The Interactive Fly

Zygotically transcribed genes

Genes Regulating Molting

Formation of the adult fly

Tissue-specific gene regulation and Ecdysone regulated gene networks

The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis

A command chemical triggers an innate behavior (pre-ecdysis, ecdysis, and postecdysis) by sequential activation of multiple peptidergic ensembles

Rescheduling behavioral subunits of a fixed action pattern by genetic manipulation of peptidergic signaling

miR-71 and miR-263 jointly regulate target genes Chitin synthase and Chitinase to control locust molting

The splice isoforms of the Drosophila ecdysis triggering hormone receptor have developmentally distinct roles

Genes regulating molting

Patterns of puffing in larval salivary gland polytene chromosome reveal the order of gene activation during the molting process. For information about puffing, see Polytene chromosomes, endoreduplication and puffing.

Tissue-specific gene regulation and Ecdysone regulated gene networks

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).

To determine whether any of the genes in these pathways are expressed as a consequence of ecdysone signaling, the EcR mutant expression data was examined for those genes that were induced during the late third instar ecdysone pulse. The induction of zeste white-3/shaggy, keren/spitz2, kuzbanian, and hedgehog are all dependent on the presence of functional EcR. The induction dynamics of the EGFR ligand gene keren/spitz2, the Notch proteolytic activation factor gene kuzbanian, and the shaggy/zeste white-3 kinase gene are similar to genes that are known direct targets of ecdysone signaling. The induction of hedgehog follows a secondary response pattern, as do genes from the E(spl) complex that are induced in response to Notch activation, although these induction kinetics are also consistent with these genes being partially activated directly by the ecdysone receptor and partially with other factors (i.e., they may be 'early-late' genes). These data show that the regulatory network controlled by ecdysone in midguts includes the activation of known components of the Wnt, EGFR, Hedgehog, and Notch pathways. Notably, ligand production for the EGF, Hedgehog, TGFβ/BMP, and Notch pathways is under control of ecdysone. The specific roles that each of these pathways plays during metamorphosis are currently unknown. These results nonetheless indicate new connections between ecdysone signaling and the activity of several other signaling pathways during the metamorphosis of this organ, either through direct targeting of the ecdysone receptor or through the actions of downstream factors (Li, 2003).

The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis

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).

Roles for brat during metamorphosis were examined because, unlike the other six 20E primary-response genes described above, a brat mutant allele is available (bratk06028) that allows an assessment of its functions during later stages of development. The bratk06028 P-element maps to the fourth exon of the brat gene. Precise excisions of this transposon result in viable, fertile animals, demonstrating that the transposon is responsible for the mutant phenotype. Lethal phase analysis of bratk06028 mutants revealed that 61% of the animals survive to pupariation, with the majority of these animals pupariating 1 to 2 days later than their heterozygous siblings. Of those mutants that pupariated, 11% died as prepupae, 8% died as early pupae, 46% died as pharate adults, and the remainder died within a week of adult eclosion. Phenotypic characterization of bratk06028 mutant prepupae and pupae revealed defects in several ecdysone regulated developmental processes, including defects in anterior spiracle eversion (29%), malformed pupal cases (15%), and incomplete leg and wing elongation (12%). Northern blot hybridization of RNA isolated from staged bratk06028 mutant third instar larvae or prepupae revealed a disruption in the 20E-regulated transcriptional hierarchy. In wild type animals, brat mRNA is induced in late third instar larvae and 10 hour prepupae, similar to the temporal profile determined by microarray analysis, with reduced levels of brat mRNA in bratk06028 mutants, consistent with it being a hypomorphic allele. βFTZ-F1 is unaffected by the brat mutation in mid-prepupae, while E74 mRNA is reduced at 10 hours after pupariation. BR-C, E93, EcR, DHR3, and L71-1 are expressed at higher levels in late third instar larvae and early prepupae, with significant upregulation of BR-C. In addition, the smallest BR-C mRNA, encoding the Z1 isoform, is under-expressed in brat mutant prepupae. It is unlikely that brat exerts direct effects on transcription since it encodes a translational regulator. Nonetheless, these effects on 20E-regulated gene expression are consistent with the late lethality of bratk06028 mutants. In particular, the rbp function provided by the BR-C Z1 isoform is critical for developmental responses to 20E, and overexpression of BR-C isoforms can lead to lethality during metamorphosis. Thus, not only are the brat mutant phenotypes consistent with it playing an essential role during metamorphosis, but it may exert this function through the regulation of key 20E-inducible genes. Efforts are currently underway to address the roles of the remaining six new 20E primary-response regulatory genes in transducing the hormonal signal at the onset of metamorphosis (Beckstead, 2005).

A command chemical triggers an innate behavior (pre-ecdysis, ecdysis, and postecdysis) by sequential activation of multiple peptidergic ensembles

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

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, 2006a).

ETH receptors are expressed in diverse ensembles of peptidergic neurons: ETH acts directly on the CNS to initiate the ecdysis behavioral sequence in moths and flies via unknown signaling pathways within the CNS. A starting point for elucidation of these downstream signaling pathways is identification of primary neuronal targets of ETH. The ETH receptor gene (CG5911), first identified in Drosophila, encodes two G protein-coupled receptors, ETHR-A and ETHR-B, via alternative splicing. In situ hybridization was used for identification of central neurons expressing ETHR-A and ETHR-B by using DNA probes specific for each receptor subtype. ETHR-A and ETHR-B transcripts were located in mutually exclusive populations of neurons distributed throughout the CNS, suggesting that two subtypes of ETH receptors likely mediate different functions. Further analysis revealed that most ETHR-A neurons are peptidergic. Neurons expressing ETHR-B have not been identified thus far (Kim, 2006a).

Multiple ensembles of ETHR-A neurons are classified according to specific neuropeptides they express. Peptides expressed in these ensembles were identified by using GAL4 transgenes under control of neuropeptide promoters to drive UAS-GFP or UAS-GCaMP expression (GAL4::GFP or GAL4::GCaMP). Expression of neuropeptides in these cells was confirmed by combining immunohistochemical staining and in situ hybridization. The first ETHR-A ensemble comprises six pairs of lateral abdominal neurons producing kinin, also known as drosokinin. These cells project axons posteriorly along the lateral edge of the neuropile and then turn anteriorly along the midline of ventral nerve cord, where they arborize and form possible central release sites. Axons of these cells also exit the CNS through nerve roots, suggesting peripheral kinin release. The second ETHR-A ensemble contains three pairs of ventrolateral FMRFamide neurosecretory cells (Tv1-3 or T1-3) in the thoracic neuromeres TN1-3. These cells project axons into the dorsomedial neurohemal organs (NHOs) specialized for peptide release into the hemolymph (Kim, 2006a).

The third class of ETHR-A neurons comprises the eclosion hormone (EH)-producing VM neurons in the brain, which project one axonal branch anteriorly into the neurohemal ring gland and a second posteriorly along the dorsal midline of the entire ventral nerve cord. The fourth ETHR-A ensemble is composed of paired dorsolateral neurons producing CCAP, MIPs, and bursicon in subesophageal, thoracic, and abdominal neuromeres (SN1-3, TN1-3, AN1-7, respectively). These cells are likely homologs of moth neurons 27/704, on the basis of their anatomy, peptide coexpression profile, and functional roles during pupal ecdysis. These neurons are referred to as Drosophila neurons 27/704 and them were subdivided on the basis of peptide coexpression. In AN1-4, CCAP is colocalized with MIPs and the heterodimeric peptide hormone bursicon (composed of burs and pburs subunits). In TN2-3 and AN5- 9, CCAP is colocalized with burs, but pburs is not expressed in these neurons. Finally, CCAP is colocalized with MIPs in large paired neurons of AN8,9, but ETHR-A expression has not been confirmed in these cells. The presence of MIP mRNA in abdominal neurons 27/704 was further confirmed by in situ hybridization (Kim, 2006a).

Ca2+ imaging of primary ETH targets in transgenic flies: Having shown that ETH receptors occur in diverse ensembles of peptidergic neurons, it was asked whether these cells are activated by ETH and whether this activity coincides with specific behavioral steps of the ecdysis sequence. Calcium dynamics in each group of ETHR-A neurons was monitored by driving expression of the GFP-based Ca2+ sensor, GCaMP, in genetically defined sets of neurons with the binary GAL4/UAS system. Ca2+ elevation induces a conformational change of GCaMP, increasing its GFP fluorescence. Using optical imaging of GFP fluorescence, [Ca2+]i dynamics of ETHR neurons were monitored, and these events were associated with each behavioral phase induced by ETH (Kim, 2006a).

An abundance of evidence indicates that the ecdysis behavioral sequence in insects is centrally patterned. In particular, the onset and duration of each behavior in the sequence (pre-ecdysis I, pre-ecdysis II, ecdysis) is the same whether observed in vivo or as fictive behavior recorded from the isolated CNS in vitro. On the basis of this evidence, [Ca2+]i dynamics of ETHR-A neuron ensembles of the isolated CNS were associated with behaviors observed in puparium-free preparations (Kim, 2006a).

FMRFamide neurons and their neurohemal endings become active early in pre-ecdysis: [Ca2+]i levels were monitored in ETHR-A/FMRFamide Tv neurons by preparing transgenic flies doubly homozygous for FMRFa-GAL4 and UAS-GCaMP. Prior to ETH1 exposure (4-6 hr prior to ecdysis), Tv cell bodies and neurohemal endings in the dorsomedial NHO exhibit low levels of basal GCaMP fluorescence (Kim, 2006a).

Exposure of the CNS to ETH1 (600 nM) elicits robust increases in calcium-associated fluorescence in cell bodies and axon terminals of all Tv neurons. At this concentration of ETH1, calcium dynamics typically are characterized by transient, spike-shaped fluctuations superimposed upon a slow upward shift of the baseline, beginning ~8 min after exposure to the peptide. This response lasts ~10-15 min, after which weaker spike-like fluctuations continue without baseline changes until the end of recordings (~40 min). It is estimated that a concentration of 600 nM ETH1 results from a dose of w0.4 pmol of the peptide in vivo. Thus the major calcium response of Tv neurons coincides with the early phase of pre-ecdysis, and weaker activity persists through ecdysis and postecdysis. In contrast, ETH2 alone (600 nM) generates Ca2+ responses after a longer delay comparable to one following exposure to 60 nM ETH1. The longer delay of Ca2+ responses after ETH2 fits with the observations of in vivo behavior, where ETH2 is a less potent agonist than ETH1. The cocktail of ETH1 and ETH2 (600 nM each) evokes Ca2+ dynamics after a delay similar to that induced by ETH1 alone. Overall, [Ca2+]i dynamics observed in Tv neurons are synchronized. In many preparations, Tv neurons from the same neuromere appear to be strongly coupled, given that they produce precisely synchronized Ca2+ dynamics. Transient Ca2+ signals are obvious in the terminal processes of Tv neurons in NHO, the release sites of FMRFamides (Kim, 2006a).

Lower concentrations of ETH1 elicit calcium dynamics after a somewhat longer delay. Interestingly, calcium dynamics are obvious first in neurohemal endings of the NHO, followed by fluctuations in cell bodies. This was particularly evident at 60 nM ETH1, where a robust calcium response in the NHO was accompanied by only a weak response in the Tv2 cell body. No calcium responses are observed in Tvs exposed to 6 nM ETH1 (Kim, 2006a).

EH neurons reach peak activity at ecdysis: VM neurons producing eclosion hormone (EH) have been implicated as primary ETH targets during fly and moth ecdysis. Expression of ETHR-A was demonstrated in VM neurons, confirming that they are primary ETH targets. To determine whether ETH elicits activity in EH neurons, transgenic flies were prepared doubly homozygous for EHup-GAL4 and UASGCaMP, that show GCaMP fluorescence only in these cells (Kim, 2006a).

EH neurons are highly sensitive to ETH1, exhibiting robust [Ca2+]i dynamics upon exposure to concentrations as low as 6 nM. No detectable fluorescence responses are observed after exposure to 0.6 nM ETH1 over a period of 50-60 min. The latency to Ca2+ responses is inversely proportional to the concentration of ETH1; higher ETH1 concentrations evoke faster responses. The cocktail of ETH1 and ETH2 (600 nM each) elicited Ca2+ responses after a ~10-15 min delay (Kim, 2006a).

Close examination of these ETH-evoked fluorescence responses reveals two components distinguished by slow and fast kinetics. The slow component is characterized by a gradual increase in baseline levels of Ca2+ followed by a decrease over 20-30 min, whereas the fast component is composed of transient, spike-like activity. Fast components have durations ranging from 5-20 s. Peak DF/F responses are quite variable, even among a group of neurons exposed to the same ETH1 concentration. No significant concentration dependence could be detected in peak response (Kim, 2006a).

Distinct subsets of neurons 27/704 are active during different phases of the ecdysis sequence: ETH-evoked Ca2+ signals of neurons 27/704 were examined in transgenic flies carrying CCAP-GAL4 and UAS-GCaMP. Use of the CCAP promoter to drive GCaMP expression resulted in a reporter pattern identical to that described previously. Upon exposure to 600 nM ETH1, distinct subsets of neurons 27/704 exhibited reproducible, stereotypic Ca2+ responses in terms of peak intensity, latency, and termination of Ca2+ dynamics. According to the magnitude of peak fluorescence intensity (peak DF/F), neurons 27/704 fall into three major groups: strong responders, weak responders, and nonresponders. The strong-responder group includes neurons 27/704 in AN1-4 (CCAP/MIPs/bursicon), AN8,9 (CCAP/ MIPs), and TN3 (CCAP). Weak responders are neurons 27/704 in SN2-3, TN1-2, and AN7 producing CCAP only. Neurons in the brain, SN1, and AN5,6 showed no reproducible Ca2+ dynamics in response to 600 nM ETH1 (Kim, 2006a).

In response to ETH1, neurons 27/704 in TN3 and AN8,9 become active within 10-15 min, whereas neurons 27/704 in AN1-4 are activated after a 15-25 min delay. Neurons in TN3 and AN8,9 are therefore activated just prior to ecdysis onset, indicating their possible roles in initiation and maintenance of ecdysis behavior. In addition, Ca2+ dynamics observed in AN8,9 neurons terminated early in postecdysis, supporting this interpretation. In contrast, Ca2+ dynamics of neurons in AN1-4 begin during ecdysis and increase in intensity during the entire postecdysis period, suggesting their roles in these events. The cocktail of ETH1 and ETH2 (600 nM each) evoked Ca2+ dynamics similar to those induced by ETH1 alone. Two groups of neurons 27/704 in abdominal neuromeres (AN1-4 versus AN8,9) exhibit differences in sensitivity to ETH and in their patterns of [Ca2+]i dynamics. It was found that 6-60 nM ETH1 activates neurons in AN1-4 (n = 4), whereas higher concentrations of ETH1 (R600 nM) are required to activate neurons in AN8,9. In addition, neurons in AN8,9 generate transient (1-2 min) Ca2+ spikes over a 15-20 min period after ETH1 activation, whereas neurons in AN1-4 generally produce slower, more persistent Ca2+ dynamics. These differences among subgroups of neurons 27/704 suggest their different functional roles during the ecdysis sequence (Kim, 2006a).

Targeted ablations of apecific ETHR neurons have behavioral consequences: To evaluate behavioral roles of specific ETHR neurons, phenotypes of the pupal ecdysis sequence were investigated in transgenic flies bearing targeted ablations of ETHR neurons, including Tv FMRFamide neurons, EH neurons, and CCAP neurons (27/704 homologs). In control flies carrying UAS-reaper and UAS-GCaMP, but lacking the GAL4 driver, pupal ecdysis was executed as in wild-type flies: pre-ecdysis (0-10 min), ecdysis (10-23 min), and postecdysis (23-100 min) (Figure 7). Given that puparium-intact animals were used, the duration of ecdysis behavior may have been overestimated. Transgenic flies bearing targeted ablations of Tv FMRFamide neurons (FMRF-KO) were generated by crossing females doubly homozygous for FMRFa-GAL4, UAS-GCaMP with homozygous UAS-reaper males. Pupal ecdysis of FMRFa-KO flies is very similar to that of control flies. Because FMRFa-GAL4 drives expression of GAL4 only in three pairs of thoracic Tv neurons and one pair of unidentified neurons in SN, FMRFa-KO flies lost only Tv neurons and not other FMRFamide neurons in the CNS. FMRFa-KO flies complete pupal ecdysis without any detectable abnormality, except that pre-ecdysis contractions appear weaker than in control flies. Pupal ecdysis of VM neuron knockout flies (EH-KO) was then examined. Behavioral analysis showed that, although they complete pupal ecdysis without any severe defects or lethality, ecdysis onset is delayed w4 min. As a result of this delay, EH-KO flies show longer pre-ecdysis than control flies. Additional parameters governing pre-ecdysis, ecdysis, and postecdysis are indistinguishable between EH-KO and control flies (Kim, 2006a).

Finally, CCAP-KO flies were generated in order to examine the functional roles of neurons 27/704 (CCAP neurons) in pupal ecdysis. As expected, CCAP-KO flies failed to initiate ecdysis contractions and could not complete head eversion. Instead, they show prolonged pre-ecdysis contractions for ~25 min, followed by weak random contractions of the abdomen (different from ecdysis and postecdysis contractions of control flies) for the next 50 min (Kim, 2006a).

Conclusions: This study has described orchestration of an innate behavior, the Drosophila pupal ecdysis sequence, by the endocrine peptide ETH. ETH release coincides with onset of behavior, and injection of ETH triggers the complete behavioral sequence, consistent with its role in ecdysis activation previously established in the moths Manduca sexta and Bombyx mori and in Drosophila larvae and adults. Absence of ETH causes lethal ecdysis deficiency, a phenotype that is rescued by ETH injection. ETH therefore functions as a 'command chemical' to orchestrate an innate behavior. Primary CNS targets of ETH was identified by using ETHR-specific in situ hybridization. ETHR-A occurs in multiple classes of peptidergic neurons producing EH, CCAP/MIPs/bursicon, FMRFamides, or kinin (Kim, 2006a).

Expression of ETHR-A was shown in VM neurons, which release EH. In response to ETH, VM neurons become active prior to ecdysis behavior and reach peak levels of activity during ecdysis. These results provide further support for a previously described positive-feedback signaling pathway between VM neurons and Inka cells. This feedback is thought to ensure depletion of ETH from Inka cells. These findings are striking because independent evidence indicates that homologous VM neurons in the moth Manduca are direct targets of ETH and that their secretory products regulate ecdysis behaviors downstream of ETH. For example, isolated EH neurons of Manduca respond to direct action of ETH with increased excitability and spike broadening. In response to ETH action, these neurons release EH, causing cGMP elevation and increased excitability in CCAP-containing neurons 27/704 of the thoracic and abdominal ganglia. CCAP and MIPs, cotransmitters produced by neurons 704, are implicated in eliciting ecdysis behavior (Kim, 2006a).

A homologous role for EH in activation of Drosophila 27/704 neurons has not been clearly demonstrated. For example, no cGMP elevation is observed in these neurons during the natural ecdysis sequence. This lack of cGMP elevation suggests that CCAP neurons are not directly targeted by EH in Drosophila. Nevertheless, EH-knockout flies exhibit a delay in ecdysis initiation, suggesting that EH may modulate excitability in 27/704 cells indirectly through release of additional factors within the CNS. It is therefore proposed that activation of EH neurons by ETH serves two purposes: (1) release of EH into the hemocoel functions as part of a positive-feedback pathway to ensure ETH depletion from Inka cells; (2) release of EH within the CNS synergizes direct ETH actions on different subsets of neurons 27/704 producing CCAP, MIPs, and bursicon, perhaps indirectly through release of downstream signals within the CNS (Kim, 2006a).

Neurons 27/704 expressing ETHR-A respond to ETH with unique patterns of Ca2+ dynamics. These neurons are subdivided by pattern of transmitter expression: CCAP/MIPs/bursicon in AN1-4; CCAP/MIPs in AN8,9; and CCAP in SN1-3, TN1-3, and AN5-7. Temporal patterns of Ca2+ dynamics were determined in each neuronal subset relevant to the behaviors observed. On the basis of these temporal patterns, it is proposed that direct action of ETH on neurons 27/704 in TN3 and in AN8,9 induces initiation and execution of ecdysis contractions and head eversion. In support of this, it is shown that ablation of CCAP neurons abolishes ecdysis contractions and head eversion. Parallel study in Manduca showed that neurons 704 expressing ETHR-A and their peptide cotransmitters, CCAP and MIPs, are implicated in control of the ecdysis motor pattern, supporting the homologous function of 27/704 neurons in Drosophila. Neurons 27/704 in AN1-4 produce CCAP, MIPs, and bursicon, and therefore a cocktail of these peptides is likely released within the CNS and into the hemolymph during postecdysis. It is suggested that centrally released peptides control postecdysis movements, whereas blood-borne CCAP/MIPs regulate heart beat and blood pressure for cuticle expansion and bursicon controls sclerotization of expanded new cuticle. Bursicon was recently identified as a heterodimeric peptide hormone regulating cuticle plasticization, sclerotization, and melanization (Kim, 2006a).

The Drosophila FMRFamide gene (FMRFa) encodes multiple FMRFamide-related neuropeptides, which are expressed in many different cell types, including neuroendocrine cells, interneurons, and perhaps motoneurons. Among these diverse FMRFamide-producing neurons, ETHR-A expression is confined to three pairs of thoracic neurosecretory neurons, Tv1-3. Results of the present study show that the Tv neurons are activated early in pre-ecdysis and that they remain active during ecdysis and postecdysis. However, FMRFa-KO flies show no differences in timing of the ecdysis behavioral sequence. Because FMRFamides enhance twitch tension of larval body-wall muscles through synaptic modulation at the neuromuscular junction, blood-borne FMRFamides released from Tv neurons likely facilitates pre-ecdysis, ecdysis, and postecdysis contractions. Thus the role of Tv neurons as primary ETH targets may be enhancement of muscle contraction during the behaviors. Further work to substantiate this is in progress (Kim, 2006a).

Expression of ETHR-A occurs in in kinin neurons of abdominal neuromeres of Drosophila. Drosophila kinin is known to be involved in water balance, but its central functions have not been described or considered. Expression of ETHR-A in kinin neurons appears to be a conserved mechanism in fly and moth; the Manduca ETHR-A is expressed in abdominal neurosecretory cells (L3,4), which produce kinins and diuretic hormones (DHs). It was further found that the isolated Manduca CNS generates the fictive pre-ecdysis motor pattern upon exposure to a cocktail of kinin and DHs. These findings suggest that ETH activates L3,4 neurons in Manduca to release kinins and DHs centrally, which initiate and execute pre-ecdysis. On the basis of the conservation between Drosophila and Manduca in spatial expression pattern of ETHR, it is proposed that ETH initiates pre-ecdysis behavior indirectly via central release of kinin in Drosophila (Kim, 2006a).

In Drosophila, pupal ecdysis is accomplished by sequential recruitment of three major behavioral units: pre-ecdysis (0-10 min), ecdysis (10-15 min), and postecdysis (15-100 min). Each behavioral unit is programmed in the CNS and sequentially activated by direct actions of ETH, which is synthesized and released from peripheral endocrine Inka cells. Around 4-5 min before pre-ecdysis onset, a sizeable portion (~50%) of Inka cells initiates secretion of ETH into the hemolymph, whereas the remaining portion completes secretion after onset of pre-ecdysis. Appearance of ETH in the hemolymph activates ETHR-A in neurons expressing neuropeptides including kinin, FMRFamides (Tv1-3), EH, or CCAP, MIPs, and bursicon, but they are not released until descending inhibition is removed at key times during the ecdysis sequence. Upon activation of ETHR, the central release of kinin initiates pre-ecdysis contractions, whereas Tv neurons secrete FMRFamides to enhance neuromuscular transmission. ETH activates neurons producing EH, CCAP, CCAP/MIPs, and CCAP/MIPs/bursicon at different times. EH cells in the brain and neurons producing CCAP in TN3 and CCAP/MIPs in AN8,9 become active ~10-13 min after pre-ecdysis initiation. EH participates in timing the activation of ecdysis neurons, whereas CCAP and MIPs from TN3 and AN8,9 control initiation and execution of the ecdysis motor program. At the end of ecdysis (25 min after pre-ecdysis onset), neurons in AN1-4 secrete a cocktail of CCAP, MIPs, and bursicon, which likely regulate postecdysis contractions and processes associated with cuticle expansion, hardening, and tanning (Kim, 2006a).

This study has mapped central ETH receptor neurons, and discovered that they comprise multiple peptidergic ensembles, which are recruited sequentially to generate each phase of the ecdysis sequence. Ensemble-specific knockout analysis supports this interpretation. Each step of the ecdysis sequence (pre-ecdysis, ecdysis, postecdysis) is driven by a central pattern generator (CPG) within the CNS in the absence of sensory input. It is known that amines and peptides can modulate and reconfigure neuronal circuits comprising CPGs so as to elicit a variety of motor patterns. It seems likely that the multiple peptidergic ensembles described in this study as targets for ETH may be involved in configuring and activating CPGs underlying each step of the ecdysis sequence (Kim, 2006a).

Processes in the brain that govern behaviors over longer time frames such as sleep, mood, sexual activities, and even learning and memory could be associated with coordinated release of neuromodulators such as peptides. Further work on activation of central peptidergic ensembles in the CNS may shed light on mechanisms underlying release of a variety of behaviors (Kim, 2006a).

Rescheduling behavioral subunits of a fixed action pattern by genetic manipulation of peptidergic signaling

The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. This study presents evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. These findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP. Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons (Kim, 2015).

Innate behaviors are stereotypic patterns of movement inherited from birth that require no prior experience for proper execution. Among such behaviors are fixed action patterns that, once initiated, run to completion independent of sensory inputs. Examples include courtship rituals, aggression displays, and ecdysis. Ecdysis represents a 'chemically-coded' behavioral sequence triggered by peptidergic ecdysis triggering hormones (ETH), which orchestrate a downstream peptidergic cascade leading to sequential activation of central pattern generators underlying patterned motor activity. The term Fixed Action Pattern (FAP) has fallen into disuse, since innate behaviors generally exhibit considerable plasticity. However the invariant nature of the ecdysis behavioral sequence makes it a clear example a classic FAP. In depth analysis of ecdysis behavior may provide a more thorough understanding of how hormones assemble and regulate behavioral circuitry in the brain, in particular circuits that operate sequentially (Kim, 2015).

ETHs are released by peripheral peptidergic Inka cells in response to declining levels of the steroid hormone 20-hydroxyecdysone. Presence of Inka cells in more than 40 species of arthropods, along with the sequence similarity of ETH peptides in diverse insect groups, suggests that ETH signaling is highly conserved in insects. Identification of the Ecdysis Triggering Hormone receptor (ETHR) gene in Drosophila melanogaster (Park, 2002) enabled elucidation of a complex downstream signaling cascade triggered by ETH. The ETHR gene encodes two functionally distinct subtypes of G protein coupled receptors (ETHR-A and -B) through alternative splicing. The presence of two ETH receptor subtypes has been observed in all insect species thus far examined (Roller, 2010). The two receptor subtypes show differences in ligand sensitivity and specificity and are expressed in separate populations of central neurons, suggesting that they have distinctive roles in ETH signaling (Kim, 2015).

A diversity of ETHR neurons in the moth Manduca sexta and fruitfly Drosophila melanogaster has been identified (Kim, 2006a; Kim, 2006b). One of the most striking properties of ETHR-A neurons is that they are virtually all peptidergic and conserved across insect orders. Groups of ETHR-A 'peptidergic ensembles' express a range of different neuropeptides, including kinins, diuretic hormone (DH), eclosion hormone (EH), FMRFamide, crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIPs), bursicon (burs and pburs), neuropeptide F (NPF), and short neuropeptide F (sNPF). It is hypothesized that released ETH acts directly on the CNS to activate these peptidergic ensembles for control of specific central pattern generator circuits that elicit stereotyped ecdysis behaviors. However evidence for direct actions of ETH on these target ensembles is yet to be reported (Kim, 2015).

Likely functions of certain ETHR-A peptidergic ensembles in Manduca have been inferred from pharmacological manipulations (Kim, 2006b). For example, serially homologous L3/4 neurons of abdominal ganglia in Manduca express a cocktail of kinins and diuretic hormones; exposure of the isolated CNS to these peptides elicits a fictive pre-ecdysis I-like motor pattern. Similarly, the IN704 peptidergic ensemble that co-expresses CCAP and MIPs is implicated in initiation of ecdysis behavior, since co-application of these two peptides elicits fictive ecdysis behavior. Homologous peptidergic ensembles in Drosophila exhibit characteristic patterns and time courses of calcium mobilization indicative of electrical activity coincident with successive steps in the ecdysis FAP (Kim, 2006a). Of particular interest are observations that bursicon, a hormone co-expressed in a subset of CCAP neurons, is required for ecdysis behavior (Kim, 2015).

This study tested hypotheses that two central peptidergic ensembles—kinin neurons and a subset of CCAP neurons (CAMB) that co-express CCAP, Allatostatin CC, Myoinhibitory peptide, and Bursicon—are direct targets of ETH and schedule pre-ecdysis and ecdysis behavior components of the ecdysis FAP, respectively in the fruit fly Drosophila. It was shown further that manipulation of ETHR expression levels and signal transduction specifically in these ensembles influences scheduling of the FAP. Finally, possible mechanisms are described underlying timing of the switch from pre-ecdysis to ecdysis behavior and a model is proposed to explain mechanistically how these behaviors are sequentially activated (Kim, 2015).

The aims of this study were to implicate identified ETHR neuronal ensembles with specific steps in the ecdysis FAP. Through genetic manipulation of all known ETHR-A central neuron ensembles and subsets thereof, kinin and CAMB neurons were implicated in scheduling of pre-ecdysis and ecdysis, respectively. The current findings confirm that these ensembles are targeted directly by ETH. A key observation is timing of calcium mobilization in kinin and CAMB ensembles following ETH application: kinin neurons mobilize calcium within minutes, while activity in CAMB neurons is delayed. CAMB neurons mobilize calcium only after kinin neuron activity ceases, some 10 minutes later. These temporal patterns of cellular activity correspond well with those of pre-ecdysis and ecdysis behaviors observed in vivo (Kim, 2015).

The findings demonstrate that the kinin peptide ensemble is necessary for proper scheduling of pre-ecdysis behavior, if not itself sufficient to elicit it. Kinin cell ablation abolishes pre-ecdysis behavior in a significant percentage (25%) of animals. The remaining 75% of individuals showed highly variable pre-ecdysis duration, ranging from 3-22 minutes, whereas duration of this behavior in control animals is tightly regulated at 9.1 ± 0.9 min. Furthermore, a 30% reduction in Leukokinin receptor expression, caused by a piggyBac-element insertion into the promoter region of the gene, also disrupts fidelity of pre-ecdysis regulation; this phenotype is rescued by precise excision of the piggyBac insertion. Finally, RNA silencing of the kinin receptor in peripheral neurons using the Peb-Gal4 driver leads to reduced intensity of the behavior and greatly increases variability of pre-ecdysis duration. This is the first report demonstrating that kinin signaling affects scheduling of the ecdysis behavioral sequence via actions on peripheral neurons. Pan-neuronal silencing of kinin receptors also disrupted pre-ecdysis scheduling, but to a lesser extent (Kim, 2015).

Manipulation of ETHR expression levels in kinin neurons also alters scheduling of pre-ecdysis behavior significantly, confirming that these neurons are targeted directly by ETH and that they play an important role in pre-ecdysis regulation. It was reasoned that knockdown of receptor expression in these neurons would lead to a lower density of ETHR in the plasma membrane, thereby reducing sensitivity to ETH and delaying onset of pre-ecdysis. These experimental results demonstrated a reduction of pre-ecdysis duration. This reduction is attributed to a delay in pre-ecdysis onset, brought about by the need for higher ETH levels for neuronal activation. Since timing of the switch to ecdysis (controlled by CAMB neurons; see below) is unaffected, pre-ecdysis duration is shortened. On the other hand, overexpression of ETHR in kinin neurons led to prolongation of pre-ecdysis duration. Following the same reasoning, this would result from premature kinin neuron activation attributable to higher sensitivity to rising ETH levels and overall longer pre-ecdysis (Kim, 2015).

Kinins were identified originally using bioassays for myotropic and diuretic functions and they have well-known actions on muscle and transport activity in epithelia. More recent studies demonstrated diverse functional roles for kinin signaling, including feeding, olfaction, and locomotory behavior. Previous works demonstrated ETHR-A expression in kinin neurons of both Drosophila and Manduca, implicating them as direct targets of ETH. Imaging studies have shown that abdominal kinin neurons in fly larvae exhibit periodic calcium oscillations under normal conditions and are involved in turning behavior. These kinin neurons project to a terminal plexus in close association to kinin receptors, suggesting it functions as a site of peptide release. Interestingly, this same ensemble of kinin neurons in the pre-pupal preparation used here showed no such periodic activity, but instead exhibited synchronized calcium oscillations activity following exposure to ETH. This difference could be unique to the pharate stage (i.e., preceding ecdysis) animal, during which insects generally are unresponsive to external stimuli. In imaging studies, ETH-induced calcium dynamics were observed to be initiated from the terminal plexus region and subsequently move anteriorly to the cell bodies. These observations suggest that this plexus serves critical functions in both sending and receiving signals. Evidence presented in this study for regulation of pre-ecdysis behavior by kinin neurons demonstrates a new function for this peptide in Drosophila, which is reinforced by previous observations in Manduca that application of kinin causes a fictive pre-ecdysis motor pattern in the isolated CNS (Kim, 2015).

How do kinin neurons function in the promotion of pre-ecdysis behavior? While manipulation of kinin signaling clearly affects behavioral intensity and duration, this study was unsuccessful in initiating pre-ecdysis through temperature-dependent activation of kinin neurons expressing either TRPM8 and TRPA1. It is concluded that, while kinin functions as a modulatory influence necessary for proper scheduling of pre-ecdysis behavior, other as yet unidentified signals are necessary for behavioral initiation (Kim, 2015).

This study has demonstrated that CAMB neurons are both necessary and sufficient for the switch from pre-ecdysis to ecdysis behavior. This conclusion rests on results from a combination of experiments. First, CAMB cell ablation abolishes the switch to ecdysis, suggesting these neurons are necessary for the switch. Failure of ecdysis initiation is attributable to bursicon deficiency, since a previous report showed clearly that expression of the bursicon gene is required for initiation of pupal ecdysis. Calcium mobilization in CAMB neurons is delayed for ~10 min after onset of activity in kinin neurons, which fits well with the ~10 min delay before appearance of ecdysis behavior following onset of pre-ecdysis behavior observed in vivo. Altered levels of ETHR expression in CAMB neurons clearly affects timing of the switch to ecdysis behavior: receptor knockdown delays the switch, whereas overexpression accelerates it. In vitro experiments confirm that altered ETHR expression levels affect timing of calcium mobilization in CAMB neurons in register with changes in behavioral timing (Kim, 2015).

Finally, this study showed that activation of CAMB neurons through temperature-sensitive TRPM8 expression initiates ecdysis behavior in vivo. Thus, CAMB neurons are both necessary and sufficient for the switch to ecdysis behavior. However, activity in CAMB neurons alone does not result in robust ecdysis behavior. Expression of ecdysis behavior with parameters corresponding to that observed in wild-type flies requires activation of the entire CCAP ensemble (Kim, 2015).

It is interesting that, in all TRPM8 activation experiments, removal of the temperature stimulus led to re-capitulation of the entire ecdysis FAP. This might be explained by positive feedback influences on the Inka cell to release ETH, possibly via EH neurons. Alternatively, the ecdysis motor circuit may exert negative feedback on the pre-ecdysis circuit, which when removed, causes a post-inhibitory rebound leading to activation of the pre-ecdysis circuit and the entire FAP. Attempts to demonstrate such negative feedback were inconclusive in this study (Kim, 2015).

CAMB neurons express a combination of CCAP, Ast-CC, MIP, and bursicon. In Manduca, application of a CCAP/MIP cocktail is sufficient to elicit fictive ecdysis behavior. It would be parsimonious to extrapolate this result to Drosophila, since both of these peptides are found in CAMB neurons. Nevertheless, in Drosophila it is clear that bursicon is a key signaling molecule necessary for ecdysis initiation. It remains to be demonstrated precisely how absence of the bursicon gene blocks the switch to ecdysis. It will be interesting to elucidate possible functional roles of co-expressed peptides in CAMB neurons CCAP, Ast-CC, MIP in activation of the motor circuitry encoding the ecdysis motor pattern (Kim, 2015).

How is timing of the switch to ecdysis determined? Since both kinin and CAMB ensembles express ETHR, one would expect ETH to activate both ensembles simultaneously. Several previous studies provide evidence for the role of descending inhibition from cephalic and thoracic ganglia in setting the delay in the switch to ecdysis behavior. This study shows that expression of pertussis toxin in CAMB neurons accelerates the switch to ecdysis, consistent with disinhibition of Gαi/o input(s). It is hypothesized that a balance of excitatory and inhibitory inputs to the CAMB neurons contributes to the delay in their activity, excitatory input coming from ETH via Gαq signaling and Gαi/o from an as yet unidentified transmitter descending from cephalic and/or thoracic ganglia. The finding that RNAi-knockdown of MIP neurons lying outside the CCAP ensemble accelerates the switch to ecdysis behavior suggests one such possible inhibitory input (Kim, 2015).

It is possible, if not likely that ETH drives both inhibitory and excitatory inputs to CAMB neurons, with ETHR-B-expressing inhibitory inputs preceding excitatory input. Such a scenario follows from the fact that sensitivity of Drosophila ETHR-B to ETH was shown to be ~450-fold higher than that of ETHR-A. Therefore, as ETH levels rise in the hemolymph, ETHR-B-expressing inhibitory neurons would be activated well before ETHR-A neurons. ETH would effectively inhibit CAMB neurons indirectly prior to direct excitation via ETHR-A activation (Kim, 2015).

Such a scenario pre-supposes that the EC50 values governing activation of ETH receptors determined previously from heterologous expression in mammalian CHO cells are valid in Drosophila neurons. Data presented in this study suggests this is so. The EC50 value for ETH1 against ETHR-A was found to be ~414 nM, while the EC50 for ETH2 was determined to be ~4.3 μM. This study applied a combination of ETH1 and ETH2, each at a concentration of 300 nM, to the isolated CNS and obtained a pattern of calcium dynamics in kinin neurons lasting for ~10 min, which matches the duration of pre-ecdysis behavior under natural conditions. Furthermore, the switch to ecdysis behavior occurs ~10 min after initiation of calcium mobilization in kinin neurons, which corresponds to timing of the switch to ecdysis behavior in vivo. Doubling concentrations of the ETH1/ETH2 cocktail reduced the duration of calcium dynamics in kinin neurons to 5.5 min and accelerated the switch to ecdysis behavior. These results make it likely that the relative sensitivities of ETHR-B and ETHR-A are as previously established and consequently activity in ETHR-B neurons would precede that of ETHR-A neurons (Kim, 2015).

This study has shown that altered levels of ETHR expression have significant consequences for timing of pre-ecdysis duration and timing of the ecdysis switch. These findings raise the possibility that scheduling of sequential steps in the ecdysis FAP may be a consequence of different sensitivities to the peptide ligand. In other words, delay in the switch to ecdysis could result from a lower density of ETHR in CAMB neurons, making them less sensitive to ETH. Possible differential sensitivity to ETH could be tested in variety of way, including assessing timing of responses to the ligand by acutely dissociated neurons and/or single cell PCR (Kim, 2015).

A mechanistic model is proposed to explain neural mechanisms underlying the Drosophila pupal ecdysis FAP (see A model depicting functional roles of kinin and CAMB neurons in scheduling of the ecdysis FAP). Principle players in orchestration of pre-ecdysis and ecdysis behaviors are the kinin and CAMB ETHR ensembles, respectively. As ETH levels rise in the hemolymph, ETHR-B neurons are activated due to their high sensitivity (EC50 ~ 1 nM). These neurons release inhibitory signals acting through Gαi/o to inhibit CAMB neurons. As ETH levels rise further, kinin neurons receive direct excitatory input from ETH signaling via ETHR-A and Gαq to mobilize calcium from intracellular stores, leading to electrical activity in these neurons. ETH acts simultaneously on CAMB neurons, but inhibition from ETHR-B neurons descending from anterior ganglia prevents them from becoming active. As inhibition wanes, CAMB neurons become active, initiating the switch to ecdysis behavior (Kim, 2015).

miR-71 and miR-263 jointly regulate target genes Chitin synthase and Chitinase to control locust molting

Chitin synthase (see Drosophila Chitin synthase)  and chitinase (see Drosophila chitinase) play crucial roles in chitin biosynthesis and degradation during insect molting. Silencing of Dicer-1 (see Drosophila Dicer-1) results in reduced levels of mature miRNAs and severely blocks molting in the migratory locust. However, the regulatory mechanism of miRNAs in the molting process of locusts has remained elusive. This study found that in chitin metabolism, two crucial enzymes, chitin synthase (CHS) and chitinase (CHT) are regulated by miR-71 and miR-263 (see Drosophila bereft) during nymph molting. The coding sequence of CHS1 and the 3'-untranslated region of CHT10 contain functional binding sites for miR-71 and miR-263, respectively. miR-71/miR-263 display cellular co-localization with their target genes in epidermal cells and directly interact with CHS1 and CHT10 in the locust integument, respectively. Injections of miR-71 and miR-263 agomirs suppresses the expression of CHS1 and CHT10, which consequently alters chitin production of new and old cuticles and results in a molting-defective phenotype in locusts. Unexpectedly, reduced expression of miR-71 and miR-263 increases CHS1 and CHT10 mRNA expression and leads to molting defects similar to those induced by miRNA delivery. This study reveals a novel function and balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation, and it provides insight into the underlying molecular mechanisms of the molting process in locusts (Yang, 2016). 

The splice isoforms of the Drosophila ecdysis triggering hormone receptor have developmentally distinct roles

In order to grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Triggering Hormone (ETH) and has been extensively studied as a model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone's neuronal targets. An alternatively spliced gene encoding a G-protein coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. This study used the recently introduced 'Trojan exon' technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. ETHRA and ETHRB were shown to be expressed in largely distinct subsets of neurons, and ETHRA- , but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. Several functionally important subsets of ETHR-expressing neurons were found including one that co-expresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks (Diao, 2016).

To characterize the neural circuit that governs ecdysis in Drosophila, this study exploited the Trojan exon technique to map and manipulate the ETH signaling pathway, and the effects of genetic and neuronal loss-of-function at were analysed at the level of the ETHR gene, its splice variants, and the cells that express them. Genetic disruption of ETHR expression phenocopies ETH loss-of-function, indicating that the ETHR gene encodes the sole receptor for ETH peptides in mediating ecdysis. Consistent with this, it was found that suppression of ETHR-expressing neurons blocks ecdysis at all developmental stages. Both the genetic and neuronal loss-of-function data further reveal distinct developmental requirements for the two ETHR isoforms and the neurons that express them, with ETHRB-, but not ETHRA-expressing neurons dispensable for larval ecdysis. Finally, it was demonstrated that ETHR-expressing neurons regulate both behavioral and physiological processes at pupal ecdysis (Diao, 2016).

Previous observations have shown that ETH initiates ecdysis at all stages in Drosophila development, but apart from work done on a small subset of neurons known to express ETHRA, the broader ecdysis circuit targeted by ETH and the functional roles of its receptors have remained largely uncharacterized. The data presented in this study confirm the importance of ETHRA in ecdysis, as previously demonstrated in Tribolium using RNAi knockdown. In addition, the Arakane study demonstrate, for the first time in any insect species, an essential function for ETHRB in that selective knockdown of ETHRB expression substantially blocks pupal ecdysis. The fact that larval ecdysis is largely unimpaired by this manipulation (together with the observation that restoration of ETHRA, but not ETHRB, expression compensates for loss of ETHR function at larval ecdysis) strongly argues that ETHRA and ETHRB have distinct functional roles and that their contribution to ecdysis is differentially dependent on developmental stage. These conclusions are consistent with the results of neuronal suppression using ETHRA- and ETHRB-specific Gal4 drivers: ETHRA-expressing neurons are required for ecdysis throughout development, whereas ETHRB-expressing neurons are required only after the larval stage. In addition, the differing phenotypes of pupal lethality seen with suppression of ETHRB- vs. ETHRA-expressing neurons argues that the two receptor isoforms mediate different processes (Diao, 2016).

The results also confirm and extend the conclusions drawn previously from in situ hybridization studies that ETHRA and ETHRB are expressed in distinct populations of neurons (Kim, 2006b). That this is also the case in the hawkmoth, Manduca suggests that this feature, like the generation of ETHRA and ETHRB splice isoforms itself, is highly conserved. The finding that ETHRA and ETHRB serve distinct functions that are mediated by different populations of neurons thus seems likely to represent an evolutionarily ancient characteristic of insect ecdysis circuits (Diao, 2016).

Interestingly, an exception to the mutually exclusive expression of ETHRA and ETHRB occurs in the Vm neurons, which secrete EH and occupy a unique role in the ecdysis circuit. EH acts in a well-characterized positive feedback loop between the Vm neurons and the ETH-secreting Inka cells. ETH and EH are initially released at low levels, but each reinforces the other's secretion to cause the later, massive release of both hormones at high levels, an event that is thought to drive the progression of the ecdysis sequence. It is possible that the ETHRB isoform, which exhibits higher sensitivity to ETH than ETHRA (Iversen, 2002; Y. Park, 2003), mediates the initial Vm response to ETH causing low-level EH release, while expression of the ETHRA isoform participates in the late, high-level response. Further work will, however, be required to test this hypothesis (Diao, 2016).

While the function of ETHRB-expressing neurons has been enigmatic, several groups of peptidergic neurons known to express ETHRA have been previously implicated in governing ecdysis. The results presented in this study refine and expand knowledge of two such cell groups, those that express CCAP or Lk (Diao, 2016).

CCAP-expressing neurons as a group have been shown to be required for head eversion, a defining event of pupal ecdysis. The results presented in this study provide direct evidence that the subset of CCAP-expressing neurons that coexpresses ETHRA is the one required for head eversion. A previous study of a late-differentiating CCAP-expressing neurons concluded that cells in abdominal neuromeres AN8-9 are required for head eversion. However, these neurons are not observed to express ETHRA mRNA, and it was surprising to likewise find them inconsistently represented in the expression patterns of ETHRMI00949- and ETHRAMI00949-Gal4 lines. The finding that both electrical suppression and ablation of these neurons leaves head eversion unimpaired forces a conclusion that CCAP-expressing neurons in AN8-9 are not responsible for this process. This conclusion is consistent with a recent report that activation of CCAP-expressing neurons that also express bursicon (and therefore do not include the AN8-9 neurons) is sufficient to induce head eversion. It is possible that the correlational nature of earlier results, which were based on the effects of stochastic ablation of subsets of all CCAP-expressing neurons, may have inadvertently suffered from sampling errors that biased the interpretation to the opposite conclusion. In general, the current demonstration that only those CCAP-expressing neurons that coexpress ETHRA block ecdysis underscores the ability of the receptor-based mapping approach described here to correctly identify critical nodes in hormonally controlled behavioral circuits (Diao, 2016).

A second critical node revealed by this analysis is the subset of ETHRA-expressing neurons that express the diuretic factor leucokinin. Based on neuronal suppression experiments using two copies of the UAS-Kir2.1 transgene, it was demonstrated that Lk-secreting neurons maintain fluid balance to support behavioral execution. It was found that ecdysis deficits induced by neuronal suppression are reversed together with fluid imbalance by feeding of tyramine suggesting that these phenotypes share a common cause in the dysregulation of fluid secretion at the level of the Malpighian tubules. A report that appeared during revision of this manuscript indicates that Lk-expressing neurons also specifically regulate the timing of pre-ecydsis behavior (Kim, 2015), which was not assayed in the current. It will be interesting to determine whether these deficits are independent of fluid imbalance and persist in tyramine-fed animals. Curiously, neither fluid imbalance nor the overt deficits in appendage extension that this study observed are described by Kim (2015) as a consequence ablating the Lk-expressing neurons. It is possible that this apparent inconsistency results from the use of different Lk-Gal4 drivers, or from the differential efficacy of neuronal suppression by UAS-Kir2.1 vs. cell killing by UAS-rpr, but in either case, the two studies support the conclusion that Lk-expressing neurons regulate physiological and/or behavioral processes important for pupal ecdysis (Diao, 2016).

Consistent with what has been previously found, this analysis suggests that peptidergic neurons, in general, are well represented within the ETHR expression pattern and are essential to the ecdysis circuit. Evidence is also provided that subsets of ETHR-expressing neurons that use the neurotransmitters acetylcholine and glutamate are functionally important. Suppression of the cholinergic subset potently blocks ecdysis at both the larval and pupal stages and may well include many neurons that also express peptides. The glutamatergic subset, however, is likely to be distinct from the cholinergic group based on the previously reported nonoverlapping expression of the cholinergic marker, ChaT, and the glutamatergic marker, VGlut (Diao, 2015). Interestingly, electrical suppression of a GABAergic subset of ETHR-expressing neurons does not result in overt ecdysis failure. It thus seems likely that inhibitory inputs previously shown to regulate the execution of different phases of the ecdysis sequence in Manduca and thought to also function in Drosophila do not derive from ETHR-expressing neurons, are not GABAergic, or are not strictly essential for ecdysis. The last possibility is favored and it is noted that this study has focused only on gross ecdysis deficits. More subtle defects that affect behavioral coordination, execution, or timing and do not result in lethality will require closer analysis. The preliminary results, however, suggest that many of the ETHR-expressing neurons identified in this study can be expected to play specific roles in ecdysis at some developmental stage (Diao, 2016).

The Trojan exon methodology used in this study to identify, manipulate, and parse the patterns of ETHR expression represents a systematic and versatile strategy for mapping functional connectivity within hormone-mediated neural circuits. In the case of the ecdysis circuit, this strategy has not only facilitated analysis of the neural substrates of behavior and physiology, but has revealed unanticipated developmental differences in the importance of the two ETHR isoforms. In the fly as in other insects, the motor patterns that mediate ecdysis vary considerably across developmental stages to accommodate differences in body plan and environmental context. However, the changes that occur in the ecdysis circuit over development remain largely unknown. The tools developed here should provide the basis for a thorough-going investigation of this, and other, important issues (Diao, 2016).


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