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Keywords:

  • Kugelzellen;
  • skein cells;
  • ultrastructure;
  • larval epidermis;
  • hylid frogs

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

An ultrastructural study was carried out on the epidermis of Agalychnis callidryas tadpoles during limb development. Larval epidermis consisted of four cell layers: basal, lower intermediate, upper intermediate, and surface or apical layers. Basal cells represented the stem compartment of intermediate cells: both belong to the skein cell (SC) lineage, described in several anuran species, on account of the conspicuous intracytoplasmic tonofilament bundles. Apical cells were secretory in nature and released mucus on the body surface. Intermediate SCs exhibited a hydrated central cytoplasm and peripheral tonofilament bundles. They closely resembled the epidermal ball-like cells, Kugelzellen (KZn) of Xenopus laevis tadpoles, and possibly shared their turgor-stiffness properties. In A. callidryas, the stratification of intermediated SCs on their stem cell layer provided the chance to study their cytodifferentiation in a suitable sequence, until basal cell differentiation shifted toward the keratinocyte lineage in premetamorphic stages. Present data assign A. callidryas to the anuran species with a constitutive SC population in larval epidermis, and demonstrate that KZn express the ultimate specialization of such cell line. SCs were arranged in the fashion of a random-rubble stone groundwork, and possessed long processes. These cytoplasmic outgrowths contained a tonofilament axial rod and held together contiguous cells. Ultrastructural findings suggest that this complex structure may impart compressive as well as sliding strengths to the larval epidermis, representing a possible adaption to the fresh water environment. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Larval epidermis of the South African clawed frog Xenopus laevis includes peculiar cells with translucent cytoplasm and a remarkable peripheral network of tonofilaments. These cells, which degenerate in the premetamorphic stages, were previously identified as Leydigschen Drüsenzellen (Pflugfelder and Schubert, 1965). Indeed, they share structural and ultrastructural similarities with Leydig cells in Urodela (Delfino et al., 2007) that are secretory in appearance, producing vesicles and granules. To avoid possible ambiguity, these cells should be properly characterized for their spheroidal shape as Kugelzellen, KZn, or ball cells (Fröhlich et al., 1977). However, they have been also described as clear cells (Fox, 1988) or “vacuoles” (Seki et al., 1989), on account of their transparent, apparently empty, cytoplasm.

Large clear cells, resembling KZn in both their shape and ultrastructural features, have also been detected with light microscope (LM) and transmission electron microscopes (TEM) in the bilayered epidermis of limbless tadpoles of the Western spade-foot frog Pelobates cultripes and the dart-arrow frog Phyllobates bicolor (Delfino et al., 2007). These two species belong to distantly related families (Pelobatidae and Dendrobatidae), branches [97] and [462] respectively, of the anuran taxonomical tree (Frost et al., 2006). Therefore, it appears that KZn are not exclusive to the clawed frog, but have an extensive phylogenetic range in this order.

Studies on tadpoles of ranid frogs: Pelophylax esculentus, formerly Rana esculenta, (Fenoglio et al., 2008) and Lithobates catesbeianus, formerly R. catesbeiana (Robinson and Heintzelman, 1987; Nishikawa et al., 1989; Izutsu et al., 1993; Kawai et al., 1994; Takada et al., 1996, Menon and Wahrman, 1999, 2001; Suzuki et al., 2001, 2002; Ishida et al., 2003) reported epidermal “skein cells” (SCs). SCs possess a transparent cytoplasm containing thick bundles of tonofilaments, detectable as “figures of Eberht” with both TEM (Robinson and Heintzelman, 1987; Nishikawa et al., 1989; Izutsu et al., 1993; Menon and Wahrman. 1999, 2001; Fenoglio et al., 2008) and fluorescence LM (Kawai et al., 1994; Suzuki et al., 2001). In recent studies on Xenopus spp., Kinoshita and Sasaki (1994a, b), Izutsu et al. (2000), Watanabe et al. (2001), Watanabe et al. (2003) described Kugelzellen as skein cells. These reports ultimately established that SCs and KZn belong to the same cell lineage, possibly in intermediate and, respectively, advanced phases of specialization.

The ontogeny of SCs is a marker of development. There is a dramatic decline of these larva-specific cells prior to metamorphosis (Yoshizato, 1992). Most information on skein cells deals with both the genotypic (Suzuki et al., 2001, 2002; Watanabe et al., 2001; Ishida et al., 2003) and phenotypic (Nishikawa et al., 1989; Kinoshita and Sasaki, 1994a) expression of specific keratin products. Ultrastructural analysis is restricted to the apoptotic degeneration during metamorphosis (Fröhlich et al., 1977; Robinson and Heintzelman, 1987) or their responses in experiments on larval epidermis (Menon and Wahrman, 1999, 2001; Fenoglio et al., 2008). Only scanty ultrastructural information is available on SC ontogeny, including their differentiation from putative stem cells. To fill this gap, we studied with the TEM premetamorphic phases of the development in Agalychnis callidryas (the red eye tree-frog) larva. Our preliminary observations of skin in tadpoles of this hylid frog indicated that we could study the sequence of SC differentiation, because there were skein cells with a dermis-surface stratification. A further goal of this paper was to extend the current taxonomic range of SC knowledge to a species not usually available for study in the laboratory.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

We studied tadpoles of Agalychnis callidryas (Cope, 1862) staged according to Gosner (1960) in the larval phases 28 (3 specimens), 40 (3 specimens), and 42 (2 specimens). This ontogenetic span is characterized by sequential hind- and forelimb development. Larval specimens were reared near Gamboa (Panama) from fertilized eggs presumably destined to degenerate under natural conditions. Usually females release fertilized eggs covered by a jelly-like coat, and glue them to the ventral surface of leaves of swamp plants, so that freshly hatched larvae drop into water pools, where they spend their life before metamorphosis. We collected egg clusters hanging over muddy ponds and, on one occasion eight tadpoles were sacrificed for observation, we released the survived, metamorphosed specimens into moist environments.

After tadpoles were sacrificed with 0.1% chlorobutanol, 9 mm2 skin strips were pre-fixed in a glutaraldehyde-paraformaldehyde mixture, in 1 M, pH 7.0 cacodylate buffer (Karnovsky, 1965). The strips were stored in the same buffer and transported to the laboratory of Comparative Anatomy, Dipartimento di Biologia Evoluzionistica (DBE), University of Florence. Skin specimens were then reduced in size, post-fixed with 1% OsO4 in cacodylate buffer, and embedded in Epon 812 following routine procedures. Epon blocks were reduced with a Mark III LKB ultrotome into 1.5 μm thick (semithin) sections that were stained with buffered toluidine blue solution and used for LM analysis. Ultrathin sections for TEM investigation were obtained with a NOVA LKB ultrotome, they were then stained with hydroalcoholic uranyl acetate followed by alkaline lead citrate solutions, and observed (80 KV) with a Philips M300 TEM (Laboratory of Plant Biology, DBE).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Larval epidermis in both Stages 28 and 40 consisted of four layers, with basal, lower intermediate, upper intermediate and surface or apical cells, respectively (Fig. 1A,C–F), resting on a 15 μm thick primordial dense dermis or “collagenous lamella” (Fig. 1A,D). Basal cells (BCs) exhibited different features according to the development stage. In stage 28 tadpoles they were ellipsoidal in shape. The mayor axis was parallel to the dermal-epidermal junction and they adhered thickly to each other, which resulted in close basal interstices (Fig. 1A). Their cytoplasm was transparent around the nucleus, and relatively dense at the periphery, but basal cells involved in mitosis exhibited a homogeneous cytoplasm (Fig. 1A). In Stage 40 larvae, the basal layer had more variable features: cells were irregularly polyhedral in shape, with nuclei located at two levels, in a pseudo-bilayered arrangement (Fig. 1E). The cytoplasm exhibited random alternating, opaque and light areas (Fig. 1E), instead of the center-periphery density pattern seen at the previous stage. Large interstices between contiguous basal cells were crossed by thin cytoplasmic processes or “prickles” (Fig. 1E). BCs exhibited low polyedral to flat shapes and homogeneously dense cytoplasm in areas involved in the down-growth of epidermal gland Anlagen with formation of the spongy dermis primordium (Fig. 1F). The loose dermis assumed features of a stromal microenvironment around glands at Stage 42, when BCs formed the germinative layer in a mature epidermis (Fig. 1G).

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Figure 1. LM features of larval skin. A–D: Stage 28; E–F: Stage 40; G: Stage 42. A: Notice apical cells, as well as intermediate and basal cells, the latter involved in mitosis (arrow). Compare with E. Arrowhead points to an upper transparent intermediate cell, separated from the external environment by the cytoplasm flap of an apical cell. B: Apical cells contain secretory vesicles (arrows) and large secondary lysosomes (autolysosomes, arrowheads). C: Mitotic pattern in apical cell (arrow). D: Intermediate cell at the body-external environment border, with degenerating nucleus (arrowhead). E: At this stage, basal cells exhibit high polyhedral shapes. Compare with A, including the light, upper intermediate cell (black arrowhead). Small arrows point to thin cytoplasmic processes (prickles); large arrows to cylindrical cytoplasm processes in transverse section; white arrowheads to dense cytoplasm areas in basal cells. F: Flat prismatic basal cells, with dense cytoplasm, are continuous with the intercalated tract (arrowhead) of a presumptive serous gland bud. G: Tadpole epidermis with advanced, adult-like features, undergoing shedding processes (arrows); two serous glands occupy the spongy dermis. c, capillary; dd, dense dermis; m, melanophore; mec, myoepithelial cell; sd, spongy dermis.

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Basal cells at both premetamorphic stages held massive bundles of tonofilaments (Eberth's figures) in their cytoplasm (Fig. 2A–C), which contributed to the dense areas detected under the LM. On the dermal side, these tonofilaments converged toward the dermo-epidermal junction, contributing to close sequences of hemidesmosomes (Figs. 2A,B, 3A). In Stage 28 tadpoles, Eberth's figures were peripheral and consisted of large, single bundles of tonofilaments with homogeneous orientation (Fig. 2A). The light cytoplasm encircling the ellipsoidal nucleus was free of tonofilaments and contained rough endoplasmic reticulum (rer) and mitochondria. Complements of the rer were either short, slender tubules or somewhat dilated cisterns, which were continuous with each other. They contained a product with high (in tubules) to low density (in dilated cisterns) (Fig. 2A). Mitochondria underwent progressive degeneration and ranged from rod-shaped to roundish, with dense and transparent matrix, respectively (Fig. 2A). In a terminal, degeneration step, the inner compartment of mitochondria with light matrix contained irregular densities along with debris from the cristae (Fig. 2A). In Stage 40 basal cells, the Eberth's figures were still conspicuous (Fig. 2B,C), although they tended to be divided into smaller bundles, as delineated by sharp changes in their orientation (Fig. 2B). The BC inner cytoplasm was consistently denser than in the previous stage, and contained tubular rer complements with an electron transparent compartment (Fig. 2B,C), along with slender, rod-shaped mitochondria with a dense matrix (Fig. 2B). Short and thin prickles emanating from the polyhedral surfaces of contiguous cells, converged reciprocally with tips provided with minute maculae adhaerentes (Fig. 2C).

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Figure 2. TEM features of basal cells. A: Stage 28; B, C: Stage 40. A: Basal cell foreshadowing features of intermediate cells: peripheral tonofilaments of Eberth's figures and inner light cytoplasm. Notice various degeneration patterns in mitochondrion (asterisk), and continuous tubular and dilated rer cisterns (facing arrowheads). B, C: Advanced basal cells exhibiting peripheral Eberth's figures with splitting tonofilament bundles (white arrowhead in B), and tubular rer in the central cytoplasm. Notice minute maculae adhaerentes at the tips of the prickles crossing the large interstice (arrows in C) and a mitochondrion with dense matrix (black arrowhead in B). hd, hemidesmosomes; rer, rough endoplasmic reticulum; tf, tonofilaments.

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Figure 3. TEM features of intermediate cells. A–D: Stage 28; E: Stage 40. A: Typical stratification of larval epidermis, including basal, low intermediate and upper intermediate cells, along with secretory apical cells. B: Adjacent upper and lower internediate cells with cross oriented tonofilaments. Notice the peripheral, labyrinthine tubule invaginating into the lower cells (arrow). C: The labyrinthine tubule openings on cell surface (arrow) alternate to desmosomes. D, E: Normal and, respectively, degenerating mitochondria. gc, glycocalix; hd, hemidesmosomes; ly, primary lysosomes; m, mucous product; tf, tonofilaments.

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In both Stages 28 and 40, intermediate cells (ICs) were flat polyhedral or ellipsoidal in shape, with the larger axis parallel to the body surface. Their main arrangement resembled the random rubble pattern of a stone groundwork (Fig. 1A,C–E). Interstices between intermediate cells were relatively wider in Stage 40 tadpoles. This situation facilitated the detection of slender (1.5 μm thick) cytoplasm processes with a cylindrical shape, as suggested by their consistent circular profiles in transverse sections (Fig. 1E). As a common trait, ICs possessed two distinctive cytoplasmic regions: a larger and transparent inner zone, and a thinner, opaque outer region (cortex) that marked their section profiles. The inner cytoplasm was remarkably lighter in upper compared with the lower intermediate cells, which were less opaque than basal cells. This situation resulted in a dermis-surface density gradient, noticeable in stage 28 tadpoles (Fig. 1A). Nuclei of ICs were central, with oval to meniscus-like shapes, and their major axis followed the orientation of the cells. In some instance, hyaline karyolisis was detected in scattered intermediate cells that reached the external environment through gaps between apical cells (Fig. 1D).

Ultrastructural traits of Intermediate cells were closely derived from the TEM patterns of basal cells in Stage 28: an electron-transparent, inner cytoplasm and a continuous, dense cortex. On the other hand, scanty or no similarities were found with basal cell features of Stage 40: a relatively dense inner cytoplasm and a discontinuous rearrangement of tonofilament. The electron-translucent, perinuclear cytoplasm became progressively prevalent from the lower to the upper IC layers, increasing in both relative extension and lightness (Fig. 3A). This prevalence was accompanied by a flattening of the nuclei and a reduction of peripheral cytoplasm thickness. However, permanence of peripheral tonofilament bundles (residual Eberth's figures) masked the cytoplasm cortex thinning (Figs. 3B,E and 4C,D). In the relatively thick peripheral cytoplasm of lower intermediate cells, a labyrinthine net of tubules was observed. These tubules invaginated from the plasma membrane (Fig. 3B,C) as suggested by their fuzzy-like lining (Fig. 3B), a continuation of the glycocalix. The labyrinthine compartments communicated with the intercellular spaces through small pores alternating with desmosomes (Fig. 3C), and amplified the cell/interstice exchange surface. Mitochondria were still characterized by the degenerative features detected at Stage 28. These features ranged from well-preserved matrix and cristae (Fig. 3D), to transparent inner chambers with discrete densities and debris from the cristae (Fig. 3E).

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Figure 4. Ultrastructure of the cylindrical processes from intermediate cells. A–F: Stage 40. Tonofilament orientation is indicated by arrows, microtubules are encircled. A, B: These processes arise as conical outgrowths. C: Notice the tortuous path of a process between intermediate cells (arrowheads); square fields are enlarged in D and E, in order to show straight and, respectively, bowed tracts of the cytoplasm process in C. F: Cytoplasmic process in transverse section. tf, tonofilaments.

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TEM observation revealed that the thin cylindrical processes in the intraepidermal interstices were relatively long projections of intermediate cell cytoplasm. These processes arose as conical outgrowths from the cytoplasm cortex, and were almost completely occupied by a thick bundle of tonofilaments (Fig. 4A), continuous with the Eberth's figures. Just after their origin, the cytoplasm outgrowths exhibited an irregular profile with slender branches (Fig. 4B). More distally from their origin, the processes assumed a cylindrical, branchless shape, penetrating interstices between intermediate cells (Fig. 4C). Their cytoplasm retained thick bundles of tonofilaments in the fashion of an axial cytoskeleton. The bundles consisted of straight, parallel filaments in longitudinal section (Fig. 4D), but short, bowed strands were seen in elbow-like tracts of the cytoplasm processes (Fig. 4E). In transverse sections, peripheral microtubules were also detected and their orientation followed the cytoplasm process axis (Fig. 4F).

Apical cells (ACs) were also similar in both 28 and 40 stages, ranging from low polyhedral to dome-shaped. Their nuclei were almost central, with lobated, elongated/meniscoid shapes, and the longer axis was aligned to the body surface (Fig. 1A–F). Usually ACs exhibited a high cytoplasm density, resulting in intensive staining with toluidine blue and metachromatic responses in vesicles at the interface with the external environment (Fig. 1B). The supranuclear cytoplasm contained large secondary lysosomes with irregular shape and discrete densities (Fig. 1B). Scattered ACs, with a less opaque cytoplasm, were seen in mitosis (Fig. 1C), and were easily distinguished from upper intermediate cells wedged into the surface epidermal layer (Fig. 1A,E), or facing the external environment (Fig. 1D).

ACs were separated from each other by large interstices, but TEM analysis disclosed that they were sharply closed at the interface with the external environment by apical junctional complexes (Fig. 5A). ACs possessed a set of active organelles: mitochondria, rer and the Golgi apparatus. Mitochondria ranged from roundish (Fig. 5B) to short rod-like in shape (Fig. 5C), with parallel cristae and electron-dense matrix. Rough endoplasmic reticulum consisted of scattered and slender complements with an irregular orientation (Fig. 5A) and held a relatively electron-opaque material (Fig. 5B). The Golgi apparatus included stacks of flat sacculi (dictyosomes), either with a parallel (Fig. 5B) or irregular orientation (Fig. 5C). Dictyosomes produced vesicles storing a moderately electron-opaque product, arranged in a mesh-like pattern with discrete densities (Fig. 5B,C). These vesicles migrated toward the skin surface, where their content was released through exocytosis (Figs. 3A and 5A).

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Figure 5. TEM features of apical cells. A: Stage 40; B,C: Stage 28. A: General view of this surface cell layer, showing low polyhedral cells, separated by large interstices. Notice secretory vesicles and a sealing junctional complex. B, C: Notice flat and relatively dilated sacculi of Golgian stacks. In C mucous maturation is also shown. ajc, apical (sealing) junctional complex; G, Golgian stack; ly, primary lysosomes; m, mI and mII, mucous product undergoing maturation; rer, rough endoplasmic reticulum.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

As summarized in Fig. 6 (A, B, and C), this investigation reduced the cytological diversity in larval epidermis of Agalychnis callidryas (Stages 28–40) into two main cell populations: surface or apical cells that secrete mucus, and basal-intermediate cells, with a remarkable cytoskeleton of tonofilaments. These cell lineages, possibly derived from the bilayered epidermis of limbless tadpoles (Delfino, 1977; Izutsu et al., 1993), were no longer present in premetamorphic (Stage 42) tadpoles, and therefore they should be regarded as larva-specific in nature (Yoshizato, 1992; Tamakoshi et al., 1998, Watanabe et al., 2001). Previous experiments with [H3] thymidine labeling (Robinson and Heintzelman, 1987), were confirmed by evidence of mitotic patterns in the surface layer at Stage 28. In situ proliferation may balance the degeneration of apical cells exposed to the external environment, as stressed by the remarkable content of secondary lysosomes (autolysosomes) in their cytoplasm. The evolution of intermediate cells also involved degeneration, following a gradient that originated from the basal layer and finished at the epidermis surface. IC population was kept in a steady state by BC mitosis that produced almost continuous layers of intermediate cells with synchronous differentiation features. At Stage 28, BCs and ICs were linked by a close sequence of steps of cytoplasm fluidation and peripheral rearrangement of the Eberth's bundles that assigned them to the constitutive skein cell line, including basal skein cells (BSCs) and intermediate skein cells (ISCs).

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Figure 6. Semi-schematic reconstruction of the larval epidermis in Agalychnis callidryas (A), with intermediate cell (B) and apical cell (C) in larger magnification. Details in the text, not at scale. ajc, apical (sealing) junctional complex; dej, dermal-epidermal junction; i, interstice; mt, microtubule; tf, tonofilaments; ti, tubular invagination.

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Applied to the skein cells, the concept of larva-specificity is not merely based on their programmed death fate in preparation for metamorphosis, but it also involves the production of specific larval keratins (Watanabe et al., 2001). Subsequently, adult keratin substituted them during ontogenesis (Kinoshita and Sasaki, 1994a; Ishida et al., 2003), which was also suggested by immuno-histochemical evidence (Suzuki et al., 2001). As observed at Stage 40, SCs were replaced by adult epidermal cells (keratinocytes) starting from the basal layer. BSCs assumed early features of adult basal cells or epidermal germinative cells (Robinson and Heintzelman, 1987) during the development of the spongy dermis (Yoshizato, 1992; Izutsu et al., 1993; Tamakoshi et al., 1998; Suzuki et al., 2001, 2002; Ishida et al., 2003), as well as cutaneous glands (Kawai et al., 1994; Menon and Wahrman, 2001; Watanabe et al., 2001). T3 hormone coordinates the processes relevant to skin ontogenesis: selective expression of keratins, both temporal (Nishikawa et al., 1992; Kinoshita and Sasaki, 1994a, b; Suzuki et al., 2001, 2002; Watanabe Y. et al., 2001; Ishida et al., 2003) and regional (Kobayashi et al., 1996); SC replacement by adult type cells (Nishikawa et al., 1989; Kinoshita and Sasaki, 1994a, b); and gland differentiation (Hayes and Gill, 1995).

Although ultrastructural data were collected in premetamorphic stages, intermediate skein cells did not display any typical trait of apoptosis, but closely resembled Kugelzellen, namely ISCs involved in an advanced involutive specialization and displacement toward the external environ (Fröhlich et al., 1977). Switching of BSCs towards the epidermal germinative line involves unbalancing their turnover, and lost Kugelzellen are replaced by keratinocytes. On account of their ultrastructural features, KZn have been considered as stiffness modules, comparable to notochordal cells (Fröhlich et al., 1977), since their tonofilament cortex may contrast the turgor pressure exerted by the hydrated, central cytoplasm. Strikingly, turgor is apparently achieved through opposite processes in the two mechanically resistive cell systems. Notochordal cells are osmotically active, and drive water uptake from their isotonic tissue environment by production and intracytoplasmic accumulation of hydrophilic glycosoaminoglycans (Adams et al., 1990). Epidermal Kugelzellen are exposed to a hypotonic environment (fresh water) and their hydration load is passive, due to impairment of osmotic regulation, as suggested by mitochondrial noxia and histochemical findings in mature KZn (Fröhlich et al., 1978). However, KZ evolution proceeds along a dermis-surface gradient and does not affect the osmoregulative role of the deeper SCs, which are capable of removing excess water.

Starting from the possible function of the specialized ISCs (or KZn) as stiffness modules, their epithelial arrangement in A. callidryas larval epidermis can be compared to a random rubble stone multi-layer (as illustrated in Fig. 6A). The foundation of a roman road (statumen, Forbes, 1993) represents a proper analogy. It is well-known that this stone assembly has adequate compressive strength but low tensile strength, because sliding shear resistance between overlaying and underlying stones is only due to frictional braking. The thin cytoplasmic processes that grow from KZn, with an orientation determined by microtubules, hold together contiguous cells both in the same and superimposed layers. Because of their thick axial rods of tonofilaments, these processes may act as links between contiguous cells, and contribute with the maculae adhaerentes to fasten epidermis against tangential forces.

Although transient in their role, KZn represent a distinctive cell lineage in the epidermis of several anuran species. Their peculiar subcellular features suggest mechanical properties related to a hypo-osmotic environment. Furthermore, Kugelzellen in A. callidryas are arranged into a peculiar multilayer architecture that may enhance single-cell performance and result in compressive as well as tensile strengths.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
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