Cornification in developing claws of the common Australian skink (Lampropholis guichenoti) (Squamata, Lacertidae)

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1 Italian Journal of Zoology ISSN: (Print) (Online) Journal homepage: Cornification in developing claws of the common Australian skink (Lampropholis guichenoti) (Squamata, Lacertidae) L. Alibardi To cite this article: L. Alibardi (2008) Cornification in developing claws of the common Australian skink (Lampropholis guichenoti) (Squamata, Lacertidae), Italian Journal of Zoology, 75:4, , DOI: / To link to this article: Copyright Unione Zoologica Italiana Published online: 19 Dec Submit your article to this journal Article views: 406 View related articles Citing articles: 15 View citing articles Full Terms & Conditions of access and use can be found at

2 Italian Journal of Zoology, December 2008; 75(4): Cornification in developing claws of the common Australian skink (Lampropholis guichenoti) (Squamata, Lacertidae) L. ALIBARDI Dipartimento di Biologia, University of Bologna, Italy (Received 1 June 2007; accepted 24 October 2007) Abstract An ultrastructural and immunocytochemical study on developing lizard claws has been done. Claws originate from an epidermal thickening covering the tip of digits under which mesenchymal cells aggregate. The outer side of the dorsal terminal scale of a digit gives origin to the unguis, and suggests that claws are modified scales sustained by the last phalanx. Beneath four seven layers of embryonic alpha-keratin cells, beta-keratin cells are differentiated but no shedding complex is formed. Beta-keratin cells extends also over the apical part of the ventral side (sub-unguis) which is mainly formed by alphakeratin cells. Initially beta-keratin filaments have a prevalent oblique orientation along the plane containing the proximal apical axis of the digit. Later bundles become more irregularly distributed. Beta-keratin cells of the unguis remain largely separated and do not merge into a syncitium like in the beta-layer of scales. The thickness of the horny layer is higher in the unguis than in the sub-unguis and decreases from the claw tip toward its basal most part. It is hypothesized that the evolution of claws occurred by the extension of the outer scale surface of the terminal scales beyond the digit tip under the influence of the apical mesenchyme in the terminal phalange. Keywords: Lizard, embryo, claw differentiation, ultrastructure, immunocytochemistry, evolution Introduction The morphological patterns of formation of skin derivatives in amniote integument such as claws and nails are not well known in non-mammalian vertebrates (Baden 1970; Chapman 1986; Wu et al. 2004). Detailed cytological studies are available only for developing nails and hoofs of a few mammalian species (Hashimoto et al. 1966; Hashimoto 1971; Baden & Kvedar 1983; Chapman 1986; Bragulla & Hirschberg 2003). In the developing digits of mammals (Hamrich 2003) and birds (Lucas & Stettenheim 1972; Spearman & Hardy 1986), claws and nails are reported to derive from the thickening of the embryonic epidermis of the dorsal tip of digits under a likely mesenchymal induction or dermal influence. The development of avian claws has been little studied and many cytological details of its formation are not known (Kerr 1919; Lucas & Stettenheim 1972; Spearman & Hardy 1985). In reptiles even less information is available on the structure and development of the claw (Maddin et al. 2007; Maddin & Reisz 2007), and only general statements are found in the literature including the suggestion that claws are modified scales (Kreisa 1979). Neither detailed structural nor ultrastructural studies are available on the development of claws and the process of cornification in the modern literature. It is known that the horny portion of reptilian claws mainly consists of hard, presumably beta-keratinized cells that form a resistant layer with a strong mechanical resistance (Gillespie et al. 1982; Marshall & Gillespie 1982). As in birds, reptilian claws possibly contain deposited minerals along with keratins which contribute to their mechanic properties (Spearman & Hardy 1985). Detailed knowledge on claw formation in any reptilian species is essential to compare the mechanisms of claw cornification between reptiles and their derived amniotes, birds and mammals (Parakkal & Alexander 1972; Alibardi 2006, 2007). Also, the Correspondence: Lorenzo Alibardi, Dipartimento di Biologia Evoluzionistica Sperimentale, University of Bologna, via Selmi 3, I Bologna, Italy. alibardi@biblio.cib.unibo.it ISSN print/issn online # 2008 Unione Zoologica Italiana DOI: / Published online 19 Dec 2008

3 328 L. Alibardi knowledge on the morphogenesis of reptilian claws may give some clues on their evolution from presumed digital scales. The present study was performed to fill the gap of our knowledge on the process of histogenesis and cornification of reptilian claws. For this purpose, the reported observations are compared to those obtained from the study of the developing avian and mammalian claws. The study also aims to analyse common aspects of the process of hard cornification in the appendages of all amniotes. Materials and methods Embryos of the Australian common skink Lampropholis guichenoti (Scincidae, Squamata), collected in a previous study on scales development, were also utilized for the study of claw morphogenesis (see details in Alibardi & Thompson 1998). Embryos from stage 34 (elongating digits joined one to another by an inter-digital membrane; n52), stage 35 (longer and more defined digits with retraction of the inter-digital membrane; n52), stage 36 (distinct and elongated digits with almost reabsorbed interdigital membrane and short claws; n53), and stage 38 (elongated, pointed but unsharped claws; n53) were utilized (see general reptilian embryonic stages summarized in Porter 1972). Digits were collected both from anterior and posterior limbs and were immediately fixed for 5 8 h in cold (0 4uC) phosphate-buffered solution (PBS; 0.1 M, ph ) containing 2.5% glutaraldehyde. After 30 min rinsing in the PBS, the tissues were immersed for min in 2% aqueous OsO 4, dehydrated by ethanol, soaked with propylene oxide and embedded in the resin Durcupan. After sectioning with an ultramicrotome, 1 2 mm semithin sections were collected on glass slides to be used for light microscope (LM) study. From apical areas of digits, ultrathin sections of nm in thickness were collected on copper grids for the ultrastructural examination. Sections were stained with uranyl acetate and lead citrate, and observed under a Philips CM-100 electron microscope. Other samples were fixed in Carnoy fluid or in 4% paraformaldehyde in 0.1M PBS ph 7.4 for 5 h, dehydrated in 80% ethanol, and embedded in the hydrophilic resin Lowicryl K4M under UV polymerization at 0 4uC, the tissue samples were sectioned with an ultramicrotome at 1 4 mm thickness (semithin sections) for light microscopy. These sections were stained with 0.5% toluidine blue staining solution. Some sections fixed in Carnoy or in 4% paraformaldehyde were used for the histochemical study for sulphydryl groups and others for the immunofluorescence study on beta-keratin distribution. In addition, from tissues fixed in 4% paraformaldehyde, nm (ultrathin sections) were collected on nickel grids for the ultrastructural study. Sulphydyl groups, indicating strong keratinization, were revealed by the Chèvremont Fréderich reaction, using the general ferric ferricyanide reactive (Troyer 1980). A beta-1 antibody, produced in rabbits against a chick scale beta-keratin and liberally provided by Dr R.H. Sawyer (Biological Science Department, University of South Carolina, Columbia, USA; see Sawyer et al. 2000) was utilized for immunodetection of this keratin. A more specific antibody produced in rats against a lizard beta-keratin was also used (Alibardi & Toni 2006b). In detail, the two antibodies were used to determine at the light and ultrastructural level this specific keratin in the corneous material accumulated in claws. For fluorescence microscopy, semithin sections were pre incubated for 30 min in 5% normal goat serum and 2% bovine serum albumin (BSA) in 0.05 M Tris/HCl buffer at ph 7.6, in order to neutralize non-specific antigenic sites in the specimens. Sections were then incubated overnight at 4uC in the BSA Tris buffer containing the primary antibody (dilutions 1:100 were used for the beta-1 antibody, and 1:50 for the lizard beta-keratin). In control trials the primary antibody was omitted. After rinsing in the BSA Tris buffer, the sections were incubated for 1 h at room temperature in the same buffer containing 1:50 anti-rabbit- or 1:50 anti-rat-igg FITC conjugated (as labelled secondary antibodies). After extensive rinsing, sections were mounted in Fluoromount (EM Sciences, USA), and observed under a Zeiss epifluorescence microscope equipped with a fluorescein filter. For immunoelectron microscopy, nm ultrathin sections were collected on nickel grids, and treated with the primary antibody as above using 1% cold water fish gelatin in 0.05 M Tris/HCl buffer to block non-specific binding sites. Anti-rabbit or antirat IgG conjugated to 10 nm large gold particles secondary antibodies (1:60 dilution in buffer, Sigma, USA, or Biocell, UK) were used as immuno-dye, respectively. Sections were observed under TEM, after 6 min of staining with 2% uranyl acetate. Results Light microscopy: structure, histochemistry and immunocytochemistry At stages 33 the tips of digits were covered with a twolayered epidermis that resembled that of the remaining

4 Claw development in common Australian skink 329 body areas. The profile of epidermal surface was mainly linear in section and no scale folding was detectable at this embryonic stage on digits (while scale anlagen were present in other body regions such as in the dorsal midtrunk and tail). At stages 34 the tip of digits (still connected by an inter-digital connective tissue) showed the initial stages of claw formation while the epidermal folds of the digit scales were not yet visible. In both frontal and longitudinal planes the epidermis on the tip of the digit showed a thickened epithelium, consisting of columnar cells covered by a flat periderm. On the basal side epidermis is closely contiguous to mesenchymal cells (Figure 1A). The chondroblasts of the forming terminal phalange reach the apex of the digit. In some sections the mesenchyme showed a condensation in this apical area and mesenchymal cells appeared in close contact with the columnar cells of the apical epidermal ridge. No reactivity for sulphydryl groups was observed in the epidermis at this stage. In longitudinal section of early elongating claws at stages 35 and 36, the dorsal surface initially resembled the outer surface of an elongated scale while the ventral side was thinner, resembling the inner side of a scale (Figure 1B, C). Most of the remaining epidermis of the digit was two-/threelayered at stage 35 and no sign of differentiation of beta-keratin cells was detected in the anlagen of the other digit scales. Beta-keratinization in claws preceded the same process later observed in the forming digital scales. Besides, in claws more layers of spindleshaped beta cells were present than in normal scales. The thick epidermis of elongating and pointing claws was multilayered in the dorsal side of the claw, with 3 5 layers of spindle-like beta cells (Figure 1C, D). The number of beta cell layers increased from the base of the claw and folded underneath the ungueal scales (the next to claw), to the tip of the claw. The pattern of stratification of beta cells on the dorsal side of the claw, in both sagittal and frontal sections, resembled the stratification of beta-keratin layer from the hinge region to the outer scale surface of embryonic scales. Beta cells showed intense blue reactivity for sulphydril groups which was absent in the other epidermal layers (Figure 1E). The epidermal fold at the base of the claws epidermis resembled the inner scale surface of an elongated scale located at the tip of digits. In the ventral side, pre-corneous keratinocytes contained keratohyalin-like granules, and the corneous layer was remarkably thinner than in the dorsal side (Figure 1C). At stages 36 and 38 the claw was grown, and its dorsal, bowed, surface was thicker than in the ventral surface (Figure 1F, G). The dorsal and ventral surfaces of the claw were deeply inserted Figure 1. A, frontal section of developing toe at stage 34. Arrowhead on the thickened apical epithelium. Bar 40 mm; B, longitudinal view of developing claw at embryonic stage 36. The bowed dorsal surface shows a thickened corneous layer (arrow). Arrowheads iondicate the folded epidermis in continuity with the inner surface of dorsal and ventral scales. Bar, 30 mm; C, detail of claw in longitudinal section showing the bowed corneous cells (double arrowhead) at the tip, in contact with the apical papilla. Arrow indicates beta-keratin cells of the corneous layer of the unguis. Arrowheads indicate keratohyalin-like granules of the subunguis. Bar, 10 m; D, dorsal frontal section at stage 36 showing the spindle-like cells of the beta-keratin layer (arrowheads). The latter becomes thin by the hinge regions (arrows) in continuation with the inner surfaces of terminal scales. Bar, 30 mm; E, intense blue staining (arrows) due to the occurrence of sulfydryl groups (-SH) in beta cells of a frontally-sectioned claw at stage 36. Bar, 20 mm; F, sagittal section of a claw at stage 38 showing the thick unguis (adouble arrowhead) and the thinner sub-unguis. The latter terminates in a hinge region (arrow) in continuation with the ventral scale. Arrowhead indicates the multi-layered embryonic epidermis undernath the tip of the sub-unguis. Bar, 30 mm; G, oblique section of claw at stage 38 showing thick, compact corneous layer (c) and differentiating cells (arrow). Bar, 25 mm. H, immunolabelled cell-layers of the unguis using the beta-lizard keratin antibody. The fluorescence is mainly localized in the differentiating beta-layer and less in the compact corneous layer. Dashes underlie the epidermis. Bar, 25 mm. Abbreviations: ap, apical papilla; c, mature corneous layer of the claw; d, dorsal; e, epidermis (ordinary); i, inner (ventral) claw surface or sub-unguis; me, mesenchyme; o, outer (dorsal) claw surface (unguis); ph, last phalange (cartilagineous); v, ventral.

5 330 L. Alibardi between the terminal scales, in continuation with their inner surfaces. At these stages, a pale periderm and other embryonic cell layers were seen externally to the dark beta-keratin layer on the surface of claws (Figure 1F). Embryonic layers of the claw appeared thicker in comparison to those present over ordinary scales of the embryo. The immunolabelling with beta-keratin antibodies showed positivity in beta cells of claws from stages 36 onward, while in the remaining epidermal layers as well as in the dermis no staining was detected (Figure 1G, H). The labelling was stronger in the transitional, pre-corneous cells than in the outer, compact horny layer. Transmission electron microscopy: Ultrastructure and immunocytochemistry The epidermis of the claw at stages 34 and 35 consisted of 4 7 layers of thin pale cells underneath the periderm which formed the keratinized embryonic epidermis (Figure 2A, B). The external periderm cell layer showed short microvilli and endocytotic vesicles (not shown). The inner periderm cells contained sparse bundles of keratin and formed a thickened cornified plasma membrane (cornified cell envelope). The following, inner 2 3 layers were more electron-dense and their cells contained numerous mm thick bundles of filaments or granules of keratin with an irregular pattern of 3 nm thick filaments, as detected at high magnification (over 70,0006). These bundles initially tended to arise in the peripheral cytoplasm along the plasma membrane. On a frontal plane of digit, bowed cells at the claw tip contained electron-dense bundles of beta-keratin with oblique to perpendicular orientation (Figure 2A C). In longitudinal section of digit, beta-keratin filaments appeared oblique or elongated and directed toward the apical tip of claws. Therefore the orientation of beta-keratin bundles deposited initially in differentiating keratinocytes appeared mainly directed parallel to the epidermal surface of the digit. Basal cells and two three layers of suprabasal keratinocytes contained ribosomes clusters, while endoplasmic reticulum profiles were scanty. Filaments as well as thin bundles of keratins were also scanty. The latter, however, increased in dimension in flat cells of upper layers of the epidermis, comprising three layers of cells before the compact horny layer (Figure 3A). In more external, precorneous cells, large bundles of dense keratin merged forming a network pattern (Figure 3B). Ribosomes and likely glycogen particles remained Figure 2. A, detail of the tip of a claw in frontal section at stage 36. Numerous differentiating beta cells accumulate bundles of beta-keratin (arrows) beneath the electron-dense corneous layer. Bar, 5 mm; B, detail of embryonic epidermis (arrow on dense thickening of the plasma membrane) whose lowermost cells accumulate keratin bundles (arrowheads) in the transition with to the first, dark beta-layer. Bar, 1 mm; C, higher magnification of the transition between the dense, maturing beta-layers (arrows on the thickened and dense plasma membranes) and the underneath differentiating beta cells (arrowhead on the beta-keratin packets/ bundles). Bar, 250 nm. Abbreviations: ba, basal (germinal) layer; ee, embryonic epidermis; cc, initial compact corneous layer; l, lipid droplet; n, nucleus; p, outer periderm; sb, suprabasal layer; tc, transitional layer. embedded within beta-keratin bundles during condensation into transitional cells. These filaments as well as large bundles of compact corneous material appeared immunolabelled with both the beta-1 and the beta-lizard antibodies, confirming light microscopic immunolocalization (Figure 3C, D). At stage 36, the more apical part of the dorsal layer of dense beta cells reaching the tip of claws (unguis) formed in section a curved line that terminated near a peg of epidermis (apical papilla) present at the very tip of the forming claw (Figures 1C, 4A). These electron-dense cells contained numerous ribosomes in a cytoplasm rich in dark beta-keratin bundles (Figure 4B). The latter were mainly oriented perpendicular or slightly oblique with respect to the apical-proximal axis of the claw. The bowed beta cells occurring at the tip of the claw were in continuation with paler,

6 Claw development in common Australian skink 331 Figure 3. A, longitudinal section of early differentiating beta cells at stage 38 containing long bundles of beta-keratin (arrows). Bar, 1 mm; B, detail of merging beta-keratin filaments (double arrowheads) in transitional cell of the unguis epidermis at stage 38. Arrows indicate mainly ribosomes that are clumped inside the keratin mass. Bar, 1 mm; C, immunogold-labelled keratin bundles (arrow) in transitional cells after immunostaining with the beta-1 antibody (b-1). Bar, 100 nm; D, immunolabelled keratin bundles using the beta-lizard antibody (b-liz). Arrows indicate desmosome remnants. Bar, 200 nm. Abbreviations: n, nucleous; k, keratin bundles; sb, suprabasal cells; tr, transitional layer; v, vesicles. pre-corneous cells in which numerous small keratin bundles (tonofilaments) were present (Figure 4C). At the tip of the claw the epidermis formed a papilla with electron-pale cells that penetrated between the bowed, dark beta-keratinocyets. Most of alphakeratin filaments in pre-corneous cells were perpendicular or irregularly oriented in relation to the dense beta-keratin bundles of the bowed cells. The apicalmost beta cells of the bowed cell layer terminated as a single layer of dark cells. These cells were surrounded by a cornified plasma membrane, the cornified cell envelope or marginal layer (Figure 4D). Figure 4. A, detail of the tip of a claw at stage 36 (position as indicated by the double arrowhead in Figure 1). The thickly arranged, apical beta cells form a bowed layer (arrows) that contact the underlying, still differentiating cells (arrowheads). Bar, 5 mm; B, detail of further bowed beta cells with numerous but still isolated bundles of beta-keratin (arrowhead). Bar, 5 mm; C, bowed dense beta cells (arrows) at the tip of claw, which contain numerous large bundles of beta-keratin (double arrowheads). Arrows indicate beta-packets in early differentiating beta-keratin cells. Bar, 2.5 mm; D, detail of mature tips of beta cells (arrow) at the tip of the claw. Bar, 1 mm. Abbreviations: ee, embryonic epidermis; db, differentiating beta-keratin (hard corneous) cells; k, addensating keratin bundles; n, nucleous; ve, vesicle. At stage 38, beneath the pale but keratinized embryonic epidermis of the unguis, the number of dense, corneous layers increased to (Figure 5A, B). A thickened (10 20 nm) cornified cell envelope surrounded the pale and evenly keratinized cells, where a few lipid-like vesicles were present. The transition between pale embryonic and definitive dense layers was marked by the formation of denser beta-keratin bundles (as suggested by the

7 332 L. Alibardi Figure 5. A, detail of outer area near the tip of the claw unguis at stage 38 (position similar to that indicated by the double arrowheads of Figure 1 F). Arrows indicate cells of the transition layers between embryonic and the stratified dense corneous layer (double arrowhead). Bar, 2 mm; B, detail of thickened plasma membranes (arrow) of embryonic epidermis. Bar, 1 mm; C, detail of transitional cell with beta-keratin bundles (arrow). The inset at higher magnification (Bar, 20 nm) shows a pale beta-keratin filament among the two arrows. Bar, 500 nm; D, obliquely sectioned cells of the opaque corneous layer of the unguis at stage 38. Beta cells remain separated (arrowhead) and some regions of their plasma membrane are remarkably dense (arrow). Bar, 500 nm; E, further detail of the above cells showing the dense membrane patches (arrows) and deposition of dense keratins toward the cell periphery. Bar, 500 nm. Abbreviations: c, dense corneous layer containing beta-keratin; dc, dense corneous material; ee, embryonic epidermis; k, keratin bundles; l, lipid droplet; pc, pale corneous material. 3 nm thick but irregular filament pattern, Figure 5C). Beneath the dense layers, two-three transitional layer and two three differentiating betakeratin layers were present. Opaque beta-keratin corneous cells contained pale regions alternated to dense regions, the latter especially concentrated along the thickened plasma membrane (Figure 5D, C). Also, beta-keratinocytes of the unguis were separated one from another by a dense plasma membrane and were not merged into a syncitium as in keratinocyets of normal scales. Few ribosomes and glycogen granules remained among the merging bundles of beta-keratin. Furthermore, a specialized shedding plane between the embryonic epidermis and the underlying beta-layers of the claw was not seen. Shedding of the embryonic epidermis therefore occurred along the boundary between embryonic and the beta-layer. The study of the corneous layer of the epidermis of the sub-unguis or ventral side of the claw at stages 36 and 38, showed 6 10 electron-dense alpha-keratinocytes of mm thickness (Figure 6A). The corneous layer in the sub-unguis was thinner than in the dorsal unguis. The roundish keratohyalinlike granules observed at stage 35, disappeared in pre-corneous cells at stage 38, and were rarely seen incorporated within narrow keratinocytes at maturity (Figure 6B, C). These granules, often contacting keratin filaments, consisted of a homogenous electron-dense material without any limiting membrane. Other organelles frequently seen within the cytoplasm of these cells included lipid droplets and pale vesicles, probably derived from the Golgi apparatus or the smooth enoplasmic reticulum (not shown). Fully differentiated alpha-keratinocytes crowded over the epidermis of the sub-unguis, and contained dense material typical for alpha-keratinocytes of ordinary scales. Some narrow keratinocytes resembled lacunar cells of normal scales (a specialized type of alphakeratinocyte), and contained sparse pale vesicles, few keratin bundles (often along the plasma membrane), and were surrounded by a nm thickened cornified cell envelope at maturity. Discussion Morphogenesis and epidermal stratification The present study provides first data on the morphogenesis and cornification of claws in a reptile. Claws are epidermal specializations that probably evolved early in tetrapod history (Maddin & Reisz 2007; Maddin et al. 2007). Claws might have originated as a by-product of digit evolution, possibly derived from the interaction between growing phalanges and the dermal epidermal components of the terminal scale of digits. It appears that, like in the developing limbs of reptiles (Raynaud 1985), also in digits an apical epidermal ridge is formed in close contact with the apical mesenchyme. This process has been also observed in the developing digit tips of turtle,

8 Claw development in common Australian skink 333 Figure 6. A, detail of stratified corneous and pre-corneous layers of sub-unguis at stage 38 (position like i in Figure 1 F). Arrows indicate small pale vesicles. The arrowhead points to a lipiddroplet. Bar, 1 mm; B, high magnification of keratohyaline-like granules in transitional cells of the sub-unguis at stage 36. Arrows indicate (alpha)keratin filaments close to the granules. Bar, 250 nm; C, numerous pale vesicles of various size (arrowhead) are seen in the cytoplasm of a pre-corneous cell of the sub-unguis at stage 38. Arrow indicates the plasmalemma of a cornified cell. The double arrowhead points to a small keratohyalin-like granules incorporated within the corneocyte. Bar, 500 nm. The inset at high magnification (Bar, 20 nm) shows a pale alpha-keratin filament among two arrows. Abbreviations: c, corneous layer (alpha); de, desmosome remnant; ee, embryonic layer; k, (alpha-) keratin bundles; n, nucleus; v, vesicle. tuatara, and alligator embryos (Alibardi, unpublished observations). The present observations, summarized in Figure 7, confirm that specific dermal epidermal interactions are needed for the morphogenesis and growth of reptilian claws, as for those of mammals (Hamrich 2003). After an initial symmetric phase at embryonic stage 33 [Figures 7(1)], the dorsal side of the terminal scale appears to elongate more rapidly of the ventral side as more differentiating and beta-keratin Figure 7. Drawing illustrating claw morphogenesis in lizard from the terminal scale of a digit (1). The asymmetric growth of the outer versus the inner side (2 4) of the terminal scales and the formation of a thicker corneous layers in the dorsal surface are shown (5,6) (see text in Discussion for more detailed explanations). Arrows in 3 and 4 indicate the formation of an apical epidermal papilla after the faster growth (curved arrow in 49) of the epidermis of the dorsal surface of the claw (curved arrows in 4 6; the double arrow suggests a faster growth of the downgrowing tip of the claw). Details of the cell structure of the claw tip are shown in 59. Mesenchymal cells are in close contact with the epidermis. Abbreviations: AS, apical (terminal) scale; AER, apical epidermal ridge; al, alpha-layer; bl, beta-layer; D, dorsal scale; DC(bK), dorsal (outer) corneous layer (unguis); EE, embryonic epidermis; FEE, folding (arrow) embryonic epidermis under the sub-unguis (neonichium); IS, inner scale surface; KH, keratohyalinlike granules; ME, mesenchyme (apical); OS, outer scale surface; PH, last phalange; V, ventral scale; VC(aK), ventral (inner) corneous layer. cells are produced. This process is similar, although of a higher extent, to that observed in normal developing scales [Figure 7(2 6)]. In fact, a faster growth of the outer scale surface of lizards has been shown using tritiated thymidine and 5-bromodeoxy-uridine, as markers of cell proliferation (Alibardi 1996, 1998a, 1998b). A higher cell proliferation also occurs in the outer surface of the claw, which turns down to form the unguis [Figure 7(5,6); Alibardi, unpublished observations]. Beta cells form a thicker and compact layer in the

9 334 L. Alibardi dorsal side of the elongating claw, and the more distal cells move apical and turn into the apical papilla [Figure 7(49)]. The claw is initially covered by an embryonic epidermis consisting of an outer periderm involved in intense exchange activity with the amniotic fluid, as well as by a multilayered inner periderm. The embryonic epidermis is a temporary tissue destined to be sloughed at hatching. A fine dispersion of alpha-keratin filaments produces a soft form of keratinization in these external cells. The distal, curved movement of the beta-layer at the tip of the claw, also occurs for the cells of the embryonic epidermis whose folded layers tend to converge toward the ventral side of the claw forming a cushion that covers the tip [Figure 7(59)]. This cushion is also observed in developing claws of other reptilian embryos as well as in birds, where it has been indicated as neonichium (in Kerr 1919). This tissue is lost at hatching, and it is believed to protect the delicate embryonic tissue and amniotic membranes from the potential tearing action of the claw before completion of embryonic development. The tip of the claw is covered by a hard beta-layer which extends over a short distance in the subunguis [Figure 7(59)]. While outer and inner periderm layers are formed in ordinary scales (Alibardi & Thompson 1998; Alibardi 2003), in claws 4 7 layers are formed before the thick, definitive corneous layer is formed underneath at later embryonic stages. A thick stratification of the embryonic epidermis has been also observed in the developing shell epidermis of turtles where a thick stratum corneum is formed (Alibardi 2003). The relationship between number of embryonic layers and thickness of the corneous layer is not known. In the claw no shedding layer is formed beneath the embryonic epidermis and the beta-layer, a different condition from that occurring in ordinary scales. In claws, superficial cells may be lost either with a slow but continuous process or with a periodical process contrasting with the periodical shedding in normal scales. The lack of a shedding complex in claws (which occurs by a yet unknown mechanism) allows for the accumulation of numerous layers of beta-keratin cells. Perhaps under the influence of the last growing phalange of the digit, the epidermis of claws gives origin to beta cells in an early stage in comparison with the more proximal digital scales. The process resembles that of the beta-layer of scales, but beta cells in claws stratum corneum do not merge into a sincitium as in normal scales (Parakkal & Alexander 1972; Maderson 1985; Landmann 1986). The ultrastructural characteristics of keratinocytes in the inner side of the claw (sub-unguis) flat shape, narrow cells that flake off relatively easily, corneous cell membrane, initial presence of keratohyaline-like granules, strong electron-density, condensation of tonofilaments and absence of betapackets, presence of lipid droplets and vesicles, presence of extracellular lipids, etc. has indicated that these cells are essentially of the alpha-type. They form a very thick but softer stratum corneum with respect to that of the unguis. The superficial cells of the sub-unguis probably desquamate periodically and superficially like in the hinge region of normal scales. The mechanical performances of lizard claw (hardness and sharpness) therefore derive from the thick and compact beta-layer that covers the unguis and the initial part of the sub-unguis (Figure 7). This corneous layer is probably slowly worn in comparison to that present the sub-unguis layers. Process of hard cornification The cornification of the unguis resembles in general the process of beta-keratinization in scales (Parakkal & Alexander 1972; Maderson 1985; Landmann 1986; Alibardi 2003; Alibardi & Toni 2006a, 2006b). Sulfydryl groups occur in remarkable amounts in keratins of differentiating beta cells of claws. Claw beta-keratin exhibits a cross-reactivity with chick (Beta-1) and lizard scales (Beta-Liz) antibodies. This indicates that in lizard claws, betakeratins share some common epitopes with betakeratins of avian and lizard scales. A first difference between claw and scale keratinization is that betakeratinocytes remain discrete cells in the stratum corneum of the claws while they merge into a syncytium in the scale. A second difference is that beta-keratins in the claw form filaments with a prevalent parallel orientation along the longitudinal plane of the digit, and therefore parallel with respect to the epidermis surface. This also occurs in keratin bundles of mammalian nail, although the latter consist of alpha-keratins glued by keratin associated proteins rather than beta-keratins (Baden 1970; Gillespie 1991; Rogers 2004). Aside the different proteins involved, cytological processess of hard corneification in mammalian and reptilian skin appendages involve similar features. A more oriented beta-keratinization probably has an improved effect on the mechanical resistance of claws. Some studies on claw keratins have demonstrated that high-sulfur beta-keratins occur in the claw (Inglis et al. 1987) while low sulfur betakeratins prevail in ordinary scales (Dalla Valle et al. 2005, 2007a, 2007b). Present histochemical

10 Claw development in common Australian skink 335 findings are consistent with occurrence of sulfydryl groups and suggest that numerous disulfuric bonds are formed in the mature corneous material, resulting in the improvement of the mechanical properties of claws. This also occurs in mammalian hard annexa such as hairs, horns and nails, where high or ultrahigh sulfur-keratin associated proteins are present (Gillespie 1991). Common features in the process of corneification in beta cells of reptilian scales as well as claws and mammalian hairs (cortex and cuticle) as well as nails, suggest that similar processes occur in these cutaneous appendages (Alibardi 2006, 2007; Alibardi & Toni 2006a). It is believed that in developing scales of reptiles and birds and in hairs of mammals some keratinassociated proteins (KAPs) surrounds the initial framework of alpha-keratin filaments. The latter are scarce in reptilian/avian corneocytes but are more abundant in mammalian keratinocytes. Intermediate filaments are eventually replaced or embedded within beta-keratins (in reptiles/birds) or into a matrix made of KAPs (in mammals). Beta-keratins and KAPs have smaller size than alpha-keratins and are different from alpha-keratin in both molecular composition and mechanism of polymerization (Gregg & Rogers 1986; Brush 1993; Fraser & Parry 1996). Therefore, proteins indicated as feather/scale-keratins and beta-keratins in reptilian scales appear as the functional equivalent of KAPs of the interfilamentous matrix in mammalian hairs and nails. Differently from hairs keratins/kaps, betakeratins produces an X-ray and ultrastructural betakeratin pattern (Maderson 1985; Landmann 1986; Fraser & Parry 1996). Mammalian KAPs comprise glycine tyrosine-rich proteins and sulfur-rich proteins with small molecular weight (7 30 kda; Gillespie 1991; Rogers 2004). Differently from feather-keratin, mammalian KAPs do not form filaments, but represent an amorphous matrix. Therefore, in the corneous material of hairs and nails the X-ray and ultrastructural 10 nm alpha-keratin patter is preserved. The present study shows that lizard, and more in general, reptilian claws derive from a modification of the terminal scale of developing digits in association with the growth of the terminal phalange. A thick and hard corneous layer of beta-keratin forms the unguis that corresponds to the outer surface of an ordinary scale while the softer sub-unguis corresponds to the inner side of the scale. Due to the derivation from specific layers of lepidosaurian epidermis, and to the specific keratin composition with respect to that of other tetrapods, lizard claws are different from the corneous claws of either amphibians (Maddin & Reisz 2007; Maddin et al. 2007) or mammals (Chapman 1986). This suggests that among tetrapods, claws are corneous structures associated with the growth of the last phalange, with a possible evolution by convergence, and with a fundamental function for prehensility, manipulative ability, offence and defence. Acknowledgments The present study was conducted in part with personal funding and by a University of Bologna 60% grant. Dr R. Arnold (University of Sydney, Australia) read the English text. References Alibardi L Scale morphogenesis during embryonic development in the lizard Anolis lineatopus. Journal of Anatomy 188: Alibardi L. 1998a. Glycogen distribution in relation to epidermal cell differentiation during embryonic scale morphogenesis in the lizard Anolis lineatopus. Acta Zoologica 79: Alibardi L. 1998b. Differentiation of the epidermis during scale formation in embryos of lizard. Journal of Anatomy 192: Alibardi L Adaptation to the land: the skin of reptiles in comparison to that of amphibians and endotherm amniotes. Journal of Experimental Zoology B 298: Alibardi L Structural and immunocytochemical characterization of keratinization in vertebrate epidermis and epidermal derivatives. International Review of Cytology 253: Alibardi L Cell interactions in barb ridges of developing chick downfeather and the origin of feather branching. Italian Journal of Zoology 74: Alibardi L, Thompson MB Epidermal differentiation in the developing scales of embryos of the Australian scincid lizard Lampropholis guichenoti. Journal of Morphology 241: Alibardi L, Toni M. 2006a. Cytochemical, biochemical and molecular aspects of the process of keratinization in the epidermis of reptilian scales. Progress in Histochemistry and Cytochemistry 40: Alibardi L, Toni M. 2006b. Immunological characterization and fine localization of a lizard beta-keratin. Journal of Experimental Zoology B 306: Baden PH The physical properties of nail. Journal of Investigative Dermatology 55: Baden HP, Kvedar JC The nail. In: Goldsmith LA, editor. Physiology, biochemistry and molecular biology of the skin, vol 1. New York-Oxford: Oxford University Press. pp Bragulla H, Hirschberg RM Horse hooves and bird feathers: Two model systems for studying the structure and development of highly adapted integumentary accessory organs The role of dermo-epidermal interface for the micro-architecture of complex epidermal structures. Journal of Experimental Zoology B 298: Brush AH The origin of feathers: A novel approach. Avian Biology 9: Chapman RE Hair, wool, quill, nail, claw, hoof and horn. In: Bereither-Hahn J, Matoltsy GA, Sylvia-Richards K, editors. Biology of the integument, vertebrates 2. Berlin: Springer-Verlag. pp

11 336 L. Alibardi Dalla Valle L, Nardi A, Belvedere P, Toni M, Alibardi L. 2007a. Sequencing beta-keratins of differentiating epidermis of snake shows they are glycine proline serine-rich proteins with an avian-like gene organization. Developmental Dynamics 236: Dalla Valle L, Naldi A, Toffolo V, Niero C, Toni M, Alibardi L. 2007b. Cloning and characterization of beta-keratins of differentiating epidermis in gecko lizards show they are glycine-proline-serine-rich proteins with some common motif with avian beta-keratins. Developmental Dynamics 236: Dalla Valle L, Toffolo V, Belvedere P, Alibardi L Isolation of a mrna encoding a glycine proline-rich betakeratin expressed in the regenerating epidermis of lizard. Developmental Dynamics 234: Frazer RDB, Parry DAD The molecular structure of reptilian keratin. International Journal of Biological Macromolecules 19: Gillespie JM The structural proteins of hair: Isolation, characterization and regulation of biosynthesis. In: Goldsmith LA, editor. Physiology, biochemistry and molecular biology of the skin. Oxford: Oxford University Press. pp Gregg K, Rogers GE Feather keratins: Composition, structure and biogenesis. In: Bereither-Hahn J, Matoltsy GA, Sylvia-Richards K, editors. Biology of the integument, vertebrates 2. Berlin: Springer-Verlag. pp Hamrich MW Evolution and development of mammalian limb integumentary structures. Journal of Experimental Zoology B 298: Hashimoto K Ultrastructure of the human toenail. II. Keratinization and formation of the marginal band. Journal of Ultrastructural Research 36: Hashimoto K, Gross BG, Nelson R, Lever WF The ultrastructure of the skin of human embryo. III. The formation of the nail in weeks old embryos. Journal of Investigative Dermatology 47: Inglis A S, Gillespie MJ, Roxburgh CM, Whittaker LA, Casagranda F Sequence of a glycine-rich protein from lizard claw: Unusual dilute acid and heptafluorobutyric acid cleavage. In: L Italien J, editor. Protein, structure and function. New York: Plenum Press. pp Kerr JG Text-book of embryology, Vol II Vertebrata. London: MacMillian and Co. pp Kreisa RJ The comparative anatomy of the integumental skeleton. In: Wake MH, editor. Hyman s comparative vertebrate anatomy, 3rd edn. Chicago: The University of Chicago Press. pp Landmann L The skin of reptiles. Epidermis and dermis. In: Bereiter-Hahn J, Matoltsy AG, Sylvia-Richards K, editors. Biology of the integument. Vol 2: Vertebrates. Berlin: Springer-Verlag. pp Lucas AM, Stettenheim PR Growth of follicles and feathers. Color of feathers and integument. In: Avian anatomy. Integument. Agriculture Handbook 362. Washington DC: US Department of Agriculture. pp Maddin HC, Musat-Marcu S, Reisz RR Histological microstructure of the claws of the African clawed frog, Xenopus laevis (Anura: Pipidae): Implications for the evolution of claws in tetrapods. Journal of Experimental Zoology B 308:1 10. Maddin HC, Reisz RR The morphology of the terminal phalanges in Permo-Carboniferous synapsids: an evolutionary perspective. Canadian Journal of Earth Sciences 44: Maderson PFA Some developmental problems of the reptilian integument. In: Gans C, Billett F, Maderson PFA, editors. Biology of reptilia. Vol. 14B. New York: John Wiley & Sons. pp Marshall PC, Gillespie JM The tryptophan-rich keratin protein fraction of claws of the lizard Varanus gouldii. Comparative Biochemistry and Physiology B 71: Parakkal PF, Alexander NJ Keratinization. A survey of vertebrate epithelia. New York: Academic Press. Porter KR Herpethology. Philadelphia: W.B. Saunders Co. Raynaud A Development of limbs and embryonic limb reduction. In: Gans C, Billett F, Maderson PFA, editors. Biology of reptilia. Vol. 15: Development B. New York: John Wiley & Sons. pp Rogers GE Hair follicle differentiation and regulation. International Journal of Developmental Biology 48: Sawyer RH, Glenn T, French JO, Mays B, Shames RB, Barnes GL, et al The expression of beta (b) keratins in the epidermal appendages of reptiles and birds. American Zoologist 40: Spearman RIC, Hardy JA Integument. In: King AS, McLelland J, editors. Form and function of birds, Vol. 3. London: Academic Press Inc. pp Troyer H Principles and techniques of histochemistry. Boston: Little Brown Co. pp Wu P, Hou L, Plikus M, Hughes M, Scehnet J, Suksaweang S, et al Evo-devo of amniote integuments and appendages. International Journal of Developmental Biology 48: