In vitro analyses of cuticular differentiation at the chromosomal level in Lucilia sericata

Abstract

The large bristles of flies sclerotize and melanize during the pupal phase well before the rest of the cuticle. Each bristle is the product of two cells, the trichogen cell and the tormogen cell. Both their nuclei exhibit analyzable polytene chromosomes. The pupal metathoracic integument of Lucilia sericata has been excised and cultivated in Schneider's Drosophila medium for extended periods of time. Among the differentiations taking place in vitro, the most obvious is the stiffening and coloring of the bristles. The in vitro differentiation very much resembles the in vivo differentiation in its timing and appearance. Explants excised early in pupal development cannot differentiate in vitro, while those excised and cultured at later stages can. Actinomycin D was administered to the culture medium and the incorporation of [3H]uridine was analyzed after long incubations to determine if this process was a reaction of dead cuticle or controlled by living cells. The analysis of chromosome structure and puffing patterns in the five polytene chromosomes of the trichogen cell after in vitro cultivation shows that transcriptional activity is not dependent on the existence of normal chromosome conformation. The degree of pycnotic reaction and puff induction of the nuclei, however, varied under different culture conditions. The capacity of this system for differentiation can be used to study in vitro chromosomal activity during the course of development.Key words: in vitro, polytene chromosomes, sclerotization, cuticle.