InteractiveFly: GeneBrief

Hr4: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Studies in several insect species have suggested the orphan nuclear receptor encoded in Drosophila by DHR4 (CG16902) may contribute to the crossregulatory nuclear receptor network during the early stages of metamorphosis (Charles, 1999, Hiruma, 2001, Weller, 2001, Chen, 2002 and Sullivan, 2003). A critical determinant of insect body size is the time at which the larva stops feeding and initiates wandering in preparation for metamorphosis. No genes have been identified that regulate growth by contributing to this key developmental decision to terminate feeding. Mutations in the DHR4 orphan nuclear receptor result in larvae that precociously leave the food to form premature prepupae, resulting in abbreviated larval development that translates directly into smaller and lighter animals. In addition, DHR4 plays a central role in the genetic cascades triggered by the steroid hormone ecdysone at the onset of metamorphosis, acting as both a repressor of the early ecdysone-induced regulatory genes and an inducer of the ßFTZ-F1 midprepupal competence factor. It is proposed that DHR4 coordinates growth and maturation in Drosophila by mediating endocrine responses to achieve critical weight during larval development (King-Jones, 2005a).

Since the original proposal by Ashburner of the hierarchical model of ecdysone action, efforts have focused on identifying the postulated ecdysone-induced repressor of the early regulatory genes. It is propose that DHR4 is at least one of these unknown factors. DHR4 mutant prepupae exhibit both an efficient early gene repression observed upon ectopic DHR4 expression in late L3 and prolonged expression of EcR, E74, E75, and DHR3. Like DHR4, DHR3 is sufficient for early gene repression upon ectopic expression in late L3; however, DHR3 is not necessary for this response, suggesting that DHR3 and DHR4 act together, in a redundant manner, to direct early gene repression (King-Jones, 2005a).

A broad survey of DHR4 regulatory targets using microarray analysis demonstrates a wider role for this factor in gene repression at puparium formation. Among these DHR4 targets are known ecdysone-regulated genes such as Lsp1γ, Ddc, and Kr-h1 as well as 13 ecdysone-regulated muscle genes identified in an earlier microarray study as coordinately downregulated at pupariation. It is concluded that DHR4 function is not limited to early gene repression but, rather, acts more widely to downregulate gene expression at the onset of metamorphosis (King-Jones, 2005a).

Metamorphic functions for DHR4 are not restricted to puparium formation but also extend to prepupal stages through its essential role in βFTZ-F1 regulation. Like DHR3, DHR4 is required for maximal expression of this midprepupal competence factor. The effects seen on EcR, E74, and E75 transcription in 10 hr DHR4 mutant prepupae and the lethal phenotypes associated with DHR4 RNAi are indistinguishable from those seen in βFTZ-F1 mutants, suggesting that most if not all of the effects of DHR4 are channeled through βFTZ-F1 at this stage in development. Thus, as originally proposed based on the timing of DHR4 expression (Charles, 1999; Hiruma, 2001; Sullivan, 2003), this factor contributes to the crossregulatory interactions among orphan nuclear receptors in prepupae. Together with DHR3 and through βFTZ-F1, DHR4 directs appropriate genetic and biological responses to the prepupal pulse of ecdysone, ensuring that this response will be distinct from that induced by the hormone several hours earlier at pupariation (King-Jones, 2005a).

Despite an abundance of food, many DHR4¹ mutants stop feeding prematurely as early L3. Three lines of evidence indicate that most of these animals do not resume feeding: (1) once larvae have left the food and initiated gut clearing, they typically do not return to the food source. They will either pupariate or die as small larvae, likely reflecting whether or not they have achieved the critical weight necessary for pupariation. (2) Unlike their control counterparts, DHR4¹ mutant L3 can pupariate successfully despite being reared on cycloheximide-containing food. Although it is possible that DHR4¹ mutants have gained the ability to inactivate this drug, the interpretation is favored that they have stopped feeding altogether. (3) In a similar experiment, rather than arresting development like wild-type L3, DHR4¹ mutant L3 maintained on a sugar-only diet pupariate as if they were experiencing complete starvation in the absence of sugar. It is concluded that DHR4 is required for the proper duration of larval feeding. Moreover, defects in feeding behavior are likely to extend to all DHR4 mutant animals because even those larvae that pupariate at the normal time display changes in gene expression indicative of starvation (King-Jones, 2005a).

Drosophila larvae display two basic forms of behavior, either foraging for food or wandering in search of a substrate for pupariation. Foraging for food is intermittent, with animals continuing to feed, while wandering larvae stop feeding, purge their gut contents, display fat body autophagy, and eventually pupariate. The late-larval behavioral patterns and increased autophagy associated with wandering are similar to the phenotypes observed in DHR4¹ mutant L3, suggesting that these animals prematurely receive the cue to wander. In support of this proposal, precociously nonfeeding DHR4¹ mutant larvae are also seen to pupariate prematurely, while their feeding counterparts do not. It is concluded that DHR4 is required for the correct timing of the signal that triggers the cessation of feeding and the onset of wandering behavior (King-Jones, 2005a).

The control of insect body size is not simply a matter of growth control via insulin signaling and nutrition but, rather, includes a more profound determinant of size, the decision of when to stop growing and initiate maturation. This decision depends on the attainment of critical weight, which is defined as the minimum weight needed to successfully initiate a normal time course to pupariation. To date, no genetic studies have addressed this level of growth control. The characterization of DHR4 mutants implicates a critical role for this orphan nuclear receptor in assessing critical weight and determining the duration of larval development. DHR4 mutants begin to pupariate ~28 hr after the L2-to-L3 molt, almost a day earlier than wild-type animals. They pupariate at random times, independent of their state of gut clearing, with variable wandering phases. Premature DHR4 mutant prepupae are small, display fat body autophagy like normal prepupae, and express ecdysone-regulated genes specific for the prepupal stage in development although they are the chronological age of third-instar larvae (King-Jones, 2005a).

Studies in Manduca and other insects have shown that the attainment of critical weight triggers a drop in juvenile hormone (JH) titer, leading to release of the neuropeptide prothoracicotropic hormone (PTTH) and subsequent release of ecdysone from the ring gland. The resulting ecdysone pulse, in turn, initiates wandering behavior, with a subsequent high-titer ecdysone pulse triggering pupariation and maturation. Interestingly, removal of the Manduca corpora allata (the gland that produces JH) leads to premature activation of this endocrine cascade, resulting in precocious pupariation and the formation of small adults. Similarly, if JH levels are artificially increased by injecting hormone, PTTH secretion is delayed, and a prolonged larval feeding phase results in the formation of giant adults. These observations implicate a central role for JH in determining the duration of larval development and adult body size. Moreover, they provide a critical endocrinological link between growth and maturation and thus a point at which DHR4 could potentially intervene (King-Jones, 2005a).

The proposal that DHR4 plays a central role in transducing responses to the attainment of critical weight fits with its temporal and spatial patterns of expression. DHR4 is expressed in the ring gland of late L2 and throughout L3, with a lower level and complex pattern of expression in the L3 CNS. Curiously, DHR4 is most abundant in the cytoplasm of the prothoracic gland and is low or absent in the corpora allata, which produces JH. Although no nuclear localization or clear corpora allata expression was observed at any of the time points examined, low levels of expression or expression within a tight temporal window cannot be excluded. Moreover, the variable levels of cytoplasmic DHR4 that observed in the larval fat body and salivary glands at later stages of development suggest that nuclear/cytoplasmic shuttling may contribute to DHR4 regulatory function. Future tissue-specific rescue experiments should help to clarify the critical neuroendocrine cell types that may confer DHR4 function. Clearly, an intriguing possibility is that DHR4 either directly or indirectly regulates JH levels or JH signal transduction, contributing to the critical decrease in JH signaling that sets the endocrine cascade in action. Alternatively, DHR4 could regulate the neuroendocrine signaling that leads to ecdysone release. Identification of the Drosophila PTTH gene would provide a critical step toward testing this possibility. The fact that DHR4 encodes an orphan nuclear receptor, and thus may be modulated by binding one or more small lipophilic compounds, provides the most direct means of integrating this transcription factor into an endocrine circuit that defines the end of larval growth and the timing of pupariation (King-Jones, 2005a).

These studies provide a genetic and molecular framework for understanding critical weight and the coordination of larval feeding and pupariation -- key aspects of insect growth control that, in the past, have only been approached through physiological studies in nongenetic organisms. Future studies of DHR4 should provide new insights into the mechanisms by which growth is coupled to maturation, a key aspect of postembryonic development in all higher organisms (King-Jones, 2005a).

REGULATION

Targets of Activity

The spatial and temporal patterns of DHR4 expression provide a framework for interpreting phenotypic studies, suggesting that the growth defects arise from neuroendocrine functions of DHR4 during larval stages and the metamorphic requirement for DHR4 arises from its ecdysone-induced expression at pupariation. These two regulatory functions of DHR4 have been examined, focusing first on its roles in regulating gene expression at the onset of metamorphosis and then moving on to the mechanisms by which DHR4 regulates growth during larval stages (King-Jones, 2005a).

The temporal pattern of DHR4 expression and the prepupal lethality of DHR4¹ mutants suggest that this gene plays a critical role in ecdysone-regulated transcription at the onset of metamorphosis. To test this hypothesis, staged mutant and control L3 and prepupae were analyzed by Northern blot hybridization using probes to detect early (EcR, E74, E75), early-late (DHR3), or midprepupal (βFTZ-F1, Imp-L1) ecdysone-regulated gene expression. The EcR, E74A, E75A, and E75B early mRNAs are submaximally induced at puparium formation in DHR4¹ mutants, with EcR, E74, E75A, and E75B also failing to be repressed at the appropriate time. DHR3 induction appears normal in DHR4¹ mutants; however, the repression of this gene is significantly impaired. βFTZ-F1 expression is highly reduced in DHR4¹ mutant prepupae, with consequent defects in E74A and E75A induction in 10 hr prepupae, phenocopying a βFTZ-F1 mutant. Imp-L1 expression, in contrast, accumulates to wild-type levels in DHR4¹ mutant prepupae, with an ~2 hr delay, demonstrating that the DHR4¹ mutation does not have a general effect on midprepupal gene expression. Similar effects on ecdysone-regulated gene expression are seen when DHR4 function is disrupted by RNAi (King-Jones, 2005a).

As a further test of a role for DHR4 in repressing early gene expression, DHR4 was ectopically expressed in late L3 at the time when EcR and the classic early puff transcripts, E74A and E75A, are initially being induced by ecdysone. These transcripts are significantly downregulated under these conditions, resulting in almost complete suppression of the early transcriptional response to the hormone. Consistent with this effect on gene expression, most hsDHR4 transformants subjected to an identical heat-treatment regime failed to initiate metamorphosis, dying as L3 (King-Jones, 2005a).

Microarray analysis of DHR4¹ mutant prepupae and heat-treated hsDHR4 L3 were used to expand understanding of DHR4 function. Total RNA was isolated from P427 control and DHR4¹ mutant prepupae staged at 0, 4, and 8 hr after pupariation, spanning the peak of DHR4 expression. Only DHR4¹ mutant prepupae with a normal appearance were selected for this study. Also, a gain-of-function study was performed using w¹¹¹⁸ or w¹¹¹⁸; hsDHR4 L3 that were heat treated at ~10 hr prior to puparium formation and harvested 6 hr later. RNA was purified from each set of animals, labeled, and hybridized to Affymetrix Drosophila genome arrays. The resultant gene lists were compared with two data sets that are enriched for ecdysone-regulated genes: genes that significantly change their level of expression between 0 and 4 hr after pupariation in P427 control animals, a time when ecdysone is known to exert global effects on gene activity, and the only published microarray study of EcR mutants, using larval midguts. These comparisons revealed a robust correlation between genes that are normally downregulated in wild-type early prepupae, or EcR-dependent genes that are downregulated in the midgut, and genes that are upregulated in DHR4¹ mutants, suggesting a central role for DHR4 in the repression of ecdysone-regulated genes (King-Jones, 2005a).

Protein Interactions

Dynamic regulation of Drosophila nuclear receptor activity in vivo

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, a `ligand sensor' system was used to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. These responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation were seen that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control (Palanker, 2006).

Nine GAL4-LBD ligand sensor lines described in this study show tissue-specific patterns of activity during development: EcR, USP, ERR, FTZ-F1, HNF4, E78, DHR3, DHR38 and DHR96. These transgenic lines will serve as valuable tools for the genetic and molecular dissection of the receptors they represent, the pathways they regulate and the upstream factors and co-factors that modulate their activity. Specifically, the data reported here show that these lines can be used to: (1) indicate tissues and stages in which the corresponding NRs are likely to function; (2) indicate where endogenous ligands and co-factors are likely to be found; (3) suggest NR biological functions; (4) suggest possible NR-NR interactions, cascades and target genes; (5) evaluate putative co-factors and ligands; (6) screen chemical compound libraries for new agonists and antagonists; and (7) screen genetically for new pathway components. The results of these studies will also provide important insights into the ligands, co-factors and functions of their vertebrate NR homologues (Palanker, 2006).

Hormonal regulation of GAL4-LBD activation in the amnioserosa and yolk

Examination of the nine active ligand sensor lines provided a number of insights into possible relationships between their corresponding NRs. For example, although each of these ligand sensors displays unique temporal and spatial patterns of activity, activation in specific tissues and stages is common to many. These common sites of LBD activity may indicate shared functions, hierarchical or physical interactions, or related ligands. Examples of tissues that represent hotspots for GAL4-LBD activation include the amnioserosa, yolk, midgut and fat body (Palanker, 2006).

Each of these tissues, and the stages at which they score positively, correlates well with the presence of putative ligands. The yolk, for example, is believed to act as a storage site for maternally provided ecdysteroids during embryogenesis. Work with other insects has shown that these ecdysteroids are conjugated in an inactive form to vitellin proteins via phosphate bridges. Around mid-embryogenesis, these yolk proteins and phosphate bonds are cleaved, thereby releasing what are presumed to be the earliest biologically active ecdysteroids in the embryo. Interestingly though, GAL4-EcR activation in the amnioserosa depends on the disembodied (dib) gene, which encodes a cytochrome P450 enzyme required in the penultimate step of Ecdysone (E) biosynthesis, suggesting that the final steps in the linear E biosynthetic pathway are required for EcR function in this tissue and contradicting the prediction that this activity would be dependent on maternal ecdysteroids and independent of the zygotic biosynthetic machinery. The mechanisms by which dib exerts this essential role in providing an EcR ligand, however, remain to be determined (Palanker, 2006).

The response of the EcR and USP ligand sensors in the adjacent amnioserosa tissue shows that active ecdysteroids are not present until the hormone reaches the amnioserosa. A recent study of yolk-amnioserosa interactions has revealed dynamic transient projections that emanate from one tissue and contact the other, suggesting that there may be functional interactions between these two cell types. It is possible that these projections mediate the transfer of lipophilic ligand precursors from the yolk to the amnioserosa. This transfer, in turn, could determine the proper timing of EcR activation in the amnioserosa, thus triggering the major morphogenetic movements that establish the body plan of the first instar larva (Palanker, 2006).

Studies of the DHR38 receptor have demonstrated that it can be activated by a distinct set of ecdysteroids from those that activate EcR, through a novel mechanism that does not involve direct ligand binding. The activation of GAL4-DHR38 that was observed in the embryonic amnioserosa is consistent with this model of DHR38 regulation. First, exogenous 20E can only weakly activate GAL4-DHR38, relative to the strong ectopic activation seen with 20E on the EcR ligand sensor. This correlates with the weak ability of 20E to activate DHR38 in cell culture transfection assays relative to the strong 20E activation of EcR. Second, the DHR38 ligand sensor is activated in the amnioserosa earlier than the EcR construct, suggesting that it is responding to a different signal. It is possible that this signal is an ecdysteroid precursor that can act on DHR38 but not EcR - paralleling the ability of DHR38 to be activated by E, the precursor to 20E, which activates EcR. This putative ecdysteroid must be produced in a manner independent of the conventional ecdysteroid biosynthetic pathway, however, since a zygotic dib mutation has no effect on GAL4-DHR38 activation in the amnioserosa. Rather, this early activation may be due to maternal ecdysteroids that are conjugated and inactive in the yolk and transferred to the amnioserosa. These studies highlight the value of combining mutations in hormone biosynthesis with ligand sensor activation as a powerful means of dissecting hormone signaling pathways. Further studies of DHR38 function and regulation in embryos could help clarify the potential significance of this distinct activation response (Palanker, 2006).

DHR3, DHR38 and HNF4 ligand sensors appear to respond to metabolic signals Interestingly, the midgut continues to be a hotspot for ligand sensor activity long after it has engulfed the yolk during embryogenesis. This seems logical, as the midgut is responsible for most lipid absorption and release, and many vertebrate NRs are involved in fatty acid, cholesterol and sterol metabolism and homeostasis. The observed restriction of ligand sensor activity to a narrow group of cells located at the base of the gastric caeca is of particular interest. This is the site where nutrients in a feeding larva are absorbed into the circulatory system. The activation of DHR3, DHR38 and HNF4 ligand sensors in this region of the gastric caeca suggests that these receptors are activated by one or more small nutrient ligands. Moreover, this suggests that the corresponding receptors may exert crucial metabolic functions by acting as nutrient sensors (Palanker, 2006).

Further evidence of metabolic functions for DHR3, DHR38 and HNF4 arises from their ligand sensor activation patterns in the embryonic yolk and larval fat body. The yolk is the main nutrient source for the developing embryo and represents an abundant source of lipids, correlating with specific activation of DHR3, DHR38 and HNF4 ligand sensors in this cell type during embryogenesis. Upon hatching into a larva, the fat body acts as the main metabolic organ of the animal, functionally equivalent to the mammalian liver. Upon absorption by the gastric caeca, nutrients travel through the circulatory system and are absorbed by the fat body, where they are broken down and stored as triglycerides, glycogen and trehalose. Once again, the efficient activation of the DHR3, DHR38 and HNF4 ligand sensors in the fat body of metabolically active third instar larvae, and lack of sensor activity in non-feeding prepupae, supports the model that the corresponding NRs operate as metabolic sensors. This proposed function is consistent with the roles of their vertebrate orthologs. Mammalian ROR, the ortholog of DHR3, binds cholesterol and plays a crucial role in lipid homeostasis. Similarly, mammalian HNF4 can bind C14-18 fatty acids, is required for proper hepatic lipid metabolic gene regulation and lipid homeostasis, and is associated with human Maturity-Onset Diabetes of the Young (MODY1). The studies described here suggest that DHR3 and HNF4 may perform similar metabolic functions in flies, defining a new genetic model system for characterizing these key NRs (Palanker, 2006).

New insights into the regulation of Drosophila xenobiotic responses

Several vertebrate NRs play a central role in xenobiotic responses by directly binding toxic compounds and inducing the expression of key detoxification enzymes such as cytochrome P450s and glutathione transferases. Ligand sensor activation observed in the gut, epidermis, tracheae or fat body could represent xenobiotic responses insofar as toxic compounds could enter the organism through any of these tissues. Directed screens that test xenobiotic compounds for their ability to activate Drosophila NR ligand sensors will provide a means of identifying potential xenobiotic receptors. Understanding these response systems, in turn, could facilitate the production of insect resistant crops and the development of more effective pesticides (Palanker, 2006).

Like its vertebrate orthologs SXR/PXR and CAR, DHR96 has been recently shown to act in insect xenobiotic responses, providing resistance to the sedative effect of phenobarbital and lethality caused by chronic exposure to DDT (King-Jones et al., 2006). DHR96 is also required for the proper transcriptional response of a subset of phenobarbital-regulated genes. DHR96 can be activated by the CAR-selective agonist CITCO, suggesting that it may be regulated in a manner similar to that of the vertebrate xenobiotic receptors. It is also interesting to note that angelicin was found to activate the USP ligand sensor fusion. Angelicin is an angular furanocoumarin that has the furan ring attached at the 7,8 position of the benz-2-pyrone nucleus. Detailed studies have shown that insects have adapted to the presence of furanocoumarins in their host plants by expressing specific cytochrome P450 enzymes that detoxify these compounds. In the black swallowtail butterfly (Papilio polyxenes), furanocoumarins induce the transcription of P450 genes through an unknown regulatory pathway, thereby aiding in xenobiotic detoxification. The observation that angelicin, and not the linear furanocoumarins 8-methoxypsoralen (xanthotoxin) or 5-methoxypsoralen (bergapten), can activate GAL4-USP suggests that NRs may mediate this detoxification response and may be capable of distinguishing between the linear and angular chemical forms. It is possible that USP may mediate this effect on its own or, more likely, as a heterodimer partner with another NR. Similarly, the activation of GAL4-USP by fenoxycarb may represent a xenobiotic response. This activation, however, is weaker and more variable than the activation observed with angelicin. Identifying other factors that mediate xenobiotic responses in Drosophila would provide a new basis for dissecting the control of detoxification pathways in higher organisms (Palanker, 2006).

ERR activity appears to be regulated by a temporally restricted and widespread signal

GAL4-ERR displays a remarkable switch in activity during mid-embryogenesis, from strong activation in the myoblasts to specific and strong activation in the CNS. The ERR ligand sensor also shows widespread transient activation in the mid-third instar, a time when larval ERR gene expression begins, together with a global switch in gene expression that prepares the animal for entry into metamorphosis 1 day later. This so-called mid-third instar transition includes upregulation of EcR, providing sufficient receptor to transduce the high titer late larval 20E hormone pulse, upregulation of the Broad-Complex, which is required for entry into metamorphosis, and induction of the genes that encode a polypeptide glue used to immobilize the puparium for metamorphosis. The signal and receptor that mediate this global reprogramming of gene expression remain undefined. The widespread activation of GAL4-ERR at this stage raises the interesting possibility that it may play a role in this transition. Moreover, given that the only ligand sensors to display widespread transient activation are EcR and USP, in response to 20E, it is possible that this response reflects a systemic mid-third instar pulse of a ERR hormone. Vertebrate members of the ERR family can bind the synthetic estrogen diethylstilbestrol and the selective ER modulator tamoxifen, as well as its metabolite, 4-hydroxytamoxifen, suppressing their otherwise constitutive activity in cell culture. This is notably different from the highly restricted patterns of ERR ligand sensor activity that was detected in Drosophila, which suggests that it does not function as a constitutive activator in vivo. Rather, it is envisioned that the patterns of ERR activation are precisely modulated by protein co-factors and/or one or more ligands to direct the dynamic shifts in activation that are detect during embryogenesis and third instar larval development. Functional studies of the Drosophila homolog of the ERR receptor family may provide a basis for understanding these dynamic shifts in LBD activation, as well as revealing a natural ligand for this NR (Palanker, 2006).

DEVELOPMENTAL BIOLOGY

Northern analysis of RNA samples isolated from staged wild-type animals reveals that DHR4 is induced in late third-instar larvae (L3), peaks in expression ~2-4 hr after puparium formation, and shifts from a 10 kb to a 9.5 kb mRNA in 4-6 hr prepupae. Upon prolonged exposure, a low level of DHR4 mRNA can also be detected in the L3 time points. The dramatic upregulation of DHR4 just prior to pupariation suggests that it is induced by the late-larval ecdysone pulse. To test this hypothesis, Northern analysis was performed using RNA samples isolated from L3 organs cultured in the presence of either 20E, the protein synthesis inhibitor cycloheximide, or both 20E and cycloheximide. DHR4 is induced 1.5 hr after 20E addition, with maximum levels of expression by 3-4 hr. This induction is delayed and reduced in the presence of cycloheximide, indicating that DHR4 is induced directly by 20E and that its maximal expression requires 20E-induced protein synthesis. This requirement is consistent with the delayed expression of DHR4 in response to the late-larval ecdysone pulse and defines its regulation as an early-late response to the hormone. Northern blot hybridization using RNA isolated from late L3 organs cultured for 90 min with different concentrations of 20E revealed that DHR4 requires at least 5 × 10^-8 M 20E for its expression and shows a half-maximal response at ~2.5 × 10^-7 M. Both values are virtually identical to those of the DHR3 and E78 early-late 20E-inducible genes (King-Jones, 2005a).

In an effort to elucidate the roles of DHR4 in growth and metamorphosis, antibodies were raised against the DHR4 protein and used to stain tissues dissected from staged larvae and prepupae. No DHR4 protein was detected during late L2 or L3 stages, except for strong constitutive expression in the ring gland. It is likely that this expression accounts for the background level of DHR4 mRNA detectable during larval stages by Northern blot hybridization. Lower DHR4 expression could also be detected in specific neurons of the central nervous system (CNS) of mid and late L3. The ring gland stain was significantly reduced in larvae expressing DHR4 dsRNA, confirming the specificity of this signal. Results were more variable in DHR4¹ mutants, however, with some larvae showing highly reduced DHR4 protein in the ring gland and other animals showing little effect. It is interesting to note that these effects on ring gland expression upon induction of RNAi or in DHR4¹ mutants correlate with the penetrance of growth defects in these genetic backgrounds, with highly penetrant effects upon RNAi and more variability in the DHR4¹ mutants, suggesting that the growth defects may arise from an endocrine function of DHR4. Close examination of the ring glands in wild-type animals reveals that DHR4 protein is most abundant in the cytoplasm of the prothoracic gland, with little or no protein in the nuclei of these cells or in the corpora allata or corpora cardiaca. The high relative level of protein in the prothoracic cell cytoplasm, however, makes it impossible to determine whether DHR4 is absent from other regions of the ring gland (King-Jones, 2005a).

The distribution of DHR4 protein expands at puparium formation to include the fat body and salivary glands. DHR4 protein is located primarily in the nucleus of these tissues, consistent with its function as a transcription factor, although some DHR4 protein can also be detected in the cytoplasm of salivary gland and fat body cells. Tissues dissected from DHR4¹ mutant prepupae show no nuclear signal, confirming the specificity of the antibody. Little or no DHR4 protein was detected in the imaginal discs, muscle, and midgut of early prepupae (King-Jones, 2005a).

Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development

Many of the 21 members of the nuclear receptor superfamily in Drosophila are transcriptionally regulated by the steroid hormone ecdysone and play a role during the onset of metamorphosis, including the EcR/USP ecdysone receptor heterodimer. The temporal patterns of expression for all detectable nuclear receptor transcripts were examined throughout major ecdysone-regulated developmental transitions in the life cycle: embryogenesis, a larval molt, puparium formation, and the prepupal-pupal transition. An unexpected close temporal relationship was found between DHR3, E75B, and betaFTZ-F1 expression after each major ecdysone pulse examined, reflecting the known cross-regulatory interactions of these genes in prepupae and suggesting that they act together at other stages in the life cycle. In addition, E75A, E78B, and DHR4 are expressed in a reproducible manner with DHR3, E75B, and betaFTZ-F1, suggesting that they intersect with this regulatory cascade. Finally, known ecdysone-inducible primary-response transcripts are coordinately induced at times when the ecdysteroid titer is low, implying the existence of novel, as yet uncharacterized, temporal signals in Drosophila (Sullivan, 2003).

Total RNA was isolated from two independent collections of embryos staged at 2-h intervals throughout the 24 h of Drosophila embryonic development. Five Northern blots were prepared using equal amounts of RNA from each time point. These blots were sequentially hybridized, stripped, and rehybridized with radioactive probes derived from each of the 21 nuclear receptor genes encoded by the Drosophila genome. This approach allowed the generation of time courses of nuclear receptor gene expression that could be directly compared between family members. The transcripts detected are consistent with reported sizes. Transcripts from eight nuclear receptor genes were not detectable during embryonic development: E75C, E78, CG16801, DHR38, DHR83, dsf, eg, and svp (Sullivan, 2003).

Transcripts from nine nuclear receptor genes can be detected at the earliest time point (0-2 h): usp, EcR-A, FTZ-F1, DHR39, DHR78, DHR96, dERR, dHNF-4, and tll. This expression is consistent with the known maternal contribution of usp, EcR, and FTZ-F1. The observation that transcripts from DHR39, DHR78, DHR96, dERR, and dHNF-4 are undetectable by the next time point examined (2-4 h) suggests that these mRNAs are maternally loaded and rapidly degraded. EcR-B and usp transcripts are induced in early embryos, up-regulated at 6-8 h after egg laying (AEL), and maintain expression through the end of embryogenesis, with down-regulation of EcR-B in late embryos. EcR-A, in contrast, is expressed for a relatively brief temporal window, at 8-14 h AEL (Sullivan, 2003).

Six nuclear receptor genes are expressed in brief intervals during midembryonic stages. DHR39 and E75A are initially induced at 4-6 and 6-8 h AEL, respectively, and peak at 8-12 h AEL. This is followed by induction of DHR3, DHR4, and E75B at 8-12 h AEL, followed by ßFTZ-F1 expression at 12-18 h AEL. DHR39 appears to exhibit an expression pattern reciprocal to that of ßFTZ-F1, with lowest levels of mRNA at 14-16 h AEL and reinduction at 16-18 h as ßFTZ-F1 is repressed. This is followed by a second peak of E75A transcription at 18-22 h AEL (Sullivan, 2003).

A second group of nuclear receptors, DHR78, DHR96, dHNF-4, and dERR, is more broadly expressed at low levels throughout embryogenesis. DHR78 accumulates above its constant low level of expression between 8 and 14 h AEL. dERR exhibits an apparent mRNA isoform switch between 14 and 18 h AEL. dHNF-4 regulation also appears complex, with two size classes of mRNA induced at approximately 8-10 h AEL. While the 4.6-kb dHNF-4 mRNA is expressed throughout embryogenesis, the 3.3-kb mRNA is down-regulated at 14-16 h AEL. This timing is consistent with observations that dHNF-4 is expressed primarily in the embryonic midgut, fat body, and Malpighian tubules. Finally, nuclear receptors known to exert essential functions in patterning the early embryo, tll, kni, and knrl, are expressed predominantly during early stages (Sullivan, 2003).

Two genes that are not members of the nuclear receptor superfamily, BR-C and E74, were also examined in this study, as transcriptional markers for ecdysone pulses during development. Unexpectedly, both of these genes are induced late in embryogenesis, several hours after the rise in ecdysone titer at 6 h AEL. An approximately 7-kb BR-C transcript is induced at 10-12 h AEL and is present through the end of embryogenesis while E74B is induced at 14-16 h AEL and repressed as E74A is expressed from 16-20 h AEL. This BR-C expression pattern is consistent with the identification of the BR-C Z3 isoform in specific neurons of the embryonic CNS (Sullivan, 2003).

First-instar larvae were synchronized as they molted to the second instar, aged and harvested at 4-h intervals throughout second-instar larval development. Two Northern blots were prepared using equal amounts of total RNA isolated from a single collection of animals. Each blot was sequentially hybridized, stripped, and rehybridized to detect nuclear receptor transcription. The following transcripts were not detectable during the second instar: E75C, dERR, CG16801, DHR83, dsf, eg, svp, tll, kn, and knrl (Sullivan, 2003).

EcR-B expression is induced in mid-second-instar larvae, but does not reach maximum levels until 68-72 h AEL, just before the molt. In contrast, usp is expressed throughout the instar. A sequential pattern of nuclear receptor expression is observed that resembles the pattern seen in midembryogenesis. DHR39 and E75A are expressed in the early second instar. This is followed by induction of E75B, E78B, DHR3, and DHR4, followed by expression of ßFTZ-F1 at the end of the instar. DHR39 again shows a pattern that is approximately reciprocal with ßFTZ-F1, with highest levels during the first half of the instar. Similarly, DHR78, DHR96, and dHNF-4 exhibit broad expression patterns throughout second-instar larval development. E74A, E75A, and DHR38 are coordinately up-regulated with EcR-B at the end of the instar, between 64-72 h AEL. Finally, an approximately 9-kb BR-C transcript is detected throughout the second-larval instar (Sullivan, 2003).

Nuclear receptor gene expression was also examined throughout the third larval instar and into the early stages of metamorphosis, encompassing the ecdysone-triggered larval-to-prepupal and prepupal-to-pupal transitions. Third-instar larvae were staged relative to the molt from the second instar and harvested at 4-h intervals throughout the 48 h of the instar. Prepupae were synchronized relative to puparium formation (±15 min) and harvested at 2-h intervals up to 16 h after puparium formation (APF). Total RNA was isolated from whole animals and analyzed by Northern blot hybridization. Five blots were prepared from two independent collections of animals. These blots were sequentially hybridized, stripped, and rehybridized to detect nuclear receptor gene expression. The following transcripts were not detectable during third-instar larval or prepupal stages: CG16801, DHR83, dsf, eg, svp, tll, kn, and knrl (Sullivan, 2003).

Most nuclear receptor genes show little or no detectable expression in early and mid-third-instar larvae, a time when the ecdysone titer is low. Similar to the pattern seen in second-instar larvae, usp is expressed at relatively low levels throughout the instar and up-regulated at puparium formation, while EcR-B is induced at approximately 100 h AEL and rapidly down-regulated at puparium formation. This is followed by a sequential pattern of nuclear receptor expression similar to that seen at earlier stages. DHR39, E75A, and E78B are induced at 116-120 h AEL, in concert with the late larval ecdysone pulse, followed by maximum accumulation of E75B, DHR3, and DHR4 at 0-4 h APF. ßFTZ-F1 is expressed from 6-10 h APF, with a pattern that is approximately reciprocal to that of DHR39. EcR-A is expressed in parallel with E75B, DHR3, and DHR4 in midprepupae, similar to their coordinate expression during embryogenesis (Sullivan, 2003).

DHR78, DHR96, and dHNF-4 continue to exhibit broad expression profiles throughout third-instar larval and prepupal development. An E75 isoform not detected in embryos or second-instar larvae, E75C, is also detectable at low levels throughout most of the third instar and up-regulated in correlation with the late-larval and prepupal pulses of ecdysone. DHR38 is detectable at very low levels in early third-instar larvae, in synchrony with the early induction of E74B and BR-C. E74B is repressed, E74A is induced, and BR-C transcripts are up-regulated in late third-instar larvae, in synchrony with the late-larval ecdysone pulse. The prepupal pulse of ecdysone occurs at 10-12 h APF, marking the prepupal-to-pupal transition. EcR-A, E75A, E78B, DHR4, dERR, E75C, dHNF-4, and E74A are all induced at 10-12 h APF, in apparent response to this hormone pulse. These results are consistent with a microarray analysis of gene expression at the onset of metamorphosis where the temporal profiles of about half of these genes have been reported (Sullivan, 2003).

Most nuclear receptors can be divided into one of four classes based on this study: (1) those that are expressed exclusively during early embryogenesis (kni, knrl, tll); (2) those that are expressed throughout development (usp, DHR78, DHR96, dHNF-4); (3) those that are expressed in a reproducible temporal cascade at each stage tested (E75A, E75B, DHR3, DHR4, FTZ-F1, DHR39), and (4) those that are undetectable in these assays (CG16801, DHR83, dsf, eg, svp) (Sullivan, 2003).

Three nuclear receptor genes appear to be expressed exclusively during early embryogenesis: kni, knrl, and tll. This restricted pattern of expression fits well with the functional characterization of these genes, which have been shown to act as key determinants of embryonic body pattern. Eight genes (usp, EcR, FTZ-F1, DHR39, DHR78, DHR96, dERR, and dHNF-4) were identified that appear to have maternally deposited transcripts and thus possible embryonic functions. Indeed, maternal functions have been defined for usp, EcR, and alphaFTZ-F1 (Sullivan, 2003).

Four nuclear receptor genes are broadly expressed through all stages examined: usp, DHR78, DHR96, and dHNF-4. dHNF-4 mRNA is first detectable at 6-10 h AEL, as the ecdysone titer begins to rise. In addition, peaks of dHNF-4 expression are seen at 0, 12, and 16 h APF, in synchrony with the E74 and E75C early ecdysone-inducible genes. These observations raise the interesting possibility that this orphan nuclear receptor is regulated by ecdysone (Sullivan, 2003).

DHR38 transcripts are difficult to detect in these assays. This is consistent with studies which used RT-PCR or riboprobes for this purpose. Nonetheless, DHR38 mRNA can be detected during third-instar larval development, consistent with the widespread expression reported in earlier studies. DHR38 expression peaks at late pupal stages, consistent with its essential role in adult cuticle formation (Sullivan, 2003).

dERR and E75C display related temporal profiles of expression that do not fit with other nuclear receptor genes described in this study. Both of these genes are specifically transcribed during prepupal development, with increases in expression at 0 and 10-12 h APF. dERR, but not E75C, is also expressed during embryogenesis, with an initial induction at approximately 6 h AEL. These increases occur in synchrony with ecdysone pulses, suggesting that these orphan nuclear receptor genes are hormone inducible, although in a stage-specific manner. Further studies of dERR regulation, as well as a genetic analysis of this locus, are currently in progress (Sullivan, 2003).

Interactions between the DHR3 and E75B orphan nuclear receptors contribute to appropriate ßFTZ-F1 regulation during the onset of metamorphosis. DHR3 is both necessary and sufficient to induce ßFTZ-F1 and appears to exert this effect directly, through two response elements in the ßFTZ-F1 promoter. E75B can heterodimerize with DHR3 and is sufficient to block the ability of DHR3 to induce ßFTZ-F1. These three factors thus define a cross-regulatory network that contributes to the timing of ßFTZ-F1 expression in midprepupae. ßFTZ-F1, in turn, acts as a competence factor that directs the appropriate genetic and biological responses to the prepupal pulse of ecdysone. The patterns of DHR3, E75B, and ßFTZ-F1 expression observed at the onset of metamorphosis are consistent with these regulatory interactions as well as the expression patterns reported in earlier studies (Sullivan, 2003).

Unexpectedly, the tight linkage of DHR3, E75B, and ßFTZ-F1 expression seen at the onset of metamorphosis is recapitulated at earlier stages, after each of the major ecdysone pulses examined, in midembryogenesis and second-instar larval development. This observation suggests that the regulatory interactions between these receptors is not restricted to metamorphosis, but rather may recur in response to each ecdysone pulse during development. It is possible that this regulatory cascade contributes to cuticle deposition, which is dependent on ecdysone signaling in embryos, larvae, and prepupae. In support of this proposal, DHR3 and ßFTZ-F1 mutants exhibit defects in larval molting, suggesting that they act together to regulate this early ecdysone response (Sullivan, 2003).

Three other orphan nuclear receptor genes, E75A, DHR4, and DHR39, are expressed in concert with DHR3, E75B, and ßFTZ-F1, after the embryonic, second-instar, and third-instar ecdysone pulses. A peak of E75A expression marks the start of each genetic cascade, correlating with the rising ecdysone titer in 6- to 8-h embryos, the first half of the second instar, and in late third-instar larvae. This is followed by DHR3, E75B, and DHR4 expression which, in turn, is followed by a burst of ßFTZ-F1 expression. E78B is expressed in synchrony with DHR4 in late second and third-instar larvae, but not in embryos. These patterns of expression raise the interesting possibility that E75A, DHR4, and E78B may intersect with the cross-regulatory network defined for DHR3, E75B, and ßFTZ-F1. E75B and E78B are related to the Rev-erb vertebrate orphan nuclear receptor and are both missing their DNA binding domain. E75B and E78B null mutants are viable and fertile, suggesting that they exert redundant regulatory functions. E75A mutants die during larval stages, with no known direct regulatory targets. DHR4 mutants have not yet been described, although recent work indicates that this gene exerts essential roles in genetic and biological responses to the late larval ecdysone pulse. Further functional studies of these nuclear receptor genes should provide insight into their possible contribution to the regulatory circuit defined by DHR3, E75B, and ßFTZ-F1 (Sullivan, 2003).

Interestingly, DHR39 displays a reproducible pattern of expression that is inversely related to that of ßFTZ-F1, defining possible repressive interactions. DHR39 and ßFTZ-F1 have a similar DNA binding domain (63% identity) and bind to identical response elements, suggesting that they may exert cross-regulatory interactions. Moreover, DHR39 can repress transcription through the same response element that is activated by ßFTZ-F1. It would be interesting to determine whether the reciprocal patterns of DHR39 and ßFTZ-F1 expression during development is of functional significance (Sullivan, 2003).

The transcription of BR-C, EcR, E74, and E75 has been extensively characterized during the onset of metamorphosis, due to their rapid and direct regulation by the steroid hormone ecdysone at this stage in development. Surprisingly, however, their expression appears to be disconnected from the high-titer ecdysteroid pulses during embryonic and second-instar larval stages. As expected, EcR is induced early in embryonic development, in coincidence with the rising ecdysone titer at 4-10 h AEL, with EcR-B transcripts appearing first followed by EcR-A. BR-C mRNA, however, is not seen until 10-12 h AEL and E74B mRNA is induced even later, at 14-16 h AEL, when the ecdysteroid titer has returned to a basal level. Both EcR-B and E74B are repressed from 16-20 h AEL as E74A and E75A are induced, a switch that has been linked to the high-titer ecdysone pulse in late third-instar larvae; however, this response occurs during late embryogenesis when the ecdysteroid titer is low. A similar observation has been made for E75A expression in the Manduca dorsal abdominal epidermis, where a brief burst of E75A mRNA is detected immediately before pupal ecdysis, after the ecdysteroid titer has returned to basal levels (Sullivan, 2003).

It thus seems likely that the second instar ecdysone pulse occurs during the first half of the instar. This profile is consistent with the early induction of E75A. EcR-B and E74A, however, are not induced until the second half of the second instar, with a peak at the end of the instar. BR-C mRNA levels remain steady throughout the second instar. Finally, EcR-B, E74B, and BR-C are induced in early to mid-third-instar larvae, a time when one or more low-titer ecdysone pulses may occur. It is curious that E74B is poorly expressed relative to E74A during embryonic and second-instar larval stages, disconnecting its expression from that of EcR. This pattern is not seen in studies that focused on the onset of metamorphosis. Taken together, the temporal profiles of early gene expression (EcR, BR-C, E74, E75A) during late embryonic and late second-instar larval stages appear to be unlinked to the known ecdysteroid pulses at these stages. This could indicate that these promoters are activated in a hormone-independent manner at these stages in the life cycle. Alternatively, these ecdysone primary-response genes may be induced by a novel temporal signal that remains to be identified (Sullivan, 2003).

Several lines of evidence indicate that 20-hydroxyecdysone is not the only temporal signal in Drosophila. A major metabolite of this hormone, 3-dehydro-20-hydroxyecdysone, was shown to be as effective as 20-hydroxyecdysone in inducing target gene transcription in the hornworm, Manduca sexta. Similarly, 3-dehydro-20-hydroxyecdysone is more efficacious than 20-hydroxyecdysone in inducing Fbp-1 transcription in the Drosophila larval fat body. A high-titer pulse of alpha-ecdysone, the precursor to 20-hydroxyecdysone, can drive the extensive proliferation of neuroblasts during early pupal development in Manduca. This is the first evidence that alpha-ecdysone is responsible for a specific response in insects. It is unlikely, however, that this signal is transduced through the EcR/USP heterodimer, which shows only very low transcriptional activity in response to this ligand. Rather, recent evidence indicates that alpha-ecdysone may activate DHR38 through a novel mechanism that does not involve direct hormone binding (Sullivan, 2003).

Studies of ecdysteroid-regulated gene expression in Drosophila have also provided evidence for hormone signaling pathways that may act independently of 20-hydroxyecdysone. Several studies have identified a large-scale switch in gene expression midway through the third larval instar, an event that has been referred to as the mid-third-instar transition. It is not clear whether this response is triggered by a low-titer ecdysteroid pulse, another hormonal signal, or in a hormone-independent manner. Similarly, the let-7 and miR-125 micro-RNAs are induced at the onset of metamorphosis in Drosophila in tight temporal correlation with the E74A early mRNA, but not in apparent response to 20-hydroxyecdysone. These studies indicate that 20-hydroxyecdysone cannot act as the sole temporal regulator during the Drosophila life cycle (Sullivan, 2003).

REFERENCES

Search PubMed for articles about Drosophila Hr4

Charles, J.-P., Shinoda, T. and Chinzei, Y. (1999). Characterization and DNA-binding properties of GRF, a novel monomeric binding orphan receptor related to GCNF and ßFTZ-F1, Eur. J. Biochem. 266: 181-190. 10542063

Chen, J. H., Turner, P. C. and Rees, H. H. (2002). Molecular cloning and induction of nuclear receptors from insect cell lines: Insect. Biochem. Mol. Biol. 32: 657-667. 12020840

Hiruma, K and Riddiford, L. M. (2001). Regulation of transcription factors MHR4 and ßFTZ-F1 by 20-hydroxyecdysone during a larval molt in the tobacco hornworm, Manduca sexta, Dev. Biol. 232: 265-274. 11254363

King-Jones, K., Charles, J. P., Lam, G. and Thummel, C. S. (2005a). The ecdysone-induced DHR4 orphan nuclear receptor coordinates growth and maturation in Drosophila. Cell 121: 773-784. 15935763

King-Jones, K. and Thummel, C. S. (2005b). Nuclear receptors--a perspective from Drosophila. Nat. Rev. Genet. 6(4): 311-23. 15803199

King-Jones, K., Horner, M. A., Lam, G. and Thummel, C. S. (2006). The DHR96 nuclear receptor regulates xenobiotic responses in Drosophila. Cell Metab. 4: 37-48. Medline abstract: 16814731

Palanker, L., et al. (2006). Dynamic regulation of Drosophila nuclear receptor activity in vivo. Development 133(18): 3549-62. Medline abstract: 16914501

Sullivan, A. A. and Thummel, C. S. (2003). Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development. Mol. Endocrinol. 17(11): 2125-37. 12881508

Weller, J., et al. (2001). Isolation and developmental expression of two nuclear receptors, MHR4 and ßFTZ-F1, in the tobacco hornworm, Manduca sexta. Insect Biochem. Mol. Biol. 31: 827-837. 11378418

date revised: 12 June 2021

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