Multiple mechanisms contribute to the avoidance of avian epidermis by sensory axons

In birds, sensory innervation of skin is restricted to dermis, with few axons penetrating into the epidermis. This pattern of innervation is maintained in vitro, where sensory neurites avoid explants of epidermis but grow readily on dermis. We have used this coculture paradigm to investigate the mechanisms that impede innervation of avian epidermis. The lack of epidermal innervation in birds has been attributed to diffusible chondroitin sulfate proteoglycans (CSPGs) secreted by the epidermis, although direct experimental evidence is weak. We found that elimination of CSPG function with either chondroitinase or neutralizing antibodies did not promote growth of DRG neurites onto epidermis in vitro, indicating that CSPGs alone are not responsible for preventing epidermal innervation. Moreover, the failure of sensory neurites to invade epidermis is not due exclusively to soluble chemorepulsive factors, since sensory neurites also avoid dead epidermis. This inhibition can be overridden, however, by coating epidermis with the growth-promoting molecule laminin, but only if the tissue is killed first. Epidermal innervation of laminin-coated epidermis is even more robust when CSPGs are also eliminated. Thus, the absence of growth-promoting or permissive molecules, such as laminin, may contribute to the failure of sensory neurites to invade avian epidermis. Together these results show that the inhibitory character of avian epidermis is complex. Cell- or matrix-associated CSPGs clearly contribute to the inhibition, but are not solely responsible.     

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using themonoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.     

Quail-duck chimeras reveal spatiotemporal plasticity in molecular and histogenic programs of cranial feather development

The avian feather complex represents a vivid example of how a developmental module composed of highly integrated molecular and histogenic programs can become rapidly elaborated during the course of evolution. Mechanisms that facilitate this evolutionary diversification may involve the maintenance of plasticity in developmental processes that underlie feather morphogenesis. Feathers arise as discrete buds of mesenchyme and epithelium, which are two embryonic tissues that respectively form dermis and epidermis of the integument. Epithelial-mesenchymal signaling interactions generate feather buds that are neatly arrayed in space and time. The dermis provides spatiotemporal patterning information to the epidermis but precise cellular and molecular mechanisms for generating species-specific differences in feather pattern remain obscure. In the present study, we exploit the quail-duck chimeric system to test the extent to which the dermis regulates the expression of genes required for feather development. Quail and duck have distinct feather patterns and divergent growth rates, and we exchange pre-migratory neural crest cells destined to form the craniofacial dermis between them. We find that donor dermis induces host epidermis to form feather buds according to the spatial pattern and timetable of the donor species by altering the expression of members and targets of the Bone Morphogenetic Protein, Sonic Hedgehog and Delta/Notch pathways. Overall, we demonstrate that there is a great deal of spatiotemporal plasticity inherent in the molecular and histogenic programs of feather development, a property that may have played a generative and regulatory role throughout the evolution of birds.     

Ephrin-A5 inhibits growth of embryonic sensory neurons

EphA-ephrin signaling has recently been implicated in the establishment of motor innervation patterns, in particular in determining whether motor axons project into dorsal versus ventral nerve trunks in the limb. We investigated whether sensory axons, which grow out together with and can be guided by motor axons, are also influenced by Eph-ephrin signaling. We show that multiple EphA receptors are expressed in DRGs when limb innervation is being established, and EphA receptors are present on growth cones of both NGF-dependent (predominantly cutaneous) and NT3-dependent (predominantly proprioceptive) afferents. Both soluble and membrane-attached ephrin-A5 inhibited growth of approximately half of each population of sensory axons in vitro. On average, growth cones that collapsed in response to soluble ephrin-A5 extended more slowly than those that did not, and ephrin-A5 significantly slowed the extension of NGF-dependent growth cones that did not collapse. Finally, we show that ectopic expression of ephrin-A5 in ovo reduced arborization of cutaneous axons in skin on the limb. Together these results suggest that sensory neurons respond directly to A-class ephrins in the limb. Thus, ephrins appear to pattern sensory axon growth in two ways-both directly, and indirectly via their inhibitory effects on neighboring motor axons.     

Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers

Skin morphogenesis occurs in successive stages. First, the skin forms distinct regions (macropatterning). Then skin appendages with particular shapes and sizes form within each region (micropatterning). Ectopic DKK expression inhibited dermis formation in feather tracts and individual buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract then were up-regulated in restricted regions following two modes: Wnt 1 and 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis; Wnt 5a and 11 intensified in the inter-tract region and interprimordia epidermis or dermis, respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordia size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated thinner buds. In cultured dermal fibroblasts, Wnt 1 and 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in forming the dermis, tracts, interbud spacing and the growth and shaping of individual buds.     

Development of "normal" dermatomes and somatotopic maps by "abnormal" populations of cutaneous neurons

During development, motor and sensory axons grow to peripheral targets with remarkable precision. Whereas much has been learned about the development of motoneuron connectivity, less is known about the regulation of cutaneous innervation. In adults, dorsal root ganglia (DRG) innervate characteristic skin regions, termed dermatomes, and their axons project somatotopically in the dorsal horn. Here, we have investigated whether cutaneous neurons are selectively matched with specific skin regions, and whether peripheral target skin influences the central connections of cutaneous neurons. To address these questions, we shifted limb buds rostrally in chick embryos prior to axon outgrowth, causing DRGs to innervate novel skin regions, and mapped the resulting dermatomes and central projections. Following limb shifts, cutaneous innervation arose from more rostral and from fewer DRGs than normal, but the overall dermatome pattern was preserved. Thus, DRGs parcel out innervation of skin in a consistent manner, with no indication of matching between skin and DRGs. Similarly, cutaneous nerves established a "normal" somatotopic map in the dorsal horn, but in more rostral segments than usual. Thus, the peripheral target skin may influence the pattern of CNS projections, but does not direct cutaneous axons to specific populations of neurons in the dorsal horn.     

Localization of unmyelinated axons in rat skin and mucocutaneous tissue utilizing the isolectin GS-I-B

The a-D-galactose specific isolectin I-B4 from Griffonia simplicifolia (GS-I-B4) labels CNS microglia and certain peripheral neurons, including a subpopulation of small dark, type B dorsal root ganglion cells, some post-ganglionic sympathetic axons, and nearly all peripheral gustatory axons. The innervation patterns of GS-I-B4 reactive sensory ganglion cells are unknown for many peripheral target tissues, including their probable primary target, the skin. The present study describes the distribution of GS-I-B4 reactive axons in hairy and glabrous hindpaw skin and in the glans penis of rats, using both single and double-labelling histochemical techniques. Neuronal processes were identified using (1) histochemistry with horseradish peroxidase conjugated GS-I-B4 or (2) immunohistochemistry against PGP 9.5 to identify all axons, and biotinylated lectin histochemistry with avidin-FITC to identify the subpopulation of GS-I-B4 reactive axons. GS-I-B4 strongly labelled unmyelinated cutaneous sensory afferents, as well as some sympathetic efferents and visceral afferents. Lectin reactive axons were seen to innervate the upper hair shaft epidermis in hairy skin, and were abundant in the shallow dermis in hairy and glabrous skin and glans penis. Lectin reactive axons were also abundant in the lamina propria and distal urethral epithelium of the penis. These results provide new evidence for the cutaneous sensory role of GS-I-B4 reactive primary afferents, as well as evidence to support the contention that the lectin is a specific marker for a subpopulation of unmyelinated axons and not simply a marker for the myelination state of an axon.     

Expression of multiple delta-protocadherins during feather bud formation

The expression of the chicken delta-protocadherin (Pcdh) subfamily was investigated in the developing feather buds of the chicken. The expression profiles of the eight investigated Pcdhs in the cells and tissues of the feather buds differ from each other. Pcdh1, Pcdh7, Pcdh8 and Pcdh10 are differentially expressed in the epidermis of the feather bud. Expression of Pcdh1 and Pcdh10 is restricted to the periderm and Pcdh17 expression to the epidermis of interbud region. Pcdh19 is mostly expressed at the anterior side of epidermis as well as in the blood vessels of the feather buds. Furthermore, Pcdh9 and Pcdh18 both are regionally expressed in the dermis of the feather bud. These results suggest that Pcdhs may play a variety of roles during avian feather bud formation.     

THE INTRAEPIDERMAL INNERVATION OF THE SNOUT SKIN OF THE OPOSSUM : A Light and Electron Microscope Study, with Observations on the Nature of Merkel's Tastzellen

The intraepidermal innervation of the snout skin of the opossum has been studied with the light and electron microscope. Numerous large nerve fibers loose their myelin sheath in the superficial dermis and pass into the epidermis. The basement membranes of the epidermis and Schwann cell become continuous at the point of entry of the neurite into the epidermis. Within the epidermis, the neurite is associated with a specialized secretory epidermal cell, termed a Merkel cell. This cell has many secretory granules apposed to the neurite. The Merkel cells are epidermal cells since they have desmosomes between them and adjacent epidermal cells. The neurite in the stratum spinosum is enveloped by Schwann cells in a manner analogous to the Schwann cell investment of unmyelinated neurites. In the upper stratum spinosum the nerve fiber evidences changes which can be interpreted as degenerative. The Merkel cell-neurite complex is interpreted as representing a sensory receptor unit.     

Naturally occurring wounds and wound healing in chick embryo wings

Summary Spontaneous cutaneous wounds occur in avian embryos (chick, duck, quail) in various prominent parts of the body, notably the elbow, the knee and the outer face of feather buds. The frequency and size and the light and electron microscopic morphology of elbow wounds in the chick embryo are described. The cutaneous lesion appears in over 80% of the embryos at around 7 days of incubation, persists through 14 days, and finally heals completely at around 16 days of incubation. No trace of the wound is visible after that age. Wound healing of these spontaneous lesions was analysed with light microscopy (using indirect immunofluorescence for the localization of type I collagen, fibronectin and laminin) and electron microscopy. The main feature of the very slow healing process, as compared with the rapid cicatrization of experimental excision wounds, appears to be a continuous damage of the healing epidermis, until, finally, definitive wound closure occurs between 14 and 16 days of incubation. In the damaged region, where the epidermis is absent, the dermis exhibits an increased density of type I collagen fibres and of fibronectin. The upper face of the bare dermis is deprived of laminin. Spontaneous lesions do not occur in isolated wings explanted on the chick chorioallantoic membrane, where the wings do not become mobile and are not in contact with the amnion. The observations and explantation experiments suggest that the skin damage is caused by friction and abrasion of the bending elbow against the amnion or the amniotic fluid.     

Expression of Hex during feather bud development

We studied proline-rich divergent homeobox gene Hex/Prh expression in the dorsal skin of chick embryo during feather bud development. Hex mRNA expression was first observed in the dorsolateral ectoderm and mesenchyme at 5 days, then in the epithelium and the dermis of the dorsal skin before placode (primordium of feather bud) formation and then was restricted to the placode and the dermis under the placode. Afterward, Hex expression was seen in the epidermis and the dermis of the posterior region of short bud. In accordance with Hex mRNA expression in the placode, Hex protein was observed in the epidermis as well as in the dermis of the placode. Immunoelectron microscopic study indicated that the protein located both in the nuclei and cytoplasm of the epidermis and the dermis at the short bud stage. The Wnt signaling pathway plays an essential role in the early inductive events in hair (Wnt3a and 7a) and feather (Wnt7a) follicles. The pattern of Hex expression in the epidermis was similar to that of Wnt7a, while little, if any, expression of Wnt7a was detected in the dermis under the placode or the dermis of the short bud compared with that of Hex, suggesting that Hex plays an important role in the initiation of feather morphogenesis.     

Competition during innervation of embryonic amphibian head skin

We have examined the initial innervation of the head skin in Xenopus laevis embryos which is by two classes of trigeminal mechanoreceptor with beaded 'free' nerve-endings. By recording receptive areas electrophysiologically and staining peripheral sensory neurites with horseradish peroxidase, we have shown that 'movement detector' neurites from one trigeminal ganglion do not normally cross the dorsal midline of the head to innervate areas of skin on the opposite side. However, if one trigeminal ganglion is removed before peripheral innervation starts, movement detector neurites from the intact side will now cross the midline to innervate contralateral skin. These observations suggest a specific competitive interaction between movement detector neurites during their innervation of head skin. The second class of receptor, 'rapid transient' detectors, have a different pattern of innervation, crossing the midline in both normal and operated animals.     

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.     

Different subsets of axonal guidance cues are essential for sensory neurite outgrowth to cutaneous and muscle targets in the dorsal ramus of the embryonic chick

The dorsal ramus nerve diverges dorsally from each spinal nerve to innervate the epaxial muscle and dermis that are derived in situ from each dermamyotome. The outgrowth of both the sensory and motor components of this nerve are sensitive to the proximity of the dermamyotome. Motoneurons display a direct target response that is not dependent upon the concurrent outgrowth of sensory neurites (Tosney: Dev. Biol. 122:540-588, 1987). Likewise, the outgrowth of sensory neurites could be directly dependent on the dermamyotome. Alternatively, sensory neurites could be dependent on motor axons that in turn require the dermamyotome for outgrowth. To distinguish between these possibilities, motor outgrowth was abolished by unilateral ventral neural tube deletion and the patterns of subsequent sensory neurite outgrowth were assessed. The cutaneous nerve branch formed in all cases. In contrast, neither of the epaxial muscle nerves formed in the absence of epaxial motoneuron outgrowth. Furthermore, sensory neurites could not be detected diverging into muscle from the cutaneous nerve or entering muscle via other novel routes. We conclude that motoneurons are essential for sensory outgrowth to epaxial muscle but not to cutaneous targets. It is clear that different subsets of navigational cues guide sensory afferents to muscle and to cutaneous destinations.     

Drm/Gremlin, a BMP antagonist, defines the interbud region during feather development

The pattern of feather buds in a tract is thought to result from the relative ratios between activator and inhibitor signals through a lateral inhibition process. We analyse the role of Drm/Gremlin, a BMPs antagonist expressed during feather pattern formation, in the dermal precursor, the dense dermis, the interbud dermis and in the posterior dermal condensation. We have altered the activity of Drm in embryonic chick skin using retroviral vectors expressing drm/ gremlin and bmps. We show that expression of endogenous drm is under the control of a feedback loop induced by the BMP pathway, and that overexpression of drm results in fusion between adjacent feather buds. We propose that endogenous BMP proteins induce drm expression in the interbud dermis. In turn, the Drm/Gremlin protein limits the inhibitory effect of BMPs, allowing the adjacent row of feathers to form. Thus, the balance between BMPs and its antagonist Drm would regulate the size and spacing of the buds.     

The specification of feather and scale protein synthesis in epidermal-dermal recombinations

Keratin proteins synthesized by dorsal or tarsometatarsal embryonic chick epidermis in heterotopic and heterospecific epidermal-dermal recombinants were analyzed by polyacrylamide gel electrophoresis and were compared to those produced by normal nondissociated dorsal and tarsometatarsal embryonic skin, as well as to those produced by control homotopic recombinants. Recombinant skins were grafted on the chick chorioallantoic membrane and grown for 8 or 11 days. Recombinants comprising dorsal feather-forming dermis formed feathers, irrespective of the origin of the epidermis. The electrophoretic band patterns of the keratins extracted from these feathers were of typical feather type. Conversely recombinants comprising tarsometatarsal scale-forming dermis formed scales, irrespective of the origin of the epidermis. The band patterns of the keratins extracted from the epidermis of these scales were of typical scale type. Heterospecific recombinants comprising chick dorsal feather-forming epidermis and mouse plantar dermis gave rise to six footpads arranged in a typical mouse pattern. In these recombinants, the chick epidermis produced keratins, the band pattern of which was of typical chick scale type. These results demonstrate that the dermis not only induces the formation of cutaneous appendages in confirmity with its regional origin, but also triggers off in the epidermis the biosynthesis of either of two different keratin types, in accordance with the regional type (feather, scale, or pad) of cutaneous appendages induced. The possible relationship between region-specific morphogenesis and cytodifferentiation is discussed in comparison with results obtained in other kinds of epithelial-mesenchymal interactions.     

Differential growth of sensory neurons in vitro in presence of dermis and epidermis. A quantitative time-lapse analysis

The influence of dermal and epidermal cells on the growth of nerve fibres from chick embryo sensory neurons was investigated in vitro. A previous quantitative analysis showed that the growth of nerve fibres is profoundly modified in the close vicinity of epidermis. This change is mainly characterized by erratic trajectories of nerve fibres resulting from numerous lateral displacements of the growth cones. In contrast, no such behaviour is observed far away from the epidermis or in the presence of dermis. In this latter case, neurites exhibit a straighter direction of extension. These observations suggest that the epidermis exerts some kind of control on the establishment of nerve fibre pattern in the dermis.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Summary Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.