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Hair follicles are complex organs of the skin, in morphological and ontogenic continuity with the epidermis. We have examined the location of desmosomal cadherins and desmosomal plaque proteins in the hair follicle of adult and fetal human scalp skin by immunohistochemistry and have established a localization "map" of the hair follicle. Using antibodies against the plaque proteins desmoplakin I and II, plakoglobin, and plakophilin 1, we have found that these occur in most, if not all hair follicle desmosomes, whereas plakophilin 2 was absent, except in the basal cells of the outer root sheath, where a weak reactivity was found. By contrast, the desmosomal cadherins were mostly differentially synthesized, displaying a complicated map. While desmocollin Dsc3 was detected in all cell types examined, Dsc1 was detected only in the outer root sheath companion cell layer and the inner root sheath, and Dsc2 showed practically a mutually exclusive presence. Desmoglein Dsg2 was observed in basal cells of the outer root sheath as well as in the central cell layers of the subinfundibular outer rood sheath, matrix cells and trichocytes, in partial overlap with the otherwise different immunopositive reactions of Dsg1 and Dsg3. We have also determined when these proteins are synthesized during fetal hair follicle development. The differential molecular composition of desmosomes is discussed in relation to possible functional differences between the individual cell types.

As part of a program designed to characterize human hair keratin genes and their expression, we present the cDNA sequences and deduced amino acid sequences of three type-II hair keratins hHb3, hHb5, and hHb6, which by virtue of their amino acid homologies are the orthologs of the previously described sheep wool keratins, K2.10, K2.12, and K.211 [29]. Amino acid sequences comparisons of these keratins, including the previously characterized human K2.9 ortholog hHb1, show extreme conservation not only in the alpha-helices but also in the aminoterminal and proximal carboxyterminal domains. They also demonstrate higher sequence relationships between hHb1, hHb3, and hHb6 as compared to hHb5, which exhibits chain-specific sequences in both the head and tail domains. In situ hybridization studies using specific 3'-probes for the four type-II hair keratins reveal sequential patterns of gene expression in human anagen follicles. Remarkably the onset of hHb5 mRNA synthesis occurs immediately above a small population of matrix cells at the base of the hair bulb and the trichocytes lining the dermal papilla. hHb5 mRNA synthesis extends upward through the matrix and ends in the lower part of the cortex of the hair shaft. In contrast, both hHb1 and hHb3 mRNA synthesis begins simultaneously in the cortex 10-15 cell layers above the apex of the dermal papilla, thus partially overlapping that of hHb5 but continuing to a point well beyond hHb5 in the upper cortex. Synthesis of hHb6 mRNA starts slightly higher than either hHb1 or hHb3 mRNA and proceeds much farther up into the keratogenous zone of the hair shaft. Our study demonstrates that the differentiation of human hair in terms of hair keratin expression begins much earlier than previously assumed, i.e. in lower matrix cells of the hair bulb. This early phase of hair differentiation is followed by a late cortical phase of terminal differentiation which comprises at least three type-II hair keratins in the zone of elongation and the keratogenous zone of the hair shaft.

Differentiation of human plantar and palmar epidermis is characterized by the suprabasal synthesis of a major special intermediate-sized filament (IF) protein, the type I (acidic) cytokeratin 9 (CK 9). Using partial amino acid (aa) sequence information obtained by direct Edman sequencing of peptides resulting from proteolytic digestion of purified CK 9, we synthesized several redundant primers by 'back-translation'. Amplification by polymerase chain reaction (PCR) of cDNAs obtained by reverse transcription of mRNAs from human foot sole epidermis, including 5'-primer extension, resulted in multiple overlapping cDNA clones, from which the complete cDNA (2353 bp) could be constructed. This cDNA encoded the CK 9 polypeptide with a calculated molecular weight of 61,987 and an isoelectric point at about pH 5.0. The aa sequence deduced from cDNA was verified in several parts by comparison with the peptide sequences and showed the typical structure of type I CKs, with a head (153 aa), and alpha-helical coiled-coil-forming rod (306 aa), and a tail (163 aa) domain. The protein displayed the highest homology to human CK 10, not only in the highly conserved rod domain but also in large parts of the head and the tail domains. On the other hand, the aa sequence revealed some remarkable differences from CK 10 and other CKs, even in the most conserved segments of the rod domain. The nuclease digestion pattern seen on Southern blot analysis of human genomic DNA indicated the existence of a unique CK 9 gene. Using CK 9-specific riboprobes for hybridization on Northern blots of RNAs from various epithelia, a mRNA of about 2.4 kb in length could be identified only in foot sole epidermis, and a weaker cross-hybridization signal was seen in RNA from bovine heel pad epidermis at about 2.0 kb. A large number of tissues and cell cultures were examined by PCR of mRNA-derived cDNAs, using CK 9-specific primers. But even with this very sensitive signal amplification, only palmar/plantar epidermis was found positive. By in situ hybridization and immunolocalization we further showed that CK 9 is only expressed in the suprabasal cell layers of this special epidermal tissue. We discuss the molecular properties of CK 9 and its cell type- and body site-specific expression in relation to the special differentiation of palmar/plantar epidermis and to diseases specific for this body site.

Biochemical analyses have previously shown that palmar and plantar epidermis, unlike the epidermis of other body sites, contain cytokeratin 9 (Mr 64,000), an unusually large acidic (type I) cytokeratin. Guinea-pig antibodies that specifically and selectively react with bovine and human cytokeratin 9 were used for the immunocytochemical identification of cytokeratin 9 in adult and fetal human epidermis from various body sites. In the epidermis of palms and soles, antibodies against cytokeratin 9 stained a high proportion of the keratinocytes in suprabasal locations. These suprabasal cytokeratin-9-positive keratinocytes were often arranged in vertical columns and concentrated around intraepidermal sweat-gland ducts, but they sometimes also formed extended continuous sheets. In contrast, another type-I component, cytokeratin(s) 10/11, was uniformly distributed among suprabasal keratinocytes. By double-labeling immunofluorescence microscopy using a monoclonal antibody against cytokeratin(s) 10/11, we found that cytokeratin 9 usually appears in cells located one or two layers above the cells in which cytokeratin(s) 10/11 is detected, indicating that most keratinocytes expressing cytokeratin 9 also express cytokeratin(s) 10/11. At other body sites, cytokeratin 9 was only detected in sparsely distributed keratinocytes localized in upper epidermal layers; these cells were scattered or formed small clusters, and often exhibited a conspicuous association with the epidermal portion of eccrine sweat-gland ducts. During human fetal development, cytokeratin 9 was first detected at week 15 of gestation in some suprabasal cells of the foot-sole epidermis and, occasionally, in basal cells. At later fetal stages, most of the cytokeratin-9-positive cells appeared in clusters that were mainly concentrated in glandular ridges and interridges. Our results show that two major types of terminally differentiating keratinocytes can be distinguished in human epidermis, i.e. those that do and those that do not express cytokeratin 9. This special program of keratinocyte differentiation identified by the presence of cytokeratin 9 appears to be related to the morphogenesis of palm and sole epidermis, where this protein is expressed early in fetal life. Possible biological functions of this subset of cytokeratin-9-positive keratinocytes are discussed.

The cytokeratin family of intermediate filament (IF) proteins can be grouped into the epithelial polypeptides ("soft alpha-keratins"), of which at least 19 exist in the various human epithelia, and the hair-type cytokeratins ("hard alpha-keratins"), which are typical of trichocytes, i.e., the living hair-forming cells. We have recently shown [34] that the hair follicles from diverse mammalian species contain a set of eight major cytokeratin polypeptides, four each of the acidic (type I) and the basic (type II) subfamily, which are different from all known epithelial cytokeratins. In addition, we have identified two new minor trichocytic cytokeratin polypeptides, designated Hax (type I) and Hbx (type II). Antibodies against trichocytic cytokeratins that do not crossreact with any of the epithelial cytokeratins have enabled us to study the expression of both kinds of cytokeratin in the various cell types of human and bovine hair follicles. Using immunofluorescence microscopy, we have observed intense reactions of trichocytic cytokeratins only in cells contributing to the forming hairs, i.e., hair shaft, medulla and cuticle, whereas immunostaining of the peribulbar matrix cells was weaker, if at all detectable. In contrast, epithelial cytokeratins were localized in both the inner and outer root sheath epithelia but, surprisingly, also in certain portions of the trichocyte column, notably cells of the cuticle, certain medullary cells, and trichocytes of the basalmost peripapillary cell layers. Cells coexpressing trichocytic and epithelial cytokeratins have been identified by double-label immunofluorescence microscopy. Epithelial cytokeratins of the inner and outer root sheath epithelia include, most remarkably, "simple-epithelium-type" cytokeratins 8, 18, and 19; these occur in certain peribulbar regions, in distinct patterns, but with variable frequencies. The occurrence of simple epithelial cytokeratins in hair follicles has also been confirmed by high-sensitivity immunoblotting of follicular polypeptides separated by gel electrophoresis. Vimentin-positive cells were abundantly interspersed (in some follicles, but not in all) between the trichocytes of the peripapillary cone, most of them probably being melanocytes. The cell-type complexity of the hair follicle and the different patterns of cytoskeletal protein expression in the various hair follicle cells are discussed in relation to the development and growth of this organ.

The hair-forming cells (trichocytes) and the mature hair contain four major trichocytic cytokeratins from each of the subfamilies, basic (Hb1-4) and acidic (Ha1-4); these are related - but not identical - to the epithelial cytokeratins. Here we show, by biochemical methods and immunofluorescence microscopy using antibodies specific for either epithelial or trichocyte cytokeratins, that the same set of hair-type cytokeratins, including two newly identified minor components, designated Hax (type I) and Hbx (type II), are also expressed in cells forming nails, in the filiform papillae of the dorsal surface of human and bovine tongue, and, most surprisingly, in some cells of the epithelial reticulum of bovine and human thymus. By double-label immunofluorescence microscopy, we also show that the expression of the two subsets of cytokeratins, i.e., the epithelial and the trichocytic ones, is not necessarily mutually exclusive, but that certain cells of hair follicles, nail matrix and bed, lingual papillae, and the nonlymphoid cell system of the thymus contain both trichocytic and certain epithelial cytokeratins. This indicates that these cells coexpress representatives of both kinds of cytokeratin. Implications of these findings with respect to problems of regulatory control of cytokeratin synthesis in tissue development and differentiation, and the possible functional meaning of the occurrence of trichocytic cytokeratins in such histologically diverse tissues, are discussed.

Cells forming hair and nail material are characterized by the synthesis of members of a particular group of alpha-keratin polypeptides (trichocytic cytokeratins. "T cytokeratins") different from epithelial cytokeratins ("E cytokeratins"). As the precursor cells to trichocytes are derived from fetal epidermal keratinocytes expressing only E cytokeratins, we have studied the patterns of expression of both T and E cytokeratins in developing human hair-and nail-forming tissues of different fetal stages, by immunocytochemistry using antibodies specific for certain T or E cytokeratins and by two-dimensional gel electrophoresis and immunoblotting. In developing hair follicles up to the early bulbous-peg stage (weeks 12-15 of gestational age), only certain E but no T cytokeratins were identified. T cytokeratins were first detected in the late bulbous-peg stage (in week-14 scalp skin) in certain cells of the central part of the hair cone. In hair-producing follicles (weeks 18-25), the lower hair matrix cells were positive for certain E cytokeratins, whereas T cytokeratins appeared in the uppermost portion of the matrix and, most prominently, in the maturing trichocytes. From the late bulbous-peg stage on. E cytokeratin antibody Ks13.1 selectively decorated the inner root sheath. In finger nail "anlagen", T cytokeratins were detected first in week 12 and 13 fetuses, specifically in cells of the lunula region. In more-advanced stages of nail formation, expression of T cytokeratins extended not only to the upper layers of the ventral nail matrix but was also found, albeit more sparsely, in cells of the whole nail-bed epithelium. Throughout these developmental stages, coexpression of T and E cytokeratins was noted in certain cells, including E cytokeratin 19. While in earlier stages E cytokeratins 10/11, characteristic of epidermal-type cornification, were noted in different regions, including the superficial stratum of the nail bed epithelium, they were later restricted to the epithelium of the proximal nail fold. The results show that terminal trichocytic differentiation starts, both in ontogeny and during the steady growth of hairs and nails, in cells expressing E cytokeratins and that coexpression of E and T polypeptides occurs in both kinds of appendages. While in the hair follicle, the change to the exclusive synthesis of T cytokeratins appears to take place relatively abruptly and simply, the development of nail structures from the ventral nail matrix seems to be more gradual and is characterized by more-complex patterns of coexpression of both kinds of cytokeratins.

Merkel cells are special neurosecretory cells which, in adult human skin, are usually very scarce. By immunofluorescence microscopy using antibodies to human cytokeratin polypeptide no. 18, we localized distinct non-keratinocyte cells in the glandular ridges of human fetal and adult plantar epidermis. Using electron and immunofluorescence microscopy, these cells were identified as Merkel cells containing typical neurosecretory granules as well as bundles of intermediate-sized filaments and desmosomes. Two-dimensional gel electrophoresis of the cytoskeletal fractions of microdissected epidermal preparations highly enriched in Merkel cells indicated the presence of cytokeratin polypeptides nos. 8, 18 and 19 which are typical of diverse simple epithelia of the human body. Double immunofluorescence microscopy showed that these human Merkel cells contain neither neurofilaments nor vimentin filaments. In human fetuses of 18-24 weeks of age, conspicuously high concentrations of Merkel cells, reaching a density of approximately 1,700 Merkel cells/mm2 skin, were found in the glandular ridges of plantar skin. The concentration decreased considerably at newborn and adult stages. Thin cell processes (up to 20 microns long) were observed in many fetal epidermal Merkel cells. In addition, we detected isolated Merkel cells deeper in the dermis (i.e. at distances of, at most, 100 microns from the epidermis) in fetal and newborn plantar skin. Our results show that Merkel cells are true epithelial cells which, however, differ profoundly from epidermal keratinocytes in their cytokeratin expression. The findings are discussed in relation to the much disputed question of the origin of Merkel cells. The present data speak against the immigration of Merkel cells from the neural crest, but rather suggest that they originate from epithelial cells of the skin, although most probably not from differentiated keratinocytes.

Human cytokeratin 13 is one of the most abundant intermediate filament (IF) proteins of many internal stratified epithelia and occurs, at least in certain cell cultures, in an O-glycosylated form binding the lectin, wheat germ agglutinin (WGA). As other groups have reported that, in the mouse, the synthesis of mRNA encoding the 47-kDa cytokeratin corresponding to human cytokeratin 13 is induced in epidermal keratinocytes during malignant transformation, we have examined the synthesis of cytokeratin 13 mRNA and protein in human epidermis and epidermal tumors, using specific cDNA probes and cytokeratin 13 antibodies. We isolated two different cDNA clones from the vulvar carcinoma cell line A-431, in which this protein is abundant: One clone seems to represent the entire mRNA, whereas the other is only a minor component and encodes a truncated cytokeratin 13 lacking most of the carboxy-terminal tail domain, probably a product of alternative, "incorrect" splicing. Comparison of the amino acid sequences with those of other cytokeratins revealed a high degree of conservation with respect to several other human type I cytokeratins, notably cytokeratin 15, and to the murine 47-kDa cytokeratin. When human epidermis and a series of benign and malignant epidermal tumors were examined with these cDNA probes and cytokeratin-13-specific antibodies we did not find an induction of expression in keratinocytes, normal or malignantly transformed, except for some scattered, sparse cytokeratin-13-positive cells and very low levels of cytokeratin 13 mRNA, detectable only with the highly sensitive polymerase chain reaction (PCR). We conclude that the gene(s) encoding cytokeratin 13 are not induced in human keratinocytes during epidermal carcinogenesis, in apparent contrast to reports of murine epidermal tumors, and we discuss possible explanations for this interspecies difference.