Developmental biologists try to elucidate how it is possible for cells, all originating from the same egg, to develop into a variety of highly specialized structures, such as muscles, skin, brain and limbs. What organizes the behavior of these cells, and how can the information encoded in the DNA account for the observed patterns and developmental processes? Cell movements and tissue flow during embryogenesis constitute a beautiful problem of bridging scales: On the microscopic scale, cells are expressing particular genes which determine their identities and also their fate during morphogenesis. These molecular determinants then lead to the macroscopic phenomena of cell movements and tissue arrangements, for which one needs a continuum description in terms of active fluids. Taking into account that the number of cells is fairly small, a complete coarse graining is not possible, and a characterization of both mesoscopic (individual cell motion) and macroscopic (flow) behavior is required for a full description. In the here presented work, a set of different experimental methods was applied to investigate the mechanical and dynamical properties of zebrafish embryonic cells and tissues. This thesis is structured as follows: In chapter 2, we introduce the fundamental concepts that are important for the study of cell motion during zebrafish embryonic development. In chapter 3, the materials and methods applied in this work are described. The experimental results of my thesis-work are presented in chapters 4-8: Chapter 4 concentrates on the physical properties of whole tissues. It is shown that tissues are viscoelastic materials. Tissue viscoelasticity is not a new concept, but this study is the first one to quantify the mechanical properties of tissues that are in actual contact in a developing embryo. In chapter 5, cell rearrangements in culture, such as cell sorting and tissue wetting are discussed. These experiments show that tissue interactions are largely determined by tissue surface and interfacial tensions. In chapter 6, an optical stretcher device is applied to measure, solely by means of laser light, the material properties of individual cells. Hereby it is shown that single cells from the two investigated tissue types differ in their mechano-physical properties. After the study of cell and tissue mechanics, the dynamics of cell migration in three dimensions in tissue aggregates and in developing zebrafish embryos is addressed: In chapter 7, 3D-cell migration in multicellular aggregates is analyzed quantitatively by studying the mean square displacement, cell velocity distribution and velocity autocorrelation. In chapter 8, we study the cell motion within the developing zebrafish embryo. By following the motion of many cells in four dimensions, we are able to generate a velocity flow profile for this cell-flow. Chapter 9 gives a brief summary of the obtained results and an outlook to future projects motivated by the presented study. The final part of this thesis are four appendices. Appendix A contains protocols and additional methods. Appendix B contains several calculations, whose results were used in the main part of this work. Appendix C contains additional data and discussions, which were excluded from the main part due to space limitations. Finally, Appendix D consists of a compact disc with 11 movies and a movie description, which serves as supplemental material to the presented data. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 650 MB: Movies - Nutzung: Referat Informationsservice der SLUB)

Das Herz eines Zebrafishs ist bemerkenswert, da es sich nach einer Verletzung vollständig regenerieren kann. Der Regenerationsprozess wird von Fibrose begleitet - der Bildung von überschüssigem Gewebe der extrazellulären Matrix (ECM). Anders als bei Säugetieren ist die Fibrose im Zebrafish nur transient. Viele Signalwege wurden identifiziert, die an der Herzregeneration beteiligt sind. Allerdings sind die Zelltypen, insbesondere Nicht-Kardiomyozyten, die für die Regulation des Regenerationsprozesses verantwortlich sind, weitgehend unbekannt. In dieser Arbeit haben wir systematisch alle Zelltypen des gesunden und des verletzten Zebrafischherzens mithilfe einer auf Mikrofluidik basierenden Hoch-Durchsatz- Einzelzell-RNA-Sequenzierung bestimmt. Wir fanden eine große Heterogenität von ECM-produzierenden Zellen, einschließlich einer Reihe neuer Fibroblasten, die nach einer Verletzung mit unterschiedlicher Dynamik auftreten. Wir konnten aktivierte Fibroblasten beschreiben und Fibroblasten-Subtypen mit einer pro-regenerativen Funktion identifizieren. Darüber hinaus haben wir eine Methode entwickelt, um die Transkriptomanalyse und die Rekonstruktion von Zell-Verwandtschaften auf Einzelzellebene zu kombinieren. Unter Verwendung der CRISPR-Cas9-Technologie führten wir zufällige Mutationen in bekannte und ubiquitär transkribierte DNA-Loci während der Embryonalentwicklung von Zebrafischen ein. Diese Mutationen dienten als zellspezifische, permanente und vererbbare “Barcodes”, die zu einem späteren Zeitpunkt erfasst werden konnten. Mit maßgeschneiderten Analysealgorithmen konnten wir dann Stammbäume der sequenzierten Einzelzellen erstellen. Mit dieser neuen Methode haben wir gezeigt, dass im sich regenerierenden Zebrafischherz ECM-produzierende Zellpopulationen entweder mit dem Epi- oder mit dem Endokardium verwandt sind. Zusätzlich entdeckten wir, dass vom Endokardium abgeleitete Zelltypen vom Wnt-Signalweg abhängig sind. / The zebrafish heart has the remarkable capacity to fully regenerate after injury. The regeneration process is accompanied by fibrosis - the formation of excess extracellular matrix (ECM) tissue, at the injury site. Unlike in mammals, the fibrosis of the zebrafish heart is only transient. While many pathways involved in heart regeneration have been identified, the cell types, especially non-myocytes, responsible for the regulation of the regenerative process have largely remained elusive. Here, we systematically determined all different cell types of both the healthy and cryo-injured zebrafish heart in its regeneration process using microfluidics based high-throughput single-cell RNA sequencing. We found a considerable heterogeneity of ECM producing cells, including a number of novel fibroblast cell types which appear with different dynamics after injury. We could describe activated fibroblasts that extensively switch on gene modules for ECM production and identify fibroblast sub- types with a pro-regenerative function. Furthermore, we developed a method that is capable of combining transcriptome analysis with lineage tracing on the single-cell level. Using CRISPR-Cas9 technology, we introduced random mutations into known and ubiquitously transcribed DNA loci during the zebrafish embryonic development. These mutations served as cell-unique, permanent, and heritable barcodes that could be captured at a later stage simultaneously with the transcriptome by high-throughput single-cell RNA sequencing. With custom tailored analysis algorithms, we were then able to build a developmental lineage tree of the sequenced single cells. Using this new method, we revealed that in the regenerating zebrafish heart, ECM contributing cell populations derive either from the epi- or the endocardium. Additionally, we discovered in a functional experiment that endocardial derived cell types are Wnt signaling dependent.

In zebrafish, transgenic labeling approaches, robust regenerative responses and excellent in vivo imaging conditions enable precise characterization of immune cell behavior in response to injury. Here, we monitored osteoblast-immune cell interactions in bone, a tissue which is particularly difficult to in vivo image in tetrapod species. Ablation of individual osteoblasts leads to recruitment of neutrophils and macrophages in varying numbers, depending on the extent of the initial insult, and initiates generation of cathepsin K+ osteoclasts from macrophages. Osteoblast ablation triggers the production of pro-inflammatory cytokines and reactive oxygen species, which are needed for successful macrophage recruitment. Excess glucocorticoid signaling as it occurs during the stress response inhibits macrophage recruitment, maximum speed and changes the macrophage phenotype. Although osteoblast loss is compensated for within a day by contribution of committed osteoblasts, macrophages continue to populate the region. Their presence is required for osteoblasts to fill the lesion site. Our model enables visualization of bone repair after microlesions at single-cell resolution and demonstrates a pro-osteogenic function of tissue-resident macrophages in non-mammalian vertebrates.

Developmental biology adresses how cells are organised into functional structures and eventually into a whole organism. It is crucial to understand the molecular basis for processes in development, by studying the expression and function of relevant genes and their relationship to each other. A gene function can be studied be creating loss-of-function situations, in which the change in developmental processes is examined in the absense of a functional gene product, or in gain-of-function studies, where a gene product is either intrinsically overproduced or ectopically upregulated. One approach for a loss-of-function situation is the creation of specific mutants in single genes, and the zebrafish (Danio rerio) has proven to be an excellent model organism for this purpose. In this thesis, I report on two forward genetic screens performed to find new mutants affecting brain development, in particular mutants defective in development and function of the midbrain-hindbrain boundary (MHB), an organiser region that patterns the adjacent brain regions of the midbrain and the hindbrain. In the first screen, I could identify 10 specific mutants based on morphology and the analysis of the expression patterns of lim1 and fgf8, genes functioning as early neuronal markers and as a patterning gene, respectively. Three of these mutants lacked an MHB, and by complementation studies, I identified these mutants as being defective in the spg locus. The second screen produced 35 new mutants by screening morphologically and with antibodies against acetylated Tubulin, which marks all axonal scaffolds, and anti-Opsin, which is a marker for photoreceptors in the pineal gland. According to their phenotype, I distributed the mutant lines into 4 phenotypic subgroups, of which the brain morphology group with 18 mutant lines was studied most intensively. In the last part of my thesis, I characterise one of these brain morphology mutants, broken heart. This mutant is defective in axonal outgrowth and locomotion, and shows a striking reduction of serotonergic neurons in the epiphysis and in the raphe nuclei in the hindbrain, structures involved in serotonin and melatonin production. Studies in other model organisms suggested a role of factors from the floor plate and the MHB in induction of the serotonergic neurons in the hindbrain, and using broken heart, I show that Fgf molecules such as Fgf4 and Fgf8 can restore partially the loss of serotonergic neurons in the mutant. I conclude that forward genetic screens are an invaluable tool to generate a pool of mutations in specific genes, which can be used to dissect complex processes in development such as brain development.

The neuromeric concept of brain formation has become a well-established model to explain how order is created in the developing vertebrate central nervous system. The most important feature of neuromeres is their compartmentalization on the cellular level: Each neuromere comprises a lineage-restricted population of cells that does not intermingle with cells from neighboring compartments. The units of the vertebrate hindbrain, the rhombomeres, serve as the best-studied examples of neuromeres. Here, the lineage restriction mechanism has been found to function on the basis of differentially expressed adhesion molecules. To date, hard evidence for the existence of other lineage restricted regions in more anterior parts of the brain is still scarce. The focus of this study is the midbrain-hindbrain boundary (mhb) region, where the juxtaposition of the mesencephalon and metencephalon gives rise to a signaling center, termed the midbrain-hindbrain or isthmic organizer. Evidence for lineage restriction boundaries in the mhb region is still controversial, with some very recent studies supporting the existence of a lineage boundary between the mesencephalon and metencephalon and others rejecting this. Here, I present data strongly supporting the existence of a compartment boundary between the posterior midbrain and anterior hindbrain territory. I base this proposition on cell-tracing experiments with single cell resolution. By connecting the traces to a molecular midbrain marker, I establish a link between cell fate and behavior. In the second part, I present a novel tissue explant method for the zebrafish that has the potential to serve numerous developmental studies, especially imaging of so far inaccessible regions of the embryo.

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The neuromeric concept of brain formation has become a well-established model to explain how order is created in the developing vertebrate central nervous system. The most important feature of neuromeres is their compartmentalization on the cellular level: Each neuromere comprises a lineage-restricted population of cells that does not intermingle with cells from neighboring compartments. The units of the vertebrate hindbrain, the rhombomeres, serve as the best-studied examples of neuromeres. Here, the lineage restriction mechanism has been found to function on the basis of differentially expressed adhesion molecules. To date, hard evidence for the existence of other lineage restricted regions in more anterior parts of the brain is still scarce. The focus of this study is the midbrain-hindbrain boundary (mhb) region, where the juxtaposition of the mesencephalon and metencephalon gives rise to a signaling center, termed the midbrain-hindbrain or isthmic organizer. Evidence for lineage restriction boundaries in the mhb region is still controversial, with some very recent studies supporting the existence of a lineage boundary between the mesencephalon and metencephalon and others rejecting this. Here, I present data strongly supporting the existence of a compartment boundary between the posterior midbrain and anterior hindbrain territory. I base this proposition on cell-tracing experiments with single cell resolution. By connecting the traces to a molecular midbrain marker, I establish a link between cell fate and behavior. In the second part, I present a novel tissue explant method for the zebrafish that has the potential to serve numerous developmental studies, especially imaging of so far inaccessible regions of the embryo.

During zebrafish gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Recent studies have identified silberblick (slb/wnt11) as a key molecule that regulates gastrulation movement through a conserved pathway, which shares significant similarity with a signalling pathway that establishes epithelial planar cell polarity (PCP) in Drosophila (Heisenberg et al., 2000; Veeman et al., 2003), suggesting a role for cell polarity in regulating gastrulation movements. However, the cellular and molecular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still not fully understood. In the first part of the thesis, the three-dimensional movement and morphology of individual cells in living embryos during the course of gastrulation were recorded and analysed using high resolution confocal microscopy. It was shown that in slb/wnt11 mutant embryos, hypoblast cells within the forming germ ring display slower, less directed migratory movements at the onset of gastrulation, which are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. The net movement direction of the cells is not changed, suggesting that slb/wnt11-mediated orientation of cellular processes serves to facilitate and stabilize cell movements during gastrulation. By using an in vitro reaggregation assay on mesendodermal cells, combined with an analysis of the endogenous expression levels and distribution of E-cadherin in zebrafish embryos at the onset of gastrulation, E-cadherin mediated adhesion was found to be a downstream mechanism regulating slb/wnt11 function during gastrulation. Interestingly, the effects of slb/wnt11 on cell adhesion appear to be dependent on Rab5-mediated endocytosis, suggesting endocytic turnover of cell-cell contacts as one possible mechanism through which slb/wnt11 mediates its effects on gastrulation movements. - Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 23 MB)- Übersicht über Inhalte siehe Dissertation S. 92 - 93"