Localization of vtiamin A by autofluorescence during induced metaplastic changes in cultures of skin

A technique was devised for following the uptake and location of vitamin A in organ cultures. Explants of 12- and 13-day embryonic mouse upper lip skin were grown for 3, 6 or 9 days in biological medium to which was added 0, 4.1 or 6.9 micrograms per ml of retinyl acetate. This form of vitamin A caused glandular morphogenesis of vibrissa follicles, and keratinization in epidermis and follicles was completely suppressed in 12-day explants and partially suppressed in 13-day explants. Frozen sections at 16 microns showed the white, nonfading fluorescence of keratin and the green, rapidly-fading fluorescence due to vitamin A which was captured by high-speed photography. Although more concentrated within lipid droplets in the dermis, the vitamin penetrated both the epidermis and the hair follicles. The ability to obtain permanent photographic records of the fading fluorescence makes this a useful method for analyzing vitamin A distribution as well as keratin distribution.     

Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

Localization of vitamin a by autofluorescence during induced metaplastic changes in cultures of skin

Summary A technique was devised for following the uptake and location of vitamin A in organ cultures. Explants of 12- and 13-day embryonic mouse upper lip skin were grown for 3,6 or 9 days in biological medium to which was added 0,4.1 or 6.9 μg per ml of retinyl acetate. This form of vitamin A caused glandular morphogenesis of vibrissa follicles, and keratinization in epidermis and follicles was completely suppressed in 12-day explants and partially suppressed in 13-day explants. Frozen sections at 16 μm showed the white, non-fading fluorescence of keratin and the green, rapidly-fading fluorescence due to vitamin A which was captured by high-speed photography. Although more concentrated within lipid droplets in the dermis, the vitamin penetrated both the epidermis and the hair follicles. The ability to obtain permanent photographic records of the fading fluorescence makes this a useful method for analyzing vitamin A distribution as well as keratin distribution. This work was supported by the National Research Council of Canada (Operating Grant to M. H. Hardy and Postgraduate Scholarship to R. Van Exan) and by the Ontario Ministry of Agriculture and Food.     

Early effects of vitamin A on protein synthesis in the epidermis of embryonic chick skin cultured in serum-containing medium

A quantitative study of the incorporation of ³H-leucine and ³⁵S-cystine by the 12-day embryonic chick epidermis during 2 days in culture in serum-containing medium, with and without added vitamin A has been made. This has shown that very soon after explantation, protein synthesis, particularly the synthesis of cyst(e)ine-rich proteins is enhanced dramatically in the epidermis cultured in the control, serum-containing medium alone. This effect is completely absent in the medium containing added vitamin A (5 IU/ml). Under these conditions ³H-leucine and ³⁵S-cystine incorporation remain relatively unchanged during the 2-day culture period.     

The proteins of the embryonic chick epidermis : I. During the normal development in ovo

As a preliminary to a study of the proteins of the embryonic chick epidermis when grown in vitro under various culture conditions, the proteins of the anterior metatarsal epidermis, from 11 days of embryonic life up to 9 days posthatching, have been studied. Carboxymethylated derivatives of the proteins extracted by a thiol reduction procedure have been analyzed by polyacrylamide gel electrophoresis. The results have shown that the differentiation of the epidermis is characterized by the appearance between days 14 and 17 of at least 11 major protein bands in the electrophoretic pattern. Two of these bands are of relatively high molecular weight protein and appear earlier than the remaining bands which form a group of closely related, low molecular weight protein species. The differentiation of the tissue also involves the disappearance from the electrophoretic pattern of all but one of the five major bands present in extracts of the 11/12-day epidermis. A study of the proteins derived from the isolated periderm of the 14-day chick embryo beak has suggested that one of the major bands in the 11/12-day metatarsal epidermal extracts may be a peridermal protein.     

Role of dehydration in changing the permeability of erythrocyte plasma membranes by freeze-thawing

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing 32Pi, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weights between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [gamma-32P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [gamma-32P]ATP and cyclic AMP-dependent protein kinase from calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Phosphorylation of keratin polypeptides

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing ³²P_i, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weight between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [γ-³²P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [γ-³²P]ATP and cyclic AMP-dependent protein kinase form calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency

Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues.     

Epidermal protein metabolism directed toward keratinization by hydrocortisone in the chick embryonic skin growing in a chemically defined medium.

The metabolism of the epidermal structural and nonstructural proteins was studied in hydrocortisone-induced in vitro keratinization of 13-day chick embryonic skin growing in a chemically defined medium. The protein metabolism of the epidermis was examined by determining the amounts of radioactivity incorporated into the fractions of reduced, S-carboxymethylated epidermal proteins (SCMEp) which were separated by polyacrylamide gel electrophoresis. A group of high molecular weight, glycine-rich derivatives of the epidermal fibrous protein called SCMEpA were found to be actively synthesized in the hydrocortisone-treated epidermis alone, while a group of undefined protein derivatives called SCMEpX was shown to be synthesized exclusively in the nontreated epidermis. Chase-culture of the prelabeled explants revealed that hydrocortisone accelerated the degradation of general proteins including SCMEpX while SCMEpA remained metabolically stable throughout the culture. Actinomycin D did not significantly affect the hydrocortisone-induced synthesis of SCMEpA but greatly inhibited that of SCMEpX of the nontreated epidermis, suggesting the induction by the steroid of relatively stable mRNA for SCMEpA. From these findings, it is concluded that hydrocortisone directed the cultured epidermis toward keratinization through acceleration of the synthesis of epidermal structural proteins and degradation of other proteins.     

Reversible inhibition by DMSO of hydrocortisone-induced keratinization of chick embryonic skin

Hydrocortisone, at a physiological concentration of 10^?8 M, induces keratinization of chick embryonic tarsometatarsal skin in a chemically defined medium in 4 days [1]. The presence of 1–4% DMSO with hydrocortisone reversibly prevented this keratinization. DMSO suppressed the appearance of epidermal structural protein, which was preferentially induced by hydrocortisone. It also suppressed hydrocortisone-induced epidermal transglutaminase activity; which was presumably responsible for polymerization and decrease in solubility of epidermal protein in keratinization, and it suppressed increase of epidermal protein. When DMSO was added to differentiated skin or added concomitantly with a higher concentration of hydrocortisone, epidermal transglutaminase activity was suppressed. Electron microscopic studies showed that hydrocortisone induced tonofilament bundles and keratinized cells with cellular envelopes, which are all characterestic of α-type keratinization of chick embryonic skin [2], and that DMSO inhibited hydrocortisone induced keratinization and kept the epidermis in an undifferentiated state. Moreover, DMSO inhibited epidermal DNA synthesis and increase in thickness of the epidermis during culture of hydrocortisone-treated skin, indicating that it suppressed cell proliferation as well as cell differentiation. DMSO by itself at 1 or 2 % did not affect epidermal cell differentiation, but suppressed cell proliferation when compared with untreated control.     

Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation

Differentiation of the epidermis during embryonic rabbit development was found to be accompanied by dramatic changes in keratin proteins. Immunofluorescent labeling with keratin antiserum revealed that the undifferentiated epithelium of 12-d embryos was already committed to making keratin proteins. At 18 d of embryogenesis, the epithelium contained keratin proteins in the molecular weight range of 40,000-59,000. The stratification of the epithelium into two cell layers at 20 d of development coincided with the appearance of a 65-kdalton keratin. When a thick stratum corneum developed at 29 d, several additional keratins became prominent, most notably the large keratins (61- and 64-kdalton) and a 54-kdalton keratin. In addition, the 40-kdalton keratin, which had been present in earlier embryonic epidermis, disappeared. Newborn epidermis resembled that of a 29-d embryonic epidermis, with the exception of the appearance or increase in concentration of two more keratin species (46- and 50-kdalton). In vitro culturing of keratinocytes from 12- and 14-d embryonic skin demonstrated that these cells contained essentially the same keratin profiles as the undifferentiated epithelium of 18-d embryos (40-59 kdalton). Keratinocytes grown from older embryos contained increased amounts of keratin, similar to the in vivo situation, but did not synthesize the high molecular weight keratins. The changes observed during embryonic epidermal differentiation appear to be recapitulated during the sequential maturation steps of adult epidermis.     

Immunocytochemistry indicates that glycine‐rich beta‐proteins are present in the beta‐layer,while cysteine‐rich beta‐proteins are present in beta‐ and alpha‐layers of snake epidermis

Immunolocalization of glycine‐rich and cysteine–glycine‐medium‐rich beta‐proteins (Beta‐keratins) in snake epidermis indicates a different distribution between beta‐ and alpha‐layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta‐keratin layers alternated with softer and pliable alpha‐keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta‐proteins (formerly beta‐keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine‐rich beta‐proteins of 12–15 kDa, is poorly or not reactive with the beta‐layer of snake epidermis. This suggests that glycine‐rich proteins similar to those present in lizards are altered during maturation of the beta‐layer. Conversely, a glycine–cysteine‐medium‐rich beta‐protein (HgGC10) of 10–12 kDa is present in beta‐ and alpha‐layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta‐ and alpha‐cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta‐proteins rich in glycine mainly accumulate on a scaffold of alpha‐keratin producing a resistant and hydrophobic beta‐layer. Conversely, beta‐proteins lower in glycine but higher in cysteine accumulate on alpha‐keratin filaments present in beta‐ and alpha‐layers producing resistant but more pliable layers.     

Identification of a Major Xylanase from <Emphasis Type="Italic">Aspergillus flavus</Emphasis> as a 14-kD Protein

Aspergillus flavus K49 secreted at least two xylanase activities when grown on a medium containing larch (wood) xylan as a sole carbon source. Enzyme activity was assayed using an agar medium containing Remazol Brilliant Blue R conjugated oat spelt xylan as substrate. Crude enzyme preparations were inhibited by Hg⁺², with an ED₅₀ of 17.5 mM and maximum inhibition of 83% at 50 mM. A concentrated sample of A. flavus K49 xylanase preparation was subjected to gel filtration chromatography on a P-30 column. A small protein peak coinciding with the major peak of xylanase activity was separated from the other secreted fungal proteins. An additional peak of xylanase activity was observed in fractions containing multiple fungal proteins. Analysis by denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of fractions containing the smaller molecular weight xylanase revealed a major and minor protein band in the vicinity of 14 kD. Analysis of these same fractions by acidic native PAGE revealed a single band. Confirmation of identity for the isolated xylanase was provided by isolation of a protein band from a SDS–PAGE gel, followed by trypsin digestion/analysis by tandem mass spectrometry. Comparison of the peptide library derived from this protein band with sequence data from the A. oryzae genomic data base provided a solid match with an endo-1,4-β-xylanase, XlnA. This identification is consistent with a low molecular weight protein associated with the major xylanolytic activity. XlnA may be a highly mobile (diffusible), plant wall hemicellulose degrading factor with significant activity during plant infection.     

Heat shock protein 25 plays multiple roles during mouse skin development

Heat shock protein (Hsp) 25 is a member of the small Hsp family. High levels of Hsp25 can be detected in skin. During adult epidermis differentiation, the concentration of Hsp25 increases as the distance of keratinocytes from the basal layer increases, in parallel with the extent of keratinization. We previously showed that Hsp25, mouse keratin (MK) 5, and MK14 participated in the formation of characteristic ring-shaped aggregates during the differentiation of the PAM212 keratinocyte cell line. We suggested that Hsp25 was involved in the disorganization of the MK5-MK14 keratin network before the establishment of the MK1-MK10 keratin network at the beginning of epidermis stratification. In this study, we have investigated the distribution of Hsp25 and keratins throughout skin development. We demonstrate that the distribution of Hsp25 and MK5 in the epidermis at the beginning of stratification and before keratinization is similar to that observed in PAM212 keratinocytes. These results indicate that there is a strong correlation between the mechanism we described ex vivo and the events taking place in vivo. Moreover, we show that Hsp25 is produced in different cell types in the epidermis and in the hair follicle at different stages of their development. Thus, our results suggest that Hsp25 is involved in more than one process during skin development.     

Putative histidin-rich proteins in the epidermis of lizards

In the stratum granulosum of mammalian epidermis, histidin-rich proteins (filaggrins) determine keratin clumping and matrix formation into terminal keratinocytes of the stratum corneum. The nature of matrix, interkeratin proteins in the epidermis of nonmammalian vertebrates, and in particular in that of reptilian, mammalian progenitors are unknown. The present biochemical study is the first to address this problem. During a specific period of the renewal phase of the epidermis of lizards and during epidermal regeneration, keratohyalin-like granules are formed, at which time they take up tritiated histidine. The latter also accumulate in cells of the alpha-keratin layer (soft keratin). This pattern of histidine incorporation resembles that seen in keratohyalin granules of the stratum granulosum of mammalian epidermis. After injection of tritiated histidine, we have analysed the distribution of the radioactivity by histoautoradiography and electrophoretic gel autoradiography of epidermal proteins. Extraction and electrophoretic separation of interfilamentous matrix proteins from regenerating epidermis 3-48 hours post-injection reveals the appearance of protein bands at 65-70, 55-58, 40-43, 30-33, 25-27, and 20-22 kDa. Much weaker bands were seen at 100, 140-160, and 200 kDa. A weak band at 20-22 kDa or no bands at all are seen in the normal epidermis in resting phase and in the dermis. In regenerating epidermis at 22 and 48 hours post-injection, little variation in bands is detectable, but low molecular weight bands tend to increase slightly, suggesting metabolic turnover. Using anti-filaggrin antibodies against rat, human, or mouse filaggrins, some cross-reactivity was seen with more reactive bands at 40-42 and 33 kDa, but it was reduced or absent at 140, 95-100, 65-70, 50-55, and 25 kDa. This suggests that different intermediate degradative proteins of lizard epidermis may share some epitopes with mammalian filaggrins and are different from keratins with molecular weight ranging from 40 to 65-68 kDa. The immunocytochemical observation confirms that a weak filaggrin-like immunoreactivity characterizes differentiating alpha-keratogenic layers in normal and regenerating tail. A weak filaggrin labeling is discernable in small keratohyalin-like granules but is absent from the larger granules and from mature keratinocytes. The present results indicate, for the first time, that histidine-rich proteins are involved in the process of alpha-keratinization in reptilian epidermis. The cationic, interkeratin matrix proteins implicated may be fundamentally similar in both theropsid-derived and sauropsid amniotes.     

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)     

Effect of vitamin A on wound epidermis during forelimb regeneration in adult newts.

The effects of vitamin A on blastemal epidermis were studied during the early postamputational period of forelimb regeneration in Triturus alpestris. Vitamin A was administered through oral intubation at a dose of 250 IU per gram of body weight per day. The results were evaluated by morphometry, histology, and autoradiography. After 7, 11 and 14 days of treatment, several alterations were observed in the wound epidermis: a) reversal of keratinization; fewer keratinized cells were counted in sections from vitamin A-treated limbs; b) decrease in the incorporation of tritiated thymidine, as judged by estimation of labeling indices; c) increased mitotic activity in the cells of the stratum germinativum, and in the middle layer of the epithelial cells, as well. The significance of these cellular effects is discussed against the relevant literature.     

Keratinization of the epidermis of the Australian lungfish Neoceratodus forsteri (dipnoi)

The differentiation of the epidermis in sarcopterigian fish may reveal some trend of keratinization followed by amphibian ancestors to adapt their epidermis to land. Therefore, the process of keratinization of the epidermis of the Australian lungfish Neoceratodus forsteri was studied by histochemistry, electron microscopy, and keratin immunocytochemistry. The epidermis is tri-stratified in a 2-3-month-old tadpole but becomes 6-8 stratified in young adults. Keratin filaments increase from basal to external cells where loose tonofilament bundles are present. This is shown also by the comparison of positivity to sulfhydryl groups and increasing immunoreactivity to alpha-keratins in more external layers of the epidermis. Two broad-spectrum anti alpha-keratin monoclonal antibodies (AE1 and AE3) stain all epidermal layers as they do in actinopterigian fish. In the adult epidermis, but not in that of the larva, the AE2 antibody (a marker of keratinization in mammalian epidermis) often immunolabels more heavily the external keratinized layers where sulfhydryl groups are more abundant. Mucous granules are numerous and concentrate on the external surface of the epidermis to be discharged and contribute to cuticle formation. Keratin is therefore embedded in a mucus matrix, but neither compact keratin masses nor cell corneous envelope were seen in external cells. It is not known whether specific matrix proteins are associated with mucus. There was no immunolocalization of the keratin-associated proteins, filaggrin and loricrin, which suggests that the epidermis of this species lacks the matrix and cell corneus envelope proteins characteristic of that of amniotes. In conclusion, while specific keratins (AE2 positive) are probably produced in the uppermost layers as in amphibian epidermis, no interkeratin, matrix proteins seem to be present in external keratinocytes of the lungfish other than mucus.     

Retinoids alter the direction of differentiation in primary cultures of cutaneous keratinocytes

The effects of vitamin A on the morphological expression of differentiation were studied in cell cultures of cutaneous keratinocytes from the newborn rat. The cells were first cultivated in a medium containing 0.11 mM calcium until a confluent monolayer had been formed. Stratification and terminal differentiation were then triggered by raising the calcium concentration of the medium to 1.96 mM ('normal' culture). The rise in the concentration of calcium was coupled with the addition of retinol (RL) of retinoic acid (RAC) to the medium to produce an excess of vitamin A (high-retinoid culture). Delipidized serum was used to produce a deficiency of vitamin A (low-retinoid culture). The tissue organization and the ultrastructure of the keratinocytes in the stratified culture were the same as those seen in conventional cultures and skin explants. These stratified cultures expressed the morphological features of the epidermis of intact skin. The addition of RL or RAC to the medium enhanced features characteristic of the secretory epithelium, such as the formation of an extensive endoplasmic reticulum, an enlargement of the Golgi zone, and an increase in the number of vacuoles. At the same time, the addition of retinoids diminished features characteristic of the terminal differentiation of the stratified squamous epithelium, such as stratification and keratinization. Deficiency of vitamin A in the medium resulted in a culture with many differentiated layers. The differentiated cells of the low-retinoid cultures contained densely packed tonofilaments and synthesized products that reacted with the monoclonal antibody AE2 that is specific for keratin peptides which are markers of epidermal differentiation. In the cell culture system that is presented here, an excess of retinoids redirected epithelial differentiation from a stratifying and keratinizing epithelium towards a secretory epithelium. This system is a useful tool for elucidating the mechanisms responsible for the effect of vitamin A on the differentiation of epithelial cells.