Serum-dependent avian skin differentiation in vitro: time sequence of induced events

Information concerning the developmental mechanisms involved in skin differentiation have been mainly derived from in vitro experiments. We have previously observed that 6-day chick embryonic thigh skin keratinizes in vitro in chicken serum-containing medium, but does not do so in chick embryo extract-containing medium. This system seems adequate in many respects for investigating the mutual regulative relationship between epithelium and mesenchyme (so called epithelio-mesenchymal interactions). We have therefore attempted better to define the time course of differentiative events and their dependence upon the time of serum administration. Skin explants have been sequentially supplemented with differentiation-stimulating or non-stimulating nutrient, removed at different intervals and examined by histological and histochemical procedures. Different epithelial and mesenchymal behaviour results according to serum supplementation time. Serum administered in the first 48 h of in vitro maintenance is unable to stimulate either the subsequent epidermal keratinization or the correlated changes in dermal histochemical pattern. On the other hand, serum-containing medium induces keratinization and changes in dermal intercellular composition (glycoproteins accumulate to a greater extent than glycosoaminoglycans, as does hyaluronic acid relative to chondroitin sulphuric acids), if added for only the second two days. In the last 48 h, serum may promote epidermal keratinization provided that in vitro incubation has been prolonged. Administration of actinomycin D with serum in the second 48 h prevents epidermal keratinization and modifies dermal histochemical reactivity, but is ineffective if added in the last 48 h. The above findings demonstrate that serum factor(s) stimulating epidermal differentiation act(s) on skin explants in the central incubation period and that correlatively mesenchyme acquires a characteristic histochemical pattern, supporting the possibility that the effect of serum may be mediated by changes in the composition of dermal ground substance.