Ecdysone receptor: Biological Overview | Evolutionary homologs | Regulation | Targets of Activity | Protein interactions | Developmental Biology | Effects of Mutation | References
Gene name - Ecdysone receptor

Synonyms -

Cytological map position - 42A

Function - Zinc finger transcription factor

Keywords: master regulator of molting

Symbol - EcR

FlyBase ID: FBgn0000546

Genetic map position - 2-{55.2}

Classification - nuclear receptor superfamily

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Induction of molting in Drosophila coincides with release from the ring gland of 20-hydroxyecdysone, also known as ecydsone. Prior to each of the larval molts, at pupariation, at pupation and during metamorphosis, hormone is released in carefully timed spurts, coinciding with major morphological transitions. (A description of these stages is give in Developmental origin of adult structures ).

Studies with other insects shows that release of Ecdysone from the ring gland is triggered by the prothoracicotropic hormone, produced by four dorsolateral neurosecretory cells of brain (see Drosophila Prothoracicotropic hormone.

Puffing is the term for changes in polytene chromosomes. The idea that puffing represents gene activity is currently 40 years old. A temporal pattern to puffing in the salivary glands of larval flies is inducible by ecdysone injection. A small number of genes react by puffing within minutes of exposure to ecdysone, and a much larger number (>100) react within hours. It is hypothesized that the time sequence of puffing represents a genetic hierarchy of gene activation. Early puffs are independent of protein synthesis while late puffs require prior protein synthesis (Ashburner, 1990).

Ecdysone receptor is induced at the beginning of the gene activation hierarchy. EcR is induced directly by ecdysone, and provides an autoregulatory loop that increases the level of receptor protein in response to the hormone ligand. EcR exists in three isoforms, each one having an different biological function. Each requires as a partner in heterodimerization the protein Ultraspiracle, the Drosophila homolog of vertebrate RXR proteins. Although ECR can bind ecdysone on its own, binding is greatly stimulated by the addition of USP. Ligand binding stabilizes the ECR-USP heterodimer and increases its affinity for binding to ecdysone response elements in the promoters of genes.

At least five other genes, and probably more than a dozen, are of critical importance to the regulatory hierarchy directed by EcR. E75 is also induced as an early gene, one that codes for another hormone receptor superfamily transcription factor with multiple protein isoforms. Binding sites for EcR exist in the promoter of E75, and EcR is required for the induction of the early response. The E75 response is self delimiting, as transcription is terminated soon after it initiates.

Several other genes act as delayed early genes, including hormone receptor superfamily genes E78B and DHR3, both of which are induced in a delayed fashion after EcR induction. Both require ecdysone-induced protein synthesis for their maximal levels of transcription, and appear to function as monomers to control expression of target genes (Horner, 1995). The delayed timing of E78B and DHR3 induction may allow EcR and E75 to perform regulatory functions before the delayed early genes become active, but the function of these genes is still unknown (Thummel, 1995).

During a second wave of puffing 4 to 6 hours after puparium formation, ßFTZ-F1 is induced as a mid-prepupal gene. FTZ-F1 maps to the 75CD mid-prepupal puff. It has been shown to interact with elements of the alcohol dehydrogenase gene and has also been implicated as an activator of fushi tarazu. ßFTZ-F1 is another complex gene with multiple isoforms; antibodies to ßFTZ-F1 detect binding to 166 loci in late prepupal salivary gland polytene chromosomes, 51 of which represent ecdysone-regulated puffs. Of 33 puffs that show increased activity after the peak of the 75CD puff, 17 show reproducible staining for ßFTZ-F1 (Lavorgna, 1993).

Experiments with cultured larval salivary glands have demonstrated that ßFTZ-F1 transcription is negatively regulated by ecdysone. In the absence of ecdysone, ßFTZ-F1 is induced. Repression is overcome as the levels of both ecdysone and EcR decrease during early-prepupal development. Thus ßFTZ-F1, through its interaction with EcR, provides a molecular mechanism for stage-specific responses to steroid hormones (Woodard, 1994).

In late prepupae, the midprepupal puffs regress and the early puffs are reinduced. In addition, a few stage-specific early puffs, typified by E93, are directly induced by ecdysone in late prepupae, but not in late larvae. The early puffs cannot be induced by ecdysone in early-prepupal salivary glands. Rather, a preceding period of protein synthesis and low ecdysone concentration is required before these puffs become competent to respond to hormone. It is suggested that one or more proteins encoded by the mid-prepupal puff genes provide the compentence for the early puffs to be induced by the prepupal ecdysone pulse; FTZ-F1 is a good candidate for this required gene (Woodard, 1994). Negative regulation is required in molting, as much as positive effects. For example, DHR78, an orphan nuclear receptor expressed throughout the early stages of metamorphosis, cannot heterodimerize with either ECR or USP but can bind to an Ecdysone receptor response element of a downstream gene in the hierarchy inhibiting the ability of ECR and USP to induce transcription (Zelhof, 1995).

In conclusion, each of these gene dyads and triads; EcR and USP, E75A, E78B and DHR3, ßFtz-F1, DRH78 and E93, is required in a sequential genetic hierarchy for the the timing of metamorphosis and the induction and repression of genes required for the differentiation process (Thummel, 1995). The complexity of the insect molting hierarchy serves as a warning for a generation of scientists who would unravel the hierarchies of mammalian development.

A role for juvenile hormone in the prepupal development of Drosophila melanogaster

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met27 larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis (Riddiford, 2010).

Insect molting and metamorphosis are governed primarily by ecdysone (used in the generic sense) and juvenile hormone (JH), with ecdysone causing molting and JH preventing metamorphosis. Juvenile hormone has a classic 'status quo' action in preventing the program-switching action of ecdysone during larval molts and in maintaining the developmental arrest of imaginal primordia during the intermolt periods. Its effects at the outset of metamorphosis, though, are more complex. Studies mainly on Lepidoptera show that for selected tissues JH needs to be present to allow them to undergo pupal differentiation, rather than undertaking a precocious adult differentiation (Riddiford, 2010).

The mechanism through which JH maintains the status quo and directs early development at metamorphosis is still poorly understood. Whether JH has one or multiple receptors, and the nature of these receptors, is still controversial. The best candidate for a receptor is the product of the Methoprene-tolerant (Met) gene, a PAS domain protein that was originally isolated in Drosophila melanogaster. In vitro transcribed and translated Met protein has been shown to bind JH with high affinity, and RNAi knock-down experiments in Tribolium castaneum show that Met is essential for mediating the status quo action of JH in this beetle (Riddiford, 2010).

In D. melanogaster, JH is thought to have no role in the onset of metamorphosis, since exogenous JH only delays but does not prevent pupariation. Although it has no apparent effect on the development of the imaginal discs, JH prevents normal adult development of the abdominal integument when given at pupariation. Internally, JH at this time affects normal reorganization of the central nervous system and development of the thoracic musculature. These effects of JH on metamorphosis do not occur in Met mutants, unless at least 100 times the dose is given. The Met27-null mutants proceed through larval development and metamorphosis apparently normally. However, if in addition, RNAi is used to suppress expression of Germ-Cell Expressed (Gce), a related bHLH protein with a high similarity to Met that heterodimerizes with it, Met-null mutants die as pharate adults. In the Met-deficient mutant, the adult eye shows a few (<12) defective ommatidia in the posterior region. Also, the females mature fewer eggs at a slower rate than do wild-type females, indicating that Met is also important for JH effects in egg maturation (Riddiford, 2010).

This study genetically allatectomized Drosophila larvae by targeting expression of a cell death gene to the corpora allata (CA), the gland that produces JH. These larvae form smaller puparia and showed precocious maturation of the visual system, but die around head eversion (Riddiford, 2010).

Although a number of studies have reported the effects of applying exogenous JH or JH mimics to Drosophila, there are only two very recent studies of the effects of manipulating endogenous JH on larval growth and metamorphosis, both of which appeared while this paper was under review. JH is normally present in the early larval instars, declines substantially during the last (third) larval stage and then returns transiently around the time of pupariation. The allatectomized (CAX) larvae undergo the expected two larval molts, but because sometimes the remains of degenerating CA cells are seen at the start of the last larval stage, nothing can be concluded about the requirements of JH for these larval molts. Recently, Jones (2010) using 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) RNAi to depress the level of JH and its farnesoid precursors in early larvae, showed that the larvae mainly die during the molt to the third instar, indicating that JH may be required for that molt (Riddiford, 2010).

The destruction of the CA by the third instar allowed examination of the role of JH during the last instar and early metamorphosis. The finding that these larvae were smaller than their CyO, UAS-grim siblings at pupariation could be explained by either the loss of JH or by the loss of the salivary glands, since these glands are also destroyed. Because dietary JH in the final instar rescued these larvae to normal size, the lack of the CA, rather than the lack of the salivary glands, is the cause of their reduced growth. Preliminary studies show that CAX larvae grow more slowly in the third instar, but the underlying basis for this retardation is not yet understood. Similarly, allatectomized third instar larvae display premature apoptosis of the fat body and downregulation of several enzymes involved in energy metabolism at the onset of wandering. These fat body effects could underlie the reduced larval growth seen in CAX larvae (Riddiford, 2010).

A major effect of the removal of JH was on the timing of events during the prepupal period. Studies on the wild silkmoth Hyalophora cecropia first showed that removal of the CA in the last larval stage resulted in the formation of a pupa with adult characteristics. Other moths, like Manduca sexta, showed more subtle responses to allatectomy, with premature adult differentiation most evident in the patterned region of the compound eye, posterior to the morphogenetic furrow. Subsequent studies on a variety of tissues in Manduca showed that the eye, the optic lobe and the ventral diaphragm each had a prolonged period of proliferation that extended from the prepupal period through early adult differentiation. This proliferation is maintained by α-ecdysone or low levels of 20-hydroxyecdysone (20E), but is terminated by high levels of 20E, which induces differentiation. These tissues are exposed to differentiation-inducing titers of 20E that occur during the larval-pupal transition early in their growth, but studies on the ventral diaphragm showed that JH 'protects' them from these high 20E levels, allowing them to continue proliferating. Removal of JH results in these tissues undergoing premature termination of tissue growth and precocious adult differentiation (Riddiford, 2010).

The response of Drosophila larvae to the loss of JH is in line with the effects seen in Manduca, and is also most evident in the developing visual system. In normal individuals, the appearance of EcR-B1 in the optic lobe and the termination of proliferation in the outer proliferation zone coincide with the ecdysteroid peak at head eversion and become more pronounced at 18 hours APF with the rise of ecdysteroid for adult differentiation. The separation of the R7 and R8 growth cones also begins about this latter time. The only one of these tissues that has been directly tested in vitro for 20E sensitivity is the optic lobe and in this case high levels of 20E do indeed suppress proliferation. It is assumed that the other processes also respond to the changing ecdysteroid levels. The lack of JH results in a heterochronic advance of these events by 10 to 12 hours, consistent with the tissues now responding to the earlier ecdysteroid peak that causes pupariation. Although the removal of JH advances these processes, it was found that the application of JH mimics delays them. Consequently, in selective tissues in Drosophila, JH acts to direct the nature of tissue responses to ecdysone (Riddiford, 2010).

The removal of JH or of one its receptors, Met, has a mixed effect on the developing visual system. No effect on proliferation or inductive events was seen in the eye disc itself, in that in CAX animals the morphogenetic furrow continues to move and similar rows of ommatidia have sent R8 axons into the medulla by 6 hours APF, as compared with controls. Likewise, in Met27 individuals there is only a slight advance (about 2 hours) in the schedule of lamina interneuron ingrowth into the medulla. However, for some of the cellular and molecular events, like the appearance of EcR-B1 and the separation of R7 from R8, there is a 10- to 18-hour advance in their occurrence. Hence, the lack of JH or of its receptor Met causes a heterochronic shift within the developing visual system with some differentiation responses being advanced relative to the normal schedule of neuronal birth and axon ingrowth. At least in the case of the photoreceptors, the effect of Met removal is largely cell autonomous, with the reduction of Met function in just those cells being sufficient to cause the precocious appearance of EcR-B1 and the early separation of R7 from R8. By contrast, the reduction of Met in lamina interneurons allowed these cells to precociously express EcR-B1 but did not affect the behavior of the R7 and R8 growth cones. This suggests that the separation of R7 and R8 is an active response of the photoreceptors, which is likely to be caused by the rising ecdysteroid titer driving adult differentiation. Although the lack of JH or Met function at the outset of metamorphosis results in the cell-autonomous expression of EcR-B1 in the photoreceptors, misexpression experiments show that the appearance of this receptor alone is not sufficient to bring about the early separation of R7 and R8. Therefore, although the upregulation of EcR-B1 is a prominent response to rising ecdysteroid titers, it is not the key change responsible for the repositioning of the receptor terminals (Riddiford, 2010).

As it is viable, the Met mutant allowed the final results of the mistiming of development in the optic lobes to be seen. No permanent effect was seen of the early separation of the R7 and R8 growth cones on the final anatomy of these projections in the medulla, or on the structure of the later neuropil. However, the lobula was grossly distorted and the normal layering of dendritic arbors disrupted. This aberrant morphogenesis also starts early, being already evident by 12 hours APF. The cellular basis for the lobula distortion, however, is not yet known (Riddiford, 2010).

Heterochronic shifts in the timing of development that extend beyond the visual system are likely to be the cause of the lethality seen in the CAX puparia. Puparia appear normal through the first 6 to 7 hours after pupariation but then abruptly undergo tissue collapse. In normal flies, the early part of metamorphosis is accomplished by a complicated replacement of histolyzing larval tissues by the growing adult tissues. Diverse tissues show individualized times of histolysis that are tied to the ecdysteroid titer. For instance, the larval midgut cells degenerate in response to the pupariation peak of ecdysone, whereas the larval salivary gland degeneration is triggered by the small rise of ecdysteroid at the end of the prepupal period. It is suspected that without JH, some of the histolysis events are mistimed, leading to the rapid death of the prepupa. It has been shown in CAX larvae that the fat body undergoes precocious programmed cell death beginning in the third larval instar. Interestingly, this lethal effect was not seen in animals in which Aug21-GAL4 drove RNAi for JH acid O-methyltransferase, the enzyme that converts JH acid to JH, in the CA (Niwa, 2008). Whether this indicates that JH acid plays a role in prepupal development or merely reflects the incomplete loss of JH in these animals is unknown (Riddiford, 2010).

All these effects of allatectomy can be rescued by JH either fed during the third instar or applied at the time of early wandering, but not at pupariation. A decline of JH III occurs in the third instar; this is followed by a peak of JH during late wandering. When JH begins to rise is unknown, as measurements were made every 24 hours. Presumably it is the lack of this JH during wandering when the ecdysteroid titer is rising and peaking that leads to the optic lobe anomalies and the premature histolysis (Riddiford, 2010).

The finding that the Met27 null mutant has the same defects in optic lobe development as are found in CAX prepupae strongly suggests that JH is acting via the Met pathway in controlling the timing of some events in the optic lobe. Accordingly, JHM treatment cannot suppress most of the premature development seen in prepupae lacking Met. However, a major difference between the CAX animals and the Met27 mutants is that the CAX prepupae died before head eversion, whereas the Met27 animals are viable. This difference is also seen in the precocious cell death of the fat body caused by allatectomy, which does not occur in the Met null mutant even in the presence of gce RNAi. Instead precocious cell death of the fat body was seen when Met was overexpressed in that tissue and the death could be suppressed by exogenous methoprene (a JH mimic). This latter finding suggests that JH would act in this case to suppress Met-mediated cell death. This idea was tested by seeing whether the removal of Met would protect the prepupa from the death caused by early allatectomy. When Met27; Aug21-GAL4>UAS-GFP/CyO females were crossed with UAS-grim males, 44% eclosed, all showing the CyO phenotype. The remainder died at head eversion, and should have been half CAX, Met-heterozygous females and half CAX, Met-null males. Another group was separated by sex prior to pupariation. Forty-nine percent of the females and 48% of the males died at head eversion. All of the adults that emerged were CyO, showing that all the CAX prepupae died regardless of whether or not they were lacking Met function (Riddiford, 2010).

These results together with the findings that JHM treatment of the Met27 mutant gave a partial rescue of the premature separation of R7 and R8, and of the decreased proliferation in the inner proliferation zone, indicate that there may be more than one receptor for JH. Thus, JH might act through multiple pathways. A major pathway involves Met, but Gce or some other mediator may serve as an alternate pathway in some tissues. A similar protective role of JH at pupation mediated by Met is found in Tribolium; injection of Met RNAi into either fourth instar larvae or final instar larvae caused the precocious appearance of adult eyes, adult antennae and other features in the resulting pupae (Riddiford, 2010).

These studies show that JH has an endogenous function in regulating Drosophila metamorphosis, a specific example being in orchestrating the timing of differentiation events in the developing visual system. These effects of JH are primarily mediated through the Met pathway. JH also is necessary for normal larval growth and has another, as yet undefined, crucial role in prepupal development that prevents death at head eversion. The latter effect is not mediated through Met, indicating that JH might act through multiple pathways (Riddiford, 2010).


GENE STRUCTURE

Genomic length - 36 kb cDNA length - 5534

Bases in 5' UTR -1068

Exons - 6

Bases in 3' UTR - 1819


PROTEIN STRUCTURE

Three protein isoforms are encoded by EcR, designated ECR-A, ECR-B1 and ECR-B2. These proteins differ in their amino-terminal sequences but contain identical DNA binding domains and ligand binding domains. The A and B1 isoforms are encoded by overlapping transcription units that have different promotors and can be separately controlled. The N-terminal amino acids of ECR-A are coded for by three exons specific to that isoform, while both the DNA binding and ligand binding domains of ECR-A are coded for by exons shared with the other two isoforms. The B1 and B2 isoforms are encoded by mRNAs that derive from the EcR-B primary transcript by alternative splicing (Talbot, 1993).

Amino Acids - 878

Structural Domains

There are two conserved domains characteristic of steroid receptor superfamily members. The more N-terminal domain is a DNA-binding domain and the more C-terminal domain is a hormone-binding domain also implicated as a protein interaction domain (Koelle, 1991). EcR is a Class II member of the nuclear receptor superfamily, classified as such on the basis of its ability to heterodimerize with RXR (in Drosophila Ultraspiracle) and a its ability to bind to direct repeats. EcR is most closely related to the vertebrate Farnesoid X receptor (Mangelsdorf, 1995).

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3." Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).


Ecdysone receptor: Evolutionary homologs | Regulation | Targets of Activity | Protein interactions | Developmental Biology | Effects of Mutation | References

date revised: 28 MAY 97  

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