Digital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, Platynereis is particularly well suited for such studies.
Whole-body gene expression pattern registration in Platynereis trochophore and nectochaete larvae is based on whole-mount in situ hybridization, confocal microscopy, and image registration. In confocal microscopy we achieve high-resolution whole-body scanning using the mounting medium 2,2'-thiodiethanol (TDE), which allows the matching of the refractive index of the sample to that of glass and immersion oil. This reduces spherical aberration and improves depth penetration. This approach allows us to scan entire whole-mount larvae stained with NBT/BCIP in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and a nuclear stain (DAPI). Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental stage. Both the acetylated-tubulin- and the nuclear-stain-based templates allow near-cellular-resolution gene expression registration.