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)

Die Lokalisierung von mRNA ist ein wichtiger regulativer Mechanismus in polarisierten Zellen und in frühen Embryonalstadien. Dort sind räumliche Muster maternaler mRNA für die korrekte Entwicklung der Körperachsen und die Spezifizierung der Keimzellen verantwortlich. Systematische Analysen dieser Prozesse wurden jedoch bisher limitiert durch einen Mangel an räumlicher und zeitlicher Auflösung von Einzelzell- Sequenzierungsdaten. Wir analysierten die Dynamik des räumlichen und zeitlichen Transkriptoms während frühen Embryonalstadien von Zebrafischen. Wir verbesserten Empfindlichkeit und Auflösung von tomo-seq und erfassten damit systematisch räumlich aufgelöste Transkriptome entlang der animal-vegetalen-Achse Embryonen im Einzell-Stadium und fanden 97 vegetal lokalisierte Gene. Außerdem etablierten wir eine Hochdurchsatz kompatible Variante der RNA-Markierungsmethode scSLAM-seq. Wir wendeten diese in Embryonen während der Gastrulation. Von den vegetal lokalisierten Genen waren 22 angereichert in Keimzellen, was eine funktionelle Rolle bei der Spezifizierung von Keimzellen nahelegt. Mit tomo-seq untersuchten wir die evolutionäre Konservierung der RNA-Lokalisierung zwischen Zebrafischen und gereiften Oozyten zweier Xenopus-Arten. Wir verglichen die lokalisierten Gene, suchten nach konservierten 3'UTR-Motiven, und fanden zum Teil überlappende Motive, was auf eine mögliche mechanistische Konservierung der Lokalisierungsmechanismen hinweist. Wir untersuchten auch RNA-Editierung von Adenin zu Inosin während der Embryonalentwicklung und in den Organen erwachsener Fische. In im Gehirn exprimierten Transkripten fanden wir 117 Editierstellen, die hauptsächlich für Ionentransporter kodieren und zum Teil zum Menschen konserviert sind. Die höchsten Editierraten konnten wir in Eierstöcken, Hoden und frühen Embryonen nachweisen, was auf eine mögliche Rolle bei der Regulierung der RNA-Stabilität hindeutet. / Subcellular localization of mRNA is an important regulatory mechanism in polarized cells. In early embryos of many species, spatial patterns of maternal mRNA are essential for the proper development of body axes and the specification of germ cells. These processes have been studied in zebrafish, but systematic analyses have been hindered by a lack of spatial and temporal information in single-cell RNA sequencing. We performed a spatial-temporal analysis of the zebrafish transcriptome during early embryonic development to systematically characterize localized mRNA and the fate of maternal transcripts until gastrulation stage. We enhanced sensitivity and resolution of the tomo-seq method and systematically acquired spatially-resolved transcriptomes along the animal-vegetal axis of one-cell stage zebrafish embryos, and found 97 genes to be localized vegetally. Furthermore, we established an in vivo and high-throughput compatible version of the single-cell RNA labeling method scSLAM-seq in gastrulation stage embryos. We followed localized transcripts until gastrulation and found transcripts of 22 of the vegetally localized genes enriched in primordial germ cells. We propose that these genes have a functional role in the early priming of the germ cell fate. To investigate the evolutionary conservation of vegetal RNA localization, we acquired tomo-seq datasets of mature oocytes of two xenopus species. We compared the pools of localized RNA and searched for conserved 3’UTR motifs. The resulting sets showed high similarity, possibly reflecting a mechanistic conservation of localization pathways. We also investigated RNA A-to-I editing during embryonic development and in organs of adult fish. Specifically, we identified 117 recoding editing sites in the brain that mainly encode for ion transporters and are partly conserved in humans. We detected the highest editing levels in ovary, testes and in early embryos, implicating a potential role in regulating RNA stability.