Avian scale development. IX. Scale formation by scaleless (sc/sc) epidermis under the influence of normal scale dermis

Unlike normal scutate scales whose outer and inner epidermal surfaces elaborate β (β-keratins) and α (α-keratins) strata, respectively, the scaleless mutant's anterior metatarsal epidermis remains flat and elaborates only an α stratum. Reciprocal epidermal-dermal recombinations of presumptive scale tissues from normal and mutant embryos have demonstrated that the scaleless defect is expressed only by the epidermis. In fact, the scaleless anterior metatarsal epidermis is unable to undergo placode formation. More recently, it has been determined that the absence of epidermal placode morphogenesis into a definitive scale ridge actually results in the establishment of a scale dermis which is incapable of inducing the outer and inner epidermal surfaces of scutate scales. Can the initial genetic defect in the scaleless anterior metatarsal epidermis be overcome by replacing the defective dermis with a normal scutate scale dermis, i.e., a dermis with scale ridges already present? Or, are the genes involved in the production of a β stratum regulated by events directly associated with morphogenesis of the epidermal placode? In the present study, we combined scaleless anterior metatarsal epidermis (stages 36 to 42) with normal scutate scale dermis (stage 40, 41, or 42) old enough to have acquired its scutate scale-inducing ability. After 7 days of growth as chorioallantoic membrane grafts, we observed grossly and histologically, typical scutate scales in these recombinant grafts. Electron microscopic and electrophoretic analyses have verified that these recombinant scales are true scutate scales. The scaleless mutation, known to be expressed initially by the anterior metatarsal epidermis, can be overcome by exposing this epidermis to appropriate inductive cues, i.e., cues that direct the differentiation of the outer and inner epidermal surfaces of the scutate scales and the production of specific structural proteins. We have determined that the time between stages 38 and 39 is the critical period during which the normal scutate scale dermis acquires these inductive abilities.     

Expression of the cell adhesion molecules, L-CAM and N-CAM during avian scale development

To examine the involvement of cell adhesion molecules in the inductive epithelial-mesenchymal interactions during avian scale development, a study of the spatiotemporal distribution of L-CAM and N-CAM was undertaken. During scutate scale development, L-CAM and N-CAM are expressed together in cells of the transient embryonic layers destined to be lost at hatching. The ongoing linkage of the cells of these layers by both CAMs sets them apart, early in development, as unique cell populations. L-CAM and N-CAM were also expressed simultaneously at the basal surface of the early germinative cells where signal transduction is presumed to occur. In spite of the differences in cell shape, adhesion, density and proliferative state between populations of epidermal placode and interplacode cells, the expression of L-CAM and N-CAM appeared to be uniform and nondiscriminating for these discrete cell lineages. The same pattern of L-CAM and N-CAM expression was observed during morphogenesis of reticulate scales that develop without placode formation. While L-CAM and N-CAM are present during the early stages of scale development and most likely function in cell adhesion, the data do not support a role for these adhesion molecules in the formation of the morphogenetically critical placode and interplacode cell populations. In both scale types, L-CAM became predominantly epithelial, and N-CAM became predominantly dermal as histogenesis occurred. Initially, N-CAM was concentrated near the basal lamina where it may be involved in the reciprocal epidermal-dermal interactions required for morphogenesis. However, as development of the scales progressed, N-CAM disappeared from the tissues. L-CAM expression continued in the epidermis and was intense on all suprabasal cells undergoing differentiation into either an alpha-stratum or beta-stratum. However, L-CAM was more prevalent on the basal cells of alpha-keratinizing regions than on the basal cells of beta-keratinizing regions.     

Avian scale development. XIII. Epidermal germinative cells are committed to appendage-specific differentiation and respond to patterned cues in the dermis.

The ability of the germinative cell population of scutate scale epidermis to continue to generate cells that undergo their appendage-specific differentiation (beta stratum formation), when associated with foreign dermis, was examined. Tissue recombination experiments were carried out which placed anterior metatarsal epidermis (scutate scale forming region) from normal 15-day chick embryos with either the anterior metatarsal dermis from 15-day scaleless (sc/sc) embryos or the dermis from the metatarsal footpad (reticulate scale forming region) of 15-day normal embryos. Neither of these dermal tissues are able to induce beta stratum formation in the simple ectodermal epithelium of the chorion, however, the footpad dermis develops an appendage-specific pattern during morphogenesis of the reticulate scales, while the sc/sc dermis does not. Morphological and immunohistological criteria were used to assess appendage-specific epidermal differentiation in these recombinants. The results show that the germinative cell population of the 15-day scutate scale epidermis is committed to generating suprabasal cells that follow their appendage-specific pathways of histogenesis and terminal differentiation. Of significance is the observation that the expression of this determined state occurred only when the epidermis differentiated in association with the footpad dermis, not when it was associated with the sc/sc dermis. The consistent positioning of the newly generated beta strata to the apical regions of individual reticulate-like appendages demonstrates that the dermal cues necessary for terminal epidermal differentiation are present in a reticulate scale pattern. The observation that beta stratum formation is completely missing in the determined scutate scale epidermis when associated with the sc/sc dermis adds to our understanding of the sc/sc defect. The present data support the conclusion of earlier studies that the anterior metatarsal dermis from 15-day sc/sc embryos lacks the ability to induce beta stratum formation in a foreign epithelium. In addition, these observations evoke the hypothesis that the sc/sc dermis either lacks the cues (generated during scutate and reticulate scale morphogenesis) necessary for terminal differentiation of the determined scutate scale epidermis or inhibits the generation of a beta stratum.     

Avian scale development. VII. Normal keratinization follows abnormal morphogenesis of reticulate scales from the “scaleless” mutant

The scutate scales are entirely missing in chick embryos homozygous for the gene, “scaleless.” Reticulate scales of this mutant are present; however, they have undergone abnormal morphogenesis into irregular mounds and crevices. The pattern of keratinization seen along the anterior metatarsus of normal embryos differs dramatically from that seen along the anterior metatarsus of scaleless embryos. In contrast, we find that the unique pattern of keratinization seen in the epidermal cells of normal reticulate scales is retained in mutant reticulate scales, even though these scales are morphologically abnormal. We believe that differences in the initial tissue interactions (which establish the inductive ability of the dermis) of these two types of scales are responsible for the differences seen in their responses to the scaleless gene. The pleiotropic nature of the scaleless gene is discussed.     

The initial expression and patterned appearance of tenascin in scutate scales is absent from the dermis of the scaleless (sc/sc) chicken.

Morphogenesis of the anterior metatarsal skin (scutate scale region), from 9.5 to 12 days of development, results in the formation of orderly patterned scale ridges. It is after the initial formation of the Definitive Scale Ridge that the characteristic outer and inner epidermal surfaces differentiate. The hard, plate-like beta stratum, with its unique beta keratins, characterizes the epidermis of the outer surface, while the epidermis of the inner surface elaborates an alpha stratum. The anterior metatarsal region of the scaleless mutant does not undergo scale morphogenesis. Therefore, scale ridges do not form nor do the outer and inner epidermal surfaces with their characteristic beta and alpha strata. We have found that the extracellular matrix molecule, tenascin, first appears in the scutate scale dermis at 12 days of development when the scale ridge is established. Tenascin is found in the dermis only under the scale ridge and is not associated with the dermal-epidermal junction. Tenascin is not found in scaleless anterior metatarsal dermis at this time. As outgrowth of the Definitive Scale Ridge takes place, tenascin distribution correlates closely with the formation of the outer epidermal surface of each scale ridge. By 16 days of development tenascin is also found in close association with the dermal-epidermal junction. Tenascin does not appear in scaleless anterior metatarsal dermis until 16 days of development and then it is randomly and sparsely distributed at the dermal-epidermal junction. Tenascin's initial appearance and pattern of distribution in the scutate scale dermis and its abnormal expression in the scaleless dermis suggest that morphogenesis plays a significant role in regulation of its expression.     

Epidermal-dermal tissue interactions between mutant foot skin and normal back skin: a comparison of the inductive capacities of scaleless low line and normal anterior foot dermis.

The inductive capacities of 9- to 16-day anterior foot dermis of scaleless low line and normal embryos were compared by recombining them with a common source of epidermis, i.e., 7-day normal back epidermis. Tissue recombinants were cultured as grafts to the chorioallantoic membrane (CAM). Both normal and scaleless low line dermis of 12 to 13 days of incubation began to lose their ability to elicit feather production in 7-day normal back epidermis. Normal foot dermis began to elicit scale production at 12 to 13 days, whereas scaleless low line anterior foot dermis maintained feather production at a low level. It is inferred that without being associated with scale placode formation, scaleless low line anterior foot dermis does not acquire specific inductive capacities related to the production of an outer scale surface in the overlying epidermis. Feather placodes do not function as surrogates of scale placodes. The difference between normal and scaleless low line anterior foot dermis in terms of specific inductive capacities related to scale production is interpreted as a secondary effect of the action of the scaleless allele in interfering with scale placode formation in the scaleless low line anterior foot epidermis.     

Avian skin development and the evolutionary origin of feathers

The discovery of several dinosaurs with filamentous integumentary appendages of different morphologies has stimulated models for the evolutionary origin of feathers. In order to understand these models, knowledge of the development of the avian integument must be put into an evolutionary context. Thus, we present a review of avian scale and feather development, which summarizes the morphogenetic events involved, as well as the expression of the beta (beta) keratin multigene family that characterizes the epidermal appendages of reptiles and birds. First we review information on the evolution of the ectodermal epidermis and its beta (beta) keratins. Then we examine the morphogenesis of scutate scales and feathers including studies in which the extraembryonic ectoderm of the chorion is used to examine dermal induction. We also present studies on the scaleless (sc) mutant, and, because of the recent discovery of "four-winged" dinosaurs, we review earlier studies of a chicken strain, Silkie, that expresses ptilopody (pti), "feathered feet." We conclude that the ability of the ectodermal epidermis to generate discrete cell populations capable of forming functional structural elements consisting of specific members of the beta keratin multigene family was a plesiomorphic feature of the archosaurian ancestor of crocodilians and birds. Evidence suggests that the discrete epidermal lineages that make up the embryonic feather filament of extant birds are homologous with similar embryonic lineages of the developing scutate scales of birds and the scales of alligators. We believe that the early expression of conserved signaling modules in the embryonic skin of the avian ancestor led to the early morphogenesis of the embryonic feather filament, with its periderm, sheath, and barb ridge lineages forming the first protofeather. Invagination of the epidermis of the protofeather led to formation of the follicle providing for feather renewal and diversification. The observations that scale formation in birds involves an inhibition of feather formation coupled with observations on the feathered feet of the scaleless (High-line) and Silkie strains support the view that the ancestor of modern birds may have had feathered hind limbs similar to those recently discovered in nonavian dromaeosaurids. And finally, our recent observation on the bristles of the wild turkey beard raises the possibility that similar integumentary appendages may have adorned nonavian dinosaurs, and thus all filamentous integumentary appendages may not be homologous to modern feathers.     

Epigenesis in developing avian scales. I. Qualitative and quantitative characterization of finite cell populations

Throughout a period from day 8.5 to day 12.5 of incubation of a chick embryo, a finite cell population of scale epidermis was characterized from various view points such as cellular organization, position, shape, area, number of constituent cells, density, and cell proliferation activity. In this study, the preparation of whole mount specimens was found to be quite valuable. On day 8.5, cells in the prospective scale region could be morphologically distinguished in the tarsometatarsus at a certain distance proximally away from the tarsometatarsal-phalangeal joint. --On day 9.25, about 1,100 cells became highly columnar in shape and densely associated, forming a placode structure. In both distally and proximally adjacent regions of this placode, the cells were semiquadrate in shape and loosely associated, leading to the formation of the interplacode structures. Such contrasting difference in cell organization between placode and interplacode was preserved from day 9.25 to day 11. During this period, both the area and number of constituent cells increased greatly in the placode and only slightly in the interplacode. However, cell proliferation activity was completely suppressed in the placode, and quite active in the interplacode. The activity in cell proliferation proved to be inversely correlated with the density of basal cells. Throughout the present study, it has been demonstrated that the early development of scale epidermis is achieved through a coordinated activity of the two discrete cell populations: the placode and interplacode.     

Differences between normal and scaleless (sc/sc) chicken epidermal cell surfaces detected with wheat germ agglutinin

Dissociated epidermal cells derived from the backskin of scaleless chick embryos (stage 34 or 35) form larger agglutinates with wheat germ agglutinin (WGA) than epidermal cells from normal embryonic skin. [³H]Acetyl WGA binding to the scaleless cells is twice as great as to normal epidermal cells. Treatment of these cells with concanavalin A (conA) results in equivalent agglutination of both mutant and normal epidermal cells, whereas neither scaleless nor normal epidermal cells are agglutinated by Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) or Ulex europeus agglutinin (UEA). This alteration in cell surface carbohydrates may be related to the failure of the scaleless mutant embryonic epidermis to undergo normal morphogenesis.     

Expression of the HB9 homeobox gene concomitant with proliferation accompanying epidermal stratification during development of chick embryonic tarsometatarsal skin

A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis revealed that, during development of chick embryonic skin, the HB9 gene was expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changed, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally (Tanaka S, Kato Y (1983b) J Exp Zool 225: 271–283). During scale ridge elongation at 12–15 days, HB9 was strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompanies epidermal stratification.     

Epigenesis in developing avian scales. III. Stage-specific alterations of the developmental program caused by 5-bromodeoxyuridine

As an approach to the study of a developmental program, 5-bromodeoxyuridine (BrdU) was administered to chick embryos in ovo at various stages of avian scale formation. This brought about stage-specific alterations in morphogenesis in the anterior tarsometatarsus such as feathered scales, from Day 6 through Day 6 1/2; feathers only, from Day 6 3/4 through Day 7 1/4; scalelessness and rudimentary scales, from Day 7 7/8 through Day 8 1/8; and partial ridge scales, from Day 8 1/8 through Day 10. The effects of BrdU were completely nullified by an excess dose of thymidine which instantly suppressed BrdU incorporation into nuclear DNA. Effects of BrdU causing scalelessness were further examined. The percentage of BrdU labeled cells was immunohistochemically detected. It increased linearly in both the epidermis and dermis, reaching nearly 100% 24 hr following its injection on Day 8. However, scale forming potency, as assayed by the area of scale epidermis on Day 11, decreased with the duration of BrdU incorporation into the cells and disproportionately dropped at 15 hr when about 50% of the cells had incorporated BrdU. Scalelessness was also produced when the period of the incorporation of BrdU exceeded 15 hr. Time sequence observations demonstrated epidermal cell shape, polarity, alignment, and packing density to be remarkably disordered so that the placode and interplacode failed to develop on Day 9 1/4. Epidermal-dermal recombinations were carried out by exchanging normal tissues with those treated with BrdU in the anterior tarsometatarsus. The results clearly showed defects in the dermis at the time of reassociation, giving rise to scalelessness.     

Apoptosis and proliferation in developing, mature, and regressing epibranchial placodes

Epibranchial placodes and rhombencephalic neural crest provide precursor cells for the geniculate, petrosal, and nodose ganglia. In chick embryos and in Tupaia belangeri, apoptosis in rhombomeres 3 and 5 helps to select premigratory precursor cells and to segregate crest cell streams derived from the even-numbered rhombomeres. Much less is known about the patterns and functions of apoptosis in epibranchial placodes. We found that, in Tupaia belangeri, combined anlagen of the otic placode and epibranchial placode 1 transiently share a primordial low grade thickening with post-otic epibranchial placodes. Three-dimensional reconstructions reveal complementary, spatially, and temporally regulated apoptotic and proliferative events that demarcate the otic placode and epibranchial placode 1, and help to individualize three pairs of epibranchial placodes in a rostrocaudal sequence. Later, rostrocaudal waves of proliferation and apoptosis extend from dorsal to ventral parts of the placodes, paralleled by the dorsoventral progression of precursor cell delamination. These findings suggest a role for apoptosis during the process of neuroblast generation in the epibranchial placodes. Finally, apoptosis eliminates remnants of the placodes in the presence of late invading macrophages.     

Origin of feathers: Feather beta (beta) keratins are expressed in discrete epidermal cell populations of embryonic scutate scales

The feathers of birds develop from embryonic epidermal lineages that differentiate during outgrowth of the feather germ. Independent cell populations also form an embryonic epidermis on scutate scales, which consists of peridermal layers, a subperiderm, and an alpha stratum. Using an antiserum (anti-FbetaK) developed to react specifically with the beta (beta) keratins of feathers, we find that the feather-type beta keratins are expressed in the subperiderm cells of embryonic scutate scales, as well as the barb ridge lineages of the feather. However, unlike the subperiderm of scales, which is lost at hatching, the cells of barb ridges, in conjunction with adjacent cell populations, give rise to the structural elements of the feather. The observation that an embryonic epidermis, consisting of peridermal and subperidermal layers, also characterizes alligator scales (Thompson, 2001. J Anat 198:265-282) suggests that the epidermal populations of the scales and feathers of avian embryos are homologous with those forming the embryonic epidermis of alligators. While the embryonic epidermal populations of archosaurian scales are discarded at hatching, those of the feather germ differentiate into the periderm, sheath, barb ridges, axial plates, barbules, and marginal plates of the embryonic feather filament. We propose that the development of the embryonic feather filament provides a model for the evolution of the first protofeather. Furthermore, we hypothesize that invagination of the epidermal lineages of the feather filament, namely the barb ridges, initiated the formation of the follicle, which then allowed continuous renewal of the feather epidermal lineages, and the evolution of diverse feather forms.     

Differences in the histogenesis and keratin expression of avian extraembryonic ectoderm and endoderm recombined with dermis

The responses of the chorionic ectoderm and allantoic endoderm (from 8-day chick embryos) to dermal induction were compared through tissue recombinants grafted onto the chorioallantoic membrane. The chorionic epithelium formed the appropriate epidermis with a fully developed stratum corneum in response to both spur and scutate scale dermises. Analysis of these recombinant epidermal tissues by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that tissue-specific expression of the alpha (alpha) and beta (beta) keratin polypeptides occurred. In addition, indirect immunofluorescence studies with antisera to alpha or beta keratins showed that the beta stratum, which characterizes the epidermis of spurs and scutate scales, was formed, and the alpha keratins were distributed as in the normal epidermal tissues. In contrast, although the allantoic endoderm became stratified in association with either spur or scutate scale dermis, a stratum corneum with a beta stratum did not develop. SDS-PAGE analysis demonstrated that while the characteristic beta keratins of scutate scales and spur were not detected, most of the alpha keratins normally elaborated by these structures were present, suggesting that even without histogenesis of a stratum corneum the expression of alpha keratins of endoderm could be regulated in a tissue-specific manner by dermis. This study also demonstrated that there are differences in the abilities of the chorionic and allantoic epithelia to respond to the same dermal cues, which may reflect earlier restrictions in their developmental potentials.     

Early development of the cranial sensory nervous system: from a common field to individual placodes

Sensory placodes are unique columnar epithelia with neurogenic potential that develop in the vertebrate head ectoderm next to the neural tube. They contribute to the paired sensory organs and the cranial sensory ganglia generating a wide variety of cell types ranging from lens fibres to sensory receptor cells and neurons. Although progress has been made in recent years to identify the molecular players that mediate placode specification, induction and patterning, the processes that initiate placode development are not well understood. One hypothesis suggests that all placode precursors arise from a common territory, the pre-placodal region, which is then subdivided to generate placodes of specific character. This model implies that their induction begins through molecular and cellular mechanisms common to all placodes. Embryological and molecular evidence suggests that placode induction is a multi-step process and that the molecular networks establishing the pre-placodal domain as well as the acquisition of placodal identity are surprisingly similar to those used in Drosophila to specify sensory structures.     

Avian scale development. X. Dermal induction of tissue-specific keratins in extraembryonic ectoderm

Epidermal-dermal tissue interactions regulate morphogenesis and tissue-specific keratinization of avian skin appendages. The morphogenesis of scutate scales differs from that of reticulate scales, and the keratin polypeptides of their epidermal surfaces are also different. Do the inductive cues which initiate morphogenesis of these scales also establish the tissue-specific keratin patterns of the epidermis, or does the control of tissue-specific keratinization occur at later stages of development? Unlike feathers, scutate and reticulate scales can be easily separated into their epidermal and dermal components late in development when the major events of morphogenesis have been completed and keratinization will begin. Using a common responding tissue (chorionic epithelium) in combination with scutate and reticulate scale dermises, we find that these embryonic dermises, which have completed morphogenesis, can direct tissue-specific statification and keratinization. In other words, once a scale dermis has acquired its form, through normal morphogenesis, it is no longer able to initiate morphogenesis of that scale, but it can direct tissue-specific stratification and keratinization of a foreign ectodermal epithelium, which itself has not undergone scale morphogenesis.     

Immunocytochemical and autoradiographic studies on the process of keratinization in avian epidermis suggests absence of keratohyalin

The process of keratinization in apteric avian epidermis and in scutate scales of some avian species has been studied by autoradiography for histidine and immunohistochemistry for keratins and other epidermal proteins. Acidic or basic alpha-keratins are present in basal, spinosus, and transitional layers, but are not seen in the corneous layer. Keratinization-specific alpha-keratins (AE2-positive) are observed in the corneous layer of apteric epidermis but not in that of scutate scales, which contain mainly beta-keratin. Alpha-keratin bundles accumulate along the plasma membrane of transitional cells of apteric epidermis. In contrast to the situation in scutate scales, in the transitional layer and in the lowermost part of the corneous layer of apteric epidermis, filaggrin-like, loricrin-like, and transglutaminase immunoreactivities are present. The lack of isopeptide bond immunoreactivity suggests that undetectable isopeptide bonds are present in avian keratinocytes. Using immunogold ultrastructural immunocytochemistry a low but localized loricrin-like and, less, filaggrin-like labeling is seen over round-oval granules or vesicles among keratin bundles of upper spinosus and transitional keratinocytes of apteric epidermis. Filaggrin-and loricrin-labeling are absent in alpha-keratin bundles localized along the plasma membrane and in the corneous layer, formerly considered keratohyalin. Using ultrastructural autoradiography for tritiated histidine, occasional trace grains are seen among these alpha-keratin bundles. A different mechanism of redistribution of matrix and corneous cell envelope proteins probably operates in avian keratinocytes as compared to that of mammals. Keratin bundles are compacted around the lipid-core of apteric epidermis keratinocytes, which do not form complex chemico/mechanical-resistant corneous cell envelopes as in mammalian keratinocytes. These observations suggest that low amounts of matrix proteins are present among keratin bundles of avian keratinocytes and that keratohyalin granules are absent.     

Region-specific expression of scutate scale type beta keratins in the developing chick beak.

This study shows that different patterns of scutate scale type beta keratins are accumulated in the three adjacent structures of the embryonic chick beak: periderm, egg tooth, and cornified beak. The cornified beak accumulates all of the beta keratins of scutate scale except pp2,3. The periderm, which is the outermost, multilayered covering of the whole embryonic beak, accumulates only beta keratins 2,3, and p2,3 of the scutate scale pattern. The egg tooth, which is the rounded elevation on the dorsal surface of the upper beak, and the embryonic claw accumulate greatly reduced levels of 2,3 and p2,3 compared to scutate scale. Like cornified beak, the claw does not accumulate pp2,3, but both tissues express a potentially new beta keratin, beta keratin 8. Neither the histidine rich "fast" proteins (HRPs), which are expressed in embryonic scutate scales and feathers, nor the avian cytokeratin associated proteins (cap-1 and cap-2), which are expressed in scutate and reticulate scales, are expressed in any of the embryonic beak structures or in the claw. The implications of these findings with regard to regulation of terminal differentiation of avian skin are discussed.