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)

During vertebrate development, gastrulation leads to the formation of three distinct germlayers. In zebrafish a central process is the delamination and the ingression of single cells from a common ancestor tissue - that will lead to the formation of the germlayers. Several molecules have been identified to regulate this process but the precise cellular mechanisms are poorly understood. Differential adhesiveness, a concept first introduced by Steinberg over 40 years ago, has been proposed to represent a key phenomena by which single hypoblast cells separate from the epiblast to form the mesendoderm at later stages. In this work it is shown that differential adhesion among the germlayer progenitor cells alone cannot predict germlayer formation. It is a combination of several mechanical properties such as cell cortex tension, cell adhesion and membrane mechanical properties that influence the migratory behavior of the constituent cells.

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During vertebrate development, gastrulation leads to the formation of three distinct germlayers. In zebrafish a central process is the delamination and the ingression of single cells from a common ancestor tissue - that will lead to the formation of the germlayers. Several molecules have been identified to regulate this process but the precise cellular mechanisms are poorly understood. Differential adhesiveness, a concept first introduced by Steinberg over 40 years ago, has been proposed to represent a key phenomena by which single hypoblast cells separate from the epiblast to form the mesendoderm at later stages. In this work it is shown that differential adhesion among the germlayer progenitor cells alone cannot predict germlayer formation. It is a combination of several mechanical properties such as cell cortex tension, cell adhesion and membrane mechanical properties that influence the migratory behavior of the constituent cells.

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)

Weanalyze the mechanical properties of three epithelial/mesenchymal cell lines (MCF-10A, MDAMB- 231, MDA-MB-436) that exhibit a shift in E-, N- and P-cadherin levels characteristic of an epithelial−mesenchymal transition associated with processes such as metastasis, to quantify the role of cell cohesion in cell sorting and compartmentalization. Wedevelop a unique set of methods to measure cell–cell adhesiveness, cell stiffness and cell shapes, and compare the results to predictions from cell sorting in mixtures of cell populations.Wefind that the final sorted state is extremely robust among all three cell lines independent of epithelial or mesenchymal state, suggesting that cell sorting may play an important role in organization and boundary formation in tumours.Wefind that surface densities of adhesive molecules do not correlate with measured cell–cell adhesion, but do correlate with cell shapes, cell stiffness and the rate at which cells sort, in accordance with an extended version of the differential adhesion hypothesis (DAH). Surprisingly, theDAHdoes not correctly predict the final sorted state. This suggests that these tissues are not behaving as immiscible fluids, and that dynamical effects such as directional motility, friction and jamming may play an important role in tissue compartmentalization across the epithelial−mesenchymal transition.

The process of cell-sorting is essential for development and maintenance of tissues. With the Differential Adhesion Hypothesis, Steinberg proposed that cellsorting is determined by quantitative differences in cell-type-specific intercellular adhesion strengths. An implementation of the Differential Adhesion Hypothesis is the Differential Migration Model by Voss-Böhme and Deutsch. There, an effective adhesion parameter was derived analytically for systems with two cell types, which predicts the asymptotic sorting pattern. However, the existence and form of such a parameter for more than two cell types is unclear. Here, we generalize analytically the concept of an effective adhesion parameter to three and more cell types and demonstrate its existence numerically for three cell types based on in silico time-series data that is produced by a cellular-automaton implementation of the Differential Migration Model. Additionally, we classify the segregation behavior using statistical learning methods and show that the estimated effective adhesion parameter for three cell types matches our analytical prediction. Finally, we demonstrate that the effective adhesion parameter can resolve a recent dispute about the impact of interfacial adhesion, cortical tension and heterotypic repulsion on cell segregation. / Der Prozess der Zellsortierung ist für die Entwicklung und Erhaltung von Geweben unerlässlich. Mit der Differentiellen Adhäsionshypothese schlug Steinberg vor, dass die Zellsortierung durch quantitative Unterschiede in den zelltypspezifischen interzellulären Adhäsionsstärken bestimmt wird. Eine Umsetzung der Differentiellen Adhäsionshypothese ist das Differentielle Migrationsmodell von Voss-Böhme und Deutsch. In diesem wurde für Systeme mit zwei Zelltypen ein effektiver Adhäsionsparameter analytisch hergeleitet, der das asymptotische Sortiermuster vorhersagt. Die Existenz und Form eines solchen Parameters für mehr als zwei Zelltypen ist jedoch unklar. Hier verallgemeinern wir analytisch das Konzept eines effektiven Adhäsionsparameters für drei und mehr Zelltypen und zeigen numerisch seine Existenz für drei Zelltypen auf der Basis von in silico Zeitreihendaten, die von einem zellulären Automaten des Differentiellen Migrationsmodells erzeugt werden. Darüber hinaus klassifizieren wir das Segregationsverhalten mithilfe statistischer Lernverfahren und zeigen, dass der geschätzte effektive Adhäsionsparameter für drei Zelltypen mit unserer analytischen Vorhersage übereinstimmt. Schließlich zeigen wir, dass der effektive Adhäsionsparameter eine kürzlich aufgekommene Diskussion über den Einfluss von Grenzflächenadhäsion, Kortikalspannung und heterotypischer Abstoßung auf die Zellsegregation lösen kann.