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Results
Since epidermis in Pelobates cultripes and Phyllobates bicolor tadpolesexhibits similar structural and ultrastructural traits, a common description has been given, unless distinctive features were observed.
LM analysis: Larval epidermis is a bilayered epithelium with inner (basal) and outer (pavement) cells that shows specific morphological traits (Fig. 1A-C). Inner cells have been ascribed to the basal layer since they contact the basement membrane (dermal-epidermal junction), regardless of their derivation from the undifferentiated epidermis cells. Basal cells rest on a relatively thick dense dermis, and are large and tall prismatic to spheroid-ellipsoidal in shape. Pavement cells are smaller and roughly cuboidal or short prismatic in shape. On the dermal side of the basal cells, tonofilaments form dense patch-bands (Fig. 1A-C) in a cytoplasm background that ranges from opaque in prismatic cells to translucent in spheroid-ellipsoidal cells. These clear cells have been regarded as mature KZn, the main target of our research. In P. bicolor they possess an obvious peripheral layer of opaque cytoplasm (cortex, Fig. 1A, B), which is thinner in translucent KZn than KZn with a relatively dense cytoplasm, sometimes engaged in mitotic processes (Fig. 1B). Cytoplasm density is also variable in pavement cells (Fig. 1) which contain mucous granules (Fig. 1B) near their microridged free surface (Fig. 1A, B).
TEM analysis: oblique sections and/or partially overlapping of contiguous cells may give the appareance of a multi-layered epidermis (Fig. 1D). A somewhat range of cytoplasm density and tonofilament content is obvious in KZn, suggesting a maturational evolution: as a rule, the denser the cytoplasm the larger the amounts of filaments, accumulating in the infranuclear cytoplasm. Beside basal tonofilament accumulations, translucent KZn contain a distinctive peripheral cytoskeleton (Fig. 1D). All KZn contact the dermal-epidermal junction (Fig. 2A-E), confirming that they belong to the basal cell layer. On the dermal side, the tonofilament bundles arch upwards, approaching to the plasma membrane (Fig. 2E) and contributing to the hemidesmosomes (Fig. 2A-E). These junctions are not found only where KZn reach the epidermal-dermal junction with thin processes (Fig. 2B). Relationships between contiguous KZn are relatively loose on the dermal side, resulting in wide interstices (Fig. 2A, D). In P. bicolor KZn, the tonofilaments in the infanuclear cytoplasm form peripheral leaf-like patches (Figs. 2B and 3A) and bands roughly parallel to the cell surface (Fig. 2B). The tonofilament patches contour exiguous compartments of the interstices, contained in invaginations of KZ surface (Fig. 2C). In P. cultripes KZnthe peripheral cytoskeleton of the infranuclear cytoplasm is less compact, forming a reticular framework (Fig. 2D).
The lateral surfaces of KZn emanate thin processes resembling irregular microvilli (Figs. 2A, B, D and 3B, D, F) as well as larger processes joined by spot desmosomes (maculae adherentes, Fig. 3E). The cytoskeleton retains differential features in the two species: tonofilament bands roughly parallel to the lateral cell surface in P. bicolor (Fig. 3B, E), but irregular micro-trabeculation networks in P. cultripes, (Fig. 3D, F, G). Submature KZn contain slender rough endoplasmic reticulum (rer) cisterns (Figs. 2E and 3A), as well as small mitochondria with dense matrix (Fig. 3A) and dilated cristae (Fig. 3C, D). In P. bicolor, an obvious cell cortex starts to appear in immature KZn (Fig. 4A), assumes its typical appearances as a dense 1-1.5 µm thick cytoplasm layer in intermediate maturational stages (Fig. 2B), and eventually thins out in translucent KZn (Figs. 2B and 3E). High nucleo-plasmatic ratio confirms that KZn with relatively dense cytoplasms are immature cells of this specialised line, although they possess rer cisterns (Fig. 4A). In P. cultripes, the biosynthesis machinery includes Golgi stacks, releasing small vesicles with moderately opaque content (Fig. 4B). In the supranuclear cytoplasm, this product forms clusters of larger, translucent vesicles (up to 1.5 µm in diameter, Fig. 4C) contiguous to opaque mucous granules in pavement cells (Fig. 4C). The mucous product displays similar, secretory phase-dependent features in the two species investigated: small (up to 0.5 μm), electron-dense granules during intracytoplasmic storage (Fig. 4C, E), and large (up to 2 µm), moderately opaque granules during release (Fig. 4C, D). Near the body surface, contiguous cells are joined together by serial desmosomes, so that the intercellular spaces are reduced to slender interstices (Fig. 4C-E). The cytoplasm of pavement cells is opaque (Fig. 4C), but with a central-peripheral density gradient (Fig. 4E) towards the skin surface (Fig. 4D).