Fine structure and immunocytochemistry of monotreme hairs, with emphasis on the inner root sheath and trichohyalin-based cornification during hair evolution

The fine structure of hairs in the most ancient extant mammals, the monotremes, is not known. The present study analyzes the ultrastructure and immunocytochemistry for keratins, trichohyalin, and transglutaminase in monotreme hairs and compares their distribution with that present in hairs of the other mammals. The overall ultrastructure of the hair and the distribution of keratins is similar to that of marsupial and placental hairs. Acidic and basic keratins mostly localize in the outer root sheath. The inner root sheath (IRS) comprises 4-8 cell layers in most hairs and forms a tile-like sheath around the hair shaft. No cytological distinction between the Henle and Huxley layers is seen as cells become cornified about at the same time. Externally to the last cornified IRS cells (homologous to the Henle layer), the companion layer contains numerous bundles of keratin. Occasionally, some granules in the companion layer show immunoreactivity for the trichohyalin antibody. This further suggests that the IRS in monotremes is ill-defined, as the companion layer of placental hairs studied so far does not express trichohyalin. A cross-reactivity with an antibody against sheep trichohyalin is present in the IRS of monotremes, suggesting conserved epitopes across mammalian trichohyalin. Trichohyalin granules in the IRS consist of a framework of immunolabeled coarse filaments of 10-12 nm. The latter assume a parallel orientation and lose the immunoreactivity in fully cornified cells. Transglutaminase immunolabeling is diffuse among trichohyalin granules and among the parallel 10-12 nm filaments of maturing inner root cells. Transglutaminase is present where its substrate, trichohyalin, is modified as matrix protein. Cornification of IRS is different from that of hair fiber cuticle and from that of the cornified layer of the epidermis above the follicle. The different consistency among cuticle, IRS, and corneous layer of the epidermis determines separation between hair fiber, IRS, and epidermis. This allows the hair to exit on the epidermal surface after shedding from the IRS and epidermis. Based on comparative studies of reptilian and mammalian skin, a speculative hypothesis on the evolution of the IRS and hairs from the skin of synapsid reptiles is presented.     

The mutant gene wellhaarig disturbs the differentiation of hair follicle cells in the mouse]

First generation (G1) hairs in mice homozygous for the wellhaarig (we) gene are wavy and shorter than in normal mice; basal regions of the hairs are deformed. Follicles of G1 hairs in mid-dorsal region of 8-, 12- and 16-days old we/we mice were examined. Huxley cells of the inner root sheath (IRS) in apical region of hair follicles appeared to be hypertrophied. Cytoplasm of these cells was not stained by basic dyes and showed no birefringence. Cytoplasm of the IRS Henle cells was not stained by basic dyes either. These data indicate that keratinization of the IRS cells is disturbed in mutant homozygotes. The layer of outer root sheath in the we/we mice was thinner than in normal mice; this is probably due to hypertrophy of the IRS cells. The structure of differentiating cells of the hair shaft in normal and mutant mice was similar. The data obtained suggest that abnormal G1 hairs in we/we mice result from disturbance in IRS cells differentiation.     

Involucrin, but not filaggrin and Kdap mRNA, expression is downregulated in 3-D cultures of intact rat hair bulbs after calcium stimulation

The hair follicle consists of several distinctive epidermal cell layers. The hair root, which undergoes keratinization, is surrounded by two sheaths: the inner root sheath (IRS) and the outer root sheath (ORS). The ORS is continuous with the basal layer of the epidermis. Its cells do not keratinize in situ, unlike IRS. We have previously demonstrated that keratinization of the ORS was prevented by contact with the IRS in hair follicle mid-segments (i.e. fragments dissected from skin at the level above the hair bulb and below the opening of the sebaceous gland duct) cultured on agarose layer. The purpose of this study was to determine whether the same applies to the hair bulb. After isolation, intact bulbs or hair bulb-derived cells were incubated in suspension in a low or high calcium medium. The level of mRNA for differentiation markers: involucrin, filaggrin, keratinocyte differentiation associated protein and trichohyalin, was studied by RealTime PCR. We observed increased Ca(2+) upregulated expression of involucrin, filaggrin, trichohyalin and Kdap in cultures of bulb-derived cells, but in hair bulbs downregulation of involucrin and trichohyalin was observed. We concluded that the inner root sheath exerts an inhibitory effect on the expression of involucrin and trichohyalin already in the hair bulbs. The observation that downregulation of involucrin expression under Ca(2+) influence occurs both in hair bulb and midsegments could simplify future experiments, since their separation does not seem to be necessary.     

Distribution of keratin and associated proteins in the epidermis of monotreme,marsupial, and placental mammals

The expression of acidic and basic keratins, and of some keratinization marker proteins such as filaggrin, loricrin, involucrin, and trichohyalin, is known for the epidermis of only a few eutherian species. Using light and high-resolution immunocytochemistry, the presence of these proteins has been studied in two monotreme and five marsupial species and compared to that in eutherians. In both monotreme and marsupial epidermis lamellar bodies occur in the upper spinosus and granular layers. Development of the granular layer varies between species and regionally within species. There is great interspecific variation in the size (0.1-3.0 microm) of keratohyalin granules (KHGs) associated with production of orthokeratotic corneous tissues. Those skin regions lacking hairs (platypus web), or showing reduced pelage density (wombat) have, respectively, minute or indiscernible KHGs, associated with patchy, or total, parakeratosis. Ultrastructural analysis shows that monotreme and marsupial KHGs comprise irregular coarse filaments of 25-40 nm that contact keratin filaments. Except for parakeratotic tissues of platypus web, distribution of acidic and basic proteins in monotreme and marsupial epidermis as revealed by anti-keratin antibodies AE1, AE2, and AE3 resembles that of eutherian epidermis. Antibodies against human or rat filaggrins have little or no cross-reactivity with epidermal proteins of other mammals: only sparse areas of wombat and rabbit epidermis show a weak immunofluorescence in transitional cells and in the deepest corneous tissues. Of the available, eutherian-derived antibodies, that against involucrin shows no cross-reactivity with any monotreme and marsupial epidermal tissues and that against trichohyalin cross-reacts only with cells in the inner root sheath and medulla of hairs. These results suggest that if involucrin and trichohyalin are present throughout noneutherian epidermis, they may have species-specific molecular structures. By contrast, eutherian-derived anti-loricrin antibodies show a weak to intense cross-reactivity to KHGs and corneous tissues of both orthokeratotic and parakeratotic epidermis in monotremes and marsupials. High-resolution immunogold analysis of loricrin distribution in immature keratinocytes of platypus parakeratotic web epidermis identifies labeled areas of round or irregular, electron-pale granules within the denser keratohyalin component and keratin network. In the deepest mature tissues, loricrin-like labeling is diffuse throughout the cytoplasm, so that cells lack the preferential distribution of loricrin along the corneous envelope that characterizes mature eutherian keratinocytes. Thus, the irregular distribution of loricrin in platypus parakeratotic tissues more resembles that which has been described for reptilian and avian keratinocytes. These observations on the noneutherian epidermis show that a stratum granulosum is present to different degrees, even discontinuous within one tissue, so that parakeratotic and orthokeratotic areas may alternate: this might imply that parakeratotic monotreme epidermis reflects the primitive pattern of amniote alpha-keratogenesis. Absent from anamniote epidermis and all sauropsid beta-keratogenic tissues, the ubiquitous presence of a loricrin-like protein as a major component of other amniote corneous tissues suggests that this is a primitive feature of amniote alpha-keratogenesis. The apparent lack of specific regionalization of loricin near the plasma membranes of monotreme keratinocytes could be an artifactual result of the immunofluorescence technique employed, or there may be masking of the antigenicity of loricrin-like proteins once they are incorporated into the corneous envelope. Nevertheless, the mechanism of redistribution of such proteins during maturation of monotreme keratinocytes is different from, perhaps more primitive, or less specialized, than that in the epidermis of eutherian mammals.     

Trichohyalin mechanically strengthens the hair follicle: multiple cross-bridging roles in the inner root shealth

Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks.     

Analysis of the sheep trichohyalin gene: potential structural and calcium-binding roles of trichohyalin in the hair follicle

Trichohyalin is a structural protein that is produced and retained in the cells of the inner root sheath and medulla of the hair follicle. The gene for sheep trichohyalin has been purified and the complete amino acid sequence of trichohyalin determined in an attempt to increase the understanding of the structure and function of this protein in the filamentous network of the hardened inner root sheath cells. Sheep trichohyalin has a molecular weight of 201,172 and is characterized by the presence of a high proportion of glutamate, arginine, glutamine, and leucine residues, together totaling more than 75% of the amino acids. Over 65% of trichohyalin consists of two sets of tandem peptide repeats which are based on two different consensus sequences. Trichohyalin is predicted to form an elongated alpha-helical rod structure but does not contain the sequences required for the formation of intermediate filaments. The amino terminus of trichohyalin contains two EF hand calcium-binding domains indicating that trichohyalin plays more than a structural role within the hair follicle. In situ hybridization studies have shown that trichohyalin is expressed in the epithelia of the tongue, hoof, and rumen as well as in the inner root sheath and medulla of the hair follicle.     

The organization of the keratin I and II gene clusters in placental mammals and marsupials show a striking similarity

The genomic database for a marsupial, the opossum Monodelphis domestica, is highly advanced. This allowed a complete analysis of the keratin I and keratin II gene cluster with some 30 genes in each cluster as well as a comparison with the human keratin clusters. Human and marsupial keratin gene clusters have an astonishingly similar organization. As placental mammals and marsupials are sister groups a corresponding organization is also expected for the archetype mammal. Since hair is a mammalian acquisition the following features of the cluster refer to its origin. In both clusters hair keratin genes arose at an interior position. While we do not know from which epithelial keratin genes the first hair keratins type-I and -II genes evolved, subsequent gene duplications gave rise to a subdomain of the clusters with many neighboring hair keratin genes. A second subdomain accounts in both clusters for 4 neighboring genes encoding the keratins of the inner root sheath (irs) keratins. Finally the hair keratin gene subdomain in the type-I gene cluster is interrupted after the second gene by a region encoding numerous genes for the high/ultrahigh sulfur hair keratin-associated proteins (KAPs). We also propose a tentative synteny relation of opossum and human genes based on maximal sequence conservation of the encoded keratins. The keratin gene clusters of the opossum seem to lack pseudogenes and display a slightly increased number of genes. Opossum keratin genes are usually longer than their human counterparts and also show longer intergenic distances.     

Perspectives on hair evolution based on some comparative studies on vertebrate cornification

Hair evolution contributed to the biological success of mammals. Hair origin from synapsid scales is speculative and requires extensive modifications of the morphogenetic process transforming lens-shaped dermis of scales into small dermal papillae in hair. Hair evolution from glands is hypothetical but is supported from studies on the signaling control of hair vs. glandular morphogenesis. Based on immunocytochemical and comparative studies, it is hypothesized that the onion-like organization of hair derived from glandular pegs which central part produced lipids and some keratin. In a following stage, involucrin, trichohyalin, and keratins were produced in the central cells of the gland and formed a solid medulla surrounded by keratinocytes of the inner root sheath. The origin of this protohair was possibly related to increased concentration of beta-catenin and other signaling molecules in epithelial cells following the evolution of a dermal papilla. The latter activated the keratogenic genes, already utilized in cells of the claws, in concentric layers of cells of the glandular peg. Lipidogenic genes were depressed. As new genes evolved in the genome of synapsids, new circular layers of keratinocytes containing specialized hard keratins and keratin-associated proteins were formed around medullary cells. The new keratinocytes probably originated the cortex separating medulla from the external cells that became the inner root sheath. The hypothesis indicates that in a following stage, the medulla was obliterated or replaced by cortical cells while the external part of the cortex formed a cuticular surface due to the different growth rate with inner root sheath cells.     

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis

The claw of lizards is largely composed of beta‐keratins, also referred to as keratin‐associated beta‐proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta‐keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha‐keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha‐keratins resembled that of beta‐keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

常见毛囊细胞角蛋白在毛囊周期中的表达研究

目的探讨常见毛囊细胞角蛋白在毛囊周期中的表达特征。 方法取毛囊发育期、生长期启动、生长期、退化期和静止期的小鼠皮肤,石蜡切片后通过免疫荧光的方法,检测细胞角蛋白Krt5、Krt6、Krt10、Krt14、Krt15和Krt19的表达情况。 结果Krt5在静止期和生长期启动表达于所有毛囊上皮细胞,在其他时期表达不一致;Krt6表达于所有时期的外根鞘细胞和内根鞘细胞;Krt10表达于生长期和退化期的毛母质和内根鞘细胞,在其他时期表达不一致;Krt14在生长期和退化期表达于所有毛囊上皮细胞,在其他时期表达不一致;Krt15和Krt19表达于毛囊发育期、生长期启动和静止期的毛囊隆突区细胞,在生长期和退化期表达不一致。 结论角蛋白作为毛囊结构或毛囊干细胞标记物仅适用于特定的毛囊周期。研究者在使用毛囊角蛋白作为标记物时,应首先明确其在毛囊周期中的表达情况。     

Trichohyalin, an intermediate filament-associated protein of the hair follicle

A precursor protein associated with the formation of the citrulline-containing intermediate filaments of the hair follicle has been isolated and characterized. The protein, with a molecular weight of 190,000, was isolated from sheep wool follicles and purified until it yielded a single band on a SDS polyacrylamide gel. The Mr 190,000 protein has a high content of lysine and glutamic acid/glutamine residues and is rich in arginine residues, some of which, it is postulated, undergo a side chain conversion in situ into citrulline residues. Polyclonal antibodies were raised to the purified protein, and these cross-react with similar proteins from extracts of guinea pig and human follicles and rat vibrissae inner root sheaths. Tissue immunochemical methods have localized the Mr 190,000 protein to the trichohyalin granules of the developing inner root sheath of the wool follicle. We propose that the old term trichohyalin be retained to describe this Mr 190,000 protein. Immunoelectron microscopy has located the Mr 190,000 protein to the trichohyalin granules but not to the newly synthesized filaments. This technique has revealed that trichohyalin becomes associated with the filaments at later stages of development. These results indicate a possible matrix role for trichohyalin.     

Expression of keratin K14 in the epidermis and hair follicle: insights into complex programs of differentiation

Keratins K14 and K5 have long been considered to be biochemical markers of the stratified squamous epithelia, including epidermis (Moll, R., W. Franke, D. Schiller, B. Geiger, and R. Krepler. 1982. Cell. 31:11-24; Nelson, W., and T.-T. Sun. 1983. J. Cell Biol. 97:244-251). When cells of most stratified squamous epithelia differentiate, they downregulate expression of mRNAs encoding these two keratins and induce expression of new sets of keratins specific for individual programs of epithelial differentiation. Frequently, as in the case of epidermis, the expression of differentiation-specific keratins also leads to a reorganization of the keratin filament network, including denser bundling of the keratin fibers. We report here the use of monospecific antisera and cRNA probes to examine the differential expression of keratin K14 in the complex tissue of human skin. Using in situ hybridizations and immunoelectron microscopy, we find that the patterns of K14 expression and filament organization in the hair follicle are strikingly different from epidermis. Some of the mitotically active outer root sheath (ORS) cells, which give rise to ORS under normal circumstances and to epidermis during wound healing, produce only low levels of K14. These cells have fewer keratin filaments than basal epidermal cells, and the filaments are organized into looser, more delicate bundles than is typical for epidermis. As these cells differentiate, they elevate their expression of K14 and produce denser bundles of keratin filaments more typical of epidermis. In contrast to basal cells of epidermis and ORS, matrix cells, which are relatively undifferentiated and which can give rise to inner root sheath, cuticle and hair shaft, show no evidence of K14, K14 mRNA expression, or keratin filament formation. As matrix cells differentiate, they produce hair-specific keratins and dense bundles of keratin filaments but they do not induce K14 expression. Collectively, the patterns of K14 and K14 mRNA expression and filament organization in mitotically active epithelial cells of the skin correlate with their relative degree of pluripotency, and this suggests a possible basis for the deviation of hair follicle programs of differentiation from those of other stratified squamous epithelia.     

Functional structure of the carpal and ventral vibrissae of the squirrel (Sciurus vulgaris)

A study is made of the histology, distribution and ultrastructure of nerve-organs in the carpal and ventral sinus hairs of the squirrel Sciurus vulgaris L. These sinus hairs were found to be typical tactile hairs. Their situation on the inner surfaces of the forelimbs, and on the ventral body wall is assumed to be useful during climbing. Five different types of nerve end-organs were found:
1. Ring-shaped end-organs, especially in the ring sinus, associated with certain cells in connectivetissue and in many cases also anchored to the hair follicle.
2. Merkelcells in the basal cell layer of the outer root sheath at the level of the ring sinus.
3. Lanceolate end-organs between the glassy membrane and the connective tissue of the ringwulst and betweenthese in the upper part of the ring sinus.
4. Encapsulated end-organs in the cavernous sinus.
5. Free thin nerveendings.     

Immunolocalization of large corneous beta‐proteins in the green anole lizard (Anolis carolinensis) suggests that they form filaments that associate to the smaller beta‐proteins in the beta‐layer of the epidermis

The distribution of large corneous beta‐proteins of 18–43 kDa (Ac37, 39, and 40) in the epidermis of the lizard Anolis carolinensis is unknown. This study analyses the localization of these beta‐proteins in different body scales during regeneration. Western blot analysis indicates most protein bands at 40–50 kDa suggesting they mix with alpha‐keratin of intermediate filament keratin proteins. Ac37 is present in mature alpha‐layers of most scales and in beta‐cells of the outer scale surface in some scales but is absent in the Oberhäutchen, in the setae and beta‐layer of adhesive pads and in mesos cells. In differentiating beta‐keratinocytes Ac37 is present over 3–4 nm thick filaments located around the amorphous beta‐packets and in alpha‐cells, but is scarce in precorneous and corneous layers of the claw. Ac37 forms long filaments and, therefore, resembles alpha‐keratins to which it probably associates. Ac39 is seen in the beta‐layer of tail and digital scales, in beta‐cells of regenerating scales but not in the Oberhäutchen (and adhesive setae) or in beta‐ and alpha‐layers of the other scales. Ac40 is present in the mature beta‐layer of most scales and dewlap, in differentiating beta‐cells of regenerating scales, but is absent in all the other epidermal layers. The large beta‐proteins are accumulated among forming beta‐packets of beta‐cells and are packed in the beta‐corneous material of mature beta‐layer. Together alpha‐keratins, large beta‐proteins form the denser areas of mature beta‐layer that may have a different consistence that the electron‐paler areas. J. Morphol. 276:1244–1257, 2015. © 2015 Wiley Periodicals, Inc.     

Keratins of the human hair follicle: "Hyperproliferative''keratins consistently expressed in outer root sheath cells in vivo and in vitro

Keratins produced by morphologically distinct compartments of the human hair folicle (hHF) were analysed and compared to those produced by cultured hHF and interfollicular keratinocytes. Five of the major keratins, the basic keratins nos. 5 and 6 (apparent mol. mass 60 and 58 kDa) and the acidic keratins nos. 14, 16, and 17 (51, 49 and 48 kDa), could be labelled in intact hHF and were found in all fractions of the outer root sheath (ORS). The other major keratins, which were not labelled under these conditions (basic-neutral hHbI and -II; 60-62 kDa and acidic hHaI and -II; 40-42 kDa) were associated with hair shaft (hHS) both in the follicle and, virtually unchanged, in the distal part of the hair. Another, previously undescribed, group of proteins with keratin-like properties exhibiting a broad pI-spectrum (basic to slightly acidic: hIC-I, -II, -III, 64-67 kDa; distinctly acidic: hIC-IV, about 54 kDa) was detected in isolated inner root sheath (IRS), in the cuticular material shed from denuded hHS, and also in nail plates. In our experiments only ORS cells grew readily in culture irrespective of their origin from peripheral (mesenchyme-adjacent) or more central ORS-cell layers. In contrast to keratinocytes from interfollicular epidermis (IFE) the cultured ORS cells expressed a keratin set virtually identical to that expressed in vivo. This set also closely resembled that expressed by IFE keratinocyte cultures. The identity of the respective keratins (nos. 5, 6, 14, 16, and 17) present in all these cells in vivo and in vitro was confirmed by tryptic peptide mapping. The data indicated that the microenvironment (in situ) directs the differentiation of ORS cells in a manner comparable to the way it is directed by conventional culture conditions, with consistent expression of the "basal" and "hyperproliferative" set of keratins. This, however, does not exclude the possibility that other types of environmentally induced response may occur, as seen for example during the reepithelialization of superficial skin wounds by ORS cells.     

The origin of citrulline-containing proteins in the hair follicle and the chemical nature of trichohyalin, an intracellular precursor.

The present studies have demonstrated that the medulla and inner root sheath cells develop within their cytoplasm a protein that is unique in composition and is present in the trichohyalin granules. The protein is rich in arginine residues, some of which undergo a side-chain conversion in situ into citrulline residues. An unusual Ca2+-dependent enzyme activity distinguishable from cross-linking transamidase has been detected in the hair follicle and will act in vitro on trichohyalin protein as the natural substrate. The conversion in vivo must occur during the time that the medullary and inner root sheath cells move up the follicle and their cytoplasm fills with cross-linked protein containing citrulline. The function of citrulline in these proteins is not understood but its formation is a major process during hair growth.     

Hair follicular cell/organ culture in tissue engineering and regenerative medicine

Hair follicles are complex organs composed of the dermal papilla (DP), dermal sheath (DS), outer root sheath (ORS), inner root sheath (IRS) and hair shaft. Development of hair follicles begins towards the end of the first trimester of pregnancy and is controlled by epidermal–mesenchymal interaction (EMI), which is a signaling cascade between epidermal and mesenchymal cell populations. Hair grows in cycles of various phases. Specifically, anagen is the growth phase, catagen is the involuting or regressing phase and telogen is the resting or quiescent phase. Alopecia is not life threatening, but alopecia often causes severe mental stress. In addition, the number of individuals afflicted by alopecia patients has been increasing steadily. Currently there are two methods employed to treat alopecia, drug or natural substance therapy and human hair transplantation. Although drug or natural substance therapy may retard the progress of alopecia or prevent future hair loss, it may also accelerate hair loss when the medication is stopped after prolonged use. Conversely, the transplantation of human hair involves taking plugs of natural hair from areas in which occipital hair is growing and transplanting them to bald areas. However, the number of hairs that can be transplanted is limited in that only three such operations can generally be performed. To overcome such problems, many researchers have attempted to revive hair follicles by culturing hair follicle cells or mesenchymal cells in vitro and then implanting them in the treatment area.     

Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins

Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co- expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.     

Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. Skin morphogenesis

Expression and the role of E- and P-cadherin in the histogenesis of the surface epidermis and hair follicles were examined using the upper lip skin of the mouse. P-cadherin is expressed exclusively in the proliferating region of these tissues, that is in the germinative layer of the surface epidermis, the outer root sheath and the hair matrix. E-cadherin is coexpressed in these layers but this molecule was also detected in non-proliferating regions such as the intermediate layer of the surface epidermis and the immature regions of the inner root sheath. Neither P- nor E-cadherin was detected in fully keratinized layers such as the horny layer of the surface epidermis, the outermost layer of the outer root sheath and the mature hair fibres. These two cadherins were not detected in dermal cells. We cultured pieces of the upper lip skin in vitro in the absence or presence of a monoclonal antibody to E-cadherin (ECCD-1) or to P-cadherin (PCD-1). In control cultures, skin morphogenesis normally occurred in a pattern whereby the hair follicles grew and dermal cells were condensed to form the dermal sheath. A mixture of ECCD-1 and PCD-1, however, induced abnormal morphogenesis in the skin in several respects. (1) The cuboidal or columnar arrangement of basal epithelial cells was distorted. (2) Hair follicles were deformed. (3) Condensation of dermal cells was suppressed, causing a homogeneous distribution of these cells. These results suggest that cadherins present in epidermal cells are involved not only in maintaining the arrangement of these cells but also in inducing dermal condensation.