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Material and methods
To refer to this article use this url: http://dpc.uba.uva.nl/ctz/vol76/nr04/art01
We analysed limbless tadpoles in the ontogenetic range 23-27, marked by development of operculum and labial teeth (Gosner, 1960). Epidermis maintains a stable structure in these stages, until cutaneous glands develop during toe morphogenesis (Delfino et al., 1991). Tadpoles of Pelobates cultripes - six specimens - and Phyllobates bicolor - eight specimens - were collected near Ciudad Real (Spain) and Medelin (Colombia), respectively, and sacrificed with 0.1% chlorobutanol. Skin strips, 9 mm2, were fixed in the Karnovsky (1965) solution, postfixed in 1% OsO4 (both in cacodylate buffer) and embedded in Epon 812. Semi-thin sections of 1.5 µm thickness (for the LM) were stained with buffered (1%, borax) toluidine blue, ultrathin sections with a hydroalcoholic saturated solution (25 mg/ml) of uranyl acetate followed by alkaline lead citrate (2 mg/ml). Ultrathin sections were observed (80 KV) with a Siemens 101 TEM.
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Fig. 1. Epidermis structure in limbless tadpoles of Phyllobates bicolor (a-b, LM micrographs) and Pelobates cultripes (c, LM micrograph; d, TEM micrograph). A. KZn and pavement cells varying in cytoplasm density. B. Mitotic pattern (encircled) in KZ and mucous granules (mg) in pavement cell. C. KZn with different cytoplasm densities and melanin granules (p). D. KZn vary considerably in both cytoplasm density and filament content. (*)(*)(*) ◊ (*) = decreasing cytoplasm density, small arrows in a and b point to microridges, arrowheads in a-d to basal tonofilament patches, large arrow in d to peripheral cytoskeleton. |
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Fig. 2. Basal tonofilament bundles and hemidesmosomes in KZn of Pelobates cultripes (a, d) and Phyllobates bicolor (b, c, e). A. Both submature (*)(*) and immature (*)(*)(*) KZn exhibit basal bundles. B. Two submature KZn (*)(*) and one mature KZ (*) with thick and thin cytoplasm ‘cortices’, respectively (opposite arrowheads); ellipse encircles a hemidesmosome-free area at the dermal-epidermal interface. C. The light area (arrowhead) is an interstitial space in a semi-tangential cell section, as suggested by the dotted line. D. In this mature KZ, tonofilaments form a micro-trabeculation (arrowheads). E. Tonofilaments converging towards the dermal side. Arrows point to thin cytoplasm processes, he = hemidesmosomes, i = interstice, rer = rough endoplasmic reticulum. |
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Fig. 3. Representative organelles and lateral portions of KZn. Pelobates cultripes (d, f, g) and Phyllobates bicolor (a, b, c, e). A. Cisterns of rough endoplasmic reticulum and mitochondria with dense matrix; arrowhead points to a tonofilament patch. B. Slender processes (arrows) from KZn and peripheral tonofilaments with a regular course (opposite arrowheads). C. Mitochondrion with dilated cristae. D. Micro-trabeculation (arrowhead ) and spheroid mitochondrion with dilated cristae (encircled); arrow points to slender cytoplasm process. E. Desmosomes (encircled) and contiguous cell cortices. F. Irregular, micro-trabeculation network; arrows point to slender cytoplasm processes. G. Detail of micro-trabeculation. |
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Fig. 4. Biosynthesis machinery in KZn (a, Phyllobates bicolor, b and c, Pelobates cultripes) and secretory activity in pavement cells (c, Pelobates cultripes, d and e, Phyllobates bicolor). A. Immature KZ with slender rough reticulum cisterns (arrows). B. Golgi stack releasing minute vesicles (arrow). C. Pavement cell storing small dense granules (mg’) and releasing a larger granule (mg), contiguous to immature KZn holding vesicle clusters (arrows). D. Serial desmosomes (arrows) and large granules (mg) near the body surface. The large, grey-gradient arrow shows a gradual increase in cytoplasm density. E. Small dense granules (mg’) and serial desmosomes (arrows). |