Atherosclerosis is one of the main diseases responsible for the high global mortality rate involving heart and blood vessel disorders. The build-up of fatty materials in the inner wall of the human artery prevents sufficient oxygen and nutrients reaching the organs of the body. Atherosclerosis is a chronic, long term condition, which develops and progresses over time; however, the disease does not present any symptoms until an advanced stage is reached, which results in potential permanent debility and sometimes sudden death. This thesis is concerned with the progression of atherosclerosis in an artery with mild stenosis that has resulted in a 30% reduction in its diameter. To this end, data on the low wall shear stress has been correlated with the atherosclerotic prone region. In a stenosed artery, this region corresponds to the separation zone that is formed distal to the lumen reduction. Atherosclerosis is a complex phenomenon, and not only involves wall shear stress, but also cellular interactions. Previous research has shown that even in the absence of wall biological effects, the blood cell distribution is strongly influenced by the hydrodynamics of the fluid. The mechanisms of blood cell distribution and the dynamic behaviour of the blood flow were investigated by developing a physical model of the stenosed artery, and by using particles to represent the presence of the blood cells. Particle Image Velocimetry system was employed and the size of particles were the 10μm and 20μm. The flow field was characterised and the particle distribution was measured. The characteristics of steady flow in the stenosed artery at Reynolds numbers of 250 and 320 revealed the importance of fluid inertia and the shear gradient distal to stenosis. Unequal distribution of the particles modelling the blood cells was observed, as more particles occupied the recirculation zones than the high shear region and central jet. The particle migration was found to depend on the particle size, particle concentration and fluid flow rates. The results suggested that the presence of similar effects in the real human arterial system may be significant to the progression of atherosclerotic plaques. At lower Reynolds number of 130, a particle depleted layer was observed at the wall region. In physiological flow the cell free layer will prevent the transport of oxygen and nitrogen oxide (NO) to the muscle tissues. A numerical method was used to simulate the flow characteristics measured in the experiment. The numerical results revealed the importance of the hydrodynamic mechanism of particle migration. Drag and lift forces were found to affect the residence time of particles in the recirculation region. The findings of this work have suggested that for a complex geometry like a large stenosed artery at physiological flow rates, hydrodynamic forces are important in cell migration in the flow separation zone. Even without biological forces, the cells migrate to the low wall shear stress region. For computational dynamics studies, this study has demonstrated the need for higher-order modelling at the cellular level in order to establish the particle migration mechanisms.

Directed cell migration is essential in both physiological and pathological situations. Many guidance cues have been extensively investigated in the past decades, to be able to regulate directional cell migration, including chemical, physiological and haptotactic cues. In the past years, we have focused on the roles of physiological electric field in the guidance of directed cell migration. It is well accepted that physiological electric fields exist both extracellularly and intracellularly with different functions, and interestingly, endogenous EFs exist in not only physiological but also pathological events. For instance, the existence of a small current in developing embryos which is also known as the endogenous electric field has been tested, such as the blastopore in Xenopus, chicken embryos, and etc. It has been also demonstrated that endogenous electric fields exist at the wound edges of injured cornea and skin. Physiological electric field is among many other guidance cues controlling an important cellular response – directed cell migration in response to stimuli, a phenomenon named electrotaxis or galvanotaxis. We and others have extensively demonstrated that physiological EFs could control directional cell migration, and that several signalling pathways are required for the regulation of such event. In the current study, we used Dictyostelium model to further explore the molecular and genetic mechanisms of how electrotaxis is controlled, by extensively investigating candidate molecules and genes in such regulation. We found that PI3K, PTEN and Ras signalling pathways are largely involved in the regulation of electrotaxis, Ras plays more dominant roles in this event in comparison with PI3K and PTEN, which only partially contributed towards the electrotactic response of the Dictyostelium cells. Asymmetric redistribution of signalling molecules are shown to play an essential role in the initiation and maintenance of the electrotactic response of the cells.

In vertebrates the cornea must maintain its transparency throughout adult life to ensure sight, and understanding the mechanisms underpinning corneal homeostasis are fundamental to developing new treatments to cure or prevent blindness. This study investigated the role the planar cell polarity pathway plays in the directed migration of adult corneal epithelial cells, in maintaining the homeostatic environment of the eye and during wound healing. RT-PCR confirmed, for the first time, the expression of multiple core PCP genes within human corneal epithelial (HCE) cells. Components of the PCP pathway were pharmacologically and genetically manipulated during wound healing of corneal epithelial cells and the importance of the downstream target JNK, and core PCP gene Vangl2, during wound healing was demonstrated. Manipulation of core PCP components was found also to directly affect the ability of HCE cells to realign and migrate in response to physical topographical cues in vitro. This study therefore indicated that PCP may regulate the directed migration of corneal epithelial cells as they travel over the basement membrane. Using conditional knockout mice the loss of Vangl2, a core PCP gene, and its effect on both planar and the apical-basal polarity of the corneal epithelium was investigated. Severe morphological defects were observed in Vangl2-null mice indicative of underlying problems in apical-basal polarity of the epithelial cells. The basement membrane of Vangl2-null cells was largely absent in vivo, which suggested that at least some of the planar defects were secondary to an unexpected failure of apical-basal polarity. This study has shown for the first time that PCP plays a crucial role in the maintenance of an adult vertebrate tissue, particularly during wound healing and maintenance of the corneal epithelium. It has also indicated a role for the core PCP gene, Vangl2, in setting up apical-basal polarity of these adult cells.

The anterior visceral endoderm (AVE) is a specialised subpopulation of the visceral endoderm (VE), a single layer simple epithelium that surrounds the extra-embryonic ectoderm and epiblast of the egg cylinder stage embryo. Initially induced at the distal tip of the egg cylinder, AVE cells undergo a stereotypic migration towards the prospective anterior, stopping at the interface between the underlying epiblast and extra-embryonic ectoderm (ExE). Previous research has shown that membrane enrichment of Dvl2 is present in the VE overlying the epiblast (Epi-VE). In this thesis I confirm the presence of planar cell polarity (pep) signalling in this region by assaying the subcellular localisation of additional core pep proteins Vangl2 and Daaml. I show that null embryos of the Nodal antagonist Lefty1 exhibit ectopic membrane enrichment of Dvl2 and a previously unreported AVE over-migration phenotype. Furthermore, using pharmacological inhibition of Nodal signalling I show that the TGF~ protein Nodal modulates pep signalling in the YE. Utilising DIe and confocal microscopy I perform detailed time-lapse analyses of the VE to quantify the dynamic cell behaviour and topology. Using this assay I show that wild-type embryos exhibit dynamic cell movement, which is regionally restricted to the Epi-VE. Analysis of Leftyl-/- and ROSA26lyn-Celsr-l mutants, both of which exhibit disrupted pep signalling and AVE over-migration phenotypes, indicates that normal VE dynamics and topology are disrupted. The results of this quantitation indicate that these mutants exhibit increased cell migration and neighbour exchange across the YE. These data show that regional restriction of movement is lost and results in the AVE over-migration phenotypes observed. Together these results show that regionally restricted pep signalling in the VE acts to modulate cell behaviour and topology, which in turn determines the regional restriction and normal end-point of AVE migration.

Immune cell migration and chemotaxis plays a key role in immune response. Further research to study the mechanisms of immune cell migration and to develop clinical applications requires advanced experimental tools. Microfluidic devices can precisely apply chemical gradient signals to cells, which is advantageous in quantifying cell migratory response. However, most existing microfluidic systems are impractical to use without specialized facilities and research skills, which hinders their broad use in biological and medical research communities. In this thesis, we integrated several new developments in microfluidic gradient generating devices, compact imaging systems, on-chip cell isolation, cell patterning, and rapid data analysis, to provide an easy-to-use and practical solution for immune cell migration and chemotaxis experiments. Using these systems, we quantitatively studied neutrophil migration for both research and clinical applications. First, we developed a compact USB microscope-based Microfluidic Chemotaxis Analysis System (UMCAS), which integrates microfluidic devices, live cell imaging, environmental control, and data analysis to provide an inexpensive and compact solution for rapid microfluidic cell migration and chemotaxis experiments with real-time result reporting. To eliminate the lengthy cell preparation from large amounts of blood, we developed a simple all-on-chip method for magnetic isolation of untouched neutrophils directly from small volumes of blood, followed by chemotaxis testing on the same microfluidic device. Using these systems, we studied neutrophil migration in gradients of different chemoattractants, such as interleukin-8 (IL-8), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and clinical sputum samples from Chronic Obstructive Pulmonary Disease (COPD) patients. Previous studies have shown that COPD is correlated with neutrophil infiltration into the airways through chemotactic migration. The thesis work is the first application of the microfluidic platform to quantitatively characterizing neutrophil chemotaxis to sputum samples from COPD patients. Our results show increased neutrophil chemotaxis to COPD sputum compared to control sputum from healthy individuals. The level of COPD sputum induced neutrophil chemotaxis was correlated with the patient’s spirometry data. Collectively, the research in this thesis provides novel microfluidic systems for neutrophil migration and chemotaxis analysis in both basic research and clinical applications. The developed microfluidic systems will find broad use in cell migration related applications. / May 2016

Cell migration is essential for embryonic development, it occurs in adult organisms during processes like wound healing and its misregulation contributes to pathological conditions such as metastasis. Despite this, most studies of cell migration have been undertaken in vitro. Ena/VASP proteins, believed to be actin anti-capping proteins, have been studied extensively in fibroblasts in vitro, and using Drosophila macrophages (hemocytes) within the developing embryo, the role of the Drosophila homologue of Mena, Ena, is investigated in vivo. Consistent with data from fibroblasts in vitro, Ena localised to regions of actin dynamics within migratory hemocytes, where this protein stimulated lamellipodial dynamics and positively regulated filopodial number and length. However, whilst overexpression of Ena/VASP proteins in fibroblasts reduced migration speeds, Ena overexpression in hemocytes dramatically increased migration speeds in three different assays. This positive regulation of migration speed closely resembled the increased motility of breast cancer cells that overexpress Mena and evidence presented here, suggests that this key difference may be explained by spatial constraints that are imposed upon cells within three dimensional environments. Indeed, such constraints prevented ruffling, a more detrimental form of retraction, in hemocytes in vivo. Furthermore, fibroblasts overexpressing Mena in vitro form membrane ruffles more frequently. Therefore Ena/VASP proteins drive migration by enhancing lamellipodial protrusion, but in certain environments these protrusions are lost as ruffles slowing migration. The method by which Ena regulates lamellipodial protrusion and migration speeds was then investigated: Ena increased Fascin-mediated actin bundling and the number of Fascin rich-actin bundles that coalesced. Analysis of individual actin bundles revealed that coalescence increased protrusion rate and that both protrusion rate and coalescence, increased cell migration speeds. This suggests that Ena facilitates an increase in cell migration by promoting the coalescence of Fascin bundles, and positions Ena as a key regulator of migration speeds in vivo.

by Yung, Kim Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 135-153). / Abstract --- p.i / Acknowledgments --- p.iv / List of content --- p.v / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Neural crest cells and cardiac neural crest cells --- p.1 / Chapter 1.2 --- The role of cardiac neural crest cells in the septation of the outflow tract --- p.5 / Chapter 1.3 --- Neural crest-related malformations --- p.8 / Chapter 1.4 --- Early changes in cardiovascular development induced by neural crest ablation --- p.11 / Chapter 1.5 --- Experimental strategies commonly employed in tracing the premigratory neural crest cells --- p.14 / Chapter 1.6 --- Objectives of the present study --- p.21 / Chapter Chapter 2 --- Location of the cardiac neural crest along the neural axis in the mouse embryo --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.29 / Chapter 2.2.1 --- Preparation of DiI --- p.29 / Chapter 2.2.2 --- Embryo collection --- p.29 / Chapter 2.2.3 --- Microinjection of DiI --- p.30 / Chapter 2.2.4 --- Isolation of tissue fragments from the lateral neural epithelium --- p.31 / Chapter 2.2.5 --- Dil labelling of the donor fragment isolated from the lateral neural epithelium --- p.32 / Chapter 2.2.6 --- Grafting of DiI labelled fragments from the lateral neural epithelium --- p.32 / Chapter 2.2.7 --- Embryo culture --- p.33 / Chapter 2.2.8 --- Examination of cultured embryos --- p.34 / Chapter 2.2.9 --- Cryosection --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Development of the cultured embryos in control and experimental groups --- p.36 / Chapter 2.3.2 --- Location of the cardiac neural crest region along the neural axis --- p.38 / Chapter 2.4 --- Discussion --- p.44 / Chapter 2.4.1 --- Development of embryos in vitro --- p.44 / Chapter 2.4.2 --- Comparison of the two methods for tracing cell migration: focal labelling and orthotopic grafting --- p.49 / Chapter 2.4.3 --- Location of the cardiac neural crest region along the neural tube --- p.53 / Chapter Chapter 3 --- Initial and terminal stages of cardiac neural crest cell migration --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Examination of the initial and terminal stages of migration of cardiac neural crest cells by haematoxylin and eosin (H&E) staining --- p.62 / Chapter 3.2.2 --- Preparation of WGA-Au --- p.62 / Chapter 3.2.3 --- Collection of embryos for microinjection of WGA-Au --- p.63 / Chapter 3.2.4 --- WGA-Au labelling of the presumptive cardiac neural crest region --- p.64 / Chapter 3.2.5 --- Embryo culture --- p.65 / Chapter 3.2.6 --- Examination of cultured embryos --- p.66 / Chapter 3.2.7 --- Silver enhancement staining --- p.66 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Initial stage of cardiac neural crest migration studied by haematoxylin and eosin staining and silver enhancement staining --- p.67 / Chapter 3.3.2 --- Terminal stage of cardiac neural crest migration studied by haematoxylin and eosin staining and silver enhancement staining --- p.69 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Wheat germ agglutinin-gold conjugate (WGA-Au) as a cell marker --- p.71 / Chapter 3.4.2 --- Initial stage for cardiac neural crest cell migration --- p.72 / Chapter 3.4.3 --- Terminal stage for cardiac neural crest cell migration --- p.74 / Chapter Chapter 4 --- Migration pathways of cardiac neural crest cells… --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and Methods --- p.82 / Chapter 4.2.1 --- Preparation of DiI --- p.82 / Chapter 4.2.2 --- Preparation of WGA-Au --- p.82 / Chapter 4.2.3 --- Embryo collection --- p.82 / Chapter 4.2.4 --- Microinjection of WGA-Au and DiI --- p.82 / Chapter 4.2.5 --- Isolation of tissue fragments from the lateral neural epithelium --- p.83 / Chapter 4.2.6 --- WGA-Au labelling of the donor fragments from the lateral neural epithelium --- p.83 / Chapter 4.2.7 --- DiI labelling of the donor neural epithelium --- p.83 / Chapter 4.2.8 --- Grafting of WGA-Au or DiI-labelled donor tissues from the lateral neural epithelium --- p.83 / Chapter 4.2.9 --- Coating of latex beads by WGA-Au --- p.83 / Chapter 4.2.10 --- Microinjection of WGA-Au-coated latex beads --- p.84 / Chapter 4.2.11 --- Embryo culture --- p.84 / Chapter 4.2.12 --- Examination of cultured embryos --- p.85 / Chapter 4.2.13 --- Silver enhancement staining of the WGA-Au labelled sections --- p.85 / Chapter 4.2.14 --- Cryosection --- p.85 / Chapter 4.3 --- Results --- p.86 / Chapter 4.3.1 --- Distribution of labelled cells after WGA-Au labelling or orthotopic grafting --- p.86 / Chapter 4.3.2 --- Distribution of labelled cells after DiI labelling or orthotopic grafting --- p.88 / Chapter 4.3.3 --- Distribution of latex beads --- p.90 / Chapter 4.4 --- Discussion --- p.92 / Chapter 4.4.1 --- Methodology --- p.92 / Chapter 4.4.2 --- Migration pathways of the cardiac neural crest cells --- p.94 / Chapter 4.4.3 --- Migration of latex beads --- p.98 / Chapter Chapter 5 --- Derivatives of cardiac neural crest cells in the developing mouse heart --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Materials and Methods --- p.110 / Chapter 5.2.1 --- DiI labelling of the cardiac neural crest region of the mouse embryo --- p.110 / Chapter 5.2.2 --- Collection of the embryonic hearts --- p.111 / Chapter 5.2.3 --- Heart organ culture --- p.111 / Chapter 5.2.4 --- Cryosectioning --- p.112 / Chapter 5.2.5 --- Paraffin wax sectioning --- p.113 / Chapter 5.2.6 --- Immunohistochemical staining --- p.113 / Chapter 5.3 --- Results --- p.118 / Chapter 5.3.1 --- Distribution of 2H3 positive cells in the heart developedin vivo --- p.118 / Chapter 5.3.2 --- Development of the heart at 10.5 d.p.c. in organ culture --- p.119 / Chapter 5.3.3 --- Distribution of DiI labelled cells in the heart one day after organ culture --- p.119 / Chapter 5.3.4 --- Distribution of 2H3 positive cells in the hearts one day after organ culture --- p.120 / Chapter 5.4 --- Discussion --- p.121 / Chapter 5.4.1 --- Relationship between 2H3 positive cells and cardiac conduction system --- p.121 / Chapter 5.4.2 --- Development of the mouse embryonic hearts in vitro --- p.123 / Chapter 5.4.3 --- Distribution patterns of the 2H3 immunopositive cellsin the hearts developed in vitro and in vivo --- p.125 / Chapter 5.4.4 --- Relationship between the DiI labelled cells and2H3 immunopositive cells --- p.125 / Chapter 5.4.5 --- Genes that express in the cardiac neural crest cells --- p.127 / Chapter Chapter 6 --- Conclusion --- p.129 / References --- p.135 / Appendix --- p.154

The gene pebble (pbl) encodes a Rho GEF required for the migration of mesoderm cells during Drosophila gastrulation. The spreading of mesoderm cells is controlled by the FGF signalling pathway acting through the FGF receptor Heartless (Htl). Pbl represents an important downstream component of this FGF pathway and activates the Rho GTPase Rac, but the regulation of Pbl by FGF signalling is unclear. Furthermore Pbl is required for the formation of the actin-myosin contractile ring during cytokinesis by activation of RhoA. The purpose of this work is to find molecular links between Pbl and the Htl signalling pathway and get insight into the localization and regulation of Pbl during mesoderm cell migration A genetic screen is carried out to find genes that interact with Pbl and are involved in mesoderm development. A gain-of-function variant of Pbl that causes defects in eye morphology was used to find genetic interactors. Results of a screen using chromosomal deletions and an EMS-based screen revealed candidates, which genetically interact with Pbl and are required for mesoderm cell migration. In addition, a structure-function analysis of the Pbl protein was performed. The data revealed an important role of the PH domain for the localization of Pbl at the cell cortex. Moreover the PH domain is indispensable for the function of Pbl in mesoderm migration. Furthermore an important role for the C-terminal tail of Pbl for the regulation of the protein was shown, which might be regulated by FGF signalling. The C-terminal tail is required for the stability of the protein outside the nucleus and it regulates the substrate preference of Pbl for Rac and Rho. Furthermore indication was found that the function of the C-terminal tail possibly is regulated by phosphorylation of Ser825 in the C-terminal tail. Mutation of this site affects the function of Pbl during mesoderm migration but not in cytokinesis. Therefore phosphorylation of the C-terminal tail might regulate or enhance the exchange activity of Pbl for Rac. The localization and function of Pbl depends on the PH domain and the C-terminal tail of Pbl. Both domains have distinct roles during Pbl function in mesoderm cell migration.

Several genes thought to confer susceptibility to dyslexia have been identified, and the purpose of this study is to 1) determine the expression pattern of one of these gene products and 2) characterize the function of the product of one of these genes, namely KIAA0319-Like (KIAA0319L), using the developing chick visual system as a model. Whole mount in situ hybridization was performed for KIAA0319L on embryonic day (E)3 – E5 and in situ hybridization on sections was performed at later stages. Engineered microRNA (miRNA) constructs targeting KIAA0319L were prepared and their specificity and efficiency for knocking down KIAA0319L were tested. miRNAs were electroporated in E5 optic tecta (OT). Embryos were sacrificed at E12. OT were removed, sectioned and analyzed. Results demonstrate that KIAA0319L is expressed in the developing chick visual system. Knockdown of KIAA0319L in the OT results in abnormal migration indicating that KIAA0319L is necessary for this process.

Several genes thought to confer susceptibility to dyslexia have been identified, and the purpose of this study is to 1) determine the expression pattern of one of these gene products and 2) characterize the function of the product of one of these genes, namely KIAA0319-Like (KIAA0319L), using the developing chick visual system as a model. Whole mount in situ hybridization was performed for KIAA0319L on embryonic day (E)3 – E5 and in situ hybridization on sections was performed at later stages. Engineered microRNA (miRNA) constructs targeting KIAA0319L were prepared and their specificity and efficiency for knocking down KIAA0319L were tested. miRNAs were electroporated in E5 optic tecta (OT). Embryos were sacrificed at E12. OT were removed, sectioned and analyzed. Results demonstrate that KIAA0319L is expressed in the developing chick visual system. Knockdown of KIAA0319L in the OT results in abnormal migration indicating that KIAA0319L is necessary for this process.