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).

GENE STRUCTURE

cDNA clone length - 6113

Exons - 4

Bases in 3' UTR - 1556

PROTEIN STRUCTURE

Amino Acids - Hr4-PA (1543 aa); Hr4-PB (1521 aa)

Structural Domains

For information about Drosophila nuclear receptors see the review by King-Jones (2005b).

A PCR approach has been used to isolate, from Bombyx mori, a cDNA encoding a novel orphan receptor (GRF) that is most closely related to Bombyx betaFTZ-F1 and to the vertebrate germ cell nuclear factor. The major GRF mRNA is detected in most tissues as an 8-kb transcript whose amount increases in concert with the circulating ecdysteroid concentration, but with a delay. The expression pattern of GRF is similar to that of the Bombyx homologue of the Drosophila early-late gene DHR3, and precedes that of betaFTZ-F1 in all stages and tissues examined. The GRF protein is thus likely to be required in many tissues, but in a temporally restricted manner suggesting that GRF has a well-defined function in the ecdysteroid-induced transcription cascade. The GRF protein binds in vitro to a single estrogen receptor half-site AGGTCA preceded by a 5'-TCA extension, and is therefore a potential co-regulator of the orphan receptors betaFTZ-F1 and DHR39 (Charles, 1999).

Fragments of EcR and USP were cloned from two insect cell lines, Sf21 and High Five cells (derived respectively from Spodoptera frugiperda and Trichoplusia ni), using a PCR-based approach employing degenerate primers designed on the basis of conserved regions of nuclear receptors, together with 5'- and 3'-RACE. An additional orphan nuclear receptor, HR4 fragment, was cloned from High Five cells. Comparison of these fragments with Manduca sexta counterparts shows that the cloned SfEcR [ecdysone receptor (EcR) from Sf21 cells] has high similarity to MsEcR-B1, whereas the cloned SfUSP [ultraspiracle (USP) from Sf21 cells] and TnUSP (USP from High Five cells) match more closely to MsUSP-2 than to MsUSP-1. The TnHR4 showed most similarity to a Bombyx mori GRF. While EcR and USP are constitutively expressed in both cell lines, HR4 is barely detectable by Northern blot analysis in High Five cells. Treatment with 20-hydroxyecdysone (20E) and agonist RH-5992 enhances transcription of EcR in both cell lines, while the transcription of USP is suppressed in High Five cells. Such suppressed USP transcription is not observed in Sf21 cells. Transcription of TnEcR can also be enhanced by ecdysone and 3-dehydroecdysone, whereas transcription of SfEcR is unchanged with these two ecdysteroid compounds. Induction of HR4 transcription is observed with 20E, RH-5992, ecdysone and 3-dehydroecdysone. The protein synthesis inhibitor, cycloheximide, superinduced expression of EcR and HR4 and restored the 20E/RH-5992-suppressed expression of TnUSP in the cells. Northern blot analysis also revealed that PCR, using degenerate USP primers, was able to amplify some other orphan nuclear receptors and their expression was inducible by 20E and RH-5992 and some of them were superinducible by cycloheximide (Chen, 2002).

During the last larval molt in Manduca sexta, a number of transcription factors are sequentially expressed. MHR4 is a transcription factor that belongs to the nuclear receptor superfamily and is a homolog of germ cell nuclear factor (GCNF)-related factors (GRFs) of Bombyx mori and Tenebrio molitor and is similar to a sequence found in the Drosophila genome. Unlike E75A and MHR3, whose mRNAs are induced when the ecdysteroid titer increases, the expression of MHR4 mRNA occurs transiently at the onset of the decline of ecdysteroid titer followed by ßFTZ-F1 mRNA expression when the ecdysteroid titer becomes low. When day 2 fourth epidermis is exposed to 20-hydroxyecdysone (20E) in vitro, MHR4 mRNA appears between 12 and 21 h, peaks at 24 h, and then declines. Using the protein synthesis inhibitors cycloheximide and anisomycin both in vivo and in vitro, it has been found that the MHR4 transcript is directly induced by 20E and requires the presence of 20E for its expression. The accumulation of MHR4 mRNA, however, does not occur until a 20E-induced inhibitory protein(s) disappears. This control of MHR4 expression is unique among the ecdysone-induced transcription factors. When the epidermis is cultured with 20E, ßFTZ-F1 mRNA is not induced until after the removal of 20E as previously found for Drosophila and the silkworm Bombyx mori. The presence of juvenile hormone had no effect on accumulation of either transcript (Hiruma, 2001).

In Drosophila, DHR3 activates ßFTZ-F1 mRNA expression and represses ecdysteroid-induced early gene expression such as that of E74A, E75A, and BRC. Yet in Manduca, relatively little MHR3 is present between 10 and 19 h after HCS when ßFTZ-F1 mRNA is increasing. Possibly MHR3 is initially activating the ßFTZ-F1 gene, but the presence of MHR4 suppresses this expression. Then when the 20E level declines below the threshold for MHR4 expression, ßFTZ-F1 mRNA can appear. This scenario would be similar to the complex control of MHR4 mRNA expression that has been shown in this study (Hiruma, 2001).

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93%-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization shows that a 7.5 kb MHR4 mRNA appears in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappears about 12 h later, then transiently reappears shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression is seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA is also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis shows that the >9.5 kb betaFTZ-F1 mRNA appears in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declines, then it disappears in the larval and pupal molts before the second peak of MHR4 appears. betaFTZ-F1 mRNA is also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs are found in the testis during the onset of spermatogenesis in the prepupal period (Weller, 2001).

date revised: 13 November 2005

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