In Drosophila and other arthropods, pulses of the steroid hormone 20-hydroxyecdysone (20E) are responsible for the temporal coordination of larval molts and metamorphosis. Physiological responses during these events can be diverse; for example during metamorphosis obsolete larval tissues are destroyed and adult structures arise from imaginal disc cells. Remarkably, these actions are carried out in a coordinated, tissue specific manner. At the site of target tissues, 20E binds to its cognate nuclear receptor triggering a cascade of gene activation. Primary 20E-inducible genes, which are directly induced by the steroid-receptor complex, are the earliest genes in the cascade to be transcribed and are insensitive to protein synthesis inhibitors. In contrast, secondary 20E-inducible genes are expressed later and are dependent on the synthesis of primary-response genes. Early studies of the 20E cascade looking at the puffing patterns of polytene chromosomes of late 3rd instar larvae in response to 20E, predicted that primary-response genes would code for proteins that are responsible both for the induction of secondary response genes as well as for the inhibition of their own transcription . Three of the most well characterized 20E-primary response genes, Eip74EF, Eip75B, and br fit this description perfectly. Furthermore, these three genes reside at chromosome cytolocations 74EF, 75B, and 2B5 respectively, which, along with approximately three other loci, exhibit rapid and dramatic puffing after exposure to 20E either naturally or artificially [2–4]. Likewise, the earliest characterized secondary response genes are found in a region (71E) that forms a distinct yet delayed puff upon 20E exposure [5, 6]. Although the precise function of these genes has yet to be determined, based on their genomic sequence they are thought to encode effector proteins which is consistent with early predictions of secondary-response gene function . Since the original characterization of these 20E-response genes, however, many examples of primary and secondary response genes with diverse functions have emerged, and many of these 20E-inducible genes do not appear to be associated with any identifiable 20E-induced puffs [7–13] underling the importance identifying individual components of the cascade for a clearer picture of 20E action.
The transcription factor complex coordinating the entire 20E-hierarchy, the 20E nuclear receptor heterodimer is composed of the vertebrate retinoid X receptor homolog, Ultraspiracle (USP) and the ecdysone receptor (EcR). EcR isoform distribution differs between tissues and typically those tissues expressing different isoforms show different responses to ecdysone at metamorphosis [14–17]. For example, immunolocalization experiments examining EcR isoform expression pattern at the onset of metamorphosis show that larval tissues destined to die during metamorphosis, such as the salivary gland, stain strongly for isoform B1 and weakly for isoform A whereas proliferating tissues, such as imaginal discs, show a reciprocal pattern staining strongly for isoform A and weakly for isoform B1 . The most recent annotation of the Drosophila genome documents five EcR isoforms that differ in sequence at the amino terminus but share common DNA- and ligand- binding domains. The ligand-binding pocket of the 20E receptor shows remarkable flexibility enabling its activation by a variety of steroidal and non-steroidal 20E analogs/agonists . For example, the ligand-binding domain of the Drosophila EcR homolog in Heliothis virescens adopts different structures in the presence of different 20E analogs .
Of the insect and plant derived analogs characterized, ponsterone A (PoA) is the most potent agonist of the EcR receptor  with an affinity approximately eight times greater than 20E . As such, PoA, which differs from 20E by the abscence of a single hydroxyl group, has been used to work out numerous kinetic and physical parameters of the EcR . Interestingly, the natural role of this and other phytoecdysteroids is still under debate, although most evidence disfavors a hormonal role in plants . Instead, it is predicted that phytoecdysteroids induce precocious molting and subsequently death in insects providing an effective defense against insect feeding . Given the increased binding affinity of PoA over 20E as well as its potential use in insect control, it is of importance to understand how this phytoecdysteroid affects transcription on a global scale.
The Drosophila Kc cell line is one of the most well documented 20E-responsive cell lines currently available and has been used in numerous studies examining the effects of 20E. In this study we use cDNA-based microarrays representing approximately 80% of the Drosophila genome to identify 20E-responsive genes in Kc167 cells. By exploiting the sensitivity of secondary-response gene transcription to protein synthesis, we are able to determine which of these genes are primary and which are secondary 20E-inducible genes. Furthermore, the transcriptional response to physiological levels of 20E is compared to that elicited under two other conditions: a 20-fold higher concentration of 20E or its plant derived structural analog, PoA. These analyses lead to the identification of 35 genes that reacted similarly to all three treatments. In addition to examining the transcriptional response to 20E in Kc cells, we were also interested in examining the response in a natural target tissue of 20E signaling, salivary glands. Salivary glands have been critical in the elucidation of the 20E signaling hierarchy through the examination of puff patterns on polytene chromosomes. By focusing on the response of the salivary gland in the isolation from other larval tissues we were able to identify many 20E-responsive genes that were not detected when whole larvae were examined . Taken together, this work provides a detailed picture of the genes involved in the ecdysone hierarchy of gene transcription in Kc167 cells and salivary glands.