Wnt11 is a key signalling molecule that regulates cell polarity/migration during vertebrate development and also promotes the invasive behaviour of adult cancer cells. It is therefore essential to understand the mechanisms by which Wnt11 signalling regulates cell behaviour. The process of vertebrate gastrulation provides an excellent developmental system to study Wnt11 function in vivo. It is known that Wnt11 mediates coordinated cell migration during gastrulation via the non-canonical Wnt pathway that shares several components with a the planar cell polarity pathway (PCP) in Drosophila. However, the mechanisms by which these PCP components facilitate Wnt11 function in vertebrates is still unclear. While in Drosophila, the asymmetric localization of PCP components is crucial for the establishment of cell polarity, no asymmetric localization of Wnt11 pathway components have so far been observed in vertebrates. To shed light on the cellular and molecular mechanisms underlying Wnt11 signalling, I developed an assay to visualize Wnt11 activity in vivo using live imaging of Wnt11 pathway components tagged to fluorescent proteins. This allowed me to determine the sub-cellular distribution of these components and to correlate the effect of Wnt11 activity with the behaviour of living embryonic cells. I found that Wnt11 locally accumulates together with its receptor Frizzled7 (Fz7) at sites of cell-cell contacts and locally recruits the intra-cellular signalling mediator Dishevelled (Dsh) to those sites. Monitoring these apparent Wnt11 signalling centres through time-lapse confocal microscopy revealed, that Wnt11 activity locally increases the persistency of cell-cell contacts. In addition, I found that the atypical cadherin Flamingo (Fmi) is required for this process. Fmi accumulates together with Wnt11/Fz7 at sites of cell-cell contact and locally increased cell adhesion, via a mechanism that appears to be independent of known downstream effectors of Wnt11 signalling such as RhoA and Rok2. This study indicates that Wnt11 locally interacts with Fmi and Fz7 to control cell-contact persistency and to facilitate coherent and coordinated cell migration. This provides a novel mechanism of non-canonical Wnt signalling in mediating cell behaviour, which is likely relevant to other developmental systems. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 50 MB: Movies - Nutzung: Referat Informationsvermittlung der SLUB)

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"