The organisation of microtubule networks into a bipolar spindle is essential for reliable chromosome segregation during cell division. A pair of centrioles surrounded by pericentriolar material (PCM), define the canonical centrosome that acts as the main microtubule organising centre (MTOC) during mitosis. In mammalian meiosis, centrioles are eliminated early on during oogenesis. Despite the absence of centrosomes, a large number of centrosomal proteins are highly expressed in mouse oocytes. Here, I characterise the localisation and function of centrosomal proteins at a previously undescribed meiotic spindle pole domain (MSPD). An initial protein screen identified a group of pericentriolar satellite proteins that localised to a previously undescribed spindle pole domain throughout meiotic maturation in mouse oocytes, including Pericentriolar material 1 protein (PCM1). This domain was distinct from spindle microtubules and the acentrosomal microtubule organising centres (aMTOCs). Initial characterisation focused on PCM1, the main centriolar satellite scaffold protein in somatic cells. Depletion of PCM1 revealed interdependence with the essential aMTOC component, Pericentrin. In the absence of PCM1, aMTOCs could no longer assemble or maintain their structural integrity. PCM1 degradation and disassembly of aMTOCs disrupted spindle assembly and reduced the total amount of nucleated microtubules throughout meiosis. In the absence of the main microtubule nucleating aMTOCs, oocytes relied on the Ran GTPase activity to form a small bipolar spindle. A similar mechanism was previously reported in human oocytes that lack prominent MTOCs. The extended centrosomal protein screen identified additional components of the MSPD. TACC3, under the regulation of Aurora-A at aMTOCs, drive assembly of the MSPD. This domain was absent in MTOC free human oocytes but a second population of TACC3 (identified in mouse oocytes) localised to the meiotic spindle and K-fibres was essential for maintaining spindle pole integrity. Establishing the Lightsheet Z.1 system for live cell imaging of human oocytes enabled us to observe the dynamic distribution of TACC3 in these oocytes. In the absence of prominent MTOCs and the MSPD, human oocytes likely rely on other spindle assembly factors and motor proteins to organise their spindle. Future work to address if the absence of the MSPD could account (in part) for the observed spindle instability in human oocytes is an exciting outlook.

Solid tumors are complex structures comprising besides the cancer cells vasculature, extracellular matrix (ECM), soluble molecules and a plethora of various other cell types. These components form a so-called tumour microenvironment. From the numerous cell types that are part of tumor microenvironment, tumor infiltrating lymphocytes (TILs) play a major role in patient prognosis. Their presence is also of major importance with regard to new biological therapies based on immune checkpoint inhibitors. Crucial role of TILs is also reflected by the new approaches in cancer diagnostics namely by Immunoscore method (currently used in clinical settings). Immunoscore is based on localization and quantification of CD3+ and CD8+ TILs in thin histological sections of tumor tissue. The question remains to which extent the information obtained from 2D slices reflects the situation in tumor microenvironment considering its spatial heterogeneity. The development of new methodological approaches allowing evaluation of histological information in 3D is the key to answer this question. The theoretical part of this work first describes the heterogeneity of the tumor microenvironment and the role of immune cells within it. Then, the role of spatial heterogeneity and its possible influence on the histopathological...

Embryonic development entails a series of morphogenetic events which require a precise coordination of molecular mechanisms coupled with cellular dynamics. Phyla such as arthropods show morphological and gene expression similarities during middle embryogenesis (at the phylotypic germband stage), yet early embryogenesis adopts diverse developmental strategies. In an effort towards understanding patterns of conservation and divergence during development, investigations are required beyond the traditional model systems. Therefore, in the past three decades, several insect species representing various insect orders have been established as experimental model systems for comparative developmental studies. Among these, the red flour beetle Tribolium castaneum has emerged as the best studied holometabolous insect model after the fruit fly Drosophila melanogaster. Unlike Drosophila, Tribolium is a short-germ insect that retains many ancestral characters common to most insects. The early embryogenesis of Tribolium shows dynamic epithelial rearrangements with an epibolic expansion of the extraembryonic tissue serosa over the embryo, the folding of the embryo in between the serosa and the second extra embryonic tissue amnion and the folding of the amnion underneath the embryo. These extensive tissues are evolutionarily conserved epithelia that undergo different tissue movements and are present in varying proportions in different insects, providing exceptional material to compare and contrast morphogenesis during early embryogenesis. However, most of the previous work on insects including Tribolium have largely focused on the conservation and divergence of gene expression patterns and on gene regulatory interactions. Consequently, very little studies on dynamic cell behaviour have been done and we lack detailed information about the cellular and tissue dynamics during these early morphogenetic events. During my PhD, I first established a live imaging and data analysis pipeline for studying Tribolium embryogenesis in 4-D. I combined live confocal and lightsheet imaging of transgenic or transiently labelled embryos with mechanical or genetic perturbations using laser ablations and gene knockdowns. Using this pipeline quantifications of cell dynamics and tissue behaviours can be done to compare different regions of the embryo as the development proceeds. In the second and third part of my thesis, I describe the actomyosin dynamics and associated cell behaviours during the stages of serosa epibolic expansion, amniotic fold formation and serosa window closure. I cloned and characterised the cellular dynamics of the Tribolium spaghetti squash gene (Tc-squash) - the non-muscle Myosin II regulatory light chain, which is the main molecular force generator in epithelial cells. Interestingly, the analysis of Tc-squash dynamics indicates a conserved role of Myosin II in controlling similar cell behaviours across short germ and long germ embryos. In the last part of the thesis, I report the dynamics of an actomyosin cable that emerges at the interface of the serosa and amnion. This cable increases in tension during development, concomitant with serosa tissue expansion and increased tensions in the serosa. It behaves as a modified purse string as it’s circumference shrinks due to a decrease in the number of cable forming cells over time. This shrinkage is an individual contractile property of the cells forming the cable. This indicates that a supracellular and contractile actomyosin cable might be functional during serosa window closure in insects with distinct serosa and amnion tissues. Further, the tension in the cable might depend on the relative proportion of the serosa, amnion and embryonic regions. Using these integrated approaches, I have correlated global cellular dynamics during early embryogenesis with actomyosin behaviours, and then performed a high-resolution analysis and perturbations of selected events. The established imaging, image processing and perturbation tools can serve as an important basis for future investigations into the tissue mechanics underlying Tribolium embryogenesis and can also be adapted for comparisons of morphogenesis in other insect embryos. More broadly, correlating the existing genetic, mechanical and biochemical understanding of developmental processes from Drosophila with species such as Tribolium, could help identify deeply conserved design principles that lead to different morphologies through differences in underlying regulation.:Page List of Tables v List of Figures vii 1 Introduction 1 1.1 Evo-Devo of insects 3 1.2 Tribolium castaneum 5 1.3 Fluorescence live imaging and lightsheet microscopy 10 1.4 Morphogenesis 15 1.5 Thesis objective 29 2 4D lightsheet imaging and analysis pipeline of Tribolium embryos 33 2.1 Standardisation of an injection protocol for sample mounting and imaging with the Zeiss LZ1 SPIM 35 2.2 Double labelling of Tribolium embryos 37 2.3 Image processing with Fiji 37 2.4 Long term timelapse imaging of Tribolium embryogenesis with SPIM 44 2.5 2D cartographic projections of 3D data as a method to visualise and analyse SPIM data 47 2.6 Summary 59 3 Cellular dynamics of the non muscle Myosin II regulatory light chain - Tc-Squash 61 3.1 Tc-Squash dynamics during Tribolium embryogenesis 64 3.2 Myosin drives basal cell closure during blastoderm cellularisation 66 3.3 Myosin shows planar polarity in the embryonic tissue 69 3.4 Myosin accumulation and apical constriction of putative germ cells at the posterior pole 71 3.5 Myosin pulses during apical constriction of mesoderm cells 74 3.6 Myosin accumulates at the extraembryonic-embryonic boundary to form a contractile supracellular cable 77 3.7 Summary 77 4 A supracellular actomyosin cable operates during serosa epiboly 79 4.1 Actin and Myosin accumulate at the extraembryonic-embryonic boundary 81 4.2 The actomyosin assembly migrates ventrally till it forms the rim of the serosa window 82 4.3 The actomyosin cable shows dynamic shape changes during serosa window closure 87 4.4 Serosa cells increase in area till circular serosa window stage 89 4.5 Tension in the serosa tissue increases during epibolic expansion 89 4.6 Serosa cells decrease their apical areas after laser ablation 92 4.7 Tension in the actomyosin cable increases during serosa epiboly 93 4.8 Myosin dynamics at the cable changes between early and serosa window stage 96 4.9 Individual cell membrane shrinkage and cell rearrangements decrease the cable circumference 98 4.10 Myosin dynamics at the cable during serosa window closure 101 4.11 Tension in the cable is not relieved after multiple laser cuts 103 4.12 Analysis of the actomyosin cable in Tc-zen 1 knockdown 105 4.13 Summary 109 5 Discussion 111 5.1 Reconstruction of insect embryogenesis using lightsheet microscopy and tissue cartography 111 5.2 Conserved Myosin II behaviours and its implications on morphogenesis across insects 114 5.3 A contractile supracellular actomyosin cable functions serosa window closure in Tribolium 119 6 Materials and Methods 123 6.1 Tribolium stock maintenance 123 6.2 RNA extraction and cDNA synthesis 124 6.3 Cloning of templates for mRNA synthesis and transgenesis 124 6.4 dsRNA synthesis for RNAi experiments 126 6.5 Capped, single stranded RNA synthesis 126 6.6 Fluorescence image acquisition 27 A Appendix 131 Bibliography 143

Laser lightsheet triangulation is a well-established optical measurement method, which is frequently used in industrial applications. With some adaptions, the technique can also be used underwater. Placing a green laser line projector and a camera at a fixed base inside a watertight housing enables flexible and accurate underwater measurements at low cost. To achieve near-orthogonal intersections on the object surface, camera and laser need to be placed oblique to the housing interface. Refraction influences have to be considered strictly in geometric modelling, with the light propagating through the air–glass and glass–water interfaces. The measurement and calibration methods presented in the paper utilize a concept of splitting the lightsheet into multiple sub-beams, which are traced, refracted, and intersected with image observations to receive 3D measurements. In a calibration step, the relative orientation between camera, laser and interfaces is determined. In a theoretical accuracy analysis, the error influences caused by the calibrated parameters is estimated with a standard deviation of 0.2 to 0.4 mm in a depth range of up to 15 cm. A prototype triangulation sensor is applied for practical measurements of two test objects with known geometry. The predicted accuracy is validated in stationary single scan measurements. The sensor is furthermore moved along the test objects, using a six-degrees-of-freedom method to determine its exterior orientation. The combination of multiple single scans results in dense point clouds with 0.3 mm standard deviation compared to a reference.

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