Role of epidermal-dermal tissue interactions in regulating tenascin expression during development of the chick scutate scale.

During normal chicken development tenascin begins to accumulate in the dermis of anterior metatarsal skin at the time of scutate scale ridge formation, and is localized in a distinct pattern along the outer scale surface. Anterior metatarsal skin from scaleless (sc/sc) embryos, which do not form scutate scales, begins to accumulate tenascin 4 days later than normal skin. This study shows that normal and scaleless anterior metatarsal dermis accumulate the same tenascin isoforms and undergo the same isoform changes in the post-hatch period, but there is less tenascin accumulated in scaleless dermis and there is no pattern to its distribution. In both normal and scaleless anterior metatarsal skin, tenascin mRNA is localized in the dermis and is distributed in the same way as the protein. Thus, scaleless skin is defective in the ability to accumulate appropriate amounts of tenascin and to maintain the tenascin in the patterned manner of normal. Recombinant skin cultures show that epidermal-dermal interactions are required for tenascin accumulation. The dermis specifies the way that tenascin is organized, but interaction with epidermis is required to maintain this organization. The epidermal role appears to be permissive because in heterotypic recombinants, neither scaleless anterior metatarsal epidermis nor normal footpad epidermis changes the way that tenascin appears in the normal anterior metatarsal dermis; and in reciprocal recombinants, normal anterior metatarsal epidermis does not change the way tenascin is accumulated in either scaleless anterior metatarsal dermis or normal footpad dermis.

Region-specific patterns of beta keratin expression during avian skin development.

The transient embryonic layers primarily composed of a periderm and subperiderm cover most regions of the chick embryo and are the first suprabasal cell layers covering the body ectoderm. This study presents evidence for regional variation in the expression of beta keratin in the embryonic layers. Here we show that the embryonic layers covering the anterior metatarsal region of the chicken hindlimb (scutate scale forming region) produce several members of the beta keratin family of polypeptides, designated beta (beta) 1-7. These specific polypeptides are later expressed in this region exclusively in the thick, cornified beta strata of mature scutate scales. In contrast to this sequence of events, the embryonic layers overlying the epidermis of the ventral foot pad (reticulate scale-forming region) and those covering the epidermis in apteric regions of the body produce beta keratin polypeptides beta 1-3 and beta 2,3, respectively, but no subsequent expression of these proteins occurs in the mature epidermises of these regions. Furthermore, we find that the embryonic layers of the skin overlying the anterior metatarsal region of birds homozygous for the mutation "scaleless" (sc/sc), which completely lack scutate scales, produce the same members of the beta keratin family, beta 1-7, as the embryonic layers and beta strata of normal scutate scales. Thus, the accumulation of specific beta keratin polypeptides in the developing anterior metatarsal region appears to occur in two distinct phases; first, an early region-specific expression in cells of the embryonic layers followed by a second phase of expression which occurs in conjunction with appendage morphogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Keratinization of the outer surface of the avian scutate scale: interrelationship of alpha and beta keratin filaments in a cornifying tissue.

The outer surface of adult Gallus domesticus scutate scale was studied as a model for epidermal cornification involving accumulation of both alpha and beta keratins. Electron-microscopic analysis demonstrated that the basal cells of the adult epidermis contained abundant lipid droplets and that filament bundles and desmosomes were distributed throughout the cell layers. Indirect immunofluorescence microscopy and double-labeling immunogold-electron microscopy confirmed that the stratum germinativum contained alpha keratin but not beta keratin. Beta keratins were first detected in the stratum intermedium and were always found intermingled with filament bundles of alpha keratin. As the differentiating cells moved into the outer regions of the stratum intermedium and the stratum corneum, the large mixed keratin filament bundles labeled increasingly more with beta keratin antiserum and relatively less so with alpha keratin antiserum. Sodium dodecyl sulfate-polyacrylamide gel analysis of vertical layers of the outer surface of the scutate scale confirmed that cells having reached the outermost layers of stratum corneum had preferentially lost alpha keratin. The mixed bundles of alpha and beta keratin filaments were closely associated with desmosomes in the lower stratum intermedium and with electron-dense aggregates in the cytoplasm of cells in the outer stratum intermedium. Using anti-desmosomal serum it was shown that these cytoplasmic plaques were desmosomes.

Identification, expression, and localization of beta keratin gene products during development of avian scutate scales.

Epidermal-dermal interactions influence morphogenesis and expression of the beta keratin gene family during development of scales in the embryonic chick. The underlying mechanisms by which these interactions control beta keratin expression are not understood. However, the present study of beta keratin gene expression during avian epidermal differentiation contributes new information with which to investigate the role of tissue interactions in this process. Using beta keratin-specific synthetic oligonucleotide probe, beta keratin mRNA was hybrid-selected from total poly A+ RNA of scutate scales. Seven beta keratin polypeptides were translated in vitro and could be identified by their positions in two-dimensional gels among the detergent-insoluble extracts of scutate scale epidermis. In vivo phosphorylation studies suggested that an additional three beta keratin polypeptides were present as phosphoproteins. The temporal appearance of beta keratin mRNA and the corresponding polypeptides was followed during scutate scale development. Polyclonal antiserum made against two of the beta keratin polypeptides was used for immunohistochemical and immunogold electron-microscopic analysis of beta keratin tissue distribution. Immunological reactivity was observed specifically along the outer scale surface in epidermal cells above the stratum germinativum. Immunogold beads were localized on 3-nm filament bundles. In situ hybridization with a beta keratin-specific RNA probe demonstrated that mRNA accumulated in the same regional manner as the polypeptides. This selective expression of beta keratin genes in specific regions of the developing scutate scale suggests that epidermal-dermal interactions provide not only for morphological events, but also for control of complex patterns of histogenesis and biochemical differentiation.

Expression of beta keratin genes during skin development in normal and sc/sc chick embryos.

The expression of RNA sequences specific for scale beta (beta)-keratins has been followed during skin development in normal and scaleless (sc/sc) embryos. Total RNA from skin at various stages (36-46) of development, as well as newly hatched chicks, was immobilized on nitrocellulose paper and hybridized with a [32P]cDNA probe to beta-keratins (pCSK-12). Sequences for beta-keratins showed patterns of expression which were specific for each genotype and scale type examined. During the development of normal scutate scales, which are characterized by the formation of a beta stratum, RNA with beta-keratin sequences first appeared at stage 40, and continued to accumulate through hatching. RNA with beta keratin sequences appeared in scaleless skin between stages 40 and 41, was greatly diminished by stage 44, and was no longer present at stage 46. In normal reticulate scales, which like scaleless skin, do not develop a beta stratum accumulation of RNA with beta-keratin sequences was limited to a brief embryonic period between stages 42 and 44. These patterns of RNA expression correlated well with the appearance of beta-keratin polypeptides, suggesting that beta-keratin synthesis may be controlled at the level of keratin mRNA transcription. Correlations between the patterns of beta-keratin expression and histological events suggest that the brief accumulation of beta-keratin mRNA in scaleless skin and normal reticulate scales is related to the formation of the subperiderm (a protective layer of cells, peculiar to embryonic skin) while the continuous accumulation of beta-keratin mRNA during scutate scale development reflects the formation of a beta stratum.

In vitro cleavage of Pr65gag by the Moloney murine leukaemia virus proteolytic activity yields p30 whose NH2-terminal sequence is identical to virion p30.

In vitro cleavage of Gazdar murine sarcoma virus Pr65gag, which has all of the antigenic determinants of Moloney murine leukaemia virus Pr65gag, i.e. p15, p12, p30 and p10, by the Moloney murine leukaemia virus proteolytic activity yielded a p30 whose partial NH2-terminal sequence was identical to Moloney murine leukaemia virus. Both [3H]leucine-labelled and unlabelled Pr65gag were used to generate the cleaved p30.

Injection of ultraviolet-damage-specific enzyme by T4 bacteriophage.

When UV-irradiated T4 bacteriophage (v(+)) infects in the presence of chloramphenicol, the phage DNA rapidly acquires single-stranded breaks proportional to the dose of UV. In contrast, when UV-irradiated T4 v(1) (radiation sensitive mutant) infects under identical conditions, the phage DNA remains integral. A series of coinfections with v(+) and v(1) phage (UV-v(1) + majority non-UV-v(+) and UV-v(+) and majority non-UV-v(1)) show that the enzyme responsible for breakage is injected by the phage. It is also demonstrated that the v(1) phage injects an inactive enzyme that delays breakage by the v(+) enzyme and interferes with subsequent repair. The cross of v(+) and v(1) phage produces mixed progeny that contain both active and inactive enzyme in a single capsid. The possible function of this breaking enzyme, necessitating injection of multiple copies, is considered.