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.     

In vivo effects of vitamin D on the proliferation and differentiation of rat keratinocytes

The hormonal form of vitamin D appears to be a physiological regulator of the epidermogenesis. While its differentiation-promoting effect is well accepted, there are conflicting reports of its action on keratinocyte proliferation. This study evaluates the specific changes induced by vitamin D treatment in the epidermis of rats nutritionally deprived of vitamin D by cell size analysis, acridine orange flowcytometry, and the immunohistochemical detection of proteins related to the different stages of differentiation (epidermal calcium binding protein and suprabasal keratins recognized by KL1 antibody) The total keratinocyte and isolated keratinocyte subpopulations were studied. Vitamin D deficiency was associated in the total population with a lower percentage of actively proliferating cells and with a lack of differentiation markers. Study of the isolated cell populations demonstrated, however, that small cells were actively proliferating, whereas they were mainly in the resting stage in the normal epidermis. Treatment with vitamin D dramatically increased cell proliferation and stimulated the appearance of differentiation markers. Some of the observed effects, such as an increase in proliferation and the appearance of epidermal calcium binding protein, were due to the normalisation of the vitamin D deficiency-induced hypocalcemia, whereas the expression of suprabasal keratins was directly dependent on vitamin D. We conclude that the action of vitamin D on the epidermis is associated with increases in both proliferation and differentiation of keratinocytes. Vitamin D itself and its resulting action on calcium homeostasis appear to contribute to the observed effects. © 1996 Wiley-Liss, Inc.     

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.     

Endogenous beta-galactoside-binding lectin expression is suppressed in retinol-induced mucous metaplasia of chick embryonic epidermis

The fate of endogenous beta-galactoside-binding lectin of chick embryo (14K type) was investigated during the course of skin differentiation. Lectin (14K) was found in keratinized epidermis and was localized mainly in the basal and intermediate cells. However, the protein lectin in the epidermis disappeared when the cultured skin was treated with vitamin A and mucous metaplasia was observed. The synthesis of lectin mRNA was also strongly suppressed by vitamin A in a concentration-dependent manner. On the other hand, in the dermis, in which the lectin was localized in the extracellular matrix, lectin expression was scarcely affected by vitamin A. These results indicated that the lectin was expressed in the keratinized epidermis but that its expression was suppressed in vitamin A-induced mucous-secreting epithelium. The suppression may be a result of a transition of the epidermal regulatory system to one of mucous-secreting epithelium. This is the first finding that 14K lectin expression might be regulated during the course of the epidermal differentiation.     

Topographical and developmental studies on target sites of 1,25 (OH2) vitamin D3 in skin

Summary Tritium-labeled 1,25 (OH₂) vitamin D₃, when injected into vitamin D-deficient adult and pregnant rats is concentrated and retained strongest in nuclei of cells in the outer root sheath of the hair, followed by the stratum granulosum, spinosum, and basale of the epidermis. In the hair follicle, in addition to the most heavily labeled outer root sheath, nuclear labeling exists also in cells of the hair bulb and of the inner root sheath, as well as in basal cells of the sebaceous gland. In contrast, cells of the dermal papilla and the connective tissue of the dermis are generally unlabeled, except for labeled cells in the outer connective tissue sheath at the infundibulum of vibrissae of 20-day fetal rats and a few scattered labeled cells in the dermis, probably macrophages. In the developing hair, in 18- and 20-day fetal rats, a distinct topographic pattern of labeled cells can be seen, which is characteristic of the different stages of hair follicle development. In the hair germ, heavily labeled cells appear first in the stratum spinosum. In the hair peg, they remain in this position in its juxtaepidermal portion; however, when a dermal papilla develops, heavily labeled cells assume a marginal position. This suggests a sequential epidermal-epidermal and mesenchymal-epidermal receptor induction. Injection of tritium labeled 25 (OH) vitamin D₃ did not show nuclear concentration in these tissues and excess unlabeled 25 (OH) vitamin D₃ — unlike excess 1,25 (OH₂) vitamin D₃ — did not prevent nuclear uptake of tritium labeled 1,25 (OH₂) vitamin D₃. The results indicate differential effects of 1,25 (OH₂) vitamin D₃ on different structures in the epidermis and dermis.Supported by US PHS grant PCM8200569     

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.     

Histological effects of vitamin A on the tail amputated tadpoles ofBufo melanostictus

Vitamin A has manifold effects on the development, growth and pattern formation of several amphibians. At the same time it causes severe embryonic malformations. The histological changes brought about by vitamin A in the tail tissues of anurans are quite amazing. A common morphological change brought about by vitamin A in tail-amputated tadpoles ofBufo melanostictus include the formation of a large bulbular mass at the distal end of the tail following tail regeneration. Histology revealed that the bulbular mass consisted of notochordal cells only. Other histological changes are: a thickening of the epidermis and the basement membrane, enlargement of the notochord and the nerve cord, thickening of the sheath covering the notochord and the myelin sheath covering the nerve cord and the disorganization of muscle bundles. The significance of such changes is discussed.     

Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.     

The proteins of the embryonic chick epidermis : II. During culture in serum-containing medium with and without added vitamin A

The proteins of the 12-day embryonic chick anterior metatarsal epidermis have been studied during growth in vitro in a serum containing medium with and without added vitamin A (5 IU/ml). The keratinization observed in the serum-containing medium alone was thus shown to be defective since only two of the proteins associated with keratinization during development in ovo were synthesized by the cultured epidermis, whereas the major group of 9 keratin protein bands was almost completely absent. The possible structural origins of these keratin protein bands is discussed in the light of these findings.In the medium containing vitamin A, synthesis of the two keratin proteins observed in the control epidermis was prevented and instead the band pattern obtained from the retinol-treated epithelium remained very similar to that of the 12-day epidermal starting material. Certain bands were increased in intensity in the presence of vitamin A, however, and in particular, the major band of the 12-day epidermis, which appears to be peridermal in origin, was present in increased amounts.     

HPLC analysis of vitamin E isoforms in human epidermis: correlation with minimal erythema dose and free radical scavenging activity

The content and composition of different vitamin E isoforms was analyzed in normal human skin. Interestingly the epidermis contained 1% alpha-tocotrienol, 3% gamma-tocotrienol, 87% alpha-tocopherol, and 9% gamma-tocopherol. Although the levels of tocotrienol in human epidermis appear to be considerably lower than reported in the hairless mouse, the presence of significant amounts of tocotrienol levels leads to speculation about the physiological function of tocotrienols in skin. Besides antioxidant activity and photoprotection, tocotrienols may have skin barrier and growth-modulating properties. A good correlation was found for epidermal alpha-tocopherol (r = 0.7909, p <.0003), gamma-tocopherol (r = 0.556, p <.025), and the total vitamin E content (r = 0.831, p <.0001) with the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging in epidermis, as assessed by electron paramagnetic resonance (EPR) spectroscopy. In human epidermis, alpha-tocopherol is quantitatively the most important vitamin E isoform present and comprises the bulk of first line free radical defense in the lipid compartment. Epidermal tocotrienol levels were not correlated with DPPH scavenging activity. The minimal erythema dose (MED), an individual measure for sun sensitivity and a crude indicator for skin cancer susceptibility, did not correlate with the epidermal content of the vitamin E isoforms. Hence it is concluded that vitamin E alone is not a determinant of individual photosensitivity in humans.     

Regulation of terminal differentiation of cultured human keratinocytes by vitamin A

Vitamin A is known to exert an important influence on epithelial differentiation. The fetal calf serum supplement of cell-culture medium contains enough of the vitamin to affect the differentiation of cultured keratinocytes derived from epidermis and from other stratified squamous epithelia. The cellular and molecular properties of the cultures are altered when the medium is supplemented with serum from which the vitamin A has been removed by solvent extraction (delipidized serum). Cell motility is reduced, the adhesiveness of cells increases and pattern formation is prevented. In both epidermal and conjunctival keratinocytes, removal of vitamin A leads to the synthesis of a 67 kd keratin characteristic of terminally differentiating epidermis and to much reduced synthesis of the 52 kd and 40 kd keratins typical of conjunctiva. These changes, both cellular and molecular, are reversed by the addition of retinyl acetate to the medium containing delipidized serum. Cell motility and pattern formation are restored, and detachment of the most mature cells from the surface of the stratified epithelium is promoted. Synthesis of the 67 kd keratin is prevented and the synthesis of the 40 and 52 kd keratins is stimulated. The nature of the keratins synthesized is regulated by the concentration of vitamin A, and each cell type adjusts its synthesis differently at a given vitamin concentration.     

Vitamin A and E tissue distribution with comparisons to organochlorine concentrations in the serum, blubber and liver of the bowhead whale (Balaena mysticetus)

Vitamin A and E concentrations were determined in liver (n = 51), blubber (n = 23) and serum (n = 53) of subsistence-hunted bowhead whales (Balaena mysticetus), between 1998 and 2001. Retinol and alpha-tocopherol were the major forms of vitamins A and E detected, respectively. Liver contained the highest mean concentrations of vitamin A, followed by epidermis, blubber, and serum. Liver also contained the highest mean concentration of vitamin E, followed by serum, epidermis, and blubber. Stratification of retinol and tocopherol was examined throughout the blubber cores collected. Retinol concentrations were significantly higher in the epidermis than in the deeper blubber layers. Tocopherol concentrations were similar for epidermis and the intermediate layer of blubber. Both the epidermis and the intermediate layer of blubber had significantly higher tocopherol concentrations than the innermost and outermost blubber layers. Vitamin A and E concentrations were investigated with respect to gender and reproductive status of females (males, non-pregnant females, pregnant females), age groups and season of harvest. Certain persistent organic contaminants are known to have a negative effect on retinol concentration in serum of pinnipeds and cetaceans. Bowhead whales have relatively low concentrations of organochlorines (OCs) in comparison to other mysticete species. The relationships between serum, liver and blubber retinol and serum and blubber OC concentrations were examined with no significant correlations noted.     

The effect of vitamin D status on cutaneous sterologenesis in vivo and in vitro

Recent studies have shown that cutaneous sterologenesis is autonomous from the influence of circulating sterols, and that the epidermis is an important site of sterologenesis which is regulated by permeability barrier requirements. In addition to barrier function, an additional, important function of the epidermis is to synthesize sterol precursors of vitamin D3. The present study was designed, first, to determine whether vitamin D status and/or circulating levels of 1,25-dihydroxyvitamin D3 might play a role in regulating cutaneous sterol synthesis in vivo and, second, whether 1,25-dihydroxyvitamin D3 modulates sterologenesis in cultured human keratinocytes. Hairless mice were maintained on a vitamin D-deficient diet in the dark and supplemented with various doses of vitamin D3/day. Despite demonstrating serum 25-hydroxyvitamin D3 levels ranging from less than 10 to 343 ng/ml, the incorporation of tritiated water into cholesterol and total nonsaponifiable lipids in both the epidermis and dermis was similar in the four groups of animals. Likewise, administration of various doses of 1,25-dihydroxyvitamin D3 to vitamin D-deficient mice resulted in serum levels of 1,25-dihydroxyvitamin D3 ranging from less than 10 to 85 pg/ml; yet, cholesterol and total nonsaponifiable lipid synthesis was similar in both the dermis and epidermis in all groups of animals. Moreover, administration of 0.6 micrograms/kg per day of 1,25-dihydroxyvitamin D3 to 'normal' vitamin D-replete mice also had no effect on cutaneous sterol synthesis. Furthermore, conversion of 7-dehydrocholesterol to cholesterol in vitamin D-deficient vs. supplemented animals did not differ. Finally, addition of 1,25-dihydroxyvitamin D3 to cultured keratinocytes over a concentration range of 10(-12)-10(-7) M did not affect sterologenesis, except at supraphysiologic doses (10(-7) M). Together, these results suggest that vitamin D status does not influence sterol synthesis in the skin.     

1,25-Dihydroxyvitamin D3 target cells in rat mammary gland

In autoradiograms of mammary glands of rats on days 18 and 20 of pregnancy and day 6 of lactation after injection of 3H 1,25-(OH)2 vitamin D3, a nuclear concentration of radioactivity is observed in alveolar and ductal cells, as well as in cells of the epidermis of the nipple. Myoepithelial cells and connective tissue cells do not concentrate the hormone in their nuclei. The nuclear radioactivity appears specific, since 1,25-(OH)2 vitamin D3 but not 25-(OH)2 vitamin D3 prevents the uptake. The results suggest the existence of nuclear receptors for 1,25-(OH)2 vitamin D3 in mammary tissues and thus complements previous biochemical evidence showing the presence of cytoplasmic receptors for the hormone in mouse mammary glands; in addition, our results allow the identification of the cell types possessing the receptors.     

Topical antioxidant vitamins C and E prevent UVB-radiation-induced peroxidation of eicosapentaenoic acid in pig skin

Eicosapentaenoic acid protects against UV-radiation-induced immunosuppression and photocarcinogenesis, but it is also prone to oxidative degradation, which may reduce or abolish its beneficial effects. The protective effect of topically applied vitamin E, vitamin C, or both against UVB-radiation-induced lipid peroxidation in the presence of eicosapentaenoic acid was investigated using an ex vivo pig skin model. Changes in the bioavailability of both antioxidants induced by UV radiation were studied in different skin compartments. The UVB-radiation dose used (25 kJ/m2) was similar to that required to induce immunosuppression in BALB/c mice. Exposure of pig skin with an epidermal eicosapentaenoic acid content of 1.0 +/- 0.3 mol% to UVB radiation resulted in an 85% increase of epidermal lipid peroxidation (P < 0.005). Topical application of vitamin E or vitamin C 60 min prior to UVB irradiation resulted in a major increase in both antioxidants in the stratum corneum and viable epidermis (P < 0.05). Vitamin E and vitamin C completely protected against UVB-radiation-induced lipid peroxidation (P < 0.005), but compared to vitamin E, a 500-fold higher vitamin C dose was needed. UVB irradiation induced a vitamin E consumption of up to 100% in the stratum corneum and viable epidermis, and a vitamin C consumption of only 21% in the stratum corneum. Simultaneously applied vitamin E and vitamin C also completely protected against UVB-radiation-induced lipid peroxidation (P < 0.05), and lower antioxidant doses were needed compared to vitamin E or vitamin C alone. In the presence of vitamin C, epidermal vitamin E was more stable upon UVB irradiation (P < 0.05), suggesting interaction between vitamin E and vitamin C. In conclusion, topically applied vitamin E and/or vitamin C efficiently protect against UVB-radiation-induced lipid peroxidation in the presence of eicosapentaenoic acid. The beneficial biological effects of eicosapentaenoic acid may therefore be improved if vitamin E and/or vitamin C are present in sufficient amounts. The ex vivo pig skin model provides a useful tool for assessing short-term biochemical effects related to UVB radiation, without the use of living experimental animals.     

Endopolyploidie und Chloroplastenzahlen in verschiedenartigen Zellen trisomer Zuckerrüben

Summary Earlier investigations were continued on cell specific effects of eight different extra chromosomes in single trisomic sugar beets (Beta vulgaris L.). The degree of endopolyploidy, which was determined approximately by means of chloroplast numbers per cell, was changed by the presence of certain extra chromosomes: it either increased (I, II, VIII) or decreased (III through VII), but in epidermis and in spongy parenchyma the change was in the same direction. Sometimes, however, the degree was found to increase in spongy parenchyma alone or to decrease in epidermis alone; this evidence is consistent with the generally low liability of the epidermis to endopolyploidization. Independently of endopolyploidy the basic number of plastids (i.e., the amount reached in a cell type under given conditions, but without endopolyploidy) was altered in certain trisomes: it was higher (with extra chromosomes III through VI) or lower (II, perhaps also I) than in eudiploid control plants, the change taking the same direction in epidermal and in spongy parenchyma cells as in guard cells.     

Vitamin E inhibits PGE2 and O2- production in rat peritoneal macrophages.

In order to examine the possible role of vitamin E on the modulation of macrophages, we investigated the effect of vitamin E on O2- and PGE2 production in macrophages. The production of both PGE2 and O2- in rat peritoneal macrophages was dose-dependently stimulated by the addition of PMA and calcium ionophore A23187. However, the macrophages obtained after intraperitoneal injection of vitamin E for six successive days showed less PGE2 and O2- production when stimulated with PMA or A23187 as compared to those of control macrophages. O2- production in control macrophages stimulated with 139 nM PMA and 1 microM A23187 as 4.2 +/- 0.3 and 3.0 +/- 0.2 nmol/min per 10(6) cells, respectively. On the other hand, O2- production by the macrophages from vitamin E-treated rats was 1.5 +/- 0.4 nmol/min per 10(6) cells when stimulated with the PMA, and was not detectable when stimulated with A23187. As for the production of PGE2, control macrophages produced 2.59 +/- 0.70 ng/30 min per 10(6) cells when stimulated with PMA and 8.96 +/- 3.26 ng/30 min per 10(6) cells with the A23187, whereas PGE2 production by the macrophages from vitamin E-treated rats was reduced to 12-20% of the control. By analyzing alpha-tocopherol content and intracellular concentration of calcium ion [( Ca2+]i) in the macrophages isolated from control and vitamin E-treated rats, vitamin E treatment augmented alpha-tocopherol content (384.7 +/- 76.1 vs. 1.2 +/- 0.4 ng/10(6) cells) and decreased free [Ca2+]i when stimulated with A23187 (652 +/- 14 vs. 1201 +/- 223 nM).     

1,25-Dihydroxyvitamin D3 stimulates specifically the last steps of epidermal differentiation of cultured human keratinocytes

Human keratinocytes grown on deepidermized dermis (DED) are able to reconstruct a morphologically normal stratified and keratinized epidermis. This culture system is suitable for studying in vitro the effects of various hormones and factors on epidermal differentiation, and the goal of the present work was to study the effect of vitamin D. We found that the hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3, produced very specific alterations in epidermal architecture in a dose-dependent manner, consisting of significant reduction of the nucleated layers of the epithelium, but not of the stratum corneum, which was instead slightly thickened. The study of stage-specific differentiation markers showed that the two extreme layers of epidermis, i.e. the basal layer and the stratum corneum, were unaffected by the hormone, but that the reduction involved specifically the intermediate differentiation compartment, i.e. the spinous and granular layers. It was shown that the reduction of the intermediate compartment provoked by 1,25-dihydroxyvitamin D3 is not due to a block in the proliferation of basal cells or to inhibition of their differentiation into suprabasal cells, but to stimulation of the terminal differentiation of suprabasal cells into corneocytes.     

The use of retinoids as probes for analyzing morphogenesis of glands from epithelial tissues

Summary Thirty-five years ago Honor Fell and Edward Mellanby were studying effects of high doses of vitamin A on skeletal development in chick embryos when they noticed that a piece of epidermis, accidentally included in an organ culture, had undergone mucous metaplasia. Further studies by Fell and others eventually led to an understanding of the important role of vitamin A in modulating epithelia in vivo. Fifteen years later another organ culture experiment showed me that excess vitamin A could also initiate the morphogenesis of branching and mucus-secreting glands from developing vibrissa follicles in upper lip skin of embryonic mice. Since then our group has shown that induction of this novel structure by naturally occurring retinoids resembles a normal embryonic induction in that it is stage-dependent, time-dependent, and irreversible. Tissue separation and recombination studies showed that isolated upper lip epidermis can form these glands when combined with retinoid-treated upper lip dermis. Untreated mouse epidermis can form similar glands after combination with chick dermis containing higher retinoid levels. The hamster cheek pouch, normally devoid of glandular structures, can also form mucous glands when treated with a retinoid, either in vivo or in vitro. Recombination studies in organ culture have now shown that mesenchyme exposed to retinoid is essential for gland morphogenesis from pouch epithelium. Evidences is accumulating that retinoic acid may even be the active morphogen in some normally developing systems.