Loren W. Knapp

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

SummaryThe 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.

Epidermis, Dermis and Appendages

The avian integument is uniquely characterized by the presence of feathers, whose distribution, structure and development have been reviewed recently on a comparative basis by Lucas and Stettenheim (1972). Brush (1974, 1975, 1980) and Brush and Wyld (1982) have used the biochemical composition of feathers in comparative and evolutionary studies of several bird species. In general, the body of most birds appears covered by feathers with obvious naked areas restricted to the feet, beak, comb and wattle. However, as discussed by Lucas and Stettenheim (1972) and Stettenheim (1972) feathers occur in tracts (pterylae) on most birds, which are separated by naked regions of skin known as apteria. The apteria may exist within individual feather tracts as the naked skin between feathers or between pterylae. In the developing chicken skin, Sengel (1976) distinguished two types of apteric regions; one which is entirely devoid of feathers (for example, the midventral apterium) and one which develops a few loosely distributed down feathers (for example, the apterium between the breast and ventral pterylae). This distinction is of importance in developmental studies where one is interested in the control of appendage formation. In a later section we will discuss some of the more recent findings on the development and structure of feathers, with special reference to the localization of keratins with indirect immunofluorescence, using non-cross-reacting antisera to both α -and s-(feather) keratins.

Evolutionary origin of the feather epidermis

The formation of scales and feathers in reptiles and birds has fascinated biologists for decades. How might the developmental processes involved in the evolution of the amniote ectoderm be interpreted to shed light on the evolution of integumental appendages? An Evo–Devo approach to this question is proving essential to understand the observation that there is homology between the transient embryonic layers covering the scale epidermis of alligators and birds and the epidermal cell populations of embryonic feather filaments. Whereas the embryonic layers of scutate scales are sloughed off at hatching, that their homologues persist in feathers demonstrates that the predecessors of birds took advantage of the ability of their ectoderm to generate embryonic layers by recruiting them to make the epidermis of the embryonic feather filament. Furthermore, observations on mutant chickens with altered scale and feather development (Abbott and Asmundson [ 1957 ] J. Hered. 18:63–70; Abbott [ 1965 ] Poult. Sci. 44:1347; Abbott [ 1967 ] Methods in developmental biology. New York: Thomas Y. Crowell) suggest that the ectodermal placodes of feathers, which direct the formation of unique dermal condensations and subsequently appendage outgrowth, provided the mechanism by which the developmental processes generating the embryonic layers diverged during evolution to support the morphogenesis of the epidermis of the primitive feather filament with its barb ridges. Developmental Dynamics 232:256–267, 2005.

Keratin immunoreactivity in the Late Cretaceous bird Rahonavis ostromi

ABSTRACT Immunohistochemical studies, supported by additional lines of evidence, suggest that original proteinaceous components of keratin claw sheath material may be preserved in the pedal ungual phalanx associated with the primitive bird, Rahonavis ostromi, from the Late Cretaceous of Madagascar. This conclusion is based upon immunohistochemical analyses, and supported by brightfield, transmission, and scanning electron microscopy, mass spectrometry, and x-ray photoelectron spectroscopy. Although keratinous structures such as hair, nails, claws, scales and feathers have been identified in the fossil record, these identifications were based on morphological similarity rather than molecular analyses. Chemical or immunological evidence for the survival of keratin has not previously been established in fossils older than ~33,000 BP. This study demonstrates immunological staining and amino acid composition consistent with the presence of fragments of beta keratin, a protein family unique to reptiles and bird...

Biochemical identification and immunological localization of two non-keratin polypeptides associated with the terminal differentiation of avian scale epidermis

SummaryThe expression of two previously uncharacterized polypeptides produced in epidermal cells of chick reticulate and scutate scales during late embryonic scale histogenesis and in hatchling birds has been studied biochemically and immunologically. These polypeptides have been identified by two-dimensional pH gradient gel electrophoresis as basic in charge, with apparent molecular weights of 20 and 23 kD, and they have been characterized immunologically and by amino acid analysis as non-keratin in nature. Monoclonal antibodies which react with both polypeptides have been used for immunohistochemical and immunogold electron-microscopic localization. Immunoreactivity was observed in suprabasal cells of reticulate scale epidermis, where it codistributed with bundles of α-type cytokeratins in the α-keratin-rich layers of epidermis known as the alpha stratum and in suprabasal cells of the outer epidermal surface of scutate scales, where it codistributed with α-and β-type keratin filament bundles in the β-keratin-rich layers of epidermis known as the beta stratum.

Developing Antibodies to Synthetic Peptides Based on Comparative DNA Sequencing of Multigene Families

Using antisera to analyze the expression of specific gene products is a common procedure. However, in multigene families, such as the beta-keratins of the avian integument where strong homology exists among the scale (ScbetaK), claw (ClbetaK), feather (FbetaK), and feather-like (FlbetaK) subfamilies, determining the cellular and tissue expression patterns of the subfamilies is difficult because polyclonal antisera produced from any one protein recognize all family members. Traditionally, researchers produced and screened multiple monoclonal antisera produced from the proteins of interest until an antiserum with sufficient specificity could be obtained. Unfortunately, this approach requires a lot of effort, and once obtained, such antisera may have limited applications. Here, we present procedures by which comparative DNA sequences of members from the beta-keratin multigene family were translated and aligned to identify amino acid domains that were conserved within the FbetaK subfamily, but which were divergent from the other subfamilies. A synthetic 23-mer peptide with the conserved amino acid sequence was generated and used to produce a polyclonal antiserum that recognizes only the FbetaK subfamily of proteins. Western blot analysis and confocal microscopy with this antiserum are now providing valuable new insights concerning the developmental and evolutionary relationships between the scale, claw, and feather proteins found in birds. This represents a powerful new approach combining techniques from molecular evolution and developmental biology to study the expression and evolution of specific members of multigene families.

Clonal variations in keratin: Intermediate filament expression by human somatic cell hybrids

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.

Chapter 4 The Experimental Manipulation of Keratin Expression and Organization in Epithelial Cells and Somatic Cell Hybrids

Publisher Summary Keratins are found in nearly all epithelial tissue types in vertebrates, and in many cases keratins constitute the majority of the proteins produced ( for e.g., skin and its derivatives). Their organization in the cytoplasm of epithelial cells has made them the fundamental elements of the cytoskeleton and established them as a part of a much larger, interrelated group of polymer-forming proteins that give rise to intermediate filaments. Experimental manipulation of epithelial cells provides a means by which these proteins and the filaments formed by them can be better understood, both in the context of basic cell biology and with regard to their importance in the clinical diagnosis of disease. This chapter discusses some of the consequences of the cytological and genetic manipulation of keratin intermediate filaments in epithelial cells and somatic cell hybrids. The approaches presented are used to investigate keratin intermediate filaments with respect to the overall structure and function of the vertebrate cytoskeleton and to relate the expression of keratin genes and the subsequent organization of keratins to specific cell behavior and growth potential. Results and interpretations from two experimental approaches are presented in the chapter in which the expression and/or organization of keratins has been altered. The first involves the analysis of drug-induced alterations of cytokeratin in cultured epithelial cells and the second focuses on the analysis of clonal variations in keratin filament expression by human somatic cell hybrids.

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

Abstract 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. [ 3 H]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.

Intermediate filament expression and lifespan potential in human somatic cell hybrids

SummaryLimited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition, low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative potential in these hybrids.