3D Imaging and Quantitative Analysis of Digestive Glands Morphogenesis in Crustacean Embryonic and Postembryonic Stages: Application of Micro-CT, Light and Electron Microscopy

The processes of morphogenesis during animal development are complex and interdependent at all organisational levels. Thus, it is beneficial to visualize morphogenesis at different scales and including 3D imaging and quantification. Here the application of a combination of complementary imaging techniques (micro-computed tomography, histology, and transmission electron microscopy), supplemented by morphometrical analyses, is presented in a study of digestive glands morphogenesis during embryonic and postembryonic development of isopod crustacean. The first pair of gland tubules is formed in mid-stage embryo from gland primordium, separated into two lobes by gland epithelium longitudinally from posterior to anterior direction. The width reduction and volume decrease of the tubules were observed from late embryo through marsupial and postmarsupial mancae stages. The second pair of gland tubules starts to form in mid-stage embryo S14 and elongates from late embryo onwards, with gradual volume increase. Epithelial cells of digestive glands are morphologically modified from cuboidal to the dome-shaped B cells and wedge-shape S cells in late marsupial mancae, which coincides with complete depletion of yolk within gland lumen. Septate junctions in gland epithelium elongate from embryos to postmarsupial mancae, while their ultrastructure does not change considerably. The most intense elongation of septate junction was evident at transition from marsupial to postmarsupial manca stage, which is consistent with release of the animal from marsupium to the external environment. Integration of data acquired by the presented imaging techniques and quantitative analyses allowed us to relate histological and ultrastructural modifications of epithelium to crucial transitions in digestive gland morphogenesis and to the key steps of animal embryonic and postembryonic development.