Imaging and analyzing neural tube during mouse embryonic development: new tools with synchrotron radiation to study this complex 3D structure

Full text: Neurulation is a critical process during embryonic development. The neural tube (NT), formed during neurulation, will differentiate into encephalic and medullar structures, thus, impact on neurulation could lead to serious consequences in newborns. The severity of these malformations varies from life impeding (e.g. anencephaly), life threatening (e.g. spina bifida) or might influence cognitive and motor ability. Also, some pathogens such as TORCH agents affects NT development in several degrees. Although severely affected embryos are easily noticed, identification of subtle malformations in a three-dimensionally complex structure like the NT is challenging using only two-dimensional conventional techniques such as histological sections, in situ hybridization and immunolabeling for optical or electron microscopy. Here we present preliminary results of an optimized 3D imaging approach, from sample preparation to analysis and quantification, addressing the NT development in mouse model. Whole embryos at 10.5 days post coitum (dpc), preserving NT structural complexity, were harvested and prepared for high resolution synchrotron microtomography at the IMX beamline. The first step in the computational workflow was to correctly separate the NT from all surrounding embryonic tissues. Superpixel-based supervised classification (using in-house developed software called Annotat3D) was applied to correctly identify NT cells in the three-dimensional volume. Next, NT segmentation was refined to fit the NT borders. Later, we developed algorithms to untwist the 3D embryo segmentation and quantify the thickness of neuroepithelium according to the embryo's antero-posterior (AP) axis. The results are promising, with 4 embryos from healthy mouse dams analyzed. In the next steps, we will validate this methodology and findings in embryos from affected dams in flavivirus infection model in order to better understand the impact of subtle malformations to postnatal life. (author)