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The cardiovascular system is the first organ to develop in vertebrates. Cardiovascular development requires the coordination of several overlapping events that give rise to and then remodel the heart and its associated vascular structures into a network that effectively distributes blood into all organs without disrupting their tissue architecture. Neuropilin 1 (NRP1) is a transmembrane receptor that is required for the normal development of the cardiovascular system. NRP1 has binding sites for two crucial vascular regulators: vascular endothelial growth factor (VEGF) and class III semaphorins (SEMA3). During my PhD, I have examined the role of NRP1 and its ligands and co-receptors in the morphogenesis of the mammalian aortic arch. This highly asymmetric vessel complex originates in the embryo from 5 pairs of pharyngeal arch arteries (PAAs), which initially form in a rostro-caudal and symmetric fashion to connect the aortic sac with the dorsal aorta, but subsequently remodel to help separate the pulmonary and systemic circulation. Accordingly, failed PAA formation or remodelling causes congenital heart disease. I have examined Nrp1-null mice to define the time window in which NRP1 acts. Moreover, I have combined wholemount immunostaining with lineage tracing in mouse embryos to distinguish whether PAA formation involves NRP1 regulation of vasculogenesis from vascular progenitors, vessel sprouting/fusion from the dorsal aorta and aortic sac or neural crest/smooth muscle cell-mediated vascular remodelling. Finally, I have analysed tissue- and ligand-specific mutants to identify the cell types that rely on NRP1 signalling. My findings suggest that NRP1 has several consecutive roles at different stages of PAA morphogenesis. In a complementary study, I have contributed to work investigating molecular mechanisms that establish vascular exclusion zones in the embryonic mouse. I have found that two distinct SEMA3 proteins converge on the PLXND1 receptor to repel sprouting vessels around epithelia to limit vascular invasion of the developing motor column and bronchiolar smooth muscle and prevent excessive vessel sprouting around somites. My findings thereby add significantly to current knowledge of the regulatory interactions that underpin normal cardiovascular development and whose dysfunction causes congenial cardiovascular diseases.

The overriding goal of this thesis was to further re ne our understanding of the genetic architecture of cardiomyopathies, Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) and Hypertrophic Cardiomyopathy (HCM). 407 patients with ARVC and 957 with HCM had 41 cardiomyopathy and other putative candidate genes sequenced. By comparing these cohorts against each other and against ethnicity and phenotype matched controls, insights were gained into the role of di erent types of genetic variants in these conditions. This in part involved utilising 4500 Whole Exome Sequences (WES) that are part of the UCLexomes consortium, an in-house dataset that aggregates a diverse set of studies. High throughput DNA sequencing technologies, WES or Whole Genome Sequencing (WGS) are revolutionizing the diagnosis and novel gene discovery for rare disorders. As the eld transitions from the early discovery for Mendelian and near Mendelian diseases to more complex and oligo-genic diseases, there is substantial bene t in being able to combine data across studies, performing the type of meta-analysis for cases and controls that have proven to be so successful for Genome-Wide Association Studies (GWAS). However, WGS and WES are substantially more a ected by sequencing errors and technical artefacts than genome-wide genotyping arrays. As a consequence, meta-analysis of sequence based association studies are often dominated by spurious associations, which result in technical limitations. Here, we show that it is possible to take advantage of the type of mixed models developed initially to control for population structure in GWAS studies, and apply these ideas to control for technical artefacts. In an attempt to ascertain the role of CNVs in HCM, these data were examined for the presence of rare causative CNVs. 12 CNVs were identi ed from an initial Read Depth approach. 4 of these were subsequently validated by CoNIFER, a bioinformatics method, and Array Comparative Genomic Hybridisation (aCGH): one large deletion in MYBPC3, one large deletion in PDLIM3, one duplication of the entire TNNT2 gene and one large duplication in LMNA. These results show that the role of CNVs in HCM is small and highlight the e ciency of this two step-strategy.

Cardiovascular diseases (CVDs) - including stroke and heart failure - are the leading cause of death worldwide. More people die from CVDs each year than any other cause. Magnetic resonance imaging (MRI) is a powerful technique which is now routinely used for imaging these diseases as it offers high-resolution anatomical detail, exquisite soft-tissue contrast and assessment of function such as tissue water content, oxygenation, metabolism, vascular blood flow and microvascular perfusion. This thesis focuses on the development of MRI techniques for use in pre-clinical animal models of cardiovascular diseases, with a focus on stroke and heart disease. Firstly, in chapter 3, the continued development of an in-house MRI sequence known as extravascular convectography (EVAC) for measuring the flow of interstitial fluid is described. A series of phantom experiments were conducted to assess the sensitivity of the sequence to slow flowing fluid. Next, an in vivo repeatability and reproducibility study was conducted before finally the technique was applied to a rat model of stroke. In chapter 4, a pair of studies was carried out using recently established, advanced cardiac imaging techniques. In the first study, CINE and late gadolinium-enhanced inversion recovery (LGE IR) imaging were used to assess cardiac structure and function in a Prox1-deficient genetic mouse model of dilated cardiomyopathy. In the second part of the chapter, a multi-parametric MRI study - incorporating CINE, LGE IR, arterial spin labeling and T2-mapping - was conducted in a mouse model of reperfused myocardial infarction to assess the extent of area-at-risk and compare with gold-standard histological staining. Finally, in chapter 5, the development of a retrospective high-temporal resolution (HTR) CINE MRI sequence for assessing cardiac diastolic function is described and compared with pulsed wave Doppler ultrasound, which is the currently-accepted standard for measuring diastolic function. The HTR-CINE sequence was established, validated and optimised in phantoms and naïve mouse hearts. Repeatability studies were then carried out to ensure the robustness of the technique before application to a mouse model of myocardial infarction. The overall aim of the research in this thesis is the development of MRI techniques for application to experimental models of cardiovascular disease.

Chromatin remodelling provides a key mechanism for the regulation of gene expression through dynamic alterations in nucleosome occupancy at promoters and enhancers. Haploinsufficiency for the ATP-dependent chromatin remodeller chromodomain-helicase-DNA-binding protein 7 (CHD7) causes human CHARGE syndrome. CHARGE is characterised by a distinct pattern of congenital anomalies, including cardiovascular malformations, and has traditionally been considered a neurocristopathy. However, a number of the congenital heart defects associated with CHARGE cannot be attributed to disruption to the neural crest alone. This thesis therefore addresses the tissue-specific requirements and roles for CHD7 during cardiogenesis. CHD7 protein is shown to be present throughout the developing heart until E13.5. Conditional ablation of Chd7 in the early cardiogenic mesoderm results in embryonic lethality due to severe cardiovascular defects. These include haemorrhaging and oedema, major venous and arterial pole malformations, and disruption to cardiac innervation and vascularisation. To further dissect tissue-specific requirements for CHD7, cardiomyocyte-, second heart field- and endothelial-specific knockdowns were also performed. Each cross results in a milder subset of the cardiac defects observed after mesodermal ablation, indicating that CHD7 is required in multiple lineages within the cardiogenic mesoderm. Microarray analysis and validation by in situ hybridisation were used to identify genes dysregulated in the heart following mesodermal Chd7 ablation. These included components of the Semaphorin and Slit-Robo signalling pathways, which have known roles in heart development. Furthermore, aberrant expression of genes involved in calcium handling within cardiomyocytes is seen. Excitation-contraction coupling is disrupted in mutant embryonic cardiomyocytes, demonstrating relevance of the gene expression changes at the cellular level. This work reveals a requirement for CHD7 in mesodermal cardiac progenitors for both inflow and outflow tract development. Novel pathways are identified downstream of CHD7 activity in the developing heart, including the extracellular Semaphorin and Slit-Robo pathways, as well as components of the excitation-contraction coupling machinery.

The immense challenge of annotating the entire mouse genome has stimulated development of cutting-edge imaging technologies in a drive for novel information. These techniques promise to improve our understanding of the genes involved in embryo development, at least one third of which have been shown to be essential. Aligning advanced imaging technologies with biological needs will be fundamental to maximising the number of phenotypes discovered in the coming years. International efforts are underway to meet this challenge through an integrated and sophisticated approach to embryo phenotyping, which will include advanced imaging tools. This thesis investigates advanced imaging methodologies and computational image analysis techniques for mouse embryo phenotyping using magnetic resonance imaging (MRI). Additionally, the novel application of an emerging method called photoacoustic imaging is demonstrated for imaging mouse embryos in utero. First, the lack of tissue staining capabilities that currently limits embryo MR imaging was addressed by investigating the MRI staining properties of two readily available contrast agents and their underlying contrast enhancement mechanisms. A methodological framework was developed for high-throughput screening of embryos using diffusion MRI and implemented to study the splotch mouse model of human neural tube defects. A validation study was carried out to comprehensively assess the accuracy of volumetric measurements generated using a computational image analysis method called segmentation propagation. Finally, an all-optical photoacoustic scanner and novel time-reversal image reconstruction algorithm were developed, enabling photoacoustic imaging of whole embryos in utero. Overall, this thesis presents advanced imaging methodologies and computational image analysis techniques that may form an essential part of the toolkit available for annotating the mouse genome and facilitate identification of novel phenotypes in the coming years.

This thesis describes research into the developmental roles of IFT80, a protein component of intraflagellar transport (IFT) complex B. Mutations in IFT80 have been shown to be causative in both asphyxiating thoracic dystrophy (ATD) and short rib polydactyly (SRP) type III. Both diseases are autosomal recessive chondrodysplasias and share clinical and radiological similarities including shortening of the long bones and constriction of the thoracic cage. A genetrapped Ift80 line was used to investigate the role of Ift80 during murine development. This mouse model is a hypomorph due to low level wildtype transcript production. Hypomorphic levels of Ift80 are often embryonic lethal highlighting a key role for Ift80 in development. In cases where Ift80gt/gt mice survive to postnatal stages they phenocopy both ATD and SRP type III by exhibiting growth retardation, shortening of the long bones, constriction of the ribcage, and polydactyly. Analysis of the Hedgehog signalling pathway, a pathway shown to be abrogated in multiple models of IFT dysfunction, showed that reduction in Ift80 expression levels leads to reduced activity of the pathway. Processing of the key transcription factor Gli3 into its repressor form was seen to be altered leading to an abnormal activator/repressor ratio which may account for the observed pathway disruption. Widespread disruption to Wnt signalling, a pathway not commonly investigated in IFT models, was also seen in this line. This defective cellular signalling was not accompanied by the loss or malformation of cilia as seen in some knockout models of other IFT component genes. Phenotypes indicative of defects in cilia structure or function such as situs inversus, cystic renal disease and retinal degeneration were also not observed. SF-TAP and SILAC proteomics approaches were used to identify protein binding partners of IFT80. These suggested IFT80 associates primarily with a non-core subset of complex B proteins. A putative novel binding partner, NUDC was also identified in these screens. NUDC is known to play a role in dynein transport along microtubules, and was shown to localise to a region near the base of the cilium. A model is proposed in which an interaction between Ift80 and Nudc influences dynein entry into the cilium, and that reduced efficiency of this interaction in Ift80gt/gt mice causes a mild abrogation of retrograde transport leading to disrupted cellular signalling and thus to the skeletal phenotypes observed.