The differentiation of the skin and its appendages. I. Normal development of papillary ridges

In the present study, the normal development of papillary ridges was studied in the volar pads of both fore and hindpaws of the opossum, Monodelphis domesticus. At birth, the developmental state of the opossum's paws is equivalent to that of a six-week human embryo. The development of papillary ridges in the opossum occurs entirely postnatally and the hindpaw lags behind the forepaw by at least four days in most developmental parameters. Papillary ridge formation is preceded by four events: skin innervation, Merkel cell differentiation, mesenchymal condensation, and epidermal proliferation. The apical pads at the tips of the digits and the interdigital pads between the heads of the metacarpals (or metatarsals) have a unique pattern of innervation and mesenchymal content as compared to the non-pad skin. Each pad is innervated by a prominent nerve trunk and axons ascend towards the epidermis providing a density of innervation that exceeds that in the non-pad epidermis. Merkel cells are absent in non-pad epidermis but present in the pads prior to the onset of formation of papillary ridges. A loose aggregation of mesenchyme forms the core of the pads and the superficial dermis is more cellular in the pads as compared to the equivalent dermis in surrounding non-pad skin. Developing papillary ridges always contained Merkel cell-axon complexes. Merkel cell axon complexes serve as the anatomical substrate of slowly adapting (SA) mechanoreceptors. The presence of these complexes during early skin differentiation is consistent with the use of the opossum's forepaw in climbing to the nipple, but also suggests other possible functions. We hypothesize that the nervous system might play a role in the timing or patterning of the formation of papillary ridges.     

The differentiation of the skin and its appendages. II. Altered development of papillary ridges following neuralectomy.

In order to test the hypothesis that the nervous system is an important determinant of skin differentiation, deletions of the left lumbosacral dorsal root ganglia (DRGs), the sources of cutaneous afferents to the left hindpaw, were performed on opossum pups at day 1 when hindpaws have just begun to be innervated. At birth, each lumbosacral DRG measures about 200 microns rostrocaudally and a deletion measuring 1 mm would span 4-5 DRGs. Following survival periods of 5-24 days, serial sections through the trunk documented partial left lumbosacral DRG deletion and a variable degree of spinal cord destruction. The blood supply to the trunk and hindpaws was preserved. Bilateral enlargement of residual DRGs was observed and regenerating skin at the site of the deletion was hyperplastic and hyperinnervated. The skin of the plantar pads of the hindpaws was studied following the neuralectomies. Statistically significant differences were observed between the left (experimental) and right (control) hindpaws. The density of innervation of the left hindpaw was reduced compared to the right hindpaw, development of papillary ridges was retarded by 3-4 days, and non-innervated Merkel cells were hypogranulated. This period of delay in ridge development is probably a reflection of the expansion of residual DRGs into the peripheral domains of deleted DRGs. The present study confirms a role for afferent nerves in the timing of cutaneous differentiation and a mutual trophic dependence between cutaneous nerves and Merkel cells in the epidermis.     

The development of receptors in the glabrous forepaw skin of pouch young opossums

To determine the state of development of mechanoreceptors during the period in which central neurol regions can be altered by peripheral lesions, the distribution of terminal neurites and specialized receptors was examined in the glabrous forepaw skin of a developmental series of pouch young opossums, using the silver stain of Sevier & Munger (1965) and electron microscopic observations. At 10 days after birth bundles of neurites approach the dermal-epidermal junction, but neither neurites nor receptors are seen in the epidermis. Some neuntes enter the epidermis by 20 days; by 25 days developing Merkel cells are seen in the epidermis; at 30 days mature Merkel cells and primordial Pacinian corpuscles are present, respectively, in the epidermis and deep dermis; at 42 postnatal days dermal papillae containing neurites were observed at the dermal-epidermal junction, which may be developing Meissner corpuscles. Numbers of neurites, and of their branchings, increase up to 60 days; as do the size, number and degree of differentiation of the Pacinian corpuscles.The critical period for the effects of peripheral lesions upon the morphology of the cuneate-gracile nuclear complex, as well as the times of initial massive synaptogenesis, cell migration and differentiation in this first and second order mechanosensory synaptic region, are completed prior to the innervation of the epidermis and the appearance of specialized mechanoreceptors. Thus input from epidermal and specialized receptors cannot be responsible for guidance of these developmental processes.     

The differentiation of the skin and its appendages. II. Altered development of papillary ridges following neuralectomy

In order to test the hypothesis that the nervous system is an important determinant of skin differentiation, deletions of the left lumbosacral dorsal root ganglia (DRGs), the sources of cutaneous afferents to the left hindpaw, were performed on opossum pups at day 1 when hindpaws have just begun to be innervated. At birth, each lumbosacral DRG measures about 200 μm rostrocaudally and a deletion measuring 1 mm would span 4–5 DRGs. Following survival periods of 5–24 days, serial sections through the trunk documented partial left lumbosacral DRG deletion and a variable degree of spinal cord destruction. The blood supply to the trunk and hindpaws was preserved. Bilateral enlargement of residual DRGs was observed and regenerating skin at the site of the deletion was hyperplastic and hyperinnervated. The skin of the plantar pads of the hindpaws was studied following the neuralectomies. Statistically significant differences were observed between the left (experimental) and right (control) hindpaws. The density of innervation of the left hindpaw was reduced compared to the right hindpaw, development of papillary ridges was retarded by 3–4 days, and non-innervated Merkel cells were hypogranulated. This period of delay in ridge development is probably a reflection of the expansion of residual DRGs into the peripheral domains of deleted DRGs. The present study confirms a role for afferent nerves in the timing of cutaneous differentiation and a mutual trophic dependence between cutaneous nerves and Merkel cells in the epidermis.     

Morphological diversity in digital skin microstructure of didelphid marsupials

The purpose of this study was to investigate how didelphid marsupials have diversified in morphology of their claws and digital pads as they evolved different foraging preferences such as terrestrial, aquatic, and arboreal feeding behaviours. Both arboreal and more terrestrial didelphids have papillary ridges on the digital pads of the fore and hindfoot. In contrast, the papillary ridges on the pedal digital skin of the water opossum Chironectes minimus have been replaced by nonoverlapping, thickened epidermal scales. Chironectes also differs from the other didelphids studied in having finger tips with reduced claws and digital pads that are covered with raised epidermal scales having projecting, finger-like cones arranged radially around the perimeter of each scale. The reduced claws and unusual digit skin microstructure of Chironectes likely improve this animal's ability to recognise and identify live animal prey under water using only its sense of touch.     

Neural modulation of cutaneous differentiation: epidermal hyperplasia and precocious hair development following partial neuralectomy in opossum pups

The original intent of the present study was to evaluate the compensatory response of the nervous system to areas of denervation. A portion of the spinal cord in the lumbosacral region of one-day opossum pups (Monodelphis domesticus) was removed by cauterization. This partial neuralectomy produced an expected compensatory response of neurons in the dorsal root ganglia, but in addition produced unexpected abnormalities of cutaneous differentiation. At 4-6 days following surgery, an increase in the thickness of the epidermis resembling glabrous palmar or plantar skin was seen. This hyperplastic epidermis appeared to be associated with an abnormally dense innervation of the dermis and epidermis. Eight days following partial neuralectomy most animals showed areas of precocious hair development. Nerve fibers were always seen in the dermis associated with these precocious hairs and were seen to penetrate the basal lamina in the region of the epidermal-hair shaft boundary. These results imply a critical role for afferent nerves in the normal development of the skin and its appendages.     

The sensory innervation of the rat rhinarium

The present study documents the characteristics of innervation of the rhinarium or hairless rat snout skin by light and electron microscopy. The outer glabrous surface is covered with a stratified squamous epithelium that forms both rete pegs and rete ridges, the latter on the inferior border near the philtrum. The glabrous skin contains numerous presumptive epidermal and dermal free nerve endings (FNE's), Merkel terminals at the base of the rete ridges and pegs, and simple, nonencapsulated corpuscles. A second region of dense innervation, found on an elevation of the inner wall of the vestibule, contains similar components of innervation, with the exception that no Merkel terminals were identified. Since no Merkel terminals were present in this area of the vestibule, intraepidermal as well as dermal FNE's could be identified with certainty. This skin is covered by a thin squamous epithelium overlying dense connective tissue. The simple corpuscles are similar to those in the rhinarium, as well as resembling those described in other species. FNE's were frequently observed intimately associated with simple corpuscles. Several examples of large FNE's with two to three layers of cytoplasmic lamellae were found, suggestive of transitional forms between FNE's and simple corpuscles. Thus, the pattern of sensory innervation in the glabrous rat snout skin is similar to that found in other furred species described to date, but in addition, the sensory innervation of ridged skin in the rat also resembles that of epidermis organized into rete pegs. This dense sensory innervation may be correlated with whisking behavior of the predominantly nocturnal rat.     

Specializations of somatosensory innervation in the skin of humpback whales (Megaptera novaeangliae)

Cetacean behavior and life history imply a role for somatosensory detection of critical signals unique to their marine environment. As the sensory anatomy of cetacean glabrous skin has not been fully explored, skin biopsy samples of the flank skin of humpback whales were prepared for general histological and immunohistochemical (IHC) analyses of innervation in this study. Histology revealed an exceptionally thick epidermis interdigitated by numerous, closely spaced long, thin diameter penicillate dermal papillae (PDP). The dermis had a stratified organization including a deep neural plexus (DNP) stratum intermingled with small arteries that was the source of intermingled nerves and arterioles forming a more superficial subepidermal neural plexus (SNP) stratum. The patterns of nerves branching through the DNP and SNP that distribute extensive innervation to arteries and arterioles and to the upper dermis and PDP provide a dense innervation associated through the whole epidermis. Some NF-H+ fibers terminated at the base of the epidermis and as encapsulated endings in dermal papillae similar to Merkel innervation and encapsulated endings seen in terrestrial mammals. However, unlike in all mammalian species assessed to date, an unusual acellular gap was present between the perineural sheaths and the central core of axons in all the cutaneous nerves perhaps as mechanism to prevent high hydrostatic pressure from compressing and interfering with axonal conductance. Altogether the whale skin has an exceptionally dense low-threshold mechanosensory system innervation most likely adapted for sensing hydrodynamic stimuli, as well as nerves that can likely withstand high pressure experienced during deep dives.     

Avian scale development. XVII: The epidermis of the scaleless (sc/sc) anterior metatarsal skin is determined, but the dermis lacks permissive cues for the patterned expression of the determined state.

Embryos homozygous for the gene scaleless (sc/sc) completely lack scutate scales and the beta strata which characterize terminal differentiation of the scale ridges located on the anterior metatarsal region of the foot. Although the sc/sc epidermis cannot undergo scale morphogenesis, it can respond to the inductive dermal ridges of normal scutate scales by generating beta strata. Recently, we discovered that the anterior metatarsal epidermis of normal embryos becomes committed to the formation of beta strata prior to morphogenesis of definitive scale ridges. Here, we examined the possibility that the sc/sc anterior metatarsal epidermis also becomes determined, i.e., committed to scutate scale-specific terminal differentiation. Experimental tissue recombinants were used to assess the ability of the sc/sc epidermis to generate beta strata. The results show that the germinative cells of the 15-day sc/sc epidermis are committed to generating beta strata, even though they have not undergone scutate scale morphogenesis. Thus, the mechanisms involved in establishing epidermal determination must differ form those regulating scale morphogenesis. In addition, we examined the formation of patterned, permissive cues in the anterior metatarsal and footpad dermises of sc/sc embryos. Analysis of recombinants showed that both the 15- and 20-day dermises from the sc/sc anterior metatarsal region fail to provide cues for beta stratum formation, when associated with the determined 15-day scutate scale epidermis. Likewise, the 15-day sc/sc footpad dermis cannot support beta stratum formation. However, 20-day sc/sc footpad dermis is able to support the generation of a few abnormally patterned beta strata, demonstrating that sc/sc dermis which has experienced even limited morphogenesis is able to provide permissive cues for the terminal differentiation of the scutate scale epidermis.     

Morphological and Histological Study on the Foot Pads of the Anatolian Bobcat (Lynx lynx)

This study was conducted to reveal the morphometric and morphological features of foot pads in the Anatolian bobcat (Lynx lynx). To achieve this objective, dissection, histological, and radiography techniques were applied to two dead materials obtained from the Republic of Turkey Ministry of Forest and Water Works Sivas Branch Manager. Digit I is radiographically rudimentary in the forepaw. The paws of the forelimb of the Anatolian bobcat have a carpal, a metacarpal, and four digital pads, while the hind feet have a metatarsal pad and four digital pads. The metacarpal pad is cone‐like, while the metatarsal pad resembles a butterfly. The digital pads in the paws of the forelimb are longer and thinner than in the paws in the hind feet. The paws in both feet are situated as binary. Through histological examination, it was determined that the skin of the foot pads consists of epidermis, dermis, and pad cushion. The epidermis is subdivided into basal, spinous, granular, lucidum, and corneum layers. The dermis of each pad consists of papillar and reticular strata containing sweat glands, elastin, collagen, and reticular fibers. Anat Rec, 301:932–938, 2018. © 2017 Wiley Periodicals, Inc.     

Three-dimensional analyses of touch domes in the hairy skin of the cat paw reveal morphological substrates for complex sensory processing

The three-dimensional morphology of the innervation of touch domes in the hairy skin folds of cat forepaws was investigated by the confocal laser scanning microscopic analyses of sections stained immunocytochemically with primary antibodies for protein gene product 9.5, neurofilament 200 and cytokeratin 20 in combination with transmission electron microscopic observations. One square centimeter of interdigital skin can contain as many as 68 touch domes. Each touch dome can have up to 150 Merkel cells and all are innervated by a single large-caliber afferent myelinated nerve fiber at the level of the palisade endings around the guard hair. It gives rise to multiple long, myelinated branches. Each final myelinated branch gives rise to several short and fine unmyelinated branches, supplying approximately 15 Merkel cell–axon complexes. Each Merkel cell is typically contacted by multiple small discoid endings instead of by a large single one. Discoid endings on separate Merkel cells were usually the distal ends of the unmyelinated branches, although, some were en-passant swellings of the branches. Only a few Merkel cell–axon complexes at the marginal zone of each territory could also be supplied by adjacent final myelinated branches. Each Merkel cell is surrounded by protrusions of keratinocytes that are penetrated by several collagen bundles of the dermis. This intricate pattern of innervation may explain the unique irregular discharges of action potentials typical for this type of mechanoreceptor.     

Ultrastructure of Merkel corpuscles and so-called “transitional” cells in the white leghorn chicken

In the chicken Merkel corpuscles are located in the dermis and consist of specialized Merkel cells, discoid nerve endings and lamellar cells. Merkel cells contain characteristic membrane-bound dense-core granules and bundles of microfilaments. Asymmetric junctions, synapse like, with thickened membranes and clusters of dense-core vesicles were observed between the Merkel cells and the nerve endings. The nerve ending is derived from myelinated nerves and sometimes contains clusters of clear vesicles. A laminar system formed by lamellar cells of the Schwann cell type encloses the Merkel cells and the nerve endings. So called “transitional” cells, showing some of the morphological features of both keratinocytes and Merkel cells, were observed in the basal layer of the epidermis. One was located partly in the epidermis and partly in the dermis. The structure of Merkel corpuscles is compared with that of Merkel cells in other tetrapods. The developmental significance of “transitional” cells and the origin of Merkel cells are discussed.     

Human Merkel cell regeneration in skin derived from cultured keratinocyte grafts.

Human Merkel cell regeneration in epidermis derived from cultured keratinocyte autografts was studied from 6 days to 6 years after transplantation. Cultured keratinocyte sheets derived from skin of the sole, axilla, groin, or scalp were transplanted to full-thickness wounds in 20 pediatric patients treated for massive burns or giant congenital nevi. Normal age- and site-matched skin as well as meshed split-thickness autografts from the same patients served as controls. Merkel cells were identified by immunohistochemistry using antibodies to cytokeratins #8 and #18. Cultured keratinocytes in vitro expressed no neuroendocrine markers, but nonspecific, simple-epithelial cytokeratin expression was observed in about 20% of cells. After transplantation, Merkel cells were identified only in cultured grafts derived from sole skin and appeared in the epidermis as early as 21 days postgrafting. Dermal Merkel cells were rarely observed, but their appearance invariably succeeded that of intraepidermal Merkel cells. Regenerated Merkel cells were never innervated, and their emergence was unrelated either spatially or temporally to epidermal reinnervation. In skin bridges of meshed split-thickness grafts, Merkel cells survived after degeneration of associated neurites, but no Merkel cells appeared within re-epithelialized interstices. Among the neuroendocrine markers tested, Merkel cells in cultured grafts, meshed skin grafts or normal pediatric skin expressed only neuron-specific enolase. They failed to stain for calcitonin, chromogranin A, Leu-7, synaptophysin, bombesin, or vasoactive intestinal polypeptide by immunohistochemistry. These findings suggest that: (a) Merkel cells derive from keratinocyte precursors which undergo neuroendocrine differentiation in the epidermis; (b) that keratinocyte stem cells are capable of undergoing Merkel cell differentiation postnatally; (c) that postnatal Merkel cell differentiation may be body-site dependent; and (d) that Merkel cell development and maintenance is independent of neural induction.     

Benign melanocytic lesions. A morphometric analysis

Nuclei of melanocytes in 10 lentigines simplices, 10 junctional, 10 compound and 10 intradermal acquired melanocytic nevi were morphometrically evaluated, at three different skin levels: epidermis, papillary and reticular dermis. The considered parameters were: area, perimeter, form Ar, and form Ell. Mean values of nuclear area and form Ell demonstrated a progressive increase from lentigines to junctional to compound to dermal nevi. This trend was also observed for each considered level. In compound nevi mean values of nuclear area showed an increase from the epidermis to the papillary dermis. In compound and intradermal nevi they showed a decrease from the papillary to the reticular dermis. Mean values of nuclear form Ell presented a continuous increase from the epidermis to the papillary and reticular dermis. Our study shows that, in the evolution of the considered melanocytic lesions (from lentigo to dermal nevi), the nuclei of melanocytes become larger and rounder. In compound nevi, epidermal nuclei were smaller and more elongated than those of the papillary dermis. In compound and intradermal nevi, superficial dermal nuclei appeared larger but less round than the deep ones. These peculiar findings have been generally referred to as a melanocytic maturation process. They are also probably due to other factors, perhaps local factors, because a similar trend is observed in some malignant melanomas.     

Use of mesenchymal stem cells for cutaneous repair and skin substitute elaboration

Human mesenchymal stem cells (MSCs) are a heterogeneous population of fibroblast-like cells, which are present in different locations, including bone marrow, adipose tissue, extra-foetal tissues, gingiva and dermis. MSCs, which present multipotency capacities, important expansive potential and immunotolerance properties, remain an attractive tool for tissue repair and regenerative medicine. Currently, several studies and clinical trials highlight the use of MSCs in cutaneous repair underlining that their effects are essentially due to the numerous factors that they release. MSCs are also used in skin substitute development. In this study, we will first discuss the different sources of MSCs actually available. We will then present results showing that bone marrow-derived MSCs prepared according to Good Manufacturing Practices and included in a dermal equivalent are able to promote appropriate epidermis growth and differentiation. These data demonstrate that bone marrow-derived MSCs represent a satisfactory alternative to dermal fibroblasts in order to develop skin substitute. In addition, MSCs could provide a useful alternative to sustain epidermis development and to promote wound healing.     

Covering the limb--formation of the integument

An organism's outermost covering, the integument, has evolved to fulfil a diverse range of functions. Skin provides a physical barrier, an environment for immunological surveillance, and also performs a range of sensory, thermoregulatory and biosynthetic functions. Examination of the skin of limb digits reveals a range of skin types including the thickened hairless epidermis of the toe pads (palmar or plantar epidermis) and thinner epidermis between the hair follicles (interfollicular epidermis) of hairy skin. An important developmental function of skin is to give rise to a diverse group of appendages including hair follicles, with associated sebaceous glands (or feathers and scales in chick), eccrine sweat glands and the nail. A key question is how does this morphological variety arise from the single‐layered epithelium covering embryonic limb buds? This review will attempt to address this question by linking the extensive morphological/anatomical data on maturation of epidermis and its appendages with (1) current research into the range, plasticity and location of the putative epidermal stems cells; (2) molecular/microenvironmental regulation of epidermal stem cell lineages and lineage choice; and (3) regulation of the differentiation pathways, focusing on differentiation of the interfollicular epidermis.     

The differentiation of repeated epilation (Er/Er) mouse mutant skin in organ culture and in grafts

Summary The homozygous repeated epilation (Er/Er) mouse mutant dies at birth and shows a variety of malformations, one of which is a skin defect.The developmental abilities of skin fragments from these Er/Er mouse embryos were studied in organ culture and in grafts performed either under the renal capsule of young mouse hosts or under the skin of mouse fetuses.In organ culture, the skin fragments differentiated in accordance with their genetic origin. The most characteristic feature was the abnormally thickened spinous layer and the formation of numerous epidermal nodules in Er/Er skin pieces removed from 13- to 16-day-old embryos, and cultured for 4 to 6 days, whereas the normal skin showed a constant layered organization. As in normal skin, keratin fibers developed within 4 to 6 days of culture. However, in contrast to normal skin, where keratin sheaths developed all over the surface of the epidermis, the Er/Er skin exhibited keratin masses inside the nodules.Combinations of mutant Er/Er epidermis with normal dermis resulted in abnormal skin differentiation, with formation of nodules similar to those observed for unseparated Er/Er skin fragments, whereas the reciprocal combination (normal epidermis with Er/Er dermis) produced normal skin differentiation. Cornified layers developed in both types of explants.Grafts of Er/Er and, for comparison, of normal skin fragments under the renal capsule or under a fetus's skin showed that the development of Er/Er skin in a normal or Er/+ host was similar to that of a normal skin. Thus, if isolated from the mutant organism, and inserted into a normal environment, the skin recovered within 6 to 7 days after transplantation and then developed normally.Our experiments suggest that the abnormal skin development of the Er/Er mutant might be caused by environmental influences.     

Can hematopoietic stem cells be an alternative source for skin regeneration?

In the past few years, the plasticity of adult cells in several post-natal tissues has attracted special attention in regenerative medicine. Skin is the largest organ in the body. Adult skin consists of epidermis, dermis and appendages such as hair and glands that are linked to the epidermis but project deep into the dermal layer. Stem cell biology of skin has been a focus of increasing interest in current life science. Committed stem cells with a limited differentiation potential for regeneration and repair of epidermis have been known for decades. Recent studies further found that adult skin tissues contain cell populations with pluripotent characteristics. Multipotent stem cells from skin with and without hair follicles, both in epidermal and dermal tissues, can differentiate and generate multiple cell lineages. Especially, the hematopoietic system in epidermal and dermal tissue, like skin, may be a local, acceptable reservoir of various adult stem cell populations. Given their easy accessibility, such stem cells can provide an experimental model not only for skin biology but also for studying the epithelial–mesenchymal cell interactions of organs other than the skin. This review presents an overview of recent advances in research into skin repair and regeneration involving stem cells from epidermis, dermis, and bone marrow. In particular, we focus on the possible use of blood stem cells as an alternative resource for research advances in skin biology.     

The skin of the whale (Balaenoptera physalus)

Skin specimens were obtained from every representative region of the body of an adult Finback whale (Balaenoptera physalus) and examined by means of various histochemical and histological techniques. The following characteristic features were found: The epidermis is exceedingly thick over the general body surfaces and varies from a maximum of 3.0 mm over the ventral surface to 2.5 mm on the back. The complex understructure of the epidermis has rete ridges oriented to the craniocaudad body axis. The papillary layer of the dermis has long and pointed papillae which are wedged into the epidermis. The sensory cutaneous nerve endings demonstrated by silver impregnation and cholinesterase consist predominantly of small Vater-Pacini corpuscles situated in the higher level of the dermis. The intricate blood capillary network, positive for alkaline phosphatase is encased in the dermal papillae. There are no hair follicles, sebaceous and sweat glands.