During insect metamorphosis, each tissue displays a unique physiological and morphological response to the steroid hormone 20-hydroxyecdysone (ecdysone). Gene expression was assayed in five tissues during metamorphosis onset. Larval-specific tissues display major changes in genome-wide expression profiles, whereas tissues that survive into adulthood display few changes. In one larval tissue, the salivary gland, a computational approach was used to identify a regulatory motif and a cognate transcription factor involved in regulating a set of coexpressed genes. During the metamorphosis of another tissue, the midgut, genes encoding factors from the hedgehog, Notch, EGF, dpp, and wingless pathways are activated by the ecdysone regulatory network. Mutation of the ecdysone receptor abolishes their induction. Cell cycle genes are also activated during the initiation of midgut metamorphosis, and they are also dependent on ecdysone signaling. These results establish multiple new connections between the ecdysone regulatory network and other well-studied regulatory networks (Li, 2003).

Developmental patterns of gene expression were studied from five different tissues and organs: central nervous system (CNS), wing imaginal disc (WD), larval epidermis and attached connective tissue (ED), midgut (MG), and salivary gland (SG), during late larval and early prepupal development when ecdysone triggers metamorphosis. At these stages of development, the five tissues display very different morphological and physiological responses to ecdysone. The wing imaginal disc responds to the hormone by initiating evagination, or unfolding, as it changes from a compact epithelial bilayer to an extended appendage. The salivary glands secrete glue proteins that are used to immobilize the puparium during metamorphosis. The cuticle attached to the larval epidermis undergoes a process of hardening and tanning to form the pupal case. The central nervous system (CNS) displays little morphological change during the late third instar ecdysone pulse, but the animal displays changes in behavior and in neurosecretory status. The two major types of cells in the larval midgut, larval epidermal cells and adult epidermal progenitor cells (midgut imaginal islands), respond in opposite ways to ecdysone. The larval epidermal cells initiate the process of programmed cell death, while the imaginal cells proliferate and form the adult midgut (Li, 2003).

One tissue, the midgut, was selected to assay during its complete metamorphosis, which occurs from 18 hr before puparium formation (BPF) to 12 hr APF. During this 30 hr period, eleven time points were examined as the larval midgut is destroyed and replaced with the adult midgut. The two major cell types present in this organ are distinguishable by size. The larval epithelial cells are large, with decondensed polyploid nuclei, and undergo programmed cell death in response to ecdysone. Embedded among the larval cells are small diploid imaginal midgut cells, which proliferate in response to the hormone to form the adult epithelial cells. Additionally, the midgut contains relatively small numbers of muscle, tracheal, and endocrine cells (Li, 2003).

In total, transcripts from a surprisingly large fraction of the genome, >30%, changed significantly during the metamorphosis of the midgut (18 hr BPF to 12 hr APF). Broad classes of temporally separable gene expression patterns are evident. These classes include sets of transcripts that rapidly decrease coincident with onset of programmed cell death in the larval cells, sets that are induced during early or late metamorphosis, and sets of transcripts expressed at highest levels during the middle period of the time course when the larval cells are in the final stages of cell death and the adult cells are rearranging to form new tissue (Li, 2003).

Within these broad classes, specific sets of genes that have related functions and show parallel expression were identified, indicating that they make up gene batteries. Six such examples, included coregulated transcripts that encode proteins found in specific macromolecular complexes, biochemical pathways, organellar functions, and structural components of the cells that compose this tissue. Transcripts encoding proteasome components increase during the ecdysone pulse that triggers the onset of cell death in larval cells. Transcripts encoding glycolytic enzymes rapidly decrease during the initiation of metamorphosis, but gradually resume expression as the imaginal cells proliferate. Vacuolar ATPases shows a pattern similar to the glycolytic enzymes, whereas tubulin- and actin-encoding transcripts peak during the intense period of imaginal cell proliferation and migration as the adult midgut is formed. Transcripts encoding structural components of the peritrophic membrane of the mature larval gut gradually decrease during its replacement with adult tissue (Li, 2003).

The expression patterns were examined of regulatory genes known to be involved in the ecdysone transcriptional hierarchy predicted to control the gene batteries that were identified. Also examined was the expression of genes with known roles in programmed cell death or cell cycle control. The expression of known ecdysone-responsive regulatory genes was consistent with previous observations in midgut. Although the larval midgut is composed of cell types that undergo divergent responses to ecdysone -- apoptosis and cell proliferation -- it was nonetheless possible to detect significant changes in transcript levels from genes encoding proteins involved in both processes. The apoptosis activator gene ark was expressed at 4 hr BPF. E93 and reaper, which encode proteins that serve as critical control points in the commitment to programmed cell death, were expressed at PF, as was the initiator caspase dronc. These midgut expression profiles were compared to those reported for salivary glands at and after 10 hr APF, when a prepupal pulse of ecdysone triggers apoptosis in that tissue; almost the entire genetic cascade was found to be similarly activated in salivary glands and midgut albeit at two distinct periods of development. However, one notable difference was observed at the top level of the cascade. In the salivary gland, E93 is activated by βFTZ-F1, whereas in the midgut the βFTZ-F1 gene is not induced until 6-8 hr after E93 is induced. The regulation of E93 therefore does not depend on βFTZ-F1 in the midgut, but must rely on another as yet unidentified factor(s). During midgut metamorphosis, developmental modulation of transcript levels were also observed for genes encoding DNA polymerases, cyclins, CDCs, and other cell cycle regulators, as well as genes encoding DNA repair proteins such as Hus1, Rad23, and PCNA/Mus209 (Li, 2003).

Which of the genes that are differentially expressed at the onset of midgut metamorphosis require ecdysone signaling? Ecdysone-dependent transcriptional activity was removed using mutant Ecdysone Receptor (EcR) alleles, rescuing null EcR mutants to the third larval instar by using a heat shock-inducible EcR transgene. Gene expression was examined in mutant midguts that were isolated from mutant animals arrested at the end of the third larval stage (stage 2a mutants). 376 (76%) of the 495 genes that are significantly induced during the onset of midgut metamorphosis (18 hr BPF to 2 hr APF) required EcR function, whereas 296 (64%) of 460 transcripts that decline significantly in level during this time period require ecdysone signaling through EcR. Thus, a very large proportion of the genes that are developmentally regulated at the initiation of metamorphosis in this organ are under the control of the transcription factors that mediate the ecdysone signal. However, it does not appear that EcR function is a general requirement for transcription, because a significant fraction of differentially expressed genes are unaffected in EcR mutant tissue (Li, 2003).

Of the several different classes of genes expressed during midgut metamorphosis, the regulation of all genes in the proteasome, tubulin/actin, and lysozyme clusters requires EcR to exhibit their normal changes in developmental expression. However, many genes in the v-ATPase cluster and nearly half the genes in the peritrophin cluster did not require EcR. The downregulation of hexokinase A, 6-phosphofructokinase, and pyruvate kinase genes in the glycolysis pathway were affected in the EcR mutants, while many others in this pathway were not. Hexokinase A, 6-phosphofructokinase, and pyruvate kinase are rate-controlling enzymes in the glycolytic pathway, indicating that their ecdysone dependence is functionally significant. The expression of the numerous known ecdysone receptor target genes such as E75, E74, broad, E23, and DHR3 required EcR as expected. The induction dynamics for the E74 and DHR3 transcription factor genes was as expected, as was their dependence on EcR. In contrast to E74 and DHR3, DHR78 has previously been described to reside upstream of EcR at the top of the ecdysone regulatory hierarchy -- the expression of EcR is dependent on the wild-type function of DHR78. However, DHR78 can also be induced by ecdysone in organ culture. The results demonstrate that DHR78 wild-type induction is indeed dependent on EcR function. Taken together, these data indicate a positive feedback loop between EcR and DHR78 during the onset of metamorphosis in the midgut (Li, 2003).

Genes encoding factors involved in cell cycle and growth control, and in DNA repair, are also under the control of EcR. In spite of the role of ecdysone in stimulating cell proliferation during metamorphosis, no cell cycle genes have previously been linked to the ecdysone regulatory hierarchy. The induction of the cell cycle regulatory genes CyclinB, cdc2, and CyclinD were all observed to be dependent on EcR function. The rapid induction of cdc2 during the late third instar ecdysone pulse is similar to that observed for direct targets of EcR. The CyclinD gene is also induced at this time, but its maximal induction occurs several hours after that observed for cdc2. Cyclin D promotes cellular growth, whereas Cyclin B/Cdc2 controls G2/M transitions in proliferative cells. The dependence of these three genes on EcR function indicates that ecdysone may control cell proliferation, at least in part, through their regulation. Coordinate with the induction of CyclinB, cdc2, and CyclinD, the induction was observed of DNA polymerase-delta and DNA repair genes such as Rad23, and PCNA/mus209. The induction of these DNA repair and synthesis genes is also EcR dependent. The expression changes of these genes may be the result of the direct action of EcR, or due to the action of factors directly controlled by the ecdysone receptor complex. It is unlikely that the increase in expression of these genes is simply due to increased numbers of proliferative cells because the total number of divisions between 18 hr BPF and PF are few, and not all cell cycle or DNA repair genes showed an increase in expression at the initiation of metamorphosis. For example, the level of CyclinJ, which is known to be required during early embryonic division cycles, is actually reduced in expression from 18 hr BPF to PF. When the expression of cell death genes was examined in EcR mutant tissue, E93 induction was observed as well as induction of the Ark caspase activator and the dronc caspase gene required wild-type function of EcR (Li, 2003).

Factors in several well-studied signaling pathways are induced during midgut metamorphosis. These include Wnt (dishevelled, armadillo, and zeste white 3), TGFβ/BMP (sara, daughters against dpp, and glass bottom boat), EGFR (torpedo/egfr, rhomboid/veinlet, vein, and keren/spitz2), and Notch pathway genes (delta, kuzbanian, suppressor of hairless, E(spl)malpha, and E(spl)mβ). All of these pathways are used during embryonic midgut development, and these data indicate they are reused during midgut metamorphosis. Genes in the Hedgehog signaling pathway (hedgehog, smoothened, and cubitus interruptus) changed significantly as well (Li, 2003).

The steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription. This study identifies the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. Genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. Evidence is provided that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. An initial characterization is presented of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes. This study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development (Beckstead, 2005).

To identify genes that alter their expression in synchrony with the late third instar and prepupal pulses of 20E, RNA was isolated from w¹¹¹⁸ animals staged at -18, -4, 0, 2, 4, 6, 8, 10, and 12 hours relative to pupariation, labeled, and hybridized to Affymetrix Drosophila Genome Arrays. The sensitivity and accuracy of the array data were determined by comparing the expression patterns of known 20E-regulated genes with previously published developmental Northern blot data. A subset of this analysis reveals that the temporal expression pattern of key regulatory genes - EcR, usp, E74A, DHR3, FTZ-F1, and DHR39 - are faithfully reproduced in the temporal arrays, as well as the 20E-regulated switch from Sgs glue genes to L71 late genes in the larval salivary glands, and the expression of representative IMP and Edg genes in the imaginal discs and epidermis. This comparison demonstrates that the microarrays accurately reflect the temporal patterns of 20E-regulated gene expression at the onset of metamorphosis and have sufficient sensitivity to detect rare transcripts such as EcR and E74A (Beckstead, 2005).

EcR mutants die during early stages of development, complicating their use for studying receptor function during metamorphosis. To circumvent this problem, a transgenic system was used that allows heat-induced expression of double-stranded RNA corresponding to the EcR common region to disrupt EcR function at puparium formation (EcRi). RNA was harvested for array analysis from EcRi animals staged at -4, 0, and 4 hours relative to pupariation. All EcRi animals formed arrested elongated prepupae, consistent with an effective block in 20E signaling and highly reduced EcR protein levels. Data obtained from these arrays were compared to the array data from control animals at the same stages of development to identify EcR-dependent genes. The initial effect of EcR RNA interference RNA (RNAi) is significant upregulation of gene expression in late third instar larvae, followed by a switch at puparium formation such that the majority of genes are not properly induced. These data are consistent with genetic studies of usp that define a critical role for this receptor in repressing ecdysone-regulated genes during larval stages, and provide further evidence that one essential function for the EcR-USP heterodimer is to prevent premature maturation through the repression of select 20E target genes during larval stages (Beckstead, 2005).

A total of 4,188 genes change their expression at least 1.5-fold in at least one time point in EcRi animals, suggesting that almost a third of all genes require EcR, either directly or indirectly, for their proper regulation at the onset of metamorphosis. This number is consistent with the 2,268 genes that have been reported to change their expression at pupariation in one of five tissues examined: midgut, salivary gland, wing disc, epidermis, and central nervous system. It is also similar to the 4,042 genes that change their expression at least 1.5-fold at pupariation in temporal arrays. Of these 4,042 genes, 2,680 are affected in EcRi animals, supporting the proposal that EcR plays a major role in coordinating transcriptional responses at the onset of metamorphosis. Not all genes that change their expression at pupariation, however, are dependent on EcR. Several such transcripts were selected for validation by Northern blot hybridization. This is consistent with an earlier microarray study of EcR-regulated genes in the larval midgut. This study found that of 955 genes that change their expression in wild-type midguts at the onset of metamorphosis, 672 genes are affected by an EcR mutation while 283 genes are unaffected, close to the proportion of EcR-independent genes identified by this study. This is also consistent with earlier studies that indicate that other signaling pathways are active at this stage in development. For example, the miR-125 and let-7 microRNAs are dramatically induced at puparium formation, in tight temporal synchrony with the 20E primary-response E74A mRNA, but do so in a manner that is independent of either 20E or EcR. Similarly, α-ecdysone, the immediate upstream precursor of 20E, has critical biological functions, can activate the DHR38 nuclear receptor, and can induce genes in Drosophila third instar larvae that are distinct from those that respond to 20E (RBB, GL and CST). The sesquiterpenoid juvenile hormone can also function with 20E to direct specific transcriptional responses during early metamorphosis. The results of the study described here, however, indicate that most genes that change their expression at the onset of metamorphosis do so in an EcR-dependent fashion, and pave the way for future studies that integrate these responses with those of other signaling pathways (Beckstead, 2005).

To identify 20E-regulated genes, wandering third instar larvae were dissected and their organs cultured in the presence of either no hormone, 20E alone, cycloheximide alone, or 20E plus cycloheximide for 6 hours. RNA extracted from these samples was analyzed on Affymetrix Drosophila Genome Arrays. Comparison of the no hormone and 20E-treated datasets led to the identification of 20E-regulated genes, while comparison of the cycloheximide dataset with data derived from organs treated with 20E and cycloheximide led to the identification of a set of genes referred to as 20E primary-response genes. In comparing these datasets, it is important to note that cycloheximide treatment alone can stabilize pre-existing mRNAs and thus mask their induction by 20E. These transcripts would not be identified by these experiments. In addition, some 20E-inducible genes are expressed at higher levels in the absence of protein synthesis, due to the lack of 20E-induced repressors. The addition of cycloheximide thus provides a means of detecting 20E-regulated transcripts that might otherwise be missed. In this study, 743 20E-regulated genes were identified, with 555 genes responding to 20E alone, 345 genes responding to 20E in the presence of cycloheximide, and 159 genes overlapping between these two datasets (Beckstead, 2005).

Comparison of the 20E-regulated genes to those genes that require EcR for their proper regulation at the onset of metamorphosis led to a final list of 20E-regulated, EcR-dependent genes. Only those genes that are upregulated by 20E in culture and downregulated in at least one of the EcRi time points, or downregulated by 20E in culture and upregulated in at least one of the EcRi time points, were considered for further analysis, leading to the identification of 479 genes. The majority of 20E-final genes that are upregulated by 20E are induced in -4 hour late larvae and/or early prepupae, in apparent response to the late larval 20E pulse, while many genes downregulated by 20E are repressed at these times. The downregulated 20E-final genes that peak in 4 to 6 hour prepupae could be repressed by 20E and thus expressed during this interval of low 20E titer (Beckstead, 2005).

EcR-dependent genes and the 20E-final gene set was compared to data from two microarray studies that examined 20E-regulated biological responses - either EcR-dependent genes expressed in the larval midgut at pupariation, or changes in gene expression that occur during 20E-induced larval salivary gland cell death. As expected, many genes that are normally downregulated in the midgut at pupariation are upregulated in the EcRi gene set (113 genes), and genes that are normally upregulated in the midgut at pupariation are downregulated in the EcRi gene set (120 genes). Similarly, significant overlaps are seen between the 20E-final set and midgut genes that change their expression at pupariation (65 genes upregulated and 10 genes downregulated). Statistically significant overlaps were also observed with genes that change their expression during salivary gland cell death, consistent with a critical role for 20E in directing this response. These correlations validate the datasets and support the conclusion that the results represent 20E responses in multiple tissues at the onset of metamorphosis (Beckstead, 2005).

An examination of these genes reveals several known key mediators of 20E signaling during development. These include three classic ecdysone-inducible puff genes, E74A, E75, and E78 , as well as Kr-h1, which encodes a family of zinc finger proteins required for metamorphosis, the DHR3 nuclear receptor gene, and Cyp18a1 . Expanding this list by including all 20E-regulated genes, results in the identification of the DHR39, DHR78, and FTZ-F1 nuclear receptor genes, as well as the L71 (Eip71E) late genes, IMP-E2, IMP-L3, Fbp-2, Sgs-1, urate oxidase, and numerous genes identified in other studies as changing their expression at the onset of metamorphosis. The identification of well-characterized 20E-regulated genes within these datasets suggests that the other genes in these lists are also likely to function in 20E signaling pathways, and thus provide a foundation to extend an understanding of 20E action in new directions (Beckstead, 2005).

In an effort to identify biological pathways that might respond to 20E at the onset of metamorphosis, EcRi and 20E-final datasets were comapred with published microarray studies of circadian rhythm, starvation, stress, and immunity. No statistically significant overlaps were seen with the circadian rhythm gene sets examined; however, significant overlaps were observed with genes that are expressed during starvation, stress, or an innate immune response. For the starvation response, genes that change their expression upon starvation for 4 hours or starvation in the presence of sugar for 4 hours were examined. 120 genes induced under these conditions that are upregulated in EcRi animals, and 90 genes that are repressed upon starvation and downregulated in EcRi animals. The starvation-regulated genes are part of an EcR-dependent switch that occurs at puparium formation, where many of the induced genes are normally downregulated at puparium formation, and many starvation-repressed genes are upregulated at puparium formation. These genes include eight members of the cytochrome P450 family, three triacylglycerol lipase genes, α-trehalose-phosphate synthase, and a fatty-acid synthase gene that are downregulated at the onset of metamorphosis, while lipid storage droplet-1, pumpless, a UDP-galactose transporter, a lipid transporter, and phosphofructokinase are upregulated at this stage. Similarly, genes that change their expression in response to oxidative or endoplasmic reticulum stress are significantly upregulated in EcRi animals at puparium formation, reflecting their normal coordinate downregulation at puparium formation, and demonstrating that this response is mediated by EcR. Within the 87 genes that overlap between the downregulated stress response genes and the upregulated EcR-dependent genes, 14 of the 17 Jonah genes were identifed that encode a family of coordinately regulated midgut-specific putative proteases. Six genes that encode trypsin family members are also within this gene set, indicating that many peptidase family members are regulated by EcR. Taken together with the data on EcR-regulated starvation genes, these results indicate that EcR plays a central role in controlling metabolic responses at pupariation, directing the change from a feeding growing larva to an immobile non-feeding pupa (Beckstead, 2005).

Genes that change their expression upon microbial infection are also significantly upregulated in EcRi animals at puparium formation, and coordinately downregulated at pupariation. Interestingly, both the Toll ligand-encoding gene dorsal and the key Toll effector gene spätzle were identified as downregulated at the onset of metamorphosis in a EcR-dependent manner, suggesting that central regulators of the Toll-mediated immune response pathway are under EcR control. In addition, well studied immune response genes are downregulated by 20E, including Cecropin C, Attacin A, Drosocin, Drosomycin, and Defensin. These observations indicate that many metabolic and immunity-regulated genes are part of the genetic program directed by 20E at the onset of metamorphosis, and that these genes are normally coordinately downregulated at puparium formation in an EcR-dependent manner (Beckstead, 2005).

All potential transcriptional and translational regulators were selected from the list of most highly induced 20E primary-response genes that are EcR-dependent and not yet implicated in 20E signaling pathways, identifying seven genes: sox box protein 14 (sox14), cabut, CG11275, CG5249, vrille, hairy, and brain tumor (brat). Northern blot hybridization was used to validate the transcriptional responses of these genes to 20E. All seven genes are induced by 20E in larval organ culture, with CG5249 displaying a very low level of expression and hairy showing only a modest approximately twofold induction. Several transcripts are increased upon treatment with cycloheximide alone, consistent with its known role in stabilizing some mRNAs. Addition of 20E and cycloheximide, however, resulted in higher levels of transcript accumulation, similar to the response seen when E74A is used as a control. Their temporal patterns of expression at the onset of metamorphosis also reveal brief bursts of transcription that correlate with the 20E pulses that trigger puparium formation and adult head eversion. These seven genes thus appear to represent a new set of 20E primary-response regulatory genes that could act to transduce the hormonal signal during metamorphosis (Beckstead, 2005).

At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. To elucidate neural substrates underlying the ecdysis sequence, neurons expressing ETH receptors (ETHRs) have been identified in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. These findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles (Kim, 2006).

Analysis of the pupal ecdysis behavioral sequence: In Drosophila, pupal ecdysis is preceded by pupariation, whereby the prepupa contracts its body into a barrel shape to form the puparium composed of the old larval cuticle. The underlying new pupal cuticle then separates from the puparium during pupal ecdysis ~12 hr later. The stereotypic nature of pupal ecdysis and reliable developmental markers make it a favorable model for the behavioral analysis and neural imaging (Kim, 2006).

Pupal ecdysis consists of three centrally patterned behavioral subunits performed sequentially: pre-ecdysis (~10 min), ecdysis (~5 min), and postecdysis (~60-70 min). The behavioral sequence was examined through the semitransparent puparium ('puparium-intact'), but it was found that the puparium obscures and places constraints on some movements. This made it particularly difficult to discriminate differences in abdominal swinging movements during ecdysis and postecdysis. Therefore, a 'puparium-free' preparation was used by surgically removing the puparium immediately after pre-ecdysis onset. The improved visibility and room for movement in this preparation allowed for a more complete analysis of natural and ETH-induced behavior. The following description of the natural pupal ecdysis sequence resulted from comparison of behaviors observed in both puparium-intact and puparium-free prepupae (Kim, 2006).

Pre-ecdysis: About 5 min after in vivo ETH release, preecdysis commences with the abrupt appearance of an air bubble at the posterior end of the prepupa (time zero). Pre-ecdysis involves anteriorly directed rolling contractions along the lateral edges of the abdomen, alternating on the left and right sides of the animal. These contractions move the air bubble anteriorly to separate pupal cuticle from the puparium. This behavior is completed within ~10 min and is followed by ecdysis behavior. Preecdysis behavior is the same in puparium-intact and puparium-free animals (Kim, 2006).

Ecdysis: In higher Diptera including Drosophila, the incipient adult head develops within the prepupal thorax. During pupal ecdysis, head eversion results from lateral swinging movements of the abdomen occurring along with anteriorly directed peristaltic contractions. In puparium-intact preparations, head eversion occurs ~1 min after the onset of ecdysis swinging and is completed within ~5s. After completion of head eversion, ecdysis contractions continue for ~15 min, facilitating expansion of wing pads and legs to their final size. The frequency of ecdysis swinging (~5 swings/min) decreases markedly after head eversion (~2 swings/min). In puparium-free animals, head eversion occurs sooner, and the duration of ecdysis behavior lasts only ~5 min. Later in ecdysis, anteriorly directed swinging contractions are often interrupted by posteriorly directed ones, indicating a transition to postecdysis (Kim, 2006).

Postecdysis: Postecdysis behavior consists of two behavioral subroutines: postecdysis swinging and stretch-compression movements of the abdomen. Postecdysis swinging occurs along with posteriorly directed peristaltic contractions and alternates with longitudinal movements of the abdomen, referred to as 'stretch-compression.' The frequency and intensity of postecdysis contractions wane gradually until they are detected mainly in the anterior part of the abdomen; they cease w100 min after pre-ecdysis onset. Postecdysis behavior concludes with compression of the pupa at the posterior end of the puparium (Kim, 2006).

ETH release coincides with initiation of the ecdysis sequence: To confirm the role of ETH in initiation of the pupal ecdysis sequence, its release from endocrine Inka cells was monitored in vivo by using time-lapse EGFP fluorescence imaging in pharate pupae (prepupae) carrying the chimeric transgene 2eth3-egfp. In this transgenic fly, EGFP is expressed as part of a fusion protein with the ETH propeptide precursor, and loss of EGFP fluorescence indicates ETH release. Because pharate pupae generally are immobile and Inka cells are located immediately below the semitransparent puparium, in situ imaging of Inka cell in intact pharate pupae is feasible. Two to three Inka cells were monitored simultaneously in each experiment (Kim, 2006).

Depletion of ETH-EGFP occurs in about 50% of monitored Inka cells shortly before pre-ecdysis onset (time zero). The time course of secretory activity for each Inka cell was variable. The mean value for the timing of ETH release was 4.5 min prior to pre-ecdysis onset, and the duration of ETH secretion was 4.4. In contrast, 40% of Inka cells showed no sign of secretory activity by pre-ecdysis onset. After initiation of pre-ecdysis contractions, it was usually impossible to continue monitoring loss of EGFP fluorescence as a result of movement artifacts. All Inka cells are depleted of ETH-EGFP by the end of ecdysis sequence (Kim, 2006).

Injection of ETH induces the ecdysis sequence: Because ETH release coincides with onset of pupal preecdysis, it was of interest to determine whether ETH injection would trigger the pupal ecdysis sequence. The Drosophila gene eth encodes a precursor producing one copy each of two peptides, ETH1 and ETH2, which share similar structure and biological activity. In vivo experiments were carried out primarily in puparium-free preparations (Kim, 2006).

Injection of ETH1 alone into pharate pupae (~1-2 hr prior to natural ecdysis) induced within 1-3 min strong pre-ecdysis contractions followed by ecdysis and postecdysis contractions sequentially. ETH-induced pre-ecdysis showed a strong dose dependence, with higher doses inducing shorter pre-ecdysis duration and higher frequency of contractions. Similar but somewhat less pronounced dose-dependent effects were observed during ecdysis behavior, whereas the frequency of postecdysis contractions showed little or no dose dependence during the first 10 min of behavior (Kim, 2006).

Injection of ETH2 was less efficacious for induction of the behavioral sequence compared to ETH1. ETH2 generated prolonged pre-ecdysis behavior lasting to 50 min or more, but no ecdysis behavior. In contrast, injection of the same dose of ETH1 (0.4 pmol) produced the complete behavioral sequence consisting of pre-ecdysis, ecdysis, and postecdysis. Higher doses of ETH2 (20 pmol) generated a behavioral sequence comparable to that induced by 4 pmol ETH1 in terms of pre-ecdysis duration and frequency of contractions. Behaviors after injecting a cocktail of ETH1 and ETH2 (0.4 pmol of each peptide) were also examined. Because the two peptides are processed from the same precursor, it is likely that these peptides are coreleased under natural conditions. The duration of the behavioral sequence induced by injection of the cocktail was similar to the naturally occurring sequence or one induced by 0.4 pmol ETH1 alone. It is estimated that a 0.4 pmol ETH injection into a prepupa w10 hr after puparium formation results in a concentration of ~300 nM in vivo (Kim, 2006).