Expression of transglutaminase activity in developing human epidermis

Epidermal transglutaminase (TGase) is known to catalyse cross-linking of several precursor proteins in the formation of cornified cell envelope at the terminal differentiation of keratinocytes. Expression of TGase activity was studied using an in situ TGase activity assay in human fetal skin samples of 49-163 days estimated gestational age (EGA). In the early two-layered epidermis (49-56 days EGA), in situ TGase activity was not observed in the periderm cells or the basal cells. In the late two-layered epidermis (57-65 days EGA), in situ TGase activity became weakly positive in the periderm cells, but not in the basal cells. In the three-layered (66-95 days EGA) and in four- or more layered (96-135 days EGA) stratified epidermis, in situ TGase activity was still restricted only to the periderm cells. After keratinization occurred in the interfollicular epidermis (163 days EGA), in situ TGase activity was expressed in the granular and cornified layers. This unique localization of TGase activity further support the hypothesis that periderm cells form cornified cell envelope during their regression process in human fetal skin development.     

Cell surface carbohydrate changes during embryonic and fetal skin development

Monoclonal antibodies to four type 2 chain carbohydrate antigens were used for immunohistochemical studies of embryonic and fetal skin. The antibodies detected N-acetyllactosamine and 3 fucosyl substitutes of this, blood group antigen H, Lex, and Ley. Periderm consistently stained for N-acetyllactosamine, Lex and Ley. The H antigen showed a variable and weak expression on peridermal cells from day 57 to day 84 estimated gestation age (EGA). After this period the H antigen was no longer expressed at peridermal cells. In the epidermis, N-acetyllactosamine was present on all cells until the age of 15 weeks EGA. After this period N-acetyllactosamine could only be demonstrated on basal cells after treatment with neuraminidase, indicating a masking of N-acetyllactosamine by sialic acid. The H antigen could not be demonstrated in the epithelium before 14 weeks EGA. At this time it appeared on spinous and granular cells in the epithelium. Lex stained both basal cells and intermediate cells positively, until keratinization around week 20 EGA. Ley is never expressed on basal cells. It is weakly expressed by intermediate cells from week 14 EGA. Our study demonstrates that N-acetyllactosamine is maximally expressed at the early stages of development, but may later be modified either by sialylation or fucosylation into blood group H or Lex, or by Ley substances, respectively. The orderly and well-defined changes observed during skin differentiation are in agreement with other studies, which have demonstrated the existence of chemically defined cell surface changes accompanying cell differentiation.     

Label-retaining cells in human embryonic and fetal epidermis

Human embryonic and fetal epidermis was examined and labeling indices (LIs) for basal, intermediate, and periderm cells were determined. The LI for fetal basal cells was 8-11% and the LI for fetal intermediate cells was 7.5-9%. The total fetal epidermal LI was 16-20%, which equaled the basal LI for embryonic epidermis. After 21 days in organ culture, only basal cells in the fetal epidermis labeled with tritiated thymidine, while both basal and intermediate cells in the embryonic epidermis labeled and the total LI for fetal and embryonic epidermal cells was the same as the adult epidermal LI (7%). The LI for periderm decreased with increasing estimated gestational age (EGA) from 9.5% at 49 days EGA to 0.4% at 85 days EGA. A subpopulation of epithelial cells that retained tritiated thymidine label and that have some of the attributes associated with stem cells has been previously demonstrated in rodents. In order to examine human embryonic and fetal epidermis for the presence of such cells, epidermis from various gestational ages were labeled and grown in organ culture for 21 days. The mean percent label-retaining cells (LRCs) for embryonic and fetal epidermis was determined. Approximately 4% of the embryonic and 2% of the fetal epidermal cells retained label for 21 days in organ culture. Embryonic LRCs were found in the basal and suprabasal layers, but fetal LRCs were found only in the basal layer. The presence of LRCs in human embryonic and fetal epidermis suggests that epithelial cell proliferation in these tissues may be regulated via a stem cell pattern of proliferation.     

Identification of bullous pemphigoid, pemphigus, laminin, and anchoring fibril antigens in human fetal skin

Human fetal skin was evaluated for sequential and regional development of several epidermal antigens. Indirect immunofluorescent methods were used to detect laminin, bullous pemphigoid antigen, pemphigus antigen, and anchoring fibril antigens identified by monoclonal antibodies AF1 and AF2. Eighty-three human fetal skin biopsies from 32 human fetuses were examined. The fetuses examined ranged from estimated gestational age (EGA) of 7-38 weeks. Laminin was present in the basement membrane zone of all the fetal tissues examined. Bullous pemphigoid antigen developed first in the palm and sole, 9 weeks EGA, and was present in all other sites by 17 weeks EGA. Pemphigus antigen was present by 11 weeks EGA. AF1 and AF2 staining was not present until 26 weeks EGA, AF1 and AF2 stained epidermal basal cells in addition to the basement membrane zone area. Comparison of human fetal skin development with basal cell carcinoma identified similarities between basal cell carcinoma and early fetal development.     

Expression of the small heat shock protein HSP 27 in developing human skin

The 27 kDa heat shock protein (HSP 27) is expressed in keratinocytes of the upper epidermal layers, and recent evidence suggests that this protein is involved in the regulation of epidermal differentiation. The expression of HSP 27 was investigated in developing human skin by immunohistochemistry utilizing a specific monoclonal antibody. We used formalin-fixed, paraffin-embedded tissue of abdominal skin obtained from 34 human fetuses ranging between 13 and 30 weeks estimated gestational age (EGA). We found that HSP 27 is not expressed in keratinocytes until week 14 EGA. At this stage staining is observed in the periderm and the upper intermediate cells but not in hair germs. During further development, HSP 27 expression correlates with increasing epidermal differentiation, i.e. shedding of the periderm and beginning of keratinization. HSP 27 expression is confined to the upper cell layers and sparse basal cells. In hair follicles, HSP 27 can be detected in the innermost cell layer of the outer root sheath and in keratinocytes of the bulge identical to what is observed in adult skin. The hair papilla, matrix cells and sebaceous glands are negative for HSP 27 and remain so during further development. In eccrine sweat glands of the 24th week EGA, HSP 27 is confined to the superficial cell layer of the sweat ducts. In the present report we demonstrate differentiation-related expression of HSP 27 in developing human skin. Further in vitro studies will address the molecular function of HSP 27 in epidermal differentiation and development.     

Prenatal exclusion of harlequin ichthyosis; potential pitfalls in the timing of the fetal skin biopsy

BACKGROUND: Harlequin ichthyosis (HI) is a severe and usually fatal congenital skin disorder with autosomal recessive inheritance. Several cases of HI prenatal diagnosis have been performed using fetal skin biopsy, mainly at around 23 weeks estimated gestational age (EGA), and reported in the literature. However, prenatal testing must be done earlier than 21 weeks EGA in several countries including Japan where the present HI families live, because termination is legally allowed only until 22 weeks EGA. OBJECTIVES: We report the successful prenatal exclusion of HI in two fetuses from two independent families and discuss the technical difficulties and potential pitfalls in the prenatal exclusion of HI at early gestation stages. METHODS: Fetal skin biopsy specimens and amniotic fluid samples at 19 and 20 weeks EGA from two fetuses at risk of HI were examined by light and electron microscopy. RESULTS: For the prenatal diagnosis in case 1, the fetal skin biopsy samples were obtained at 20 weeks EGA and showed normal keratinization in the hair canals; no abnormalities were observed in the keratinized cells. In case 2, the interfollicular epidermis and the hair follicles in the samples obtained at 19 weeks EGA had not differentiated enough to show proper keratinization. However, lamellar granules were normally formed in the inner root sheath cells of the late bulbous hair pegs. From these ultrastructural findings, the case 1 fetus was diagnosed as unaffected with HI, and the case 2 fetus was diagnosed as unlikely to be affected. Subsequently, both were born as healthy, unaffected babies. CONCLUSIONS: The timing of biopsies at 19 weeks EGA is not ideal for fetal skin biopsy because the samples are not always sufficiently differentiated for the prenatal diagnosis of HI. However, morphological observations of lamellar granules gives us important additional information useful for HI prenatal diagnosis.     

Ontogeny and regional variability of keratin 2e (K2e) in developing human fetal skin: a unique spatial and temporal pattern of keratin expression in development

Keratin 2e (K2e) is expressed in the upper spinous and granular cells of adult epidermis. A highly specific polyclonal antibody was made against a C-terminal peptide of K2e and used to observe K2e expression at different developmental stages. At 12.5 weeks estimated gestational age (EGA) K2e was detected in trunk skin in scattered cells in the intermediate layer. At 13.5 weeks EGA, greater numbers of intermediate cells were stained with variable intensity, and staining in this pattern increased with age. Epidermal sheets from 14 weeks EGA showed that K2e + cells were excluded from developing hair follicles. At 135 days EGA, the following regional patterns were observed: in cheek, trunk, dorsal and ventral knee, elbow and dorsal hand there was moderate to intense staining of upper intermediate keratinocytes excluding cells of the hair canals and sweat ducts. The periumbilical region distinctly lacked K2e staining, while more distal areas showed increasing numbers of K2e + cells. The earliest expression of K2e was at 10 weeks EGA in the presumptive nail bed of developing digits. By 13.5 weeks EGA this pattern had shifted to the proximal nail fold, and K2e was absent in the nail bed. K2e was excluded from developing sweat glands and ducts and from developing hair follicles at the hair germ and early peg stages. By 15 weeks EGA in the fetal hair follicle small groups of cells were K2e + and by 19 weeks K2e + cells were seen at the level of the matrix. Some overlap in staining was detected for K2e with K10, and in palmar skin with K9; however, mostly the filamentous staining patterns for these keratins were distinctive. This study shows that the complex patterns of temporal and regional expression of K2e differ from known patterns for other epidermal keratins and suggest different regulation and function for this epidermal keratin.     

Regional development of Langerhans cells and formation of Birbeck granules in human embryonic and fetal skin.

The regional development of Langerhans cells (LC) and the formation of Birbeck granules (BG) were examined in human embryonic and fetal skin. Samples were obtained from multiple anatomic sites and stained with anti-CD36, anti-CD1a, and anti-HLA-DR antibody as well as Lag antibody specifically reactive to BG and some vacuoles of human LC. In the first trimester, CD36+ dendritic epidermal cells were identified before the appearance of CD1a+ cells and Lag+ cells. Some of the former co-expressed HLA-DR antigens but not CD1a antigens. In the second trimester, regional variations in LC development were observed. Epidermal LC of palms and soles reached a peak in number in the first trimester but were rarely detected after 18 weeks estimated gestation age (EGA), whereas, in other regions, their number increased with age. In the second trimester, CD1a+ cells and Lag+ cells were also identified in the epidermis, although Lag+ cells appeared later than CD1a+ cells. The Lag+ cells until 17 weeks EGA showed a variety of staining intensities and immunoelectron microscopy revealed that they contained various amounts of Lag-reactive BG. Flow cytometric analysis showed that relative amounts of Lag antigens in LC increased during the second trimester and that fetal LC of 18 weeks EGA expressed the same amounts of HLA-DR, CD1a, and Lag antigens as did adult human LC. In the dermis, in the second trimester, numerous CD36+ cells and HLA-DR+ cells were found, whereas CD1a+ cells and Lag+ cells were rarely detected. Taken together, it is suggested that HLA-DR+ dendritic cells acquire CD1a+ antigens first and then form BG after migration to the epidermis and that fetal LC are phenotypically mature in the second trimester.     

Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis

Background

NF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes.

Methods

The expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization.

Results

The expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins.

Conclusions

These results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes.     

Changing patterns of localization of putative stem cells in developing human hair follicles

In rodents, the hair follicle stem cells lie in a well-defined bulge in the outer root sheath; however, the bulge as a stem cell site of human hair follicle epithelium is still controversial. Epidermal stem cells are thought to express high levels of beta1 integrin and low levels of E-cadherin and beta- and gamma-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of beta1 integrin subunits, E-cadherin, and beta- and gamma-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells, possible stem cells, were localized to the entire hair germ (65-84 d EGA) and later to the outermost cells of hair peg (85-104 d EGA). In the bulbous hair peg (105-135 d EGA) and in the differentiated lanugo hair follicle (>135 d EGA), they were settled in the bulge and the outermost layer of the outer root sheath. This sequential localization was similar to that of cells rich in epidermal growth factor receptor expression and positive with keratin 19, a putative marker of epidermal stem cells. In addition, these beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells showed similar, undifferentiated morphologic features by electron microscopy. This information of ontogenic localization of possible hair follicle stem cells contributes to the further understanding of mechanisms of human hair follicle morphogenesis and supports the idea that the human fetal hair follicle bulge is a site of stem cells for follicular epithelium.     

Hsp27 expression coincides with epidermal stratification during human epidermal morphogenesis

Heat shock protein 27 (Hsp27), apart from its protective function in response to stress, is implicated in the regulation of cell growth, differentiation and apoptosis. Data on the expression of Hsp27 in the developing human epidermis are sparse and partially conflicting. Thus, the purpose of the present study was to investigate Hsp27 expression during the morphogenesis of human epidermis. Skin biopsies and dispase-separated epidermal sheets obtained from 7 human embryos (7 and 8 weeks estimated gestational age, EGA), from 79 human fetuses (9-23 weeks EGA) and from 10 healthy adult volunteers were investigated by immunohistochemistry and Western blotting, respectively. The earliest detection of Hsp27 expression was found by immunohistochemistry at the 12th week EGA (basal and intermediate layer) and by Western blotting at the 9th week EGA. From the 16th to the 23rd week EGA immunoreactivity was not detectable in the basal layer, whereas in the overlying layers it revealed a differentiation-related pattern. The simultaneous onset of epidermal stratification and Hsp27 expression (9th week EGA) and the alterations of the latter in the subsequent stages of development, suggest that this stress protein may be involved in the molecular events underlying human epidermal morphogenesis.     

Ontogeny of structural components at the dermal-epidermal junction in human embryonic and fetal skin: the appearance of anchoring fibrils and type VII collagen

The ontogeny and composition of the dermal-epidermal junction (DEJ) in developing human embryonic and fetal skin was studied at progressive stages of gestation by immunofluorescence microscopy and immunocytochemistry using transmission electron microscopy (TEM). The DEJ of embryonic skin at 5 weeks estimated gestational age (EGA) was a simple basement membrane zone limited to the basal cell plasma membrane, lamina lucida, and lamina densa. A network of reticular collagen fibrils (reticular lamina) was deposited beneath the lamina densa by 6 weeks. Coincident with the onset of increased complexity in epidermal and dermal structure, at the time of the embryonic to fetal transition, the DEJ displayed additional components that were markers of maturation. At 7-8 weeks EGA, fine filamentous structures extended from the DEJ into the reticular lamina. By 9 weeks EGA hemidesmosomes and banded anchoring fibrils were recognizable, although distributed sparsely at the DEJ. With increasing gestational age, these structures displayed greater electron density and structural completeness. By the end of the first trimester, the DEJ appeared ultrastructurally similar to that of mature skin. Weak immunofluorescent labeling demonstrated the presence of type VII collagen at the DEJ by 8 weeks EGA. From 10-12 weeks EGA immunofluorescent labeling of the DEJ for type VII collagen was distinctly punctate, while immunoperoxidase labeling observed by TEM was linear, continuous, and sublamina densa in position. With ongoing gestation the immunofluorescent labeling became increasingly stronger at the DEJ. Thus, type VII collagen was present at the DEJ in the zone immediately beneath the lamina densa, before the appearance of mature anchoring fibrils but coordinate with the appearance of fine filamentous, unbanded structures, and appeared to increase with the development and accumulation of anchoring fibrils.     

Periderm cells form cornified cell envelope in their regression process during human epidermal development.

Terminally differentiated stratified squamous epithelium forms a lining of the plasma membrane called the cornified cell envelope, a thick layer of several covalently cross-linked precursor proteins including involucrin, small proline-rich proteins, and loricrin. Their cross-linking isodipeptide bonds are formed by epidermal transglutaminases 1-3. Material from lamellar granules is attached on the extracellular surface of corneocytes during the keratinization process. The formation of cornified cell envelope and sequential expression of major cornified cell envelope precursor proteins, transglutaminases, and 25 kDa lamellar granule-associated protein were studied in human embryonic and fetal skin. Ultrastructurally, membrane thickening has already started in periderm cells of the two-layered epidermis and an electron-dense, thickened cell envelope similar to cornified cell envelope in adult epidermis is observed in periderm cells at the three-layered and later stages of skin development. In the two-layered epidermis (49-65 d estimated gestational age), immunoreactivities of involucrin, small proline-rich proteins, all the transglutaminases, and lamellar granule-associated protein were present only in the periderm. In the three-layered epidermis and thereafter (66-160 d estimated gestational age), loricrin became positive in the periderm cells, transglutaminases extended to the entire epidermis, and lamellar granule-associated protein was detected in intermediate cells as well as periderm cells. Immunoelectron microscopy demonstrated that both major cornified cell envelope precursor proteins, involucrin and loricrin, were restricted to the cornified cell envelope in periderm cells at this stage of development. After 160 d estimated gestational age, the periderm had disappeared and cornified cell envelope proteins and lamellar granule-associated proteins were expressed in the spinous, granular, and cornified cells and transglutaminases were detected in the entire epidermis. These findings indicate that cornified cell envelope precursor proteins, transglutaminases, and lamellar granule-associated proteins are expressed in coordination in periderm cells during human epidermal development and suggest that periderm cells form cornified cell envelope in the process of regression.     

Intraepidermal formation of Merkel cells in xenografts of human fetal skin

An experimental transplantation model using human fetal skin was applied to approach the question of the embryologic origin of human Merkel cells. Palmar and plantar skin from five fetuses, between 8 and 11 weeks of estimated gestational age (EGA), was xenografted to subcutaneous beds of nude mice. After 4 or 8 weeks of growth, biopsies were taken from these xenografts and examined for the presence of Merkel cells, using immunocytochemistry with antibodies specific for simple epithelial-type cytokeratins and neuron-specific enolase (NSE) as well as using electron microscopy. Skin from the same fetuses at the time of transplantation was screened in the same way. In all fetuses, no (or very scarce) epidermal Merkel cells were detected at the transplantation time, but in all cases abundant epidermal Merkel cells of apparent human origin were found after 4 or 8 weeks in xenograft culture. Dermal nerve fibers, as recognized by neurofilament antibodies, were scarce or essentially absent in the xenografts. These results indicate that Merkel cells regularly develop in epidermis dissected and xenografted in an early fetal stage, although the dissection implies the interruption of the dermal nerves. The results strongly support the notion of the origin of Merkel cells from epidermal precursor cells. The apparent absence of dermal Merkel cells and dermal nerve fibers in the xenografts suggests that the presence of dermal sensory nerve fibers may be required for the dropping off of epidermal Merkel cells into the upper dermis, which occurs in normal fetal development.     

The fine structure of developing human epidermis: light, scanning, and transmission electron microscopy of the periderm.

Eight stages in the development of the human embryonic and fetal periderm have been established, primarily on the basis of surface morphology, major changes in epidermal stratification, and differentiation. The changes in the periderm observed with the scanning electron microscope have been correlated with and supplemented by cytologic studies with the transmission electron microscope in the periderm and other epidermal layers. Light microscopy was used to determine what proportion of the epidermal thickness is accounted for by the periderm and what relationship individual periderm cells have with underlying cells. The results yield a comprehensive, three-dimensional image of the human epidermis during development and support a concept of the periderm as a layer of "dynamic" cells which project superficial blebs, expand in surface area, then regress at the onset of keratinization, leaving only cellular remnants associated with the adult type epidermis.     

Prenatal diagnosis of lethal junctional epidermolysis bullosa by fetal skin biopsy

A 22-year-old woman, whose first infant had died of lethal junctional epidermolysis bullosa (JEB), requested prenatal diagnosis for her third pregnancy. At 20 weeks gestation, fetal biopsy was performed under direct vision by fetoscopy. A semithin section of epon-embedded skin showed dermo-epidermal separation at the light microscopic level. Electron microscopy revealed the site of separation to be within the lamina lucida of the epidermal basement membrane (EBM). Indirect immunofluorescence on a 5 microns cryostat specimen of skin showed a complete absence of GB3 monoclonal antibody immunostaining at the EBM compared with a control 18 week old normal fetal skin sample. The diagnosis was therefore made that the fetus was affected with lethal JEB and a prostaglandin termination performed. The diagnosis was confirmed by further studies on the aborted fetus. 54 cases of prenatal diagnosis of various types of epidermolysis bullosa performed at Institute of Dermatology over the last 10 years are briefly reviewed. Several social and practical problems to launch prenatal diagnosis in Japan are also discussed.     

Skin lipid content during early fetal development

Although little is known about changes in the lipid composition of the skin during fetal development, information regarding the developmental sequence of fetal skin lipid content could be important for understanding the emergence of epidermal barrier function, as well as providing baseline criteria for prenatal diagnosis of certain inherited disorders of cornification. In these studies, epidermis was separated from dermis in fetal skin samples ranging from 50 to 140 d, estimated gestational ages (EGA), and its lipid composition was analyzed by quartz rod microchromatography/flame-ionization and thin layer chromatography. Lipid biochemical data were correlated with developmental milestones observed by electron microscopy (morphologic studies). The lipid composition of epidermal and dermal fractions from skin samples between 50 and 110 d EGA was similar, with both tissues exhibiting a predominance of free sterols and phospholipids. After 110 d EGA dermis became enriched in triglycerides, corresponding to the progressive development of adipocytes after this time. EGA epidermis after 110 d was enriched not only in triglycerides, but also sterol esters. Moreover, ceramides and glycosphingolipids also became increasingly prominent, changes that were greatest in epidermis from older fetuses and from cephalad regions. These changes in epidermal lipid composition corresponded morphologically to the progressive emergence of both folliculocentric epidermal cornification and sebaceous gland development.     

Ultrastructural localization of binding sites of sera from patients with linear IgA bullous dermatosis

The localization of the antigen recognized by IgA basement membrane zone (BMZ) antibodies from patients with linear IgA bullous dermatosis (LABD) has not been established. The aim of our study was to find out the binding sites for IgA-BMZ antibodies in LABD in adults and children and, for comparison, the binding sites for IgA antibodies in IgA cicatricial pemphigoid (IgA-CP). Our series comprised 21 sera from adult LABD, 4 sera from childhood LABD, and 2 sera from IgA-CP. The studies were performed using the sodium chloride split-skin method and indirect immunoelectron microscopy (IEM) with the use of the pre-embedding immunoperoxidase techinique on two substrates: monkey oesophagus and normal human skin. Of the 27 sera, 24 reacted with the epidermis (19 from adult, 4 from childhood LABD and 1 from IgA-CP) and at the electron microscopic level labelled the upper part of the lamina lucida (LL) and/or hemidesmosomes, and 2 reacted with the dermis (1 from typical adult LABD and 1 from IgA-CP) and labelled the sublamina densa (SLD) region. Two sera were negative in IEM. In conclusion, the study indicated that the localization of the antigens is similar in adult and childhood LABD, and in IgA-CP.Part of this work was presented during the Second Tricontinental Meeting of the Japanese Society for Investigative Dermatology, the Society for Investigative Dermatology and the European Society for Dermatological Research. October 1993, Kyoto, Japan     

Purinergic receptors are part of a signaling system for keratinocyte proliferation, differentiation, and apoptosis in human fetal epidermis

We have investigated the expression of P2X5, P2X7, P2Y1, and P2Y2 receptor subtypes in 8- to 11-wk-old human fetal epidermis in relation to markers of proliferation (proliferating cell nuclear antigen (PCNA) and Ki-67), keratinocyte differentiation (cytokeratin K10 and involucrin), and markers of apoptosis (TdT-mediated dUTP nick end labeling (TUNEL) and anti-caspase-3). Immunohistochemistry showed that each of the four receptors was expressed in spatially distinct zones of the developing epidermis: P2Y1 receptors were found in the basal layer, P2X5 receptors were predominantly in the basal and intermediate layers, and both P2Y2 and P2X7 receptors were in the periderm. Colocalization experiments suggested different functional roles for these receptors. P2Y1 receptors were found in fetal keratinocytes positive for PCNA and Ki-67, suggesting a role in proliferation. P2X5 receptors double labeled with differentiated fetal keratinocytes that were positive for cytokeratin K10, suggesting a role in differentiation. P2X7 receptors colocalized with anti-caspase-3 antibody and were also expressed in periderm cells positive for TUNEL, suggesting a role in periderm cell apoptosis. P2Y2 receptors were found only in periderm cells and may have a role in chloride and fluid secretion into the amniotic fluid.     

Expression profiles of cell-cell and cell-matrix junction proteins in developing human epidermis

Abstract The spatial distribution of a panel of cell junction proteins was studied in developing human epidermis by confocal laser scanning microscopy. The results demonstrated that many of the cell junction proteins were expressed in early two-layered embryonic epidermis, but their subcellular distribution displayed marked changes during development. Specifically, desmosomal proteins, desmoplakin, desmocollin and desmoglein, adherens junction components, E-cadherin, ·-catenin and vinculin, and an actin-binding protein ·-actinin were expressed as early as 8 weeks of estimated gestational age (EGA). Type IV collagen and ‚1 and ‚4 integrins were also present. At this early developmental stage, the epidermis is known to comprise two layers of cells, the basal layer and the peridermal layer. In addition to being present in cell-cell contacts, desmosomal antigens and E-cadherin were unexpectedly localized to the basal aspect of basal cells in samples at 8 weeks. On the other hand, talin, which in adult skin is localized to the dermal-epidermal junction, could not be detected until 12 weeks. These results suggest that the separation of cell membranes to the basal and apicolateral compartments does not occur before the maturation of the basement membrane zone. At 8 weeks EGA, gap junction antigen connexin 43 was expressed in scarce spots in cell-cell contacts of basal cells. In samples of 11–21 weeks EGA, the density of desmosomal and adherens junction markers as well as connexin 43 increased in cell-cell junctions, together with the appearance of the intermediate cell layer and beginning of stratification of the epidermis. Received: 15 May 2000 / Revised: 7 August 2000 / Accepted: 1 February 2001