Three-dimensional microscopy and image analysis methodology for mapping and quantification of nuclear positions in...

Organogenesis involves extensive and dynamic changes of tissue shape during development. It is associated with complex morphogenetic events that require enormous tissue plasticity and generate a large variety of transient three-dimensional geometries that are achieved by global tissue responses. Nevertheless, such global responses are driven by tight spatio-temporal regulation of the behaviours of individual cells composing these tissues. Therefore, the development of image analysis tools that allow for extraction of quantitative data concerning individual cell behaviours is central to study tissue morphogenesis. There are many image analysis tools available that permit extraction of cell parameters. Unfortunately, the majority are developed for tissues with relatively simple geometries such as flat epithelia. Problems arise when the tissue of interest assumes a more complex three-dimensional geometry. Here, we use the endothelium of the developing zebrafish dorsal aorta as an example of a tissue with cylindrical geometry and describe the image analysis routines developed to extract quantitative data on individual cells in such tissues, as well as the image acquisition and sample preparation methodology.This article is part of the Theo Murphy meeting issue 'Mechanics of development'.