Mass-isolated imaginal discs of Drosophila melanogaster form a chitin-containing pupal procuticle In vitro. Optimal procuticle deposition occurs when the discs are incubated for 4-6 hr with 0.5-1.0 μg/ml of 20-hydroxyecdysone and then with less than 0.05 μg/ml of 20-hydroxyecdysone. The formation of the chitin-containing procuticle is demonstrated using three independent assays: with fluorescene-conjugated cuticle proteins that bind to chitin; by electron microscopy; by incorporation of [3H]glucosamine into a chitin fraction. Synthesis and deposition of pupal cuticle proteins are also demonstrated. Incorporation of [3H]glucosamine into chitin is sensitive to inhibitors of protein, RNA and chitin synthesis, but has little sensitivity to inhibitors of DNA synthesis, and dolichol-dependent glycosylation. © 1982.
Fristrom, J. W., Doctor, J., Fristrom, D. K., Logan, W. R., & Silvert, D. J. (1982). The formation of the pupal cuticle by Drosophila imaginal discs in vitro. Developmental Biology, 91(2), 337–350. https://doi.org/10.1016/0012-1606(82)90040-9