The Importance of Mesenchymal Factors in the Differentiation of Chick Epidermis I

The Importance of Mesenchymal Factors in the Differentiation of Chick Epidermis I. The Differentiation in Culture of the Isolated Epidermis of the Embryonic Chick and its Response to Excess Vitamin A by C. B. MC LOUGH LIN1 From the Strangeways Research Laboratory, Cambridge WITH FOUR PLATES INTRODUCTION IT is well established that in the developing chick the underlying mesenchyme initiates the appearance of specific epidermal derivatives, e.g. feathers (Sengel, 1956), claws (Cairns & Saunders, 1954), and the preen gland (Gomot, 1958). On the other hand, it is not yet known to what extent the epidermis is independent of mesenchymal intervention for its basic differentiation into a stratified, squamous epithelium. Sobel's (1958) work on the 8-day chick pituitary suggests that the differentiation and multiplication of certain epithelial cells cannot proceed in the absence of mesenchymal elements. She found that the isolated epithelial cells of the hypophysial rudiment survived but were unable to differentiate or multiply; when associated with perichondrial fibroblasts, however, they resumed mitosis and produced typical a and ft cells. In the first part of the present investigation, experiments were made to see whether the embryonic epidermis, like the hypophysial epithelium, requires the presence of fibroblasts to enable it to grow and differentiate, or whether it can proliferate, acquire its characteristic squamous structure and keratinize, when isolated and cultivated in the absence of connective tissue. It has been shown (Fell & Mellanby, 1952; Fell, 1957) that when embryonic chicken skin is cultivated in medium containing excess vitamin A (7-5-22-5 i.u./ml. of medium), the epidermis fails to keratinize and is transformed into a columnar, mucus-secreting epithelium which sometimes becomes ciliated. In view of the fact that excess vitamin A also produces striking changes in the intercellular material of cartilage (Fell & Mellanby, 1952; Fell & Thomas, 1960), it seemed possible that in the skin explants the action of the vitamin might be primarily on the dermal connective tissue and that the epithelial metaplasia might be a secondary effect. 1 Author's address: Strangeways Research Laboratory, Wort's Causeway, Cambridge, U.K. [J. Embryol. exp. Morph. Vol. 9, Part 3, pp. 370-384, September 1961] ISOLATED EPIDERMIS 371 The second part of this study was undertaken to test this hypothesis; the effect of excess vitamin A on isolated epidermis in culture has been examined, to find whether the mucous change would appear in the absence of the dermal fibroblasts. The results show that the isolated epidermis is capable of keratinizing and of responding to excess vitamin A by mucus secretion, but that it cannot grow in the absence of mesenchyme. MATERIAL AND METHODS Material For a histological study of the normal differentiation of chick skin in vivo, the entire limb-buds of 4- and 5-day embryos and fragments from the thigh and shank regions of 8-, 10-, 12-, 13-, and 17-day embryos were used. Cultures were made from the skin of 5-day limb-buds. Tissue culture Cultures of intact skin. The development in culture of the intact skin was studied in 26 explants fixed at intervals of 1 to 11 days. The tissue was grown by Maximow's double coverslip method in a medium composed of 2 parts of cock's plasma and 1 part of embryo extract; the extract consisted of 1 part of embryo mince (13-day chick) and 2 parts of Tyrode's solution. The explants were fed with fresh medium after 2 days' growth and every 4th day were transplanted to a fresh coverslip, a procedure that prevented undue spreading of the tissue. Cultures of isolated epidermis. Epidermis was isolated from the mesenchyme as follows. The hind limb-buds were removed from the 5-day embryo and their apical ridges cut off and discarded; the rest of each bud was cut transversely, but somewhat obliquely, into 4 wedge-shaped pieces which were washed for 4 minutes in Ca-Mg-free Tyrode's solution and then transferred to a 3 per cent, solution of crude trypsin (BDH) in Ca-Mg-free Tyrode (Moscona, 1952). After about 10 minutes the epidermis began to separate from the mesenchyme; the process was hastened by gently rocking the dish containing the fragments, and completed by teasing the delicate sheets of epidermis free with fine, blunted glass needles. As each sheet of epidermis was detached, it curled into a tight roll with the periderm outwards; to inactivate the trypsin these rolls were transferred to a dish of horse serum diluted with an equal volume of Tyrode. After 15 minutes in the serum, the fragments were explanted on Maxi- mo w double coverslips in a mixture of 2 parts plasma and 1 part embryo extract; the epithelium degenerated when a higher concentration of extract was used. The explants were fed and transplanted as described above, and were fixed after 1 to 10 day's cultivation. Sixty-two explants of isolated epidermis grown in normal medium were examined. During tryptic digestion a sticky transparent mucoid material oozed out of the mesenchyme and adhered firmly both to the mesenchyme cells and to the 5584.9 Bb 372 C. B. McLOUGHLIN—DIFFERENTIATION OF epithelium (Plate 1, fig. 1), with the consequence that the isolated epidermis sometimes carried with it a wisp of this material through the entire procedure to explantation. On prolonged exposure to trypsin the mucoid gradually dissolved. This is the same intercellular material whose importance to the reaggregation of separated cells has been established by Moscona (1961). As described below, it was also found to affect the orientation of epidermal cells. For the experiments with vitamin A, synthetic vitamin A alcohol was dissolved in ethanol and added to the plasma so as to give a concentration of either 2-5 or 6-6 i.u. vitamin A/ml, in the culture medium. The solution of vitamin A in ethanol was always such that the concentration of ethanol in the plasma was 002 per cent. An equal amount of ethanol was added to the plasma of the control cultures. Eighteen explants were grown in vitamin A medium. Histology Tissues were fixed in 3 per cent, acetic Zenker's solution or in Carnoy's fluid, and sections were stained with Meyer's acid haemalum and eosin or alcian blue (0-5 per cent, in distilled water, 20 seconds), by Mallory's azan method, Heiden- hain's iron haematoxylin, or the periodic-acid/Schiff method (PAS). RESULTS Normal development in vivo of the limb-bud epidermis The epidermis of the limb-bud of a 5-day chick embryo is a delicate 2-layered structure composed of an outer periderm (epitrichium) and an inner basal layer (Plate 1, fig. 2). The basal cells, which will finally produce the entire keratinizing epidermis, are seated on an extremely thin basement membrane which stains with the PAS technique. They are stellate and arranged in a single irregular layer between the basement membrane and the periderm (Plate 1, figs. 2, 3); their mitotic rate is high. The cells of the periderm are extremely flattened and can be seen best in oblique sections (Plate 1, fig. 3); they are capable of division and so, unlike the more distal layers of older epidermis, they may be independent of the basal layer for their replenishment. A periderm is present in chick embryos from at least as early as 2 days' incubation until it is sloughed from the stratum corneum in the 18-day embryo. By contrast with the stellate structure of the basal layer, the peridermal cells are firmly attached to each other, so that from the time of their appearance to about 12 days, when the gaps between the basal cells are finally closed, the periderm is the only uninterrupted cytoplasmic barrier inter- posed between the interior of the embryo and the amniotic fluid. In several places the epidermis is thickened and its cells assume a typical columnar or cuboidal epithelial arrangement. These areas of epithelial thickening ISOLATED EPIDERMIS 373 are the apical ridge whose important morphological function is well known (Saunders, 1948), an axillary columnar thickening which participates in the moulding of the axilla (Saunders, 1953), and a cuboidal tract which runs along the anterior and posterior borders of the limb-bud and is continuous with both the axillary and apical thickening. This last becomes obvious by its greater rigidity when the epidermis is removed from the mesenchyme. These charac- teristics are better observed in 4-day limb-buds than in those of 5 days in which the epidermis has become much thinned owing to the rapid growth of the rudiment. The presence of these epithelial thickenings is correlated with localized thickenings of the basement membrane, which are both observed under the microscope and inferred from the fact that these regions are the last to be freed by tryptic digestion during the separation of the epithelium from the mesenchyme. Till 12 days in vivo the epidermis remains 2-layered, since all its expansion is at first absorbed in covering the rapidly growing limb, and is later localized in the feather-germs which appear at about the 7th day. The basal layer gradually assumes a typical columnar epithelial arrangement, but the gaps between the originally stellate cells are not finally closed till the 12th day. From this stage onwards the epidermis begins to form squamous layers, but even at 17 days true keratinized epithelium, in which the nuclei have completely disappeared, is not present in thigh skin though there is a very thin layer on the scales of the shank. A stratum granulosum is generally thought to be absent from chick epidermis, but a thin layer of flattened cells containing rounded granules which take up Meyer's acid haematoxylin is visible in oblique sections of 17-day thigh epidermis.

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