Compartmentalisation and metabolism are two universal features observed among all living organisms. Their emergence and harmonious functioning are considered critical for the emergence of life. In an attempt to understand the chemical processes that may have resulted in the emergence of life, origin(s)-of-life researchers have investigated 1. possible mechanisms of prebiotic compartmentalisation and 2. plausible protometabolic reactions as precursors to metabolic chemistry. Historically, these two aspects of origin(s)-of-life research have been investigated in isolation. However, there is a growing consensus regarding the necessity of investigating prebiotic compartmentalisation and protometabolic reactions in tandem. Prebiotic compartmentalisation via formation of lipid vesicles have long been regarded as a general route for protocell formation. However, modern views on protocell formation suggest that such structures might be insufficient, in the absence of specialised transport proteins, to efficiently create suitable environments for sustaining out-of-equilibrium reactions, necessary for the emergence of life. This, coupled with the discovery of membrane-less organelles, formed through liquid-liquid phase separation (LLPS) in modern cells, and their ever increasingly identified roles in biology, has sparked renewed interest among origin(s)-of-life researchers in the synergy between prebiotic reactions and Oparin’s hypothesis of prebiotic compartmentalisation via coacervation. To date, most of the work, investigating the effect of coacervation on chemical reactions, have focused on biochemical reactions catalysed by complex biomolecules such as proteinaceous enzymes and ribozymes. Moreover, historically, the phenomenon of complex coacervation has primarily been associated with long-chain polymers. However, recent demonstrations of complex coacervation among several prebiotically relevant metabolites necessitates the need for developing experimental strategies that can comprehensively investigate complex coacervation, in terms of phase diagrams, involving such prebiotically relevant metabolites. Such understanding is essential to enhance the design of experiments that aim to combine protometabolic reactions and complex coacervation. To this end, the research presented in this thesis, at first, demonstrates a highthroughput screening methodology for complex coacervate formation, in chapter 2, using automated liquid handling strategies and random forest classifier machine learning algorithm based classification of bright-field microscopy images. This methodology has then been utilised to obtain the conditions in which oxidised and reduced nicotineamide adenine dinucleotide (NAD+ and NADH) form coacervate with 50-mer poly-lysine and poly-arginine. Further, the precipitation properties of 50-mer poly-arginine in sodium bicarbonate solution have been investigated. In chapter 3, incorporation of complex coacervation in three different protometabolic reactions in three different ways have been exhibited. In “Protometabolic NAD+ reduction using pyruvate”, NADH, which is the product of the reaction, participates in coacervation with 50-mer poly-arginine. In “Coenzyme A catalysed peptide ligation”, the catalyst of the reaction, coenzyme A contributes to coacervation with 10-mer polyarginine. In “Heme catalysed oxidation of Amplex Red”, the coacervate is formed form polymers, such as carboxymethyl dextran and poly-diallyldimethylammonium, that are not reactants, products or catalyst of the studied reaction. It has been further shown that the incorporation of coacervation, to “Protometabolic NAD+ reduction using pyruvate”, results in the enhancement of NADH formation by 2.5 times in comparison to the amount of NADH produced in the absence of any poly-cations. Preliminary results also suggest that the presence of coacervation resulted in the suppression of both “Coenzyme A catalysed peptide ligation” and “Heme catalysed oxidation of Amplex Red”. This study elucidates an unprecedented methodology for screening of coacervate formation as well as highlighting the various ways in which coacervation of prebiotically relevant metabolites can be embedded into protometabolic reactions. Further, this study also sheds light on the influence of coacervation on protometabolic reactions, revealing patterns that were previously primarily observed in biochemical reactions catalysed by enzymes (or ribozymes).

Despite the advances in modern technology, unraveling the genome evolution of an organism or even groups of several species remains a challenging task. Up-to-date cytogenetics and computational approaches enable the investigation of genomes from the nucleotide sequence up to the chromosomes, yet, drawing conclusions about evolutionary and mechanistic processes remains far from being trivial. The crop sugar beet (<i>Beta vulgaris</i> subsp. <i>vulgaris</i>) and its wild relatives form a well-suited group of plants (members of the Amaranthaceae family) to demonstrate the possibilities and limits that both, cytological and computational genomics, possess in addressing open questions on the genome in a phylogenetic context and in all its conformations: from its organization in chromosomes right down to the loss and gain of genes and the composition of repetitive DNA sequences. Since the two beet genera <i>Beta</i> and <i>Patellifolia</i> comprise diploid as well as polyploid species, genomic variability between them is not only based on DNA sequence differences, but on changes in the chromosome number as well. In the frame of this work, using microscopic approaches (outlined in chapter II), it was determined that all beets share a base chromosome number of x = 9. Differing properties between cultivated and wild beet accessions are the result of polyploidization and changes in the DNA sequence rather than a restructuring of the chromosomes. In chapter III, the focus is on the tetraploid wild beet <i>Beta corolliflora</i>, whose polyploidization likely led to the development of many tolerances against adverse environmental conditions. Since its ancestry remained unresolved for a long time, five different bioinformatics tools have been developed and complemented with cytogenetics to unravel its parental relationships. As an ‘autopolyploid’ hybrid descending from closely related <i>Beta macrorhiza</i> accessions, <i>B. corolliflora</i> occupies an intermediate position within the spectrum of auto- to allopolyploidy. Today’s breeding endeavors aim for the (re-)introduction of genes from wild beet into cultivated beet accessions to improve crop species in the face of changing cultivation conditions. Yet, such efforts are impeded due to crossing barriers, reflected in the separation of the beet species into three distinct gene pools. Chapter IV aims to identify repetitive DNA sequences that may be involved in speciation and formation of these gene pools. For this, genome data has been generated for a panel of 17 different beet accessions and was analyzed bioinformatically as well as experimentally, using long and short read technology, fluorescent <i>in situ</i> and Southern hybridization. The overall repeat content was found to correlate with the beet genome sizes and whereas some repeats are well conserved among the beet species, the specificity of others mirrors the split into the three beet gene pools. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools with uniform centromeres in the primary and tertiary gene pool and patchwork centromeres in the secondary gene pool, likely contributing to the barriers in beet breeding. Furthermore, endogenous sequences of viral origin were also detected in all beet genomes with specific elements for the different beet gene pools. As for <i>B. vulgaris</i>, these endogenous pararetroviruses were found to contribute to the host’s defense against other (putatively harmful) viruses (chapter V). In summary, this thesis demonstrates the synergistic potential of integrating computational and cytological genomics for a comprehensive genome analysis of beets that can be transferred to any other species panel. Combining both approaches enables to unlock a deeper understanding of the genetic makeup and evolution of the species of interest, in particular with regard to the impact of repetitive elements. / Trotz des technologischen Fortschritts bleibt die Entschlüsselung der Evolution eines Genoms oder gar der Genome mehrerer Arten eine anspruchsvolle Aufgabe. Moderne zytogenetische und computergestützte Ansätze ermöglichen die Untersuchung von Genomen von der Nukleotidsequenz bis hin zu den Chromosomen. Trotzdem ist es alles andere als trivial, daraus Rückschlüsse auf evolutionäre und mechanistische Prozesse zu ziehen. Die Zuckerrübe (<i>Beta vulgaris</i> subsp. <i>vulgaris</i>) und ihre wilden Verwandten stellen eine Pflanzengruppe aus der Familie der Amaranthaceae dar, die sich gut dafür eignet, Möglichkeiten und Grenzen der zytologischen sowie computergestützten Genomik aufzuzeigen. Bei der Beantwortung offener Fragen zum Genom in einem phylogenetischen Kontext werden dabei all seine Ausprägungen in Betracht gezogen: von der Genomorganisation in Chromosomen bis hin zum Verlust und Erhalt von Genen und der Zusammensetzung repetitiver DNA-Sequenzen. Da die beiden Rübengattungen <i>Beta</i> und <i>Patellifolia</i> sowohl diploide als auch polyploide Arten umfassen, beruht die genomische Variabilität zwischen den Rübengenomen nicht nur auf Unterschieden in der DNA-Sequenz, sondern auch auf Veränderungen in der Chromosomenzahl. Im Rahmen der vorliegenden Arbeit wurde mit Hilfe mikroskopischer Methoden (siehe Kapitel II) festgestellt, dass alle Rüben eine Basischromosomenzahl von n = 9 aufweisen. Unterschiedliche Eigenschaften zwischen kultivierten und wilden Rüben-Akzessionen sind das Ergebnis von Polyploidisierung und Veränderungen in der DNA-Sequenz und nicht von chromosomalen Umstrukturierungen. In Kapitel III liegt der Fokus auf der tetraploiden Wildrübe <i>Beta corolliflora</i>, deren Polyploidisierung wahrscheinlich zahlreiche Toleranzen gegenüber widrigen Umweltbedingungen bedingt. Da ihre Abstammung lange Zeit ungeklärt blieb, wurden fünf verschiedene bioinformatische Methoden entwickelt und zytogenetisch komplementiert, um die Elternspezies zu entschlüsseln. So handelt es sich bei <i>B. corolliflora</i> wahrscheinlich um eine „autopolyploide“ Hybride, die von eng verwandten <i>Beta macrorhiza</i>-Akzessionen abstammt und eine Zwischenform im Spektrum der Polyploidie darstellt. Heutige Züchtungsansätze zielen auf die (Wieder-)Einführung von Genen aus Wildrüben in kultivierte Rübensorten ab, um die Kulturarten angesichts der sich ändernden Anbaubedingungen widerstandsfähiger und/oder ertragreicher zu machen. Solche Bemühungen werden jedoch durch Kreuzungsbarrieren eingeschränkt, die sich in der Gruppierung der Rübenarten in drei verschiedene Genpools widerspiegeln. Kapitel IV zielt darauf ab, repetitive DNA-Sequenzen zu identifizieren, die möglicherweise an Adaption und Artbildung beteiligt sind. Genomdaten wurden für 17 verschiedene Rüben-Akzessionen generiert und sowohl bioinformatisch als auch experimentell mittels Fluoreszenz-<i>in situ</i>- und Southern-Hybridisierung analysiert. Der Gesamtgehalt an repetitiven DNA-Sequenzen korreliert mit der Genomgröße der Rübenakzessionen. Während einige repetitive DNA-Sequenzen zwischen den Rübenarten konserviert sind, spiegelt die Spezifität anderer die Aufteilung in die drei Rübengenpools wider. Insbesondere die Satelliten-DNA variiert beträchtlich zwischen den Rübengenomen, was zur Entwicklung unterschiedlicher chromosomaler Strukturen in den drei Genpools geführt hat: Der primäre und tertiäre Genpool sind hierbei durch einheitlichen Zentromere gekennzeichnet, während sich der sekundäre Genpool durch eine individuelle Zentromerzusammensetzung auszeichnet, die von Chromosom zu Chromosom verschieden sein kann. Dies trägt wahrscheinlich zu den Hindernissen im Zuge der Rübenzucht bei. Außerdem wurden in allen Rübengenomen endogene Sequenzen viralen Ursprungs nachgewiesen, die für die verschiedenen Rübengenpools spezifisch sind. Für <i>B. vulgaris</i> wurde festgestellt, dass diese endogenen Pararetroviren zur Verteidigung gegen andere (ggf. schädliche) Viren beitragen (Kapitel V). Zusammenfassend zeigt diese Arbeit das synergistische Potenzial einer Integration der computergestützten und zytologischen Genomik für eine umfassende Genomanalyse von Rüben auf, die auf jedes andere Artenpanel übertragen werden kann. Die Kombination beider Ansätze ermöglicht ein tieferes Verständnis des Genoms und der Evolution der betreffenden Art, insbesondere in Hinblick auf den Einfluss repetitiver DNA-Sequenzen.

No description available.

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitor Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging with single-molecule fluorescence correlation spectroscopy, we demonstrate that Fgf8a, which is produced at the embryonic margin, propagates by diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a-mediated patterning. Manipulation of the extracellular Fgf8a levels alters the signaling outcome, thus establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, by hindering Fgf8a diffusion, we demonstrate that extracellular diffusion of the protein from the source is crucial for it to achieve its morphogenic potential.

Goal-directed behavior requires integrated mental representations of perceptions and actions. The neurophysiological underpinnings of these processes, however, are not yet understood. It is particularly undetermined, which oscillatory activities in which brain regions are involved in the management of perception-action representations. We examine this question with a focus on response inhibition processes and show that the dynamics of perception-action representations reflected in theta band activity (TBA) are particularly evident in the supplementary motor area and the occipito-temporal cortex. Mental representations coded in alpha band activity (ABA) during perception-action integration are associated with the occipito-temporal cortex. Crucially, perception-action representations are exchanged between theta and alpha frequency bands. The results imply that ABA functions as dynamic top-down control over binding, retrieval and reconfiguration processes during response inhibition, which in turn are reflected by TBA. Our study thus highlights how the interplay of oscillatory activity enables the management of perception-action representations for goal-directed behavior.

Objective: A wide range of cardiac diseases is associated with inflammation. “Inflamed” heart tissue is infiltrated with pro-inflammatory macrophages which extensively secrete matrix metalloproteinase 9 (MMP9), a regulator of extracellular matrix turnover. As MMP9 is released from macrophages in a latent form, it requires activation. The present study addresses the role of cardiomyocytes in the course of this activation process. Methods and results: In mono- and co-cultures of pro-inflammatory rat macrophages (bone marrow-derived and peritoneal) and cardiomyocytes (H9C2 cell line) gelatin zymography demonstrated that activated macrophages robustly secreted latent pro-MMP9, whereas cardiomyocytes could not produce the enzyme. Co-culturing of the two cell species was critical for pro-MMP9 activation and was also accompanied by processing of cardiomyocyte-secreted pro-MMP2. A cascade of pro-MMP9 activation was initiated on macrophage membrane with pro-MMP2 cleavage. Namely, pro-inflammatory macrophages expressed an active membrane type 1 MMP (MT1MMP), which activated pro-MMP2, which in turn converted pro-MMP9. Downregulation of MT1MMP in macrophages by siRNA abolished activation of both pro-MMP2 and pro-MMP9 in co-culture. In addition, both cell species secreted MMP13 as a further pro-MMP9 activator. In co-culture, activation of pro-MMP13 occurred on membranes of macrophages and was enhanced in presence of active MMP2. Using incubations with recombinant MMPs and isolated macrophage membranes, we demonstrated that while both MMP2 and MMP13 individually had the ability to activate pro-MMP9, their combined action provided a synergistic effect. Conclusion: Activation of pro-MMP9 in a co-culture of pro-inflammatory macrophages and cardiomyocytes was the result of a complex interaction of several MMPs on the cell membrane and in the extracellular space. Both cell types contributed critically to pro-MMP9 processing.

The freshwater pearl mussel Margaritifera margaritifera is an endangered bivalve which is usually regarded as sedentary, although individual movement has been observed both vertically and horizontally. Little is known about the causes and rates of mussel movement. The objective of this study was to test the effect of microhabitat characteristics on the horizontal movement distance and rates of freshwater pearl mussels. A total of 120 mussels (length range 40–59 mm) were marked individually with passive integrated transponder tags, placed in stream microhabitats differing in their sediment composition and monitored biweekly over a period of 10 weeks. Mussels situated in sand-dominated habitats had a significantly higher mean movement rate (3.2 ± 4.2 cm/day, mean ± SD) than mussels situated in gravel-dominated (1.9 ± 2.7 cm/day) or stone-dominated habitats (1.8 ± 3.2 cm/day). The direction of the movements appeared random; however, an emigration from sandy habitats was observed, probably to avoid dislodgment from these hydraulically unstable habitats. This study demonstrates that freshwater pearl mussels can actively emigrate from unsuitable microhabitats. Once suitable streams with respect to physical, chemical, and biological quality were identified, it is therefore only necessary to identify suitable mesohabitats (area of 10–30 m²) when reintroducing or relocating mussels.

Cognition is the mechanism by which animals acquire, process, store, and act on information from the environment, including perception, learning, memory, and decision-making (Shettleworth 2009a). Comparative cognition aims to investigate how cognitive skills are distributed across taxa, and thus find the socio-ecological challenges that might explain this distribution (Healy et al. 2009; MacLean et al. 2012). As a result, comparative cognition allows researchers to find variations in problem-solving abilities, better understand the processes that lead to the emergence of complex cognition, and understand the evolutionary challenges that are linked to the emergence of specific skills (Barrett et al. 2002; Bueno-Guerra and Amici 2018; Dunbar 1992; Harcourt et al. 1988; MacLean et al. 2012; Shettleworth 2009a). In their natural environment, animals face a variety of ecological and social challenges. Theories of cognitive evolution suggest that these challenges have favoured the emergence of cognitive skills that allow individuals to better cope with the requirements they encounter in their ecological niches, like foraging or interacting with conspecifics (Ashton et al. 2018; Holekamp 2007; Tomasello and Call 1997). Therefore, cognitive skills usually evolve when individuals face specific problems, either in a physical context (i.e., cognitive processes used to solve physical problems) or in a social context (i.e., cognitive processes used to interact with social partners). In my thesis, I focus on physical cognition and object understanding, which includes how animals deal with inanimate objects and what they understand about their spatial, temporal, and causal relations. One reason why some species may have evolved different cognitive abilities is that they face specific ecological challenges which the evolution of complex cognition, and thus greater behavioural flexibility, allows to overcome more efficiently. These ecological challenges include, for example, dietary breadth or domestication. Moreover, social challenges may also constitute a strong selection pressure for the evolution of enhanced cognitive abilities. When social life is complex, individuals must use flexible cognitive strategies to recognize other individuals, keep track of their relationships, and predict, coordinate and manipulate their behaviours. Unlike objects, social partners are reactive, unpredictable and respond with different behaviours to one's actions; therefore, social complexity would present high cognitive challenges and strong selection pressures for the evolution of cognition. Social challenges include, for example, living in large groups, frequently engaging in social interactions, or showing fission-fusion dynamics. Apart from interspecific variation, cognition also varies across conspecific individuals, depending on factors such as sex, age, rank, social integration or early life experiences. In addition to these characteristics, personality traits (i.e., interindividual behavioural differences that are consistent over time and across contexts) may also influence performance on cognitive tasks. In this project, I used ungulates as a model to directly test socio-ecological hypotheses about the evolution of cognition. Ungulates are an ideal model to test cognitive abilities from a comparative perspective. First, they show an impressive diversity of socio-ecological traits, which allows for a reliable contrast of different evolutionary hypotheses. Second, there are very few studies that have examined the relationship between cognition and socio-ecological traits in ungulates, and the largest majority have used neuroanatomical proxies for cognitive skills. Third, although ungulates are economically important for humans, we still know little about their cognition, and about the cognitive enrichments that might be used to improve their welfare. This thesis aimed to test variation in different cognitive skills across several ungulate species, to assess the ecological, social and individual factors that best explain inter- and intra-specific variation in their cognitive abilities. I used well-established experimental procedures to test individuals´ object permanence, short-term memory, causality, understanding of object properties, gravity and quantity discrimination skills. Further, I tested individuals´ neophobic reactions to new objects. All tests were carried out on captive individuals in the zoos of Leipzig (Germany) and Barcelona (Spain), and partially in other European zoos. I tested the following species in one or more studies: goats (Capra aegagrus hircus), llamas (Lama glama), guanacos (Lama guanicoe), Grevy’s zebras (Equus grevyi), Chapman’s zebras (Equus burchelli chapmanni), rhinos (Diceros bicornis michaeli), giraffes (Giraffa camelopardalis rothschildi), European bisons (Bison bonasus), Forest buffalos (Syncerus caffer nanus), oryx (Oryx dammah), dromedaries (Camelus dromedarius), red deer (Cervus elaphus), barbary sheep (Ammotragus lervia), Przewalski horses (Equus ferus przewalskii), and sheep (Ovis aries). In summary, 15 different ungulate species were tested in well-established experimental procedures to test individuals´ object permanence, short-term memory, causality, understanding of object properties, gravity and quantity discrimination skills. Further, individuals´ neophobic reactions to new objects were tested. In the first paper on neophobia, I tested neophobic responses to novel objects in different ungulate species and found differences both within and across species. In particular, more socially integrated individuals were more neophobic than less central ones, showing a higher latency to approach food that was closer to a novel object. Further, Barbary sheep were less neophobic than all the other species and spent a higher proportion of time close to novel objects. In the second paper, I tested whether higher levels of fission-fusion dynamics predict better cognitive skills. I found Grevy´s zebras (which are characterized by higher fission-fusion dynamics) to perform better than Chapman´s zebras in tasks requiring inference and quantity discrimination skills. In the third paper, I tested the cognitive skills of ungulates in different object-understanding tasks and showed that all species were able to understand that objects continue to exist even when they are out of sight, remember the location of objects after delays of up to 60 seconds (short-term memory), and infer the location of the food from the presence or lack of sound produced when shaking containers. I found that subjects, across species, had some understanding of object properties and gravity, being able to locate food behind one of the two occluders based on their shape and inclination, and searching for falling food in the correct location. In the fourth paper, individuals performed above chance levels in most conditions of the Numerosity and Size tasks, in which they had to rely on item number and size to maximize food intake. Overall, I detected inter- and intra-specific variation across the cognitive tasks performed. The socio-ecological factors that explained the inter-specific variation were domestication (i.e., species that have been selected for living in close relationships with humans), group size (i.e., the actual size of the tested group) and fission-fusion dynamics (i.e., individuals living in groups frequently splitting into subgroups of varying size and composition). Apart from inter-specific variation, I could also detect inter-individual variation, with socially more integrated individuals being more neophobic. Taken together, the results of my thesis show that different inter- and intra-specific factors lead to variation in cognitive skills between ungulate species.

Gastrulation involves a complex series of morphogenetic processes that unfold in precise coordination alongside tissue genetic patterning. During the gastrulation of Drosophila melanogaster, a monolayer of cells known as the blastoderm undergoes a sequence of tissue rearrangements that reveal axis determination and cell fates. The Drosophila model provides a comprehensive toolkit for studying embryogenesis, offering insights into its streamlined developmental program, which efficiently produces the final organism while maintaining robustness. This prompts an exploration of the mechanisms that coordinate individual morphogenetic events. Active forces, generated by cytoskeletal-driven cell shape changes, exert themselves on the viscoelastic blastoderm tissue, resulting in passive transmission. Traditional analysis involved studying these events at molecular, cellular, and tissue scales. However, the emergence of single-plane illumination microscopy, enabling whole volume (in toto) imaging of developing embryos with high temporal and spatial resolution, has reshaped this approach. This advancement in microscopy allows us to investigate how morphogenetic events synergise or hinder one another and how the embryo integrates this information to coordinate gastrulation. A recent development in gastrulation research involves identifying regions of heightened friction between the cells and the eggshell within the embryo. Integrins expressed near the midgut primordium have been found to mediate this interaction, challenging the notion that cell movement is the sole driving force of morphogenesis. Instead, static regions emerge as vital contributors to mechanical stability and the preservation of left-right symmetry during germ band extension. This role of integrins is not confined to Drosophila alone, as similar findings have been observed in the beetle Tribolium castaneum, emphasizing their role in orchestrating tissue flows and raising questions about their conservation. Consequently, my focus centered in understanding the physical forces underlying gastrulation, encompassing their interplay, balance, and conflicts in the developing embryo. I considered not only the active forces generated by cell shape changes, but also the passive forces transmitted in the surrounding tissue and the friction generated by the interaction with the physical constraint of the eggshell. In the initial chapter of this thesis, I delved into a lesser-explored morphogenetic event: the formation of the cephalic furrow. This transient tissue fold delineates the head from the trunk but differs from other invaginations in that it does not give rise to further differentiated structures. Instead, it unfolds later in development without leaving a trace of its origin. By employing in toto imaging alongside genetic and photo-manipulation techniques, I investigated the influence of the active forces driving the furrow formation on the head-trunk boundary tissue, and the consequences upon their absence. I found that the invagination is driven by active forces that propagate in the surrounding tissue. By genetic inhibition of the furrow, the presence of ectopic buckling was confirmed in the head-trunk boundary, appearing in between mitotic domains in a rather stochastic fashion. Next, I assessed the impact of further morphogenetic events in the head-trunk boundary tissue using whole embryo imaging and quantitative strain analysis. From my observations, I was able to conclude that the cephalic furrow primes tissue folding to dissipate forces coming from two sources, the local mitotic domains' expansion and the remote germ band compression. These discoveries suggest that the cephalic furrow and its genetic patterning may have evolved in response to the coalition of compressive forces in the head-trunk boundary tissue. The second chapter focused on understanding the ensemble of tissue flows while considering the newfound importance of static regions, mediated by the integrin subunit scab. The possibility to visualise the whole embryo upon mechanical photo-manipulation and integrin mutation, allowed me to uncover how enhanced friction's localised forces contribute to the directionality of tissue movements during the posterior midgut invagination. I was able to verify that cell-to-shell attachment helps maintaining the speed and direction of germ band extension, a highly mechanically unstable process. At the same time, I explored the effects of two milder expression sites of scab not addressed in the past on the mid-dorsal and ventral-anterior region of the embryo. I determined that the first contributes to the shift of the cephalic furrow posteriorly, which is required to allow mitotic domains divisions in the dorsal side of the head. Upon integrin depletion, ectopic buckling in between mitotic domains was observed, resembling our results in the first chapter. Lastly, thanks to whole volumetric imaging, the newly found expression site in the ventral-anterior region was determined to stabilise the head in face of torque generation of the deviating germ band. These results indicate a potential early safety mechanisms against symmetry breaking in the unstable stages of germ band extension, before posterior midgut invagination. In the concluding chapter, I explored the origins of left-right organismal symmetry instability during germ band extension. Analysing cartographic projections of the whole blastoderm surface, I discovered an inherent chirality identified in symmetric embryos at both the cellular and local tissue levels. By quantification of geometric features, along with tissue strain and curl rates, I found significant and consistent differences in all specimens in a specific region of the embryo in the lateral posterior side. These finding raises questions about the potential role of early chiral determinants in embryogenesis, that could translate local asymmetries into organismal twisting. In summary, this thesis underscores the significance of a multi-scale and interdisciplinary approach to embryonic development. The view of genetic patterning setting up the canvas for morphogenesis is taken to the next level, considering this canvas as an active material that needs fine coordination of single cellular events and the forces generated by them. These forces, in turn, shape molecular components and gene expression, culminating in a dynamic picture of a somewhat unstable, though robust, equilibrium of embryonic development.:I Introduction 1 Tissue morphogenesis 2 Embryonic development of Drosophila melanogaster 3 Modern methods for volumetric imaging 4 Project motivation and questions II Results 5 The role of the cephalic furrow. 6 Integration of tissue flows and static regions 7 Left-Right (a)symmetry 8 Discussion 9 Appendix

Through a slow developmental process driven by learning and neural maturation, humans can flexibly combine a limited number of sounds into words and then into sequences (i.e., ‘combined utterances’), following syntactical rules. This enables the creation of an infinite number of utterances to convey an unlimited range of information. Whether this capacity is unique to humans or evolved from related species remains uncertain. Comparative research with non-human primates (hereafter: primates) is crucial for understanding this. In contrast to humans, the development of primate repertoires appears to be largely innate and fixed from birth. However, there is some evidence of limited vocal plasticity in certain primate species, with developmental changes linked to acoustic variations and usage learning, likely influenced by social dynamics. A recent study has revealed that adult chimpanzees, one of our closest living relatives, possess an unusual complex vocal repertoire with hundreds of non-random vocal utterances. This lead me question: are these hundreds of vocal utterances innate and present from birth or do they develop through ontogeny? If so, what processes underlie this vocal development, and do they mirror those seen in the development of human language? Therefore, this thesis aims to investigate the ontogeny of vocal sequence acquisition and their potential function in chimpanzees, using a full repertoire approach. To this aim, I conducted 9 months of focal animal sampling on 98 wild chimpanzees (0-55 years old), living in Ivory Coast. I analysed 11,397 vocal utterances from 1,807.3 hours of vocal recordings. First, I found that chimpanzees require about 10 years to reach the adult repertoire, which extends beyond the age when single vocal units emerge. The developmental trajectory of vocal sequence acquisition aligned with key developmental social milestones, supporting social complexity as a driver of vocal complexity. Moreover, neuro-muscular maturation also appears to drive vocal development. Second, I found that the combinatorial flexibility, ordering and re-combinatorial patterns are limited at birth but increase with age. Last, I found that vocal sequences may function to routinely convey combined information about juxtaposed daily life events, a crucial step in the evolution of generalised combinatorial communication. This thesis underscores the importance of studying entire vocal repertoires, including sequences, and their development. It enhances our understanding of chimpanzee communication and suggests likely shared origins and functions of complex communication systems, ultimately contributing to our knowledge of the evolution of human language.