figures of eberth in the amphibian larval epidermis

JOURNAL OF MORPHOLOGY 212337-97 (1992)

Figures of Eberth in the Amphibian Larval Epidermis HAROLD FOX Department of Biology, Medawar Building, University College, London WClE 6BT, England

ABSTRACT Figures of Eberth are prominent extensive filamentous structures in the basal epidermal cells of larval amphibians. They are compared and contrasted qualitatively and quantitatively in a number of species of the three groups of living amphibians.

Fully developed Figures consist of massive skeins of tonofilaments oriented in three dimensions and hinged on hemidesmosomes within the cell. The overall appearance of the Figures is similar in anurans, urodeles and Ichthyo- phis among the apodans. However, in terms of size and number per unit length of the proximal cell margin, the hemidesmosomes and the thickness or their emergent skeins in anurans and Ichthyophis differ significantly from those parameters in urodeles, a feature that is presumably independent of cell size. Figures are poorly developed or missing in embryos of Typhlonectes, which has no larval stage in its life history.

These ubiquitous skeletogenous structures in the aquatic larval amphibians, among other things, could be protective of underlying delicate tissues and act as a stabilizer in bodily movement during swimming. They could also serve as a reserve of cytokeratin for use during later cellular division and sloughing.

The Figures of Eberth are named after C.J. Eberth (1866), who described them in frog larvae, although Rudneff independently dis- covered them a year earlier (Chapman and Dawson, '61). Since then, the Figures, or similar structures in the epidermal cells of amphibian larvae, have been widely investi- gated. Reviews of classical work by light mi- croscopy have been published by Weed ('34) and Cameron ('36) and on the fine structure by Chapman and Dawson ('611, who described the Figures in the tail of Rana clami- tuns, as well as by Fox and Whitear ('86).

Figures of Eberth are prominent filamentous structures in the basal epidermal cells of the head, body, tail, and limbs of larval amphibians. They also occur in fish, in the same cells, of embryonic salmonids, leptoceph- alous larvae, juveniles such as elvers of An- guilla, and in lampreys-the skein cells, where they persist in adults (Whitear, '86).

Figures are formed from skeins (or bundles) of tonofilaments, which extend in three dimensions in the cell. They join hemidesmosomes, the so-called bobbins of Weiss and Ferris ('54), structures composed of densities of the membrane, or attachment plaques, on the proximal cell margin, fused with an over-

lying layer of looped tonofilaments (Kelly, '66).

Tonofilaments are synthesized by ribo- somes within the cell. They appear early in larval life, reach peak size during late prometamorphosis, and are absent after metamorphosis in anurans (Weed, '34; Fox, '85b; Fox and Whitear, '861, in urodeles (Kelly, '66; Lavker, '72; Warburg and Lewin- son, '77), and in Ichthyophis among the apodans (Welsch and Storch, '73: Fox, '83, '86a,b, '87; Breckenridge et al., '87). By contrast, Figures are retained throughout life in neotenic urodeles such as Ambystoma mexicanum (Fahrmann, '71a) and Proteus anguinus (Fox and Durand, '90).

The following account deals with the overall structure of the Figures of Eberth and provides measurements of some of their specific components in a number of larvae of the three groups of living amphibians, including the neotenic Proteus anguinus and among the Apoda, Ichthyophis kohtuoensis and embryos of the viviparous Typhlonectes compressicaudus.

One may pose the following questions. Are the Figures present in larvae of the three groups of amphibians similar in structure and, if not, how do they differ? Are Figures

D 1992 WILEY-LISS, INC.

88 H. FOX

present in basal epidermal cells of the entire animal, or is there regional variability in their distribution? Are Figures present in embryonic stages? Can any indication of Fig- ure function be inferred from their morphology?

The results show that the Figures are similar in structure and appearance in specimens typical of the three groups examined. However, in terms of size and number of hemidesmosomes per cell margin, and their emergent skein thickness, the Figures of Ich- thyophis conform with those of anurans. Such parameters of urodelan Figures differ significantly from those of the other two groups. There is, indeed, some modest variability in the distribution of the Figures, and embryos of Typhlonectes, a viviparous apodan, have poorly developed Figures in their skin during embryogenesis.

The presence of the Figures, and the pat- tern and arrangement of their constituent skeins in aquatic larvae, suggests that they play some role (among others) in swimming movements and body elasticity.

MATERIALS AND METHODS

Figures of Eberth were examined in larvae of the following specimens: Anura-Xenopus laeuis stages 55-57 and 57-59 (Nieuwkoop and Faber, '56) tentacle skin, and stage 59 back skin; Bufo bufo stages lV,, and lV,, (Cambar and Gipouloux, '56) body skin; Rana temporaria (after Rana dalmatina) (Cambar and Marrot, '54) body skin; Urodela-Sala- mandra salamandra well-developed larva, stage unknown, flank skin and adult tail skin; Pleurodeles waltl stage 49 (Gallien and Durocher, '57) hindlimb skin; Proteus anguinus (7% mo-5 yr old (Fox and Durand, '90) cephalic skin and a described adult body skin; Apoda-Ichthyophis kohtaoensis larva A (Fox, '87) body skin; embryonic Typhlonectes compressicaudus stages 1113, 17-27 mm long, l l l , , 32-53 mm long, and lV,, 43-65 mm long, of appearance like a small adult (Delsol et al., '81, '83) body skin.

Skin of the anurans and of Salamandra and Pleurodeles was fixed ice-cold in a mix- tu re of glutaraldehyde and osmic acid (Hirsch and Fedorko, '68). Proteus skin was fixed in cold 2.5% glutaraldehyde for 2-5 hr and then 4% osmic acid. The skin of Zchthyo- phis was fixed in 3.6% glutaraldehyde for 2 hr, followed by 4% osmic acid. Embryonic skin of Typhlonectes was fixed in Karnovsky fixative (Karnovsky, '65), a mixture of glutaraldehyde and formaldehyde, and postfixed

in 1% osmic acid in Sorenson buffer. Fixed skin was dehydrated in graded alcohols (and, in most cases, propylene oxide) and embed- ded in Epon. Blocks were sectioned ran- domly, usually at right angles to the skin surface, with a diamond knife, and suitable silver-gray sections were double-stained in uranyl acetate and lead citrate. Viewing was under a JEOL JEM CX 100 electron micro- scope.

Though various species were fixed differ- ently (vide supra), the resulting profiles of organelles in the epidermis (and dermis also, see Fox and Whitear, ,901, appeared normal in all cases, without any apparent signs of shrinkage or necrosis. Compared with illus- trations of comparable Figure components and with limited data on their measurements (vide infra), Figures of the present work are likewise similar. Random measurements of the tonofilaments (intermediate filaments 7-12 nm thick), in different species of the present work, conformed to their normal di- ameter. Thus the measurements should at least provide some fair indication of the sizes of the components in the described species of the three groups of amphibians.

Standard Student's t-tests of significance, P < 0.01, were used to compare the measurements obtained.

RESULTS Anura

Among the anurans, the Figures of Eberth in the basal cells of Rana and Bufo older larval stages are particularly well developed (Fig. la-d). They may show lesser development in Xenopus (Fig. le), where they are known to be variable in different regions of the body (Fox and Whitear, '86). Random individual skeins of tonofilaments, when distinguishable within the Figure mass, show a range of thickness of 0.5-2.5 km; they are of comparable &mensions in Figures of other groups (Table 1). Frequently, however, constituent skeins are not demarcated amid the tonofilamentous mass because of merging, as shown in Figure l a , although individual skeins join the hemidesmosomal plaques (Fig. le). Joining of individual skeins to hemidesmosomes is seen particularly well in urodeles (Figs. lg,h, 2c).

Hemidesmosomes are elongate structures in anuran larvae, especially in the basal cells of Rana and Bufo (Fig. la,c). Skeins may arch to join adjacent hemidesmosomes or those farther away (Chapman and Dawson, '61) (Fig. Id). Arching can occur on the same

FIGURES OF EBERTH IN AMPHIBIAN SKIN 89

elongate hemidesmosome, a feature recog- nized especially in Rana (Fig. lb) notwithstanding the fact that the individual hemidesmosomes are often closely apposed on the proximal cell margin to form a multistruc- ture.

In larval tentacles of Xenopus, at stages 55-57, hemidesmosomes are occasionally seen associated with small concentrations of tonofilaments (Fig. 10, an arrangement comparable to that seen in very early stages of development of the Figures, at steps C-D of Fox and Whitear ('86). These structures do not develop any further, and there are no Figures of Eberth in the epidermis of tentacles of later larval stages 57-59.

Urodela Basal cells of urodeles (and Ichthyophis)

are larger than those of anurans (Fox, '83); thus the Figures appear to be more extensive in specimens of Salamandra, Proteus, and Ichthyophis than in anurans, though generally they are similar in appearance.

Among urodeles, Pleurodeles, at stage 49, is 21 mm long and has only developed two digits on the hindlimbs. In this young larva, presumably the Figures in the flattened basal cells of the hindlimb are not at peak development, for the filament content is loose, and the skeins were generally thinner than those of the other species (Fig. lg).

Figures are well developed in newly hatched larvae of Salamandra (Warburg and Lewin- son, '77). In Salamandra of the present work, separate skeins of the Figures are often not distinct. However, when demarcated, they are of comparable thickness to those in other groups (see Table 1). Figures are extensive and well developed in the larva (Fig. lh). Typically in these non-neotenous urodeles, Figures disappear during metamorphosis and hence are absent in adults (Fig. 2a).

Well-developed Figures are present in the cephalic and body skin of all Proteus specimens examined, as shown in Figure 2b. As in the other amphibians, the basal cells include polysomes, mitochondria, and often pinocytotic vesicles at the proximal cell margin. Usually the rough endoplasmic reticulum (rER) is not well developed in the cells. How- ever, younger Proteus specimens occasionally display an extensive rER, oriented at right angles to the proximal cell margin (Fig. 2d).

Individual skeins, when recognizable, are variable in thickness (see Table 1); those joining the hemidesmosomes frequently lead-

ing almost perpendicularly into the body of the Figures (Fig. 2c). This alignment could well be common in other Figures among different species examined, but presumably only obvious in specifically oriented sections. There is arching of skeins between hemidesmosomes.

Apoda The Figures of Ichthyophis are already

present in skin of the 3-cm-long larva (Welsch and Storch, '73). Likewise, the Fig- ures are well developed in the 4.5-cm larva (Fox, '87) and also in the older stage A larva of the present work (Fig. 2e). Apart from numerical differences between the various components of the Figures of Ichthyophis and the urodeles (vide infra and Table 11, their general appearance is similar to that of Proteus Figures. Pinocytotic vesicles are prominent at the cell margin, and individual skeins are frequently hard to distinguish amid the body of the merged filaments. Likewise, arching of skeins between the hemidesmosomes occurs (Fig. 20.

In all stages of Typhlonectes examined, the epidermis is composed of two to three layers of flattened cells displaying a microvillous surface, similar to that of the uterine Chtho- nerpeton embryo (Fox, '86b). Basal cells of stages 11 1, and 11 1, embryos have no obvious Figures of Eberth, although frequently there are incipient membrane densities on the cell margin (Fig. 2g,h). Membrane densities in cells of stages 111, and 1V are about 30 nm thick, as in other groups, and there are about 20 of them per 10-p. cell length, as in those of anurans and Ichthyophis.

In stage l l l , , skeins, if recognizable, are hardly developed and only about 0.08-0.09 km thick (see Table 1). A small filament content may be associated with adjacent membrane densities, but the Figures are poorly developed, in contrast to those in comparable larval stages of the other amphibi-

In general, therefore, the Figures of Eberth in the different amphibians examined are substantial, except for those in the young Pleurodeles and the embryonic Typhlonectes. Indeed, apart from the nucleus, they are the most prominent structures in the basal cells. In most cases, demarcated skeins, within the body of the Figures, whatever their orienta- tion, are usually thicker than those that di- rectly join the hemidesmosomes.

ans.

90 H. FOX

Figure 1


Statistical comparisons of Figure components in the three groups

As shown in Table 1, the mean length of the hemidesmosomes and the mean thickness of their emergent skeins of anurans, as a group, are significantly greater than those of urodeles, as a group. Although there is some evidence of fusion of adjacent hemidesmosomes, particularly in the anurans (Fig. lb), most of the hemidesmosomes are clearly longer than those of urodeles, as shown in comparable electron micrographs. The length in anurans is about twice as long. The greater thickness of their emergent skeins, again about twice that of the urodeles, corresponds to the elongation of the hemidesmosomes.

There are significantly fewer hemidesmosomes per 10-km length of the basal cell margin in anurans than in urodeles. Anurans display about one-half as many as urodeles. The membrane (plaque) density in urodeles, however, is significantly thicker than that of anurans, although the overall thickness of

Fig. 1. a: Rana temporaria stage 46, body skin. Area of extensive Figures of Eberth (FE) in a basal cell (BC), with elongate hemidesmosomes (HD). There is arching of skeins of tonofilaments on the same hemidesmosome (arrow). Scale bar = 1 pm. b: Rana temporaria stage 46, body skin. Elongate hemidesmosome that could well rep- resent a fusion of several hemidesmosomes-see the fine gaps (arrowed) at the junctions. In this case, arching of skeins (A) is between adjacent hemidesmosomal plaques. Scale bar = 0.5 pm. c: Bufo. bufo stage lV,,, body skin. Proximal region of a basal cell with Figures and elongate hemidesmosomes (HD). Individual skeins are not seen amid the Figures, but emergent skeins (ES) are recognizable leaving the hemidesmosomes. There are only few polysomes and a sparse rER (rough endoplasmic reticulum). Scale bar = 1 pm. d Bufo bufo stage lV,,, body skin. Arching of skeins (A) between the adjacent hemidesmosomes. Scale bar, 1 pm. e: Xenopus laeuis stage 59, back skin. Area of Figures (FE). Note the arching (A) of the emergent skeins (ES) between adjacent hemidesmosomes (HD). CO, collagen of the basement lamella. Scale bar = 0.5 fim. f: Xenopus laeuis stage 55-57, tentacle skin. Proximal region of a basal epidermal cell with membrane densities (plaques) (HP) and a slight concen- tration of tonofilaments (T) nearby, the only sign of Figures in the tentacles. Scale bar = 0.5 pm. g: P. waltl stage 49, hind-limb skin. Young Figures showing emergent skeins (ES) joining the hemidesmosomes at the cell margin. Note that the membrane plaques (HP) of the anuran Xenopus tentacle are longer than those of the urodelan Pleurodeles. Compare Figs. f and g. Scale bar = 0.5 pm. h: Salamandra salamandra larva; body skin. Proximal region of a basal cell. The individual skeins of tonofilaments of the Figures are not distinctly demarcated, except where they (ES) are associated with the hemidesmosomes. The membrane plaques are distinct from the region of the contiguous overlying tonofilaments, which adhere and loop against the plaque. Scale bar = 0.5 pm.

the hemidesmosomes is similar in both groups. Indeed, this parameter did not differ significantly among all three groups of amphibians examined.

Comparison between anurans and Zchthyo- phis revealed no significant differences between the means of the various parameters tested. Apart from hemidesmosomal length, measurements of all other components closely agreed in dimension size.

Comparison between urodeles and Zchthyo- phis showed that there are significant differences in the measurements of all components of the Figures tested, except for the overall hemidesmosome thickness, which is about the same (vide supra). In Zchthyophis, the hemidesmosomal length is significantly longer, and the emergent skeins are significantly thicker than those in urodeles. As in anurans, there are significantly fewer hemidesmosomes per 10-km cell length in Zchthyophis but the mean membrane density of the plaque is significantly thicker in urodeles (Table 1).

DISCUSSION

Previously, Fox and Whitear ('86) showed that, in anuran larvae, the development of the Figures of Eberth is preceded by an in- crease in the content of polyribosomes and rER in the basal cells. A filamentous zone at the proximal margin of the basal cell joins the newly formed membrane densities, the first definitive form of the Figures. Thereaf- ter in older larvae, the rER is reduced, and the Figures are composed of large skeins of tonofilaments that extend in three dimensions around the nucleus filling much of the cell.

Where skeins join the membrane densities, or attachment plaques, constituent tonofilaments loop either outside or within the plaque, or perhaps terminate there. Plaques of hemidesmosomes in larval Triturus toro- sus are ellipsoidal in shape, in contrast to the round form of plaques of desmosomes (Kelly, '66). At late metamorphosis of anurans (step G of Fox and Whitear ('86), cytokeratin aggregates appear during regression of the Fig- ures. These aggregates were not found among the Figures of the urodeles and Ichthyophis in the present work; possibly they occur in their Figures during metamorphosis.

In Rana catesbeiana, whose larval stage may be prolonged, Robinson and Heintzel- man ('87) reported that the ventral epidermis of larvae, albeit lacking surface keratin, resembles that of adult skin in thickness.

92 H. FOX

TABLE 1. Means and standard errors (SE) of uariousparameters of the Figures ofEberth in different amphibian species’

HD length HD Th. Mb. Th No. HDilO Fm Sk. Th. at Sk. Th. Species (pm) (d (nm) cell length HD (wm) in Fig. (km)

Anura Xenopus (St. 59) Bufo (St. lV,J Bufo (St. lV,J Rana (St. 46) Rana (St. 49)

Urodela Salamandra (Larva) Pleurodeles (St. 49) Proteus (7% mo) Proteus (1 yr-1 yr 3 mo) Proteus (1 yr-7 mo) Proteus (4 yr old) Proteus (5 yr old) Proteus (Adult)

Ichthyophis (Larva) Typhlonectes (St. 111,) Typhlonectes (St. 111,) Typhlonectes (St. lV,)

Apoda

Group measurements All Anura

All Urodela

0.25 f 0.023 (6)

0.42 f 0.066 (8)

0.36 2 0.064 (6)

0.60 f 0.086 (9)

0.32 f 0.190 (8)

0.18 f 0.021 (7)

0.21 f 0.018 (9)

0.17 f 0.022 (4)

0.15 f 0.007 (12)

0.18 2 0.025 (13)

0.20 ‘f 0.019 (11)

0.18 f 0.021 (9)

0.22 f 0.015 (11)

0.29 f 0.029 (7)

0.16

0.14

-

0.41 c 0.036 (37)

0.19 f 0.006 (76)

121 f 5.3 (5)

120 f 4.2 (5)

137 f 4.5 (6)

129 f 2.6 (7)

122 t 4.6 (6)

124 f 5.5 (5)

138 2 6.2 (5)

127 f 4.0 (2)

118 f 4.7 (9)

126 f 1.2 (7)

117 f 2.4 (8)

121 f 2.3 (6)

118 f 5.0 (7)

121 f 4.8 (5) -

-

-

127 f 2.1 (29)

122 f 1.7 (49)

28 2 0.8 (5)

26 2 1.0 (5)

26 2 1.6 (6)

26 2 1.4 (7)

28 2 0.7 (6)

29 -c- 2.0 (5)

32 % 2.0 (5)

39 ? 1.0 (2)

33 2 1.2 (9)

33 2 1.2 (7)

36 2 1.5 (8)

40 -t- 0.6 (6)

33 -c- 1.8 (7)

28 2 2.1 (5)

30

30

-

27 f 0.44 (29)

34 ? 0.70

26 f 3.1 (5)

20 f 1.1 (6)

20 f 1.1 (6)

13 f 1.8 (5)

30 ? 1.3 (7)

37 f 1.5 (10)

27 f 2.7 (5)

33 ? 4.1 (3)

37 f 2.7 (7)

39 f 0.8 (6)

45 ? 1.4 (5)

42 2 3.1 (5)

33 2 2.2 (5)

24 2 2.4 (6) -

20

20

20 f 1.0 (29)

37 f 1.1

0.19 f 0.013 (5)

0.27 f 0.025 (6)

0.26 2 0.029 (6)

0.25 k 0.047 (6)

0.33 f 0.067 (6)

0.15 f 0.010 (6)

0.14 f 0.010 (5)

0.15 f 0.020 (3)

0.14 f 0.010 (9)

0.14 f 0.010 (6)

0.13 f 0.010 (6)

0.13 -c 0.010 (7)

0.13 2 0.010 (6)

0.23 k 0.020 (6) -

0.08-0.09

0.26 f 0.018 (29)

0.14 f 0.004

0.7-0.9

1.0-1.5

0.5-2.0

0.8-1.6

1.2-2.5

0.6-0.9

0.6-0.7

0.7-1.4

0.63.0

0.7-3.0

0.5-0.8

0.8-2.0

1.0-2.0

0.7-0.9

-

-

-

0.5-2.5

0.4-3.0

‘Measurements of the individual species of anurans, urodeles and apodans, and of grouped anurans and urodeles are listed. The number of components measured is given in brackets; the skein thicknesses within the Figures (last column) are of random distinguishable examples. Abbreviations: HD, hemidesmosome; Mb, membrane; Sk, skein; Th, thickness.

Explanation ofresults. The results demonstrate a fairly clear uniformity of the measurements of each parameter, within and between species of anurans and urodeles, further emphasized in many cases by the small SE of their means. The elongate hemidesmosomes and their reduced numbericell length in anurans, compared with those in urodeles, are apparent: the differences are significant (P < 0.01). So are the differences in thickness of the emergent skeins of tonofilaments from the hemidesmosomes, which are superior in anurans.

Skein thickness, when clearly demarcated within the main body of the Figures, is variable, though of comparable dimensions in the different groups. Range: 0.5-2.5 pm (Anura); 0.4-3.0 pm (Urodela); and 0.7-0.9 pm (Ichthyophis).

Means of the parameters of Ichthyophis generally are intermediate between those of the other two Orders, though in most cases they differ significantly from those in urodeles (see details in the text).

Indeed, there are well developed poison glands with ducts, structures that usually originate and differentiate during climax (Fox, ’86a). The larval skin also possesses three to four layers of “skein cells,” which have Eberth- like bundles of tonofilaments, and the basal cells have true Figures of Eberth, with their

associated hemidesmosomes, that disappear during metamorphosis. The skein cells further develop to form the apical border of the skin; the Figures in the basal cells have disappeared by the end of metamorphosis.

Figures of Eberth are present in most basal epidermal cells of anuran larvae, including


those of the operculum, where in Rana pipiens and Rana japonica (Reichel, '76; Sasaki et al., '83) the hemidesmosomes appear from illustration to be well developed and elongate, as in the R. temporaria and Bufo of the present work. However, there are some regional omissions. Among anurans, Figures are not present in the tentacles of Xenopus (Ovalle, '79; Tachibana et al., '80). In the present work, Figures of the tentacles show an early degree of development by stages 55-57, but this development does not pro- ceed any further, and in later stages, as in 57-59, there are no signs of Figures in the basal cells. Again, Figures are not developed in the external gill filaments of Rana pipiens at Shumway stage 23 (Shumway, '401, after which time the gills degenerate (Michaels et al., '71). Among the urodeles, Figures were not mentioned by Jarial ('89) in his descrip- tion of the external gills of Ambystoma mexicanum. However, Greven ('80) reported their absence, together with the associated hemidesmosomes, in the gills of Salamandra salamandra larvae. Figures are absent in the balancer of the larval Pleurodeles, although they occur in adjacent head skin (Fox, '85a). The external gills of apodan larval Ichthyo- phis kohtaoensis and I . paucisulcus do not possess Figures (Welsch and Storch, '73; Welsch, '81).

In the Amphibia, Figures of Eberth have disappeared by the time metamorphosis has been completed but they are retained in the neotenous urodeles (Dent, '68; Fahrmann, '71a; Jurd, '85), including Proteus (Fox and Durand, '90; and the present work). They probably occur in the skin of Necturus maculosus, for Lindinger ('84) described the basal epidermal cells as having cytoplasm densely packed with tonofilaments that form a cytoskeleton joining the hemidesmosomes and desmosomes.

The disappearance of the Figures appears to be induced by thyroid hormones during metamorphosis (Fahrmann, '71b; Fox, '85b), when the circulatory hormonal level peaks (Larras-Regard, '85; Rosenkilde, '85). The skin of Proteus shows only a modest response to 1:107 triiodothyronine (T,) (Fox and Du- rand, 'go), although the thyroid gland prod- ucts of Proteus can influence other species (Jurd, '85). Presumably in phylogeny, Pro- teus tissues have become (or have remained), generally resistant to metamorphic change mediated by the thyroid hormones.

Previously, in an 8-cm-long larva of Rana clamitans, Chapman and Dawson ('61) reported that the attachment points (basal plasma membrane thickenings) are 0.3-0.4 km long and 25 nm thick. These measurements agree with those found in anurans of the present work. However, the dense filamentous layer plus the plaque (overall thickness of the hemidesmosome), of 85 nm, which these investigators reported, is somewhat less than the 120 nm (approx.) in overall thickness of hemidesmosomes for the anurans, and also the urodeles and Ichthyophis, of the present work (see Table 1). Possibly, occa- sional slight obliquity in sectioning might have exaggerated the hemidesmosomal thickness in these specimens, although this parameter varied very little among the three groups of the present work.

Among the present quantitative results, the thicker skeins from the longer hemidesmosomes of anurans and Ichthyophis might well have been expected. Desmosomal and hemidesmosomal plaques are a specific focus for adhesion and binding of tonofilaments (Skerrow, '86). Therefore, the greater the size of the plaque, the greater the focal area available to which the filaments can adhere, with a resulting thicker skein.

Epithelial cells of urodeles and dipnoans are the largest among the vertebrates (Szar- ski, '68, '76). Those of the skin of urodeles and Ichthyophis are of comparable dimensions and the cells of anurans are about one- half the size (Fox, '83). Nevertheless, measurements of the various Figure components showed them to be similar in anurans and Ichthyophis despite the profound differences in their cell size. By contrast, all components measured, except for the uniform thickness of the hemidesmosomes of both groups, dif- fered significantly from those of urodeles. Such differences recorded, which appear to be independent of cell size, presumably evolved in amphibian phylogeny from a common ancestral structural arrangement.

Gestation of embryos of Typhlonectes in the mother takes about 6 months. Delsol et al. ('81, '83) described 14 stages of development, birth occurring at stage lV,, although stages lV, and lV, are similar to those of small adults. Stage lV, corresponds to the stage of beginning metamorphosis. A trans- parent ventral epidermis permits a clear view of the internal viscera. Perhaps the poorly developed Figures of Typhlonectes in part account for this transparency. I t is of interest

94 H. FOX

Figure 2


that Typhlonectes, which is the only viviparous species among the amphibians described, has no free-swimming larva in its life history; its embryos were the only forms without any well differentiated Figures in their skin. How- ever, although Figures are barely developed in the embryonic skin of Typhlonectes, they are present nevertheless in the body skin of Salamandra salamandra, an intrauterine form (Greven, 'BO), although in this case there is a free-swimming larval stage (War- burg and Lewinson, '77). Perhaps ovovivipar- ity in the Salamandra is fairly recent in its phylogenetic history; one possible function of the Figures, as an aid during swimming, is retained in the aquatic larva.

Earlier workers believed that the Figure tonofilaments were intracellular continua- tions of the epidermis that bound it to the epidermis (Saguchi, '13; Weed, '34). Cam- eron ('36) thought the Figures were a se- creted substance used for dermal connective tissue. Chapman and Dawson ('61) consid- ered that the attachment points (hemidesmosomes), on the basal cell margin, aided in giving rigidity to the cell. The filamentous cytoskeleton could provide intracellular ten- sile support (Kelly, '66). More recently, Lane and Whitear ('80) concluded that comparable basal epidermal skein cells, of the free-

Fig. 2. a: Salamandra salamandra adult, tail skin. Basal epidermal cell (BC). There are no Figures of Eberth. Scale bar = 1 pm. b Proteus anguinus 4-year-old specimen, cephalic skin. Basal cell with massive Figures (FE) of merged skeins. Note the numerous short hemidesmosomes (HD) along the proximal cell margin, typical of urodeles. There is little rER. Mitochondria (M) surround a more lucent granular area around the nucleus (N). Scalebar = 1 pm. c:Proteus anguinus 4-year-old specimen, cephalic skin. Skeins of tonofilaments (ES), from the membranous plaques (HP) of the hemidesmosomes (HD), lead almost perpendicularly into the body of the Figures. Scale bar = 0.5 pm. d Proteus anguinus 1 year-3 month- old specimen, cephalic skin. An extensive rER extends from the proximal cell margin amid the Figures of Eberth. CO, dermal collagen; M, mitochondrion. Scale bar = 1 pm. e: Zchthyophis kohtaoensis larva, body skin. Massive Figures of Eberth (FE) in a basal epidermal cell. Skeins (ES) from the hemidesmosomes (HD) merge into the mass of tonofilaments. Polysomes and a rER are scarce. Scale bar = 1 pm. f: ~ch~hyophzs kohtaoensis larva, body skin. Arching of skeins (A) between adjacent hemidesmosomes (HD). Scale bar = 0.5 pm. g: Typhlonectes compressicaudus stage 111, embryo, body skin. Basal cell proximal region with some rER. There are membrane densities (plaques) (HP) but the Figures of Eberth are absent. Scale bar = 0.5 km. h: Typhlonectes compressicaudus stage 111, embryo, body skin. Basal cell proximal region with membrane densities (HP). There are no Figures of Eberth in the basal cell. Scale bar = 0.5 pm.

swimming adult lampreys, act as a support against cellular distortion and protect the epidermal germinal cells.

Microvilli together with the ridges on the skin of embryonic and larval amphibians form an extensive area for respiratory activity. In addition, the surface mucus and tight junctions provide a semipermeable barrier, limit- ing movement of water and metabolites into the epidermis (Fox, '86a; Katz, '86). The ubiquity of the Figures in aquatic larvae of amphibians, including neotenous forms, as well as their large size and similar appearance in the different species, likewise are presumably of functional importance. One might speculate that the Figures serve for some or all of the following functions: (1) protect underlying delicate tissues such as capillaries and nerves, in a larval skin only two to four layers thick and without surface keratin, although the collagenous basement lamella could also be protective; (2) provide flexibility and elasticity to the body and/or assist recoil during movements, such as swimming, and prevent distortion (see Lane and Whitear, '80); (3) contribute to the rigidity of the base of the epidermis and the mainte- nance of a stable form (see Chapman and Dawson, '611, notwithstanding the mitotic activity of the basal germinative layer (Weed, '34); (4) form a barrier to any excessive influx of fluid and/or chemical substances into the body interior; and (5) serve as a reservoir of tonofilaments to be available for dividing cells and other extra cell layers, during metamorphosis (Weed, '34; Robinson and Heintzel- man, '87). In addition, cytokeratin aggregates, found in anurans and derived from condensed tonofilaments during Figure regression, might be recycled material to be used as a keratin reserve during sloughing of surface cells at climax (Fox and Whitear, '86).

It would seem unlikely that the Figures are of importance in the fibrogenesis of the underlying basement membrane (Singer and Sal- peter, '6 1).

However, until further investigation eluci- dates the functional significance of this im- pressive morphological structure, its purpose will remain speculative.

ACKNOWLEDGMENTS

Thanks for skin material are due to J . Durand in Paris, for Proteus; to J.M. Ex- brayat in Lyon, for Typhlonectes; to C. Her- bert in Portsmouth, for Xenopus; and to U. Welsch in Munich, for Ichthyophis. Discus-

96 H. FOX

sions in past years on the subject with Mary Whitear are acknowledged. Pat Ferguson and Roy Mahoney provided valuable technical as- sistance.

LITERATURE CITED Breckenridge, W.R., S. Nathanael, and L. Pereira (1987)

Some aspects of the biology of lchthyophis glutinosus (Amphibia: Gymnophiona). J . 2001. Lond. 211~434- 449.

Cambar, R., and J.D. Gipouloux (1956) Table chronologique du developpement embryonnaire et larvaire du crapaud commun: Bufo bufo L. Bull. Biol. 90~198- 217.

Cambar, R., and B. Marrot (1954) Table chronologique du developpement de la grenouille agile ( R a m dalmatina Bon). Bull. Biol. 88~168-177.

Cameron, J.A. (1936) The bodies of Eberth as a source of connective tissue ground substance in Rana catesbeiana Shaw. J. Morphol. 60:279-285.

Chapman, G.B., and A.B. Dawson (1961) Fine structure of the larval anuran epidermis with special reference to the figures of Eberth. J . Biophys. Biochem. Cytol. 10~425435.

Delsol, M., J. Flatin, J.M. Exbrayat, and J. Bons (1981) Developpement de Typhlonectes compressicaudus, Am- phibien apode vivipare. Hypotheses sur sa nutrition embryonnaire et larvaire par un ectotrophoblaste. C.R. Acad. Sci. Paris 293.281-285.

Delsol, M., J. Flatin, J.M. Exbrayat, and J . Bons (1983) Developpement embryonnaire de Typhlonectes compressicaudus (Dumeril et Bibron, 1841). Constitution d’un ectotrophoblaste. Bull. SOC. Zool. Fr. 108:680-681.

Dent, J.N. (1968) Survey of amphibian metamorphosis. In W. Etkin and L. Gilbert (eds.): Metamorphosis: A Problem in Developmental Biology. New York Apple- ton-Century-Crofts, pp. 271-311.

Eberth, C.J. (1866) Zur Entwicklung der Gewebe im Schwanz der Froschlarven. Arch. Mikrobiol. Anat. 2t490-506.

Fahrmann, W. (1971a) Die Morphodynamik der Epider- mis des Axolotis (Siredon mexicanum Shaw) unter den Einfluss von exogen applizierten Thyroxin. 1. Die Epi- dermis des neotenous Axolotls, 2. Mikrosk. Anat. For- sch. 83:472-506.

Fa rmann , W. (1971b) Die Morphodynamik der Epider- mis des Axolotls (Siredon mexicanum Shaw) unter den Einfluss von exogen applizierten Thyroxin. 11. Die Epidermis wahrend der Metamorphosis. Z. Mikrosk. Anat. Forsch. 83.535468.

Fox, H. (1983) The skin of lchthyophis (Amphibia: Cae- cilia): An ultrastructural study. J . Zool., Lond. 199~223- 248.

Fox, H. (1985a) Balancer fine structure of the Pleurode- les larva. Acta Zool. (Stockh.) 66r97-110.

Fox, H. (198513) Changes in amphibian skin during larval development and metamorphosis. In M. Balls and M. Bownes (eds.): Metamorphosis. Oxford: Clarenden Press, pp. 59-87.

Fox, H. (1986a) The skin of Amphibia. In J . Bereiter- Hahn, A.G. Matoltsy, and K.S. Richards (eds.): Biology of the Integument, Vol. 2. Heidelberg: Springer, pp.

Fox, H. (1986b) Early development of caecilian skin with special reference to the epidermis. J. Herpetol. 20t154- 167.

Fox, H. (1987) On the fine structure of the skin of larval, juvenile and adult Ichthyophis (Amphibia: Caecilia). Zoomorphology 107~67-76.

Fox, H., and J. Durand (1990) An examination of the effect of thyroid hormone on the skin of the neotenic

78-135.

Proteus anguinus. Eur. Arch. Biol. (Brux.) 101.269- 284.

Fox, H., and M. Whitear (1986) Genesis and regression of the figures Eberth and occurrence of cytokeratin aggregates in the epidermis of anuran larvae. Anat. Em-

Fox, H., and M. Whitear (1990) Cellular elements in the dermis and collagen remodelling during larval life of amphibians. Arch. Histol. Cytol. 53r381-391.

Gallien, L., and M. Durocher (1957) Table chronologique du developpement chez Pleurodeles waltlii Micha. Bull. Biol. 91t97-114.

Greven, H. (1980) Ultrastructural investigations of the epidermis and the gill epithelium in the intrauterine larvae of Salamandra salamandra (L) (Amphibia, Urodela). Z. Mikrosk. Anat. Forsch. 94:196-208.

Hirsch, J., and M.E. Fedorko (1968) Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and “post- fixation” in uranyl acetate. J . Cell. Biol. 38:615-627.

Jarial, M.S. (1989) Fine structure of the epidermal Ley- &g cells in the axolotl Ambystoma mexicanurn in rela- tion to their function. J . Anat. 167t95-102.

Jurd, R.D. (1985) Haematological and immunological “metamorphosis” in neotenous urodeles. In M. Balls and M. Bownes (eds.): Metamorphosis. Oxford: Clar- enden Press, pp. 313-331.

Katz, U. (1986) The role of amphibian epidermis in osmoregulation and the adaptive response to changing environment. In J . Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards (eds.): Biology of the Integument. Vol. 2. Heidelberg: Springer, pp. 427498.

Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron micros- copy. J . Cell. Biol. 27~137A.

Kelly, D.E. (1966) Fine structure of desmosomes, hemidesmosomes and an adepidermal globular layer in developing newt epidermis. J . Cell. Biol. 285-72 .

Lane, E.B., and M. Whitear (1980) Skein cells in lamprey epidermis. Can. J . Zool. 58:450455.

Larras-Regard, E. (1985) Hormonal determination of neoteny in facultative neotenic urodeles. In M. Balls and M. Bownes (eds.): Metamorphosis. Oxford: Claren- dan Press, pp. 294-312.

Lavker, R.M. (1972) Fine structure of the newt epidermis. Tissue Cell. 4:663-675.

Lindinger, M.I. (1984) Fine structure of the abdominal epidermis of the adult mud puppy Necturus maculosus (Rafinesque). Cell. Tissue Res. 238~395405.

Michaels, J.E., J.T. Albright, and D.I. Patt (1971) Fine structural observations on cell death in the epidermis of the external gills of the larval frog, Rana pipiens. Am. J . Anat. 132:301-318.

Nieuwkoop, P.D., and J . Faber (1956) Normal Table of Xenopus laeuis (Daudin). 2nd Ed. Amsterdam: North- Holland.

Ovalle, W.K. (1979) Neurite complexes and Merkel cells in larval tentacles of Xenopus laeuis. Cell. Tissue Res. 204~233-241.

Reichel, P. (1976) Differentiation of the opercular integument in Ranapipiens. Differentiation 5:75-83.

Robinson, H., and M. Heintzelman (1987) Morphology of ventral epidermis ofRana catesbeiana during metamorphosis. Anat. Rec. 217~305-317.

Rosenkilde, P. (1985) The role of hormones in the regula- tion of amphibian metamorphosis. In M. Balls and M. Bownes (eds.): Metamorphosis. Oxford: Clarenden Press, pp. 221-259.

Saguchi, S. (1913) Ueber Mitochondrien (Chondri- okonten) und mitochondriale Strange (sog. Eberthsche intrazellulare Gebilde) in den Epidermiszellen der

bryo]. 174:73-82.


Anurenlaryen nebst Bemerkungen uber die Frage der Epidermis Cutisgrenze. Arch. Mikr. Anat. 83:177-244.

Sasaki, H., H. Takahama, I. Horiguchi, and K. Watanabe (1983) Electron-microscopical study of the operculum in anuran tadpole after extirpation of the right fore- limb during metamophosis. Cell. Tissue Res. 232~513- 537.

Shumway, W. (1940) Stages in the normal development of Rana pipiens. 1. External form. Anat. Rec. 78: 139- 147.

Singer, M., and M. Salpeter (1961) The bodies of Eberth and associated structures in the skin of the frog tadpole. J . Exp. Zool. 147t1-19.

Skerrow, C. (1986) Desmosomal protein. In J . Bereiter- Hahn, A.G. Matoltsy, and K.S. Richards (eds): Biology of the integument. Vol. 2. Heidelberg: Springer, pp. 762-787.

Szarski, H. (1968) Evolution of cell size in lower vertebrates. In T. Orvig (ed.): Current Problems in Lower Vertebrate Phylogeny. Noble Symp. Vol. 4. Stockholm: Almavist & Wiksell. DD. 445453.

Szarski’, H. (1976) Celi size and nuclear DNA content in

Tachibana, T., Y. Sakakura, and Y. Nawa (1980) Merkel vertebrates. Int. Rev. Cytol. 44r92-111.

cell differentiation in the developing tentacle of Xeno- pus Zaeuis. Acta. Anat. Nippon 55:588-599.

Warburg, M.R., and D. Lewinson (1977) Ultrastructure of epidermis of Salamandra salamandra followed through ontogenesis. Cell. Tissue Res. 181r369-393.

Weed, I.G. (1934) Cytological studies of the epidermis of Ranapipiens and Rana damitans tadpoles with special reference to the figures of Eberth. J . Morphol. 56:213- 219.

Weiss, P., and W. Ferris (1954) Electronmicrograms of larval amphibian epidermis. Exp. Cell Res. 6.546449.

Welsch, U. (1981) Fine structural and enzyme histochem- ical observations on the respiratory epithelium of the caecilian lungs and gills: A contribution to the under- standing of the vertebrate respiratory epithelium. Arch. Histol. Jap. 44: 117-133.

Welsch, U., and V. Storch (1973) Die FeinStruktur ver- hornter und nichtverhornter ektodermaler Epithelien und der Hautdrusen embryonaler und adulter Gymno- phionen. Zool. Jb. Anat. 90r323-342.

Whitear, M. (1986) The skin of fishes, including cyclos- tomes. In J . Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards (eds.): Biology of the Integument. Vol. 2. Heidelberg: Springer, pp. 8-64.

figures of eberth in the amphibian larval epidermis

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