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Progression of metazoans through different developmental programs requires temporal control that is achieved by molecules originating from endocrine tissues that diffuse throughout the whole body of the animal to coordinate program execution by activating cell specific gene expression patterns. These programs then define cascades of successive, distinct developmental stages or the choice between alternative fates for the same stage. A model for this developmental control is found in the nematode C. elegans, where environmental cues signal through insulin and TGF-beta cascades to regulate the daf-12/nuclear hormone receptor (NHR) ligand synthesis that then coordinates organism wide developmental timing and fate choice. For cell intrinsic aspects of C. elegans temporal control of development, microRNAs play an important role but their connection to organism wide endocrine control is unknown. This work shows how the DAF-12/NHR directly activates let-7 family microRNAs during the L3 stage to repress L2 stage activator hbl-1 to prevent L2 stage programs from reoccurring. The interaction of upstream transcription factors with the downstream cis-regulatory elements in promoters of the let-7 family microRNAs are further analyzed in detail and identify potential DAF-12 coregulators that might connect daf-12 endocrine signaling also to later stage developmental control. These observations are the first to integrate microRNAs into establishedendocrine signaling cascades. In addition they reveal specific details about how organism wide upstream, endocrine signaling pathways induce downstream cell intrinsic changes of gene expression and developmental progression. This work postulates a "molecular switch" that actively drives stage transitions, consisting of a NHR that directly activates microRNAs to actively repress mediators of old stages while directly activating translation of protein coding genes mediating the new stage.

Biological tubes possessing a curvilinear form and a hollow interior exist in most multicellular eukaryotes. In Eumetazoa, the tubes usually comprise an eminently complex network and enable the transport and exchange of fluids and gases between tissues and organs, but also between organisms and their environment. Thus, tubular structures are both morphologically and physiologically integral parts of the animals. Based on a genetic screen for novel factors involved in heart tube differentiation and morphogenesis in Drosophila, the identified mutants were subdivided into several classes: cardiac hyperplasia (kuz and mam, both involved in the Notch-dependent cardiomyocyte specification, Publication 1); impaired cytokinesis (pav and tum, both components of the centralspindlin complex); a single ptc mutant showing a “truncated” heart (Publication 2); and a single loss-of-function mutant displaying reduced lumen diameter in epithelial tubes and perturbed secretion of ECM-components. The latter allele was mapped to the gene locus gartenzwerg (garz) that encodes a large ArfGEF. Due to its novelty, garz was selected as a central part of the thesis (Publication 3). Although garz seems to be expressed ubiquitously, its transcripts are abundant in active secreting cells of tubular structures. Moreover, mutations of garz abolish Golgi-integrity, cause massive retention of secretory cargo in the ER and arrest the apical transport of lipids and ECM molecules. As a consequence, lumen of the salivary glands and trachea fail to expand and show a decreased diameter. The observed phenotypes in tracheal network and salivary glands phenocopy those of COPI/COPII-subunits as well as actin-dependent secretion mutants, suggesting the underlying mechanism might be common. Thus, it is supposed that proper tubulogenesis needs Garz for initiation of the Arf1-COPI machinery. Furthermore, Golgi-based post-translational modifications, targeted sorting of vesicles, outward transport of proteins, or directed membrane delivery all depend on the secretory pathway, and such processes are essential in establishing polarized cells which build the tubular structures. In conclusion, this mechanism seems to be neither restricted to tubulogenesis nor specific to Drosophila. Due to the presence of garz homologues in every eukaryotic genomes, the Arf1/COPI based secretory pathway may play a universal role in metazoan development.

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)

Mutationen von Transkriptionsfaktoren (TF) betreffen nicht nur die Funktion des TFs, sondern auch die Expression seiner Zielgene und liegen häufig angeborenen Entwicklungsdefekten zugrunde. Über 20 Mutationen in HOXD13, einem TF der die Entwicklung der Extremitäten kontrolliert, sind bisher als Ursache verschiedenartiger Extremitätenfehlbildungen entdeckt worden. Eine molekularbiologische Grundlage für die Vielgestaltigkeit der HOXD13-Mutationen ist jedoch unbekannt. Die bisherigen Methoden zur funktionellen Charakterisierung von TF-Mutationen ermöglichten eine lediglich eingeschränkte Interpretation der molekularen Pathomechanismen. Die kürzlich entwickelte ChIP-seq Methode ermöglicht eine umfassende, funktionelle Charakterisierung eines TFs. In dieser Arbeit wurde eine Methode etabliert, um eine Vielzahl von Transkriptionsfaktoren und TF-Mutationen systematisch zu untersuchen. Zur Validierung wurden zwei neue Punktmutationen in HOXD13, p.Q317K und p.R298Q, charakterisiert. Beide Mutationen betreffen die DNA-bindende Domäne von HOXD13, rufen aber stark unterschiedliche Fehlbildungen hervor. Die Ergebnisse zeigen, dass die HOXD13Q317K Mutante eine veränderte Sequenzspezifität aufweist, welche nun jener eines anderen TFs, PITX1, ähnelt. Auch genomweit zeigt HOXD13Q317K ein Bindungsprofil, welches eher PITX1 als HOXD13wt entspricht. Durch weitere, unabhängige Analysen und Experimente wurde bestätigt, dass die p.Q317K Mutation HOXD13 in einen TF mit PITX1-ähnlichen Eigenschaften verändert. Die HOXD13R298Q-Mutante zeigt eine weitgehend unveränderte Bindungssequenz gegenüber HOXD13wt, jedoch eine veränderte Zusammensetzung der genomischen Bindestellen. Dies weist, in Kombination mit dem humanen Phänotyp auf einen dominant-negativen Pathomechanismus dieser Mutanten hin. Zusammengenommen zeigt diese Arbeit durch die Erhebung von experimentellen Daten, dass klar unterscheidbare molekularbiologische Mechanismen den HOXD13Q317K- und HOXD13R298Q-Mutationen zugrunde liegen. / Mutations in transcription factors (TF) do not only affect the function of the TF, but also the expression of its target genes and are frequently underlying congenital malformations. More than 20 distinct pathogenic mutations in HOXD13, a TF controlling limb development, have been associated with a broad range of limb malformations. However, a molecular basis underlying the variability of HOXD13-associated phenotypes remains elusive. To date, the experimental methods used to functionally characters TF mutations have allowed only limited insights into the underlying molecular pathomechanisms. The recently developed ChIP-seq technology has proven to be a powerful method to profile the binding characteristics of TFs; however a number of technical hurdles hinder its application for functional characterization of mutant TFs. This work describes the establishment of a ChIP-seq approach to investigate a wide spectrum of TFs and TF mutations. The approach was applied to characterize two previously unknown missense mutations in HOXD13, p.Q317K and p.R298Q, which both alter the DNA-binding domain of HOXD13 but cause very different disease phenotypes. The results show that the HOXD13Q317K mutant has an altered sequence specificity that resembles the recognition sequence of another TF, PITX1. Further, the genome-wide binding pattern of HOXD13Q317K shifts towards a more PITX1-like binding pattern. Even further analysis and viral overexpression in chicken limb buds confirm that the mutation partially converts HOXD13Q317K into a TF with PITX1-like properties. The HOXD13R298Q has a largely unchanged sequence specificity, but an altered composition of genomic binding sites. This, in combination with the human phenotype, indicates that the mutant might act in a dominant-negative manner. Collectively, this work shows through generation of direct experimental evidence, that clearly distinct molecular mechanisms underlie the pathogenicity of HOXD13Q317K and HOXD13R298Q mutations.

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