Ecdysone receptor


Targets of Activity

Ecdysteroid signaling in insects is mediated by the ecdysone receptor complex, which is composed of a heterodimer of the Ecdysone receptor and Ultraspiracle. The DNA binding specificity plays a critical role in defining the repertoire of target genes that respond to the hormone. The determination of the preferred core recognition motif by a binding site selection procedure is described. The consensus sequence consists of a perfect palindrome of the heptameric half-site sequence GAGGTCA that is separated by a single A/T base pair. No binding polarity of the Ecdysone receptor/Ultraspiracle heterodimer to the core recognition motif is observed. This core motif mediates the highest level of ligand-induced transactivation when compared to a series of synthetic ecdysone response elements and to the natural element of the Drosophila hsp27 gene. This is the first report of a palindromic sequence being identified as the highest affinity DNA binding site for a heterodimeric nuclear hormone receptor complex. Evidence is presented that the ligand of the Ecdysone receptor preferentially drives Ultraspiracle from a homodimer into a heterodimer. This mechanism might contribute additionally to a tight control of target gene expression (Vogtli, 1998).

The steroid hormone 20-hydroxyecdysone, acting through the Ectydsone receptor directs Drosophila metamorphosis by activating a series of genetic regulatory hierarchies. ETS domain transcription factors encoded by the ecdysone-inducible E74 early gene, E74A and E74B, act at the top of these hierarchies to coordinate the induction of target genes. These E74 isoforms were ectopically expressed to understand their regulatory functions during the onset of metamorphosis. E74 can regulate its own transcription, most likely through binding sites within its gene. Ectopic expression of E74B can partially repress the E78B and DHR3 orphan receptor genes, suggesting a role for E74 in the appropriate timing of early-late gene expression. Furthermore, E74A is both necessary and sufficient for E78B induction, implicating E74A as a key regulator of E78B expression. Consistent with studies of E74 loss-of-function mutations, it is found that E74B is a potent repressor of late gene transcription. Ectopic E74B completely prevents L71-1 and L71.6 induction in newly formed prepupae. E74A is sufficient to prematurely induce the L71-1 late gene. However, ectopic expression of both Broad and E74A activators in an E74B mutant background is not sufficient to prematurely induce all late genes, indicating that other factors contribute to this regulatory circuit. E74A and E74B transcription is precisely coordinated by dynamic changes in ecdysone concentration. E74B is induced by a low ecdysone concentration and repressed by higher hormone concentations. The ecdysone concentration required for 50% maximal E74B repression is similar to that required for 50% maximal E74A induction. Thus each rise in ecdysone titer directs an obligate switch in E74 isoforms. It is suggested that the presence of E74B protein counteracts Broad complex A1 and other activators that might be present, directly preventing late gene induction until the end of larval development when E74B is repressed and E74A is induced. The ETS DNA-binding domain shared by E74A and E74B allows these factors to oppositely regulate the same target genes with distinct temporal specificity, permitting the tight coupling of rises in ecdysone titer to the induction of secondary-response genes. It is interesting to note that a similar switch between negative and positive ETS domain transcription factors has been described during Drosophila eye and ventral ectoderm development, directed by the opposing effects of yan and pointed. These observations demonstrate that the steroid-triggered switch in E74 transcription factor isoforms plays a central role in the proper timing of secondary-response gene expression (Fletcher, 1997).

The E74 gene is responsible for the early ecdysone-inducible puff at position 74EF and encodes two related DNA-binding proteins which appear to play a regulatory role in the hierarchy. E74A is expressed in a wide variety of late-third instar tissues, suggesting that it plays a broad pleiotropic role in response to the hormone. In early prepupae, when the overall levels of E74A mRNA are decreasing, relatively high levels of E74A RNA persist in the gut, peripodial membranes of the imaginal discs, and proliferation centers of the brain. The spatial distribution of nuclear E74A protein correlates with the RNA distribution with the single exception that no E74A protein can be detected in the proliferation centers of the brain. There is also a temporal discrepancy between E74A mRNA and protein accumulation. The peak of E74A protein induced by the late larval ecdysone pulse follows the peak of E74A mRNA by approximately 2 h. This delay is not seen in 10 h prepupae, when the next pulse of ecdysone induces the simultaneous expression of E74A mRNA and protein. The unusually long and complex 5' leader in the E74A mRNA may regulate its translation (Boyd, 1991).

Pulses of ecdysone at the end of Drosophila larval development dramatically reprogram gene expression as they signal the onset of metamorphosis. Ecdysone directly induces several early puffs in the salivary gland polytene chromosomes that, in turn, activate many late puffs. Three early puffs, at 2B5, 74EF, and 75B, have been studied at the molecular level. Each contains a single ecdysone primary-response gene that encodes a family of widely expressed transcription factors. The 63F early puff is significantly different from the previously characterized early puff loci. First, the 63F puff contains a pair of ecdysone-inducible genes that are transcribed in the larval salivary glands: E63-1 and E63-2. Second, E63-1 induction in late third instar larvae appears to be highly tissue-specific, restricted to the salivary gland. Third, E63-1 encodes a novel Ca(2+)-binding protein related to calmodulin. The discovery of an ecdysone-inducible Ca(2+)-binding protein provides a foundation for integrating steroid hormone and calcium second messenger signaling pathways and generates an additional level for potential regulation of the ecdysone response (Andres, 1995).

The expression of Hormone-receptor-like in 78 in larval salivary glands allowed for the identification of potential regulatory targets by antibody staining of polytene chromosomes. Whereas no binding sites can be detected in polytene chromosomes prepared from Hr78 mutant larvae, multiple stained sites could be detected in polytene chromosomes prepared from wild-type mid-third instar larvae. Hr78 protein can bind to a subset of 20E receptor binding sites in vitro, suggesting that Hr78 might function at the top of the ecdysteroid regulatory hierarchies. In order to determine if Hr78 exhibits a similar binding specificity in vivo, polytene chromosomes were stained with antibodies directed against either Hr78 or Ultraspiracle. The staining pattern of Usp is identical to that of EcR, and thus indicative of sites bound by the 20E receptor. Some sites are bound primarily by the EcR or Hr78, while the majority of sites are bound by both proteins, consistent with an overlap in their binding specificity. In order to map the sites bound by Hr78, salivary glands were dissected from newly formed white prepupae, when Hr78 protein is most abundant, and polytene chromosome preparations were stained with anti-Hr78 antibodies. Over 100 Hr78 binding sites have been identified, many of which correspond to ecdysteroid regulated puff loci (Fisk, 1998).

The steroid hormone ecdysone induces a precise sequence of gene activity in Drosophila melanogaster salivary glands in late third larval instar larvae. The acquisition of competence for this response does not result from a single event or pathway but requires factors that accumulate throughout the instar. Individual transcripts become competent to respond at different times and their expression is differentially affected in ecd1, dor22 and BR-C mutants. ecd1 mutants are deficient in ecdysone. dor encodes a protein with a zinc-finger like motif. The induction of early-late transcripts, originally assumed to necessarily follow early transcripts, is partially independent of early transcript activation. Attempts to inhibit the synthesis of regulatory proteins reveal transcript-specific superinduction effects. Furthermore these inhibitors lead to the induction of betaFTZ-F1 and E93 transcripts at levels normally found in prepupal glands. These studies reveal the complexity of the processes underlying the establishment of a hormonal response (Richards, 1999).

In 90-hour salivary glands, all intermolt transcripts, especially EcR and E74B are higher in the presence of cycloheximide. In 100-h glands the effect is less markded although expression ratios were between 2.5 and 3.5 for both cycloheximide and anisomycin. Unexpectedly, E75C transcripts, normally difficult to detect, are dramatically increased in the presence of both inhibitors at 100-h. There is no effect of cyclohexaminde for the early transcript E74A in 90-h glands, while E75A transcripts in the same glands show a 5-fold superinduction. At 100 h, both E74A and E75A ratios are close to 1, as would be expected for an induction that is independent of protein synthesis. In contrast, the inhibitors cause a striking superinduction of E75B transcripts both at 90 and 100 h. DHR3 also undergoes superinduction. A number of mechansims have been suggested to explain the superinduction phenomenon. These include: (1) the existence of a labile repressor, whose rapid turnover is sensitive to inhibitors; (2) the protection of transcripts from degradation either by physical association with inhibitors or by the inhibition of labile mRNases; (3) the inhibition of early protein synthesis to prevent the negative feed-back loop postulated to repress early transcript synthesis and (4) the inhibitor acting as an inducer at concentrations below those necessary to block protein synthesis. Since effects are transcript specific, different combinations of mechanisms may function at each locus (Richards, 1999).

The crooked legs gene is induced by ecdysone during the onset of metamorphosis. CROL mRNA can be detected in mid-third instar larvae, consistent with the expression of beta-galactosidase in the CNS of crol4418 flies at this stage in development. The level of CROL mRNA then increases in late third instar larvae, in parallel with the high titer ecdysone pulse that triggers puparium formation. The levels of crol transcription decrease to low levels in mid-prepupae and then rise significantly in 12 hour prepupae, following the ecdysone pulse that triggers head eversion. This correspondence between the rises in ecdysone titer and the induction of crol transcription are consistent with crol being an ecdysone-inducible gene, subject to regulation by the Ecdysone receptor. To test this hypothesis more directly, salivary glands were dissected from mid-third instar larvae and cultured for 4 hours in the absence or presence of ecdysone. RNA was then isolated and crol transcription was analyzed by northern blot hybridization. This study revealed that CROL mRNA levels are induced approximately two-fold by ecdysone, similar to the level of induction seen in vivo in late third instar larvae. A similar induction of crol transcription is seen in cultures of mixed larval organs treated with ecdysone. These observations support the hypothesis that crol transcription is inducible by ecdysone, but the relatively low level of induction suggests that other factors may contribute to this regulation (D'Avino, 1998).

The Larval serum protein-2 gene (Lsp-2) is uniquely expressed from the beginning of the third instar to the end of adult life in the fat body tissue. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. A single functional ecdysone response element (EcRE) is localized at position -75 relative to the Lsp-2 transcription initiation site. A 27-bp sequence harboring the EcRE bounds both the Drosophila ecdysone receptor and Ultraspiracle in a cooperative manner (Antoniewski, 1995).

Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster encodes one of the major hexameric hemolymph proteins of third-instar larvae that is also a major component of adult serum. Regulated transcription of Lsp-2 results in high-level, ecdysone-stimulated expression throughout the larval fat body and low-level, spatially restricted expression in the adult fat cells. To localize cis-acting regulatory sequences responsible for the stage- and tissue-specific activity at Lsp-2, the expression of Lsp-2-lacZ fusion genes was studied by P element-mediated germline transformation of Drosophila. A 230 base pair larval enhancer, which includes an ecdysone response element (EcRE), specifically targets gene activity to the larval fat body. Although the adult mode of Lsp-2 expression depends on the larval enhancer, additional negative regulatory elements dictate both tissue-specificity and unique spatial restriction within the adult fat body. The implications of these findings for the identification of fat body-specific gene regulatory units in other insects are discussed (Benes, 1996).

The ß3 tubulin gene is regulated by ecdysone through the EcR. Sequences within a kilobase upstream of the transcriptional start site, and 360 bp from within the first intron are involved in hormone regulation. The nucleotide sequence of the intronic region contains several Ecdysone receptor response element consensus sequences (Tourmente, 1993).

A fundamental unresolved question in endocrinological research is how systemic signals like pulses of steroid hormones are converted into a variety of tissue- and stage-specific responses. The existence of three different Ecdysone receptor isoforms, which are differentially expressed in larval and imaginal tissues, may provide the first clue for the differential regulation of these responses. Sgs genes are salivary gland secretion protein genes, regulated in Drosophila by the molt cycle. Two regulatory elements were identified in the upstream region of the Drosophila Sgs-3 gene that are both able to bind the Ecdysone receptor (EcR/USP) and the product of the fork head gene. Interestingly, only one of the EcR/USP binding sites is able to recognize in vitro-translated EcR/USP, which provides evidence for the existence of different receptor forms having different DNA binding specificities. Deletions of the elements leads to a reduced accumulation of Sgs-3 mRNA without altering the temporal expression profile of the gene. The data are consistent with the hypothesis that the Ecdysone receptor directly contributes to the transcriptional activation of Sgs-3 by binding to at least one of the two elements. Since the Sgs-4 gene is also controlled by a functional EcR/USP binding site, a direct participation of EcR/USP in the formation of regulatory complexes may be of general importance for the hormonal control of Sgs genes (Lehmann, 1997).

In Drosophila, peaks of the titer of the steroid hormone ecdysone act as molecular signals that trigger all the major developmental transitions occurring along the life cycle. The EcR/USP heterodimer, known to constitute the functional ecdysone receptor, binds with high affinity to specific target sequences. The target sequences, known as ecdysone response elements (EcREs) still remain to be fully characterized at both the molecular and functional levels. In order to investigate the properties of EcREs composed of directly repeated half-sites (DRs), an analysis was carried out of the binding properties of the ng-EcRE, a DR element located within the coding region of ng-1 and ng-2, two highly homologous genes mapping at the ecdysone-regulated 3C intermolt puff. The ng-EcRE contacts the Ecdysone receptor through its directly repeated half-sites spaced by 12 bp, and this element may interact efficiently with at least three Drosophila orphan receptors, namely DHR38, DHR39 and beta FTZ-F1. Interestingly, DHR38 is bound alone or in combination with USP, providing the first evidence that the EcR-USP and DHR38-USP may directly compete for binding to a common response element. These results suggest that EcREs composed of widely spaced DRs may contribute to the establishment of extensive cross-talk between nuclear receptors, thus modulating ng-1 and ng-2 intermolt expression (Crispi, 1998).

The three yolk protein genes (yps) of Drosophila are expressed in the ovary and fat body of the adult female. Their levels of expression in the fat body depend upon both juvenile hormone (JH) and 20-hydroxyecdysone (20E). For 20E, induction of reporter gene expression in males was assayed. For JH, this was followed by upregulation of the genes in nutritionally deprived females, which express yolk proteins (YPs) at very low levels. 20E inducible sites are present upstream of yp3; the sites are located 3' and within the coding sequence of yp3 introns that can respond to 20E. There are also sites in the intergenic spacer between yp1 and yp2. Evidence for repressors is also found upstream of the yp genes, suggesting that downstream 20E inducible elements may be important in vivo. There appears to be a difference between different constructs in males in the response to 20E in the fat body of the thorax and in the abdomen. It is not clear whether those sequences which respond to 20E are genuine ecdysone response elements (i.e., binding sites for the ecdysone receptor) or if the effect is indirect. JH upregulation of YPs is observed using only native yp genes as reporters, suggesting that this hormone may act either on intron sequences or on yp coding sequences, or perhaps by influencing stability of the yp mRNA (Bownes, 1996).

A 23 bp sequence from the ecdysone-inducible hsp27 promoter has been shown to function as an Ecdysone response element that can confer 20-fold ecdysone inducibility on an ecdysone-nonresponsive promoter. There are also EcR-binding sites in the ecdysone-induced E74 gene (Koelle, 1991).

BmEcR from the commercial silkmoth, Bombyx mori, is a functional ecdysone receptor. Upon dimerization with BmCF1 (the silkmoth homolog of Drosophila Usp), BmEcR binds the radiolabeled steroid ligand 125I-iodoponasterone A with Kd = 1.1 nM, rendering it indistinguishable from that exhibited by DmEcR/DmUSP. BmEcR/BmCF1 forms a specific complex with an ecdysone response element (EcRE) derived from the Drosophila heat shock protein 27 (hsp27) gene promoter; as with DmEcR/DmUSP, formation of this complex is stimulated by the presence of 20-hydroxyecdysone. BmEcR can substitute for DmEcR in an EcR-deficient Drosophila tissue culture line, stimulating trans-activation of an ecdysone-inducible reporter gene construct. Thus, BmEcR and BmCF1 are the functional counterparts of DmEcR and DmUSP, respectively and, despite considerable sequence divergence between the Drosophila and Bombyx proteins, the counterparts are (at least qualitatively) functionally equivalent (Swevers, 1996).

Larval midgut and salivary gland histolysis are stage-specific steroid-triggered programmed cell death responses. Larval salivary glands can be maintained for many hours in organ culture, providing an ideal opportunity to study the hormonal requirements for a variety of responses to ecdysone, including glue secretion, polytene chromosome puffing and specific gene regulation. The majority of glands cultured in the presence of ecdysone for 7 hours show a strong nuclear acridine orange stain indicating that salivary gland cell death is an ecdysone-triggered response. In vivo, dying larval midgut and salivary gland cell nuclei become permeable to the vital dye acridine orange; their DNA undergoes fragmentation, indicative of apoptosis. Crawling mid-third instar larvae were injected with ecdysone. Such injected larvae pupariate within 6-8 hours, and midguts isolated from these larvae show a uniform nuclear acridine orange staining, indicative of the onset of programmed cell death. The histolysis of these tissues can be inhibited by ectopic expression of the baculovirus anti-apoptotic protein p35, implicating a role for caspases in the death response. Coordinate stage-specific induction of the Drosophila death genes reaper (rpr) and head involution defective (hid) immediately precedes the destruction of the larval midgut and salivary gland. In addition, the diap2 anti-cell death gene is repressed in larval salivary glands as rpr and hid are induced, suggesting that the death of this tissue is under both positive and negative regulation. diap2 is repressed by ecdysone in cultured salivary glands under the same conditions that induce rpr expression and trigger programmed cell death. These studies indicate that ecdysone directs the death of larval tissues via the precise stage- and tissue-specific regulation of key death effector genes (Jiang, 1997).

In order to test if held out wings is transcriptionally regulated by ecdysone, third larval instar tissues were isolated before the increase in ecdysone titer. They were cultured for 8 hours, and a physiologically high titer of ecdysone was added to cultures for varying periods of time. RNA was extracted from the tissues, electorphoresed, transferred to a membrane, and hybridized with a radiolabelled how probe. how is induced by ecdysone in cultured organs (Baehrecke, 1997).

Inducible expression of double-stranded RNA directs specific genetic interference in Drosophila

The introduction of double-stranded RNA (dsRNA) can selectively interfere with gene expression in a wide variety of organisms, providing an ideal approach for functional genomics. Although this method has been used in Drosophila, it has been limited to studies of embryonic gene function. Only inefficient effects have been seen at later stages of development. When expressed under the control of a heat-inducible promoter, dsRNA interfers efficiently and specifically with gene expression during larval and prepupal development in Drosophila. Expression of dsRNA corresponding to the EcR ecdysone receptor gene generates defects in larval molting and metamorphosis, resulting in animals that fail to pupariate or prepupae that die with defects in larval tissue cell death and adult leg formation. In contrast, expression of dsRNA corresponding to the coding region of the betaFTZ-F1 orphan nuclear receptor has no effect on puparium formation, but leads to an arrest of prepupal development, generating more severe lethal phenotypes than those seen with a weak betaFTZ-F1 loss-of-function allele. Animals that express either EcR or betaFTZ-F1 dsRNA show defects in the expression of corresponding target genes, indicating that the observed developmental defects are caused by disruption of the genetic cascades that control the onset of metamorphosis. These results confirm and extend understanding of EcR and betaFTZ-F1 function. They also demonstrate that dsRNA expression can inactivate Drosophila gene function at later stages of development, providing a new tool for functional genomic studies in Drosophila (Lam, 2000).

EcR encodes three protein isoforms, designated A, B1, and B2, which can function together with the Drosophila RXR homolog, USP, as receptors for ecdysone. Some isoform-specific EcR mutations result in a small number of escapers that arrest development at the onset of metamorphosis, indicating that this receptor plays a key role in transducing the ecdysone signal at this stage. To assess null mutant phenotypes for EcR during the onset of metamorphosis, P-element transformants were established that use the heat-inducible hsp70 promoter to drive expression of a snapback dsRNA corresponding to the coding region shared by all EcR isoforms (Lam, 2000).

Two lines, hs-EcRi-11 and hs-EcRi-42, were examined in detail. A single 30 minute heat treatment in mid-third instar larvae results in a high degree of lethality in both lines. An additional heat treatment 6 hours later increases the severity of this phenotype in hs-EcRi-11 transformants, resulting in predominantly larval lethality. The hs-EcRi-11 animals express high levels of heat-induced EcR dsRNA and display reduced levels of EcR protein. High levels of EcR dsRNA were expressed following heat treatment. Two sequential heat treatments, at ~18 and 12 hours before puparium formation, generate the most severe lethal phenotypes in hs-EcRi-11 animals. A more detailed characterization of this transformant line was performed using the double 30 minute heat treatment regime. The majority of hs-EcRi-11 animals (81%) arrested development as stationary late third instar larvae. Although these animals stop moving, as do wild-type late third instar larvae, they fail to shorten their body to form the characteristic shape of a prepupa and die several days later. Some animals attempted to pupariate (13%), but formed long prepupae that occasionally failed to tan, and died 1-2 days later. A small number of animals (6%) died as pupae with defects in leg elongation. Dissection of hs-EcRi-11 prepupae revealed normal larval midguts in newly-formed prepupae, but the midguts fail to progress beyond the stage normally seen in prepupae that are 2 hours. The midgut fails to contract and undergo cell death, resulting in persistence of the gastric caeca and proventriculus. In addition, the adult midgut, which normally surround the dying larval cells by 12 hours after puparium formation, do not appear to form. Similarly, larval salivary glands are still present 24 hours after puparium formation in hs-EcRi-11 animals, indicating a block in their normal cell-death response. Therefore, expression of EcR dsRNA at ~18 and 12 hours before puparium formation has a profound effect on the ability of animals to enter metamorphosis in response to the late larval pulse of ecdysone (Lam, 2000).

Ecdysone exerts its effects on development through a well-defined cascade of ecdysone-regulated gene expression. The hormone directly induces a small set of primary-response genes, including the E74 and E75 early genes. These genes encode transcription factors that regulate downstream secondary-response late genes. In the salivary glands, the late larval pulse of ecdysone triggers a switch in late gene expression, repressing the glue genes and inducing the L71 late genes, along with more than 100 late puffs visible in the giant polytene chromosomes. To understand the molecular basis of EcR interference by dsRNA, the patterns of ecdysone-regulated gene expression in staged hs-EcRi-11 late larvae and early prepupae were examined. As expected, the levels of EcR mRNA were significantly reduced in these animals, consistent with the reduced levels of EcR protein detected by western blot analysis. Ecdysone-induced E74A and E75A transcription is significantly reduced in hs-EcRi-11 late third instar larvae. Repression of E74B, which is linked to E74A induction, also fails to occur in these animals. Consistent with these defects in early ecdysone-induced regulatory gene expression, the switch in salivary gland late genes is effectively blocked. The Sgs-4 glue gene is not repressed at puparium formation and the L71-6 late gene is not induced. Moreover, reaper mRNA fails to accumulate to normal levels in hs-EcRi-11 mid-prepupae, consistent with the observed defects in ecdysone-triggered cell death (Lam, 2000).

Taken together, these phenotypes correspond closely to those described for loss-of-function EcR mutations. A small number of null mutants that are missing only the EcR-B isoforms survive to the third larval instar and attempt to initiate metamorphosis, forming stationary larvae that resemble the predominant lethal phenotype of hs-EcRi-11 animals. EcR-B null mutants, however, undergo larval cuticle apolysis, a process that fails to occur in hs-EcRi-11 animals. This observation is consistent with the proposal that EcR dsRNA would inactivate all EcR isoforms, and would therefore generate more severe phenotypes than those seen in the absence of only the B isoform (Lam, 2000).

Consistent with this proposal, null mutants for all three EcR isoforms that are rescued to the third larval instar by ectopic expression of an EcR-B2 cDNA not only fail to pupariate properly, but also display a block in larval cuticle apolysis similar to that in hs-EcRi-11 animals. These partially rescued EcR mutants also show little or no expression of ecdysone-inducible early proteins, consistent with a block in the late larval genetic cascade. These effects resemble the transcriptional defects observed in hs-EcRi animals. It is interesting to note that heat treatment of unstaged hs-EcRi-11 larvae results in molting defects that resemble those seen in EcR loss-of-function mutants, indicating that this construct can also be used to define EcR functions during larval development. Taken together, the phenotypes of hs-EcRi-11 animals are entirely consistent with a specific and effective block in EcR function during later stages in the life cycle (Lam, 2000).

Functional analysis of regulatory elements controlling the expression of the ecdysone-regulated Drosophila ng-1 gene

The steroid hormone ecdysone controls multiple aspects of insect development, including larval molts and metamorphosis, and can induce specific genetic responses in different tissues. The regulatory elements directing the expression of ng-1, an ecdysone-regulated Drosophila gene showing a highly specific developmental expression profile, have been identified by transgenic analysis. An ecdysone-responsive element located within the ng-1 coding region is necessary for high-level gene expression, whereas the gene's spatial and temporal expression profile is fully controlled by a distinct upstream regulatory region. This region binds a set of transcriptional factors, including the Fkh regulatory protein, which can potentially modulate the ecdysone genetic regulated response (Crispi, 2001).

For many ecdysone-regulated genes, the definition of the regulatory role that an ecdysone-responsive element can play in the determination of a specific genetic expression profile is often complicated by the dense organization of regulatory sequences responsible for the hormonal response and the developmental expression within short upstream regions acting as multifunctional regulatory domains. This type of organization, where interacting regulatory elements are usually tightly clustered or even superimposed, is common to many target genes in both invertebrate and vertebrate systems, and is often referred as a Hormone Response Unit (HRU). The results reported here reveal that the regulatory sequences directing the expression of the ecdysone-regulated ng-1 gene are organized quite differently. In fact, ng-EcRE deletion or sequence alterations results in a marked reduction of the amount of the ng-1::lacZ fused transcripts, but does not change substantially the developmental expression profile of the mutant transgenes. In addition, the cis-acting regulatory elements responsible for ng-1 tissue and stage-specific expression do not map in close association with the ng-EcRE, but are clustered within a distinct upstream regulatory region acting as a salivary gland enhancer in third instar larvae. Given that the ng-EcRE, in conjunction with its close flanking sequences, functions as an autonomous developmental enhancer, the results obtained here strongly imply that its activity can be widely modulated by additional regulatory elements, and suggest a critical role for context-dependent and combinatorial factor interactions in setting the specificity of its hormonal response. Within the ng-1 upstream enhancer, the cis-acting elements responsible for the temporal expression profile could not be distinguished from those involved in determining the tissue specificity. In fact, while the complete region directs a salivary-gland specific expression in third instar larvae, all the smaller subregions tested fail to activate the expression of the reporter gene. It is plausible that the tight linkage of protein binding sites within this regulatory region might cause this effect, given that, as determined by EMSA analysis, a set of at least five factors binds specifically at this regulatory domain. Thus, full-level activation of ng-1 may depend on a concerted action of EcR binding at the ng-EcRE and on a set of transcription factors, which includes the Drosophila Fkh protein, bound elsewhere, at the upstream control region. At the transcriptional level, the modulation of the ng-1 activity may be accomplished by the ng-EcRE in at least two different ways. (1) ng-EcRE/ecdysone-receptor complex can directly influence the binding or the activity of components of the transcription machinery; (2) receptor binding may alter chromatin structure locally, thus removing constraints imposed by chromatin conformation to the access of regulatory transcription factors at the upstream regulatory domain. Both hypotheses are consistent with the finding that the upstream regulatory region, although at a reduced level, is by itself able to properly activate the transcription of the reporter gene. However, given that the presence or functional integrity of the ng-EcRE, although relevant to the accumulation of ng-1 mRNA, is not strictly required either for the initiation or for the maintenance of ng-1 expression, it is also possible that ecdysone binding at the ng-EcRE might trigger alternative effects, such as modulation of the ng-1 mRNA half-life. Ecdysone-mediated regulation of the mRNA stability has already been described for the Sgs-3, Sgs-7 and Sgs-8 glue gene mRNAs, whose half-lives decline upon addition of ecdysone to cultured salivary glands. Thus, the possibility that ecdysone-mediated ng-1 regulation might occur, at least in part, at the post-transcriptional level cannot be excluded, and the peculiar location of the ng-EcRE within the ng-1 transcribed region makes this possibility even more intriguing. The recent finding that a ng-EcRE RNA probe is able to form multiple complexes when incubated with nuclear salivary gland extracts adds further support to this hypothesis (Crispi, 2001).

Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis

Small hydrophobic hormones like steroids control many tissue-specific physiological responses in higher organisms. Hormone response is characterized by changes in gene expression, but the molecular details connecting target-gene transcription to the physiology of responding cells remain elusive. The salivary glands of Drosophila provide an ideal model system to investigate gaps in knowledge, because exposure to the steroid 20-hydroxyecdysone (20E) leads to a robust regulated secretion of glue granules after a stereotypical pattern of puffs (activated 20E-regulated genes) forms on the polytene chromosomes. A convenient bioassay for glue secretion is described in this study, and this bioassay is used to analyze mutants in components of the puffing hierarchy. 20E mediates secretion through the EcR/USP receptor, and two early-gene products, the rbp+ function of BR-C and the Ca2+-binding protein E63-1, are involved. Furthermore, 20E treatment of salivary glands leads to Ca2+ elevations by a genomic mechanism, and elevated Ca2+ levels are required for ectopically produced E63-1 to drive secretion. The results presented establish a connection between 20E exposure and changes in Ca+ levels that are mediated by Ca2+-effector proteins, and thus establish a mechanistic framework for future studies (Biyasheva, 2001).

SgsDelta3-GFP transgenes were used to monitor glue secretion. SgsDelta3-GFP is expressed in a pattern identical to that of the endogenous Sgs3 gene, and the fusion protein is properly sorted, secreted, and expelled from the salivary gland in a manner identical to that of the endogenous glue mix. The chromophore of this fusion protein is stable for several days and can be used to observe glue synthesis and secretion in living animals and dissected tissues. This allows for the easy performance of genetic screens for mutations that affect exocytosis. Finally, the SgsDelta3-GFP transgenes are important staging tools, allowing one to monitor development of third-instar larvae more precisely than current 'blue food' methods. This is possible because one can select individual larvae with specific patterns of 'green glue' in their salivary glands resulting from a developmentally regulated physiological stimulus. Thus, introducing an SgsDelta3-GFP transgene into the genetic background should allow for a more reliable method of selecting mass quantities of similarly aged animals for physiological and biochemical analyses (Biyasheva, 2001).

Although the majority of data presented here focuses on the secretion of glue granules by the premetamorphic pulse of ecdysteroids, there are reports that speculate that a small ecdysteroid pulse induces a series of developmental events, including glue gene induction during the mid-third instar (90-100 h AEL). However, the idea that 20E acts through the EcR/USP heterodimer to mediate these changes has recently been questioned, and the results presented here substantiate the challenge. l(3)ecd1 mutants, severely compromised but probably not devoid of circulating ecdysteroids, synthesize SgsDelta3-GFP. In addition, animals depleted of Ultraspiracle (Usp), EcR-B1, or EcR-B2 also express the fusion gene. However, since neither the EcR nor the usp mutants tested remove all protein (EcR-A remains intact; Usp is truncated in usp2 mutants, and some leaky expression might occur from the hs-usp construct), the caveat that glue gene induction requires only a small amount of ecdysteroid-EcR/USP signaling must be considered. It is also noted that the studies reporting that BR-C null mutants do not produce glue, are consistent with the observation that an SgsDelta3-GFP transgene is not expressed in npr1 animals (Biyasheva, 2001).

It has also been reported that the mid-instar developmental transitions fail to occur in DHR78 (Hr78) orphan nuclear receptor mutants. This observation has led to the speculation that a hormone signal, working through DHR78, is needed for these events. However, in this study DHR78 mutants synthesize SgsDelta3-GFP and escapers that pupariate secrete normally, despite using two different DHR78 alleles and two different SgsGFP stocks. One explanation for this observation is that genetic modifiers necessary for the mid-instar phenotype were lost or gained when the Sgs-GFP tester stocks were generated. However, the lethal phase (mid-third instar) associated with the DHR78 parental stocks is extended to the late third-instar/early prepupal period after several generations of reproduction and growth on media. This raises the interesting possibility that part of the reported DHR78 phenotype is environmentally or nutritionally influenced (Biyasheva, 2001).

Using classical genetic approaches and RNAi technology, it has been shown that the ecdysteroid receptor, consisting of EcR and Usp, is required for glue secretion. Since limited secretion is detected in EcR-B1 and usp mutants, RNAi was used to establish their absolute requirement. This new technology is a rapid and simple approach to compromise candidate genes, to determine their requirement in secretion, especially when mutants are either not available or die early in development. Using osmotic shock to introduce double-stranded RNA into cultured glands greatly increases the utility of this method (Biyasheva, 2001).

Salivary glands cultured with 20E secrete glue, whereas glands cultured in the presence of both 20E and cycloheximide fail to secrete. This suggests that protein synthesis is required for glue secretion; however, no single early or early-late product, thus far identified, has been found to be necessary for this secretion. The rbp+ function of the BR-C early locus, however, is required for secretion to occur in a timely manner. These mutants secrete SgsDelta3-GFP, but the process is delayed by at least 4 h. Although the rbp5 lesion is expected to be an amorph (all BRC-Z1 proteins are truncated before the DNA-binding domain), it does produce a small protein with some reported function (Biyasheva, 2001).

Also of interest are the mutations that eliminate 2Bc and E74B. These result in animals that are unable to expel secreted glue onto the ventral body surface. Since there must be a neural signal and pharyngeal muscle contractions to expel glue, these mutants may possess a neural and/or muscular defect. This would agree with previous studies that some BR-C functions are important for correct muscle attachment, and that E74B is required for head eversion (Biyasheva, 2001).

Because mutations in early and early-late genes fail to block secretion, two possible explanations come to mind: (1) either the early products tested are functionally redundant or (2) additional hormone-responsive genes required for secretion have not yet been identified. These possibilities can now be tested using double-mutant combinations, mutant and RNAi combinations, and ectopic expression studies like those conducted with E63-1 (Biyasheva, 2001).

Calcium levels are elevated after 2 h of 20E treatment, and a Ca2+ signal is necessary for secretion induced by ectopic expression of the E63-1 early protein. These results demonstrate that 20E acts genomically to upregulate at least two classes of proteins: those that increase cytoplasmic Ca2+ levels, and those (E63-1 and others) that mediate exocytosis in response to the Ca2+ signal (Biyasheva, 2001).

A conditional rescue system reveals essential functions for the ecdysone receptor( EcR) gene during molting and metamorphosis in Drosophila

In Drosophila, pulses of the steroid hormone ecdysone trigger larval molting and metamorphosis and coordinate aspects of embryonic development and adult reproduction. At each of these developmental stages, the ecdysone signal is thought to act through a heteromeric receptor composed of the EcR and USP nuclear receptor proteins. Mutations that inactivate all EcR protein isoforms (EcR-A, EcR-B1, and EcR-B2) are embryonic lethal, hindering analysis of EcR function during later development. Using transgenes in which a heat shock promoter drives expression of an EcR cDNA, temperature-dependent rescue of EcR null mutants has been employed to determine EcR requirements at later stages of development. EcR is required for hatching, at each larval molt, and for the initiation of metamorphosis. In EcR mutants arrested prior to metamorphosis, expression of ecdysone-responsive genes is blocked and normal ecdysone responses of both imaginal and larval tissues are blocked at an early stage. These results show that EcR mediates ecdysone signaling at multiple developmental stages and implicate EcR in the reorganization of imaginal and larval tissues at the onset of metamorphosis (Li, 2000).

Although initially normal in appearance and behavior when rescued to the third larval instar, EcR mutants exhibit substantial delays in progression through this stage and fail to pupariate. EcR mutants are delayed in initiating wandering behavior and in extreme cases may persist in larval form for up to 7 days, rather than the normal 2 days, following the second to third larval molt. Appearance of internal tissues is consistent with an early block in ecdysone responses in all tissues examined. Leg imaginal discs fail to elongate, gastric caeca shortening is arrested, and the larval salivary glands persist in EcR mutants. In addition, proliferation of midgut imaginal cells is not initiated in EcR mutants. These results suggest that EcR is required for most or all ecdysone-regulated developmental events during early metamorphosis, including imaginal disc morphogenesis, destruction of larval tissues through programmed cell death and proliferation of imaginal cells (Li, 2000).

The phenotype of rescued EcR null mutants reported here differs in several respects from mutants lacking only EcR-B1 functions. EcR-B1 mutants fail to pupariate, but successfully complete the larval/pupal apolysis, an event that is blocked in rescued EcR null mutants. Mutants that lack EcR-B1 and EcR-B2 due to deletion of the EcR-B transcription start site show a similar phenotype to EcR-B1 mutants, suggesting that EcR-A is sufficient to trigger larval/pupal apolysis. As shown by the elongation and fusion of leg imaginal discs, EcR-B1 mutants initiate normal ecdysone response in the imaginal discs, a class of tissue that expresses high levels of EcR-A, whereas ecdysone-triggered responses are defective in these mutants in tissues that express high levels of EcR-B1. These observations, and the finding that disc elongation and eversion in the rescued EcR null mutants described here are blocked at an early stage, are consistent with the model that EcR-A and EcR-B1 trigger distinct developmental responses to ecdysone. Interestingly, however, proliferation of midgut imaginal cells is blocked at an earlier stage in EcR null mutants than in EcR-B1 mutants, suggesting that either EcR-A or EcR-B2 may also participate in the control of midgut imaginal cell proliferation in response to ecdysone (Li, 2000).

Western analysis of extracts from rescued EcR mutants using antibodies directed against products of five ecdysone-responsive genes shows that expression of three early response genes are largely (BR-C) or completely (E74A, E75B) abolished. Expression of the early-late gene DHR3 and the mid-prepupal response gene betaFTZ-F1 are also severely affected. These results show that expression of ecdysone-responsive genes early in metamorphosis is dependent on EcR. The results confirm and extend to the whole animal earlier findings from analysis of larval salivary gland polytene chromosomes that transcriptional puffing of ecdysone-responsive genes is blocked in EcR-B1 mutants. The retention of low levels of BR-C 91 and 81 kDa products in EcR mutants is consistent with the incomplete block to BR-C puffing previously seen in EcR-B1 mutants (Li, 2000).

Induction of the early-late Ddc gene during Drosophila metamorphosis by the ecdysone receptor

During Drosophila metamorphosis, the 'early-late' genes constitute a unique class regulated by the steroid hormone 20-hydroxyecdysone. Their induction is comprised of both a primary and a secondary response to ecdysone. Previous work has suggested that the epidermal expression of the dopa decarboxylase gene (Ddc) is likely that of a typical early-late gene. Most of the Ddc activity (>90%) is found in the epidermal cells where dopamine metabolites promote pigmentation and cross-linking of the cuticle. Some Ddc activity (about 5%) is also found in the central nervous system where it produces dopamine and serotonin, which serve as neurotransmitters. The level of Ddc activity remains relatively constant in the central nervous system throughout development, but peaks of activity are observed at each of the five molts in the epidermis. Accumulation of the Ddc transcript in the epidermis is rapidly initiated in the absence of protein synthesis, which implies that the ecdysone receptor plays a direct role in induction. However, full Ddc expression requires the participation of one of the transcription factors encoded by the Broad-Complex. An ecdysone response element (EcRE) that contributes to the primary response has been characterized. Using gel mobility shift assays and transgenic assays, a single functional EcRE, located at position -97 to -83 bp relative to the transcription initiation site, has been identified. This is the first report of an EcRE associated with an early-late gene in Drosophila. Competition experiments indicated that the affinity of the Ddc EcRE for the ecdysone receptor complex is at least four-fold less than that of the canonical EcRE of the hsp27 gene. Using in vitro mutagenesis, it was determined that the reduced affinity of the EcRE resided at two positions where the nucleotides differed from those found in the canonical sequence. The ecdysone receptor, acting through this EcRE, releases Ddc from a silencing mechanism, whose cis-acting domain has been mapped to the 5'-upstream region between -2067 and -1427 bp. Deletion of this repressive element results in precocious expression of Ddc in both epidermis and imaginal discs. Thus, epidermal Ddc induction at pupariation is under the control of an extended genomic region that contains both positive and negative regulatory elements (Chen, 2002).

Ligand-dependent de-repression via EcR/USP acts as a gate to coordinate the differentiation of sensory neurons in the Drosophila wing

Loss of function of either the ecdysone receptor (EcR) or Ultraspiracle (USP), the two components of the ecdysone receptor, causes precocious differentiation of the sensory neurons on the wing of Drosophila. It is proposed that the unliganded receptor complex is repressive and that this repression is relieved as the hormone titers increase at the onset of metamorphosis. The point in development where the receptor complex exerts this repression varies for different groups of sensilla. For the chemosensory organ precursors along the wing margin, the block is at the level of senseless expression and is indirect, via the repressive control of broad expression. Misexpressing broad or senseless can circumvent the repression by the unliganded receptor and leads to precocious differentiation of the sensory neurons. This precocious differentiation results in the misguidance of their axons. The sensory precursors of some of the campaniform sensilla on the third longitudinal vein are born prior to the rise in ecdysone. Their differentiation is also repressed by the unliganded EcR/USP complex but the block occurs after senseless expression but before the precursors undertake their first division. It is suggested that in imaginal discs the unliganded EcR/USP complex acts as a ligand-sensitive 'gate' that can be imposed at various points in a developmental pathway, depending on the nature of the cells involved. In this way, the ecdysone signal can function as a developmental timer coordinating development within the imaginal disc (Schubiger, 2005).

The ecdysone signal is transmitted via the ecdysone receptor to activate a number of direct target genes. It has generally been assume that the hormone and its receptor activate a hierarchy by activating early genes that then activate the many late genes. A large body of work based primarily on larval tissues has supported this model. Thus when the ecdysone receptor is non functional, the first step in the cascade fails, the early genes are not activated, and the tissues are unable to undergo a metamorphic response. In imaginal discs it has been shown that loss of function of USP leads to the inability to activate early genes, such as DHR3, EcR and E75B, but also results in precocious differentiation, rather than in a failure to initiate a particular metamorphic response. It has now been demonstrated that loss of EcR function in the wing discs gives similar results (precocious BR-Z1 expression and sensory neuron differentiation) to the ones reported for loss of USP function, and it is concluded that the unliganded EcR/USP heterodimer is the functional repressor. Thus at least some processes at the onset of metamorphosis are not controlled by the ecdysone-induced hierarchy, but rather through the relief of the repressive function of the unliganded EcR/USP complex once the ecdysone titers rise. The importance of this repressive function of the unliganded receptor is further demonstrated by experiments using a dominant negative EcR, which does not bind the hormone, and as a consequence repression cannot be relieved and target genes are not expressed. The repressive role proposed for the unliganded ecdysone receptor complex would also explain why loss-of-function USP clones, in general, result in the differentiation of normal adult bristle organs since loss of receptor function would only control the timing of differentiation. Such an interpretation is supported by early pigmentation of abdominal bristles in usp3 clones that has been observed on occasions. In vivo studies of activation by EcR/USP suggest that activation plays a major role in the metamorphic response of larval tissues but has only a minor role in the development of the imaginal discs. It remains to be seen which processes are activated by the ecdysone hierarchy and which by loss of the repressive actions of the unliganded receptor (Schubiger, 2005).

Adult chemosensory neurons on the wing margin undergo precocious differentiation in loss-of-USP clones. To understand which step is repressed by the EcR/USP complex the early expression patterns of a set of genes involved in neuron differentiation was examined in such mutant clones. In the absence of USP function the early pattern of Achaete (AC) expression in the margin is unaffected. In contrast, both Neur (as visualized by A101) and Sens are expressed in usp mutant cells before they are detected in the surrounding wild-type tissue. In vitro experiments revealed that A101 expression that is already on at the time cultures were set up, remains on through the culture period, but that there is a block by EcR/USP at the level of sens expression that prevents the maturation of the SOPs of the chemosensory neurons in the wing margin. The block is released once the hormone titers rise (Schubiger, 2005).

The repressive function of the unliganded receptor does not act directly on the genes tested. The block of SOP differentiation is controlled through BR-Z1, and br function is required for the activation of sens, a gene necessary and sufficient for sensory organ differentiation. Thus expressing BR-Z1 or Sens early in the margin allows the inhibition from the unliganded ecdysone receptor to be by-passed and the sensory neurons in the margin to differentiate precociously. When Sens is misexpressed it was found that clusters of extra neurons differentiate in the region of high driver expression. This is in agreement with reports that high levels of Sens activate the proneural genes and promote the formation of SOPs. By contrast, when BR-Z1 is misexpressed in the margin, a more normal pattern of sensory neuron arrangement is observed, that is very similar to what is observed in loss-of-function USP or EcR cells. This indicates that BR-Z1 does not induce the formation of SOPs but rather causes the up-regulation of Sens in cells that have already committed to the SOP fate. Occasionally expressing BR-Z1 in the margin leads to the differentiation of a sensory neuron in the posterior margin, normally devoid of neurons. It is possible that in such a situation BR-Z1 misexpression can at times lead to sufficiently high expression of Sens to cause SOP differentiation (Schubiger, 2005).

Loss-of-function of BR demonstrates the requirement for BR to activate the high levels of Sens in the SOPs, as well as the low levels in the posterior margin, but without molecular data it is not known if br is directly activating sens. Since BR-Z1 normally appears later than the initial low expression of Sens, it is proposed that early Sens expression is most probably controlled by BR-Z2. BR-Z2 is expressed shortly after the molt to the third instar, and ectopic BR-Z2 expression induces low levels of Sens. BR-Z3, which also induces Sens when ectopically expressed may induce low levels of Sens as well, but since BR-Z3 is normally expressed at very low levels in the wing disc, it is thought that Br-Z3 plays a minor role. BR-Z1 then is needed for the accumulation of Sens in the mature SOPs (Schubiger, 2005).

In summary the above genetic interactions suggest that unliganded EcR/USP represses BR expression that is required for sens activation and the formation of the mature SOPs in the margin (Schubiger, 2005).

The SOPs are born in a specific temporal sequence in the wing disc. The first SOPs arise in the third instar, 20-30 hours before pupariation; they include GSR, ACV and L3-2 along the third vein. The SOPs of the margin arise later, at 10-12 hours before pupariation, so they are at a very different stage from that of the early born SOPs at the time metamorphosis begins. Since the unliganded receptor is acting as a repressor it is postulated that the block must be occurring at different times during the progression of sensory organ differentiation for these two groups of sensilla. Based on genetic studies, the ecdysone-sensitive arrest for the chemosensory sensilla of the margin occurs in the up-regulation of Sens, since the SOP is undergoing maturation. For the early born sensilla, however, Sens levels are already elevated before the rise in 20E and are not dependent on BR function. For these early born sensilla the ecdysone-sensitive arrest occurs after high Sens expression but prior to the division of the SOP. It is thought that for different sets of sensilla the imposition of an ecdysone-sensitive arrest at different points in development is important to coordinate the differentiation of the sensilla. Such a mechanism would ensure that the outgrowing axons begin to elongate in a choreographed manner leading to the correct axon pathways and to their finding of the correct targets in the CNS according to their physiological function. This idea is supported by the observation that the axons of sensilla forced to differentiate precociously by the absence of a functional ecdysone receptor or by early expression of BR-Z1 or Sens often take abnormal routes (Schubiger, 2005).

Ecdysone is also acting as a timer for the formation of the chordotonal and Johnston's organs as well as for the initiation of the morphogenetic furrow. These structures arise early in the third instar (80 hours after egg laying) and appear to be under the control of the small ecdysone peak at that time. In the case of the leg chordotonal organ, ecdysone appears to be controlling the proneural gene atonal (ato). It is not known yet if this control also occurs via de-repression as is seen for the wing (Schubiger, 2005).

The subsequent progression of the morphogenetic furrow is also dependent on ecdysone. This action of ecdysone has been proposed not to occur via EcR. However, loss of USP leads to an advancement of the furrow and precocious differentiation of the photoreceptors. It has been reported that the progression of the morphogenetic furrow, as well as the timing of differentiation of the chordotonal organs in the leg, are controlled by the insulin receptor (InR)/Tor pathway, with increased InR signaling leading to precocious differentiation. In the wing margin, by contrast, increasing or decreasing InR signaling does not affect the timing of differentiation of the chemosensory neurons. Thus there must be multiple temporal control mechanisms for sensory structures. The current results have demonstrated repression of sensory organs by the unliganded ecdysone receptor at the end of the third instar, but do not rule out additional steps controlled by ecdysone or other factors. It remains to be elucidated which timer(s) is used when, and for which sensory structures (Schubiger, 2005).

In holometabolous insects functional larval tissues are replaced by the differentiating imaginal ones. The endocrine system is acting on larval tissues composed of differentiated cells that are thus in an equivalent state to initiate programs such as cell death and neuronal remodeling. Here EcR/USP's role is activational. For the differentiation of the imaginal tissues the endocrine system faces a varied cellular landscape where some cells may still be dividing while other have begun to differentiate. In these tissues the unliganded receptor acts as a repressor to interrupt the sequence of differentiation at different points in order to coordinate the response to the rising 20E titers. Release of repression by 20E may therefore function as a 'gate' at the onset of metamorphosis and thus would enable development of imaginal tissues to be coordinated and tightly controlled by the rising ecdysone titers. In metamorphosing amphibians a similar situation is seen with functional larval tissues such as the tail and the gills dying and adult limbs and lungs developing in response to thyroid hormone. It would not be surprising to find that the thyroid hormone receptor is activational in the larval tissues but that the forming adult tissues are controlled through de-repression (Schubiger, 2005).

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).

The steroid hormone ecdysone controls systemic growth by repressing dMyc function in Drosophila fat cells

How steroid hormones shape animal growth remains poorly understood. In Drosophila, the main steroid hormone, ecdysone, limits systemic growth during juvenile development. This study showed that ecdysone controls animal growth rate by specifically acting on the fat body, an organ that retains endocrine and storage functions of the vertebrate liver and fat. This study demonstrates that fat body-targeted loss of function of the Ecdysone receptor (EcR) increases dMyc expression and its cellular functions such as ribosome biogenesis. Moreover, changing dMyc levels in this tissue is sufficient to affect animal growth rate. Finally, the growth increase induced by silencing EcR in the fat body is suppressed by cosilencing dMyc. In conclusion, the present work reveals an unexpected function of dMyc in the systemic control of growth in response to steroid hormone signaling (Delanoue, 2010).

The growth rate and the duration of juvenile growth are two key parameters that determine the size of the animal at the time of maturation. These two parameters are coupled during the juvenile period to determine organismal size at maturation by mechanisms that are not yet understood. Recent work has established that the two hormonal systems controlling these parameters, ecdysone and insulin/IGF, have antagonistic actions that set up the larval growth rate. The present study demonstrates that the fat body is the unique relay for ecdysone-induced growth inhibition, and provides molecular and genetic evidence that inhibition of dMyc function by ecdysone signaling in fat cells plays a key role in this control (Delanoue, 2010).

Paradoxically, a positive, cell-based role for ecdysone was observed in growth and proliferation during larval stages. Indeed, clonal loss of function for EcR induces a reduction of cell size despite an increase in PI3K activity and dMyc expression. This indicates that ecdysone signaling is autonomously required for optimal cell growth, despite its negative action on the growth rate at the systemic level. These results are in line with previous studies indicating that ecdysone/EcR/Usp and some of their downstream targets are required for cell-cycle progression and tissue growth. Ex vivo culture experiments using dissected discs from Bombyx mori have shown that an optimal concentration of 20E is required for proper disc growth that is 6- to 10-fold lower than the 20E concentration required to stimulate the molting cycle. This suggests that different ecdysone concentrations are required for cell-autonomous growth induction and nonautonomous growth inhibition. It is likely that elevated 20E levels like those attained at the end of larval development are required for the inhibition of dMyc expression in the fat body, leading to systemic growth inhibition (Delanoue, 2010).

These data indicate that the fat body is central in the regulation of organismal growth by ecdysone. Previous studies have established that this organ acts as a nutrient sensor and coordinates global growth according to nutrition conditions. Therefore, it appears positioned at a crossroad, allowing extrinsic and intrinsic inputs to be integrated and translated into a coordinated control of body growth. These results indicate that dMyc, but not IIS, is required in the fat body for transducing the growth effects of EcR signaling. Previous work showed that TOR signaling, and not IIS, controls the nutrition sensor that operates in fat body cells. Although not required in fat cells for either of these controls, IIS is downregulated in peripheral tissues, and this regulation contributes to the systemic growth inhibition observed under both conditions. This suggests that the fat body remotely controls the expression/production/secretion/activity of the circulating Dilps that activate dInR. It was recently demonstrated that under conditions of abundant nutrients, the fat body emits a 'secretion signal' that triggers the release of Dilps from brain cells (Géminard, 2009). The molecular links proposed between TOR signaling and dMyc position dMyc as a downstream effector of TOR signaling, playing a role in the transcriptional activation of target genes. This and the current data suggest the possibility that both nutrition- and ecdysone-induced signals converge on a Myc-dependent mechanism in fat cells leading to the systemic regulation of growth by general IIS (Delanoue, 2010).

Both dMyc and TOR signaling control ribosome biogenesis and protein translation initiation. In addition to its role as an energy reservoir, the fat body, like the mammalian liver, is one of the most active secreting tissues, and a large majority of the hemolymph proteins are synthesized in this tissue. This secreting function is thought to be essential for larval growth and is likely to be sensitive to ribosome quantity. Therefore, the fat body could control systemic growth either via the production of specific secreted factors according to ribosomal abundance, or via the control of hemolymph protein concentration that would in turn regulate growth. The current studies should help discriminate between these different hypotheses (Delanoue, 2010).

It was observed that altering EcR signaling in the fat body markedly modifies the activities of many IIS signaling actors such as PI3K, AKT, and dFoxO. This study demonstrates that this regulation does not contribute to the control of organismal growth, suggesting that the function of 20E-induced IIS repression at the end of larval development is restricted to metabolic effects, such as the arrest of carbohydrate and lipid storage, and to the induction of autophagy (Delanoue, 2010).

Changing ecdysone circulating level influences dMyc expression in the fat body but not in any other larval tissues. Therefore, the EcR-dependent control of dMyc expression is specific to this tissue. No consensus binding sites were detected for EcR/Usp in the dMyc promoter region. This suggests that dMyc is not a direct target of EcR-mediated gene repression, but rather that EcR signaling controls expression of a fat-specific downstream component that is itself responsible for adipose dMyc transcriptional regulation (Delanoue, 2010).

Myc has been recently shown to promote oxidative phosphorylation as well as glycolysis through coordinate transcriptional control of the mitochondrial metabolic network (Zhang, 2007). This metabolic regulation by dMyc has not been studied in the present work, but it is conjectured that rising ecdysone levels at the end of the juvenile period influence both translational and metabolic activities regulated by dMyc in the fat body, a regulation that could have important consequences on the energy homeostasis of the animal during this important developmental transition (Delanoue, 2010).

In conclusion, the present work reveals an unexpected role for dMyc in the systemic control of growth. It was demonstrated that the fat body-specific activity of dMyc is the target of EcR signaling and is involved in a remote regulation of growth through IIS in peripheral tissues (Delanoue, 2010).

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode C. elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. This study used Drosophila S2 cells to analyze the effects of 20-HE on Drosophila microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNAi knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNA, it was shown that nanomolar concentrations of 20-HE primes cells to subsequently express microRNA when exposed to micromolar levels of 20-HE. The role microRNA plays in the established relationship between 20-HE and the induction of innate immunity was examined. The 3'UTR of the antimicrobial peptide diptericin was found to have a let-7 binding site and let-7 was found to represses translation from this site. It is concluded that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNA control of antimicrobial peptide translation (Garbuzov, 2010).

Conserved microRNA miR-8 controls body size in response to steroid signaling in Drosophila

Body size determination is a process that is tightly linked with developmental maturation. Ecdysone, an insect maturation hormone, contributes to this process by antagonizing insulin signaling and thereby suppressing juvenile growth. This study reports that the microRNA miR-8 and its target, u-shaped (USH), a conserved microRNA/target axis that regulates insulin signaling, are critical for ecdysone-induced body size determination in Drosophila. The miR-8 level is reduced in response to ecdysone, while the USH level is up-regulated reciprocally, and miR-8 is transcriptionally repressed by ecdysone's early response genes. Furthermore, modulating the miR-8 level correlatively changes the fly body size; either overexpression or deletion of miR-8 abrogates ecdysone-induced growth control. Consistently, perturbation of USH impedes ecdysone's effect on body growth. Thus, miR-8 acts as a molecular rheostat that tunes organismal growth in response to a developmental maturation signal (Jin, 2012).

This study reveals the mechanism by which ecdysone suppresses insulin signaling and thereby decelerates larval growth. During larval development, ecdysone regulates the levels of miR-8 and its target, USH, a PI3 kinase inhibitor, through the EcR downstream pathway, and quantitative regulation of miR-8 by ecdysone leads to determining the final fly size. Since the ecdysone level is low during the larval stage not engaged in the molting process, a mild response of miR-8 increase by EcR inhibition in this stage would be expected. Notably, however, it was found that this increase in miR-8 level by EcRDN is constantly sustained throughout the third instar larval stage, which is the period of exponential growth. Interestingly, among early response genes of EcR signaling, E74 and BR-C are also persistently expressed during this period; these gene products repress miR-8 expression at the transcriptional level. Thus, throughout the third instar larval period, EcR downstream signaling keeps miR-8 (and, concomitantly, insulin signaling) under control. Because the duration of this regulation lasts several days, the effect of miR-8 modulation accumulates and manifests a significant impact on final body size. This cumulative effect accounts for the effect of ecdysone signaling on body size despite only modest changes in the miR-8 level. When EcR signaling was hampered throughout the larval stage, leading to a sustained increase of miR-8, the final body size became noticeably bigger (Jin, 2012).

This function of miR-8 in shaping body size provides a novel example in which an animal uses the inherent ability of miRNAs in fine-tuning target gene expression. Previously, several studies have shown that such a strategy has been used with miRNAs in diverse biological contexts. Specific examples include maintaining the optimal level of miRNA target proteins, which is critical for organismal survival, and setting the thresholds of target gene activity to prevent inappropriate development. The current data show the application of this type of strategy in a continuous process of organismal growth. By tuning the activity of insulin signaling, miRNAs could regulate organismal growth and ensure the attainment of appropriate body size. It is currently unclear whether similar regulatory mechanisms exist in other organisms. However, in humans and rodents, the miR-200 family of miRNAs are predominantly expressed in organs such as the pituitary, thyroid, testes, ovary, and breast, most of which are major target organs of steroid hormones. Moreover, the miR-200 family of miRNAs are significantly down-regulated by the estrogen hormone in breast cancer cells and uterus tissues, suggesting that the miR-200 family may also be controlled by steroids in mammals. It would be interesting to investigate whether a comparable regulatory axis of steroid hormone/miR-200/insulin signaling is conserved through metazoan evolution (Jin, 2012).

A view through a chromatin loop: insights into the ecdysone activation of early genes in Drosophila

The early genes are a key group of ecdysone targets that function at the top of the signaling hierarchy. In the presence of ecdysone, early genes exhibit a highly characteristic rapid and powerful induction that represents a primary response. Multiple isoforms encoded by early genes then coordinate the activation of a larger group of late genes. While the general mechanism of ecdysone-dependent transcription is well characterized, it is not known whether a distinct mechanism governs the hormonal response of early genes. Previous work has found that one of the Drosophila early genes, E75, harbors multiple functional ecdysone response elements (EcREs). This study extends the analysis to Broad and E74 and has found that EcRE multiplicity is a general feature of the early genes. Since most of the EcREs within early gene loci are situated distantly from promoters, the chromosome conformation capture method was used to determine whether higher order chromatin structure facilitates hormonal activation. For each early gene chromatin loops were detected that juxtapose their promoters and multiple distant EcREs prior to ecdysone activation. These findings suggest that higher order chromatin structure may serve as an important mechanism underlying the distinct response of early genes to ecdysone (Bernardo, 2014).

Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle

MicroRNA-mediated post-transcriptional regulations are increasingly recognized as important components of the circadian rhythm. This study identified microRNA let-7, part of the Drosophila let-7-Complex, as a regulator of circadian rhythms mediated by a circadian regulatory cycle. Overexpression of let-7 in clock neurons lengthens circadian period and its deletion attenuates the morning activity peak as well as molecular oscillation. Let-7 regulates the circadian rhythm via repression of Clockwork Orange (Cwo). Conversely, upregulated cwo in cwo-expressing cells can rescue the phenotype of let-7-Complex overexpression. Moreover, circadian Prothoracicotropic hormone (PTTH) and Clock-regulated 20-OH ecdysteroid signalling contribute to the circadian expression of let-7 through the 20-OH Ecdysteroid receptor. Thus, this study has found a regulatory cycle involving PTTH, a direct target of Clock, and PTTH-driven miRNA let-7 (Chen, 2014).

Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

During development cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. This study examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. A cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa. In the wing, ecdysone signaling at the larva to puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva to puparium pulse during early metamorphosis, Broad expression plummets allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing (Guo, 2016).

This study presents a model for how the pulse of ecdysone at the larval to pupal transition impacts the cell cycle dynamics in the wing during metamorphosis. Ecdysone signaling at the larva to puparium transition induces Broad, which in turn represses Stg to generate a temporary G2 arrest, which synchronizes the cell cycle in the wing epithelium. As ecdysone levels decline, Broad expression plummets, allowing Stg to be re-activated resulting in a pulse of cdc2 activity that promotes a rapid G2/M progression during the final cell cycle in the wing. This ultimately culminates in the relatively synchronized cell cycle exit at 24h APF, coinciding with the second large pulse of ecdysone. This second pulse in the pupa activates a different set of transcription factors (not Broad), promoting the acquisition of terminal differentiation characteristics in the wing. In this way, two pulses of ecdysone signaling can both synchronize the final cell cycle by a temporary G2 arrest and coordinate permanent cell cycle exit with the acquisition of terminal differentiation characteristics in the wing (Guo, 2016).

Over 30 years ago it was shown that 20-HE exposure in Drosophila tissue culture cells induces a cell cycle arrest in G2-phase. This response appears to be shared among 3 different cell lines, Cl-8, Kc and S2. This study shows that in Kc cells, pulsed 20-HE exposure also leads to a G2 arrest followed by rapid cell cycle re-entry after 20-HE removal and a subsequent prolonged G1. This cell cycle response to a pulse of 20-HE is reminiscent of the cell cycle changes that occur during early metamorphosis in the pupal wings and legs (Guo, 2016).

It is worth considering why Kc and S2 cells, which are thought to be derived from embryonic hemocytes would exhibit a similar cell cycle response to 20-HE to the imaginal discs. Relatively little is known about how ecdysone signaling impacts embryonic hemocytes, although recent work suggests that ecdysone signaling induces embryonic hemocyte cell death under sensitized conditions. More is known about larval hemocytes, which differentiate into phagocytic macrophages and disperse into the hemolymph during the first 8h of metamorphosis. Ecdysone is involved in this maturation process, as lymph glands of ecdysoneless (ecd) mutants fail to disperse mature hemocytes and become hypertrophic in the developmentally arrested mutants. This suggests that the high levels of systemic ecdysone signaling at the larval-puparium transition mediates a switch from proliferation to cell cycle arrest and terminal differentiation for lymph gland hemocytes during metamorphosis. Without ecdysone signaling, hemocytes may continue to proliferate and fail to undergo terminal differentiation leading to the hypertrophic lymph gland phenotype observed. Interestingly, while the loss of broad also prevents proper differentiation of hemocytes similar to loss of ecd, loss of broad does not lead to the hypertrophy observed in ecd mutants. Further studies will be needed to examine whether the ecdysone induced cell cycle arrest in larval hemocytes occurs in the G2 phase, or whether their cell cycle arrest proceeds via a similar pathway to that shown in this study for the wing (Guo, 2016).

Multiple lines of evidence suggest that the ecdysone receptor complex in the larval wing acts as a repressor for certain early pupa targets and that the binding of ecdysone to the receptor relieves this repression. For example loss of EcR by RNAi or loss of the EcR dimerization partner USP, de-represses ecdysone target genes that are high in the early pupal wing such as Broad-Z1 and βFtz-F1. The EcR/USP heterodimer also cooperates with the SMRTR co-repressor in the wing to prevent precocious expression of ecdysone target genes such as Broad-Z1. Consistent with the hypothesis that a repressive EcR/USP complex prevents precocious expression of Broad-Z1 and thereby a precocious G2 arrest, inhibition of SMRTR can also cause a G2 arrest. Thus, in the context of the early pupal wing, it is proposed that the significant pulse of ecdysone at the larval to puparium transition relieves the inhibition of a repressive receptor complex, leading to Broad-Z1 activation. Consistent with this model, high levels of Broad-Z1 in the larval wing lead to precocious neural differentiation at the margin and precocious inhibition of stg expression in the wing pouch. Interestingly, a switch in Broad isoform expression also occurs during the final cell cycle in the larval eye, such that Broad-Z1 becomes high in cells undergoing their final cell cycle and entering into terminal differentiation. However in this case, Broad-Z1 expression is not associated with a G2 arrest and occurs in an area of high Stg expression, suggesting the downstream Broad-Z1 targets in the eye may be distinct or regulated differently from those in the wing (Guo, 2016).

The ecdysone receptor has also been shown to down regulate Wingless expression via the transcription factor Crol at the wing margin, to indirectly promote CycB expression. While a loss of EcR at the margin decreased CycB protein levels, the effects of EcR loss on CycB levels in the wing blade outside of the margin area were not obvious. It is suggested that in the wing, the role for EcR outside of the margin acts on the cell cycle via a different mechanism through stg. Consistent with a distinct mechanism acting in the wing blade, over-expression of Cyclin B in the early prepupal wing could not promote increased G2 progression or bypass the prepupal G2 arrest. Instead the results on the prepupal G2 arrest are consistent with previous findings that Stg is the rate-limiting component for G2-M cell cycle progression in the fly wing pouch and blade (Guo, 2016).

In order to identify the gene expression changes in the wing that occur in response to the major peaks of ecdysone during metamorphosis, RNAseq was performed on a timecourse of pupal wings. Major changes were observed in gene expression in this tissue during metamorphosis. In addition, known ecdysone targets were identified that are affected differently in the wing during the first larval-to-pupal ecdysone pulse and the second, larger pulse at 24h APF. Ecdysone signaling induces different direct targets with distinct kinetics. Furthermore specific targets, for example Ftz-F1 can modulate the expression of other ecdysone targets, to shape the response to the hormone. Thus, it is expected that a pulse of ecdysone signaling leads to sustained effects on gene expression and the cell cycle, even after the ecdysone titer returns to its initial state. These factors together with the differences in the magnitude of the ecdysone pulse may contribute to the differences in the response to the early vs. later pulses in the wing (Guo, 2016).

Ecdysone signaling can also affect the cell cycle and cell cycle exit via indirect mechanisms such as altering cellular metabolism. This is used to promote cell cycle exit and terminal differentiation in neuroblasts, where a switch toward oxidative phosphorylation leads to progressive reductive divisions, (divisions in the absence of growth) leading to reduced neuroblast cell size and eventually terminal differentiation. Although reductive divisions do occur in the final cell cycle of the pupa wing, this type of mechanism does not provide a temporary arrest to synchronize the final cell cycle in neuroblasts as is see in wings. Importantly, a striking reduction is seen in the expression of genes involved in protein synthesis and ribosome biogenesis in the wing during metamorphosis, consistent with the lack of cellular growth. Instead the increased surface area of the pupal wing comes from a flattening, elongation and apical expansion of the cells due to interactions with the extracellular matrix creating tension and influencing cell shape changes. This is also consistent with the findings that a significant number of genes associated with protein targeting to the membrane are increased as the wing begins elongation in the early pupa. Further studies will be needed to determine whether the changes in expression of genes involved in ribosome biogenesis and protein targeting to the membrane are controlled by ecdysone signaling, or some other downstream event during early wing metamorphosis (Guo, 2016).

Perhaps the most interesting and least understood aspect of steroid hormone signaling is how a diversity of cell-type and tissue-specific responses are generated to an individual hormone. Cell cycle responses to ecdysone signaling are highly cell type specific. For example abdominal histoblasts, the progenitors of the adult abdominal epidermis, become specified during embryogenesis and remain quiescent in G2 phase during larval stages. During pupal development, the abdominal histoblasts must be triggered to proliferate rapidly by a pulse of ecdysone to quickly replace the dying larval abdominal epidermis. This is in contrast to the behavior of the wing imaginal disc, where epithelial cells undergo asynchronous rapid proliferation during larval stages, but during metamorphosis the cell cycle dynamics become restructured to include a G2 arrest followed by a final cell cycle and entry into a permanently postmitotic state, in a manner coordinated with tissue morphogenesis and terminal differentiation (Guo, 2016).

How does the same system-wide pulse of ecdysone at the larval to puparium transition lead to such divergent effects on the cell cycle in adult progenitors? Surprisingly it seems to be through divergent effects on tissue specific pathways that act on the same cell cycle targets. In the abdominal histoblasts the larval to puparium pulse of ecdysone triggers cell cycle re-entry and proliferation via indirect activation of Stg, by modulating the expression of a microRNA miR-965 that targets Stg. This addition of the microRNA essentially allows ecdysone signaling to act oppositely on the same cell cycle regulatory target as Broad-Z1 does in the wing. Thus, tissue specific programs of gene regulatory networks can create divergent outcomes from the same system- wide hormonal signal, even when they ultimately act on the same target (Guo, 2016).

Continued: see Targets of Activity part 2/2

Interactive Fly, Drosophila Ecdysone receptor: Biological Overview | Evolutionary homologs | Regulation | Protein interactions | Developmental Biology | Effects of Mutation | References

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