Expression of the 43 kDa papain inhibitor during human fetal skin development

The presence of the 43 kDa papain inhibitor protein in the skin of 40 fetuses with the gestational age varying from 9 to 40 weeks was studied. Immunohistochemistry showed the first evidence of the 43 kDa papain inhibitor in the acral parts, mostly in the nail bed, at the 14th week when the epidermis in all other sites was not stained. Staining of the follicular infundibulum as well as some parts of the uppermost epidermis and keratin layer was observed from 17 to 40 weeks. The staining was most intensive at 20-30 weeks gestational age, and later the intensity gradually decreased so that in the full-term newborn the 43 kDa papain inhibitor was hardly detectable or absent. It was concluded that the 43 kDa papain inhibitor is present in fetal skin at the stage of stratification (9-14 weeks) only in the nail region and its volar surroundings. It appears at the stage of interfollicular keratinization (14-24 weeks) in the uppermost epidermis, especially in the appendical openings and its amount seems the decrease during the stage of interfollicular keratinization (24 weeks--full-term). The expression of 43 kDa inhibitor protein is temporally related to that of filaggrin.     

Fibronectin distribution in epithelial and associated tissues of the rat

Summary Specific antiserum was used to investigate the distribution of the extracellular glycoprotein, fibronectin, in rat skin and tongue tissue by light and electron microscopy with immunofluorescence and immunoperoxidase techniques. We conclude that fibronectin is absent from stable, differentiated parts of tissues, such as the sebaceous glands or the matrix, medulla, cortex, and cuticles of the hair and the inner and outer root sheaths, or even in tissues in which there is some cell movement, such as the epidermis. It is, however, characteristic of sites at which cell division is occurring in contact with an extracellular scaffolding, such as basement membrane or loose connective tissue. Conspicuous examples were in the glassy membrane and connective tissue sheath associated with the follicular epithelium, the basement membrane underlying vascular endothelial cells, the connective tissues surrounding and investing nerve and muscle fibre bundles, and the dermal connective tissue where fibronectin was often associated closely with collagen fibres.At the basement membrane of the dermal/epidermal junction, fibronectin occurred at the plasma membrane of the basal cells and in the lamina lucida area. There was no correlation with specific areas of cell-substrate adhesion, such as the hemidesmosomes. The endoplasmic reticulum of fibroblasts stained strongly suggesting that these cells represent a major site of synthesis.     

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.     

Cutaneous lipid synthesis during late fetal development in the rat

Lipid synthesis in fetal skin may be important both for the development of a mature epidermal permeability barrier and for growth. In these studies, we measured cutaneous cholesterol, sphingolipid and fatty acid synthesis during the critical period of epidermal barrier development in fetal rats to determine whether barrier function influences synthetic rates. In addition, the activities of HMG CoA reductase, serine palmitoyl transferase and acetyl coenzyme A carboxylase were evaluated. In whole skin, synthesis of cholesterol, ceramide, sphingomyelin and fatty acid decreased from day 17 to day 21 of gestation, as did the activity of HMG CoA reductase, serine palmitoyl transferase and acetyl coenzyme A carboxylase. In both the epidermis and dermis, a decrease in cholesterol, ceramide, sphingomyelin and fatty acid synthesis was measured over days 19–21 of gestation. Epidermal HMG CoA reductase activity also decreased over this same time period. In summary, epidermal and dermal synthetic rates and enzyme activity were highest early in gestation when the barrier was least competent and decreased as competence was achieved. Since other studies with mature animals have revealed that epidermal synthetic rates and enzyme activity are highest when barrier disruption is maximal, enhanced epidermal lipid synthesis precedes the estabilishment of a competent barrier in both fetal and mature rodents.     

Formation of epidermal and dermal Merkel cells during human fetal skin development

The origin of Merkel cells is still a matter of debate, specifically the question of whether they are derived from epithelial cells of the epidermis or from immigrated neural crest cells. As an argument for the latter hypothesis the occurrence of dermal, nerve-associated Merkel cells in human fetal skin has often been mentioned. Therefore, we analyzed the distribution of Merkel cells in epidermis and dermis of plantar skin of human embryos and fetuses, ranging in gestational age between 7 and 17 weeks. Merkel cells were identified by immunocytochemistry on frozen sections using antibodies against simple epithelium-type cytokeratins and by electron microscopy. In the 17-week-old fetus, 17% of the total cutaneous (epidermal and dermal) Merkel cells were located in the upper dermal compartment, whereas in the 14-week-old fetus only 3.9% of the Merkel cells were dermal, including some cells that seemed to be in the process of traversing the dermal-epidermal junction. Thirteen-week-old fetuses showed even fewer dermal Merkel cells. Twelve-week-old fetuses exhibited 660 epidermal Merkel cells per 100 mm total section length, but none in the upper or deep dermis. In 7- to 9-week embryos, no Merkel cells were recognized. However, at this stage, but not in later stages, the basal cells of the plantar epidermis expressed certain simple epithelium-type cytokeratin polypeptides. These results speak against an invasion of Merkel cells or putative neural crest-derived precursor cells into the epidermis via a dermal passage. They suggest that in plantar skin Merkel cells arise, between weeks 8-12, from precursor stages of epithelial cells of the early fetal epidermis which still express simple epithelium-type cytokeratins. The results further suggest that in subsequent stages of skin development some epidermal Merkel cells detach from the epithelium and migrate into the upper dermis where some of them may associate with small nerves.     

Relationship of adhesion molecules expression with epithelial differentiation markers during fetal skin development

Background:  Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV).
Methods:  Using immunohistochemistry expression of E- and P-cadherins, integrins beta-1 and -4, CK10, CK14 and INV was assessed in skin fragments of 10 human fetuses (gestational weeks ranged from 4 to 24, all weighing up to 500 g).
Results:  At initial phases of development, integrins beta-1 and -4 and E- and P-cadherins were present on epithelial cell membranes in all layers. CK14 and CK10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E-cadherin was detected in all layers, but the cornified stratum and P-cadherin were observed in the lower layers. CK14 was expressed in basal layer, CK10 in suprabasal stratum and INV was observed in cornified layer.
Conclusions:  Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.     

Expression of macrophage migration inhibitory factor in rat skin during embryonic development

Abstract:  We have previously shown that human epidermal keratinocytes express macrophage migration inhibitory factor (MIF) mRNA, and immunohistochemical studies showed that MIF is expressed in human epidermis. To explore the possible pathophysiological roles of MIF in skin during rat fetal development, we examined the expression patterns of MIF during rat epidermal development using Northern blot analysis and in situ hybridization. Expression of MIF mRNA was first detected by in situ hybridization in the developing epidermis and hair germ cells from embryonic day (ED) 16. From ED 19, moderate levels of MIF expression were detected in the epidermis and epithelial sheath cells of growing hair follicles. In postnatal rat skin, higher MIF expression was detected in the epidermis and hair follicles on postnatal day 3. These observations were also confirmed by Northern blot analysis. Immunohistochemical analysis with an anti-MIF antibody showed a similar distribution to that of the mRNA. Our results suggest that MIF is associated with epidermal and hair follicle development.     

Expression of caspase-14 and keratin-19 in the human epidermis and appendages during fetal skin development

Caspase-14 is a seemingly non-apoptotic caspase involved in keratinocyte differentiation and cornification of the skin. Keratin-19 is an epithelial marker and a potential marker of epidermal stem cells that is expressed during human fetal skin development. We examined the immunohistochemical expression of caspase-14 in relation to CK-19 in the human fetal skin during development and perinatally, to assess their role in human skin maturation. Skin samples were received at autopsy. In the fetal epidermis, caspase-14 was predominantly expressed in the more differentiated layers, gradually disappearing from the basal layer toward term. By contrast, keratin-19 expression gradually decreased with epidermal maturation through gestation (rho = ?0.949; p = 0.0001) and was a marker of the germinative layers. Keratin-19 was preserved in scarce basal cell nests at term and postnatally. Caspase-14 and keratin-19 were inversely expressed in the differentiating epidermal layers through gestation (p < 0.0001). Concerning the appendages, in hair follicles and sebaceous glands, caspase-14 located preferentially in the more differentiated layers of the inner root sheath, whereas keratin-19 was expressed in the outer sheath. Eccrine sweat glands showed a variable pattern of caspase-14 and keratin-19 expression. In conclusion, caspase-14 emerged as a marker of human skin differentiation during development, while keratin-19 marked the germinative epithelial layers in the fetal epidermis and appendages and possibly the nests of epidermal stem cells.     

Fibronectin in the skin of patients with allergic dermatitis

In the skin of healthy subjects linear deposits were found of fibronectin at the dermoepidermal junction and in the walls of blood vessels, and reticular deposits in the papillary and reticular layers of the skin. In the epidermis of 12 out of 51 patients with allergic contact dermatitis fibronectin deposits were found in relation to Langerhans cells and keratinocytes. This was not observed in the epidermis in healthy subjects. The authors suggest that fibronectin on the surface of Langerhans cells and keratinocytes may play a role in antigen presentation.     

Fibronectin expression determines skin cell motile behavior

Mouse keratinocytes migrate significantly slower than their human counterparts in vitro on uncoated surfaces. We tested the hypothesis that this is a consequence of differences in the extracellular matrix (ECM) that cells deposit. In support of this, human keratinocyte motility was markedly reduced when plated onto the ECM of mouse skin cells, whereas the latter cells migrated faster when plated onto human keratinocyte ECM. The ECM of mouse and human keratinocytes contained similar levels of the α3 laminin subunit of laminin-332. However, mouse skin cells expressed significantly more fibronectin (FN) than human cells. To assess whether FN is a motility regulator, we used small interfering RNA (siRNA) to reduce the expression of FN in mouse keratinocytes. The treated mouse keratinocytes moved significantly more rapidly than wild-type mouse skin cells. Moreover, the FN-depleted mouse cell ECM supported increased migration of both mouse and human keratinocytes. Furthermore, the motility of human keratinocytes was slowed when plated onto FN-coated substrates or human keratinocyte ECM supplemented with FN in a dose-dependent manner. Consistent with these findings, the ECM of α3 integrin-null keratinocytes, which also migrated faster than wild-type cells, was FN deficient. Our results provide evidence that FN is a brake to skin cell migration supported by laminin-332-rich matrices.     

Keratin expression in Merkel cells of fetal rat skin.

The cytokeratin expression of Merkel cells in fetal rat skin was studied by light- and electron microscopy. Employing a pre-embedding staining method, 2 monoclonal anti-keratin antibodies (RCK-102, MA-902) were shown to stain Merkel cells specifically. Neighbouring keratinocytes were unstained. The staining reaction seems to be based on the expression of 52.5 kD cytokeratin.     

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.     

The distribution of Merkel cells in human fetal and adult skin.

Merkel cells (MCs) have been a subject of investigation in human and animal studies for over a century, but their origin, function(s), and exact distribution in human skin remain largely unknown. The objectives of the present study were to quantify these cutaneous neuroendocrine cells in fetal and adult human skin, using an immunohistochemical marker (neuron-specific enolase) and morphometric methods. Our results indicate that, in postnatal life, MC are sparsely distributed along the undersurface of epidermal and occasionally adnexal epithelium in an approximate range of 0.1-1.0 MCs per centimeter of basal zone. They are most numerous on volar skin and least on genital skin, and on the face they have a primarily perifollicular orientation. Fetal samples have shown that MCs appear between the 15th and 18th week of gestation and are most abundant on volar skin. They appear to arise within the epidermis, and are initially numerous and later diminish with increasing gestational age. Their predominance in intrauterine life suggests a functional role in growth and development.     

Changes of the activities of enzymes involved in prostaglandin synthesis in rat skin during development and aging

Summary The developmental changes of enzymes involved in prostaglandin (PG) synthesis were investigated in rat skin from birth to 1.5 years old. In all stages of development, the activities of PG-synthesizing enzymes were found in 100,000xg supernatants of homogenates of rat skin, and PGD₂ was the major PG among those formed from PGH₂ in the presence of 1 m glutathione (GSH). The PGD synthetase activity in rat skin at birth was 2.14 nmol/min per mg protein, increasing to the highest level (3.69 nmol/min per mg protein) at 3 weeks after birth and then gradually decreasing up to 1.5 years old. The activities of PGE₂ and PGF₂ synthetases in rat skin were almost unchanged during development and aging. In contrast, the activity of GSH-S-transferase was at its lowest level at birth and gradually increased, reaching a plateau at 3 weeks after birth and remaining relatively constant during the development. The increase of PGD synthetase activity in 3-week-old rats was mainly due to the increase of specific activity of PGD synthetase in the epidermis, which was separated from the dermis by heat treatment (55° C, 30s). Immunohis-tochemical study, using (rat spleen PGD synthetase)-specific antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, increased in the epidermis in 3-week-old rats. These results suggest that a change of PGD₂ synthetase activity during aging of the skin is closely related to the development of ATPase⁺ Langerhans cells in the epidermis.     

Physiologic distribution and differentiation of melanocytes in human fetal and neonatal skin equivalents

There is evidence that epidermal keratinocytes play a critical role in melanocyte position and differentiation in the epidermis, although little is known about the molecular mechanisms involved. We have used an in vitro skin equivalent as a model system in which to study keratinocyte/melanocyte interactions in both fetal and neonatal skin. Because the skin equivalent model has been shown to closely simulate the morphologic and biochemical features of differentiated epidermis we hypothesized that the factors that influence melanocyte position and differnetiation would also function in this system. Localization of melanocytes in skin equivalents, using the monoclonal antibody HMB-45, established that melanocytes in fetal skin equivalents are grouped and distributed both basally and suprabasally, whereas melanocytes in neonatal skin equivalents are singly distributed among basal epidermal keratinocytes, similar to the distributions of fetal and neonatal melanocytes, respectively, in vivo. Similarly, in fetal and neonatal skin equivalents the patterns of expression of a number of melanoma/melanocyte-associated antigens closely parallels that seen in vivo. These results suggest that the skin equivalent model is an excellent system in which to study the dynamic factors that regulate melanocyte migration, proliferation, and differentiation during ontogeny and post-natal differentiation of the skin.     

Densities,distribution and phenotypic expression of T cells in human fetal skin

T cells are present in normal adult human skin, but their occurrence in fetal skin is unknown. T cell and Langerhans cell (LC) populations were studied using single or double immunohistochemical staining on cryostat-section. Skin samples taken from different body regions of 17 fetuses ranging from 18 to 30 weeks estimated gestational-age (w-EGA), were examined. In all specimens but one, we did not find any epidermal T cell. In contrast, dermal CD3⁺ T cells occurred at all w-EGA. The density of these cells increased with increasing age. Double staining showed that CD3⁺ T cells were predominantly CD4⁺/CD45RA⁺. On the other hand, LC, as assessed by CD1a expression, was evenly distributed within the interfollicular epidermis and papillary dermis at all gestational ages. Analysis of T cell and LC density in different body regions did not show significant topographic differences. We suggest that lack of epidermal T cells, although the LC network was fully represented, might reflect the scarce opportunity of fetal LC to contact foreign antigens in utero.