The Ecdysoneless (ecd

Ring glands dissected from homozygous l(3)ecd1 ts wandering larvae and upshifted in vitro to the restrictive temperature, 29°C, synthesize abnormally low quantities of ecdysteroid. Nevertheless, ecd1 ring glands retain the ability to respond at 29°C to an extract prepared from wild-type larval neural tissues that presumably contain prothoracicotropic hormone (PTTH), although both basal and stimulated levels of synthesis are lower than those in wild-type ring glands. Extracts prepared from ecd1 neural tissue exhibit an unusually high level of PTTH activity. Mutant ring glands downshifted in vitro to the permissive temperature after removal from larvae maintained at 29°C regain the ability to produce normal basal and stimulated ecdysteroid levels. Collectively, these experiments demonstrate that the ecd1 mutation disrupts the physiology of the ring gland at 29°C autonomously and may also interfere with PTTH release. Article: The ecdysoneless temperature-sensitive [l(3)ecd11 mutation of Drosophila melanogaster was isolated originally as a conditional larval lethal that exhibits drastically lower ecdysteroid levels when reared at a restrictive temperature, 29°C (Garen et al., 1977). Nonetheless, the developmental time lag between temperature upshift and reduction of ecdysteroid titer, as well as the autonomous expression of the gene in imaginal tissues, indicates that ecd1 may act indirectly to disrupt the ecdysteroid-synthesizing capacity of the ring gland (Redfern and Bownes, 1983; Sliter, 1986). Despite these complexities, it is apparent that ecd1 larvae ultimately show a reduced ecdysteroid titer, probably caused by impaired function of the larval ring gland (Redfern and Bownes, 1983). Further, mutant individuals shifted to 29°C for up to 48 hr can be rescued by a subsequent shift back to 18°C, indicating that the mutant effects of ecd1 are reversible and do not lead to immediate and permanent dysfunctions of larval tissues, particularly the ring gland (Redfern and Bownes, 1983; Sliter, 1986). The present study aims to extend previous observations concerning the effect of the ecd1 mutation upon the ability of the ring gland to synthesize ecdysteroids in vitro and to determine whether the mutation exerts an autonomous effect upon ring gland function and/or prothoracicotropic hormone (PTTH) synthesis or action. The latter hormone initiates the molting cycle by stimulating ecdysteroid synthesis in the prothoracic gland cells of the ring gland and a reduced titer of PTTH leads to decreased ecdysteroid synthesis (see Gilbert et al., 1981). MATERIALS AND METHODS Stock maintenance. All larvae utilized in these experiments were reared on standard agar food medium under a constant light, 18°C regime until the time of experimentation, unless otherwise noted. Eggs were collected at 4hr periods from individuals homozygous for ecd1 st red e ca. These adults were the selected progeny of mass crosses between individuals of a conditional balanced lethal stock (TM6/ecd1 st red e ca) at the permissive temperature, 18°C. Homozygous individuals can be distinguished by the orange eye color that results from the interaction of the recessive eye color marker mutations and wild-type halteres (TM6 carries Ubx 67b ) and e). Constant selection for the parents of tested progeny was necessary because genetic modifiers accumulate within a few generations in a homozygous population, even at 18°C. Similar egg collections were made from members of a wild-type Canton-S strain. Dissection and incubation methods. For the experiments to measure the response of an in vitro temperature upshift, individual ring glands or brain-ventral ganglion-ring gland complexes were dissected from early wandering larvae at 18°C and placed in 15-μl drops of Grace's medium preincubated at 18 or 29°C in accordance with a previously developed protocol for another dipteran (Roberts et al., 1984). Larvae were taken as they wandered off the food medium and those with swollen salivary glands were discarded. After either 2 or 4 hr of incubation in vitro, a 10-μl sample was removed from the culture medium and the ecdysteroid content quantified by radioimmunoassay (RIA) (Warren et al., 1984). The data are expressed in ecdysone equivalents. It should be noted that the general term ecdysteroid is used here although it appears that ecdysone is the major product of the Sarcophaga ring glands (Bollenbacher et al., 1976) and Manduca prothoracic glands (King et al., 1974). In the case of Drosophila ring glands, however, the in vitro incubation products include ecdysone, 20deoxymakisterone, and an unidentified ecdysteroid (Redfern, 1984; Warren, Henrich, and Gilbert, unpublished