A new method for observing the formation of tissues with complex geometries
The dorsal aorta of the zebrafish embryo imaged by a fluorescence microsope combined with a confocal microscope. The 3D position of each nuclei was determined using a software. The red and blue spheres represent the positions of the artery and vein nuclei, respectively.
Philos Trans R Soc Lond B Biol Sci Sept. 24, 2018
Sept. 25, 2018
Throughout the development of the embryo, the shape of tissues and organs changes significantly. In order to observe the evolution of complex structures with cylindrical geometry, such as the aorta, the digestive tract or even the lungs and kidneys, researchers from Julien Vermot's team at the IGBMC (CNRS/Inserm/University of Strasbourg) have developed a new sample preparation methodology for acquiring three-dimensional (3D) microscopy data and for the following image analyses. This work was published on September 24 in the journal Philisophical Transactions B.
Tissues must constantly adapt their shape during embryonic development, which requires a high degree of tissue plasticity and generates a wide variety of transient three-dimensional geometries. Several image analysis tools are available to extract parameters of cells composing such structures. Unfortunately, most of them are developed for tissues with relatively simple geometries, such as flat epithelia. Problems arise when the tissue of interest assumes a more complex 3D geometry.
In this study, Julien Vermot's team chose to study the tissue enveloping the inner surface of developing zebrafish dorsal aorta as an example of a tissue with cylindrical geometry. With the support of the IGBMC Imaging Platform, researchers have developed a new sample preparation method and image analysis routine, which enables the study of the vascular tissue architecture and the position of cells that compose it.
First, in collaboration with Gilles Charvin's team, the researchers printed a 3D mould allowing them to create chambers where the zebrafish embryos can be immobilized in the same position at each observation. Small cavities at the bottom of the mould accommodate the small yolk-sac* allowing the embryos to rest perfectly on their side, thus ensuring that the antero-posterior axis is always oriented in the same direction. To visualize the dorsal aorta, fluorescently labelled embryos were imaged using 2-photon microscopy combined with confocal microscopy. For each embryo analysed, a stack of images was acquired at different depths to completely encompass the vessel. Finally, the researchers used their image analysis routine to extract quantitative data that can be averaged and clustered over multiple embryos.
By repeating such analyses at different embryonic stages, the researchers could observe the distribution of cells during the maturation of the developing dorsal aorta. This innovative method could be applied to other tissues with cylindrical architecture and provide new knowledge on tissue dynamic.
*reserves in a nutritious way present at the beginning of life and located under the embryo of zebra fish
This study was funded by the Foundation for Medical Research, the ANR, the Lefoullon Delalande Foundation and the European Molecular Biology Organization.