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Ultrastructural and imunohistochemical features of the epidermal cells in amphioxus Branchiostoma lanceolatum

Introduction: Epidermis is a surface organ which protects animal from the environmental effects and from the pathogens. In vertebrate family, the epidermis of terrestrial animals is keratinized while in aquatic forms and amphibians can produce mucus. Amphioxus, a cephalochordate, is an important animal model in the study of vertebrate (chordate) phylogeny. Objectives: Epidermal cells of amphioxus (Branchiostoma lanceolatum L.) were investigated morphologically by transmission electron microscopy (TEM) and using immunohistochemistry and immunofluorescence for detection of a major cytoskeletal and extracellular matrix proteins. Materials & methods: The study was done on ten adult amphioxus specimens fresh-caught in the Adriatic Sea. After fixation, dehydration and embedding in paraffin blocks, paraffin sections of 6 μm thick were mounted on glass slides. For immunohistochemistry and immunofluorescence, slides were deparaffinized and rehydrated, then heated in a citrate buffer. Protein blocking buffer was used to exclude unspecific staining. The sections were incubated with primary antibodies according to their own protocols. For immunohistochemistry, DAB staining was used, and for immunofluorescence the sections were incubated with suitable secondary antibodies for one hour. For transmission electron microscopy, small pieces of tissue were fixed in 3.5% paraformaldehyde during 24 hours on 4 °C, and afterwards in 3% glutaraldehyde for 2 hours. The post fixation was done in 2% osmium tetroxide and tissue was embedded in Epoxy resin and cut transversally. Ultrathin sections, stained with uranyl acetate and lead citrate, were observed under Zeiss EM 10A electron microscope. Results: Epidermis of the amphioxus consisted of one-layered columnar epithelium. These epithelial cells were characterized by voluminous nucleus, a well-developed supranuclear Golgi apparatus, abundant vesicles and apical secretory vesicles surrounded by cytokeratin filaments. A single row of smaller vesicles also appeared close to the basal cell membrane. Short microvilli were seen on the apical domain of the epidermal cells. The plasma membrane of adjacent cells strongly intertwined on its lateral domains in the form of finger-like protrusions. Positive immunolocalization of hyaluronic acid and collagen type I was observed in apical domain of plasma membrane of amphioxus, respectively. This positive signal probably refers to the secretory granules in the apical domain of the epidermal cell shown on the TEM. Immunolocalization of aggrecan was seen around the cell nucleus, deep below the apical domain, suggesting that aggrecan is not located in secretory granules. A strong signal to cytokeratin 10 was present throughout the cytoplasm of the epidermal cells. Conclusion: Due to its morphology and histochemical composition, epidermal cells of amphioxus may contribute to course surface protection and the mucous layer in amphioxus may serve as a physical barrier between the body and its environment.

Epidermis ; Amphioxus ; Immunohistochemistry ; Immunofluorescence ; Transmission electron microscopy

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