Computational protocol: The Notch pathway in the annelid Platynereis: insights into chaetogenesis and neurogenesis processes

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Protocol publication

[…] Image registration of three-dimensional gene pattern scans based on the DAPI nuclear labelling were performed using the open-source software package ITK4.0 and a collection of specific scripts developed in the Jékely Lab []. All patterns were registered to average anatomical reference templates (48 and 72 hpf). Both of these reference templates were calculated using a total of 40 DAPI-labelled larva three-dimensional stacks. The complete protocol for unbiased anatomical reference calculation and the reference files themselves are available upon request. Average patterns for a given gene were calculated using at least five individual larvae and gene pattern comparisons were performed using ImageJ and Imaris. [...] Bright field images were taken on a Leica microscope. Classical confocal images were taken with a Leica SP5 confocal microscope. Adjustments of brightness, contrast and Z projections were performed using the ImageJ software. Confocal images for registration procedure were taken in a Zeiss LSM 710 confocal microscope. Three-dimensional surface of expression patterns and associated videos were performed using, respectively, the surface and animation tools of Imaris. Co-localization stacks of expression patterns were obtained with the Coloc tool of Imaris and co-localization images were then edited on ImageJ. Live imaging of the 24 hpf FM4-64 labelled embryos was performed on a Zeiss LSM 780 confocal microscope with a 63× water immersion objective. Z-stacks (z = 0.53 µM) of several whole embryos were performed every hour (using the multiposition option). The figure panels were compiled using Adobe Illustrator and Adobe Photoshop software. […]

Pipeline specifications

Software tools ImageJ, Imaris, Coloc, Adobe Illustrator
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Platynereis dumerilii, Drosophila melanogaster, Caenorhabditis elegans