Fetal Skin Development Research Articles

Expression of Amphiregulin Is Regulated in Cultured Human Keratinocytes and in Developing Fetal Skin

Previous studies have indicated that amphiregulin is a major autocrine factor for human keratinocytes. To evaluate the possibilities that amphiregulin could function in fetal skin morphogenesis and contribute to the growth regulation of epidermis, immunostaining with a specific anti-amphiregulin monoclonal antibody was observed at different stages of fetal skin development, and the results were compared with neonatal and adult skin specimens and cultured neonatal keratinocytes. Immunoreactive amphiregulin was readily detected in the periderm and basal epidermal layers of embryonic epidermis but became gradually less detectable in the periderm concurrent with an increase in staining of the spinous layer as it developed during the fetal period. Basal and spinous keratinocyte expression of amphiregulin was predominantly cytoplasmic, but with punctate nuclear foci, and this pattern persisted into the neonatal period. At all developmental stages, epithelial and mesenchymal cells of the follicle were reactive, often in a nuclear pattern. Dermal mesenchymal cells were increasingly reactive in late fetal skin, but the staining decreased postnatally. In adult skin only randomly scattered nuclei of spinous keratinocytes and follicular structures such as the inner root sheath were stained. Examination by scanning laser confocal microscopy of cultured neonatal keratinocytes showed a nonrandom distribution of amphiregulin to the peripheral cytoplasm and plasma membranes at the outer perimeter of cell colonies, with much less reactivity of apposed keratinocyte membranes at interior sites. Nuclei were heterogeneously stained. Amphiregulin reactivity declined at higher cell densities. These data indicate that expression of amphiregulin is regulated in vitro and developmentally during cutaneous morphogenesis.

Matrilysin (PUMP) Correlates with Dermal Invasion During Appendageal Development and Cutaneous Neoplasia

Matrix-degrading metalloproteinases play a major role in tissue remodeling. Recent studies have shown that enzymes of this class are constitutively expressed primarily by stromal cells and not by epithelium. Here we present immunohistochemical evidence that matrilysin is localized within epidermal cells in developing skin and in tumor cells of cutaneous malignancies. The expression of matrilysin protein in developing fetal skin (6-15 weeks) is localized primarily to the germinative basal cell layer of fetal epidermis and early appendageal buds. The buds continue to express matrilysin during mesenchymal invasion. As development progresses (15-19 weeks) matrilysin is concentrated only in cells at the distal portion of the invading follicular and sweat gland appendageal cords. In adult skin, matrilysin was localized specifically to the outer root sheath of the hair follicles and the secretory cells of the eccrine glands but was absent in the epidermis. Nodulocystic, keratotic, adenoid basal cell carcinomas (BCCs) did not express matrilysin. In contrast, in the more aggressive morpheaform (infiltrative) BCCs and recurrent BCCs, matrilysin was localized at the tumor-stromal interface. In squamous cell carcinomas matrilysin was present in tumor cells at the stromal interface surrounding the tumor nests. The demonstration of matrilysin protein in germinal basal cells during fetal skin development and its presence in tumor cells at the stromal junction suggests that this enzyme may contribute to the proteolytic activity associated with cell-extracellular matrix interactions during appendageal development and tumor invasion.

Expression of Type II Collagen at the Middle Stages of Chick Embryonic and Human Fetal Skin Development

Using in situ hybridization techniques and RNase protection assays, type II collagen mRNA was transiently detected in the epidermis of chick embryonic skins during days 9-15 after fertilization, with a maximum expression at day 11. Immunohistochemical studies demonstrated that deposition of type II collagen was also transiently localized at the subepidermal region during days 10-15. Type II collagen gene and gene product concomitantly started to decline preferentially at the region where feather buds were being formed on day 12, and thereafter diminished at the region between feather buds. Using immunohistochemical methods, type II collagen was also detected in human fetal scalp skin at 17-23 fetal weeks at the subepidermal region, excluding the region beneath the hair follicles. These results indicate that the lack of type II collagen expression is related to the development of feather and hair at a certain stage of chick embryonic and human fetal skin development.

Transient expression of type II collagen at the middle stages of chick embryonic and human fetal skin development

Transient expression of type II collagen at the middle stages of chick embryonic and human fetal skin development

Expression of the Hox 2.2 homeobox gene in murine embryonic epidermis

Abstract The expression of the Hox 2.2 gene was studied in mouse fetal skin by in situ hybridization with an antisense RNA probe derived from the homeobox region of this gene. In contrast to the expression of Hox 2.2 in spinal cord, which is strongest in 11-day embryos, and is greatly diminished by day 14 and day 17, the signal for Hox 2.2 in skin could be not be detected in 11-day epidermis, was barely detectable on day 14, became strong on day 17, and decreased in new-born animals (day 19). RNase protection assays using Hox 2.2 homeobox-containing and 3′flanking region probes confirmed that the signals detected in 17-day fetal skin by in situ hybridization represent Hox 2.2 transcripts, and that the message is expressed throughout the day 15 to day 18 period during which the epidermis is undergoing terminal differentiation. RNase protection analysis also revealed two alternatively spliced forms of the Hox 2.2 mRNA are present throughout fetal skin development. Northern gel analysis of 17-day fetal skin using a Hox 2.2 homeobox-containing probe at high stringency showed two bands of 1.6 and 1.9kb, respectively. The 1.9 kb band was greatly enhanced by hybridization at reduced stringency, suggesting the expression of additional homeobox genes with homology to Hox 2.2. These results suggest that the Hox 2.2 homeobox gene plays a role in epidermal development.

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 infundibulumes 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 interfolicular 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.

Development of Human Fetal Skin Transplanted to the Nude Mouse

Thirty-five human fetal skin (HFS) grafts were transplanted to nude mice for 7 to 70 d and evaluated histologically with 64 biopsies. The estimated gestational ages (EGA) of the grafts at the time of the transplantation ranged from 8 to 19 weeks. The maturation of the engrafted fetal skin was evaluated by assessing epidermal, dermal, and appendage development. Within the nude mouse, the HFS demonstrated progression in stratification and maturation of the epidermis. The dermis increased in depth, adding fibrovascular stroma and adipose tissue. The appendages demonstrated invagination, differentiation, and progression of organogenesis. Subcutaneously placed grafts showed the same rate of HFS development as HFS in utero. The grafts transplanted to the surface of the nude mice and exposed to air demonstrated an acceleration of development. We conclude that HFS transplanted to the nude mouse is an effective in vivo model for maintaining and altering HFS maturation.

Retention of differentiated characteristics in human fetal keratinocytes in vitro

Many of the morphologic and biochemical changes that occur during human fetal skin development have been described, yet there has been little experimental analysis of the processes that regulate the development of human fetal skin. This is due in part to difficulties in culturing human fetal epidermal keratinocytes. We have successfully cultured fetal keratinocytes in two different in vitro systems; in a serum-free keratinocyte growth medium (KGM) on tissue culture plastic and cocultured with dermal fibroblasts as spheroidal aggregates. To characterize these fetal keratinocytes in vitro we have assessed their ability to express several markers of epidermal differentiation. Human fetal keratinocytes grown on plastic in KGM stratify and express some of the components of the differentiated epidermis, such as involucrin and the high molecular weight keratins. However, these keratinocytes co-express keratins and vimentin and do not form a structured basement membrane. More characteristics of fetal skin are preserved in mixed aggregates of epidermal keratinocytes and dermal fibroblasts, including epidermal stratification, synthesis of basement membrane components, tissue-specific expression of intermediate filaments, involucrin, and expression of high molecular weight keratins. The maintenance of human fetal epidermal keratinocytes in these two in vitro systems and their ability to express many differentiated characteristics suggests that these cultures will be valuable for studies of the molecular mechanisms that regulate the regionally specific differentiation of the human fetal epidermis.

Characterization of Epidermal Keratins during Fetal Pig Skin Development

Annals of the New York Academy of SciencesVolume 556, Issue 1 p. 480-482 Characterization of Epidermal Keratins during Fetal Pig Skin Development C. A. SIGNOR, C. A. SIGNOR Ciba-Geigy Corporation, Pharmaceuticals Division, Ardsley, New York 10502Search for more papers by this authorT. KURIHARA-BERGSTROM, T. KURIHARA-BERGSTROM Ciba-Geigy Corporation, Pharmaceuticals Division, Ardsley, New York 10502Search for more papers by this author C. A. SIGNOR, C. A. SIGNOR Ciba-Geigy Corporation, Pharmaceuticals Division, Ardsley, New York 10502Search for more papers by this authorT. KURIHARA-BERGSTROM, T. KURIHARA-BERGSTROM Ciba-Geigy Corporation, Pharmaceuticals Division, Ardsley, New York 10502Search for more papers by this author First published: June 1989 https://doi.org/10.1111/j.1749-6632.1989.tb22543.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume556, Issue1Heparin and Related Polysaccharides: Structure and ActivitiesJune 1989Pages 480-482 RelatedInformation

Morphometric and statistical analyses describing the in utero growth of human epidermis.

Epidermal development of human embryonic and fetal skin from the lower limb was studied using morphometric and statistical methods. Epidermal growth, as defined by an increase in epidermal thickness and the number of cell layers, occurred in three distinct stages during the first and second trimesters. The first growth spurt occurred between 5 and 13 weeks estimated gestational age (EGA) and was followed by a plateau phase with little change in epidermal thickness from 14 to 21 weeks, after which the epidermis began to increase in height again. The periderm reached its maximal height by approximately 13 weeks EGA, and by 25 weeks was shed into the amniotic fluid. Thus, within a five-month period (5 to 25 weeks EGA) the epidermis changed from a single cell layer less than 10 micron thick to a 10 to 12-cell layer, keratinized epithelium greater than 60 micron thick. In contrast, epidermis from adult lower limb consisted of about 25 cell layers and was almost 75 micron in thickness. The age-related differences in epidermal thickness probably reflect changes in cell size and shape more than changes in the directional movement (apically vs. laterally) of proliferating keratinocytes, because the addition of cell layers throughout development was relatively constant. During the plateau phase, when there is a rapid increase in fetal growth rate, the suprabasal keratinocytes become more flattened, thereby allowing for the addition of new cell layers while maintaining a relatively constant epidermal thickness.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Structural and histochemical aspects of epidermis development of fetal porcine skin.

Epidermal development of fetal porcine skin was studied in fetuses from 41 days of gestation until birth with scanning and electron microscopy techniques as well as histochemical methods, including immunohistochemistry. The porcine fetus develops a relatively thick and solid multilayered cover of epidermal cells, which is not lost before birth. It consists of tightly packed cells of the periderm and the stratum intermedium. The periderm cells are totally filled with filamentous proteins; in the intermediate cells, the filamentous proteins are concentrated in the cell periphery, forming a thick marginal zone. Immunohistochemically, the cytofilaments could be identified as cytokeratins of lower and higher molecular weights. The first thin stratum corneum lamellae are formed below the stratum intermedium at about 80-85 days of gestation.

Expression of epidermal keratins and filaggrin during human fetal skin development.

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Fibronectin Distribution During the Development of Fetal Rat Skin

Fibronectin distribution during fetal rat skin development has been studied immunocytochemically at the light and electron microscope level from 16 days of gestation to birth. The dermal-epidermal junction, the dermis, and connective tissue around developing muscle were shown by light microscopy to be heavily stained throughout this period. The development of hair follicles from about 18 days onward was not associated with any consistent change in fibronectin distribution. The heavy staining of the upper dermis was associated with a high density of mesenchymal cells, and immunoelectron microscopy revealed fibronectin on the surface of many of these cells and in association with the surrounding fine collagen fibrils. At the dermal-epidermal junction, both follicular and interfollicular, fibronectin was localized mainly in the plasma membrane and lamina lucida regions of the basement membrane, and there was also staining associated with the underlying fine collagen fibrils. These observations are further evidence for the proposed role of fibronectin as a mediator of the cell-matrix interactions which are of importance for tissue development and maintenance.

Regional Development of the Human Epidermis in the First Trimester Embryo and the Second Trimester Fetus (Ages Related to the Timing of Amniocentesis and Fetal Biopsy)

The epidermis was sampled from multiple body sites of whole human embryos and fetuses of 45- and 65-days, 16- and 21-weeks estimated gestation age to determine whether a regionally dependent, variable rate of interfollicular epidermal development exists. Surface characteristics and thickness of the epidermis were evaluated by scanning electron and light microscopy, respectively. It was concluded that all epidermal development proceeded simultaneously during the first trimester with the exception of the foot which was more advanced in both thickness and state of differentiation. During the second trimester the epidermis of both the head and the foot showed more precocious development, particularly in relation to the special sense organs and to the plantar surface. The interfollicular epidermis of the majority of the body, however, was approximately equivalent in the state of development. These data have potential relevance to prenatal diagnosis of inherited skin disease from amniocentesis and/or fetal biopsy specimens; the present survey of the total epidermal surface will allow one to predict the types of skin-derived cells that should be present in the amniotic fluid at a given age, and to evaluate a fetal biopsy from one region and be confident that it is an accurate index of fetal skin development, age and status in general.

The effect of hydrocortisone on the response of fetal rat skin in culture to ultraviolet irradiation.

1. The effect of hydrocortisone on the development of fetal rat skin in organ culture, and on its repair after exposure to a mixed beam from a mercury lamp, are described. 2. The addition of hydrocortisone (7.5 microg/ml) to the culture medium (HC medium) caused accelerated differentiation and keratinisation of the epidermis followed by atrophic changes as in vivo. 3. Explants were grown for 2 days in either normal or HC medium and then irradiated with light from an Hanovia lamp. 4. Irradiation of the control explants produced severe necrosis in both epidermis and dermis and much disorganisation of the dermal intercellular material; 2 days after exposure the s. corneum with adherent cellular debris had become either completely detached from the denuded dermis, or raised to form a fluid-filled blister. Epidermal regeneration had begun by the 4th day after irradiation and was usually complete by the 6th day. 5. Hydrocortisone modified the response to irradiation as follows: (1) reduced and retarded cellular breakdown, (2) prevented vesication, (3) preserved the organisation of the dermal intercellular material, (4) hastened epithelialisation, (5) accelerated the differentiation of the new epidermis. Effects (2), (3), and (4) were probably secondary to (1). 6. Experiments with various light filters showed that the effective wavelengths for producing lesions in the skin explants were those below 3000 A. 7. It is suggested that the beneficial effect of hydrocortisone on the repair of irradiated skin explants might be due, at least in part, to a reduced proteolytic activity in the damaged tissue through a stabilising action of the hormone on the lysosomes. 8. The relationship of these findings to clinical observations is discussed.