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Experiments on skin development in amniotes have shown conclusively that the pattern of cutaneous appendages is determined by the dermis. The following concept of feather pattern formation is proposed. The diameter of feather rudiments and the dermal cell density inside the dermal feather condensations are genetically fixed, region-specific parameters. The first feather rudiment is established along the middorsal line (in the lumbar region) as soon as a sufficient width of dense dermis is available. This occurs at about 6 1/2 days in the chick embryo. The next rudiments are formed in front of and behind the first one, within the longitudinal band of dense dermis. They are laid down as close as possible to the first one, given rudiment diameter and dermal condensation cell density. In other words, as they form, the middorsal rudiments are tangent circles.. Later they become separated from one another due to longitudinal growth. As progressively more dense dermis becomes available to the sides of the middorsal row of rudiments, new rudiments are being laid down lateral to the middorsal row, in successive longitudinal rows, until, by 9 or 10 days, all the available dense dermis has been used up. Again, the rudiments in the lateral rows, given their diameter and dermal condensation cell density, develop as close as possible to those of the preceding row, i.e. tangentially to two neighbouring rudiments of the preceding row, thus generating a hexagonal pattern.

Collagen types I and III were purified from the skin of 3-or 7-week-old chickens, collagen type IV from bovine skin or EHS mouse tumour, fibronectin from human serum, and laminin from EHS mouse tumour. Antibodies were produced in rabbits or sheep, and used in indirect immunofluorescence on frozen sections of 9-to 16-day-old normal or mutant (scaleless) chick-embryo foot skin. In normal scale-forming skin and inscaleless skin, the distribution of anti-laminin and anti-type IV collagen label was uniform along the dermal-epidermal junction and showed no stage-related variations, except for fluorescent granules located in the dermis of early scale rudiments. By contrast, in normal scale-forming skin, the density of anti-types I and III label decreased in the dermis within scale rudiments, whereas it gradually increased in interscale skin. Conversely, anti-fibronectin label accumulated at a higher density within scale rudiments than in interscale skin. In the dermis of thescaleless mutant, anti-types I and III label and antifibronectin label were distributed evenly: the density of anti-collagen label increased with age, while that of antifibronectin decreased and almost completely vanished in 16-day-old skin, except around blood vessels. The microheterogeneous distribution of some extracellular matrix components, namely interstitial collagen types I and III and fibronectin, is interpreted as part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of scales. The even distribution of these components in mutantscaleless skin is in agreement with this view. Basement membrane constituents laminin and type-IV collagen do not appear to be part of the dermal morphogenetic message.

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.

The shape, distribution, and orientation of peridermal cells were examined in the dorsolumbar skin of 7 1 2 - day chick embryos. Most feather rudiments of middorsal and lateral rows showed a marked cephalocaudal polarity. A similar polarity was found in the prospective rudiments of skin areas lateral to the last-formed row. On the cranial slope and apex of rudiments, cells are convex and predominantly elongated at right angles with respect to the cephalocaudal axis, whereas on the caudal slope, most cells are flat, polygonal, surrounded by a border-line ridge, and oriented predominantly with their long axis parallel to the cephalocaudal axis. The significance of this pattern is discussed in view of the fact that the epidermis is the determinant tissue in feather orientation.

Feet of chicks are normally covered with scales. Injection of retinoic acid into the amniotic cavity of 10-day chick embryos causes the formation of feathers on the foot scales. To elucidate whether retinoic acid affects primarily the epidermis or the dermis, heterotypic dermal-epidermal recombinants of tarsometatarsal skin were tested as to their morphogenetic capacity, when grafted to the chick chorioallantoic membrane. Recombinants involving treated epidermis and untreated dermis formed feathered scales, while the reverse recombinants of untreated epidermis and treated dermis led to the formation of scales only. Likewise the association of treated tarsometatarsal dermis with untreated epidermis from a non-appendage-forming region (the midventral apterium) resulted in the formation of scales only. These results show that retinoic acid affects primarily the epidermis. Further insight into the mechanism of dermal-epidermal interaction was gained by heterotopic recombinations of early (8.5- and 10-day) untreated tarsometatarsal dermis with epidermis from the midventral apterium. These recombinants formed scales, proving that tarsometatarsal dermis is endowed with scale-forming properties as early as 8.5 days of incubation. Finally, it is concluded that retinoic acid acts on the chick foot epidermal cells by temporarily inhibiting their scale placode-forming properties, allowing their latent feather placode-forming properties to be expressed.

Two months after transplantation of human skin onto the nude mouse, excisional wounds were made through the entire thickness of the skin, at the center of the graft, using a 2-mm punch. At various time intervals thereafter, ranging from 2 days to 9 weeks, the graft sites were harvested and processed for an immunohistological study. With a monoclonal antibody directed against HLA-ABC antigens, it was demonstrated that the healing epidermis is of human origin. Moreover, with three different monoclonal antibodies directed against human keratins, named respectively AE1, AE3, and KL1 and with an anti-involucrin antiserum, it is reported that the keratinization and involucrin distribution patterns observed in normal human epidermis are reconstituted, 2 months after transplantation, in the major part of the grafted epidermis, undergo changes during the reepithelialization process, and are restored in the healed epidermis 9 weeks after injury. This study indicates that the nude mouse/human skin model could be a valuable tool to study a major aspect of regeneration such as the reepidermization of human skin without recourse to human volunteers.

The distribution of glycosaminoglycans (GAGs) was studied in embryonic chick skin, using alcian blue staining with critical electrolyte concentration and glycanase treatment, immunofluorescence and transmission electron microscopy. Light microscopy revealed an uneven distribution of sulphated and non-sulphated GAGs at all stages of feather development. Along the dermal-epidermal junction and throughout the depth of the dermis, staining was stronger inside the feathers than in the interplumar skin. With increasing MgCl2 concentration, the decrease in stain intensity along the dermal-epidermal junction was stronger in interplumar skin than inside feather structures, indicating that sulphated GAGs are more abundant within feathers than in interplumar skin. The same differential sensitivity to electrolyte concentration was noted in the dermis, except at the feather placode stage, when labelling inside the dermal condensation was virtually wiped out at 0.6 M MgCl2 and higher concentrations, whereas it persisted in the surrounding dermis up to 0.8 M MgCl2, indicating that the dermal condensation contains a larger amount of hyaluronate than non-feather-forming dermis. Enzyme treatment of sections with Streptomyces hyaluronidase as compared with those treated with chondroitinase ABC corroborated these findings. Immunofluorescent detection of heparan sulphate proteoglycan revealed the presence of the antigen along the dermal-epidermal junction at all stages of feather development, with peaks of brightness in discrete spots of feather structures. Electron microscopy revealed the presence of ruthenium red and tannic acid positive material in the dermal-epidermal junctional zone and inside the dermis. The density of marked granules was somewhat higher in intraplumar than in interplumar regions. These observations demonstrate that certain sulphated and non-sulphated GAGs are distributed in a microheterogeneous manner, which appears to be related to the morphogenetic events of feather development. They are discussed in view of the possible role these components might play in dermal-epidermal interactions. They strengthen the notion, already gained from previous studies on the localization of interstitial collagens and fibronectin, that extracellular matrix components play an important structural and informative role in organogenesis.

A new approach in the understanding of the role of the two molting hormones, α- ecdysone and β- ecdysone , in disc development is described. A new culture medium has been developed which allows discs, with either hormone, to undergo all steps of metamorphosis. Time-lapse cinematographic and scanning electron microscopic analysis of the mechanism of evagination led to the conclusion that cells probably do not move extensively with respect to one another. In contrast, changes in cell shape appear to play a major role in this process.

Resume L'etude des mucopolysaccharides dans les ebauches et les germes plumaires a ete realisee, chez l'embryon de poulet, en associant la coloration au Bleu alcian et l'histoautoradiographie avec le S35O4Na2 comme traceur; ces deux techniques se completent, l'une apportant la precision morphologique, l'autre la specificite histochimique. Avant l'apparition des ebauches plumaires (stade 0), les mucopolysaccharides sont surtout abondants dans le derme mesenchymateux. Leur repartition uniforme ne permet pas de situer l'emplacement des futures ebauches plumaires. Des l'apparition des ebauches plumaires (stade 1), on observe d'abord un enrichissement en mucopolysaccharides du massif lenticulaire mesenchymateux de l'ebauche. Ensuite, a la fin du stade 1, les mucopolysaccharides se concentrent a la base et autour du massif lenticulaire, tandis qu'ils disparaissent progressivement a l'apex de l'ebauche mesenchymateuse. Au moment ou les germes plumaires acquierent un plan de symetrie bilaterale (stade 2), les mucopolysaccharides sont localises dans la moitie cephalique de la base du germe. Par la suite, au stade de l'invagination du germe plumaire, les mucopolysaccharides sont confines a l'emplacement de la future papille dermique de la plume.

Resume La pteryle spinale de l'embryon de Poulet de 11 a 12 jours d'incubation (st. 36 H.H.) a ete decrite. L'irradiation localisee aux rayons X et le marquage aux grains de carbone entre 2 et 3 jours d'incubation (7 a 33 paires de somites) ont permis de dresser la carte des territoires presomptifs de la pteryle spinale. Une correspondance entre les plumes initiales axiales ou para-axiales et les somites a ete etablie. En raison de la croissance axiale dysharmonique, la peau subit, par rapport aux tissus sous-jacents, un deplacement en direction caudale, particulierement grand dans les regions cervicale anterieure et lombo-sacro-caudale. La pteryle spinale n'est pas metamerisee. Le nombre des plumes initiales qui se forment dans chacune de ses six regions depend de l'allongement relatif que subit chacune d'elles entre le stade ou s'individualisent ses somites et celui de 11 a 12 jours d'incubation. Plus cet allongement est grand, plus le nombre de plumes par segment est eleve : 2,3 plumes initiales dans la region cervicale anterieure et une seulement dans les regions lombaire, sacree et caudale. La correspondance stricte entre les plumes initiales et les vertebres de ces trois dernieres regions est vraisemblablement fortuite. Les facteurs responsables de l'arrangement des plumes ont probablement leur siege dans les tissus cutanes eux-memes.

The skin of vertebrates is composed of two tissues of distinct embryological origin and of different histological structure, the dermal mesenchyme and the epidermal epithelium. During gastrulation the precursors of these two tissues are brought into superposition by the morphogenetic movements: the presumptive epidermis on top, and the presumptive dermis underneath. Progressively, a specific junctional structure is built between the two tissues, the basement membrane.

Actin isoform expression may change during development, and in certain physiological, experimental and pathological situations. It is accepted that during sarcomeric (skeletal and cardiac) muscle development, the alpha-skeletal and alpha-cardiac isoforms of actin accumulate rapidly at the onset of muscle fibre formation, while there is a rapid fall in the expression of nonmuscle (beta and gamma) actin isoforms. Here we show that, before birth, both skeletal and myocardial cells express significant amounts of alpha-smooth muscle actin mRNA and protein. This expression is transient and disappears over the 1-7 days following birth. Our findings show that the program regulating actin isoform expression in sarcomeric muscle development is complex and that alpha-smooth muscle actin participates in this process.

Collagen types I and III were purified from the skin of 3- or 7-week chickens and fibronectin from human serum. Antibodies were raised in rabbits and used in indirect immunofluorescence on frozen sections of 5- to 16-day chick embryo feather-forming skin. Prior to the formation of dense feather-forming dermis, anticollagen fluorescence was confined to a thin underlining of the dermal-epidermal junction (DEJ), while antifibronectin label was retained on loosely dispersed material in the predermal mesenchyme. Dense feather-forming dermis was characterized by loosening of the anti-collagen label along the DEJ, by its spreading throughout the thickness of dermis, and by an overall densification of antifibronectin label. Feather formation coincided with a decrease of anti-collagen and an increase of antifibronectin label density in the dermal feather condensations and in the core of outgrowing feather buds. By contrast, density of anti-collagen-labeled material was highest and anti-fibronectin-labeled material was lowest in interplumar and glabrous skin. In slanting feather buds and feather filaments, distribution of anti-collagen-labeled material exhibited a type-specific cranial-caudal asymmetry. The microheterogeneous distribution of extracellular matrix components might constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of cutaneous appendages.

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.

In the chick embryo, large scales (scuta) form on days 9 –11 on the anterior face of the shank and the upper surface of the toes; smaller scales (scutella) appear on day 11 on the posterior face of the shank; most of the smallest scales (reticula) form on days 12 and 13 on the plantar surface of the foot. A single injection of 125 μg of retinoic acid (RA) into the amniotic cavity caused the formation of feathers on the foot scales in locations and percentages differing according to the age of embryos at the time of treatment. Injection at 10 days caused feathers to form on the feet in 57 % of the embryos, almost all of which bore feathers on their scuta and scutella; a few reticula were occasionally affected. When treated at 11 days, 48 % of embryos had feathered feet, the tarsometatarsal scutella and digital reticula being most frequently affected. Treatment at 12 days resulted in feathers on the feet of 15 % of the embryos, all of which bore feathers on the reticula only, while the scuta and scutella were not affected. It was concluded that the foot skin regions which were affected by RA treatment were those in which scale morphogenesis was starting or about to start at the time of injection. These regions contained pre-placodal or placodal stages of scuta and scutella, and the preelevation or elevation stages of reticula.

The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.

Cytochalasin B (1 μg/ml) completely inhibited the evagination of isolated leg imaginal discs cultured in vitro in a synthetic medium (ME) containing α-ecdysone (3 μg/ml). In discs precultured for 6 hr in medium ME without the drug, then transferred to cytochalasin B-containing medium, continuation of evagination was stopped immediately. The inhibition of evagination was completely reversible, provided pretreatment with cytochalasin B did not exceed 8 hr. Results are discussed in view of what is known on the effect of cytochalasin B on other developmental systems. Findings are compatible with the primary action of the drug being an alteration of cell surface properties, thus bringing to light the importance of these properties in the course of normal imaginal disc evagination.

Publisher Summary This chapter discusses the organogenesis and arrangement of cutaneous appendages in birds. The skin of the vertebrate embryo, and of birds, is a suitable system for the analysis of the factors of differentiation. It is composed of two distinct tissues of different developmental history, the interplay of which results in the construction of the cutaneous covering with its varied appendages—feathers and scales in birds. One of the constituents of the skin is derived from the mesodermal layer, that is, the dermis. The other component, namely, the epidermis, stems from the ectodermal layer. During early ontogeny, the ectodermal layers become epithelial in their histological structure, whereas the mesodermal component acquires a mesenchymal conformation. The organized superposition of epithelium and mesenchyme gives rise to the skin as such and its derivatives. The epidermis all over the embryo appears to be a neutral covering and carries only the necessary information for the realization of minute cytological features. It also contains the necessary biochemical equipment for the synthesis of the keratin molecules. The orientation of feathers is determined by the ectodermal component of skin. The phenotypic traits that depend on the ectoderm can never be overruled by mesodermal influence.

THE embryonic skin of birds is the site of morphogenetic interactions between the epidermis and dermis leading to the formation of cutaneous appendages1–3. Recombinations of the dermis and epidermis from the dorsal feather-forming region and the tarsometatarsal scale-forming area show that the dermis plays a decisive part in the regional differentiation of the skin1,2. The development of epidermal structures such as feathers and scales is generally determined by the regional origin of the associated dermis. In specific heterochronous combinations, however, the epidermis tends to impose the nature of its differentiation according to its own regional quality. An example of this occurs when the dorsal feather-forming 6–7 day old epidermis is associated with tarsometatarsal scale-forming 11–12 day old dermis. In such combinations a conflicting situation appears to be created which leads to the formation of either feathers and scales or feathers alone. Because the production of scales alone is not observed in these conditions, it seems that feather morphogenesis predominates over scale morphogenesis.