird2 model, with unshredded nestlet only, the results showed that all three genotypes could build good nests and no significant differences for the three genotypic groups were obtained confirming that

Gtf2ird2 does not affect fine-motor skills.

5.5.2 Behaviour Suckling is a crucial maternal behaviour. During early days, between postnatal days 1 to

14, suckling mother’s milk is the only source of food for the pups and thus the most important means of survival. My study has established that Gtf2ird2 deletion significantly affects suckling; homozygous mothers suckled the pups the most whilst wildtype mothers suckled the pups the least. Overall maternal behaviour was calculated as the mean sums

133 of suckling and nursing scores on postnatal days 6, 10 and 14. Gtf2ird2 homozygous mothers exhibited significantly higher maternal behaviour compared to both the heterozygous and wildtype mothers. This result is in agreement with the overtly sociable demeanour observed in WBS indivduals (Mervis et al. 2000; Pober 2010; Weisman et al.

2015) as suckling is a means of bonding and like the WBS indivduals the homozygous mothers in my study spent most time bonding with the pups and nurturing them significantly more than the wildtype mothers. The results obtained for suckling showed significance only for the first set of observation which implies that after 4 hours of separation from the pups, the homozygous mothers were socially deprived and thus tend to be more attentive towards their litters once reunited with them. This upregulated suckling behaviour resulted in the litters of homozygous mothers weighing significantly more than litters of wildtype mothers.

Litter size is an important factor that influences maternal behaviours in mice (Mendl 1988;

Chiang et al. 2002). Whilst some studies have shown that mothers rearing smaller litters

(2 to 5 pups) spend more time in maternal care than those with larger litters (8 or more pups) who spent more time eating and drinking (Priestnall 1972), other studies have shown that maternal care is influenced more by the genetic differences in mothers rather than litter size (Chiang et al. 2002). This study has established that maternal behaviour is influenced by both the underlying genetic makeup of the mice and the litter size, but some behaviours are influenced only due to the litter size. Suckling for instance was affected by both the genotype of the mother and the number of pups she had (p=0.013). When the size of all the litters were compared to overall suckling behaviour of mothers, irrespective of maternal genotype, a line of best fit with a positive gradient was obtained showing that there is a positive correlation between the number of pups in a litter and the time the mother spent suckling. This further affirms the hyper sociable nature of homozygous mothers as despite of litter size they were significantly maternal and more responsive towards their pups. Litter size also affected time spent licking (p<0.001) which was not

134 directly affected by maternal genotype. Pups in a bigger litter also experienced more social play, play fight (p=0.024), compared to pups in a smaller and/or single offspring litter. However, maternal genotype did not directly affect this play fighting behaviour in offspring.

Differences between the three genotypic groups were not generally significant for time spent retrieving pups, nest building, resting alone, eating and drinking, grooming, and doing other activities. During pup retrieval, even though all females were capable of following a perfectly ‘normal’ pattern of retrieval and efficiently gathered the scattered pups back to the nest, on day 10 only, homozygous mothers dropped the pups most frequently during the process compared to wildtype or heterozygous mothers. This might be due to the increased weights of the pups as a result of increased suckling on day 10 thus making it harder for the mothers to retrieve the pups on day 10 compared to day 6.

Moreover, behavioural observations suggest that the results of pup retrieval for day 14 should not be taken in to consideration as the pups start having clearer vision by day 14 and become mobile and walk back to the nest on their own. The pup retrieval result for day 10 in the homozygous mice might be explained by physical constraints, such as subtle craniofacial abnormalities, that the deletion causes to the mice; however I cannot be certain as the results were significant for day 10 only and no craniofacial or skull measurements were taken. Although the Gtf2ird2 KO mice did not have any visible craniofacial abnormality in skull and jaw as observed in the Gtf2ird1 homozygous mice

(Figure-1.6, page 39) (Tassabehji et al. 2005; Schneider et al. 2012) it would be worth investigating this further in the future to see if Gtf2ird2, like Gtf2ird1 and Gtf2i (Tassabehji et al. 2005; Enkhmandakh et al. 2009; Pober 2010) could be a genetic determinant of mammalian craniofacial development.

5.5.3 Filial Cannibalism Besides taking care of the offspring and providing them with food, shelter and warmth, mice are reported to be infanticidal under certain circumstances (Weber and Olsson

135 2008). Whilst the majority of mice are infanticidal towards unrelated pups (McCarthy 1990) female mice sometimes kill own offspring as a response to stress or to reduce the litter size when food is restricted (Elwood et al. 1990). Wild mice have been reported to be more likely to exhibit infanticide (Jakubowski and Terkel 1982; McCarthy and Saal 1985).

During our study when the breeding cages were set and pregnant females were separated, it was observed that around two days after the birth of the pups, 41% of the heterozygous mothers were cannibalistic towards their entire litter; each litter had on average five to six pups. Compared to the heterozygous mothers, only 5% wildtype and

6% homozygous mothers were cannibalistic towards their entire litters. As there was no scarcity of food provided in the lab and as the cannibalistic mothers killed the entire litter rather than a few pups, it can be assumed that the cannibalistic behaviour was a response to stress. It was ensured at all times during breeding to the end of the study that all animals are handled exactly the same way (Animal Maintenance, Page 53); just before birth to six days after birth of litter the cages were not cleaned to prevent any disturbances. Even though the exact source of stress is unknown, it can be assumed that handling the pups and mother for weighing regularly and separating them from each other for four hours might be the likely cause as this phenotype has not been seen in previous breeding experiments on this model (Tassabehji personal communication). From the behavioural studies I found that Gtf2ird2 homozygous mothers suckled their pups the most compared to heterozygous and wildtype mice. This increased nurturing tendency could be due to the heightened Oxt expression (172% expression relative to wildtype), which dampened any stress related behaviours. This might be a possible reason as to why they did not exhibit significant infanticidal behaviour whilst the heterozygous females did.

5.5.4 Growth Weight of the litters was recorded from the day they were born (D1) and thereafter on day

6, 10, 14 and 21. Unlike Gtf2ird1 homozygous mice (Tassabehji et al. 2005), Gtf2ird2

136 homozygous pups had similar weight and size to that of wildtypes. However, this model showed a significantly increased body weight in litters of homozygous Gtf2ird2 mice compared to litters of wildtypes, since postnatal day 6. This observation correlates with increased suckling behaviour of homozygous Gtf2ird2 mothers.

5.5.5 Neuroendocrinology The neuroendocrine data support the hypothesis that Gtf2ird2 gene deletion elevates maternal behaviour through upregulation of Oxt in the PVN. Oxt plays key roles in mediating social behaviours (Donaldson and Young 2008) especially maternal care behaviour (Murgatroyd and Nephew 2013) in a number of species. The significance of Oxt in maternal care behaviour regulation was first established in the early 80's through a series of intravenously administered oxytocin and oxytocin antagonists (Pedersen et al.

1994; Pedersen and Prange 1979).

My study found a significant upregulation of Oxt mRNA levels in the PVN and SON of

Gtf2ird2 knockout mice. Oxt is primarily expressed in the magnocellular neurones of hypothalamic PVN and SON (Gimpl and Fahrenholz 2001). The upregulation of Oxt in this mouse model indicates the potential role of the Gtf2ird2 gene in mediating social symptoms of Williams-Beuren Syndrome. In fact Oxt has been found to be dysregulated in

WBS indivduals (Dai et al. 2012). The study by Dai et al. (2012) not only measured significantly high median levels of oxytocin in WBS versus controls but also saw a less marked increase in vasopressin. Avp and Oxt are on the same chromosomal locus and are both involved in affecting social phenotypes (Bosch and Neumann 2010). Although a significant Oxt expression was observed in the PVN of Gtf2ird2 homozygous mice, no differences were seen for Avp mRNA expression (Figure-5.12). My results thereby indicate the involvement of the Gtf2ird2 gene in mediating Oxt levels in WBS indivduals.

Oxtr expression was predicted to be upregulated along with Oxt expression (Murakami et al. 2011; Murgatroyd and Nephew 2013; Rich et al. 2014) in the PVN and SON. However,

137 a significantly reduced level of Oxtr mRNA was seen in the PVN samples of homozygous

Gtf2ird2 mice and no differences in Oxtr expression levels were observed in the SON.

Oxtr mRNA has been previously found to be down-regulated in human myometrial cells exposed to high levels of Oxt for 48 hours (Phaneuf et al. 1997). Gtf2ird2 mother mice in my study gave birth to litters around 48 to 72 hours prior to tissue collection and were all subject to suckling their litters at end point; both factors significantly increasing basal Oxt levels (Gimpl and Fahrenholz 2001; Nagasawa et al. 2012). My analysis show that

Gtf2ird2 gene deletion further elevates this Oxt level thus I propose that there might be an indication of a transcriptional suppression and/or desensitisation mechanism which results in a lower expression of Oxtr mRNA in the PVN of these homozygotes. In the PVN oxytocin is synthesised and is released via exocytosis in to nerve terminals of posterior pituitary gland (Viero et al. 2010). Labour and lactation are key stimulators of oxytocin release (Neumann et al. 1993) which then binds to oxytocin receptors and triggers an increase in intracellular calcium ion levels (Dayanithi et al. 2000). Research on myometrium has provided evidence of oxytocin receptor desensitisation from prolonged period of upregulated oxytocin levels (Adachi and Oku 1995; Phaneuf et al. 1997;

Phaneuf et al. 1998; Phaneuf et al. 2000; Robinson et al. 2003). The characteristic features of this desensitisation include reduced oxytocin-regulated trigger of phospholipase C–calcium pathway and significant down-regulation of the mRNA encoding oxytocin receptor (Phaneuf et al. 1994; Phaneuf et al. 1998). Studies on cultured human myocytes have found an oxytocin induced reduction in oxytocin receptor protein (Adachi and Oku 1995) but no change was found in oxytocin receptor protein levels in myometrium cells (Phaneuf et al. 1997). Thus, from these studies the Oxtr mRNA down- regulation in the homozygous Gtf2ird2 brain tissue samples are indicative of an oxytocin- induced desensitisation; further study investigating binding profile of oxytocin to the receptor and measuring the level of oxytocin receptor protein will help confirm this hypothesis. Studies on Oxtr have also found an effect of gonadal steroids and cytokines

138 (Soloff and Fernstorm 1987; Insel et al. 1993; Klein and Fahrenholz 1994; Gimpl and

Fahrenholz 2001) on Oxtr expression. Further research investigating the expression of these proteins in the Gtf2ird2 mouse model would be required to elucidate this.

Comprehensive studies of rodent models have shown over the years that Oxt not only mediates maternal care behaviour (Champagne et al. 2001) but also maternal aggression profiles (Bosch et al. 2004; Raugnath et al. 2005; Bosch and Neumann 2011). In fact infanticidal behaviours in Oxt knockout models are commonly observed (Raugnath et al.

2005). I found a significantly reduced level of Oxt in my Gtf2ird2 heterozygous samples compared to homozygous samples (not wildtypes). This might hence be indicative of an increased level of stress causing infanticidal behaviours in these mothers which were also found to be cannibalistic to their whole litters (Figure-5.9). From the results of my studies I hypothesise that partial deletion of Gtf2ird2 may mimic similar cannibalistic behaviours as seen in Oxt knockout models (Raugnath et al. 2005). Full deletion of both copies of

Gtf2ird2 induces hyper social behaviours in individuals. These social mice therefore were not affected by the frequent disturbances such as regular weight measures and separation from pups.

Corticotrophin releasing hormone (Crh), which increases in response to stress, inhibits maternal care in rats (Pedersen et al. 1991) and primates (Saltzman et al. 2011) and suppresses maternal aggression in mice (Gammie et al. 2004). To clarify whether dysregulated Crh levels might be a potential cause of the stress behaviours in these heterozygous mothers I further measured the mRNA expression levels of Crh mRNA in

PVN and SON in all three genotypic mice. Although no significant differences were found, hemizygous Gtf2ird2 gene deletion might be affecting other stress signalling pathways.

5.6 Conclusions

Gtf2ird2, a family member of the Gtf2i and Gtf2ird1 transcription factor genes is involved in cognition and behaviour in WBS indivduals (Porter et al. 2012). Due to the low

139 prevalence of WBS indivduals with a bigger deletion, which includes Gtf2ird2, very few studies to date have investigated the functions of the gene (Palmer et al. 2012; Porter et al. 2012). My study has identified the role of Gtf2ird2 in important social scenarios and provides the first evidence of maternal studies on a single-gene knockout mouse model of

Gtf2ird2. Unlike Gtf2ird1 homozygous mice, although there are no apparent physical differences between the wildtype and homozygous Gtf2ird2 mice, a significant difference was seen for important social behaviours. Gtf2ird2 knockout mothers bonded with their litters significantly more than wildtype and heterozygous mothers during the behavioural observations. The four hour separation protocol made these hyper-social homozygous mice socially deprived thus at reunion with pups they suckled and nurtured them significantly higher than either wildtype or heterozygous mothers. This hyper sociability and enhanced empathising behaviour is in line with social symptoms of WBS indivduals

(Morris et al. 2006). My study has further found the neuroendocrine reason behind this highly social behaviour; Gtf2ird2 gene knockout elevated oxytocin expression in the PVN region of the brains. This research thus provides a comprehensive understanding of how the Gtf2ird2 gene may be involved in social symptoms of WBS. The study also observed an infanticidal profile in heterozygous female mice, reminiscent of oxytocin knockout mice, thus further highlighting the importance of studying the role of this gene in social phenotypes.

140

6. Indirect genetic effects contribute to maternal and pup weight traits during early development

Weight data collection for this study was carried out by S. Lyst and QTL mapping was conducted by N. Sharmin.

141 6.1 Abstract

Indirect genetic effects (IGEs) are defined by the influence of genes expressed in one individual on the phenotypes of an interacting individual. IGEs may make an important contribution to phenotypic traits. Previously, they were ignored by being referred to as environmental effects but ongoing research on trait selection and evolution has highlighted its importance as an additional component of the environment. There is still a lack of empirical evidence of IGEs on developmental traits. Here I implemented a quantitative genetics approach to investigate the IGEs of variable BXD genotype on weight data of B6 mothers and offspring. I mapped quantitative trait loci (QTL) responsible for the variation in C57BI/6J (B6) phenotype as a function of the BXD genotype using the webportal www.genenetwork.org. My study has identified three significant QTL on BXD litter genotype influencing weight changes in B6 mothers during provisioning. Multiple suggestive QTL were identified on BXD maternal genotype affecting B6 litter growth. The candidate genes identified in these QTL are involved in important cellular regulatory functions and weight traits. This study provides evidence of IGEs on weight traits in mice and highlights the importance of investigating IGEs further.

142 6.2 Introduction

An understanding of evolutionary and ecological processes, for example response of traits to selection, requires a clear understanding of the genotype to phenotype relationship for complex traits (Mutic and Wolf 2007). When studying the genotype-phenotype relationship, the established paradigm has been to directly map an individual’s genotype to its own phenotype (Falconer and Mackay 1996). These effects are thus referred to as

‘direct genetic effects’. However, in populations of interacting individuals genes expressed in a focal individual can have its phenotypic effects in other individuals. These effects are referred to as ‘indirect genetic effects’ (IGEs) (Wolf et al. 1998). The presence of IGEs modifies the definition of genetic architecture which is the relationship between the genotype and the phenotype thus complicating evolutionary genetics. Hence in order to have a clear understanding of the genetic architecture and their contribution to evolutionarily significant genetic variation it is important to obtain an understanding of

IGEs (Mutic and Wolf 2007).

Historically most research on IGE has concentrated on maternal effects, that is, how the environments provided by mothers affect the offspring; although, IGEs can result from interaction with relatives or even unrelated individuals as well (Wolf et al. 1998). Maternal effects result in IGEs whenever the traits that contribute to the environment provided by the mother are heritable. Studies of maternal effects have shown that the maternal genotype often accounts for as much as fifty-percent or even more of the variance in characters expressed early in life (Wolf et al. 1998). Comparatively, the variance accounted for by the direct effect of the offspring’s own genotype varies between ten to fifty percent (Mousseau and Fox 1999). Thus, for many early developmental characteristics, the phenotype of a focal individual can be affected by maternal effects to a large degree; this has been shown by a number of evolutionary models of maternal effects

(Cheverud and Moore 1994; Wolf et al. 1998; Hager et al. 2008).

143 This study focussed on quantifying IGEs by analysing weight data (General Methods,

Page 58) collected from recombinant inbred BXD mouse system and B6 line during early development of pups. The BXD system is the largest reference panel in mammals with over five million separating single nucleotide polymorphisms (SNPs) allowing analysis of complex systems genetics models (Wolf and Brodie 1998). Recombinant inbred sets are named by joining the abbreviation of each parental strain with an ‘X’. Thus, strains derived from crossing a B6 female and a DBA/2J male are members of the BXD set. The B6 line is genetically identical; hence by mapping phenotypic traits of B6 mice as a function of

BXD genotype, IGE can be established.

Quantitative trait loci (QTL) are regions in genomes that account for the variation in quantitative traits. QTL mapping is a statistical method that links phenotypic data to genotypic data in order to explain how variation in complex traits arises due to underlying genetic basis (Kearsey 1998). Through mapping these QTL, I aimed to test the hypothesis that IGEs differentially affect traits in a focal individual. A previous study in our lab, where whole BXD litters were cross-fostered with B6 litters, has identified two loci in BXD maternal genotype that affected growth of B6 pups during the first week of life (Gini 2014).

To further analyse this effect, in this study through creating mixed litters of BXD and B6 genotypes and cross-fostering B6 pups with BXD mother and vice versa, the aim was to identify the candidate QTL, responsible for variation in growth of the B6 pups.

Furthermore, by mapping weight changes in B6 mothers as a function of BXD litter genotype I investigated the IGEs of litter genotype on maternal provisioning.

6.3 Methods

The aim of this study was to computationally analyse IGEs through identifying candidate

QTL, causing variation in phenotype of B6 pups reared by BXD mother. Using QTL mapping tools, identification of QTL will establish IGEs of BXD genotype on the weight traits of B6 mice.

144 32 BXD lines were taken and reciprocally mixed with B6 pups to form mixed litters such that each consisted of some BXD pups which are genetically variable and some genetically uniform B6 pups (Figure-6.1). Cross-fostering litters resulted in the B6 pups in the mixed litters being nursed by BXD mothers and the BXD pups being nursed by the B6 mothers. This allowed any genetic variation in the BXD lines on the B6 traits to be measured. Litters of BXD and B6 mothers were cross-fostered within 24 hours of birth.

Weight of the litters was recorded prior to cross-fostering after which weights of both the mothers and pups were recorded on postnatal days 6, 10, 14 and 21. On days 6, 10 and

14 mothers were separated from the pups for four hours and then re-joined for two hours

(Hager and Johnstone 2003). Short term weight changes during the separation and reunion were recorded on these days.

Figure-6.1: Experimental design. Different BXD strains and B6 mothers adopt cross-fostered mixed litters of BXD and B6 offspring. BXD strains are represented by different shades of grey and B6 is represented by black colour. A BXD female and B6 female nursed mixed litters of respective BXD strain and B6 pups.

145 6.3.1 QTL Mapping

My aim was to find out whether any variation in the B6 phenotype was due to the underlying variation in the BXD genotype. Thus I controlled for any other factors that could affect the B6 phenotype; this was done by calculating the residuals through GLMs on

SPSS.

Using the webportal www.genenetwork.org, variation in B6 traits was mapped as a function of the corresponding BXD genotype. Genenetwork uses a specific interface in which the data from a given trait can be entered, and a linkage between differences in traits and genetic markers, called likelihood ratio statistic (LRS), can be calculated as a function of markers across the genome (Wang and Yeh 2003). The LRS will be high if there is a large difference in the phenotype for example between mice with a B versus D allele at a given chromosomal locus. Genenetwork also carries out significance testing of the QTL which gives a measure of whether the QTL is significant or suggestive for that specific trait with respect to the significant and suggestive thresholds (Figure-6.2). The significant threshold is an estimated LRS value that conforms to a probability of five percent rejecting the null hypothesis that there is no linkage anywhere in the genome; the suggestive threshold is set to a probability of 63 percent of falsely rejecting the null hypothesis. These thresholds are computed by evaluating the distribution of the highest

LRS scores generated by a set of 2000 random permutations of line means. A peak or high LRS on a specific chromosome for my data located the QTL.

Genenetwork uses a number of mapping tools for the purpose of mapping potential QTL. I used interval mapping in genenetwork to investigate the existence of QTL causing variation in specific traits (growth of pups, weight at weaning). Interval mapping uses an estimated genetic map as the framework for the location of QTL (Figure-6.2). The intervals that are defined by ordered pairs of markers are searched in increments and statistical methods, such as linear regression, are used to test whether a QTL is likely to be present at the location within the interval or not. Interval mapping, as defined by Lander

146 and Botstein (1989), statistically tests for a single QTL at each increment across the ordered markers in the genome (Lander and Botstein 1989). The results of the tests are expressed as LRS scores, which compare the evaluation of the likelihood function under the null hypothesis (no QTL) with the alternative hypothesis (QTL at the testing position) for the purpose of locating probable QTL. Figure-6.2 shows an example of interval mapping output.

7 1

3 2

5 4

Figure-6.2: Example of an interval mapping output. The map is an output for a trait on chromosome 1 of maternal genome. The line 1 shows the significant LRS score and line 2 shows suggestive LRS score, calculated by permutation tests. The blue plot labelled 3 represents the LRS score obtained for the trait being investigated. The green and red plots labelled 4 and 5 represent the additive effect of loci, with red meaning the B6 allele increases the trait value at that location, and green meaning the DBA allele increases the trait value. The scale in green on the right shows the trait value due to DBA allele or B6 allele. The orange seismograph labelled 6 indicates the density of markers in the BXD set. The plot labelled 7 represents the known genes corresponding in this location of the chromosome.

6.3.2 Identification of Candidate Genes

The QTL mapped were recorded as either significant or suggestive; suggestive QTL approximately 50 percent higher than suggestive threshold were taken in to consideration.

Specific genes related to the trait under investigation were identified by using QTLminer

(http://www.genenetwork.org/webqtl/main.py?FormID=qtlminer) and mouse genome informatics (MGI) (http://www.informatics.jax.org/allele). QTLminer creates a list of genes using QTL interval as an input (Alberts and Schughart 2010). The chromosome number

147 and start and end positions of the QTL (in megabases) are entered in the program which then forms a list of all the genes within the interval along with information about the genes such as name, description, genomic position and pathways in which the genes are involved (Alberts and Schughart 2010). Furthermore, QTLminer helped in highlighting the genes with non-synonymous SNPs, deletions or insertions in adipose tissue, muscle or liver. These tissues were selected due to their relevance to the weight and growth data under investigation. The genes prioritised by QTLminer were then cross-checked against the MGI database resources. Like QTLminer, MGI creates a list of genes using inputs on chromosomal location of the QTL. But they do so by searching against their international database resources on laboratory mice and integrate the information with biological and genetic data (Bult et al. 2010). Thus the results obtained from MGI not only highlighted the name and description of the genes but also information on published research on gene expression studies (Bult et al. 2010). In summary QTL positions were logged in to

‘Phenotypes, alleles and Diseases Query’ in MGI which gave a list of all the relevant genes and results of knockout studies on them. Among the list of genes were the ones recorded from QTLminer. Thus by comparing the outcome of QTLminer and MGI the genes relevant to the traits were identified as candidate genes.

148 6.4 Results

In this study I investigated the IGEs of BXD maternal and litter genotype on respective B6 litter and mother’s traits. The traits that I was interested in were growth of litters and subsequent weight changes in mothers during early development. I particularly investigated weekly weight gain in B6 pups reared by BXD mothers from postnatal days 1 to 21. I also studied short term weight changes in B6 pups on days 6, 10 and 14. These measures helped to understand IGEs of genes expressed in BXD mothers and siblings on growth of B6 pups and weight changes in B6 mothers. For monitoring effects of BXD litter genotype on B6 maternal phenotype, I mainly studied weekly changes in B6 mother’s weight and short term weight changes when rearing mixed litters of BXD and B6 pups.

Using GLM in SPSS I found significant effects of litter size (GLM; F1,64=5.53; p=0.022), number of B6 and BXD pups, and weight of BXD mother (GLM; F1,64=5.06; p=0.028) on the weekly growth of B6 pups. The residuals obtained from these models were used to map QTL in BXD mother’s genotype that controlled weekly weight gain in B6 pups.

Similarly, effects of litter size (GLM; F1,64=0.50; p=0.427), BXD foster mother’s weight

(GLM; F1,64=0.64; p=0.527) and B6 pup’s initial weight (GLM; F1,64=0.02; p=0.899) on short term weight changes of B6 pups was measured and although no significant effects were found the raw data was directly mapped in genenetwork (www.genenetwork.org).

The main covariates controlled for in measures of weight changes in B6 mothers was food consumption (GLM; F1,64=3.950; p=0.02), initial weight (GLM; F1,64=8.56; p=0.05) and litter size (GLM; F1,64=6.98; p=0.011).

When residuals of these models were used to map IGEs, I found no QTL for weekly growth of B6 pups (Figure-6.3). A suggestive QTL was found for short term weight changes of B6 pups on day 14 and three significant QTL was found for weight changes of

B6 mothers as a function of BXD litter genotype.

149

Figure-6.3: Interval mapping output of weekly growth (P1-6) of B6 pups reared by BXD mother. The interval map output shows an example of the maps obtained for weekly growth of B6 pups reared by BXD mother with BXD siblings. The map is an output of week 1 (day 1-6). No QTL reached the significant threshold (LRS score 17.16) or suggestive threshold (LRS score 10.73).

Short term weight changes, in B6 pups, on day 14 was influenced by a suggestive QTL in

BXD mother’s chromosome number 18 (Figure-6.4). This QTL consists of three candidate genes which not only showed high numbers of SNPs between BXD strains in the interval maps but also directly relate to the trait. Sap130, Bin1, and Stard4 genes show high expression in adipose and muscle tissues in QTL miner and when investigated further using MGI, studies on these genes revealed a direct relation to body weight and feeding behaviours (Muller et al. 2003; Riegelhaupt et al. 2010).

Figure-6.4: Interval mapping of short term weight changes in B6 pups on day 14 The interval mapping output for the trait, short term weight changes in B6 pups on day 14 identified one suggestive QTL, on chromosome 18, shown by the arrow; LRS score 17. The threshold for suggestive LRS was 11.08 and significant LRS was 18.46. The suggestive QTL on chromosome 17 was not considered as it did not exceed the suggestive threshold by more than fifty percent.

150 Weekly weight change in B6 mothers between postnatal day 1 and 6 was controlled by two significant QTL on BXD litter genotype; one on BXD chromosome six and the other on

13. Both of these QTL surpassed the significant LRS threshold (16.44) (Figure-6.5) and consist of one good candidate gene each. A QTL on chromosome 6, Creb3l2, also known as BBF2H7 encodes a transcriptional factor involved in endoplasmic reticulum stress response (Saito et al. 2009) and Hrh2 locus on chromosome 13 encodes a histamine receptor protein (Nomura et al. 2013).

Figure-6.5: Interval mapping of B6 maternal weekly weight change (P 1-6) The interval mapping output for the trait, weekly weight change in B6 mother between postnatal day 1 and 6 identified two significant QTL, on chromosome 6 (LRS score 17.2) and 13 (LRS score 20.4), shown by the arrows. The threshold for suggestive LRS was 10.51 and significant LRS was 16.44.

BXD litter genotype also had a significant QTL on chromosome nine that affected short term weight change of B6 mothers on postnatal day 6 (Figure-6.5). This locus has two candidate genes both directly relevant to the trait; Col5a3 gene encodes Collagen V protein which is highly expressed in the adipose tissue (Huang et al. 2011) and Dnmt1 gene which encodes the enzyme DNA methyltransferase (Gaudet et al. 2003). A suggestive QTL on BXD chromosome eight was also found for this trait (Figure-6.6) however, QTL miner and MGI databases did not identify any relevant candidate genes in this region.

151

Figure-6.6: Interval mapping output of B6 maternal short term weight change on day 6 The interval mapping output for the trait, short term weight change in B6 mother on postnatal day six identified one significant QTL on chromosome 9 (LRS score 18.44) and two suggestive QTL one on chromosome eight (LRS score 15) and one on chromosome 16 (LRS score 12.8). The threshold for suggestive LRS was 10.91 and significant LRS was 18.44. The suggestive score on chromosome eight was taken in to account but no relevant genes were found in this location. The suggestive QTL in chromosome 16 was not taken in to account because of low LRS scores.

The above results highlight an interesting pattern of QTL for B6 maternal weight traits.

Both maternal weight traits were affected only during the first six postnatal days showing that indirect effect of BXD litters genotype is at peak during this time. Conversely, although BXD mother’s genotype affected short term weight gain in B6 pups only on day

14, I found a few weak suggestive loci on day 6 affecting weekly growth of B6 pups

(Table-6.1). Table-6.1 (Page 152) shows all the identified QTL with LRS scores higher than the suggestive threshold (p-value=0.63). The QTL were found to be distributed along the whole genome rather than clustered, and have mostly narrow peaks. The additive effect of alleles derived from the B6 parental strains on the phenotypes in question are evenly distributed between positive and negative. No suggestive or significant QTL were found for weekly weight change in B6 pups between days 1 to 6 and 14 to 21 and short term weight change in B6 pups on day 10 thus these traits have not been listed in the table.

152

Table-6.1: List of suggestive and significant QTL. The Table shows the list of QTL identified its LRS score, additive effect of the B6 or DBA allele, location of the QTL and the trait affected. All QTL listed in the Table reached the suggestive threshold except the ones marked with * which reached the significant threshold for that interval map. All QTL highlighted in bold were taken in to consideration and investigated further.

153 6.5 Discussion

An indirect genetic effect is a genetic effect of an individual on traits of other individuals

(Griffing 1967; Moore et al. 1997; Wolf et al. 1998). IGEs may also arise when one individual modifies the environment experienced by other individuals, and thus contribute to a component of the environmental variation that underlies phenotypic variation (Wolf et al. 1998). Thus in the past such external influences have traditionally been categorised as being environmental effects and have been ignored (Mutic and Wolf 2007). In recent years, however, much attention has been given to IGEs and its evolutionary significance on phenotypic traits (Bijma 2014). The primary aim of my research was to understand

IGEs of variable BXD genotype on weight traits of B6 mice in a mother-offspring nurturing environment. I not only investigated the maternal effects of BXD mother’s genotype on growth of B6 litters, but also studied the effect of BXD litter genotype on the weight of B6 foster mothers.

Maternal effects are the most frequently considered IGEs, where the environment provided by the mother significantly affects traits in the offspring (Moore et al. 1997; Wolf et al. 1998; Wolf 2003). Yet, in our study none of the offspring’s weight traits correlated significantly with loci on the maternal genome. I did not find any strong QTL for growth of

B6 pups during early upbringing however a suggestive QTL was found for weight changes on postnatal day 14 when weaning conflict is at peak. From day 14 mothers start reducing milk provisioning and pups commence eating solid food (Kikusui et al. 2009). This is an interesting finding as altered weaning can strongly affect important physiological and behavioural traits such as body weight (Fuchs 1981), play fighting (Shimozuru et al.

2007), anxiety and aggression related traits (Nakamura et al. 2003; Kikusui et al. 2004;

Kanari et al. 2005). I found three very interesting genes on BXD chromosome 18 which correlated with weight gain in B6 pups on day 14. Sap130, Bin1, and Stard4 are protein coding genes involved in developmental pathways. Sap130 encodes spliceosome- associated protein 130, which is involved in regulation of proteins linked to protein

154 degradation (Cordero-Espinoza and Hagen 2013). Protein degradation is crucial for maintaining cell signalling and transcription and thus overall cellular functions (Petroski and Deshaies 2005). Sap130 is relevant to weight traits because of its sequence homology to DDB1, knockout of which results in severe growth deficits (Wakasugi et al.

2007). Similarly knockout studies on Bin1 gene have indicated its crucial role in mouse development (Muller et al. 2003). Bin1 is one of the four BAR-protein encoding genes involved in multiple cellular processes such as endocytosis and transcription (Muller et al.

2003). A study on a knockout mouse model of Bin1 has found perinatal lethality and cardiomyopathy (Muller et al. 2003) and in-vitro studies have found an impaired myoblast differentiation due to inhibition of Bin1 gene in mice (Lee et al. 2002). Whilst Bin1 is involved in muscle development, Stard4 directly controls body weight by regulating cholesterol levels in the body (Rodriguez-Agudo et al. 2008; Mesmin et al. 2011). Stard4 encodes steroidogenic acute regulatory protein (StAR)D4 which is a member of the StAR related lipid transfer family (Riegelhaupt et al. 2010). A knockout model of Stard4 showed significantly smaller body weights compared to wildtype litter mates (Riegelhaupt et al.

2010) in mice; this highlights the importance of this locus on weight trait of B6 pups.

Despite the finding of this suggestive QTL, I expected to get a significant QTL for maternal effects of BXD maternal genotype on B6 litter weight traits. This is because previous studies, both theoretical and empirical, have found significant correlation of maternal effects on growth related traits (Wolf et al. 2002; Wolf and Cheverud 2012). In fact the study by Wolf et al. (2002) found four maternal effects QTL in an inbred mouse strain which accounted for 31.5% variance in litter growth during early upbringing (Wolf et al.

2002). A reason behind the lack of significant maternal effects QTL in my study could be as a result of possible sibling effects which are analogous to maternal effects. Since the

BXD mother and offspring genotypes are correlated, sibling effects may appear as maternal genetic effects. However, GLM did not find any significant effect of BXD sibling

155 genotype on B6 pup weights (GLM; F1,64=0.59; p=0.827), thus it can be assumed that there are no possible effects from sibling genotypes.

On the other hand, like the previous study in our lab (Gini 2014), I found multiple significant QTL of BXD litter genotype affecting maternal weight traits. Studies have shown the correlation between maternal body weight and litter weight (Barnet and

Dickson 1984). Thus it may be advantageous for BXD pups to influence maternal weight traits. Trivers (1974) had predicted that offspring may influence parental provisioning and this has been demonstrated by a number of studies on parent-offspring conflict (Agrawal et al. 2001; Hager and Johnstone 2003; Cui et al. 2004). The QTL found in my study thereby play important roles in determining maternal provisioning and highlight the importance of IGEs of litter genotype on parental strategies.

I found two significant QTL for weekly weight change in B6 mothers between postnatal day 1 and 6. These days are crucial in development and mother-offspring interaction as mice being altricial animals depend fully on the mother for thermoregulation and feeding during the first 14 days. The first QTL found for this trait maps to chromosome six in the

BXD litter genotype. The identified candidate gene Creb3l2 encodes an endoplasmic reticulum stress transducer protein, under-expression of which leads to cell apoptosis

(Saito et al. 2009). Conversely Hrh2 on chromosome 13 encodes a widely expressed histamine receptor protein, H2 (Nomura et al. 2013). In the stomach, H2 receptors play key role in controlling gastric inflammation (Nomura et al. 2013) and polymorphism in the protein has even been linked to schizophrenia (Mancama et al. 2002). The next significant

QTL identified two candidate genes which map to BXD chromosome nine and correlates with short term weight change in B6 mothers on postnatal day 6. Col5a3 gene is highly expressed in white adipose tissue, pancreatic islets and skeletal muscle (Huang et al.

2011). A study on a knockout mouse model of the gene has discovered lower dermal fat in the knockout female mice followed by resistance to high fat diet (Huang et al. 2011).

Male and female knockout mice were diabetic and had defective skeletal muscles

156 compared to wildtypes (Huang et al. 2011). The functions of this gene are directly in line with the weight traits under investigation. The next candidate gene, Dnmt1 encodes the enzyme DNA methyltransferase (Gaudet et al. 2003). Reduced DNA methylation leads to tumour formation (Li and Zhang 2014) thus decreased Dnmt1 expression leads to T cell lymphoma in mutant mice by promoting chromosomal instability (Gaudet et al. 2003).

6.6 Conclusion

In a social environment, IGEs play an important contribution to phenotypic traits (Hager et al. 2008). My study has shown that maternal effects and IGEs do have an impact on weight traits in mothers and offspring. This study has further highlighted the specific QTL in BXD genotype controlling the variations in phenotypic traits of B6 mothers and offspring and has discussed the genes identified in these loci. These candidate genes play important roles in development, cell processes and growth (Wakasugi et al. 2007;

Riegelhaupt et al. 2010; Huang et al. 2011) thereby emphasising the importance of studying IGEs further. The results are in line with previous studies in our lab (Gini 2014) and have found abundant evidence for IGEs of the pup genome on maternal provisioning traits. The evidence of some maternal effects, although suggestive, and significant litter genotype IGEs confers the expectation that both mothers and pups contribute to shaping the level and form of provisioning (Trivers 1974).

157

7. General Discussion

158

In classical Mendelian genetics, a mutation in the DNA sequence is predicted to cause impairments in the gene function leading to characteristic well-defined phenotypes

(Antonarakis and Beckmann 2006). However, even specific mutations may lead to a wide array of symptoms due to effects of other genes and environment (Hunter 2005). The type of mutation may also cause variation in phenotypic traits by, for example, leading to a partial or total loss of gene function. Studying variations in phenotypes arising from mutations in genes can help us to understand the roles of genes in physiological traits. In genetic disorders, assessing the contribution of mutation in specific genes to changes in phenotypes is a primary method for measuring gene functions (Mickle and Cutting 2005).

Genotype-phenotype correlation studies provide a good background in the understanding of gene functions. Random mutagenesis and site-directed mutagenesis are commonly used techniques for measuring this genotype-phenotype relationship (Shortle et al. 1981; Edelheit et al. 2009). The genetic architecture of a trait can also be defined by mapping genes in specific parts of the genome that contribute to quantitative traits

(Lander and Bostein 1989).

Using such site-directed mutagenesis techniques and QTL mapping, the aim of my PhD was to understand the contribution of specific genes and loci to disease phenotypes and developmental traits. Gtf2ird1 and Gtf2ird2 are frequently deleted in Williams-Beuren

Syndrome cases and have been linked to a number of symptoms observed in WBS including craniofacial abnormalities (Tassabehji et al. 2003; Tassabehji et al. 2005), motor deficits (Schneider et al. 2012) and impaired social behaviours (Young et al. 2007; Porter et al. 2012). My thesis has given a detailed overview of WBS, its clinical features and research on disorder models. In Chapters One and Two, I have discussed previous studies on Gtf2ird1 and have explained the purpose of my research and phenotypes seen in the knockout mouse model of the gene. In Chapter Three, I have explained the phenotypes observed in a knockout mouse model of Gtf2ird2 gene and its relevance to

WBS. In Chapter Four, I took a quantitative genetics approach to study genotype-

159 phenotype correlations by identifying candidate genes involved in quantitative developmental traits. Here I discuss if and how the aims of my PhD have been achieved and what future directions can be taken from the findings of my research. The subheadings denote the questions asked in the aims and objectives sections (Page 41 and 48).

7.1 Gtf2ird1

7.1.1 Are homozygous female mice more social and empathic than heterozygous and wildtype Gtf2ird1 females?

This question was raised from the highly social and empathic behaviours seen in WBS indivduals from a young age (Mervis et al. 2000). Previous studies on knockout mouse models of Gtf2ird1 have also found increased rates of social interactions (Young et al.

2008; Enkhmandakh et al. 2009) and social drive (Skitt 2012) in the homozygous mice compared to wildtypes. My studies measured this social interaction profile by recording maternal care behaviours of the Gtf2ird1 female mice towards litters during the early developmental phase. My first study, Chapter One, recorded the maternal behaviours of

Gtf2ird1 homozygous and wildtype female mice towards litters of own genotype

(homozygous or wildtype) and heterozygous genotypes. The results of this study found a reduced maternal care pattern in homozygous mice compared to wildtypes during the early postnatal days but found elevated levels of maternal behaviour towards the start of weaning. Physical problems in homozygous mice were found to interfere with optimal maternal provisioning. Thus, it was hypothesised from this study that physiological disabilities in the Gtf2ird1 knockouts impair maternal social behaviours yet the mice were empathic and provisioned the litters when weaning conflict was at its peak. The litter genotype was not controlled for in this experiment thus in my second study, Chapter Two, a detailed experimental design with defined litter genotype was implemented. The maternal behaviours of heterozygous Gtf2ird1 female mice were also recorded in this

160 study alongside additional behavioural measures (Page 56). The results of this study,

Chapter Two, confirmed and expanded on the findings from Chapter One and have discussed in detail how different physiological problems in the Gtf2ird1 homozygous mice relate to the different maternal care patterns observed; this includes impaired pup retrieval and licking behaviours from craniofacial abnormalities and impaired eating behaviour from possible metabolic problems and/or thermoregulatory defects. In summary, Gtf2ird1 homozygous females in my studies did not show an elevated social profile compared to heterozygous and wildtype female mice but they showed greater empathy towards litters than wildtypes as they provisioned litters more at the beginning of weaning when mothers usually reduce their nurturing behaviour (Cramer et al. 1990). Litters reared by homozygous mothers also demonstrated a comparable proportional growth to the litters reared by wildtype and heterozygous mothers thus suggesting that Gtf2ird1 homozygous females are capable of overcoming their physiological disabilities and nurture their offspring successfully. The prolonged eating behaviours and lack of maternal care during the observationas are also indicative of attention-shifting deficits in the Gtf2ird1 homozygous mice. This is in line with studies on WBS and Down syndrome individuals

(Brown et al. 2003; Karmiloff-Smith et al. 2012; Dimintriou et al. 2015) which found that

WBS individuals are unable to shift their attention to different visual stimuli at a time and instead spend prolonged time focusing on individual objects.

7.1.2 Does offspring genotype affect maternal behaviour?

To understand if litter genotype alone has a significant effect on maternal behaviours, in

Chapter two, I cross-fostered homozygous, heterozygous and wildtype Gtf2ird1 litters to

B6 mothers and monitored any variation in maternal behaviours. No significant differences were observed in the mean maternal behaviours of B6 mothers thus it can be concluded that litter genotype was not a significant determinant of behavioural phenotypes. The cross-fostered Gtf2ird1 litters were obtained from a CBA X B6 background and thus share

161 high genetic similarity to the B6 mothers. A previous study has found minimal differences in maternal behaviours of B6 mothers towards cross-fostered pups of same strain (Van

Der Veen et al. 2008). Comparison of maternal behaviours of B6 mice to other strains, such as DBA/2J, has consistently found better retrieval, nursing and grooming behaviours in the B6 mice (Shoji and Kato 2006; Van Der Veen et al. 2008). In this study the B6 mothers nursing knockout, heterozygous and wildtype Gtf2ird1 pups showed similar and a very normal pattern of maternal behaviours. This indicates that the B6 mice rearing knockout pups did not differentiate between knockout pups from wildtype pups. Since whole Gtf2ird1 litters were cross-fostered to each of the B6 mice instead of mixed genotypic litters it is harder to conclude if the B6 mice would otherwise be able to recognise a knockout pup, with physical deformities, from wildtype or heterozygous pups.

An interesting aspect of this study was the attempt to understand whether maternal care by B6 mice can shape behaviours of Gtf2ird1 litters. Studies on inbred strains or knockout models often tend to overlook the effect of maternal care on offspring behaviours (Van

Der Veen et al. 2008). Cross-fostering litters of knockout mice to wildtype mothers can induce significant behavioural changes in the knockout offspring (Weller et al. 2003) due to the enhanced maternal provisioning by the wildtype mothers compared to knockouts. I found similar results in my disease model and this has been discussed further below.

7.1.3 Do maternal effects of wildtype and homozygous mothers play an important role in mediating maternal behaviours of mutant females in the next generation?

Early life experiences are a key determinant of traits throughout life (Lindstorm 1999).

Whilst influences of maternal effects and environment on phenotypic traits such as reproductive success are well documented (Marshall and Uller 2007), there is a lack of studies investigating maternal effects on disorder phenotypes. When parental behaviour affects development of offspring, it serves as a non-genetic mode of transmission (Francis et al. 1999). Since variation in maternal care profile leads to significant behavioural and

162 neuroendocrine changes in offspring (Liu et al. 1997; Caldji et al. 1998; Francis et al.

1999), I aimed to determine whether such effects can alter behavioural phenotypes of the disorder model. My study had compared maternal behaviours of knockout, heterozygous and wildtype Gtf2ird1 female mice reared by B6 or knockout mother in the previous generation (Figure-11 and 12, Pages 78 and 80). Chapter one and two had established diminished maternal behaviours in knockout mothers. Thus, I assumed that knockout and wildtype females reared by knockout mothers will show diminished maternal provisioning compared to knockout and wildtype females reared by B6 mothers, despite underlying genetic factors. I can conclude from these results that maternal effects play a significant role in balancing the effects of the Gtf2ird1 deletion on overall maternal behaviours.

Although direct genetic effects of the gene deletion led to knockout females demonstrating impaired nurturing behaviours compared to wildtypes, being cared for by B6 mice instead of knockouts improved the nursing and attentive behaviours in the knockout, heterozygous and wildtype mice as adults. This suggests that maternal effects can influence genotypic effects as diverse as disorder phenotypes thus playing an indirect genetic contribution to developmental traits.

7.1.4 Future Directions

In summary my research on the Gtf2ird1 mouse model has established that deletion of this gene causes variable physiological impairments which significantly alter maternal behaviours. The studies have highlighted a few symptoms which should be investigated further. This includes increased appetite in the knockout mice. During the behavioural observations the knockout Gtf2ird1 females were found to be foraging and feeding for a significant period of time yet these mice weighed less and were a smaller size than the willdtypes. Impaired eating behaviours can be due to stressful events that cause the release of corticotrophin-releasing hormones (CRH) from the PVN of the hypothalamus

(Sominsky and Spencer 2014). Regular handling and separation from the pups were the

163 most likely stressful events during the experiments. Glucocorticoids released from the hypothalamus can also influence appetite by interacting with several appetite inducing targets including insulin release (Asensio et al. 2004; Dallman et al. 2004). Diabetes from insulin resistance is common in WBS indivduals (Stagi et al. 2014) thus by measuring the levels of CRH and glucocorticoids in this mouse model will help understand any contribution of the Gtf2ird1 deletion in this symptom. Towards the end of my behavioural experiment on the Gtf2ird1 mice, ten homozygous, heterozygous and wildtype females were bred again and on postnatal day 3, brain and serum tissues were collected with the aim of measuring the levels of Crh, Oxt, and Avp in this knockout model. Due to the time restriction of my project I was unable to complete the molecular experiments on the

Gtf2ird1 model but this will be the immediate next step on this mouse model. Regardless of an increased appetite and feeding behaviour, the Gtf2ird1 knockout mice did not gain any weight. This might be indicative of an impaired metabolism in these mice or defects in thermoregulatory mechanism, as discussed in Chapter two, thus future studies investigating the metabolic profile of this mouse model are necessary. Metabolic activity could be analysed by monitoring the breathing rate and measuring consumption of oxygen and production of carbondioxide and by assessing the fat and muscle mass of the mice using magnetic resonance imaging.

Increase in appetite can be a response to stress (Schneider et al. 2012; Sominsky and

Spencer 2014) which in this mouse model most likely resulted from regular handling and prolonged social deprivation from four hours of pup separation. Behavioural observations on alternative days without pup separation protocol and no regular handling might be able to give an understanding of how these mice behave when left undisturbed. However, it can be argued that this will not be a true representation of behaviours as social separation from litters is common in the wild during which mothers forage and gather nesting material. A comparison of behavioural data from the existing protocol and an ‘undisturbed’

164 protocol might help to understand whether handling act as frequent stressors and alter maternal provisioning behaviours.

Canales et al. (2014) have recently established hearing deficits in a Gtf2ird1 mouse model. My Gtf2ird1 homozygous mice showed a delayed maternal responsivity in the pup retrieval task. It will be interesting to investigate whether this symptom might be as a result of hearing impairments in these mice. Pup vocalisations are an important mediator of maternal response (Cohen-Salmon et al. 1985) especially during the early days after birth when pups lack fur and subcutaneous fat and hence tend to cool very rapidly and constantly seek maternal attention (Portfors 2007). Recording pup vocalisations and subsequent maternal behaviours might be a good future experiment to investigate this symptom further. To investigate if the mouse model has profound sensorineural hearing loss, Preyer’s reflex could be tested by subjecting the mice to a sudden loud noise for example from a handclap (Jero et al. 2001); this test will however be unable to detect less severe auditory dysfunctions.

Oxt is a crucial regulator of social behaviours. Dai et al. (2009) have found compelling evidence of an increased Oxt and Avp levels in blood samples of WBS indivduals. Thus measuring levels of expression of Oxt, Oxtr and Avp genes in the PVN and SON of this

Gtf2ird1 mouse model is an important study to be conducted in the dissected brain and serum tissues. Currently there is no evidence of mechanisms of genes deleted in WBS in regulating Oxt and Avp levels but, the importance of studying the role played by Gtf2ird1 and Gtf2i genes in neurohormone regulation has been raised (Dai et al. 2009).

Another important aspect that needs to be addressed is how maternal effects of B6 and

Gtf2ird1 knockout upbringing influenced maternal care patterns in our study (Chapter-2).

Comparing DNA methylation patterns between the foster mother and offspring might be able to answer this. Weaver et al. (2004) found an effect of licking frequency by female rats on DNA methylation of glucocorticoid receptors in the offspring. When they cross- fostered the litters to mothers showing reduced licking frequency, an opposite effect was

165 found (Weaver et al. 2004; Meaney 2005). Thus, using the collected brain tissue samples of my Gtf2ird1 model, we can investigate whether phenotypic changes from maternal effects were as a result of underlying epigenetic effects in the offspring.

7.2 Gtf2ird2

The Gtf2ird2 gene is deleted in only a small number of WBS cases (Porter et al. 2012).

Because of this, there has been a lack of research on this gene thus our knowledge of this third member of the GTF gene family was limited. My research has focussed on understanding the function of Gtf2ird2 by conducting behavioural and molecular studies on a knockout mouse model of the gene. The aim of the study was to understand if

Gtf2ird2 regulates fine-motor skills, hyper-social behaviours and neurohormone levels in the mice. This section discusses the results I found and compares the phenotypes in

Gtf2ird2 knockout model to the Gtf2ird1 model.

7.2.1 Does Gtf2ird2 affect nest building skills?

Individuals with WBS frequently have fine-motor coordination problems (Gagliardi et al.

2007). When this symptom was investigated in a Gtf2ird1 mouse model, significantly impaired fine-motor skills were demonstrated by the homozygous mice (Schneider et al.

2012). This was tested by adapting a protocol from Deacon (2006) which entailed scoring the quality of nests constructed by the mice and comparing the scores between the three genotypes. The same experiment was repeated for the Gtf2ird2 mouse model in Chapter three in which no significant differences between the quality of nests built by homozygous, wildtype and heterozygous mice was found. Thus this study demonstrated that Gtf2ird2, unlike Gtf2ird1, is not a determinant of fine-motor skills. Moreover, neither of the Gtf2ird2 homozygous mice showed obvious misalignment of jaws or craniofacial deformities as seen in the Gtf2ird1 homozygous mice (Figure-6, Page 39).

166 7.2.2 Is Gtf2ird2 involved in hyper sociability in female mice?

My research on Gtf2ird2 is the first reported work on a knockout mouse model of this gene. I used the same behavioural protocol that was used for my Gtf2ird1 study in

Chapter two except the litters in this study were not cross-fostered. Litter genotype was aimed to be kept fairly constant by breeding each of the three genotypic females

(homozygous, heterozygous and wildtype) with heterozygous males. The results of my study found a pronounced hyper sociability in the homozygous mothers when they were reunited with the pups after four hours of separation. Thus, I hypothesised that deletion of

Gtf2ird2 seems to regulate behavioural phenotypes in this knockout model especially when the mice were socially deprived for a few hours. The elevated nurturing behaviour in the homozygous mothers compared to wildtypes is more evident from the growth profile of the litters. Litters of homozygous mothers weighed significantly more than litters of wildtype mothers from postnatal day 10 to weaning (Figure-27). A study by Porter et al.

(2012) has found a role played by Gtf2ird2 in behaviour, brain development and executive functioning defined by cognitive processing involving memory and problem solving skills in

WBS indivduals. It was concluded from this study that deletion of all three Gtf genes lead to severe neurological phenotypes in the indivduals (Porter et al. 2012). My study showed evidence of possible involvement of the Gtf2ird2 gene in behavioural traits. Furthermore, in comparison to the Gtf2ird1 knockout no apparent physiological abnormalities were observed in this mouse model. For example, Gtf2ird2 homozygous mice had similar feeding and eating patterns to that of the wildtypes and all three genotypic adults had similar size and body weights with no distinct craniofacial abnormalities. It was thus possible to conduct an experiment where the experimenter was blind to the genotype of the mice.

An interesting phenotype that has however been highlighted in this mouse model is the rate of filial cannibalism demonstrated by heterozygous Gtf2ird2 female mice. I assumed that stress from frequent handling and weighing is the most likely trigger for such

167 behaviours however I aimed to find some neuroendocrine explanation for this by measuring Oxt and Crh gene expression levels in the brains; Oxt and Crh are involved in maternal aggression related traits (Nephew et al. 2009).

7.2.3 Does Gtf2ird2 regulate expression levels of neurohormones in the hypothalamic brain regions of the Gtf2ird2 mouse model?

At the endpoint of the behavioural study, expression levels of oxytocin (Oxt), oxytocin receptor (Oxtr), vasopressin (Avp) and corticotrophin releasing hormone (Crh) genes were measured in brain tissues collected from the Gtf2ird2 mice. The main purpose of measuring these social neuropeptides was firstly to understand if the enhanced nurturing behaviours in the Gtf2ird2 homozygous mice were due to an underlying neuroendocrine mechanism and in addition to analyse why heterozygous mice showed such filial cannibalistic behaviour. Oxt, Avp and Crh are key regulators of complex social behaviours, attachment and stress related responses (Heinrichs, and Domes 2008).

Oxytocin and vasopressin proteins are synthesised in the PVN and SON of the hypothalamus which also secrete corticotrophin releasing hormone (Pirnik et al. 2004). In stressful events, oxytocin dampens behavioural and neuroendocrine responses and enhances approach behaviour (Pedersen 1997; Carter 1998; Insel and Young

2001; Young et al. 2002). My study has found remarkably similar results. The levels of Oxt mRNA in the PVN of homozygous Gtf2ird2 mothers were very high (172% expression relative to wildtype) whereas the levels were equally low in the heterozygous PVNs (69% expression relative to wildtype). This suggests that Gtf2ird2 could affect behavioural responses by regulating Oxt expression. Full deletion of Gtf2ird2 in the homozygous females increases Oxt expression, whilst partial deletion of the gene lowers the expression in the heterozygous mice (but not compared to the wildtypes). Overexpression of Oxt in the homozygous mice elevated their social approach and nurturing behaviours whilst dampening their response to stress. In contrast, the lower expression of Oxt in the

168 heterozygous mice could have removed this dampening effect of Oxt and resulted in an enhanced response to stress, most likely from the regular handling and litter separation protocol as this phenotype was otherwise not seen in normal breeding programs

(Tassabehji, person communication). Surprisingly no statistically significant differences were found for Oxt expression levels in the SON however a similar trend of high expression (154% expression relative to wildtype) in homozygous mice was observed

(Figure-32, Page 130). Interestingly expression of Oxtr was significantly lower (52% expression relative to wildtype) in the PVN of homozygous mice than in wildtype PVNs.

This could be indicative of oxytocin-regulated desensitisation of oxytocin receptors

(Phaneuf et al. 1997) but current evidence of this mechanism has only been found in human myometrial cells and myocytes (Adachi and Oku 1995; Phaneuf et al. 1998;

Robinson et al. 2003).

No differences in expression were found for either Avp or Crh. Both of these neuropeptides are involved in modulating aggression and anxiety related traits (Smagin et al. 2001; Young and Wang 2004) thus I assumed an impairment in the expression of these genes to be one of the likely mediators of the behaviours observed in this mouse model. No change in the levels of Avp and Crh in this Gtf2ird2 mouse model suggests that

Gtf2ird2 has no regulatory mechanism for Avp and Crh expression. However, a point to be considered in this measure is that all mice were lactating and suckling their litters at the time of tissue collection. Lactation and suckling are factors that have been repeatedly found to affect Avp and Crh levels (Lightman and Young 1989; Neumann et al. 1993) thus the results obtained in my study might not be a true representation of the expression levels of these genes, especially as a modulator of stress.

7.2.4 Future directions

The results of my study on this Gtf2ird2 mouse model have highlighted a number of phenotypes which require further investigation to fully understand the contribution of the

169 gene in the disease phenotypes. The first possible phenotype to be studied further is craniofacial abnormalities in this mouse model. Although no severe or visually obvious craniofacial abnormalities were found in the Gtf2ird2 mouse model, the finding of impaired pup retrieval on postnatal day 10 may be suggestive of subtle defects. On postnatal day

10, homozygous Gtf2ird2 mothers dropped their pups multiple times during the retrieval process; similar phenotypes were found for Gtf2ird1 homozygous mice in Chapter two. A computerised tomography scan is a potential method that can be used to identify subtle craniofacial defects, if any, in the skull of these mice.

The behavioural protocol used in my study has clearly highlighted an upregulated social behaviour in this Gtf2ird2 model. However, there are two aspects which might help improve this understanding. Firstly, implicating a cross-fostering protocol to ensure that all three genotypic mothers nurse genetically uniform litters will help control for any effects of litter genotype. The breeding plan used in this study did aim to maintain a fairly constant litter genotype, but, a cross-fostering design might help in solidifying the findings.

Secondly, an ‘undisturbed’ behavioural protocol as described above in the ‘future directions’ section of my Gtf2ird1 research (Page 162) will confirm whether regular handling and weighing are the triggers of filial cannibalistic stress responses in the heterozygous Gtf2ird2 mothers. I did not find any impairment in underlying Avp and Crh expression levels in the heterozygous mothers possibly due to the collection of tissues when suckling. Thus measures of expression levels in non-lactating females might give a better understanding of an effect of the Gtf2ird2 gene deletion on regulation of Avp and

Crh genes.

I did find interesting results for Oxt and Oxtr gene expressions in the homozygous and heterozygous Gtf2ird2 PVNs. It would be important to measure the levels of these mRNA in the collected serum samples of this mouse model and also administering doses of oxytocin in the heterozygous females to see if infanticide can be prevented. This might show exaggerated social behaviours by compensating for the possible down-regulated

170 Oxt gene expression due to hemizygous Gtf2ird2 deletion. To confirm whether reduced

Oxtr mRNA levels in homozygous PVN were because of desensitisation of oxytocin receptors from the significantly elevated Oxt expression, binding of oxytocin to the receptor and level of oxytocin receptor protein should be measured in the Gtf2ird2 brain tissue samples. Prior studies on myometrium (Adachi and Oku 1995; Phaneuf et al. 1997) can be used as references to establish this desensitisation effect on brain tissues. A study investigating the amygdala region of this mouse model is an important next step. WBS indivduals have been found to have an increased amygdala volumes contributing to their highly approachable behaviours and facial recognition capabilities (Martens et al. 2009). I have cryosectioned the central and medial amygdalas of the Gtf2ird2 mice. Investigating this region further will help identify any contribution of the Gtf2ird2 gene in the regulation of variation in amygdala of WBS indivduals.

7.3 Gtf2ird1 and Gtf2ird2 as mouse models to study WBS

My study has highlighted the importance of studying the Gtf genes and their contribution to WBS symptoms. The WBS symptoms that have been highlighted in these mouse models are social behaviours, growth, craniofacial features, fine-motor skills and impaired oxytocin levels (Morris et al. 1988; Mass and Belostoky 1993; Dai et al. 2009; Morris

2013). Studies on Gtf2i mouse models have been found to have retarded growth along with abnormal craniofacial features (Enkhmandakh et al. 2009). Previous studies on

Gtf2ird1 have found growth deficits, craniofacial impairments (Tassabehji 2005), poor fine- motor skills (Schneider et al. 2012) and hyper sociability (Young et al. 2008) in knockout mouse models. Gtf2ird2 gene has been linked to executive functioning in humans (Porter et al. 2012). My study has confirmed growth deficits and severe craniofacial defects in the

Gtf2ird1 mouse model along with a pronounced appetite and reduced nursing behaviours in homozygous mice. Gtf2ird1 mice struggled with daily activities including eating solid food, licking themselves and pups, constructing nests as well as retrieving pups because

171 of their severely misaligned jaws and very poor fine-motor skills. These homozygous mice also showed a delayed start to the pup retrieval process indicating a lack of perception to the needs of their offspring. A likely cause might be hearing impairments in these homozygous Gtf2ird1 mice (Canales et al. 2014) which are reflective of mild to moderate hearing loss in WBS indivduals (Pober 2010). In spite of these phenotypes, litters nurtured by homozygous mothers had very similar growth rates to litters nursed by wildtype mothers thus indicating empathetic traits in these mothers who are able to overcome their disabilities to care for their litters. WBS indivduals show an increased empathy and concern towards individuals (Tager-Flusberg and Sullivan 1999) and have an increased likelihood to approach to help when they see an individual in pain (Mervis et al. 2000). My

Gtf2ird1 mice thus mimic this personality trait by nurturing for the pups even more at the time when otherwise normal female mice start to separate themselves from their litter.

In contrast to Gtf2ird1, Gtf2ird2 homozygous mice did not show any severe physical defects. The homozygous mothers were extremely nurturing and sociable and suckled their litters significantly more frequently than wildtypes. These mice had intact fine-motor skills and showed normal patterns of eating and drinking behaviours. The results did indicate possible subtle craniofacial problems but further analysis is required to draw any conclusions. My study has found a possible mechanism of the Gtf2ird2 gene in regulating

Oxt expression. Dai et al. (2009) found elevated oxytocin and vasopressin levels in WBS indivduals, no research to date has further investigated this or found any link between genes deleted in WBS and this phenotype. My study hence provides insight into the phenotypes of a knockout model of Gtf2ird2 and the first correlation between a gene deleted in WBS and underlying neuroendocrine mechanisms.

It is important to remember that mice with a total loss of one gene may not give a precise gene dosage found in WBS (Osborne 2010). A previous study on loss of the whole WBS region in a mouse model did mirror some clinical features such as impaired motor coordination and cardiovascular defects (Li et al. 2009) but lacked some key symptoms

172 including the social phenotypes. A single gene mouse model is thus very promising because it allows the correlation of a specific gene to specific phenotypes. Mouse models also provide a sound base for therapeutic interventions. In the future, my studies on WBS mouse models might help in pre-clinical testing of therapies aimed at combatting disinhibited social behaviours, deficient fine-motor skills and irregular neuropeptide levels in WBS indivduals.

7.4 Indirect genetic effects

IGE in a mother-offspring interaction environment has been established in two of my studies; first in Chapter Two where maternal effects were found to alter disease phenotypes of a WBS mouse model and secondly in Chapter four where I found four loci in BXD mice accounting for phenotypic changes in B6 mice. Here I discuss how the aims of Chapter four were reached.

7.4.1 Are there maternal effects of BXD maternal genotype on growth of B6 pups and IGEs of BXD litter genotype on weight changes of B6 mothers?

I did not find any significant QTL for maternal effects of BXD genotype on traits of B6 pups. I did however find a suggestive QTL on postnatal day 14. Conversely I found three significant QTL on BXD litter genotype affecting weight changes in B6 mother between postnatal days 1 and 6. The lack of significant maternal effects QTL is a surprising result and contradicts the importance of maternal influences on traits of offspring during the early postnatal days (Wolf et al. 2002). Maternal effects QTL experience a reduced selection pressure because of their prevalence in only females (Wade et al. 2009) and should hence evolve at a faster rate and have a stronger effect on traits. A lack of significant maternal effects QTL in my study might be as a result of close link between the

BXD and B6 genotypes. The BXD mice having been generated from crossing B6 and

DBA mice (Chapter-4, page 141) have reduced genetic variation and in a controlled lab

173 environment, where food is abundant, variation in maternal provisioning may not be strong enough to be picked up by QTL analysis. In such a moderately variable environment, phenotypic differences can be lower than normal (Lott 1991). However, suggestive QTL are indicative of presence of multiple QTL with small effects which should be investigated further. The presence of multiple significant QTL in offspring genotype affecting maternal weight traits agrees with Trivers theory on parental provisioning being influenced by offspring (Trivers 1974). Offspring can influence parental investment and provisioning by producing signals to which the parents respond either actively or passively (Bergstrom and Lachmann 1998; Johnstone 1999). Differences in the signals produced within a litter are controlled by environmental and genetic factors (Agrawal et al. 2001) but the genetic contribution is not well explored (Kölliker et al. 2000). The genes identified in my study might hence give some examples of the genes in BXD litters involved in affecting maternal traits, however, whether these genes are directly linked to signals produced by offspring have not been explored.

7.4.2 IGE and maternal effects QTL and functions

The QTL identified for maternal effects and IGEs of offspring genotypes have been mapped to chromosomes 18, 6, 13 and 9. The suggestive QTL in chromosome 18 of the

BXD maternal genotype regulates short-term weight changes in B6 pups at the start of weaning on postnatal day 14. The three highlighted candidate genes, Sap130, Bin1 and

Stard4, in this region are involved in protein degradation, transcription, and cholesterol regulation. Studies on knockout models of these genes and genes homologous to them have found an effect on body weights (Lee et al. 2002; Wakasugi et al. 2007; Riegelhaupt et al. 2010).

The QTL on chromosomes 6 and 13 control weight changes of B6 mothers during the early postnatal days 1 to 6. Creb3l2 and Hrh2 genes in these regions regulate important cellular proteins such as endoplasmic reticulum transducer protein and histamine receptor

174 protein, and thus control processes such as cell homeostasis (Saito et al. 2009), gastric inflammation (Nomura et al. 2013) and symptoms seen in schizophrenia (Mancama et al.

2002). The QTL on chromosome nine in BXD litter genotype correlates to short-term weight changes in B6 mothers on day 6. Two candidate genes were identified in this region; Col5a3 and Dnmt1. Whilst Col5a3 directly control weight traits in individuals

(Huang et al. 2011), Dnmt1 is involved in tumour genesis (Li and Yi 2014).

7.4.3 Future directions

Further studies are required to understand why a lack of maternal effects QTL was found and also to establish any sibling effects on traits. Tests on a variety of BXD populations and different environmental conditions might help explain whether the results on maternal effects were due to limitations of the experiment or because of a true lack of the maternal effects QTL in the BXD mice studied. There are over a thousand known genotypic recombination patterns in the BXD strain (Shifman et al. 2006); in my study 32 BXD lines were used. Thus, studying more BXD lines might help in finding maternal effect QTL with stronger effects on offspring growth. Sibling effects can be tested by increasing the number of BXD pups versus B6 pups and mapping any differences in IGEs. Another important future project will be to combine the results of this study with results on behavioural data collected from this mouse model. This will give a better understanding of how maternal provisioning correlates with weight traits and whether there are any common loci accounting for changes in both maternal behaviours and corresponding weight traits in pups.

175

8. Final Conclusion

176

The main goal of my PhD was to achieve a broad understanding of how behavioural and developmental phenotypes can be affected by genetic and indirect genetic factors. In order to accomplish this, during the majority of the project I concentrated on deciphering the roles played by two genes, Gtf2ird1 and Gtf2ird2, deleted in the multi-system, developmental disorder Williams-Beuren syndrome. The genetics underlying the unique hyper sociability aspect of this disorder was explored in my research by studying mouse models of Gtf2ird1 and Gtf2ird2. Also, data collected from recombinant inbred BXD mice were used to map QTL causing indirect genetic effects on growth and weight changes.

I found significant effects of Gtf2ird1 and Gtf2ird2 deletion on physiological traits in mice.

The results of my studies are in line with existing literature on the contribution of these genes to WBS symptoms. My studies have however found novel results which highlight the importance of studying Gtf genes in detail. My studies on Gtf2ird1 have found an array of physiological deficits in the mice which significantly impaired their behavioural responses. Gtf2ird1 knockout mice still showed increased empathy as reflected by enhanced maternal provisioning compared to wildtype and heterozygous mothers.

Furthermore, maternal effects on disorder phenotypes in the next generation have broadened our knowledge of the variable effects of the environment provided by mothers.

My studies in the Gtf2ird2 mouse model have given an insight in to the role played by this gene in regulating social behaviours in WBS. An increase in oxytocin mRNA levels from

Gtf2ird2 deletion has demonstrated the contribution of genes deleted in WBS on regulating neurohormone levels and ultimately social behaviours. Therefore, a major conclusion of my thesis is that the transcriptional regulator genes Gtf2ird1 and Gtf2ird2 play very important roles in the disease phenotypes of WBS; it is now crucial to conduct further studies focussing on how individual genes deleted in WBS affect neuroendocrine hormonal levels.

177 My study on indirect genetic effects (IGEs) (Chapter-4) has demonstrated that IGEs go beyond maternal effects and found genes expressed in offspring which influenced weight changes in mothers. Thus, studying direct or indirect effect of genes helps us to understand the underlying molecular mechanisms of diseases and eventually how disease phenotypes can be managed. The studies on WBS are not only limited to understanding WBS phenotypes, but also can be applied to understanding the causes of overlapping diseases with similar traits or sporadic occurrences in the population.

178 References

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205

Appendices

206 Appendix-1: Protocols

This section provides the detailed, step-wise protocols that were used in the experiments conducted during the PhD.

Protocols for behavioural studies

Setting up breeding cages:

1. Obtain a new individually ventilated cages (Tecniplast 30x10x10cm) containing aspen woodchip bedding (Datesand Group Ltd, Manchester UK)

2. Add cotton nesting material, 200g food and one water bag.

3. Weigh females and males using Sartorius Entris 2202-1S Toploader Balance 2202g x 0.01g (Cole-Parmer)

4. Place females in male’s cage. 2 females and 1 male in each cage.

5. Make sure the females and males are numbered properly (Mouse ID – batch and ear punch code) and record which cage contains which male and females.

6. Set up two breeding cages a day. This will help stagger birth.

207 7. For studies with cross-fostering design (Chapter-2), set up 2 breeding cages of Gtf2ird1 and two breeding cages for C57Bl/6J mice on each day.

8. Mark the date in calendar and check for pregnancy signs regularly after 15 days.

9. Pregnancy should be checked by weighing females. Separate females when they weigh >25g (This is usually about 8-10g heavier than they are at the start, and with most you can see a rounded belly).

Separating pregnant females:

1. Once pregnancy signs are confirmed separate females from the breeding cage to a new cage.

2. Ensure that female in new cage has water, bedding, 2 nestlets and 200 g food.

3. Put ‘Do not feed’ label on the cage. (We want to monitor food consumption)

4. Do not cull any males until you are sure they will not be used for next experiment.

5. If none of the females in a breeding cage gets pregnant after 20 days since first set, replace the male with one from a successful mating cage.

6. Once littered, record: Number of pups born, weight of litter, day born. Put ‘Do not feed or clean’ label on the cage.

7. Record in calendar the day pups are born as postnatal day 1.

Phenotyping maternal behaviours:

1. Place heat mats on the table and turn them on.

2. Get a new cage with woodchip bedding.

3. Take female out, weigh and place in the new cage but with food and water from home cage (exchange lids). Put a small paper label (pre-prepared with mouse ID) to identify the female.

4. Weigh pups and put back in the home cage (remove filter below green plastic lid to allow air to circulate).

208 5. Put home cage, with pups, on heat mat. Put mother’s cage in the cage rack. Record the time of mother-pup separation.

6. Wait 20 minutes in between before repeating these steps for next set of litters.

7. After 4 hours of mother-pup separation: • weigh food • weigh mother • weigh pups • replace pups into home cage, far from nest. • replace mother into cage near the nest and start stopwatch • Record time taken to retrieve all pups, as well as ticking individual retrievals. • At 20sec make first tick on record sheet not on 0 second. • Carry out 15 minutes of undisturbed behavioural observations. • Behaviours are recorded using focal animal sampling and instantaneous recording with a recording interval of 20seconds (Martin et al. 1993). • Behaviour in bold in the ethogram (Figure-39 and 40) recorded every 20seconds. (not more than 3 ticks per minute per square). • Behaviour in italics in the ethogram (Figure-39 and 40) are recorded whenever observed. • Prevent any sudden noise during the behavioural observations, such as banging doors or talking. Put Do Not Disturb on the lab door. • After the 15 minutes behavioural observation, put the home cage back to the cage rack and record the time. The new cage that was used to separate mother can be reused three to four more times before sending to be cleaned.

8. After 2 hours of mother-pup reunion, take the home cage out of cage rack and weigh food, pups and mother.

9. For the study in chapter-3, a second 15 minutes behavioural observation was carried out using the same protocol (step 7). For chapter-2, pup retrieval was tested after the 2 hours reunion instead of during the 15minutes behavioural observation.

Nesting Experiment:

1. Start this experiment the day you are going to separate pregnant females from each breeding cage to individual cages.

2. Weigh 3.0g of nestlets (record weight and make sure this amount of nesting material is given to every female once separated).

3. Place nesting material in cage at 9am.

4. Leave overnight and score nest at 9am next day (see scoring system 0-5).

5. Take a picture of the nest.

6. Remove all the nesting material that is not shredded and weigh it. Then return it to the cage.

7. Scoring system (Adapted from Deacon 2006):

209

Nests are assessed the next morning on a 1-5 rating scale. Any untorn nestlet pieces are weighed (brush off loose material and sawdust). Definition untorn = ~ 0.1 g, i.e. ~4% of a Nestlet.  Score 1: Nestlet not noticeably touched (>90% intact).  Score 2: Nestlet partially torn up (50-90% remaining intact).  Score 3: Mostly shredded but often no identifiable nest site: < 50% of the Nestlet remains intact but < 90% is within a quarter of the cage floor area, i.e. material is not gathered into a nest but spread around the cage. Note: the material may sometimes be in a broadly defined nest area but the critical definition is that > 50% has been shredded.  Score 4: An identifiable, but flat nest: > 90% of the Nestlet is torn up, the material is gathered into a nest within a quarter of the cage floor area, but the nest is flat, with walls higher than mouse body height (curled up on its side) on less than 50% of its circumference.  Score 5: A (near) perfect nest: > 90% of the Nestlet is torn up, the nest is a crater, with walls higher than mouse body height on more than 50% of its circumference.  Where criteria do not agree split the difference. For example a perfect nest with an unshredded 1g piece might get a score of 4.5.

210 Number: Female strain & Weight: Offspring number and strain: Litter size &weight:

Date: Day: Time: CONTACT: Naorin Sharmin Email: [email protected] SUPERVISOR: Reinmar Hager Email: [email protected]

Behaviour 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ∑ Maternalbehaviour care Nestbuild Nursing suckling Resting Sniffing Pup retrieve Figure Mode

No. of times dropped - F at pups’ quadrant used for Chapter Ethogram 39: licking self Autogroom Feed, drink Resting Other active Offspringbehaviour Feed in nest outside Rest in nest outside

Other active -

Suck attempt 1

playfight autogroom allogroom 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 measurements in [g]; 1 column = 3 bouts = 1min; behaviours in italics are recorded when they occur, bold marked behaviours are recorded on the beep Mode: L=Left foot, R=Right foot, S=Scruff, H=head

211 Number: Female strain & Weight: Offspring number and strain: Litter size &weight:

Date: Day: Time: CONTACT: Naorin Sharmin Email: [email protected] SUPERVISOR: Reinmar Hager Email: [email protected]

Behaviour 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ∑ Maternalbehaviour care Nestbuild Figure Nursing

suckling - 40: Ethogram used for Chapters 2&3 2&3 used for Chapters Ethogram 40: Resting Sniffing Pup retrieve Dddddddddddddddddddddddddddddddddddddddddddddd Mode Dddddddddddddddddddddddddddddddddddddddddddddd No. of times dropped dddddddddddddddddddddddddddddddddddddddddddddd F at pups’ quadrant licking self Autogroom Feed, drink Resting Other active Offspringbehaviour Feed in nest outside Rest in nest outside Other active Suck attempt playfight autogroom allogroom 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 measurements in [g]; 1 column = 3 bouts = 1min; behaviours in italics are recorded when they occur, bold marked behaviours are recorded on the beep Mode: L=Left foot, R=Right foot, S=Scruff, H=head

213

212 Protocols for quantifying neuropeptide gene expression

Cryosectioning brain regions:

Cryostat model used Leica 1800.

1. Switch the cryostat on, the day before or 2 hours prior to commencing and set temperature to -21°C.

2. Carry brain from -80°C freezer in dry ice.

3. Put the brain inside the machine to acclimatise at -21°C along with: tweezers, scalpel, microscope slides, sample corers 0.6mm and 1mm.

4. In the meantime set your staining kit: take 4 small staining tubs with lids.

Arrange next to each other. Pour Cresyl violet solution (0.5%) in the 1st tub. Water in the 2nd tub. 70% Ethanol in the 3rd Tub and 100% Ethanol in the 4th tub.

5. Take five 1.5ml eppendorfs and label the lid and the side with Mouse ID, Genotype and Brain Region. The 6 different brain regions to be punched are: PVN, SON, Amygdala Central, Amygdala Medial, Hippocampus C1, Hippocampus C2-C3 (in one eppdendorf).

6. Cut off the hippocampus region of the brain. Do that by holding the brain still using tweezers and cut using a blade.

7. Then put ‘Cryomatrix’/glue on to one of the platforms kept on the left hand side of the machine.

213

8. When the cryomatrix is still transparent using tweezers to hold the brain put the brain on the glue with the flat side at the bottom and the optic bulbs pointing up.

9. Press the brain down inside the glue and leave it like that till the glue solidifies to a white round circle. Check that the brain is rigid in the glue by using tweezers to lightly move the brain. If unstable put more glue on and around till stable or take the brain out and start again.

10. Put the platform with the brain on to the holding slot. Using the long silver screw/knob pointed by yellow arrow make the hold between the platform and slot tight. Using the black knob pointed by red arrow fix the position of the holding slot by moving it up or down and then tightens it.

11. Now that the position of the brain is secured the distance between the brain and the blade has to be fixed.

12. Press >> and then to move the brain closer to the blade or to move the brain away from the blade.

214

13. Once in the optimal position (just touching the blade), unlock the rotating hand tool and start cutting 10μ (microns) slices.

14. Keep on slicing till you see the anterior commissure which shapes like a ‘moustache’ on the brain. Anterior commissure usually starts off as two white circles on both hemispheres which gradually fuse together to form a line which looks like a moustache (circled by arrow below).

Figure on the left from: Paxinos and Franklin (2001)

15. When the ‘moustache’ appears try to take slides by putting the glass lid down. Adjust the distance between glass lid and blade to get perfect cuts.

16. Using a microscope slide pick up the brain slice by pressing the slide on to the slice. Multiple slices can be taken in one slide.

17. Take the slide out of the machine and put your hand below the slide to warm the brain slices. You will see the white brain slices becoming transparent.

215 18. Put the slide on a heat block for 2 minutes. You will see that the transparent brain slices become off white in colour. This is when the slide should be stained.

19. Staining: - Dip the slide in cresyl violet stain for 1-2mins - Wash in water till all extra stain droplets wash off - Dip in 70% alcohol for 2-3 minutes OR rinse vigorously by stirring the slide inside the alcohol. You will see the brain slices start to lose the dark purple stain. - Dip in 100% alcohol. Repeat the previous step until the brain slices become light purple in colour. - Check under microscope.

20. Similarly keep slicing, staining and checking under microscope till PVN and SON are visible.

21. Using the 0.6mm sample corer, punch through the PVN and collect the region in an eppendorf. Wipe the corer with cloth and collect SON regions.

22. Cut a few more slices till Amygdala is reached and finally the hippocampus.

216

Figure

41: Flow diagram of of cryo diagram Flow 41:

sectioning step sectioning

21 8

217

Figure

- 42: Flow diagram of of step histology diagram Flow 42:

19 218 RNA extraction protocol:

Directzol mini prep kit from Zymo Research was used for this step.

1. Homogenise tissue (brain samples) in 40µl Trizol reagent (Life technologies) and vortex till tissue dissolves completely.

2. Add further 360µl Trizol reagent and vortex.

3. Centrifuge (Thermo Scientific) at maximum speed for 3 minutes

4. Transfer supernatant in to RNase free tube (eppendorf)

5. Add equal volumes of 100% Ethanol to the sample and mix by vortexing

6. Load the mixture in to Zymo-spin column in a collection tube and centrifuge for 1 minute at maximum speed.

7. Transfer the column in to new collection tube and discard the collection tube containing flow-through.

8. Add 400µl of RNA wash buffer and centrifuge for 30seconds at maximum speed. Discard the flow through.

9. For each sample Add the reagents in order; Make a master mix first:

10. Add 80 µl of the DNase I reaction mix to the column directly and incubate for 15mins at room temperature (leave on bench).

11. Centrifuge for 30seconds at maximum speed.

12. Add 400µl of RNA Pre-Wash buffer to each column and centrifuge for 1 minute at maximum speed.

13. Discard flow through and repeat step 12 and discard flow through.

14. Add 700µl RNA Wash Buffer to the column and centrifuge for 1 minute maximum speed. Discard flow through.

219 15. Centrifuge again for an additional 2 mins maximum.

16. Transfer the column carefully in to a RNase free tube (eppendorf).

17. Add 50µl of RNase free water directly in to the column and centrifuge for 1min at maximum speed.

18. Keep the supernatant i.e. the RNA.

19. Nano drop (Thermo Scientific Nanodrop™ 1000 Spectrophotometer) to measure purity and concentration.

20. Store RNA in -80°C freezer.

cDNA extraction protocol:

High Capacity RNA-to-cDNA Kit was used for this step (Applied Biosystems, UK, Cat. 4387406). Specific quantities required by each cDNA synthesis reactions are shown in table below.

1. The concentrations of RNA samples were obtained from Nanodrop reading and were used to calculate required volume of RNA for reverse transcription to cDNA. RNA volume (μl) = 40 ng / [sample concentration (ng/μl)] Water volume (μl) = 9 μl – RNA volume (μl)

2. All reagents must be kept on ice, mixed using vortex and briefly centrifuged prior to commencing this procedure.

3. Make up a master mix of 10µl of 2X RT buffer 1 µl of 20X enzyme mix X No. of samples.

4. Aliquot 11 µl of the master mix into each tube or well.

5. Add the appropriate amount of RNA and molecular biology-grade H2O for each sample to make a total volume of 20 µl per reaction, as shown in table below.

6. Briefly centrifuge to spin down the contents and to eliminate any air bubbles.

7. Incubate in a thermal cycler (Verity 4) set at 60 min at 37°C followed by 5 min at 95°C (to stop the reaction) and held at 4°C.

8. Nanodrop cDNA and then store at -20°C until required.

The quantities of reagents required for each cDNA synthesis reaction

220

qPCR protocol:

1. Run 20µl volume reactions in MX3000p machine

2. Turn machine and computer on

3. In the computer go to MXpro program. Click SYBR Green Dissociation Curve Click Instruments Lamp ON (‘Lamp warm’ sign should come) Give 20-30mins for lamp to warm up.

4. Take reagents out and put on ice. Vortex SYBR Master mix and primers and centrifuge briefly before use.

5. Label tubes: Eppendorfs for Master mixes and qPCR 8 strip tubes. Note: qPCR tubes should NOT be labelled at the top. Only label at the edge or else laser will be unable to penetrate the bottle once in the machine.

6. Prepare Master Mix . Make extra reaction for negatives and at least one more extra reaction for pipetting errors in the MMix.

7. Once pipetting is done flick tubes back to front with your fingers and then centrifuge. Put samples in the machine.

8. In the computer go to Plate Setup: -Select the wells where you put the test samples only (no negatives) then go to Well Type and select Unknown -Under Collect Fluorescence Data select ROX and SYBR -Under Reference Dye scroll down and select: ROX

221 -Now select the wells where you put the negatives then go to Well Type and select NTC (no template control), under Fluorescence Data select ROX and SYBR and Under Reference Dye scroll down and select: ROX.

9. Go to Thermal Profile and edit the qPCR cycles:

10. Click start and save in designated folder under Williams Document. Machine will run for approximately 1hour 40 mins.

11. Click ‘Turn Lamp Off’ at the end of the run (unless you use the machine again on the same day) and turn the switch off from the back.

222 Appendix-2: Primers

Primer spans:

Oxytocin, Oxt

Location: Chromosome 2: 130,576,173-130,577,054 forward strand.

Gene: This transcript is a product of gene ENSMUSG00000027301

This gene has 1 transcript (splice variant)

Transcript: Oxt-001 ENSMUST00000028764

Name Transcript ID Length Protein Biotype CCDS RefSeq Flags

Oxt- 537 125 aa NM_011025 GENCODE

ENSMUST00000028764 Protein CCDS16745 001 bp (view) NP_035155 basic coding qPCR mouse primers:

F: TGGCTTACTGGCTCTGACCT

R: AGGCAGGTAGTTCTCCTCCTG

Key:

R 1 ATCACCTACAGCGGATCTCAGACTGAGCACCATCGCCATGGCCTGCCCCAGTCTCGCTTG ...... ATGGCCTGCCCCAGTCTCGCTTG ...... -M--A--C--P--S--L--A--C

61 CTGCCTGCTTGGCTTACTGGCTCTGACCTCGGCCTGCTACATCCAGAACTGCCCCCTGGG 24 CTGCCTGCTTGGCTTACTGGCTCTGACCTCGGCCTGCTACATCCAGAACTGCCCCCTGGG 8 --C--L--L--G--L--L--A--L--T--S--A--C--Y--I--Q--N--C--P--L--G

121 CGGCAAGAGGGCTGTGCTGGACCTGGATATGCGCAAGTGTCTCCCCTGCGGCCCGGGCGG 84 CGGCAAGAGGGCTGTGCTGGACCTGGATATGCGCAAGTGTCTCCCCTGCGGCCCGGGCGG 28 --G--K--R--A--V--L--D--L--D--M--R--K--C--L--P--C--G--P--G--G

223 181 CAAAGGACGCTGCTTCGGACCAAGCATCTGCTGCGCGGACGAGCTGGGCTGCTTCGTGGG 144 CAAAGGACGCTGCTTCGGACCAAGCATCTGCTGCGCGGACGAGCTGGGCTGCTTCGTGGG 48 --K--G--R--C--F--G--P--S--I--C--C--A--D--E--L--G--C--F--V--G

Y Y 241 CACCGCCGAGGCGCTGCGCTGCCAGGAGGAGAACTACCTGCCTTCGCCCTGCCAGTCTGG 204 CACCGCCGAGGCGCTGCGCTGCCAGGAGGAGAACTACCTGCCTTCGCCCTGCCAGTCTGG 68 --T--A--E--A--L--R--C--Q--E--E--N--Y--L--P--S--P--C--Q--S--G

301 CCAGAAGCCCTGCGGGAGCGGAGGCCGCTGCGCCGCCACAGGCATCTGCTGCAGCCCGGA 264 CCAGAAGCCCTGCGGGAGCGGAGGCCGCTGCGCCGCCACAGGCATCTGCTGCAGCCCGGA 88 --Q--K--P--C--G--S--G--G--R--C--A--A--T--G--I--C--C--S--P--D

R 361 TGGCTGCCGCACAGACCCCGCCTGCGACCCTGAGTCTGCCTTCTCGGAGCGCTGAGCCCA 324 TGGCTGCCGCACAGACCCCGCCTGCGACCCTGAGTCTGCCTTCTCGGAGCGCTGA..... 108 --G--C--R--T--D--P--A--C--D--P--E--S--A--F--S--E--R--*-.....

S R 421 CTTTCTGGGAATACCTTTAGCGCGCTTCCTTCGTTCCCCATGGCCACTGCCAGAAAAAAA ......

481 AAAAAAAAAGAAAAGAAAAGAAAAGAAAAGAAAAATAAAGTAGATTTCCTCTTCAAA ......

......

Oxytocin receptor, Oxtr primer

Location: Chromosome 6: 112,473,684-112,489,808 reverse strand. Gene: This transcript is a product of gene ENSMUSG00000049112 This gene has 1 transcript (splice variant)

Transcript: Oxtr-201 ENSMUST00000053306

Lengt Protei Name Transcript ID Biotype CCDS RefSeq Flags h n 388 NM_00108114 Oxtr ENSMUST0000005330 4568 aa Protei CCDS3958 GENCOD 7

-201 6 bp (view n 8 E basic NP_001074616 ) coding

qPCR mouse primers: F: ACGTCAATGCGCCCAAAGAA R: CGACGACTCAGGACGAAGG

Key:

224

** S ** 1 CGCGGTTGGTCATGGAGGGCACGCCCGCAGCCAACTGGAGTATCGAGTTGGACCTCGGGA ...... ATGGAGGGCACGCCCGCAGCCAACTGGAGTATCGAGTTGGACCTCGGGA ...... -M--E--G--T--P--A--A--N--W--S--I--E--L--D--L--G--

61 GTGGAGTGCCACCAGGGGCGGAGGGTAACCTCACGGCCGGGCCGCCACGACGCAACGAGG 50 GTGGAGTGCCACCAGGGGCGGAGGGTAACCTCACGGCCGGGCCGCCACGACGCAACGAGG 17 S--G--V--P--P--G--A--E--G--N--L--T--A--G--P--P--R--R--N--E--

121 CCCTGGCGCGCGTGGAGGTGGCGGTCCTGTGTCTCATACTGTTCCTGGCTCTGAGTGGCA 110 CCCTGGCGCGCGTGGAGGTGGCGGTCCTGTGTCTCATACTGTTCCTGGCTCTGAGTGGCA 37 A--L--A--R--V--E--V--A--V--L--C--L--I--L--F--L--A--L--S--G--

181 ACGCGTGCGTGCTGCTGGCGCTGCGTACGACGCGCCACAAGCACTCGCGCCTCTTCTTTT 170 ACGCGTGCGTGCTGCTGGCGCTGCGTACGACGCGCCACAAGCACTCGCGCCTCTTCTTTT 57 N--A--C--V--L--L--A--L--R--T--T--R--H--K--H--S--R--L--F--F--

Y 241 TCATGAAGCACCTGAGCATCGCCGACCTGGTGGTGGCCGTGTTCCAGGTTCTCCCGCAGC 230 TCATGAAGCACCTGAGCATCGCCGACCTGGTGGTGGCCGTGTTCCAGGTTCTCCCGCAGC 77 F--M--K--H--L--S--I--A--D--L--V--V--A--V--F--Q--V--L--P--Q--

301 TGCTGTGGGACATCACCTTCCGCTTCTACGGGCCCGACCTGCTGTGTCGTCTGGTCAAAT 290 TGCTGTGGGACATCACCTTCCGCTTCTACGGGCCCGACCTGCTGTGTCGTCTGGTCAAAT 97 L--L--W--D--I--T--F--R--F--Y--G--P--D--L--L--C--R--L--V--K--

361 ACTTGCAGGTGGTGGGCATGTTCGCCTCCACCTACCTGCTGTTGCTGATGTCGCTCGACC 350 ACTTGCAGGTGGTGGGCATGTTCGCCTCCACCTACCTGCTGTTGCTGATGTCGCTCGACC 117 Y--L--Q--V--V--G--M--F--A--S--T--Y--L--L--L--L--M--S--L--D--

421 GCTGCCTGGCCATCTGCCAGCCGCTGCGCTCACTGCGCCGCCGAACCGACCGCCTGGCGG 410 GCTGCCTGGCCATCTGCCAGCCGCTGCGCTCACTGCGCCGCCGAACCGACCGCCTGGCGG 137 R--C--L--A--I--C--Q--P--L--R--S--L--R--R--R--T--D--R--L--A--

481 TGCTGGCGACGTGGCTCGGCTGCCTGGTGGCCAGCGTGCCGCAGGTGCACATTTTCTCGC 470 TGCTGGCGACGTGGCTCGGCTGCCTGGTGGCCAGCGTGCCGCAGGTGCACATTTTCTCGC 157 V--L--A--T--W--L--G--C--L--V--A--S--V--P--Q--V--H--I--F--S--

541 TGCGCGAAGTGGCGGACGGCGTCTTCGATTGCTGGGCGGTCTTCATCCAGCCCTGGGGAC 530 TGCGCGAAGTGGCGGACGGCGTCTTCGATTGCTGGGCGGTCTTCATCCAGCCCTGGGGAC 177 L--R--E--V--A--D--G--V--F--D--C--W--A--V--F--I--Q--P--W--G--

601 CCAAGGCCTACGTCACGTGGATCACGCTCGCCGTCTACATTGTACCGGTCATCGTGCTGG

225 590 CCAAGGCCTACGTCACGTGGATCACGCTCGCCGTCTACATTGTACCGGTCATCGTGCTGG 197 P--K--A--Y--V--T--W--I--T--L--A--V--Y--I--V--P--V--I--V--L--

661 CCGCCTGCTATGGTCTCATCAGCTTCAAGATCTGGCAGAATCTGCGACTCAAGACGGCAG 650 CCGCCTGCTATGGTCTCATCAGCTTCAAGATCTGGCAGAATCTGCGACTCAAGACGGCAG 217 A--A--C--Y--G--L--I--S--F--K--I--W--Q--N--L--R--L--K--T--A--

S W 721 CCGCGGCGGCAGCTGCCGAGGGGAGTGACGCAGCCGGTGGAGCTGGCCGTGCGGCGTTGG 710 CCGCGGCGGCAGCTGCCGAGGGGAGTGACGCAGCCGGTGGAGCTGGCCGTGCGGCGTTGG 237 A--A--A--A--A--A--E--G--S--D--A--A--G--G--A--G--R--A--A--L--

781 CACGGGTCAGTAGTGTCAAGCTTATCTCCAAGGCCAAAATCCGCACAGTGAAGATGACCT 770 CACGGGTCAGTAGTGTCAAGCTTATCTCCAAGGCCAAAATCCGCACAGTGAAGATGACCT 257 A--R--V--S--S--V--K--L--I--S--K--A--K--I--R--T--V--K--M--T--

841 TCATCATTGTTCTGGCCTTCATCGTGTGCTGGACGCCTTTCTTCTTCGTGCAGATGTGGA 830 TCATCATTGTTCTGGCCTTCATCGTGTGCTGGACGCCTTTCTTCTTCGTGCAGATGTGGA 277 F--I--I--V--L--A--F--I--V--C--W--T--P--F--F--F--V--Q--M--W--

901 GCGTCTGGGACGTCAATGCGCCCAAAGAAGCTTCTGCCTTCATCATTGCCATGCTCTTGG 890 GCGTCTGGGACGTCAATGCGCCCAAAGAAGCTTCTGCCTTCATCATTGCCATGCTCTTGG 297 S--V--W--D--V--N--A--P--K--E--A--S--A--F--I--I--A--M--L--L--

961 CCAGCCTCAACAGCTGCTGCAACCCATGGATCTACATGCTCTTCACGGGCCATCTCTTCC 950 CCAGCCTCAACAGCTGCTGCAACCCATGGATCTACATGCTCTTCACGGGCCATCTCTTCC 317 A--S--L--N--S--C--C--N--P--W--I--Y--M--L--F--T--G--H--L--F--

1021 ACGAACTCGTGCAGCGCTTCCTCTGCTGCTCTGCTCGGTACCTGAAGGGCAGCCGGCCTG 1010 ACGAACTCGTGCAGCGCTTCCTCTGCTGCTCTGCTCGGTACCTGAAGGGCAGCCGGCCTG 337 H--E--L--V--Q--R--F--L--C--C--S--A--R--Y--L--K--G--S--R--P--

Y Y 1081 GAGAGACGAGCATTAGCAAGAAAAGCAACTCCTCCACCTTCGTCCTGAGTCGTCGCAGCT 1070 GAGAGACGAGCATTAGCAAGAAAAGCAACTCCTCCACCTTCGTCCTGAGTCGTCGCAGCT 357 G--E--T--S--I--S--K--K--S--N--S--S--T--F--V--L--S--R--R--S--

R 1141 CGAGTCAGAGGAGCTGTTCTCAACCATCCTCGGCATGAGCCATAGGCCTGGCCCACCAGG 1130 CGAGTCAGAGGAGCTGTTCTCAACCATCCTCGGCATGA...... 377 S--S--Q--R--S--C--S--Q--P--S--S--A--*-......

Y 1201 CCAGCCGCTGGGTGGTGTAGCCGCCGATCTTCCCCTCTGGTGGCTGTGCATGGAGCTGTA ......

S Y S 1261 TATGGTGCCTATTGATTGGTTTGCATCCCTCCTGTCCTTGGGCTGGTTAGATTCTGTGTC ......

226 Arginine Vasopressin, Avp

Location: Chromosome 2: 130,580,620-130,582,554 reverse strand.

Gene: This transcript is a product of gene ENSMUSG00000037727

This gene has 1 transcript (splice variant)

Transcript: Avp-001 ENSMUST00000046001

Name Transcript ID Length Protein Biotype CCDS RefSeq Flags

Avp- 577 168 aa NM_009732 GENCODE

ENSMUST00000046001 Protein CCDS16746 001 bp (view) NP_033862 basic coding

qPCR mouse primers:

F: TCGCCAGGATGCTCAACAC

R: TTGGTCCGAAGCAGCGTC

Key:

1 ACAGTGCCCACCTATGCTCGCCAGGATGCTCAACACTACGCTCTCCGCTTGTTTCCTGAG ...... ATGCTCGCCAGGATGCTCAACACTACGCTCTCCGCTTGTTTCCTGAG ...... -M--L--A--R--M--L--N--T--T--L--S--A--C--F--L--S

Y M 61 CCTGCTGGCCTTCTCCTCCGCCTGCTACTTCCAGAACTGCCCAAGAGGCGGCAAGAGGGC 48 CCTGCTGGCCTTCTCCTCCGCCTGCTACTTCCAGAACTGCCCAAGAGGCGGCAAGAGGGC 16 --L--L--A--F--S--S--A--C--Y--F--Q--N--C--P--R--G--G--K--R--A

121 CATCTCTGACATGGAGCTGAGACAGTGTCTCCCCTGCGGCCCGGGCGGCAAAGGACGCTG 108 CATCTCTGACATGGAGCTGAGACAGTGTCTCCCCTGCGGCCCGGGCGGCAAAGGACGCTG 36 --I--S--D--M--E--L--R--Q--C--L--P--C--G--P--G--G--K--G--R--C

R R 181 CTTCGGACCAAGCATCTGCTGCGCGGACGAGCTGGGCTGCTTCGTGGGCACCGCCGAGGC 168 CTTCGGACCAAGCATCTGCTGCGCGGACGAGCTGGGCTGCTTCGTGGGCACCGCCGAGGC 56 --F--G--P--S--I--C--C--A--D--E--L--G--C--F--V--G--T--A--E--A

227 Y 241 GCTGCGCTGCCAGGAGGAGAACTACCTGCCCTCGCCCTGCCAGTCCGGCCAGAAGCCCTG 228 GCTGCGCTGCCAGGAGGAGAACTACCTGCCCTCGCCCTGCCAGTCCGGCCAGAAGCCCTG 76 --L--R--C--Q--E--E--N--Y--L--P--S--P--C--Q--S--G--Q--K--P--C

R Y 301 CGGGAGCGGGGGCCGCTGCGCCGCCGTGGGCATCTGCTGCAGCGACGAGAGCTGCGTGGC 288 CGGGAGCGGGGGCCGCTGCGCCGCCGTGGGCATCTGCTGCAGCGACGAGAGCTGCGTGGC 96 --G--S--G--G--R--C--A--A--V--G--I--C--C--S--D--E--S--C--V--A

K S 361 CGAGCCCGAGTGCCACGACGGTTTTTTCCGCCTCACCCGCGCTCGGGAGCCAAGCAACGC 348 CGAGCCCGAGTGCCACGACGGTTTTTTCCGCCTCACCCGCGCTCGGGAGCCAAGCAACGC 116 --E--P--E--C--H--D--G--F--F--R--L--T--R--A--R--E--P--S--N--A

R 421 CACACAGCTGGACGGCCCTGCTCGGGCGCTGCTGCTAAGGCTGGTACAGCTGGCTGGGAC 408 CACACAGCTGGACGGCCCTGCTCGGGCGCTGCTGCTAAGGCTGGTACAGCTGGCTGGGAC 136 --T--Q--L--D--G--P--A--R--A--L--L--L--R--L--V--Q--L--A--G--T

M 481 ACGGGAGTCCGTGGATTCTGCCAAGCCCCGGGTCTACTGAGCCATCGCCCCCACGCCTCG 468 ACGGGAGTCCGTGGATTCTGCCAAGCCCCGGGTCTACTGA...... 156 --R--E--S--V--D--S--A--K--P--R--V--Y--*-......

541 CCCCTACAGCATGGAAAATAAACTTTTAAAAACTGCA ......

Corticotrophin releasing hormone, Crh

Location: Chromosome 3: 19,693,401-19,695,396 reverse strand.

Gene: This transcript is a product of gene ENSMUSG00000049796

This gene has 1 transcript (splice variant)

Transcript: Crh-201 ENSMUST00000061294

Name Transcript ID Length Protein Biotype CCDS RefSeq Flags

Crh- 1320 187 aa NM_205769 GENCODE

ENSMUST00000061294 Protein CCDS17258 201 bp (view) NP_991338 basic coding

qPCR mouse primers: F: GCAAATGCTGCGTGCTTTCT R: GTTAGGGGCGCTCTCTTCTC

Key:

228

1 AGGCAAATGCTGCGTGCTTTCTGAAGAGGGTCGACATTATAAAATCTCACTCCAGGCTCT ......

61 GGTGTGGAGAAACTCAGAGCCCAAGTACGTTGAGAGACTGAAGAGAAAGGGAAAAGGCAA ......

** 121 AAGAAAAAAAGAAGAGAAAGGAGAAGAGGAAGAAAACCTGCAGGAGGCATCCTGAGAGAA ......

Y 181 GTCCCTCTGCAGAGGCAGCAGTGCGGGCTCACCTACCAAGGGAGGAGAAGAGAGCGCCCC ......

241 TAACATGCGGCTGCGGCTGCTGGTGTCCGCGGGCATGCTGCTGGTGGCTCTGTCGTCCTG ....ATGCGGCTGCGGCTGCTGGTGTCCGCGGGCATGCTGCTGGTGGCTCTGTCGTCCTG ....-M--R--L--R--L--L--V--S--A--G--M--L--L--V--A--L--S--S--C

S 301 CCTGCCTTGCAGGGCCCTGCTCAGCAGGGGATCCGTCCCCCGAGCGCCGCGGGCCCCGCA 57 CCTGCCTTGCAGGGCCCTGCTCAGCAGGGGATCCGTCCCCCGAGCGCCGCGGGCCCCGCA 19 --L--P--C--R--A--L--L--S--R--G--S--V--P--R--A--P--R--A--P--Q

361 GCCCTTGAATTTCTTGCAGCCGGAGCAGCCCCAGCAACCTCAGCCGGTTCTGATCCGCAT 117 GCCCTTGAATTTCTTGCAGCCGGAGCAGCCCCAGCAACCTCAGCCGGTTCTGATCCGCAT 39 --P--L--N--F--L--Q--P--E--Q--P--Q--Q--P--Q--P--V--L--I--R--M

421 GGGTGAAGAATACTTCCTCCGCCTGGGGAATCTCAACAGAAGTCCCGCTGCTCGGCTGTC 177 GGGTGAAGAATACTTCCTCCGCCTGGGGAATCTCAACAGAAGTCCCGCTGCTCGGCTGTC 59 --G--E--E--Y--F--L--R--L--G--N--L--N--R--S--P--A--A--R--L--S

481 CCCCAACTCCACGCCCCTCACCGCGGGTCGCGGCAGCCGCCCCTCGCACGACCAGGCTGC 237 CCCCAACTCCACGCCCCTCACCGCGGGTCGCGGCAGCCGCCCCTCGCACGACCAGGCTGC 79 --P--N--S--T--P--L--T--A--G--R--G--S--R--P--S--H--D--Q--A--A

541 GGCTAACTTTTTCCGCGTGTTGCTGCAGCAGCTGCAGATGCCTCAGCGCTCGCTCGACAG 297 GGCTAACTTTTTCCGCGTGTTGCTGCAGCAGCTGCAGATGCCTCAGCGCTCGCTCGACAG 99 --A--N--F--F--R--V--L--L--Q--Q--L--Q--M--P--Q--R--S--L--D--S

R S

229 601 CCGCGCGGAGCCGGCCGAACGCGGCGCCGAGGATGCCCTCGGTGGCCACCAGGGGGCGCT 357 CCGCGCGGAGCCGGCCGAACGCGGCGCCGAGGATGCCCTCGGTGGCCACCAGGGGGCGCT 119 --R--A--E--P--A--E--R--G--A--E--D--A--L--G--G--H--Q--G--A--L

661 GGAGAGGGAGAGGCGGTCGGAGGAGCCGCCCATCTCTCTGGATCTCACCTTCCACCTTCT 417 GGAGAGGGAGAGGCGGTCGGAGGAGCCGCCCATCTCTCTGGATCTCACCTTCCACCTTCT 139 --E--R--E--R--R--S--E--E--P--P--I--S--L--D--L--T--F--H--L--L

M M 721 GCGGGAAGTCTTGGAAATGGCCCGGGCAGAGCAGTTAGCTCAGCAAGCTCACAGCAACAG 477 GCGGGAAGTCTTGGAAATGGCCCGGGCAGAGCAGTTAGCTCAGCAAGCTCACAGCAACAG 159 --R--E--V--L--E--M--A--R--A--E--Q--L--A--Q--Q--A--H--S--N--R

781 GAAACTGATGGAGATTATCGGGAAATGAAATGTTGCGCTTGGCCAAAACGATTCTGCATT 537 GAAACTGATGGAGATTATCGGGAAATGA...... 179 --K--L--M--E--I--I--G--K--*-......

18s Ribosomal RNA/ Rn18s (Housekeeping gene primer) LOCUS NR_003278 1870 bp rRNA linear ROD 31- JAN-2014 DEFINITION Mus musculus 18S ribosomal RNA (Rn18s), ribosomal RNA. ACCESSION NR_003278 VERSION NR_003278.3 GI:374088232 KEYWORDS RefSeq. SOURCE Mus musculus (house mouse) ORGANISM Mus musculus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Mus; Mus. REFERENCE 1 (bases 1 to 1870) ORIGIN 1 acctggttga tcctgccagg tagcatatgc ttgtctcaaa gattaagcca tgcatgtcta 61 agtacgcacg gccggtacag tgaaactgcg aatggctcat taaatcagtt atggttcctt 121 tggtcgctcg ctcctctcct acttggataa ctgtggtaat tctagagcta atacatgccg 181 acgggcgctg accccccttc ccgggggggg atgcgtgcat ttatcagatc aaaaccaacc 241 cggtgagctc cctcccggct ccggccgggg gtcgggcgcc ggcggcttgg tgactctaga 301 taacctcggg ccgatcgcac gccccccgtg gcggcgacga cccattcgaa cgtctgccct 361 atcaactttc gatggtagtc gccgtgccta ccatggtgac cacgggtgac ggggaatcag 421 ggttcgattc cggagaggga gcctgagaaa cggctaccac atccaaggaa ggcagcaggc 481 gcgcaaatta cccactcccg acccggggag gtagtgacga aaaataacaa tacaggactc 541 tttcgaggcc ctgtaattgg aatgagtcca ctttaaatcc tttaacgagg atccattgga 601 gggcaagtct ggtgccagca gccgcggtaa ttccagctcc aatagcgtat attaaagttg 661 ctgcagttaa aaagctcgta gttggatctt gggagcgggc gggcggtccg ccgcgaggcg

230 721 agtcaccgcc cgtccccgcc ccttgcctct cggcgccccc tcgatgctct tagctgagtg 781 tcccgcgggg cccgaagcgt ttactttgaa aaaattagag tgttcaaagc aggcccgagc 841 cgcctggata ccgcagctag gaataatgga ataggaccgc ggttctattt tgttggtttt 901 cggaactgag gccatgatta agagggacgg ccgggggcat tcgtattgcg ccgctagagg 961 tgaaattctt ggaccggcgc aagacggacc agagcgaaag catttgccaa gaatgttttc 1021 attaatcaag aacgaaagtc ggaggttcga agacgatcag ataccgtcgt agttccgacc 1081 ataaacgatg ccgactggcg atgcggcggc gttattccca tgacccgccg ggcagcttcc 1141 gggaaaccaa agtctttggg ttccgggggg agtatggttg caaagctgaa acttaaagga 1201 attgacggaa gggcaccacc aggagtgggc ctgcggctta atttgactca acacgggaaa 1261 cctcacccgg cccggacacg gacaggattg acagattgat agctctttct cgattccgtg 1321 ggtggtggtg catggccgtt cttagttggt ggagcgattt gtctggttaa ttccgataac 1381 gaacgagact ctggcatgct aactagttac gcgacccccg agcggtcggc gtcccccaac 1441 ttcttagagg gacaagtggc gttcagccac ccgagattga gcaataacag gtctgtgatg 1501 cccttagatg tccggggctg cacgcgcgct acactgactg gctcagcgtg tgcctaccct 1561 gcgccggcag gcgcgggtaa cccgttgaac cccattcgtg atggggatcg gggattgcaa 1621 ttattcccca tgaacgagga attcccagta agtgcgggtc ataagcttgc gttgattaag 1681 tccctgccct ttgtacacac cgcccgtcgc tactaccgat tggatggttt agtgaggccc 1741 tcggatcggc cccgccgggg tcggcccacg gccctggcgg agcgctgaga agacggtcga 1801 acttgactat ctagaggaag taaaagtcgt aacaaggttt ccgtaggtga acctgcggaa 1861 ggatcattaa //

231 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F, Atp5j (Housekeeping gene primer)

Location: Chromosome 16: 84,827,866-84,835,625 reverse strand.

Gene: This transcript is a product of gene ENSMUSG00000022890

This gene has 12 transcripts (splice variants)

Transcript: Atp5j-001 ENSMUST00000023608

Name Transcript ID Length Protein Biotype CCDS RefSeq Flags

Atp5j- 809 108 aa NM_016755 GENCODE

ENSMUST00000023608 Protein CCDS28283 001 bp (view) NP_058035 basic coding qPCR mouse primers: F: TATTGGCCCAGAGTATCAGCA R: GGGGTTTGTCGATGACTTCAAAT

Key:

1 GCGGCCGCGGGGGGGGACAAGCCCCGGAACCAGGCCGCCCGCGCGGGCCCGCGCTCCCCG ......

N 61 CCAGCCCGCGTCCGCAACTCACCAGCTCCGGAATTCGTCCCGTGGCCCTAGCCCGCGCTT ......

R 121 CCACAGCCGGCTGGGAACGGCGGCGGCGCGGGCTCCAGGTACAGCGCCTCTCCGGGCGAG ......

181 CCGCGCCGCTCCCGCGAGTAGCAGGAGGCGTCCGGTCGCAGACTCCCTTCGAGGCGCTTC ......

R 241 CTGTCCGGTGAGCGTCGAACGACTGAAGCCGCGGCCCATAGTGCCTTGCGATGGCGGGTA ......

232 301 GGCGTGTGTAGGCGGAGCCAGGGCCGGAAGTAGAACGGTGGCGGCGGCGGTGACTCTGGC ......

361 AGCTCGGGACTCAGTGCAAGTACAGAGACTCAGCCATGGTTCTGCAGAGGATCTTCAGGC ...... ATGGTTCTGCAGAGGATCTTCAGGC ...... -M--V--L--Q--R--I--F--R--

Y 421 TCTCCTCTGTCCTTCGGTCAGCAGTCTCTGTGCATTTGAAGAGGAACATTGGTGTTACAG 26 TCTCCTCTGTCCTTCGGTCAGCAGTCTCTGTGCATTTGAAGAGGAACATTGGTGTTACAG 9 L--S--S--V--L--R--S--A--V--S--V--H--L--K--R--N--I--G--V--T--

481 CTGTGGCCTTTAATAAGGAACTTGATCCTGTACAGAAACTCTTCGTGGACAAGATAAGAG 86 CTGTGGCCTTTAATAAGGAACTTGATCCTGTACAGAAACTCTTCGTGGACAAGATAAGAG 29 A--V--A--F--N--K--E--L--D--P--V--Q--K--L--F--V--D--K--I--R--

541 AGTACAAATCAAAGCGACAGGCATCTGGAGGACCTGTTGATATTGGCCCAGAGTATCAGC 146 AGTACAAATCAAAGCGACAGGCATCTGGAGGACCTGTTGATATTGGCCCAGAGTATCAGC 49 E--Y--K--S--K--R--Q--A--S--G--G--P--V--D--I--G--P--E--Y--Q--

601 AAGATCTGGACAGAGAGCTTTATAAGCTTAAACAAATGTATGGTAAAGGAGAGATGGATA 206 AAGATCTGGACAGAGAGCTTTATAAGCTTAAACAAATGTATGGTAAAGGAGAGATGGATA 69 Q--D--L--D--R--E--L--Y--K--L--K--Q--M--Y--G--K--G--E--M--D--

R R 661 CATTTCCTACCTTCAAATTTGATGATCCCAAATTTGAAGTCATCGACAAACCCCAGTCCT 266 CATTTCCTACCTTCAAATTTGATGATCCCAAATTTGAAGTCATCGACAAACCCCAGTCCT 89 T--F--P--T--F--K--F--D--D--P--K--F--E--V--I--D--K--P--Q--S--

Y 721 GAGGAACATACAAAATCCATGTGGTAATTTGTCATGAATTAGTTGTACAACTAATCAAAA 326 GA...... *-......

781 AATTCAAATAAACATTCATTTCACAGTTA

233 Appendix-3: qPCR amplification efficiency graphs

This section shows the amplification efficiency graphs for all the test genes and housekeeping genes.

Oxt Mean Ct values 35.00 y = -3.2026x + 29.163 R² = 0.8545 30.00 E = 105.23% 25.00 20.00 15.00

Mean Ct Ct Value Mean 10.00 5.00 0.00 -0.5 0 0.5 1 1.5 2 Log cDNA Conc.

Oxtr Mean Ct Values y = -3.4662x + 33.138 40.00 R² = 0.9928 35.00 94.31% 30.00 25.00 20.00 15.00

MeanCtValue 10.00 5.00 0.00 -0.5 0 0.5 1 1.5 2 Log cDNA Conc.

234 Avp Mean Ct Values y = -3.2769x + 29.378 35.00 R² = 0.9914 30.00 101.91% 25.00 20.00 15.00

10.00 MeanCtValue 5.00 0.00 -0.5 0 0.5 1 1.5 2 Log cDNA Conc.

Crh Mean Ct Values y = -3.4672x + 32.929 40.00 R² = 0.9905 35.00 E= 94.28% 30.00 25.00 20.00 15.00

MeanCtValue 10.00 5.00 0.00 -0.5 0 0.5 1 1.5 2 Log cDNA Conc.

Atp5j Mean Ct Values y = -3.4073x + 28.478 35.00 R² = 0.985 30.00 E= 96.56% 25.00 20.00 15.00

10.00 MeanCtValue 5.00 0.00 -0.5 0 0.5 1 1.5 2 Log cDNA Conc.

235 Rn18s Mean Ct values y = -3.0769x + 12.575 14.00 R² = 0.9913 12.00 E= 111.35% 10.00 8.00 6.00

MeanCtValue 4.00 2.00 0.00 -0.5 0 0.5 1 1.5 Log cDNA Conc.

Figure-43: Calculating efficiency of primers. The graphs show the linear plots drawn using the mean CT values obtained for the serial dilutions of qPCR optimisation. The slope of this plot was used to calculate the amplification efficiencies of the Oxt, Oxtr, Avp, Crh, Atp5j and Rn18s primers.

236 Appendix-4: Graphical representation of behavioural data from Chapter-2

Maternal behaviours of Gtf2ird1 mice reared by B6 mothers

Gtf2ird1 deletion affects maternal behaviour Suckling:

Day6: GLM; F2,29=16.09;p<0.001; Day10: GLM; F2,29=14.67;p<0.001; Day14: GLM; F2,29=66.11;p<0.001

Gtf2ird1 deletion affects maternal behaviour Nursing:

Day6: GLM; F2,29=7.21;p=0.003; Day10: GLM; F2,29=11.51;p<0.001; Day14: GLM; F2,29=0.32;p=0.727

Gtf2ird1 deletion does not affect maternal behaviour Sniffing:

Day6: GLM; F2,29=1.97;p=0.159; Day10: GLM; F2,29=2.39;p=0.112; Day14:GLM; F2,29=0.53;p=0.595.

237

Gtf2ird1 deletion affects maternal behaviour Time spent with pups:

Day6: GLM; F2,29=2.17;p=0.142; Day10: GLM; F2,29=21.66;p<0.001 Day14: GLM; F2,29=33.31;p<0.001.

Gtf2ird1 deletion affects maternal behaviour Retrieval time:

Day6: GLM; F2,29= 4.51;p=0.022; Day10: GLM; F2,27=3.22;p=0.058 Day14: GLM; F2,27=0.28;p=0.760

Gtf2ird1 deletion does not affect Autogrooming:

Day6: GLM; F2,29=0.98;p=0.391; Day10: GLM; F2,29=0.28;p=0.973 Day14: GLM; F2,29=2.47;p=0.104

238 Gtf2ird1 deletion affects Other Activities:

Day6: GLM; F2,29=3.94;p=0.032. Day10: GLM; F2,29=4.05;p=0.030. Day14: GLM; F2,29=5.11;p=0.013.

Gtf2ird1 deletion affects Feeding:

Day6: GLM; F2,29=0.83;p=0.447; Day10: GLM; F2,29=15.82;<0.001 Day14: GLM; F2,29=14.40;p<0.001

Gtf2ird1 deletion does not affect Drinking:

Day6: GLM; F2,29=1.58;p=0.224; Day10: GLM; F2,29=2.35;=0.115 Day14: GLM; F2,29=2.97;p=0.069

239 Maternal behaviours of Gtf2ird1 mice reared by knockout mothers

This section shows the graphs plotted from all the behavioural data collected during the PhD.

Gtf2ird1 deletion affects maternal behaviour Suckling:

Day6: GLM; F2,52=14.86; p=0.16; Day10: GLM; F2,56=8.08; p <0.0001 Day14: GLM; F2,56=14.86; p<0.0001

Gtf2ird1 deletion affects maternal behaviour Nursing:

Day6: GLM; F2,52=1.81; p=0.158; Day10: GLM; F2,56=3.80; p=0.015. Day14: GLM; F2,56=3.42; p=0.023.

Gtf2ird1 deletion does not affect maternal behaviour Sniffing:

Day6: GLM; F2,51=0.62;p=0.606; Day10: GLM; F2,55=1.19;p=0.321; Day14: GLM; F2,55=3.15;p=0.052

240 Gtf2ird1 deletion affects maternal behaviour Time spent with pups:

Day6: GLM; F2,52=3.29;=0.028; Day10: GLM; F2,56=4.82;p=0.005 Day14: GLM; F2,56=11.46;p<0.0001.

Gtf2ird1 deletion affects maternal behaviour Retrieval time:

Day6: GLM; F2,57=0.47;p=0.070; Day10: GLM; F2,57=1.64;p=0.019 Day14: GLM; F2,57=2.83;p=0.046.

Gtf2ird1 deletion does not affect Autogrooming:

Day6: GLM; F2,57=0.56;p=0.64; Day10: GLM; F2,57=2.04;p=0.12 Day14: GLM; F2,57=0.26;p=0.86.

241 Gtf2ird1 deletion affects Other Activities:

Day6: GLM; F2,57=2.74;p=0.05; Day10: GLM; F2,57=4.21;p=0.009 Day14: GLM; F2,57=0.25;p=0.07.

Gtf2ird1 deletion affects Feeding:

Day6: GLM; F2,55=4.85;p=0.901; Day10: GLM; F2,56=14.93;p=0.000 Day14: GLM; F2,56; p<0.0001.

Gtf2ird1 deletion does not affect Drinking:

Day6: GLM; F2,55=0.99;p=0.405; Day10: GLM; F2,56=3.38;p=0.24 Day14: GLM; F2,56=1.83;p=0.153.

242

Appendix-5: Graphical representation of behavioural data from Chapter-3

Gtf2ird2 deletion does not affect the quality of nests built and the percentage of nestlet shredded:

5 100

3 shredded Nest Score Nest 0 1 Wt Het Homo Wt Het Homo %of nestlet Maternal Genotype Maternal Genotype

Quality of nests built (Wald Chi-square=0.136, p=0.712). Percentage of nestlet shredded (Wald Chi-square=0.00, p=0.995).

Gtf2ird2 deletion affects maternal behaviour Suckling:

(n)

(n) 40 40

Day6 30 30 Day14 20 20 10 10 0 0 wt het homo Wt Het Homo

suckled of No. times Maternal Genotype suckled of No. times Maternal Genotype

Day 6: GLM; F2,30=0.07;p=0.932 and day 14 GLM; F2,30=1.11;p=0.343.

Gtf2ird2 deletion does NOT affect short term weight gain in pups:

3 3

(g) 2

(g)

2 (g)

1 1 1

Day6 Day10

0 0 Day14 0 Short term weight gain gain weight term Short

Wt Het Homo gain weight term Short Wt Het Homo Wt Het Homo Short term weight gain gain weight term Short Maternal Genotype Maternal Genotype Maternal Genotype

Day 6: GLM; F2,31=0.10;p=0.902. Day 10: GLM; F2,31=1.12; p=0.339; Day 14: GLM;

F2,31=0.12;p=0.888.

243 Gtf2ird2 deletion does NOT affect maternal behaviour nursing:

Day6: GLM;F2,33=1.29;p=0.289. Day10: GLM;F2,33=2.21;p=0.125. Day14: GLM; F2,33=0.09;p=0.909.

Gtf2ird2 deletion does NOT affect maternal behaviour sniffing:

6 10 10

5 (n)

5 (n) (n)

10 10

14 14

Day6 Day 0 0 0 Day

Wt Het Homo Wt Het Homo Wt Het Homo

No. of times Sniffed Sniffed of No. times No. of times Sniffed Sniffed of No. times Sniffed of No. times Maternal Genotype Maternal Genotype Maternal Genotype

Day6: GLM;F2,33=0.72;p=0.495. Day10:GLM;F2,33=0.36;p=0.701. Day14:GLM; F2,33=2.87;p=0.071.

Gtf2ird2 deletion does NOT affect maternal behaviour pup retrieval:

15 15

10 10

(n)

5 5 5

Day6 Day14 0 Day10 0 0

Wt Het Homo No. of pups retrieved retrieved of No. pups

No. of pups retrieved retrieved of No. pups Wt Het Homo Wt Het Homo No. of pups retrieved retrieved of No. pups Maternal Genotype Maternal Genotype Maternal Genotype

Day6: GLM; F2,33=1.75; p=0.190. Day10:GLM; F2,33=2.68;p=0.083. Day14: GLM;F2,33=0.26; p=0.769.

Gtf2ird2 deletion does NOT affect maternal behaviour licking: 30 30

20 20

20 (n)

(n)

(n) 10 10

Day14 Day6 0 Day10 0

No. of pups licked licked of No. pups No. of pups licked licked of No. pups No. of pups licked licked of No. pups Wt Het Homo Wt Het Homo Wt Het Homo Maternal Genotype Maternal Genotype Maternal Genotype

244

Day6: GLM;F2,33=0.56;p=0.577. Day10:GLM;F2,33=0.635;p=0.536. Day14: GLM; F2,33=0.16;p=0.848.

Gtf2ird2 deletion does NOT affect maternal behaviour nest building:

(n)

(n) 10

Day6 Day10 5 4 5Day14 2 0

built nest built 0 0

built nest built

No. of times mother mother of No. times

No. of times mother mother of No. times No. of times mother mother of No. times Wt Het Homo Wt Het Homo Wt Het Homo Maternal Genotype Maternal Genotype Maternal Genotype

Day6: GLM; F2,33=1.715; p=0.196. Day10: GLM; F2,33=1.36;p=0.270. Day14: GLM; F2,33=2.236;p=0.123

Gtf2ird2 deletion does NOT affect feeding and drinking behaviour of mother:

10 10

)

(n)

5 5

Day6 5

Day10 Day14

0 0 0

and drank drank and and drank drank and

Wt Het Homo drank and No. of times mother fed mother times of No.

No. of times mother fed mother times of No. Wt Het Homo No. of times mother fed mother times of No. Wt Het Homo Maternal Genotype Maternal Genotype Maternal Genotype

Day6: GLM;F2,33=1.61;p=0.216. Day10: GLM;F2,33=1.934;p=0.161. Day14: GLM;

F2,33=1.05;p=0.362.

Gtf2ird2 deletion does NOT affect autogrooming:

(n) 10 6

10 (n) (n)

Day10 Day 6 Day 5 Day14 5 2

0 0 0

No. of times mother mother times of No.

autogroomed autogroomed autogroomed autogroomed

Wt Het Homo Wt Het Homo autogroomed Wt Het Homo

Maternal Genotype Maternal Genotype Maternal Genotype Day6: GLM; F2,33=1.079; p=0.352. Day10: GLM; F2,33=0.234;p=0.792. Day14: GLM; F2,33=1.419;p=0.256

245

Gtf2ird2 deletion does NOT affect other activities:

did 50 40

Day 6(n) Day (n)

20 (n) 20

Day10 Day14

0 activities other 0 0

did did

did other activities activities other did No. of times mother mother times of No.

Wt Het Homo Wt Het Homo mother times of No. Wt Het Homo other activities activities other No. of times mother mother times of No. Maternal Genotype Maternal Genotype Maternal Genotype

Day6: GLM;F2,32=0.27;p=0.766. Day10: GLM;F2,32=4.53;p=0.18; Day14: GLM; F2,32=0.99;p=0.382.

Gtf2ird2 deletion does NOT affect the number of times mother drops pups during

retrieval on Day 6 and 14:

4 (n) 4

3 14 Day 6(n) Day 2 Day 2 1

0 0

No. of times mother mother times of No.

No. of times mother mother times of No. dropped pups pups dropped

Wt Het Homo pups dropped Wt Het Homo

Maternal Genotype Maternal Genotype Day6: GLM; F2,33=0.33;p=0.721. Day14: GLM; F2,33=0.29;p=0.748

Gtf2ird2 deletion significantly affects the total amount of time mother spent with pups

on day 10 and 14:

15 15 (Mins) (Mins) * * 10 10 10 5 5 0 0 0

Wt Het Homo Wt Het Homo Wt Het Homo Time spent on Day14 on spent Time Day10 on spent Time Maternal Genotype Maternal Genotype Maternal Genotype

Day6(Mins) on spent Time

Day6: GLM; F2,32=67.59; p=0.711. Day10: GLM; F2,32=5.74;p=0.007. Day14: GLM; F2,32=3.57; p=0.040.

246 Appendix-6: QTL maps from Chapter-4

Figure: QTL maps of weekly weight change of B6 pups which were nursed by BXD mothers. Panel A: No suggestive or significant QTL was found for weight change between day 1 to 6. Panel B: Two low suggestive QTL were found for weight change between day 6 to 10. Panel C: Two low suggestive QTL were found for weight change between day 10 to 14. Panel D: No suggestive or significant QTL was found for weight change between day 14 to 21. This shows that there was no IGEs of BXD maternal genotype on weekly growth patterns of B6 pups.

247

248