Developmental stages of the climbing gecko Tarentola annularis with special reference to the claws, pad lamellae, and subdigital setae.

Studying the in ovo mode of development of squamates has the advantage of allowing easy access to embryos without surgically compromising gravid females. Despite the non-ophidian squamates being a very diverse lineage of reptiles, embryonic tables for individuals of this group are very few. Here, I present the first in ovo embryonic table for a basal multi-scansored, pad-bearing gecko, Tarentola annularis. In this gecko, only the III and IV digits bear claws. Eleven embryonic stages are described based on chronological development of morphological characteristics. In contrast to other previously studied geckos, this species exhibits a longer incubation period. Comparison with other squamates, embryonic development of T. annularis is an indicative of a conserved developmental strategy. Interestingly, the clawless digits of this gecko do exhibit claws during the first half of embryonic development. Thus, regression of claws in these digits could be an advantage of studying this particular taxon, as it raises the question, to be answered in future study, of which mechanisms could be responsible for such claw regression. Before hatching, the outer periderm layer sloughs revealing the functional setae. The present study provides not only a model for pentadactyl limbs and digit development, but also an example of a unique developmental phenomenon, as represented by claw regression.

The Trichohyalin-Like Protein Scaffoldin Is Expressed in the Multilayered Periderm during Development of Avian Beak and Egg Tooth.

Scaffoldin, an S100 fused-type protein (SFTP) with high amino acid sequence similarity to the mammalian hair follicle protein trichohyalin, has been identified in reptiles and birds, but its functions are not yet fully understood. Here, we investigated the expression pattern of scaffoldin and cornulin, a related SFTP, in the developing beaks of birds. We determined the mRNA levels of both SFTPs by reverse transcription polymerase chain reaction (RT-PCR) in the beak and other ectodermal tissues of chicken (Gallus gallus) and quail (Coturnix japonica) embryos. Immunohistochemical staining was performed to localize scaffoldin in tissues. Scaffoldin and cornulin were expressed in the beak and, at lower levels, in other embryonic tissues of both chickens and quails. Immunohistochemistry revealed scaffoldin in the peridermal compartment of the egg tooth, a transitory cornified protuberance (caruncle) on the upper beak which breaks the eggshell during hatching. Furthermore, scaffoldin marked a multilayered peridermal structure on the lower beak. The results of this study suggest that scaffoldin plays an evolutionarily conserved role in the development of the avian beak with a particular function in the morphogenesis of the egg tooth.

Evaluation of changes in architecture of the stratum internum of the hoof wall from fetal, newborn, and yearling horses.

OBJECTIVE: To evaluate morphologic changes of the stratum internum of hooves from near-term fetal, newborn, and yearling horses. ANIMALS: Feet from 27 near-term equine fetuses, 19 newborn foals, and 8 yearlings. PROCEDURES: Primary epidermal laminae (PEL) of the stratum internum were examined for evidence of architectural changes. RESULTS: In near-term fetuses, the PEL had a homogeneous appearance and symmetric distribution around the hoof wall with no significant differences in PEL density between the toe and quarters. However after birth, branched laminae at the toe formed within the first few weeks, which significantly increased PEL density at the toe, compared with the quarters. In yearlings, morphology of the PEL differed from that in younger foals and the PEL density was significantly greater at the toe than the quarters. The PEL density at the toe and medial and lateral quarters was significantly different from each other, as these PEL densities appeared to have been associated with conformation. No significant differences in PEL densities between forefeet and hind feet were detected in any group. CONCLUSIONS AND CLINICAL RELEVANCE: Findings indicate that the stratum internum of the inner hoof wall undergoes several morphologic changes shortly after birth. The PEL become branched with a greater PEL density at the toe than the quarters. In an asymmetric foot, more PEL were associated with the sloping side than the steep side of the foot. Findings suggested that PEL growth may also occur by bifurcation as well as by mitosis from the coronet and that wall stress may be associated with increased PEL density.

Fetal development of the segment-specific papillary body in the equine hoof.

Fetal development of the unique papillary body and its localized peculiarities in the equine hoof are described based on the study of 51 fetuses, nine newborn foals, and five adult horses. The shape and dimensions of the dermal papillae and lamellae have a formative influence on the structure and physical quality of the corneous hoof capsule with its horn tubules and lamellae. The size and arrangement of these horn structures determine the mechanical quality of hoof horn. Proper horn quality is a prerequisite for the various functions of the hoof capsule, such as protecting the living dermis supporting the hoof capsule, shock absorption, and formation of the suspensory apparatus of the distal phalanx. Development of the segment-specific papillary body is initiated by the increasing mitotic activity of the epidermal cells invaginating the dermal surface, thus forming dermal microridges. These microridges are transformed into single dermal papillae, which are arranged in rows, or enlarged to become primary and secondary dermal lamellae. The formation of a segment-specific papillary body enables the increasing keratinization ratio in the hoof epidermis and the formation of the characteristic tubular and lamellar horn responsible for the special mechanical properties of hoof horn.

Ultrasonographic characterization of ovarian events and fetal gestational parameters in two southern black rhinoceros (Diceros bicornis minor) and correlation to fecal progesterone.

A tremendous potential exists for the application of transrectal ultrasonography as a tool to enhance the captive management of endangered species. Reproductive study of two southern black rhinoceros (Diceros bicornis minor) females was performed daily to every other day for a approximately 60 day period to document ovarian changes, and three times weekly in early pregnancy to once monthly in late pregnancy in order to characterize changes in fetal parameters throughout gestation. All ovarian and fetal anatomical structures were measured in millimeters. The mean (+/- SD) length of the estrous cycle or interovulatory period was 26 +/- 1.4 days (n=2 cycles). Follicular growth rate of a dominant follicle was approximately 3 mm/day once the follicle reached 35 mm in diameter. Ovulation was observed to occur at a mean (+/- SD) follicular diameter of 49.5 +/- 2.6 mm (n=4) and within 48 to 72 h after observed estrus (n=2). Large ovarian structures [mean (+/- SD) diameter of 71.7 +/- 2.9 mm; n=3], considered analogous to equine anovulatory hemorrhagic follicles, were observed to form in the winter months and suggest seasonal periods of reduced fertility. Fecal progesterone assays confirmed ultrasonographic events. Although preliminary, the results of fetal sexing are presented and compared to the horse. Our data indicate that fetal eye or fetal foot diameter measurements can be used to accurately predict gestational age from about 2 months to term, providing useful information to managers of both captive and wild rhino populations. The ability to identify and quickly release animals in late term pregnancy in the wild and thereby reduce-abortions and neonatal mortalities in holding bomas is one potential practical conservation benefit of the fetal age predictive models.

[Development of hoof cartilage with special considerations of its ossification]. / Zur Entwicklung des Hufknorpels unter besonderer Berücksichtigung der Hufknorpelverknöcherung.

The pre- and perinatal development of the hoof cartilage is described concerning the histological structure and surrounding vessels. Beginning in the third month of fetal development, the anlage of the hoof cartilage is still present in typical shape and location. It is built out of mesenchymal connective tissue. During further fetal development, the connective tissue cells will differentiate into two cell populations, fibroblasts and chondroblasts. Vessels, traversing the hoof cartilage, are surrounded by loose connective tissue, which will partially develop fibrocartilage. At birth, hoof cartilage consists of hyaline cartilage, which is disrupted by vessels, embedded in fibrous connective tissue and/or fibrocartilage. The development of the hoof cartilage is the base to understand its structure and the predisposing locations for its ossification.

Frizzled6 deficiency disrupts the differentiation process of nail development.

Nails protect the soft tissue of the tips of digits. The molecular mechanism of nail (and claw) development is largely unknown, but we have recently identified a Wnt receptor gene, Frizzled6 (Fzd6), that is mutated in a human autosomal-recessive nail dysplasia. To investigate the action of Fzd6 in claw development at the molecular level, we compared gene expression profiles of digit tips of wild-type and Fzd6(-/-) mice, and showed that Fzd6 regulates the transcription of a striking number of epidermal differentiation-related genes. Sixty-three genes encoding keratins (Krts), keratin-associated proteins, and transglutaminases (Tgms) and their substrates were significantly downregulated in the knockout mice. Among them, four hard Krts, Krt86, Krt81, Krt34, and Krt31; two epithelial Krts, Krt6a and Krt6b; and Tgm 1 were already known to be involved in nail abnormalities when dysregulated. Immunohistochemical studies revealed decreased expression of Krt86, Krt6b, and involucrin in the epidermal portion of the claw field in the knockout embryos. We further showed that Dkk4, a Wnt antagonist, was significantly downregulated in Fzd6(-/-) mice along with Wnt, Bmp, and Hh family genes; and Dkk4 transgenic mice showed a subtly but appreciably modified claw phenotype. Thus, Fzd6-mediated Wnt signaling likely regulates the overall differentiation process of nail/claw formation.

Restorative regeneration of digital tips in the African clawed frog (Xenopus laevis daudin).

Localized, specialized structures are carried on the tips of digits of many amniote and certain non-amniote tetrapods. The pes of some members of Pipidae represents a rare example among tetrapods of differential expression of digital tip form (clawed vs. non-clawed digits). As a step towards understanding how such localized forms are generated and maintained, we conducted a series of amputation experiments and observed, through the process of regeneration, the potential for reconstitution, at the tissue and organ level, of the different digit tip morphologies. Results of this study reveal that immediately following metamorphosis specialized digit tip structures are regenerated with a high degree of structural and spatial accuracy by a process that essentially replicates normal development in recently metamorphosed Xenopus laevis froglets. Furthermore, this regenerative capacity is maintained in juveniles 4 months beyond metamorphosis, and also in adults of 1 year or more in age, indicating that metamorphosis-specific conditions do not exclusively facilitate regeneration of digit tips. In addition, regenerative capacity is maintained through repeated bouts of amputation and regeneration, indicating deep-seated digit identity and retention of the distinct digit tip developmental programs within the digits. Together, these data suggest that the developmental programing responsible for the formation of the discrete digital tip morphologies is located regionally within each digit, and that it is retained through time. Our results suggest that Xenopus can serve as a model organism for exploring the molecular underpinnings of digit tip formation because regeneration leads to morphologically identical structures to those of the original digit tips.

Claw development and cornification in the passeraceous bird zebrafinch (Taeniatopygia guttata castanotis).

The histogenesis and cornification of claws in zebrafinch embryos has been analyzed. At 10-12 days post-deposition, the epidermis at the tip of the toes forms placode-like anlage associated with a mesenchymal condensation and with a terminal phalange. Claws seem to be modified scales, the dorsal side of which becomes the unguis whereas a ventral scale is the origin of the sub-unguis. At 14-15 days, numerous keratinocytes form the unguis, the corneous layer of which becomes thicker than in the sub-unguis and accumulates beta-keratin and lipids. Keratin bundles are mainly directed toward the tip of the claw and have a prevalent parallel orientation. Unguis corneocytes are thicker and accumulate more beta-keratin than corneocytes of the sub-unguis. Mature corneocytes become partially fused in a compact corneous layer at 17-18 days, near hatching. During growth of the unguis, the embryonic epidermis and beta-keratin cells curve over the tip of the claw and localize in the ventral part of the claw, forming the claw pad. The latter is shed at hatching leaving the pointed claw made of harder corneous layers in the unguis side of the claw.

[The deciduous hoof capsule (Capsula ungulae decidua) of the equine fetus and newborn foal]. / Die hinfällige Hufkapsel (Capsula ungulae decidua) des Pferdefetus und neugeborenen Fohlens.

The term Eponychium is used to describe the deciduous hoof capsule in veterinary-medical and embryological literature. In other aspects of veterinary medicine, the term is generally reserved for the perioplic corium of the permanent hoof. In order to clarify this double usage, the structure and origin of the hoof epidermis from 10 equine fetus at different stages of development and 4 newborn foals were investigated and described using light microscopical techniques. Epidermal tubules and lamellae are already present in the non-cornified fetal hoof epidermis. These structures, along with the formation of a white line, allow this epidermis to be divided into the same segments as are commonly used when referring to the permanent hoof. The greatest part of the deciduous hoof epidermis consists of the sole and frog, with significant portions forming in the coronary corium and that of the hoof wall as well. The perioplic corium only makes up insignificant portions of the fetal hoof capsule, however. Between the second half of the gestation period and birth, this deciduous (primary) hoof capsule is continually being replaced from below by newly-forming, cornified permanent hoof capsule. On the basis of its structure and origin, we therefore suggest that the term "deciduous hoof capsule (Capsula ungulae decidua)" be used as a replacement for the word "Eponychium" when referring to the primary hoof epidermis.

Development and evolution of the mammalian limb: adaptive diversification of nails, hooves, and claws.

Paleontological evidence indicates that the evolutionary diversification of mammals early in the Cenozoic era was characterized by an adaptive radiation of distal limb structures. Likewise, neontological data show that morphological variation in distal limb integumentary appendages (e.g., nails, hooves, and claws) can be observed not only among distantly related mammalian taxa but also among closely related species within the same clade. Comparative analysis of nail, claw, and hoof morphogenesis reveals relatively subtle differences in mesenchymal and epithelial patterning underlying these adult differences in distal limb appendage morphology. Furthermore, studies of regulatory gene expression during vertebrate claw development demonstrate that many of the signaling molecules involved in patterning ectodermal derivatives such as teeth, hair, and feathers are also involved in organizing mammalian distal limb appendages. For example, Bmp4 signaling plays an important role during the recruitment of mesenchymal cells into the condensations forming the terminal phalanges, whereas Msx2 affects the length of nails and claws by suppressing proliferation of germinal epidermal cells. Evolutionary changes in the form of distal integumentary appendages may therefore result from changes in gene expression during formation of mesenchymal condensations (Bmp4, posterior Hox genes), induction of the claw fold and germinal matrix (shh), and/or proliferation of epidermal cells in the claw matrix (Msx1, Msx2). The prevalence of convergences and parallelisms in nail and claw structure among mammals underscores the existence of multiple morphogenetic pathways for evolutionary change in distal limb appendages.

Fetal development of the white line (Zona alba) of the equine hoof.

The fetal development of the white line (Zona alba) in the equine hoof is described. Its specific structure of lamellar and interlamellar horn, which in turn is composed of cap and terminal horn, is formed in the second half of the hoof's fetal development. In equine fetuses with a crown-rump length of less than 550 mm, the hoof capsule lacks a 'characteristic' white line since no borders between stratum medium, stratum internum and sole horn are discernible. In the hoof of an equine fetus with a crown-rump length of 550 mm, a narrow white line has taken shape. Its shallow lamellae are arranged like arcades. Between the horn lamellae lie the polyhedral cells of the interlamellar horn. Up until birth, the height of the horn lamellae and, therefore, the width of the white line increases significantly. In the white line of the hoof of newborn foals, the terminal horn contains horn tubules with a characteristic architecture.

On the development of the papillary body in the feline claw.

The pre- and post-natal development of the feline claw was studied in 22 feline fetuses with a crown-rump length (CRL) ranging from 40 to 160 mm, in six kittens up to an age of 22 days, and in four adult cats. In fetuses up to a CRL of 75 mm, the characteristic shape of the feline claw was developed. Segment-specific dermal modifications in the various segments, especially the dorsal ridge, started to develop in fetuses with a CRL between 75 and 105 mm. Modifications of the papillary body in the different claw segments took place in the last third of prenatal development and were continued postnatally. At first, the basal lamina became wavy, followed by the formation of small dermal microridges, which would be enlarged to dermal ridges and lamellae. In the claw of adult cats, the papillary body was very small. The dermal tissue of the proximal part of the coronet formed low microridges with short papillae originating on and between these low ridges. In the wall segment, dermal microridges were formed which were arranged in a parallel fashion, and in the distal part of the wall, short dermal micropapillae arose on the crest of each microridge. In the sole segment, thin dermal lamellae were developed. The sequence of papillary body development and the varying conformations of the papillary body in the different segments of the feline claw are compared to those in the nail, the canine claw and hooves.

Long-range, nonautonomous effects of activated Notch1 on tissue homeostasis in the nail.

Using an antibody directed against gamma-secretase-generated antigen unique to activated Notch1, we mapped Notch1 activation strictly to suprabasal cells in epidermis, nail matrix, and other skin appendages during normal development. The consequences of Notch1 activation in keratinizing nail cells were investigated in a transgenic mouse model. Ectopic activation of Notch1 in postmitotic cells within the nail keratogenous zone resulted in longer nails. BrdU labeling revealed an increased number of mitotic cells in transgenic nails. The matrix and keratogenous zone expanded distally due to the increase in cell numbers. The mitosis-promoting effects by a gene product expressed exclusively in postmitotic cells indicates a long-range effect of transgenic Notch1 on regulation of nail homeostasis. We demonstrate that activation of Notch1 in the keratogenous zone resulted in ectopic activation of Wnt signaling, the first such evidence in vertebrates. However, we detected little or no beta-catenin activation in proliferating matrix cells, indicating that Wnt is at most an indirect mediator of Notch-induced proliferation. These data support the existence of a novel, cell-nonautonomous role for Notch in maintaining homeostasis of stratified squamous epithelia by indirectly promoting mitosis in basally located cells.

Horse hooves and bird feathers: Two model systems for studying the structure and development of highly adapted integumentary accessory organs--the role of the dermo-epidermal interface for the micro-architecture of complex epidermal structures.

Accessory organs of the integument are locally modified parts of the potentially feather-bearing skin in birds (e.g., the rhamphotheca, claws, or scales), and of the potentially hairy skin in mammals (e.g., the rhinarium, nails, claws, or hooves). These special parts of the integument are characterised by a modified structure of their epidermal, dermal and subcutaneous layers. The developmental processes of these various integumentary structures in birds and mammals show both similarities and differences. For example, the development of the specialised epidermal structures of both feathers and the hoof capsule is influenced by the local three-dimensional configuration of the dermis. However, in feathers, in contrast to hooves, the arrangement of the corneous cells is only partially a direct result of the particular arrangement and shape of the dermal surface of the papillary body. Whereas the diameter of the feather papilla, as well as the number, length, and width of dermal ridges on the surface of the feather papilla influence the three-dimensional architecture of the feather rami, there is no apparent direct correlation between the dermo-epidermal interface and the development of the highly ordered architecture of the radii and hamuli in the feather vane. In order to elucidate this morphogenic problem and the problem of locally different processes of keratinisation and cornification, the structure and development of feathers in birds are compared to those of the hoof capsule in horses. The equine hoof is the most complex mammalian integumentary structure, which is determined directly by the dermal surface of the papillary body. Perspectives for further research on the development of modified integumentary structures, such as the role of the dermal microangioarchitecture and the selective adhesion and various differentiation pathways of epidermal cells, are discussed.