(A, B) RNA in situ hybridization of stage 14 (A) and stage 16 (B) embryos with the ouib antisense RNA probe. Dorsal views are shown. ouib signal was detected in the primordia of PG cells (arrows). An image with sense RNA probe is shown in S1 Fig (C, D) in situ hybridization of third instar larval brain-ring gland complexes with the ouib antisense (C) and sense (D) RNA probes. ouib signal is detected in the ring gland including the PG cells (arrow). (E) The expression levels of ouib in several tissues quantified by qRT-PCR (N = 3). Total RNA was prepared from wandering third instar larvae. BR, brain; RG, ring gland; ID, imaginal disc; IN, intestine; FB, fat body; SG, salivary gland. Error bars indicate the .
AB - Pulses of ecdysteroids direct Drosophila through its life cycle by activating stage- and tissue-specific genetic regulatory hierarchies. Here we show that an orphan nuclear receptor, DHR78, functions at the top of the ecdysteroid regulatory hierarchies. Null mutations in DHR78 lead to lethality during the third larval instar with defects in ecdysteroid-triggered developmental responses. Consistent with these phenotypes, DHR78 mutants fail to activate the mid-third instar regulatory hierarchy that prepares the animal for metamorphosis. DHR78 protein is bound to many ecdysteroid- regulated puff loci, suggesting that DHR78 directly regulates puff gene expression. In addition, ectopic expression of DHR78 has no effects on development, indicating that its activity is regulated posttranslationally. We propose that DHR78 is a ligand-activated receptor that plays a central role in directing the onset of Drosophila metamorphosis.
AB - Ecdysteroids act initially by binding to nuclear and possibly also extranuclear receptors. The presence and expression of these receptors in the insect brain was investigated in the present study as a means of defining these neurons involved in ecdysteroid-regulated processes at different developmental stages. Early in the fifth larval stadium of Manduca sexta, when endogenous ecdysteroid levels are low, receptors for ecdysteroids in cerebral neurons are either absent or present at low levels. Receptors can be reliably detected only on day 0 and are not found again until day , at the beginning of the commitment peak in the ecdysteroid titer, when they occur in a small stage-specific population of cells. At this time, ecdysteroid receptors are found mainly in nuclei but are also observed at low levels in cytoplasm. By day , ecdysteroid receptors are exclusively nuclear, and the number of target cells has increased dramatically in several brain regions, including those with known neurosecretory cell groups. This population and organization of ecdysteroid target cells is constant up to day 6, after which time the number of target neurons declines. By day , only 10% of the number of labelled neurons seen on days - remain in peripheral areas. In the pupal brains, ecdysteroid receptors reappear in a new population of neurons. The results indicate changes in the genomic regulation of a varying neuron population by ecdysteroids during fifth stadium development.