Immunolocalization of Some Epidermal Proteins and Glycoproteins in the Growing Skin of the Australian Lungfish (Neoceratodus forsteri)

Abstract

Here we report the immunolocalization of mucin, nestin, elastin and three glycoproteins involved in tissue mineralization in small and large juveniles of Neoceratodus forsteri. Both small and larger juvenile epidermis are mucogenic and contain a diffuse immunolabeling for nestin. Sparse PCNA-labeled cells, indicating proliferation, are found in basal and suprabasal epidermal layers. No scales are formed in small juveniles but are present in a 5 cm long juvenile and in larger juveniles. Elastin and a mineralizing matrix are localized underneath the basement membrane of the tail epidermis where lepidotriches are forming. The latter appears as "circular bodies" in cross sections and are made of elongated cells surrounding a central amorphous area containing collagen and elastin-like proteins that undergo calcification as evidenced using the von Kossa staining. However, the first calcification sites are the coniform teeth of the small juveniles of 2-3 cm in length. In the superficial dermis of juveniles (16-26 cm in length) where scales are formed, the spinulated outer bony layer (squamulin) of the elasmoid scales contains osteonectin, alkaline phosphatase, osteopontin, and calcium deposits that are instead absent in the underlying layer of elasmodin. In particular, these glycoproteins are localized along the scale margin in juveniles where scales grow, as indicated by the presence of PCNA-labeled cells (proliferating). These observations suggest a continuous deposition of new bone during the growth of the scales, possibly under the action of these mineralizing glycoproteins, like in the endoskeleton of terrestrial vertebrates.

Keywords: evolution; immunolabeling; lungfish; skin; structure; ultrastructure.

Figure 1

DAPI immunofluorescence (A,B) and histology (C–F, toluidine blue stain) of the skin and oral epithelium of the head in a 2.5 cm long larva. (A) head featuring a thin epidermis and thicker oral epithelium (arrowheads). Bar, 100 μm. (B) Close up to the tip of the jaw showing the invaginating teeth epithelium (arrowhead) and forming epithelial ampullae (sensory organs). Bar, 20 μm. (C) particular of the forming teeth (arrows) in the maxilla and jaw. Bar, 50 μm. (D) Close up on a pointed tooth (arrow) with mineralized layer (enamel). Bar, 10 μm. (E) three forming and mineralized teeth (arrows) in the jaw. Bar, 10 μm. (F) Dark precipitate of calcium (arrows) in the mineralized layer of two teeth in the maxilla. Bar, 10 m. Legends: jct, cartilage of the jaw (Mekel); mb, mandible (jaw); mct, cartilage of the maxilla (palatoquadrate); te, telencephalon.

Figure 2

Histology (A) and immunofluorescence (B–G) of small and larger juvenile skin. (A) Thin epidermis with accumulating melanophores (arrowheads) in the dermis. Bar, 10 μm. (B) Small juvenile skin with external mucin immunoreactivity (arrowhead) and a mucous cell (arrow). Mucin-reactive dots are also seen along the basement membrane, suggesting presence of mucine-like glycoproteins. Bar, 10 μm Bar, 10 μm. (C) Thick epidermis of large juvenile containing PCNA-labeled nuclei (arrowheads). Bar, 20 μm. (D) diffuse nestine immunofluorescence in keratinocytes of small fish epidermis. Bar, 10 μm. (E) Immunonegative section. Bar, 10 μm. (F) Weak nestin immunofluorescence in thick epidermis of larger juvenile fish. Bar, 20 μm. (G) Negative control section of larger juvenile epidermis. Bar, 10 μm. Legends: de, dermis; e, epidermis; gl, likely forming gland. Dashes underline the epidermis.

Figure 3

Histology (A,B) and immunolabeling (C–E) of the skin in N. forsteri specimen 5 cm long. (A) Cross-section of multistratified tail epidermis showing sparse mucous- secreting cells (arrows) using toluidine blue stain. Arrowheads indicate cells surrounding “circular bodies” located beneath the basement membrane, bordered by the cytoplasmic elongation of pigment cells (double arrowhead). Bar, 20 μm. The inset (bar, 10 μm) shows the numerous fibroblasts (osteoblasts, arrowheads) surrounding the amorphous material and the central, pale region (arrow). (B) Close up on two “circular bodies” after PAS-methylene blue staining. The central region of the bodies is blue (arrow) while fibroblasts (arrowheads) surround a weakly pink (PAS-positive) fibrous material. Bar, 10 μm. (C) Dermal fibroblasts (arrowheads) surrounding circular bodies containing a central region immunoreactive for elastin (arrow). Bar, 20 μm. (D) Higher magnification of two circular bodies with central elastin immunostaining (arrow). Bar, 10 μm. (E) immunonegative control (arrow). Bar, 20 μm. Legends: d, dermis; e, epidermis. Dashes underline the epidermis.

Figure 4

Histology (toluidine blue) and von Kossa histochemistry (dark) of the tail skin in a 5 cm long larva. (A) Cross section showing the two sides of the epidermis with the “circular bodies” (lepidotrichia) located underneath. Bar, 20 μm. The inset (bar, 10 μm) shows a dark granulation derived from the von Kossa reaction for calcium. The central cores (arrowheads) are uncalcified. (B) Longitudinal section showing two lepidotrichia associated with numerous osteoblasts (arrows) underneath the epidermis. Bar, 10 μm. (C) Detail of a lepidotrichium with osteoblasts (arrowheads) surrounding the pale fibrous region (fi). Bar, 10 μm. (D) two terminal lepidotriches tips (arrows) contacting the epidermis. Bar, 10 μm. (E) longitudinal section of weakly mineralized (dark areas) lepidotrichium. Bar, 10 μm. (F) Two tips of weakly mineralized lepidotriches (arrows) terminating on the epidermis. Bar, 10 μm. (G) two heavily calcified lepidotriches (arrows) in cross section (roundish bodied). Bar, 10 μm. (H) A longitudinal section of a heavily calcified lepidotrichium. Bar, 10 μm. Legends: de, dermis; e, epidermis; fi, fibrous component of the lepidotrichia.

Figure 5

Histological sections of the skin of 5 cm long specimen (A) and of juveniles’ (B–I) skin. (A) Histological section showing a thin epidermis, loose dermis, spinulae (arrowheads), and the underlying thin fibrous layer (elasmodin, arrows) of the scale. Toluidine blue stain. Bar, 20 μm. (B) PAS-stained section showing intensely reactive mucus cells (arrowheads) and numerous melanophores in the dermis. The spinulae of the outer layer (squamulina) are surrounded by PAS-positive material (arrows). Bar, 20 μm. (C) Spinulated layer (denticles, arrows) immunolabeled for osteonectin (ONC). Arrowheads indicate cells associated to the denticles. The indicative position of dermal scales is shown in the associated drawing. Bar, 20 μm. (D) Detail of bony denticles immunolabeled for osteonectin and the associated cells (arrows indicating the nuclei). Bar, 10 μm. (E) detail on one bony denticle. Toluidine blue stain. Bar, 5 μm. (F) immunolabeled denticle for osteonectin (only TRITC immunofluorescence). Bar, 5 μm. (G) Osteopontin (OPT) labeling of denticles (arrows). A weaker immunofluorescence is also seen in the surrounding connective tissue and in the elasmodin layer of the scale. Arrowheads indicate cells associated to the denticles. Bar, 10 mm. (H) Immunolabeling of the spinulated layer (arrows) for alkaline phosphatase (ALP). Arrowheads indicate the nuclei of associated cells. Bar, 10 μm. (I) immunonegative control section (CO) with nuclei of the cells (arrowheads) associated to the spinulae. Bar, 10 μm. Legends: de, dermis; e, epidermis; el, elasmodin (layer); sp, spinulae or denticles (squamulin).

Figure 6

Histology (A–C) and immunofluorescence (D,F) of juvenile skin. (A) juvenile of 16 cm featuring an entire scale (arrows) located underneath the epidermis. Arrows indicate the outer or limiting layer formed by bony spinulae. Asterisks indicate a pocket-like area (arrowheads) occupied by loose dermal connective tissue. Bar, 20 μm. (B) Elasmoid scale with its definitive tilted disposition in the dermis. Asterisks indicate the loose dermal pocket associated with the spinulated bony surface. The arrows indicate the marginal regions (anterior, right, and posterior on the left) of the scale. Bar, 30 μm. The inset (bar, 20 μm) shows a detail of the cells (arrow) accumulated at the margin of the scale. (C) Detail of central region of a scale showing nuclei of cells (arrowheads) associated with the bony denticles of the scale. Asterisks label the soft dermal layer of the scale pocket. Bar, 20 μm. (D) Intense immunolabeling for osteonectin (arrows) in the spinulated layer located at the margin of the scale (dashes). Bar, 20 μm. (E) PCNA- immunolabeled cells (nuclei, arrow) located at the margin of a scale (dashed). Bar, 10 μm). (F) same section with merged fluorescence for DAPI (double- labeling pink nuclei, arrow). Bar, 10 μm. Legends: d, dermis; e, epidermis; el, elasmodin (fibrous basal layer of the scale); sp, bony spinulae (denticles) of the outer layer of the scale.

Figure 7

Von Kossa staining for calcium deposition (black) in 5 cm long specimen (A–C) and juveniles (D–F). Von Kossa stain (dark) and wekly methylene blue stain. (A) Formation of denticles of squamulin (arrows) on a thin scale. Bar, 10 μm. (B) Close up of a pointed denticle with initial calcification (dark, arrowhead). Bar, 5 μm; (C) Larger denticle with initial calcification (arrowhead). Bar, 5 μm. (D) low magnification and panoramic view of a longitudinal section showing four calcified scales in their squamulin (denticles) layer. Bar, 20 μm. (E) Close up of a calcified scale (arrow on denticles). Bar, 10 μm. (F) detail on the marginal area of a scale with calcified denticles (arrow). Bar, 10 μm. Legends: de, dermis; e, epidermis; el, elasmodin; sca, scales.