The presence of Ecd in the late-third instar ring gland is consistent with the steroid deficiency for which ecd1 was originally identified. The ability to induce puparium formation by feeding the non-pupariating ecd1 larvae at 29°C with 20-hydroxyecdysone (20E) (Garen, 1997; Redfern, 1983; Berreur, 1984), suggested that low steroid levels might be the primary cause of arrest at this stage. To test whether the non-conditional ecd mutants could also be rescued by dietary hormone, homozygous second instar ecd2 and ecdl(3)23 larvae were fed with 20E. The feeding of ecd1 larvae at 29°C served as a positive control: 50 µg/ml and 250 µg/ml 20E doses induced pupariation in 26 out of 100, and in 36 out of 100, ecd1 homozygotes, respectively. By contrast, none of 600 ecd2, or 250 ecdl(3)23, larvae progressed beyond their lethal phase when fed 20E. These results strongly imply that ecdysone deficiency alone does not account for the second instar lethality of these mutants. In support of this view, the whole-body ecdysteroid content was not significantly different between ecd2/ecd2 (0.61±0.13 pg/animal) and ecd+ (0.48±0.08 pg/animal) first instar larvae (Gaziova, 2004).
To address the problem of ecd requirement directly, ecd expression was targeted to the steroidogenic part of the ring gland using transgenic UAS-ecd activated by a Gal4 driver, Feb36. As was expected from the ability of exogenous 20E to rescue pupariation of ecd1 homozygotes at 29°C, Ecd expressed under Feb36 allowed formation of defective puparia in around 25% UAS-ecd, ecd2/ecd1 larvae upshifted to 29°C. The ectopic Ecd presence in the ring gland, evident during the second instar, should therefore restore the impaired hormone synthesis and at least postpone the arrest of ecd-null mutants, if disrupted ecdysone production was the sole cause of their death. However, the Feb36-driven Ecd was insufficient to advance UAS-ecd, ecd2/ecd2 larvae even to the second molt. By contrast, the same UAS-ecd construct expressed under a ubiquitous actin-Gal4 driver allowed ecd2 homozygotes to reach adulthood (Gaziova, 2004).
The failure to rescue non-conditional ecd mutants with Ecd targeted to the ring gland, or by 20E feeding, correlates with the absence of Ecd from the ring gland before the third instar. Taken together, the data show that ecd is autonomously required in other organs before it is needed for ecdysone synthesis. To identify the tissue-specific requirement, Ecd was expressed using several other Gal4 drivers. Ecd driven by the patched (ptc) promoter provided a partial rescue: a single copy of ptc-Gal4 allowed ecd2 homozygotes to molt to the third instar; two copies supported formation of defective but tanned prepupae (Gaziova, 2004).
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