Developmental mechanisms underlying differential claw expression in the autopodia of geckos
© Khannoon et al.; licensee BioMed Central. 2015
Received: 21 January 2015
Accepted: 11 March 2015
Published: 10 April 2015
The limb and autopodium are frequently employed to study pattern formation during embryonic development, providing insights into how cells give rise to complex anatomical structures. With regard to the differentiation of structures at the distal tips of digits, geckos constitute an attractive clade, because within their ranks they exhibit multiple independent occurrences of claw loss and reduction, these being linked to the development of adhesive pads. The developmental patterns that lead to claw loss, however, remain undescribed. Among geckos, Tarentola is a genus characterized by large claws on digits III and IV of the manus and pes, with digits I, II, and V bearing only vestigial claws, or lacking them entirely. The variable expression of claws on different digits provides the opportunity to investigate the processes leading to claw reduction and loss within a single species.
Here, we document the embryonic developmental dynamics that lead to this intraspecifically variable pattern, focusing on the cellular processes of proliferation and cell death. We find that claws initially develop on all digits of all autopodia, but, later in development, those of digits I, II, and V regress, leading to the adult condition in which robust claws are evident only on digits III and IV. Early apoptotic activity at the digit tips, followed by apoptosis of the claw primordium, premature ossification of the terminal phalanges, and later differential proliferative activity are collectively responsible for claw regression in particular digits.
Claw reduction and loss in Tarentola result from differential intensities of apoptosis and cellular proliferation in different digits, and these processes have already had some effect before visible signs of claw development are evident. The differential processes persist through later developmental stages. Variable expression of iteratively homologous structures between digits within autopodia makes claw reduction and loss in Tarentola an excellent vehicle for exploring the developmental mechanisms that lead to evolutionary reduction and loss of structures.
KeywordsTarentola Apoptosis Regression Proliferation
The tips of the digits of amniote vertebrates are governed by a molecular developmental program distinct from that which controls their more proximal portions [1,2]. Digit tip formation takes place when FGF (fibroblast growth factor) signaling ceases in the apical ectodermal ridge (possibly in response to initiation of Wnt signaling), and a secondary signal takes over . Candidate genes involved in the digit tip program have been identified (see Casanova et al.  for a review), including bone morphogenetic proteins (BMPs), BAMBI, noggin, and the Wnt pathway [3-6]. As end organs, amniote digit tips are primitively endowed with a keratinous claw, although modifications of this are also expressed as nails and hooves in some mammals.
The claw, in both reptiles and mammals, begins as a thickening of the overlying epithelium, with a fold developing at its proximal edge [7,8]. In lizards, the keratinous sheath of the claw has been described as being a modified terminal scale  that expresses a greater intensity and earlier onset of keratinization than do other scales of the digit. As claw development proceeds, the distalmost non-ungual dorsal scale grows over the fold, forming a hinge region, thereby internalizing the claw primordium . This step is followed by the production of keratins from stratified keratinocytes . Thymidine labeling indicates that proliferation occurs along the whole length of the claw epidermis . Greater proliferation of the unguis, comprised of tough β-keratins , relative to the sub-unguis, composed of more pliable α-keratins, has been suggested to generate the curvature of the claw .
The absence of a keratinized integumentary capping at the distal end of the digits is of sporadic occurrence throughout amniotes and may be associated with modified functional roles of the digit tips. Such absence may apply to all digits, or may be regionally evident, with some, but not all, digits of an autopodium, exhibiting this state. There may even be differences between positionally equivalent digits of the manus and pes.
Within the Gekkota, multiple occurrences of claw reduction and loss are evident, its prevalence being much higher than in any other lineage of normal-limbed lizards (those not exhibiting patterns of limb reduction) . All of the instances within the Gekkota occur in taxa that possess a subdigital adhesive system. The finer details of how claws are used during locomotion are yet to be elucidated , but they are employed, in some circumstances, to interact with asperities in rough surfaces to promote grip [12,13]. They are also used in digging, grasping of conspecifics (as in mating), and may even be involved in the manipulation of eggs at the time of oviposition, when the hind feet are used to roll the eggs on the ground to coat them with particles  or to place them into contact with surfaces to which they stick .
Mapping the instances of claw reduction and loss (Russell AP: The foot of gekkonid lizards: a study in comparative and functional anatomy. Unpublished PhD. Thesis, 1972) within the Gekkota onto the phylogeny published by Gamble et al.  reveals ten separate occurrences. Seven of these occur within the Gekkonidae: (1) In the Pachydactylus radiation (Pachydactylus, Elasmodactylus, Chondrodactylus, Colopus, Rhoptropus) all taxa exhibit either complete absence of claws on all digits or their reduction to vestigial, needle-like structures. The manus is generally entirely clawless (except in some digging species of Pachydactylus, in which minute claws are present), and the pes generally carries vestigial, needle-like claws (the presence of which is in some cases restricted to digits III and IV), which may be sexually dimorphic (being larger and more robust in females). (2) In the clade containing Homopholis, Blaesodactylus, and Geckolepis, these three genera bear robust claws on digits II to V of the manus and pes, whereas digit I carries a minute claw or is clawless. Within a species, manual digit I may lack a claw while pedal digit I may display a minute one. (3) In Phelsuma (including Rhoptropella), all digits are clawless or carry vestigial, needle-like claws. Digit I, manus and pes, is itself vestigial in this taxon. (4) In Ebenavia, all digits are clawless. (5) In the Gekko clade (Gekko, Pseudogekko, Luperosaurus, Ptychozoon, Lepidodactylus, Hemiphyllodactylus, Gehyra, Perochirus), digits II to V of the manus and pes carry robust claws. Digit I of the manus and pes may bear a minute claw or exhibit no presence of this. In Perochirus, diminution of the claw on manual and pedal digit I is accompanied by vestigialization of the digit itself. (6/7) Although the genus Hemidactylus generally displays robust claws on all digits, two species, H. greefii and H. brasiliana, independently show reduction of the claw to a small structure in manual digit I, and pedal digit I is also clawless in the latter.
Within the Phyllodactylidae, (8) Tarentola is characterized by having digits III and IV of the manus and pes strongly clawed, whereas digits I, II, and V of the autopodia carry either minute, needle-like claws (pes) or lack them entirely (manus). In some species, pedal digit I is also entirely without a claw.
Within the Diplodactylidae, there are two independent instances of claw reduction. (9) In Crenadactylus, all digits are clawless. (10) In Pseudothecadactylus, digit I of the manus and pes lacks a claw while all other digits are robustly clawed.
These multiple instances of claw reduction and loss are suggestive of functional differences between digits  and furnish the opportunity for investigating developmental mechanisms by which claw reduction and loss come about. The phyllodacylid genus Tarentola presents a particularly attractive vehicle for this because it exhibits the retention of fully developed claws on digits III and IV of the manus and pes alongside vestigialization or complete absence of claws on adjacent digits. This taxon thus permits the investigation of the development of full claw expression, vestigialization, and complete absence within and between autopodia of a single species. Despite our knowledge of the incidence of claw loss in geckos, an understanding of the developmental patterns that lead to claw loss have not been described.
We herein explore the timing and interaction of developmental processes bringing about full expression, reduction, or complete absence of the keratinized claw sheath in Tarentola annularis. In doing so, we also explore developmental processes in the underlying ungual phalanx. We predict that all digits will exhibit identical initial stages of claw formation that commence at the same time on all digits, and that regression of claw sheath expression and regression of the underlying ungual phalanx will subsequently occur in those digits that ultimately exhibit only minute claws or are completely devoid of them. In relation to this, we hypothesize that apoptosis (programmed cell death) plays an important role in the reduction and elimination of the claw sheath rudiments and portions of the underlying phalanx, as it does in the removal of additional tooth placodes in the mouse , the removal of non-functional tooth germs in the gecko Paroedura , and in the reduction of whole digits in some mammals . Furthermore, we hypothesize that apoptosis will be accompanied by a reduction in proliferation of generative cells in the claw sheath and ungual phalanx, as has been noted in the vestigialization of murine tooth germs and in loss of digits in skinks [21-23]. The alternative to the processes outlined above is that those digits displaying claw reduction or loss do so by initiating the developmental processes leading to claw sheath formation late or not at all. By understanding the mechanisms by which complex structures are lost, we aim to provide an insight into how variation can be generated during evolution, allowing for adaptation to specialized functions.
For gross morphological, histological, and immunohistochemical observations, adult, juvenile, and embryonic specimens of Tarentola annularis were assembled. For gross morphological observations, a series of juvenile (N = 9) and adult (N = 12) individuals were obtained from breeders in Egypt, sacrificed, fixed in neutral buffered formalin, and stored in 70% ethanol. To obtain embryos, gravid female T. annularis (N = 37) were maintained in laboratory cages and eggs, when laid, were carefully collected and transferred to plastic boxes containing perlite and incubated at a constant temperature of 30°C and a humidity of 85% to 90%. All animals were treated according to the refinement principles of respect, care, and minimization of suffering, and all work was conducted after being approved by the Committee on the Ethics of Animal Experiments, Zoology Department, Faculty of Science, Fayoum University. Euthanasia protocols used were conducted according to ethical concepts of diminishing animal pain, as laid out by the UK Home Office.
Embryos at different developmental stages were removed from their eggs and sacrificed by decapitation after cooling on ice. Digits from selected developmental stages (Additional file 1: Table S1) were chosen for examination: for ease of comparison, the Hamburger and Hamilton  embryonic stage equivalent for squamates  is provided. Digits that, in the adult state, bear fully developed, reduced or no claw sheaths were compared employing the techniques outlined below.
Manūs (N = 9) and pedes (N = 9) of ethanol-preserved juveniles and adults of T. annularis and of various embryonic stages (N = 3 to 5 for each stage) were examined using a Nikon 50i microscope and Nikon DS-5 M camera head and camera control unit (Nikon, Tokyo, Japan) and a Leica stereo microscope (MZFiii) and Leica DFC300 camera (Leica, Solms, Germany).
Scanning electron microscopy
Samples of manūs and pedes of ethanol-preserved selected embryonic stages (Additional file 1: Table S1) of T. annularis (N = 2 for each of stages 35, 36, 37, and 38) were dehydrated using graded series of ethanols, culminating with immersion in 100% ethanol (2× 30 min); placed in specimen capsules containing 100% ethanol and critical-point dried; mounted on aluminum stubs using copper double-sided tape and silver conductive paint, and coated with gold/palladium (20-nm thick film) in a Polaron SC515 SEM coating system. Specimens were examined in a JEOL6400 SEM (JEOL Ltd., Akishima-shi, Japan).
Samples of clawed, reduced-clawed, and unclawed digits were processed for histology (stages 35, N = 7; stage 36, N = 7; stage 37, N = 5). Following fixation in 10% neutral buffered formalin, samples were washed in distilled water and then in phosphate-buffered saline (PBS) and subsequently transferred to 70% ethanol. They were then dehydrated by taking them through a graded series of ethanols, culminating in three changes of 100% ethanol (Sigma-Aldrich, St. Louis, MO, USA), cleared in three changes of Histoclear (National Diagnostics, Atlanta, GA, USA), and infiltrated with three to four changes of molten paraffin wax (Thermo Scientific Raymond Lamb, Thermo Fisher Scientific, Waltham, MA, USA) (57°C) prior to embedding. Frontal and sagittal sections of 8 μm were cut on a Leica microtome (RM2245). Sections were subjected to trichrome staining (sirus red, alcian blue, Ehrlich’s hematoxylin) , and the stained sections were mounted with DPX (BDH). Sections were viewed on a Nikon Eclipse 80i microscope and photographed using a Nikon Digital Sight Camera.
In order to view proliferating cells, immunohistochemistry for PCNA (proliferating cell nuclear antigen) was performed on paraffin wax sections (stage 36, N = 7; stage 38, N = 2) that had been dewaxed in Histoclear and dehydrated through an ethanol series to PBS. PCNA detects cells in G1, S, G2, and M phase. Slides were immersed in proteinase K 20 μg/ml in PBS for 15 min and subsequently treated in 3% H2O2 for 10 min. An antigen retrieval step was performed using pH6 0.01 M citric acid at 95°C for 5 to 10 min. Samples were left to cool, washed in PBS, and then permeabilized in 0.05% trypsin in PBS. PCNA staining was then carried out using a PCNA labeling kit (Life Technologies 93–1143, Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. The color reaction was performed using a DAB kit (Vector Labs, Burlingame, CA, USA) following the manufacturer’s instructions. Samples were washed in PBS before being counterstained with 1% alcoholic eosin (Sigma) for 30 s, washed in 90%, 95%, and 100% ethanol, and mounted in DPX (BDH). Sections were viewed on a Nikon Eclipse 80i microscope and photographed using a Nikon Digital Sight Camera.
In order to identify cells undergoing programmed cell death, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining was employed. Paraffin wax sections (stage 36, N = 7) were dewaxed in Histoclear and rehydrated in decreasing ethanol concentrations, then washed with PBS. They were then immersed in freshly diluted protein digestion enzyme (Proteinase K; 65 μl/5 cm2) for 15 min. Following this, they were washed in dH2O, then quenched in 3% H2O2 in PBS for 5 min at RT, then washed in two or three changes of PBS. A TUNEL apoptosis detection kit (Millipore S7100, Millipore, Billerica, MA, USA) was used to label cells following the manufacturer’s instructions. A 75 μl/5 cm2 of equilibrium buffer was then applied for at least 10 s at RT. A 55 μl/5 cm2 of working strength TDT enzyme was then pipetted onto the sections, and they were incubated in a humidified chamber for 1 h at 37°C. Following this, a stop/wash buffer was applied for 10 min at RT. The sections were then washed in three changes of PBS, and subsequently 65 μl/5 cm2 of anti-digoxignenin conjugate was applied for 30 min in a humidified chamber at RT. The color reaction, counterstaining, mounting, and photographing were as for PCNA (above).
To identify cells early on in the apoptotic pathway, localization of activated caspase 3 was investigated. Paraffin wax sections (stage 35, N = 6) were dewaxed in Histoclear, rehydrated in decreasing ethanol concentrations (as above), and then washed in PBS. They were then subjected to an antigen retrieval wash for 30 min in Tris-EDTA buffer (pH 9.0) at 95°C. Subsequently, the samples were left for 90 min to cool to RT and were then subjected to a 10-min wash in PBS, followed by 20 min in PBS with 0.5% Triton® X-100 (Sigma-Aldrich, T9284). Slides were then transferred to a covered moist humidified chamber, and 150 μl of blocking solution (10% goat serum and 1% BSA in PBS with 0.1% Triton® X-100) was added to each slide and left for 1 h at RT. Sections were then incubated overnight at 4°C with activated rabbit caspase-3 primary antibody (1 in 200) in blocking solution in the humidified chamber. Three 1-h washes in PBS were then undertaken, followed by incubation for 1 h with Alexa-fluor 568 goat anti-rabbit (1 in 500) in blocking solution (as above) at RT in the humidified chamber. Sections were then subjected to six 5-min washes in PBS in a Coplin jar wrapped in aluminum foil to omit light. Sections were mounted with Fluoroshield™ with DAPI (Sigma-Aldrich) mounting medium, cover slipped, and imaged on a Zeiss photomicroscope (Axioskop 2) (Zeiss, Jena, Germany).
Nuclear staining using DAPI was employed to visualize the nuclei in the developing claw. Parraffin wax sections (stage 35, N = 5; stage 36, N = 4) were dewaxed, rehydrated, and mounted with Fluoroshield™ with DAPI (Sigma-Aldrich) mounting medium and cover slipped. Slides were placed in a lightproof, dry chamber and stored at 4°C prior to photographing with a Zeiss fluorescence microscope (Axioskop 2).
Claw distribution in Tarentola annularis
Claw development in T. annularis
By the end of stage 36, the digital scales were more pronounced, and the distalmost mid-sagittal dorsal scale had grown over the base of the claw epithelium, creating a hinge (Figure 5G,H). The difference between the distalmost phalanx of the clawed and unclawed/reduced-clawed digits was even more pronounced at this stage (Figure 5E-J). The nail-like scale that overlies the unclawed/reduced-clawed digit tips (Figure 1B,C,F) was evident as a large, distallyextensive developing scale at stage 36 of development (Figure 5H). Interestingly, the distalmost region of the ungual phalanx of the unclawed/reduced-clawed digits (Figure 5H) initiated endochondral ossification earlier than that of the clawed digits (Figure 5G,K). Ossification of the tip of the distalmost region of the ungual phalanx of the clawed digits was not observed until the latter part of stage 37 (Figure 5L).
Reduction and loss of claws through apoptosis and proliferative changes
Our investigations reveal that in Tarentola annularis, claws initially develop on all five digits of the manus and pes, that even at their earliest stages of detection, there are differences in cellular activity in the distal tips of the digits, and that the claws on digits I, II, and V regress during the later stages of development. Reduction and loss of these claws appears to result from a combination of several processes. Even before the claw sheaths begin to differentiate from the surface epithelium of the digit tips, differences were evident in the shape of the distalmost phalanges, associated with elevated levels of cell death in the mesenchyme of those digits in which the claw will be reduced or lost. This greater intensity of cell death may be responsible for the reduced size and altered form of the terminal phalanx of these digits as development progresses. Once the claw sheath had initiated, the position of the epithelial fold differed between the clawed and clawless/reduced-clawed digits. The groove of the fold was located more proximally in the clawed digits, and the enlarged claw extended distally from this. In the clawless/reduced-clawed digits, the fold gives rise to a nail-like scale in the dorsal midline (Figure 1B,C,F) that roofs and internalizes the majority of the elongated ungual phalanx and the majority of the reduced claw sheath where this persists.
High rates of proliferation were associated with the initial development of the epithelium of both the clawed and clawless/reduced-clawed digits, but proliferation was diminished in the clawless/reduced claw epithelium during later stages. High levels of apoptosis were observed in the unclawed/reduced-clawed digits, with cells being removed from within and around the forming claw sheath. Given the large number of dying cells observed, it is likely that apoptosis is the central mechanism leading to reduction and loss of the developing claws.
In the clawed digits, at the same stage (stage 36), very little endochondral ossification was evident in the ungual phalanx, which later developed into a large distal element supporting the fully developed claw sheath on digits III and IV. In contrast, the terminal phalanx of the clawless/reduced-clawed digits was much smaller and the cartilage had been replaced by bone at its distal tip. Such early onset of endochondral ossification may be associated with the relatively reduced size of this skeletal element on the unclawed/reduced-clawed digits. Apoptosis was also observed in the terminal phalanx associated with the disappearing claw. Cell death in this region could be responsible for further thinning and attenuation of the ungual phalanx, or it could be a consequence of the early endochondral ossification . A few apoptotic cells were associated with the large terminal phalanx of the clawed digits at later stages as these started to undergo ossification, but these were mainly associated with the perichondrium. It is therefore unclear whether apoptosis or early endochondral ossification is the major driver of the reduction of this phalanx.
Apoptosis is a well-known mechanism for bringing about the elimination or reduction of structures during development. For example, in tooth development, apoptosis is associated with the removal of vistigial tooth germs, enamel knot signaling centers, and loss of ameloblasts [19,28]. During digit development, the interdigital webbing is removed as a result of apoptosis, allowing the digital rays to be freed . If this process fails or is suppressed, syndactyly occurs [29,30], which is a natural occurrence in taxa that exhibit webbed digits . Recently, it has been shown that apoptosis is responsible for the loss of whole digits in the horse, camel, and three-toed jerboa, although not for digit reduction in the pig . In the case of the jerboa, the loss of digits appears to have co-opted the pathways normally used to remove interdigital tissue in the limb, as the transcription factor Msx2 overlaps with cell death in both cases, and Bmp4 is upregulated . Whether a similar correlation of Bmp4, Msx2, and cell death occurs in the regressing gecko claw digits provides an interesting avenue for future exploration, as Msx2 has been shown to affect the length of murine nails and claws by suppressing proliferation of germinal epithelium . In skinks, changes in the timing of Shh expression have been highlighted as leading to digit loss, with reduced proliferation of digit mesenchyme resulting in digit regression . Study of the molecular signals involved in gecko claw development and regression are therefore necessary for identifying whether the same conserved pathways (Bmps, Shh, Fgfs) are utilized to sculpt different aspects of the limb at different stages of development.
The relationship between the phalanx and the overlying claw sheath is unclear. In the process of claw reduction and loss during development, both structures appear to be involved, but whether one triggers changes in the other is unknown. It seems likely that the initially smaller protrusion at the tip of the unclawed/reduced-clawed digits constrains the region that will develop into the claw, and the thinner, elongate form of the terminal phalanx leads to the formation of a narrower claw sheath that caps this skeletal element. The resulting smaller claw sheath either persists in an abbreviated form in the pes or is lost entirely in the manus through further apoptosis, and these events are accompanied by an arrest of cell proliferation.
The digit field is formed by a small apical dermal-epidermal region at the tip of the limb bud , with an active dermis in contact with a thickened epidermis. After the ungual phalanx begins development, the terminal epidermis commences to thicken, yielding the first sign of the claw sheath. A possible influence on the process of claw sheath morphogenesis by the perichondral cells of the ungual phalanx was advanced by Alibardi . He proposed that close contact between the epithelium and the ungual phalangeal chondrocytes might trigger the earlier production of keratins (tough β-keratins on the dorsal side of the claw and pliable α-keratins on its ventral side) in the forming claw sheaths than in the other scales of the digits. The keratinocytes of the developing epidermis of the claw sheath lie in direct contact with the mesenchymal cells of the ungual phalanx .
Like those of reptiles, mammalian claws derive from a thickening of the embryonic epidermis of the dorsal tips of digits. However, in mammals, the growth of claws and nails occurs by the continuous production of new keratinocytes from a proximal germinal matrix [7,10,34-39]. This has been confirmed by following digit regeneration in the mouse . Proliferation in mammals is confined to the base of the developing nail/claw. Our results accord with the findings of Alibardi  that proliferation in geckos occurs throughout the claw epithelium. Significant differences therefore exist between claw development in reptiles and mammals.
Our prediction that all digits would exhibit identical early stages of claw formation and that there would be regression of claw sheath expression in those digits that ultimately exhibit only minute claws or their complete absence is generally supported. However, it is evident that subtle differences between digits that will ultimately bear fullydeveloped claws and those on which claws will be reduced or absent are evident from the earliest stages of detection. Also supported is our suggestion that apoptosis plays an important role in the reduction and elimination of the claw sheath rudiments. Our prediction that apoptosis is accompanied by a reduction in proliferation of generative cells is also confirmed. Differential developmental timing of a common set of processes leads to a variety of outcomes and also results in the reshaping of the ungual phalanx and its associated sheath in those digits displaying claw reduction or loss.
Robust, curved claws that cap the ends of the digits of the manus and pes constitute the primitive condition for lizards. Among lizards exhibiting fully developed (non-reduced) limbs, only in the Gekkota is the reduction and absence of claws prevalent . This reduction and loss is always associated with taxa that possess a subdigital adhesive system (including lineages within these that have secondarily lost this system ). Claw reduction and loss has occurred independently at least ten times within the Gekkota (see Introduction). Furthermore, reduction and loss of claws is generally associated with elongation of the ungual phalanx. In the Gekko, Homopholis, Hemidactylus (Gekkonidae), and Pseudothecadactylus (Diplodactylidae) clades, this is restricted to digit I of the manus and pes. It has been argued that this is associated with a trade-off of functional capabilities of digit I , and that claw reduction/loss and the reconfiguration of the ungual phalanx is implicated in increasing the available subdigital adhesive pad area.
The function of the needle-like vestigial claws (especially on the pedal digits) that are retained in Tarentola and other taxa remains unknown, but their sexually dimorphic form (being more robust in females) suggests that they may be involved in the manipulation and placement of eggs at the time of laying (see above; [14,15]).
The mechanisms underlying claw reduction and loss in Tarentola annularis, as presented in this contribution, may underlie claw reduction in other lineages. This must remain tentative, however, because for mammals, it has been shown that different mechanisms yield ostensibly the same general pattern of digit loss . Differential claw and terminal phalanx expression among digits is seemingly related to the surface area of the subdigital adhesive pads that can be carried by particular digits and thus can be regarded as an evolutionary trade-off between functional demands. On certain digits, reduced claws may retain some but not all of their original functional roles; and in some taxa, this differentiation of roles is manifested in sexual dimorphism. Since Tarentola exhibits full expression, reduction, and complete loss of claws in the same individual, it constitutes an excellent vehicle for investigating differential developmental mechanisms that lead to evolutionary reduction and loss of structures.
Tarentola is unusual in that it retains fully developed claws only on digits III and IV of the manus and pes. Thus, digits I, II, and V likely benefit from enhanced adhesive pad area in association with the reduction or loss of the claws and the elongation of the ungual phalanges. The retention of fully developed claws on digits III and IV likely assists in attachment to the rocky surfaces typically occupied by members of this genus . More needs to be known about the functional role of the individual digits in various activities, including locomotion on various surfaces, and during egg deposition.
bone morphogenetic protein and activin membrane-bound inhibitor
bone morphogenetic protein
bovine serum albumin
di-n-butyl phthalate in xylene
fibroblast growth factor
homeobox transcription factor muscle segment homeobox 2
proliferating cell nuclear antigen
terminal deoxynucleotidyl transferase
terminal deoxynucleotidyl transferase dUTP nick end labeling
Thanks to Olda Zahradnicek for discussions. ER Khannoon is funded by Fayoum University. Anthony Russell receives financial support from the Natural sciences and Engineering Research Council of Canada (Discovery Grant A 9745) and the Faculty of Science, University of Calgary. AS Tucker is funded by the Wellcome Trust and Medical Research Council. We are grateful to two anonymous reviewers who were of great assistance in helping us to improve the content of this paper.
- Sanz-Ezquerro JJ, Tickle C. Fgf signalling controls the number of phalanges and tip formation in developing digits. Curr Biol. 2003;13:1830–6.View ArticlePubMedGoogle Scholar
- Casanova JC, Sanz-Ezquerro JJ. Digit morphogenesis: is the tip different? Dev Growth Differ. 2007;49:479–91.View ArticlePubMedGoogle Scholar
- Grotewold L, Plum M, Dildrop R, Peters T, Rüther U. Bambi is coexpressed with Bmp-4 during mouse embryogenesis. Mechan Develop. 2001;100:327–30.View ArticlePubMedGoogle Scholar
- Pikus M, Wong WP, Lin J, Wang X, Jiang TX, Chuong CM. Morpho-regulation of ectodermal organs: integument pathology and phenotypic variations in K14-Noggin engineered mice through modulation of bone morphogenetic protein pathway. Am J Pathol. 2004;164:1099–114.View ArticleGoogle Scholar
- Kimura S, Saitsu H, Schaumann BA, Shiota K, Matsumoto N, Ishibashi M. Rudimentary claws and pigmented nail-like structures on the distal tips of the digits of Wnt7a Mutant Mice: Wnt7a suppresses nail-like structure development in mice. Birth Defects Res A Clin Mol Teratol. 2010;88(6):487–96.View ArticlePubMedGoogle Scholar
- Cui CY, Klar J, Georgii-Heming P, Fröjmark AS, Baig SM, Schlessinger D, et al. Frizzled6 deficiency disrupts the differentiation process of nail development. J Invest Dermatol. 2013;133:1990–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Hamrich MW. Development and evolution of the mammalian limb: adaptive diversification of nails, hooves, and claws. Evol Dev. 2001;3:355–63.View ArticleGoogle Scholar
- Alibardi L. Development, comparative morphology and cornification of reptilian claws in relation to claws (sic) evolution in tetrapods. Zoology. 2009;78:25–42.Google Scholar
- Alibardi L. Microscopic analysis of lizard claw morphogenesis and hypothesis on its evolution. Acta Zool-Stockholm. 2008;89:169–88.View ArticleGoogle Scholar
- Alibardi L. Autoradiographic observations on developing and growing claws of reptiles. Acta Zool-Stockholm. 2010;91:233–41.View ArticleGoogle Scholar
- Russell AP, Bauer AM. The appendicular locomotor apparatus of Sphenodon and normal-limbed squamates. In: Gans C, Gaunt AS, Adler K, editors. Biology of the Reptila, Volume 21 Morphology I. Ithaca, New York: Society for the Study of Amphibians and Reptiles; 2008. p. 1–465.Google Scholar
- Crandell KE, Herrel A, Mahmood S, Losos JB, Autumn K. Stick or grip? Co-evolution of adhesive toepads and claws in Anolis lizards. Zoology. 2014;117:363–9.View ArticlePubMedGoogle Scholar
- Russell AP, Johnson MK. Between a rock and a soft place: microtopography of the locomotor substrate and the morphology of the setal fields of Namibian day geckos (Gekkota: Gekkonidae: Rhoptropus). Acta Zool-Stockholm. 2014;95:299–318.View ArticleGoogle Scholar
- Henkel FW, Schmidt W. Geckos: Biologie, Haltung, und Zucht. Stuttgart: Ulmer Verlag; 1991.Google Scholar
- Bauer AM. Geckos: the animal answer guide. Baltimore: The Johns Hopkins University Press; 2013.Google Scholar
- Gamble T, Greenbaum E, Jackman TR, Russell AP, Bauer AM. Repeated origin and loss of adhesive toe pads in geckos. PLoS One. 2012;7:e39429.View ArticlePubMed CentralPubMedGoogle Scholar
- Russell AP, Bauer AM. Digit I in pad-bearing gekkonine geckos; alternate designs and the potential constraints of phalangeal number. Mem Queensland Mus. 1990;29:453–72.Google Scholar
- Matalova E, Svandova E, Tucker AS. Apoptotic signaling in mouse odontogenesis. OMICS. 2012;16(1–2):60–70.View ArticlePubMed CentralPubMedGoogle Scholar
- Zahradnicek O, Horacek I, Tucker AS. Tooth development in a model reptile: functional and null generation teeth in the gecko Paroedura picta. J Anat. 2012;221(3):195–208.View ArticlePubMed CentralPubMedGoogle Scholar
- Cooper K, Sears KE, Uygur A, Maier J, Stephen-Backowski K, Brosnahan M, et al. Patterning and post-patterning modes of evolutionary digit loss in mammals. Nature. 2014;511:41–5.View ArticlePubMed CentralPubMedGoogle Scholar
- Peterkova R, Peterka M, Viriot L, Lesot H. Dentition development and budding morphogenesis. J Cran Genet Dev Bio. 2000;20:158–72.Google Scholar
- Peterkova R, Churava S, Lesot H, Rothova M, Prochazka J, Peterka M, et al. Revitalization of a diastemal tooth primordium in Spry2 null mice results from increased proliferation and decreased apoptosis. J ExpZool B. 2009;312:292–308.Google Scholar
- Shapiro MD, Hanken J, Rosenthal N. Developmental basis for evolutionary digit loss in the Australian lizard Hemiergis. J ExpZool B. 2003;297B:48–56.Google Scholar
- Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951;88:49–92.View ArticlePubMedGoogle Scholar
- Wise PAD, Vickaryous MK, Russell AP. An embryonic staging table for in ovo development of Eublepharis macularius, the Leopard Gecko. Anat Record. 2009;292:1198–212.View ArticleGoogle Scholar
- Bancroft JD, Gamble M. Theory and practice of histological techniques. 5th ed. Edinburgh: Churchill Livingstone; 2002.Google Scholar
- Gibson G. Active role of chondrocyte apoptosis in endochondral ossification. Microsc Res Techniq. 1988;43:191–204.View ArticleGoogle Scholar
- Matalova E, Tucker AS, Sharpe PT. Death in the life of a tooth. J Dent Res. 2004;83:11–6.View ArticlePubMedGoogle Scholar
- Yokouchi Y, Sakiyama J, Kameda T, Iba H, Suzuki A, Ueno N, et al. BMP-2/-4 mediate programmed cell death in chicken limb buds. Development. 1996;122:3725–34.PubMedGoogle Scholar
- Zou H, Niswander L. Requirement for BMP signaling in interdigital apoptosis and scale formation. Science. 1996;272:738–41.View ArticlePubMedGoogle Scholar
- Hurle JM, Garcia-Martinez V, Gañan Y, Climent V, Blasco M. Morphogenesis of the prehensile autopodium in the common chameleon (Chamaeleochamaeleo). J Morphol. 1987;194:187–94.View ArticleGoogle Scholar
- Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat Genet. 2000;24:391–5.View ArticlePubMedGoogle Scholar
- Tickle C. The early history of the polarizing region: from classic embryology to molecular biology. Int J Dev Biol. 2002;46:847–52.PubMedGoogle Scholar
- Zaias N, Alvarez J. The formation of the primate nail plate. An autoradiographic study in squirrel monkey. J Invest Dermatol. 1968;51:120–36.View ArticlePubMedGoogle Scholar
- Norton LA. Incorporation of thymidine-methyl-H3 and glycine- 2-H3 in the nail matrix and bed of humans. J Invest Dermatol. 1971;56:61–8.View ArticlePubMedGoogle Scholar
- Baden HP, Kvedar JC. The nail. In: Goldsmith LA, editor. Physiology, biochemistry and molecular biology of the skin, vol. 1. New York, NY: Oxford University Press; 1983. p. 697–711.Google Scholar
- 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. Heidelberg: Springer-Verlag; 1986. p. 293–312.View ArticleGoogle Scholar
- De Berker D, Angus B. Proliferative compartments in the normal nail unit. Brit J Dermatol. 1996;135:555–9.View ArticleGoogle Scholar
- Hamrich MW. Evolution and development of mammalian limb integumentary structures. J Exp Zool B. 2003;298:152–63.View ArticleGoogle Scholar
- Muller TL, Ngo-Muller V, Reginelli A, Taylor G, Anderson R, Muneoka K. Regeneration in higher vertebrates: limb buds and digit tips. Cell Dev Biol. 1999;10:405–13.View ArticleGoogle Scholar
- Higham TE, Birn-Jefferey A, Collins C, Hulsey CD, Russell AP. Adaptive simplification and the evolution of gecko locomotion: morphological and biomechanical consequences of losing adhesion. Proc Natl Acad Sci USA. 2015;112:809–14.View ArticlePubMedGoogle Scholar
- Loveridge A. Revision of the African lizards of the family Gekkonidae. Bull Mus Comp Zool Harvard College. 1947;98:1–469. + 7 plates.Google Scholar
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