The tricellular vertex-specific adhesion molecule Sidekick facilitates polarised cell intercalation during Drosophila axis extension.

In epithelia, tricellular vertices are emerging as important sites for the regulation of epithelial integrity and function. Compared to bicellular contacts, however, much less is known. In particular, resident proteins at tricellular vertices were identified only at occluding junctions, with none known at adherens junctions (AJs). In a previous study, we discovered that in Drosophila embryos, the adhesion molecule Sidekick (Sdk), well-known in invertebrates and vertebrates for its role in the visual system, localises at tricellular vertices at the level of AJs. Here, we survey a wide range of Drosophila epithelia and establish that Sdk is a resident protein at tricellular AJs (tAJs), the first of its kind. Clonal analysis showed that two cells, rather than three cells, contributing Sdk are sufficient for tAJ localisation. Super-resolution imaging using structured illumination reveals that Sdk proteins form string-like structures at vertices. Postulating that Sdk may have a role in epithelia where AJs are actively remodelled, we analysed the phenotype of sdk null mutant embryos during Drosophila axis extension using quantitative methods. We find that apical cell shapes are abnormal in sdk mutants, suggesting a defect in tissue remodelling during convergence and extension. Moreover, adhesion at apical vertices is compromised in rearranging cells, with apical tears in the cortex forming and persisting throughout axis extension, especially at the centres of rosettes. Finally, we show that polarised cell intercalation is decreased in sdk mutants. Mathematical modelling of the cell behaviours supports the notion that the T1 transitions of polarised cell intercalation are delayed in sdk mutants, in particular in rosettes. We propose that this delay, in combination with a change in the mechanical properties of the converging and extending tissue, causes the abnormal apical cell shapes in sdk mutant embryos

Advances in quantitative microscopy

Microscopy allows us to peer into the complex deeply shrouded world that the cells of our body grow and thrive in. With the emergence of automated digital microscopes and software for anlysing and processing the large numbers of image that they produce; quantitative microscopy approaches are now allowing us to answer ever larger and more complex biological questions. In this thesis I explore two trends. Firstly, that of using quantitative microscopy for performing unbiased screens, the advances made here include developing strategies to handle imaging data captured from physiological models, and unsupervised analysis screening data to derive unbiased biological insights. Secondly, I develop software for analysing live cell imaging data, that can now be captured at greater rates than ever before and use this to help answer key questions covering the biology of how cells make the decision to arrest or proliferate in response to DNA damage. Together this thesis represents a view of the current state of the art in high-throughput quantitative microscopy and details where the field is heading as machine learning approaches become ever more sophisticated.Open Acces

The role of GLI2 in human basal cell carcinoma tumourigenesis

PhDAbnormal Sonic Hedgehog (SHH) signalling leads to increased transcriptional activation of its downstream effector, GLI2, which is implicated in the pathogenesis of a variety of human tumours, including human basal cell carcinoma (BCC). However, little is known about the molecular mechanisms underlying the tumourigenic role of GLI2 in human skin keratinocytes. This study examines the effects of inducible and stable expression of constitutively active GLI2 (GLI2:N) oncogenic transcription factor, on immortalised human epidermal keratinocytes. It is shown here that GLI2:N overexpressing N/TERT keratinocytes display gene expression patterns and phenotypic characteristics reminiscent of those observed in human BCC in vivo. It is also shown for first time, that expression of GLI2:N in N/TERT keratinocytes is sufficient to induce accumulation of binucleated/tetraploid cells as evidenced by an increase in G2/M phase of the cell cycle and binucleate cell counting, and to promote polyploidy and aneuploidy, in the absence of increased cell death or apoptosis. This cell cycle deregulation is accompanied by strong activation of anti-apoptotic protein BCL-2 and simultaneous suppression of important cell-cycle regulators such as 14-3-3σ and CDK inhibitor p21WAF1/CIP1, with no change in p53 protein levels, indicating uncontrolled proliferation of cells with ploidy abnormalities and/or DNA damage. Consistently, it is shown that p21WAF1/CIP1 protein is also absent in human BCC tumours and that forced overexpression of GLI2:N renders human keratinocytes resistant to apoptosis mediated by ultraviolet B (UVB, 290-320 nm), one of the most important etiological factors in BCC formation. Karyotype analysis of GLI2:N N/TERT keratinocytes further demonstrates that overexpression of GLI2:N induces numerical (tetraploidy, polyploidy, aneuploidy) and structural instability in N/TERT keratinocytes including chromosomal translocations and double minute chromosomes. Furthermore, β-catenin activation is the most common alteration observed during aberrant WNT signalling, and is often implicated in the development of human carcinogenesis and metastasis. In this study it is shown that GLI2:N induction induces nuclear accumulation of β-catenin in keratinocyte cell culture and in the basal layer of organotypic skin rafts, similar to human BCCs. In addition, several WNT genes were found to be upregulated upon GLI2:N induction, while β-catenin transcriptional activity is increased upon stable and conditional expression of GLI2:N. Overall these data give new insights for the possible mechanisms that mediate the tumourigenic potential of GLI2

Determining a role for the calcium-sensing receptor (CaR) in pulmonary development

In the adult, changes in free ionized plasma calcium concentration (Ca2+ G) are monitored by the G-protein-coupled, extracellular calcium-sensing receptor, CaR, but whether CaR plays a role in lung development is unknown. CaR has the potential to be a key regulator of Ca2+ dependent cell fate during development. It is hypothesized that extracellular calcium is an important extrinsic factor that modulates the intrinsic lung developmental programme, through activation of the CaR. CaR is expressed in the developing mouse lung in the pseudoglandular phase, from embryonic day 10.5 (El0.5), with a peak of expression at El2.5 and a subsequent decrease by El8, after which the receptor is absent. Lung branching morphogenesis in vitro is sensitive to Ca2+ G, being negatively modulated by the higher, fetal (i.e., 1.7 mM) Ca2+ 0 yet optimal at physiological adult Ca2+ Q (i.e., 1.05-1.2 mM). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high Ca2+G on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances lung distension, fluid secretion and intracellular calcium signalling. Conditions which are restrictive to branching and fluid secretion can be rescued by manipulating Ca2+ 0 in the culture medium. Lung explant cultures from the current mouse model of CaR inactivation respond in a similar manner to Ca2+ Q and the calcimimetic R-568. These results indicate the presence of expression of a functional CaR splice variant, which is detected at El 1.5, 12.5 and 15.5 in CaR knockout lungs. The observations presented here support a novel role for the CaR in preventing hyperplastic lung disease in utero and present two potential models for its mode of action within this system.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Determining a role for the calcium-sensing receptor (CaR) in pulmonary development

In the adult, changes in free ionized plasma calcium concentration (Ca2+ G) are monitored by the G-protein-coupled, extracellular calcium-sensing receptor, CaR, but whether CaR plays a role in lung development is unknown. CaR has the potential to be a key regulator of Ca2+ dependent cell fate during development. It is hypothesized that extracellular calcium is an important extrinsic factor that modulates the intrinsic lung developmental programme, through activation of the CaR. CaR is expressed in the developing mouse lung in the pseudoglandular phase, from embryonic day 10.5 (El0.5), with a peak of expression at El2.5 and a subsequent decrease by El8, after which the receptor is absent. Lung branching morphogenesis in vitro is sensitive to Ca2+ G, being negatively modulated by the higher, fetal (i.e., 1.7 mM) Ca2+ 0 yet optimal at physiological adult Ca2+ Q (i.e., 1.05-1.2 mM). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high Ca2+G on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances lung distension, fluid secretion and intracellular calcium signalling. Conditions which are restrictive to branching and fluid secretion can be rescued by manipulating Ca2+ 0 in the culture medium. Lung explant cultures from the current mouse model of CaR inactivation respond in a similar manner to Ca2+ Q and the calcimimetic R-568. These results indicate the presence of expression of a functional CaR splice variant, which is detected at El 1.5, 12.5 and 15.5 in CaR knockout lungs. The observations presented here support a novel role for the CaR in preventing hyperplastic lung disease in utero and present two potential models for its mode of action within this system

A Modular and Open-Source Framework for Virtual Reality Visualisation and Interaction in Bioimaging

Life science today involves computational analysis of a large amount and variety of data, such as volumetric data acquired by state-of-the-art microscopes, or mesh data from analysis of such data or simulations. The advent of new imaging technologies, such as lightsheet microscopy, has resulted in the users being confronted with an ever-growing amount of data, with even terabytes of imaging data created within a day. With the possibility of gentler and more high-performance imaging, the spatiotemporal complexity of the model systems or processes of interest is increasing as well. Visualisation is often the first step in making sense of this data, and a crucial part of building and debugging analysis pipelines. It is therefore important that visualisations can be quickly prototyped, as well as developed or embedded into full applications. In order to better judge spatiotemporal relationships, immersive hardware, such as Virtual or Augmented Reality (VR/AR) headsets and associated controllers are becoming invaluable tools. In this work we present scenery, a modular and extensible visualisation framework for the Java VM that can handle mesh and large volumetric data, containing multiple views, timepoints, and color channels. scenery is free and open-source software, works on all major platforms, and uses the Vulkan or OpenGL rendering APIs. We introduce scenery's main features, and discuss its use with VR/AR hardware and in distributed rendering. In addition to the visualisation framework, we present a series of case studies, where scenery can provide tangible benefit in developmental and systems biology: With Bionic Tracking, we demonstrate a new technique for tracking cells in 4D volumetric datasets via tracking eye gaze in a virtual reality headset, with the potential to speed up manual tracking tasks by an order of magnitude. We further introduce ideas to move towards virtual reality-based laser ablation and perform a user study in order to gain insight into performance, acceptance and issues when performing ablation tasks with virtual reality hardware in fast developing specimen. To tame the amount of data originating from state-of-the-art volumetric microscopes, we present ideas how to render the highly-efficient Adaptive Particle Representation, and finally, we present sciview, an ImageJ2/Fiji plugin making the features of scenery available to a wider audience.:Abstract Foreword and Acknowledgements Overview and Contributions Part 1 - Introduction 1 Fluorescence Microscopy 2 Introduction to Visual Processing 3 A Short Introduction to Cross Reality 4 Eye Tracking and Gaze-based Interaction Part 2 - VR and AR for System Biology 5 scenery — VR/AR for Systems Biology 6 Rendering 7 Input Handling and Integration of External Hardware 8 Distributed Rendering 9 Miscellaneous Subsystems 10 Future Development Directions Part III - Case Studies C A S E S T U D I E S 11 Bionic Tracking: Using Eye Tracking for Cell Tracking 12 Towards Interactive Virtual Reality Laser Ablation 13 Rendering the Adaptive Particle Representation 14 sciview — Integrating scenery into ImageJ2 & Fiji Part IV - Conclusion 15 Conclusions and Outlook Backmatter & Appendices A Questionnaire for VR Ablation User Study B Full Correlations in VR Ablation Questionnaire C Questionnaire for Bionic Tracking User Study List of Tables List of Figures Bibliography Selbstständigkeitserklärun

Directional migration of the neural crest: an interplay between contact inhibition of locomotion and co-attraction

Collective cell migration is recognised as a common feature of cell movement in vivo. Despite its importance for both morphogenesis and malignant progression, little is known about how directional and coordinated cell movements are regulated collectively. An interesting example of collective migration in vivo is the migration of Neural Crest (NC) cells. NC cells are highly migratory and multipotent embryonic cells, which migrate with remarkable directionality and coordination. In this thesis it is proposed that a mayor force in allowing NC collective directionality is given by local cell-cell interactions. Two main interactions, a repulsive and an attractive one, are identified here and their role in NC migration is analysed. First, it is shown that Contact Inhibition of Locomotion (CIL) is essential for NC migration. These cells collapse their protrusions upon contact with others and polarise towards cell-free regions leading to cell dispersion. Also, it is shown that the non-canonical Wnt signalling is crucial in this process as its members localise at the site of contact. This leads to activation of RhoA and inhibition of cell protrusions in this region. These results provide one of the rst examples of CIL in vivo and establish a novel role for non-canonical Wnt signalling. Second, a longer-range attractive interaction is also described here in a novel mechanism termed coattraction. It is shown in silico, in vitro and in vivo that when CIL is combined with coattraction, directional collective movements emerge instead of the simple dispersion allowed by CIL. Surprisingly, it was found that the anaphylatoxin/chemoattractant C3a and its receptor C3aR mediate NC coattraction. Finally, it is proposed that CIL and coattraction act together to allow cell collectives, such as the NC, to self-organise allowing a more effcient response to external signals such as chemoattractants and restrictive cues