@article {45719, title = {The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes.}, journal = {Journal of Cell Science}, volume = {127}, year = {2014}, month = {2014 Oct 15}, pages = {4381-95}, abstract = {

Inflammatory cells acquire a polarized phenotype to migrate towards sites of infection or injury. A conserved polarity complex comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization. However, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in three-dimensional (3D) environments in vivo. Using genetically encoded bioprobes and high-resolution live imaging, we reveal the existence of F-actin oscillations in the trailing edge and constant repositioning of the microtubule organizing center (MTOC) to direct leukocyte migration in wounded medaka fish larvae (Oryzias latipes). Genetic manipulation in live myeloid cells demonstrates that the catalytic activity of aPKC and the regulated interaction with PAR-3 and PAR-6 are required for consistent F-actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho kinase (also known as ROCK or ROK) activation. We propose that the PAR complex coordinately controls cytoskeletal changes affecting both the generation of traction force and the directionality of leukocyte migration to sites of injury.

}, issn = {1477-9137}, doi = {10.1242/jcs.146217}, author = {Crespo, Carolina and Vernieri, Claudio and Keller, Philipp J and Garr{\`e}, Massimiliano and Bender, Jeffrey R and Wittbrodt, Joachim and Pardi, Ruggero} } @article {2027, title = {Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy.}, journal = {Nature Methods}, volume = {7}, year = {2010}, month = {08/2010}, pages = {637-42}, abstract = {

Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality, and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.

}, keywords = {Animals, Drosophila melanogaster, Embryo, Microscopy, Nonmammalian, Zebrafish}, issn = {1548-7105}, doi = {10.1038/nmeth.1476}, author = {Keller, Philipp J and Schmidt, Annette D and Santella, Anthony and Khairy, Khaled and Zhirong Bao and Wittbrodt, Joachim and Stelzer, Ernst H K} } @article {1681, title = {Nlcam modulates midline convergence during anterior neural plate morphogenesis.}, journal = {Developmental Biology}, volume = {339}, year = {2010}, month = {2010 Mar 1}, pages = {14-25}, abstract = {

During development, different cell types must undergo distinct morphogenetic programs so that tissues develop the right dimensions in the appropriate place. In early eye morphogenesis, retinal progenitor cells (RPCs) move first towards the midline, before turning around to migrate out into the evaginating optic vesicles. Neighbouring forebrain cells, however, converge rapidly and then remain at the midline. These differential behaviours are regulated by the transcription factor Rx3. Here, we identify a downstream target of Rx3, the Ig-domain protein Nlcam, and characterise its role in regulating cell migration during the initial phase of optic vesicle morphogenesis. Through sophisticated live imaging and comprehensive cell tracking experiments in zebrafish, we show that ectopic expression of Nlcam in RPCs, as is observed in Rx3 mutants, causes enhanced convergence of these cells. Expression levels of Nlcam therefore regulate the migratory properties of RPCs. Our results provide evidence that the two phases of optic vesicle morphogenesis: slowed convergence and outward-directed migration, are under different genetic control. We propose that Nlcam forms part of the guidance machinery directing rapid midline migration of forebrain precursors, where it is normally expressed, and that its ectopic expression upon loss of Rx3 imparts these migratory characteristics upon RPCs.

}, keywords = {Animals, Base Sequence, Body Patterning, Cell Adhesion Molecules, Chick Embryo, DNA Primers, Electrophoretic Mobility Shift Assay, Homeodomain Proteins, Morphogenesis, Neural Plate, Protein Binding, Zebrafish}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2009.12.003}, author = {Brown, Katherine E and Keller, Philipp J and Ramialison, Mirana and Rembold, Martina and Stelzer, Ernst H K and Loosli, Felix and Wittbrodt, Joachim} } @article {1687, title = {Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.}, journal = {Science}, volume = {322}, year = {2008}, month = {2008 Nov 14}, pages = {1065-9}, abstract = {

A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides "digital embryos," that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo{\textquoteright}s cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.

}, keywords = {Algorithms, Animals, beta Catenin, Biological, Body Patterning, Cell Division, Cell Nucleus, Computer-Assisted, Databases, Embryo, Embryonic Development, Endoderm, Factual, Fluorescence, Germ Layers, Image Processing, Mesoderm, Microscopy, Models, Motion Pictures as Topic, Mutation, Nonmammalian, Software, Zebrafish}, issn = {1095-9203}, doi = {10.1126/science.1162493}, author = {Keller, Philipp J and Schmidt, Annette D and Wittbrodt, Joachim and Stelzer, Ernst H K} }